Feeds:
Posts
Comments

Posts Tagged ‘aerobic glycolysis’


Therapeutic Implications for Targeted Therapy from the Resurgence of Warburg ‘Hypothesis’

Writer and Curator: Larry H. Bernstein, MD, FCAP 

(Note that each portion of the discussion is followed by a reference)

It is now a time to pause after almost a century of a biological scientific discoveries that have transformed the practice of medicine and impacted the lives of several generations of young minds determined to probe the limits of our knowledge.  In the century that we have entered into the scientific framework of medicine has brought together a difficult to grasp evolution of the emergence of human existence from wars, famine, droughts, storms, infectious diseases, and insect born pestilence with betterment of human lives, only unevenly divided among societal classes that have existed since time immemorial. In this short time span there have emerged several generations of physicians who have benefited from a far better medical education that their forebears could have known. In this expansive volume on cancer, we follow an incomplete and continuing challenge to understand cancer, a disease that has become associated with longer life spans in developed nations.

While there are significant improvements in the diagnosis and treatment of cancers, there is still a personal as well as locality factor in the occurrence of this group of diseases, which has been viewed incorrectly as a “dedifferentiation” of mature tissue types and the emergence of a cell phenotype that is dependent on glucose, reverts to a cancer “stem cell type” (loss of stemness), loses cell to cell adhesion, loses orderly maturation, and metastasizes to distant sites. At the same time, physician and nurses are stressed in the care of patients by balancing their daily lives and maintaining a perspective.

The conceptual challenge of cancer diagnosis and management has seemed insurmountable, but owes much to the post World War I activities of Otto Heinrich Warburg. It was Warburg who made the observation that cancer cells metabolize glucose by fermentation in much the way Pasteur 60 years earlier observed fermentation of yeast cells. This metabolic phenomenon occurs even in the presence of an oxygen supply, which would provide a huge deficit in ATP production compared with respiration. The cancer cell is “addicted to glucose” and produced lactic acid. Warburg was awarded the Nobel Prize in Medicine for this work in 1931.

In the last 15 years there has been a resurgence of work on the Warburg effect that sheds much new light on the process that was not previously possible, with significant therapeutic implications.  In the first place, the metabolic mechanism for the Warburg effect was incomplete even at the beginning of the 21st century.  This has been partly rectified with the enlightening elucidation of genome modifications, cellular metabolic regulation, and signaling pathways.

The following developments have become central to furthering our understanding of malignant transformation.

  1. There is usually an identifiable risk factor, such as, H. pylori, or of a chronic inflammatory state, as in the case of Barrett’s esophagus.
  2. There are certain changes in glucose metabolism that have been unquestionably been found in the evolution of this disease. The changes are associated with major changes in metabolic pathways, miRN signaling, and the metabolism geared to synthesis of cells with an impairment of the cell death cycle. In these changes, mitochondrial function is central to both the impaired respiration and the autophagy geared to the synthesis of cancer cells.

The emergence of this cell prototype is characterized by the following, again related to the Warburg effect:

  1. Cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis
  2. The mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis.
  3. Cancer cells tend to express a partially inhibited splice variant of pyruvate kinase (PK-M2), leading to decreased pyruvate production.
  4. The two proteins that mediate pyruvate conversion to lactate and its export, M-type lactate dehydrogenase and the monocarboxylate transporter MCT-4, are commonly upregulated in cancer cells leading to decreased pyruvate oxidation.
  5. The enzymatic step following mitochondrial entry is the conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex. Cancer cells frequently exhibit increased expression of the PDH kinase PDK1, which phosphorylates and inactivates PDH. This PDH regulatory mechanism is required for oncogene induced transformation and reversed in oncogene-induced senescence.
  6. The PDK inhibitor dichloroacetate has shown some clinical efficacy, which correlates with increased pyruvate oxidation. One of the simplest mechanisms to explain decreased mitochondrial pyruvate oxidation in cancer cells, a loss of mitochondrial pyruvate import, has been observed repeatedly over the past 40 years. This process has been impossible to study at a molecular level until recently, however, as the identities of the protein(s) that mediate mitochondrial pyruvate uptake were unknown.
  7. The mitochondrial pyruvate carrier (MPC) as a multimeric complex that is necessary for efficient mitochondrial pyruvate uptake. The MPC contains two distinct proteins, MPC1 and MPC2; the absence of either leads to a loss of mitochondrial pyruvate uptake and utilization in yeast, flies, and mammalian cells.

A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth

John C. Schell, Kristofor A. Olson, Lei Jiang, Amy J. Hawkins, et al.
Molecular Cell Nov 6, 2014; 56: 400–413.
http://dx.doi.org/10.1016/j.molcel.2014.09.026

In addition to the above, the following study has therapeutic importance:

Glycolysis has become a target of anticancer strategies. Glucose deprivation is sufficient to induce growth inhibition and cell death in cancer cells. The increased glucose transport in cancer cells has been attributed primarily to the upregulation of glucose transporter 1 (Glut1),  1 of the more than 10 glucose transporters that are responsible for basal glucose transport in almost all cell types. Glut1 has not been targeted until very recently due to the lack of potent and selective inhibitors.

First, Glut1 antibodies were shown to inhibit cancer cell growth. Other Glut1 inhibitors and glucose transport inhibitors, such as fasentin and phloretin, were also shown to be effective in reducing cancer cell growth. A group of inhibitors of glucose transporters has been recently identified with IC50 values lower than 20mmol/L for inhibiting cancer cell growth. However, no animal or detailed mechanism studies have been reported with these inhibitors.

Recently, a small molecule named STF-31 was identified that selectively targets the von Hippel-Lindau (VHL) deficient kidney cancer cells. STF-31 inhibits VHL deficient cancer cells by inhibiting Glut1. It was further shown that daily intraperitoneal injection of a soluble analogue of STF-31 effectively reduced the growth of tumors of VHL-deficient cancer cells grafted on nude mice. On the other hand, STF-31 appears to be an inhibitor with a narrow cell target spectrum.

These investigators recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation–like mechanism. These compounds target Glut1 and are efficacious in vivo as anticancer agents. A novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule.

A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell Growth In Vitro and In Vivo

Yi Liu, Yanyan Cao, Weihe Zhang, Stephen Bergmeier, et al.
Mol Cancer Ther Aug 2012; 11(8): 1672–82
http://dx.doi.org://10.1158/1535-7163.MCT-12-0131

Alterations in cellular metabolism are among the most consistent hallmarks of cancer. These investigators have studied the relationship between increased aerobic lactate production and mitochondrial physiology in tumor cells. To diminish the ability of malignant cells to metabolize pyruvate to lactate, M-type lactate dehydrogenase levels were knocked down by means of LDH-A short hairpin RNAs. Reduction in LDH-A activity resulted in stimulation of mitochondrial respiration and decrease of mitochondrial membrane potential. It also compromised the ability of these tumor cells to proliferate under hypoxia. The tumorigenicity of the LDH-A-deficient cells was severely diminished, and this phenotype was reversed by complementation with the human ortholog LDH-A protein. These results demonstrate that LDH-A plays a key role in tumor maintenance.

The results are consistent with a functional connection between alterations in glucose metabolism and mitochondrial physiology in cancer. The data also reflect that the dependency of tumor cells on glucose metabolism is a liability for these cells under limited-oxygen conditions. Interfering with LDH-A activity as a means of blocking pyruvate to lactate conversion could be exploited therapeutically. Because individuals with complete deficiency of LDH-A do not show any symptoms under ordinary circumstances, the genetic data suggest that inhibition of LDH-A activity may represent a relatively nontoxic approach to interfere with tumor growth.

Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance

Valeria R. Fantin Julie St-Pierre and Philip Leder
Cancer Cell Jun 2006; 9: 425–434.
http://dx.doi.org:/10.1016/j.ccr.2006.04.02

The widespread clinical use of positron-emission tomography (PET) for the detection of aerobic glycolysis in tumors and recent findings have rekindled interest in Warburg’s theory. Studies on the physiological changes in malignant conversion provided a metabolic signature for the different stages of tumorigenesis; during tumorigenesis, an increase in glucose uptake and lactate production have been detected. The fully transformed state is most dependent on aerobic glycolysis and least dependent on the mitochondrial machinery for ATP synthesis.

Tumors ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumors and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumors leads to enhanced, oxygen-independent glucose usage and a lactate based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumor growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualized cancer therapy based on the determination of metabolic changes in tumors that might enable the targeted inhibition of invasion and metastasis.

Other important links between cancer-causing genes and glucose metabolism have been already identified. Activation of the oncogenic kinase Akt has been shown to stimulate glucose uptake and metabolism in cancer cells and renders these cells susceptible to death in response to glucose withdrawal. Such tumor cells have been shown to be dependent on glucose because the ability to induce fatty acid oxidation in response to glucose deprivation is impaired by activated Akt. In addition, AMP-activated protein kinase (AMPK) has been identified as a link between glucose metabolism and the cell cycle, thereby implicating p53 as an essential component of metabolic cell-cycle control.

Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted

S Langbein, M Zerilli, A zur Hausen, W Staiger, et al.
British Journal of Cancer (2006) 94, 578–585.
http://dx.doi.org:/10.1038/sj.bjc.6602962

The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DJm) and low expression of the K+ channel Kv1.5, both contributing toapoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DJm, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

Cancer progression and its resistance to treatment depend, at least in part, on suppression of apoptosis. Although mitochondria are recognized as regulators of apoptosis, their importance as targets for cancer therapy has not been adequately explored or clinically exploited. In 1930, Warburg suggested that mitochondrial dysfunction in cancer results in a characteristic metabolic phenotype, that is, aerobic glycolysis (Warburg, 1930). Positron emission tomography (PET) imaging has now confirmed that most malignant tumors have increased glucose uptake and metabolism. This bioenergetic feature is a good marker of cancer but has not been therapeutically pursued..

The small molecule DCA is a metabolic modulator that has been used in humans for decades in the treatment of lactic acidosis and inherited mitochondrial diseases. Without affecting normal cells, DCA reverses the metabolic electrical remodeling that we describe in several cancer lines (hyperpolarized mitochondria, activated NFAT1, downregulated Kv1.5), inducing apoptosis and decreasing tumor growth. DCA in the drinking water at clinically relevant doses for up to 3 months prevents and reverses tumor growth in vivo, without apparent toxicity and without affecting hemoglobin, transaminases, or creatinine levels. The ease of delivery, selectivity, and effectiveness  make DCA an attractive candidate for proapoptotic cancer therapy which can be rapidly translated into phase II–III clinical trials.

A Mitochondria-K+ Channel Axis Is Suppressed in Cancer and Its Normalization Promotes Apoptosis and Inhibits Cancer Growth

Sebastien Bonnet, Stephen L. Archer, Joan Allalunis-Turner, et al.

Cancer Cell Jan 2007; 11: 37–51.
http://dx.doi.org:/10.1016/j.ccr.2006.10.020

Tumor cells, just as other living cells, possess the potential for proliferation, differentiation, cell cycle arrest, and apoptosis. There is a specific metabolic phenotype associated with each of these conditions, characterized by the production of both energy and special substrates necessary for the cells to function in that particular state. Unlike that of normal living cells, the metabolic phenotype of tumor cells supports the proliferative state. Aim: To present the metabolic hypothesis that (1) cell transformation and tumor growth are associated with the activation of metabolic enzymes that increase glucose carbon utilization for nucleic acid synthesis, while enzymes of the lipid and amino acid synthesis pathways are activated in tumor growth inhibition, and (2) phosphorylation and allosteric and transcriptional regulation of intermediary metabolic enzymes and their substrate availability together mediate and sustain cell transformation from one condition to another. Conclusion: Evidence is presented that demonstrates opposite changes in metabolic phenotypes induced by TGF-β, a cell transforming agent, and tumor growth-inhibiting phytochemicals such as genistein and Avemar, or novel synthetic antileukemic drugs such as STI571 (Gleevec).  Intermediary metabolic enzymes that mediate the growth signaling pathways and promote malignant cell transformation may serve as high efficacy nongenetic novel targets for cancer therapies.

A Metabolic Hypothesis of Cell Growth and Death in Pancreatic Cancer

Laszlo G. Boros, Wai-Nang Paul Lee, and Vay Liang W. Go
Pancreas 2002; 24(1):26–33

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of kidney cancer. Here, we integrated an unbiased genome-wide RNA interference screen for ccRCC survival regulators with an analysis of recurrently overexpressed genes in ccRCC to identify new therapeutic targets in this disease. One of the most potent survival regulators, the monocarboxylate transporter MCT4 (SLC16A3), impaired ccRCC viability in all eight ccRCC lines tested and was the seventh most overexpressed gene in a meta-analysis of five ccRCC expression datasets.

MCT4 silencing impaired secretion of lactate generated through glycolysis and induced cell cycle arrest and apoptosis. Silencing MCT4 resulted in intracellular acidosis, and reduction in intracellular ATP production together with partial reversion of the Warburg effect in ccRCC cell lines. Intra-tumoral heterogeneity in the intensity of MCT4 protein expression was observed in primary ccRCCs.

MCT4 protein expression analysis based on the highest intensity of expression in primary ccRCCs was associated with poorer relapse-free survival, whereas modal intensity correlated with Fuhrman nuclear grade. Consistent with the potential selection of subclones enriched for MCT4 expression during disease progression, MCT4 expression was greater at sites of metastatic disease. These data suggest that MCT4 may serve as a novel metabolic target to reverse the Warburg effect and limit disease progression in ccRCC.

Clear cell carcinoma (ccRCC) is the commonest subtype of renal cell carcinoma, accounting for 80% of cases. These tumors are highly resistant to cytotoxic chemotherapy and until recently, systemic treatment options for advanced ccRCC were limited to cytokine based therapies, such as interleukin-2 and interferon-α. Recently, anti-angiogenic drugs and mTOR inhibitors, all targeting the HIF–VEGF axis which is activated in up to 91% of ccRCCs through loss of the VHL tumor suppressor gene [1], have been shown to be effective in metastatic ccRCC [2–5]. Although these drugs increase overall survival to more than 2 years [6], resistance invariably occurs, making the identification of new molecular targets a major clinical need to improve outcomes in patients with metastatic ccRCC.

Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target

Marco Gerlinger, Claudio R Santos, Bradley Spencer-Dene, et al.
J Pathol 2012; 227: 146–156
http://dx.doi.org:/10.1002/path.4006

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression.

Metastatic cancer is characterized by reprogramming of cellular metabolism leading to increased uptake of glucose for use as both an anabolic and a catabolic substrate. Increased glucose uptake is such a reliable feature that it is utilized clinically to detect metastases by positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) with a sensitivity of >90% [1]. As with all aspects of cancer biology, the details of metabolic reprogramming differ widely among individual tumors. However, the role of specific signaling pathways and transcription factors in this process is now understood in considerable detail. This review will focus on the involvement of hypoxia-inducible factor 1 (HIF-1) in both mediating metabolic reprogramming and responding to metabolic alterations. The placement of HIF-1 both upstream and downstream of cancer metabolism results in a feed-forward mechanism that may play a major role in the development of the invasive, metastatic, and lethal cancer phenotype.

O2 concentrations are significantly reduced in many human cancers compared with the surrounding normal tissue. The median PO2 in breast cancers is 10 mmHg, as compared with65 mmHg in normal breast tissue. Reduced O2 availability induces HIF-1, which regulates the transcription of hundreds of genes that encode proteins involved in every aspect of cancer biology, including: cell immortalization and stem cell maintenance; genetic instability; glucose and energy metabolism; vascularization; autocrine growth factor signaling; invasion and metastasis; immune evasion; and resistance to chemotherapy and radiation therapy.

HIF-1 is a transcription factor that consists of an O2 regulated HIF-1a and a constitutively expressed HIF-1b subunit. In well-oxygenated cells, HIF-1a is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (PHD2), which uses O2 and a-ketoglutarate as substrates in a reaction that generates CO2 and succinate as byproducts. Prolylhydroxylated HIF-1a is bound by the von Hippel–Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1a for proteasomal degradation (Figure 1a). Asparagine 803 in the transactivation domain is hydroxylated in well-oxygenated cells by factor inhibiting HIF-1 (FIH-1), which blocks the binding of the coactivators p300 and CBP. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by substrate (O2) deprivation and/or the mitochondrial generation of reactive oxygen species (ROS), which may oxidize Fe(II) present in the catalytic center of the hydroxylases.

The finding that acute changes in PO2 increase mitochondrial ROS production suggests that cellular respiration is optimized at physiological PO2 to limit ROS generation and that any deviation in PO2 – up or down – results in increased ROS generation. If hypoxia persists, induction of HIF-1 leads to adaptive mechanisms to reduce ROS and re-establish homeostasis, as described below. Prolyl and asparaginyl hydroxylation provide a molecular mechanism by which changes in cellular oxygenation can be transduced to the nucleus as changes in HIF-1 activity.

HIF-1: upstream and downstream of cancer metabolism

Gregg L Semenza
Current Opinion in Genetics & Development 2010, 20:51–56

This review comes from a themed issue on Genetic and cellular mechanisms of oncogenesis Edited by Tony Hunter and Richard Marais

http://dx.doi.org:/10.1016/j.gde.2009.10.009

Hypoxia-inducible factor 1 (HIF-1) regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, genetic instability, vascularization, metabolic reprogramming, autocrine growth factor signaling, invasion/metastasis, and treatment failure. In animal models, HIF-1 overexpression is associated with increased tumor growth, vascularization, and metastasis, whereas HIF-1 loss-of-function has the opposite effect, thus validating HIF-1 as a target. In further support of this conclusion, immunohistochemical detection of HIF-1a overexpression in biopsy sections is a prognostic factor in many cancers. A growing number of novel anticancer agents have been shown to inhibit HIF-1 through a  variety of molecular mechanisms. Determining which combination of drugs to administer to any given patient remains a major obstacle to improving cancer treatment outcomes.

Intratumoral hypoxia The majority of locally advanced solid tumors contain regions of reduced oxygen availability. Intratumoral hypoxia results when cells are located too far from a functional blood vessel for diffusion of adequate amounts of O2 as a result of rapid cancer cell proliferation and the formation of blood vessels that are structurally and functionally abnormal. In the most extreme case, O2 concentrations are below those required for survival, resulting in cell death and establishing a selection for cancer cells in which apoptotic pathways are inactivated, anti-apoptotic pathways are activated, or invasion/metastasis pathways that promote escape from the hypoxic microenvironment are activated. This hypoxic adaptation may arise by alterations in gene expression or by mutations in the genome or both and is associated with reduced patient survival.

Hypoxia-inducible factor 1 (HIF-1) The expression of hundreds of genes is altered in each cell exposed to hypoxia. Many of these genes are regulated by HIF-1. HIF-1 is a heterodimer formed by the association of an O2-regulated HIF1a subunit with a constitutively expressed HIF-1b subunit. The structurally and functionally related HIF-2a protein also dimerizes with HIF-1b and regulates an overlapping battery of target genes. Under nonhypoxic conditions, HIF-1a (as well as HIF-2a) is subject to O2-dependent prolyl hydroxylation and this modification is required for binding of the von Hippel–Lindau tumor suppressor protein (VHL), which also binds to Elongin C and thereby recruits a ubiquitin ligase complex that targets HIF-1a for ubiquitination and proteasomal degradation. Under hypoxic conditions, the rate of hydroxylation and ubiquitination declines, resulting in accumulation of HIF-1a. Immunohistochemical analysis of tumor biopsies has revealed high levels of HIF-1a in hypoxic but viable tumor cells surrounding areas of necrosis.

Genetic alterations in cancer cells increase HIF-1 activity In the majority of clear-cell renal carcinomas, VHL function is lost, resulting in constitutive activation of HIF-1. After re-introduction of functional VHL, renal carcinoma cell lines are no longer tumorigenic, but can be made tumorigenic by expression of HIF2a in which the prolyl residues that are subject to hydroxylation have been mutated. In addition to VHL loss-of-function, many other genetic alterations that inactivate tumor suppressors

Evaluation of HIF-1 inhibitors as anticancer agents

Gregg L. Semenza
Drug Discovery Today Oct 2007; 12(19/20).
http://dx.doi.org:/10.1016/j.drudis.2007.08.006

Hypoxia-inducible factor-1 (HIF-1), which is present at high levels in human tumors, plays crucial roles in tumor promotion by upregulating its target genes, which are involved in anaerobic energy metabolism, angiogenesis, cell survival, cell invasion, and drug resistance. Therefore, it is apparent that the inhibition of HIF-1 activity may be a strategy for treating cancer. Recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories. The future success of these efforts will be a new class of HIF-1-targeting anticancer agents, which would improve the prognoses of many cancer patients. This review focuses on the potential of HIF-1 as a target molecule for anticancer therapy, and on possible strategies to inhibit HIF-1 activity. In addition, we introduce YC-1 as a new anti-HIF-1, anticancer agent. Although YC-1 was originally developed as a potential therapeutic agent for thrombosis and hypertension, recent studies demonstrated that YC-1 suppressed HIF-1 activity and vascular endothelial growth factor expression in cancer cells. Moreover, it halted tumor growth in immunodeficient mice without serious toxicity during the treatment period. Thus, we propose that YC-1 is a good lead compound for the development of new anti-HIF-1, anticancer agents.

Although many anticancer regimens have been introduced to date, their survival benefits are negligible, which is the reason that a more innovative treatment is required. Basically, the identification of the specific molecular features of tumor promotion has allowed for rational drug discovery in cancer treatment, and drugs have been screened based upon the modulation of specific molecular targets in tumor cells. Target-based drugs should satisfy the following two conditions.

First, they must act by a described mechanism.

Second, they must reduce tumor growth in vivo, associated with this mechanism.

Many key factors have been found to be involved in the multiple steps of cell growth signal-transduction pathways. Targeting these factors offers a strategy for preventing tumor growth; for example, competitors or antibodies blocking ligand–receptor interaction, and receptor tyrosine kinase inhibitors, downstream pathway inhibitors (i.e., RAS farnesyl transferase inhibitors, mitogen-activated protein kinase and mTOR inhibitors), and cell-cycle arresters (i.e., cyclin-dependent kinase inhibitors) could all be used to inhibit tumor growth.

In addition to the intracellular events, tumor environmental factors should be considered to treat solid tumors. Of these, hypoxia is an important cancer-aggravating factor because it contributes to the progression of a more malignant phenotype, and to the acquisition of resistance to radiotherapy and chemotherapy. Thus, transcription factors that regulate these hypoxic events are good targets for anticancer therapy and in particular HIF-1 is one of most compelling targets. In this paper, we introduce the roles of HIF-1 in tumor promotion and provide a summary of new anticancer strategies designed to inhibit HIF-1 activity.

New anticancer strategies targeting HIF-1

Eun-Jin Yeo, Yang-Sook Chun, Jong-Wan Park
Biochemical Pharmacology 68 (2004) 1061–1069
http://dx.doi.org:/10.1016/j.bcp.2004.02.040

Classical work in tumor cell metabolism focused on bioenergetics, particularly enhanced glycolysis and suppressed oxidative phosphorylation (the ‘Warburg effect’). But the biosynthetic activities required to create daughter cells are equally important for tumor growth, and recent studies are now bringing these pathways into focus. In this review, we discuss how tumor cells achieve high rates of nucleotide and fatty acid synthesis, how oncogenes and tumor suppressors influence these activities, and how glutamine metabolism enables macromolecular synthesis in proliferating cells.

Otto Warburg’s demonstration that tumor cells rapidly use glucose and convert the majority of it to lactate is still the most fundamental and enduring observation in tumor metabolism. His work, which ushered in an era of study on tumor metabolism focused on the relationship between glycolysis and cellular bioenergetics, has been revisited and expanded by generations of tumor biologists. It is now accepted that a high rate of glucose metabolism, exploited clinically by 18FDGPET scanning, is a metabolic hallmark of rapidly dividing cells, correlates closely with transformation, and accounts for a significant percentage of ATP generated during cell proliferation. A ‘metabolic transformation’ is required for tumorigenesis. Research over the past few years has reinforced this idea, revealing the conservation of metabolic activities among diverse tumor types, and proving that oncogenic mutations can promote metabolic autonomy by driving nutrient uptake to levels that often exceed those required for cell growth and proliferation.

In order to engage in replicative division, a cell must duplicate its genome, proteins, and lipids and assemble the components into daughter cells; in short, it must become a factory for macromolecular biosynthesis. These activities require that cells take up extracellular nutrients like glucose and glutamine and allocate them into metabolic pathways that convert them into biosynthetic precursors (Figure 1). Tumor cells can achieve this phenotype through changes in the expression of enzymes that determine metabolic flux rates, including nutrient transporters and enzymes [8– 10]. Current studies in tumor metabolism are revealing novel mechanisms for metabolic control, establishing which enzyme isoforms facilitate the tumor metabolic phenotype, and suggesting new targets for cancer therapy.

The ongoing challenge in tumor cell metabolism is to understand how individual pathways fit together into the global metabolic phenotype of cell growth. Here we discuss two biosynthetic activities required by proliferating tumor cells: production of ribose-5 phosphate for nucleotide biosynthesis and production of fatty acids for lipid biosynthesis. Nucleotide and lipid biosynthesis share three important characteristics.

  • First, both use glucose as a carbon source.
  • Second, both consume TCA cycle intermediates, imposing the need for a mechanism to replenish the cycle.
  • Third, both require reductive power in the form of NADPH.

In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1, not shown). Although our emphasis on the Warburg effect reflects the focus of the field, we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell.

The Tumor Microenvironment Selects for Altered Metabolism One compelling idea to explain the Warburg effect is that the altered metabolism of cancer cells confers a selective advantage for survival and proliferation in the unique tumor microenvironment. As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that provides multiple solutions to hypoxic stress (reviewed in Kaelin and Ratcliffe, 2008). Because a decreased dependence on aerobic respiration becomes advantageous, cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis (the formation of new blood vessels) by upregulating several factors, including most prominently vascular endothelial growth factor (VEGF).

Blood vessels recruited to the tumor microenvironment, however, are disorganized, may not deliver blood effectively, and therefore do not completely alleviate hypoxia (reviewed in Gatenby and Gillies, 2004). The oxygen levels within a tumor vary both spatially and temporally, and the resulting rounds of fluctuating oxygen levels potentially select for tumors that constitutively upregulate glycolysis. Interestingly, with the possible exception of tumors that have lost the von Hippel-Lindau protein (VHL), which normally mediates degradation of HIF, HIF is still coupled to oxygen levels, as evident from the heterogeneity of HIF expression within the tumor microenvironment. Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF. Other molecular mechanisms are likely to be important, such as the metabolic changes induced by oncogene activation and tumor suppressor loss.

Oncogene Activation Drives Changes in Metabolism Not only may the tumor microenvironment select for a deranged metabolism, but oncogene status can also drive metabolic changes. Since Warburg’s time, the biochemical study of cancer metabolism has been overshadowed by efforts to identify the mutations that contribute to cancer initiation and progression. Recent work, however, has demonstrated that the key components of the Warburg effect—

  • increased glucose consumption,
  • decreased oxidative phosphorylation, and
  • accompanying lactate production—
  • are also distinguishing features of oncogene activation.

The signaling molecule Ras, a powerful oncogene when mutated, promotes glycolysis (reviewed in Dang and Semenza, 1999; Ramanathan et al., 2005). Akt kinase, a well-characterized downstream effector of insulin signaling, reprises its role in glucose uptake and utilization in the cancer setting (reviewed in Manning and Cantley, 2007), whereas the Myc transcription factor upregulates the expression of various metabolic genes (reviewed in Gordan et al., 2007). The most parsimonious route to tumorigenesis may be activation of key oncogenic nodes that execute a proliferative program, of which metabolism may be one important arm. Moreover, regulation of metabolism is not exclusive to oncogenes.

Cancer Cell Metabolism: Warburg & Beyond

Hsu PP & Sabatini DM
Cell  Sep 5, 2008; 134, 703-705
http://dx.doi.org:/10.1016/j.cell.2008.08.021

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and redistribution among biosynthetic pathways. This results in a proliferating phenotype with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited de novo fatty  acid   synthesis   and   TCA   cycle   glucose   oxidation. This  primarily nonoxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment.

Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-13C2]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

Metabolic Biomarker and Kinase Drug Target Discovery in Cancer Using Stable Isotope-Based Dynamic Metabolic Profiling (SIDMAP)

László G. Boros, Daniel J. Brackett and George G. Harrigan
Current Cancer Drug Targets, 2003, 3, 447-455 447

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150 kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, while silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.

A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila , and Humans

Daniel K. Bricker, Eric B. Taylor, John C. Schell, Thomas Orsak, et al.
Science Express 24 May 2012
http://dx.doi.org:/10.1126/science.1218099

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.

Review ADAR Enzyme and miRNA Story: A Nucleotide that Can Make the Difference 

Sara Tomaselli, Barbara Bonamassa, Anna Alisi, Valerio Nobili, Franco Locatelli and Angela Gallo
Int. J. Mol. Sci. 19 Nov 2013; 14, 22796-22816 http://dx.doi.org:/10.3390/ijms141122796

The fermented wheat germ extract (FWGE) nutraceutical (Avemar™), manufactured under “good manufacturing practice” conditions and, fulfilling the self-affirmed “generally recognized as safe” status in the United States, has been approved as a “dietary food for special medical purposes for cancer patients” in Europe. In this paper, we report the adjuvant use of this nutraceutical in the treatment of high-risk skin melanoma patients. Methods: In a randomized, pilot, phase II clinical trial, the efficacy of dacarbazine (DTIC)-based adjuvant chemotherapy on survival parameters of melanoma patients was compared to that of the same treatment supplemented with a 1-year long administration of FWGE. Results: At the end of an additional 7-year-long follow-up period, log-rank analyses (Kaplan-Meier estimates) showed significant differences in both progression-free (PFS) and overall survival (OS) in favor of the FWGE group. Mean PFS: 55.8 months (FWGE group) versus 29.9 months (control group), p  0.0137. Mean OS: 66.2 months (FWGE group) versus 44.7 months (control group), p < 0.0298. Conclusions: The inclusion of Avemar into the adjuvant protocols of high-risk skin melanoma patients is highly recommended.

Adjuvant Fermented Wheat Germ Extract (Avemar™) Nutraceutical Improves Survival of High-Risk Skin Melanoma Patients: A Randomized, Pilot, Phase II Clinical Study with a 7-Year Follow-Up

LV Demidov, LV Manziuk, GY Kharkevitch, NA Pirogova, and EV Artamonova
Cancer Biotherapy & Radiopharmaceuticals 2008; 23(4)
http://dx.doi.org:/10.1089/cbr.2008.0486

Cancer cells possess unique metabolic signatures compared to normal cells, including shifts in aerobic glycolysis, glutaminolysis, and de novo biosynthesis of macromolecules. Targeting these changes with agents (drugs and dietary components) has been employed as strategies to reduce the complications associated with tumorigenesis. This paper highlights the ability of several food components to suppress tumor-specific metabolic pathways, including increased expression of glucose transporters, oncogenic tyrosine kinase, tumor-specific M2-type pyruvate kinase, and fatty acid synthase, and the detection of such effects using various metabonomic technologies, including liquid chromatography/mass spectrometry (LC/MS) and stable isotope-labeled MS. Stable isotope-mediated tracing technologies offer exciting opportunities for defining specific target(s) for food components. Exposures, especially during the early transition phase from normal to cancer, are critical for the translation of knowledge about food components into effective prevention strategies. Although appropriate dietary exposures needed to alter cellular metabolism remain inconsistent and/or ill-defined, validated metabonomic biomarkers for dietary components hold promise for establishing effective strategies for cancer prevention.

Bioactive Food Components and Cancer-Specific Metabonomic Profiles

Young S. Kim and John A. Milner
Journal of Biomedicine and Biotechnology 2011, Art ID 721213, 9 pages
http://dx.doi.org:/10.1155/2011/721213

This reviewer poses the following observation.  The importance of the pyridine nucleotide reduced/oxidized ratio has not been alluded to here, but the importance cannot be understated. It has relevance to the metabolic functions of anabolism and catabolism of the visceral organs.  The importance of this has ties to the pentose monophosphate pathway. The importance of the pyridine nucleotide transhydrogenase reaction remains largely unexplored.  In reference to the NAD-redox state, the observation was made by Nathan O. Kaplan that the organs may be viewed with respect to their primary functions in anabolic or high energy catabolic activities. Thus we find that the endocrine organs are largely tied to anabolic functioning, and to NADP, whereas cardiac and skeletal muscle are highly dependent on NAD. The consequence of this observed phenomenon appears to be related to a difference in the susceptibility to malignant transformation.  In the case of the gastrointestinal tract, the rate of turnover of the epithelium is very high. However, with the exception of the liver, there is no major activity other than cell turnover. In the case of the liver, there is a major commitment to synthesis of lipids, storage of fuel, and synthesis of proteins, which is largely anabolic, but there is also a major activity in detoxification, which is not.  In addition, the liver has a double circulation. As a result, a Zahn infarct is uncommon.  Now we might also consider the heart.  The heart is a muscle syncytium with a high need for oxygen.  Cutting of the oxygen supply makes the myocytes vulnerable to ischemic insult and abberant rhythm abnormalities.  In addition, the cardiomyocyte can take up lactic acid from the circulation for fuel, which is tied to the utilization of lactate from vigorous skeletal muscle activity.  The skeletal muscle is tied to glycolysis in normal function, which has a poor generation of ATP, so that the recycling of excess lactic acid is required by cardiac muscle and hepatocytes.  This has not been a part of the discussion, but this reviewer considers it important to remember in considering the organ-specific tendencies to malignant transformation.

Comment (Aurelian Udristioiu):

Otto Warburg observed that many cancers lose their capacity for mitochondrial respiration, limiting ATP production to anaerobic glycolytic pathways. The phenomenon is particularly prevalent in aggressive malignancies, most of which are also hypoxic [1].
Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines [2-3]. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines [4].
Hypoxia reduces the ability of cells to maintain their energy levels, because less ATP is obtained from glycolysis than from oxidative phosphorylation. Cells adapt to hypoxia by activating the expression of mutant genes in glycolysis.
-Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.
The most direct induction of apoptosis caused by hypoxia is determined by the inhibition of the electron carrier chain from the inner membrane of the mitochondria. The lack of oxygen inhibits the transport of protons and thereby causes a decrease in membrane potential. Cell survival under conditions of mild hypoxia is mediated by phosphoinositide-3 kinase (PIK3) using severe hypoxia or anoxia, and then cells initiate a cascade of events that lead to apoptosis [5].
After DNA damage, a very important regulator of apoptosis is the p53 protein. This tumor suppressor gene has mutations in over 60% of human tumors and acts as a suppressor of cell division. The growth-suppressive effects of p53 are considered to be mediated through the transcriptional trans-activation activity of the protein. In addition to the maturational state of the clonal tumor, the prognosis of patients with CLL is dependent of genetic changes within the neoplastic cell population.

1.Warburg O. On the origin of cancer cells. Science 1956; 123 (3191):309-314
PubMed Abstract ; Publisher Full Text

2.Giardina TM, Steer JH, Lo SZ, Joyce DA. Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages. Biochim Biophys Acta 2008, 1777(2):118-129. PubMed Abstract | Publisher Full Text

3. Horimoto M, Resnick MB, Konkin TA, Routhier J, Wands JR, Baffy G. Expression of uncoupling protein-2 in human colon cancer. Clin Cancer Res 2004; 10 (18 Pt1):6203-6207. PubMed Abstract | Publisher Full Text

4. Randle PJ, England PJ, Denton RM. Control of the tricarboxylate cycle and it interactions with glycolysis during acetate utilization in rat heart. Biochem J 1970; 117(4):677-695. PubMed Abstract | PubMed Central Full Text

5. Gillies RJ, Robey I, Gatenby RA. Causes and consequences of increased glucose metabolism of cancers. J Nucl Med 2008; 49(Suppl 2):24S-42S. PubMed Abstract | Publisher Full Text

Shortened version of Comment –

Hypoxia induces a stochastic imbalance between the numbers of reduced mitochondrial species vs. available oxygen, resulting in increased reactive oxygen species (ROS) whose toxicity can lead to apoptotic cell death.
Mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration-.generation in the presence of intact tricarboxylic acid (TCA) enzyme.
One mitochondrial adaptation to increased ROS is over-expression of the uncoupling protein 2 (UCP2) that has been reported in multiple human cancer cell lines. Increased UCP2 expression was also associated with reduced ATP production in malignant oxyphilic mouse leukemia and human lymphoma cell lines.
Severe hypoxia causes a high mutation rate, resulting in point mutations that may be explained by reduced DNA mismatch repairing activity.

Read Full Post »


The Colors of Respiration and Electron Transport

Reporter & Curator: Larry H. Bernstein, MD, FCAP 

 

 

Molecular Biology of the Cell. 4th edition

Electron-Transport Chains and Their Proton Pumps
http://www.ncbi.nlm.nih.gov/books/NBK26904/

Having considered in general terms how a mitochondrion uses electron
transport to create an electrochemical proton gradient, we need to
examine the mechanisms that underlie this membrane-based energy-conversion process. In doing so, we also accomplish a larger purpose.
As emphasized at the beginning of this chapter, very similar chemi-
osmotic mechanisms are used by mitochondria, chloroplasts, archea,
and bacteria. In fact, these mechanisms underlie the function of nearly
all living organisms— including anaerobes that derive all their energy
from electron transfers between two inorganic molecules. It is therefore
rather humbling for scientists to remind themselves that the existence
of chemiosmosis has been recognized for only about 40 years.

mitochondria

mitochondria

 

Overview of The Electron Transport Chain

Overview of The Electron Transport Chain

We begin with a look at some of the principles that underlie the electron-transport process, with the aim of explaining how it can pump protons
across a membrane.

Although protons resemble other positive ions such as Na+ and K+
in their movement across membranes, in some respects they are unique.
Hydrogen atoms are by far the most abundant type of atom in living
organisms; they are plentiful not only in all carbon-containing
biological molecules, but also in the water molecules that surround
them. The protons in water are highly mobile, flickering through the
hydrogen-bonded network of water molecules by rapidly
dissociating from one water molecule to associate with its neighbor,
as illustrated in Figure 14-20A. Protons are thought to move across a
protein pump embedded in a lipid bilayer in a similar way: they
transfer from one amino acid side chain to another, following a
special channel through the protein.

Protons are also special with respect to electron transport. Whenever
a molecule is reduced by acquiring an electron, the electron (e -) brings
with it a negative charge. In many cases, this charge is rapidly
neutralized by the addition of a proton (H+) from water, so that
the net effect of the reduction is to transfer an entire hydrogen atom,
H+ + e – (Figure 14-20B). Similarly, when a molecule is oxidized,
a hydrogen atom removed from it can be readily dissociated into
its constituent electron and proton—allowing the electron to
be transferred separately to a molecule that accepts electrons,
while the proton is passed to the water. Therefore, in a membrane
in which electrons are being passed along an electron-transport
chain, pumping protons from one side of the membrane to
another can be relatively simple. The electron carrier merely
needs to be arranged in the membrane in a way that causes it to
pick up a proton from one side of the membrane when it accepts
an electron, and to release the proton on the other side of the
membrane as the electron is passed to the next carrier molecule
in the chain (Figure 14-21).

protons pumped across membranes ch14f21

protons pumped across membranes ch14f21

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f21.gif

Figure 14-21

How protons can be pumped across membranes. As an electron
passes along an electron-transport chain embedded in a lipid-bilayer
membrane, it can bind and release a proton at each step.
In this diagram, electron carrier B picks up a proton (H+)
from one (more…)

e_transfer

e_transfer

The Redox Potential Is a Measure of Electron Affinities

In biochemical reactions, any electrons removed from one
molecule are always passed to another, so that whenever one
molecule is oxidized, another is reduced. Like any other chemical r
eaction, the tendency of such oxidation-reduction reactions, or
redox reactions, to proceed spontaneously depends on the free-
energy change (ΔG) for the electron transfer, which in turn
depends on the relative affinities of the two molecules for electrons.

Because electron transfers provide most of the energy for living
things, it is worth spending the time to understand them. Many
readers are already familiar with acids and bases, which donate
and accept protons (see Panel 2-2, pp. 112–113). Acids and bases
exist in conjugate acid-base pairs, in which the acid is readily
converted into the base by the loss of a proton. For example,
acetic acid (CH3COOH) is converted into its conjugate base
(CH3COO-) in the reaction:

Image ch14e3.jpg

In exactly the same way, pairs of compounds such as NADH and
NAD+ are called redox pairs, since NADH is converted to NAD+
by the loss of electrons in the reaction:

Image ch14e4.jpg

NAD+_NADH

NAD+_NADH

NADH is a strong electron donor: because its electrons are held
in a high-energy linkage, the free-energy change for passing its
electrons to many other molecules is favorable (see Figure 14-9).
It is difficult to form a high-energy linkage. Therefore its redox
partner, NAD+, is of necessity a weak electron acceptor.

The tendency to transfer electrons from any redox pair can be
measured experimentally. All that is required is the formation
of an electrical circuit linking a 1:1 (equimolar) mixture of the
redox pair to a second redox pair that has been arbitrarily selected
as a reference standard, so the voltage difference can be measured
between them (Panel 14-1, p. 784). This voltage difference is
defined as the redox potential; as defined, electrons move
spontaneously from a redox pair like NADH/NAD+ with a low
redox potential (a low affinity for electrons) to a redox pair like
O2/H2O with a high redox potential (a high affinity for electrons).
Thus, NADH is a good molecule for donating electrons to the
respiratory chain, while O2 is well suited to act as the “sink” for
electrons at the end of the pathway. As explained in Panel 14-1,
the difference in redox potential, ΔE0′, is a direct measure of
the standard free-energy change (ΔG°) for the transfer of an
electron from one molecule to another.

Proteins of inner space

Proteins of inner space

energetics-of-cellular-respiration

energetics-of-cellular-respiration

Box Icon

Panel 14-1

Redox Potentials.

Electron Transfers Release Large Amounts of Energy

As just discussed, those pairs of compounds that have the most negative
redox potentials have the weakest affinity for electrons and therefore
contain carriers with the strongest tendency to donate electrons.
Conversely, those pairs that have the most positive redox potentials
have the strongest affinity for electrons and therefore contain carriers
with the strongest tendency to accept electrons. A 1:1 mixture of NADH
and NAD+ has a redox potential of -320 mV, indicating that NADH has
a strong tendency to donate electrons; a 1:1 mixture of H2O and ½O2
has a redox potential of +820 mV, indicating that O2 has a strong
tendency to accept electrons. The difference in redox potential is
1.14 volts (1140 mV), which means that the transfer of each electron
from NADH to O2 under these standard conditions is enormously
favorable, where ΔG° = -26.2 kcal/mole (-52.4 kcal/mole for the two
electrons transferred per NADH molecule; see Panel 14-1). If we
compare this free-energy change with that for the formation of the
phosphoanhydride bonds in ATP (ΔG° = -7.3 kcal/mole, see Figure 2-75), we see that more than enough energy is released by the oxidization
of one NADH molecule to synthesize several molecules of ATP from
ADP and Pi.

 Phosphate dependence of pyruvate oxidation

Phosphate dependence of pyruvate oxidation

Living systems could certainly have evolved enzymes that would
allow NADH to donate electrons directly to O2 to make water in the reaction:

Image ch14e5.jpg

But because of the huge free-energy drop, this reaction would proceed
with almost explosive force and nearly all of the energy would be released
as heat. Cells do perform this reaction, but they make it proceed much
more gradually by passing the high-energy electrons from NADH to
O2 via the many electron carriers in the electron-transport chain.
Since each successive carrier in the chain holds its electrons more
tightly, the highly energetically favorable reaction 2H+ + 2e – + ½O2
→ H2O is made to occur in many small steps. This enables nearly half
of the released energy to be stored, instead of being lost to the
environment as heat.

Spectroscopic Methods Have Been Used to Identify Many Electron
Carriers in the Respiratory Chain

Many of the electron carriers in the respiratory chain absorb visible
light and change color when they are oxidized or reduced. In general,
each has an absorption spectrum and reactivity that are distinct enough
to allow its behavior to be traced spectroscopically, even in crude mixtures.
It was therefore possible to purify these components long before their
exact functions were known. Thus, the cytochromes were discovered
in 1925 as compounds that undergo rapid oxidation and reduction in
living organisms as disparate as bacteria, yeasts, and insects. By observing
cells and tissues with a spectroscope, three types of cytochromes were
identified by their distinctive absorption spectra and designated
cytochromes a, b, and c. This nomenclature has survived, even though
cells are now known to contain several cytochromes of each type and
the classification into types is not functionally important.

The cytochromes constitute a family of colored proteins that are
related by the presence of a bound heme group, whose iron atom
changes from the ferric oxidation state (Fe3+) to the ferrous oxidation
state (Fe2+) whenever it accepts an electron. The heme group consists
of a porphyrin ring with a tightly bound iron atom held by four nitrogen
atoms at the corners of a square (Figure 14-22). A similar porphyrin ring
is responsible for the red color of blood and for the green color of
leaves, being bound to iron in hemoglobin and to magnesium in
chlorophyll, respectively.

The structure of the heme group attached covalently to cytochrome c ch14f22

The structure of the heme group attached covalently to cytochrome c ch14f22

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f22.jpg

Figure 14-22. The structure of the heme group attached covalently
to cytochrome c.

Figure 14-22

The structure of the heme group attached covalently to cytochrome c.
The porphyrin ring is shown in blue. There are five different
cytochromes in the respiratory chain. Because the hemes in different
cytochromes have slightly different structures and (more…)

Iron-sulfur proteins are a second major family of electron carriers. In these
proteins, either two or four iron atoms are bound to an equal number of
sulfur atoms and to cysteine side chains, forming an iron-sulfur center
on the protein (Figure 14-23). There are more iron-sulfur centers than
cytochromes in the respiratory chain. But their spectroscopic detection
requires electron spin resonance (ESR) spectroscopy, and they are less
completely characterized. Like the cytochromes, these centers carry one
electron at a time.

structure of iron sulfur centers ch14f23

structure of iron sulfur centers ch14f23

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f23.jpg

Figure 14-23. The structures of two types of iron-sulfur centers.

Figure 14-23

The structures of two types of iron-sulfur centers. (A) A center of the
2Fe2S type. (B) A center of the 4Fe4S type. Although they contain
multiple iron atoms, each iron-sulfur center can carry only one
electron at a time. There are more than seven different (more…)

The simplest of the electron carriers in the respiratory chain—and
the only one that is not part of a protein—is a small hydrophobic
molecule that is freely mobile in the lipid bilayer known as ubiquinone,
or coenzyme Q. A quinone (Q) can pick up or donate either one or
two electrons; upon reduction, it picks up a proton from the medium
along with each electron it carries (Figure 14-24).

quinone electron carriers ch14f24

quinone electron carriers ch14f24

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f24.jpg

Figure 14-24. Quinone electron carriers.

Figure 14-24

Quinone electron carriers. Ubiquinone in the respiratory chain picks
up one H+ from the aqueous environment for every electron it accepts,
and it can carry either one or two electrons as part of a hydrogen atom
(yellow). When reduced ubiquinone donates (more…)

In addition to six different hemes linked to cytochromes, more than
seven iron-sulfur centers, and ubiquinone, there are also two copper
atoms and a flavin serving as electron carriers tightly bound to respiratory-chain proteins in the pathway from NADH to oxygen. This pathway
involves more than 60 different proteins in all.

As one would expect, the electron carriers have higher and higher
affinities for electrons (greater redox potentials) as one moves along
the respiratory chain. The redox potentials have been fine-tuned
during evolution by the binding of each electron carrier in a particular
protein context, which can alter its normal affinity for electrons. However,
because iron-sulfur centers have a relatively low affinity for electrons,
they predominate in the early part of the respiratory chain; in contrast,
the cytochromes predominate further down the chain, where a higher
affinity for electrons is required.

The order of the individual electron carriers in the chain was
determined by sophisticated spectroscopic measurements (Figure 14-25),
and many of the proteins were initially isolated and characterized as
individual polypeptides. A major advance in understanding the
respiratory chain, however, was the later realization that most of
the proteins are organized into three large enzyme complexes.

path of electrons ch14f25

path of electrons ch14f25

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f25.gif

Figure 14-25. The general methods used to determine the path of
electrons along an electron-transport chain.

Figure 14-25

The general methods used to determine the path of electrons along
an electron-transport chain. The extent of oxidation of electron
carriers a, b, c, and d is continuously monitored by following their
distinct spectra, which differ in their oxidized and (more…)

The Respiratory Chain Includes Three Large Enzyme Complexes
Embedded in the Inner Membrane

Membrane proteins are difficult to purify as intact complexes
because they are insoluble in aqueous solutions, and some of
the detergents required to solubilize them can destroy normal
protein-protein interactions. In the early 1960s, however, it
was found that relatively mild ionic detergents, such as deoxycholate,
can solubilize selected components of the inner mitochondrial
membrane in their native form. This permitted the identification
and purification of the three major membrane-bound respiratory
enzyme complexes in the pathway from NADH to oxygen (Figure 14-26).
As we shall see in this section, each of these complexes acts as an
electron-transport-driven H+ pump; however, they were
initially characterized in terms of the electron carriers that
they interact with and contain:

mitochondrial oxidative phosphorylation

mitochondrial oxidative phosphorylation

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f26.gif

Figure 14-26. The path of electrons through the three respiratory
enzyme complexes.

Figure 14-26

The path of electrons through the three respiratory enzyme complexes.
The relative size and shape of each complex are shown. During the
transfer of electrons from NADH to oxygen (red lines), ubiquinone
and cytochrome c serve as mobile carriers that ferry (more…)

The NADH dehydrogenase complex (generally known as complex I)
is the largest of the respiratory enzyme complexes, containing more
than 40 polypeptide chains. It accepts electrons from NADH and
passes them through a flavin and at least seven iron-sulfur centers
to ubiquinone. Ubiquinone then transfers its electrons to a second
respiratory enzyme complex, the cytochrome b-c1 complex.

The cytochrome b-c1 complex contains at least 11 different
polypeptide chains and functions as a dimer. Each monomer
contains three hemes bound to cytochromes and an iron-sulfur
protein. The complex accepts electrons from ubiquinone
and passes them on to cytochrome c, which carries its electron
to the cytochrome oxidase complex.

The cytochrome oxidase complex also functions as a dimer; each
monomer contains 13 different polypeptide chains, including two
cytochromes and two copper atoms. The complex accepts one electron
at a time from cytochrome c and passes them four at a time to oxygen.

The cytochromes, iron-sulfur centers, and copper atoms can carry
only one electron at a time. Yet each NADH donates two electrons,
and each O2 molecule must receive four electrons to produce water.
There are several electron-collecting and electron-dispersing points
along the electron-transport chain where these changes in electron
number are accommodated. The most obvious of these is cytochrome
oxidase.

An Iron-Copper Center in Cytochrome Oxidase Catalyzes Efficient
O2 Reduction

Because oxygen has a high affinity for electrons, it releases a
large amount of free energy when it is reduced to form water.
Thus, the evolution of cellular respiration, in which O2 is
converted to water, enabled organisms to harness much more
energy than can be derived from anaerobic metabolism. This
is presumably why all higher organisms respire. The ability of
biological systems to use O2 in this way, however, requires a
very sophisticated chemistry. We can tolerate O2 in the air we
breathe because it has trouble picking up its first electron; this
fact allows its initial reaction in cells to be controlled closely by
enzymatic catalysis. But once a molecule of O2 has picked up one
electron to form a superoxide radical (O2 -), it becomes dangerously
reactive and rapidly takes up an additional three electrons wherever
it can find them. The cell can use O2 for respiration only because
cytochrome oxidase holds onto oxygen at a special bimetallic
center, where it remains clamped between a heme-linked iron
atom and a copper atom until it has picked up a total of four electrons.
Only then can the two oxygen atoms of the oxygen molecule be
safely released as two molecules of water (Figure 14-27).

Figure 14-27. The reaction of O2 with electrons in cytochrome oxidase.

Figure 14-27

The reaction of O2 with electrons in cytochrome oxidase. As indicated,
the iron atom in heme a serves as an electron queuing point; this
heme feeds four electrons into an O2 molecule held at the bimetallic
center active site, which is formed by the other (more…)

The cytochrome oxidase reaction is estimated to account for 90%
of the total oxygen uptake in most cells. This protein complex is
therefore crucial for all aerobic life. Cyanide and azide are extremely
toxic because they bind tightly to the cell’s cytochrome oxidase
complexes to stop electron transport, thereby greatly reducing
ATP production.

Although the cytochrome oxidase in mammals contains 13
different protein subunits, most of these seem to have a subsidiary
role, helping to regulate either the activity or the assembly of the
three subunits that form the core of the enzyme. The complete
structure of this large enzyme complex has recently been determined
by x-ray crystallography, as illustrated in Figure 14-28. The atomic
resolution structures, combined with mechanistic studies of the effect
of precisely tailored mutations introduced into the enzyme by genetic
engineering of the yeast and bacterial proteins, are revealing the
detailed mechanisms of this finely tuned protein machine.

Figure 14-28. The molecular structure of cytochrome oxidase.

Figure 14-28

The molecular structure of cytochrome oxidase. This protein
is a dimer formed from a monomer with 13 different protein
subunits (monomer mass of 204,000 daltons). The three colored
subunits are encoded by the mitochondrial genome, and they
form the functional (more…)

Electron Transfers Are Mediated by Random Collisions in
the Inner Mitochondrial Membrane

The two components that carry electrons between the three
major enzyme complexes of the respiratory chain—ubiquinone
and cytochrome c—diffuse rapidly in the plane of the inner
mitochondrial membrane. The expected rate of random collisions
between these mobile carriers and the more slowly diffusing
enzyme complexes can account for the observed rates of electron
transfer (each complex donates and receives an electron about
once every 5–20 milliseconds). Thus, there is no need to postulate
a structurally ordered chain of electron-transfer proteins in the
lipid bilayer; indeed, the three enzyme complexes seem to exist as
independent entities in the plane of the inner membrane, being
present in different ratios in different mitochondria.

The ordered transfer of electrons along the respiratory chain
is due entirely to the specificity of the functional interactions
between the components of the chain: each electron carrier is
able to interact only with the carrier adjacent to it in the sequence
shown in Figure 14-26, with no short circuits.

Electrons move between the molecules that carry them in
biological systems not only by moving along covalent bonds
within a molecule, but also by jumping across a gap as large
as 2 nm. The jumps occur by electron “tunneling,” a quantum-
mechanical property that is critical for the processes we are
discussing. Insulation is needed to prevent short circuits that
would otherwise occur when an electron carrier with a low redox
potential collides with a carrier with a high redox potential. This
insulation seems to be provided by carrying an electron deep
enough inside a protein to prevent its tunneling interactions
with an inappropriate partner.

How the changes in redox potential from one electron carrier
to the next are harnessed to pump protons out of the mitochondrial
matrix is the topic we discuss next.

A Large Drop in Redox Potential Across Each of the Three Respiratory
Enzyme Complexes Provides the Energy for H+ Pumping

We have previously discussed how the redox potential reflects
electron affinities (see p. 783). An outline of the redox potentials
measured along the respiratory chain is shown in Figure 14-29.
These potentials drop in three large steps, one across each major
respiratory complex. The change in redox potential between any
two electron carriers is directly proportional to the free energy
released when an electron transfers between them. Each enzyme
complex acts as an energy-conversion device by harnessing some
of this free-energy change to pump H+ across the inner membrane,
thereby creating an electrochemical proton gradient as electrons
pass through that complex. This conversion can be demonstrated
by purifying each respiratory enzyme complex and incorporating
it separately into liposomes: when an appropriate electron donor
and acceptor are added so that electrons can pass through the complex,
H+ is translocated across the liposome membrane.

Figure 14-29. Redox potential changes along the mitochondrial
electron-transport chain.

Figure 14-29

Redox potential changes along the mitochondrial electron-transport
chain. The redox potential (designated E′0) increases as electrons
flow down the respiratory chain to oxygen. The standard free-energy
change, ΔG°, for the transfer (more…)

The Mechanism of H+ Pumping Will Soon Be Understood in
Atomic Detail

Some respiratory enzyme complexes pump one H+ per electron
across the inner mitochondrial membrane, whereas others pump
two. The detailed mechanism by which electron transport is coupled
to H+ pumping is different for the three different enzyme complexes.
In the cytochrome b-c1 complex, the quinones clearly have a role.
As mentioned previously, a quinone picks up a H+ from the aqueous
medium along with each electron it carries and liberates it when it
releases the electron (see Figure 14-24). Since ubiquinone is freely
mobile in the lipid bilayer, it could accept electrons near the inside
surface of the membrane and donate them to the cytochrome b-c1
complex near the outside surface, thereby transferring one H+
across the bilayer for every electron transported. Two protons are
pumped per electron in the cytochrome b-c1 complex, however, and
there is good evidence for a so-called Q-cycle, in which ubiquinone
is recycled through the complex in an ordered way that makes this
two-for-one transfer possible. Exactly how this occurs can now be
worked out at the atomic level, because the complete structure of
the cytochrome b-c1 complex has been determined by x-ray
crystallography (Figure 14-30).

Figure 14-30. The atomic structure of cytochrome b-c 1.

Figure 14-30

The atomic structure of cytochrome b-c 1. This protein is a dimer.
The 240,000-dalton monomer is composed of 11 different protein
molecules in mammals. The three colored proteins form the
functional core of the enzyme: cytochrome b (green), cytochrome (more…)

Allosteric changes in protein conformations driven by electron
transport can also pump H+, just as H+ is pumped when ATP
is hydrolyzed by the ATP synthase running in reverse. For both the
NADH dehydrogenase complex and the cytochrome oxidase complex,
it seems likely that electron transport drives sequential allosteric
changes in protein conformation that cause a portion of the protein
to pump H+ across the mitochondrial inner membrane. A general
mechanism for this type of H+ pumping is presented in Figure 14-31.

Figure 14-31. A general model for H+ pumping.

Figure 14-31

A general model for H+ pumping. This model for H+ pumping
by a transmembrane protein is based on mechanisms that are
thought to be used by both cytochrome oxidase and the light-driven
procaryotic proton pump, bacteriorhodopsin. The protein
is driven through (more…)

H+ Ionophores Uncouple Electron Transport from ATP Synthesis

Since the 1940s, several substances—such as 2,4-dinitrophenol—
have been known to act as uncoupling agents, uncoupling electron
transport from ATP synthesis. The addition of these low-molecular-weight organic compounds to cells stops ATP synthesis by mitochondria
without blocking their uptake of oxygen. In the presence of an
uncoupling agent, electron transport and H+ pumping continue at
a rapid rate, but no H+ gradient is generated. The explanation for
this effect is both simple and elegant: uncoupling agents are lipid-
soluble weak acids that act as H+ carriers (H+ ionophores), and
they provide a pathway for the flow of H+ across the inner mitochondrial
membrane that bypasses the ATP synthase. As a result of this short-
circuiting, the proton-motive force is dissipated completely, and
ATP can no longer be made.

Respiratory Control Normally Restrains Electron Flow
Through the Chain

When an uncoupler such as dinitrophenol is added to cells,
mitochondria increase their oxygen uptake substantially because
of an increased rate of electron transport. This increase reflects
the existence of respiratory control. The control is thought to
act via a direct inhibitory influence of the electrochemical proton
gradient on the rate of electron transport. When the gradient is
collapsed by an uncoupler, electron transport is free to run unchecked
at the maximal rate. As the gradient increases, electron transport
becomes more difficult, and the process slows. Moreover, if an
artificially large electrochemical proton gradient is experimentally
created across the inner membrane, normal electron transport
stops completely, and a reverse electron flow can be detected in
some sections of the respiratory chain. This observation suggests
that respiratory control reflects a simple balance between the
free-energy change for electron-transport-linked proton pumping
and the free-energy change for electron transport—that is, the
magnitude of the electrochemical proton gradient affects both
the rate and the direction of electron transport, just as it affects
the directionality of the ATP synthase (see Figure 14-19).

Respiratory control is just one part of an elaborate interlocking
system of feedback controls that coordinate the rates of glycolysis,
fatty acid breakdown, the citric acid cycle, and electron transport.
The rates of all of these processes are adjusted to the ATP:ADP ratio,
increasing whenever an increased utilization of ATP causes the ratio
to fall. The ATP synthase in the inner mitochondrial membrane,
for example, works faster as the concentrations of its substrates
ADP and Pi increase. As it speeds up, the enzyme lets more H+ flow
into the matrix and thereby dissipates the electrochemical proton
gradient more rapidly. The falling gradient, in turn, enhances the
rate of electron transport.

Similar controls, including feedback inhibition of several key enzymes
by ATP, act to adjust the rates of NADH production to the rate of
NADH utilization by the respiratory chain, and so on. As a result of
these many control mechanisms, the body oxidizes fats and sugars
5–10 times more rapidly during a period of strenuous exercise than
during a period of rest.

Natural Uncouplers Convert the Mitochondria in Brown Fat into
Heat-generating Machines

In some specialized fat cells, mitochondrial respiration is normally
uncoupled from ATP synthesis. In these cells, known as brown fat
cells, most of the energy of oxidation is dissipated as heat rather
than being converted into ATP. The inner membranes of the large
mitochondria in these cells contain a special transport protein that
allows protons to move down their electrochemical gradient, by-
passing ATP synthase. As a result, the cells oxidize their fat stores
at a rapid rate and produce more heat than ATP. Tissues containing
brown fat serve as “heating pads,” helping to revive hibernating animals
and to protect sensitive areas of newborn human babies from the cold.

Bacteria Also Exploit Chemiosmotic Mechanisms to Harness Energy

Bacteria use enormously diverse energy sources. Some, like animal
cells, are aerobic; they synthesize ATP from sugars they oxidize to
CO2 and H2O by glycolysis, the citric acid cycle, and a respiratory
chain in their plasma membrane that is similar to the one in the
inner mitochondrial membrane. Others are strict anaerobes, deriving
their energy either from glycolysis alone (by fermentation) or from an
electron-transport chain that employs a molecule other than oxygen
as the final electron acceptor. The alternative electron acceptor can
be a nitrogen compound (nitrate or nitrite), a sulfur compound
(sulfate or sulfite), or a carbon compound (fumarate or carbonate),
for example. The electrons are transferred to these acceptors by a
series of electron carriers in the plasma membrane that are comparable
to those in mitochondrial respiratory chains.

Despite this diversity, the plasma membrane of the vast majority of
bacteria contains an ATP synthase that is very similar to the one in
mitochondria. In bacteria that use an electron-transport chain to
harvest energy, the electron-transport pumps H+ out of the cell and
thereby establishes a proton-motive force across the plasma membrane
that drives the ATP synthase to make ATP. In other bacteria, the
ATP synthase works in reverse, using the ATP produced by glycolysis
to pump H+ and establish a proton gradient across the plasma
membrane. The ATP used for this process is generated by
fermentation processes (discussed in Chapter 2).

Thus, most bacteria, including the strict anaerobes, maintain a proton
gradient across their plasma membrane. It can be harnessed to drive
a flagellar motor, and it is used to pump Na+ out of the bacterium via
a Na+-H+ antiporter that takes the place of the Na+-K+ pump of
eucaryotic cells. This gradient is also used for the active inward transport
of nutrients, such as most amino acids and many sugars: each nutrient is
dragged into the cell along with one or more H+ through a specific symporter
(Figure 14-32). In animal cells, by contrast, most inward transport across
the plasma membrane is driven by the Na+ gradient that is established by the
Na+-K+ pump.

Figure 14-32. The importance of H+-driven transport in bacteria.

Figure 14-32

The importance of H+-driven transport in bacteria. A proton-motive force
generated across the plasma membrane pumps nutrients into the cell and
expels Na+. (A) In an aerobic bacterium, an electrochemical proton gradient
across the plasma membrane is produced (more…)

Some unusual bacteria have adapted to live in a very alkaline
environment and yet must maintain their cytoplasm at a physiological
pH. For these cells, any attempt to generate an electrochemical H+
gradient would be opposed by a large H+ concentration gradient in
the wrong direction (H+ higher inside than outside). Presumably for
this reason, some of these bacteria substitute Na+ for H+ in all of their
chemiosmotic mechanisms. The respiratory chain pumps Na+ out of
the cell, the transport systems and flagellar motor are driven by an
inward flux of Na+, and a Na+-driven ATP synthase synthesizes
ATP. The existence of such bacteria demonstrates that the principle
of chemiosmosis is more fundamental than the proton-motive force
on which it is normally based.

Summary

The respiratory chain in the inner mitochondrial membrane contains
three respiratory enzyme complexes through which electrons pass on
their way from NADH to O2.

Each of these can be purified, inserted into synthetic lipid vesicles,
and then shown to pump H+ when electrons are transported through it.
In the intact membrane, the mobile electron carriers ubiquinone and
cytochrome c complete the electron-transport chain by shuttling between
the enzyme complexes. The path of electron flow is NADH → NADH
dehydrogenase complex → ubiquinone → cytochrome b-c1 complex →
cytochrome c → cytochrome oxidase complex → molecular oxygen (O2).

The respiratory enzyme complexes couple the energetically favorable
transport of electrons to the pumping of H+ out of the matrix. The
resulting electrochemical proton gradient is harnessed to make ATP
by another transmembrane protein complex, ATP synthase, through
which H+ flows back into the matrix. The ATP synthase is a reversible
coupling device that normally converts a backflow of H+ into ATP
phosphate bond energy by catalyzing the reaction ADP + Pi → ATP,
but it can also work in the opposite direction and hydrolyze ATP to
pump H+ if the electrochemical proton gradient is sufficiently reduced.
Its universal presence in mitochondria, chloroplasts, and procaryotes
testifies to the central importance of chemiosmotic mechanisms in cells.

By agreement with the publisher, this book is accessible by the search
feature, but cannot be browsed.

Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis,
Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989,
1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith
Roberts, and James D. Watson .

Read Full Post »


Introduction to Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP

 

Humans, mammals, plants and animals, and eukaryotes and prokaryotes all share a common denominator in their manner of existence.  It makes no difference whether they inhabit the land, or the sea, or another living host. They exist by virtue of their metabolic adaptation by way of taking in nutrients as fuel, and converting the nutrients to waste in the expenditure of carrying out the functions of motility, breakdown and utilization of fuel, and replication of their functional mass.

There are essentially two major sources of fuel, mainly, carbohydrate and fat.  A third source, amino acids which requires protein breakdown, is utilized to a limited extent as needed from conversion of gluconeogenic amino acids for entry into the carbohydrate pathway. Amino acids follow specific metabolic pathways related to protein synthesis and cell renewal tied to genomic expression.

Carbohydrates are a major fuel utilized by way of either of two pathways.  They are a source of readily available fuel that is accessible either from breakdown of disaccharides or from hepatic glycogenolysis by way of the Cori cycle.  Fat derived energy is a high energy source that is metabolized by one carbon transfers using the oxidation of fatty acids in mitochondria. In the case of fats, the advantage of high energy is conferred by chain length.

Carbohydrate metabolism has either of two routes of utilization.  This introduces an innovation by way of the mitochondrion or its equivalent, for the process of respiration, or aerobic metabolism through the tricarboxylic acid, or Krebs cycle.  In the presence of low oxygen supply, carbohydrate is metabolized anaerobically, the six carbon glucose being split into two three carbon intermediates, which are finally converted from pyruvate to lactate.  In the presence of oxygen, the lactate is channeled back into respiration, or mitochondrial oxidation, referred to as oxidative phosphorylation. The actual mechanism of this process was of considerable debate for some years until it was resolved that the mechanism involve hydrogen transfers along the “electron transport chain” on the inner membrane of the mitochondrion, and it was tied to the formation of ATP from ADP linked to the so called “active acetate” in Acetyl-Coenzyme A, discovered by Fritz Lipmann (and Nathan O. Kaplan) at Massachusetts General Hospital.  Kaplan then joined with Sidney Colowick at the McCollum Pratt Institute at Johns Hopkins, where they shared tn the seminal discovery of the “pyridine nucleotide transhydrogenases” with Elizabeth Neufeld,  who later established her reputation in the mucopolysaccharidoses (MPS) with L-iduronidase and lysosomal storage disease.

This chapter covers primarily the metabolic pathways for glucose, anaerobic and by mitochondrial oxidation, the electron transport chain, fatty acid oxidation, galactose assimilation, and the hexose monophosphate shunt, essential for the generation of NADPH. The is to be more elaboration on lipids and coverage of transcription, involving amino acids and RNA in other chapters.

The subchapters are as follows:

1.1      Carbohydrate Metabolism

1.2      Studies of Respiration Lead to Acetyl CoA

1.3      Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

1.4      The Multi-step Transfer of Phosphate Bond and Hydrogen Exchange Energy

Complex I or NADH-Q oxidoreductase

Complex I or NADH-Q oxidoreductase

Fatty acid oxidation and ETC

Fatty acid oxidation and ETC

Read Full Post »


Larry H Bernstein, MD, FCAP, Author and Curator

Chief, Scientific Communication

Leaders in Pharmaceutical Intelligence

with contributions from JEDS Rosalis, Brazil
and Radislov Rosov, Univ of Virginia, VA, USA

A Brief Curation of Proteomics, Metabolomics, and Metabolism

This article is a continuation of a series of elaborations of the recent and
accelerated scientific discoveries that are enlarging the scope of and
integration of biological and medical knowledge leading to new drug
discoveries.  The work that has led us to this point actually has roots
that go back 150 years.  The roots go back to studies in the mid-nineteenth century, with the emergence of microbiology, physiology,
pathology, botany, chemistry and physics, and the laying down of a
mechanistic approach divergent from descriptive observation in the
twentieth century. Medicine took on the obligation to renew the method
of training physicians after the Flexner Report (The Flexner Report of
1910 transformed the nature and process of medical education in America
with a resulting elimination of proprietary schools), funded by the Carnegie
Foundation.  Johns Hopkins University Medical School became the first to
adopt the model, as did Harvard, Yale, University of Chicago, and others.

The advances in biochemistry, genetics and genomics, were large, as was
structural organic chemistry in the remainder of the centrury.  The advances
in applied mathematics and in instrumental analysis opened a new gateway
into the 21st century with the Human Genome Project, the Proteome Library,
Signaling Pathways, and the Metabolomes – human, microbial, and plants.

shall elaborate on how the key processes of life are being elucidated as
these interrelated disciplines converge.  I shall not be covering in great
detail the contribution of the genetic code and transcripton because they
have been covered at great length in this series.

Part I.  The foundation for the emergence of a revitalized molecular
biology 
and biochemistry.

In a series of discussions with Jose des Salles Roselino (Brazil) over a
period of months we have come to an important line of reasoning. DNA
to protein link goes from triplet sequence to amino acid sequence. The
realm of genetics. Further, protein conformation, activity and function
requires that environmental and microenvironmental factors should be
considered (Biochemistry).  This has been opened in several articles
preceding this.

In the cAMP coupled hormonal response the transfer of conformation
from protein to protein is paramount. For instance, if your scheme goes
beyond cAMP, it will show an effect over a self-assembly (inhibitor
protein and protein kinase). Therefore, sequence alone does not
explain conformation, activity and function of regulatory proteins.
Recall that sequence is primar structure, determined by the translation
of the code, but secondary structure is determined by disulfide bonds.
There is another level of structure, tertiary structure, that is molded by
steric influences of near neighbors and by noncovalent attractions
and repulsions.

A few comments ( contributed by Assoc. Prof. JEDS Roselino) are in
order to stress the importance of self-assembly (Prigogine, R. A
Marcus, conformation energy) in a subject that is the best for this
connection. We have to stress again that in the cAMP
coupled hormonal response the transfer of conformation from
protein to protein is paramount. For instance, in case the
reaction sequence follows beyond the production of the
second messenger, as in the case of cAMP, this second
messenger will remove a self-assembly of inhibitor protein
with the enzyme protein kinase. Therefore, sequence alone
does not explain conformation, activity and function of
regulatory proteins. In this case, if this important mechanism
was not ignored, the work of Stanley Prusiner would most
certainly have been recognized earlier, and “rogue” proteins
would not have been seen as so rogue as some assumed.
For the general idea of importance of self-assembly versus
change in covalent modification of proteins (see R. A Kahn
and A. G Gilman (1984) J. Biol. Chem.  259(10), pp 6235-
6240. In this case, trimeric or dimeric G does not matter.
“Signaling transduction tutorial”.
G proteins in the G protein coupled-receptor proteins are
presented following a unidirectional series of arrows.
This is adequate to convey the idea of information being
transferred from outside the cell towards cell´s interior
(therefore, against the dogma that says all information
moves from DNA to RNA to protein.  It is important to
consider the following: The entire process is driven by
a very delicate equilibrium between possible conform-
ational states of the proteins. Empty receptors have very
low affinity for G proteins. On the other hand, hormone
bound receptors have a change in conformation that
allows increasing the affinity for the G-trimer. When
hormone receptors bind to G-trimers two things happen:

  1. Receptors transfer conformation information to
    the G-triplex and
  2. the G-triplex transfers information back to the
    complex hormone-receptor.

In the first case , the dissociated G protein exchanges
GDP for GTP and has its affinity for the cyclase increased,
while by the same interaction receptor releases the
hormone which then places the first required step for the
signal. After this first interaction step, on the second and
final transduction system step is represented by an
opposite arrow. When, the G-protein + GTP complex
interacts with the cyclase two things happen:

  1. It changes the cyclase to an active conformation
    starting the production of cAMP as the single
    arrow of the scheme. However, the interaction
    also causes a backward effect.
  2. It activates the GTPase activity of this subunit
    and the breakdown of GTP to GDP moves this 
    subunit back to the initial trimeric inactive
    state
     of G complex.

This was very well studied when the actions of cholera toxin
required better understanding. Cholera toxin changes the
GTPase subunit by ADP-ribosilation (a covalent and far more
stable change in proteins) producing a permanent conformation
of GTP bound G subunit. This keeps the cyclase in permanent
active conformation because ADP-ribosilation inhibits GTPase
activity required to put an end in the hormonal signal.

The study made while G-proteins were considered a dimer still
holds despite its limited vision of the real complexity of the
transduction system. It was also possible to get this very same
“freezing” in the active state using GTP stable analogues. This
transduction system is one of the best examples of the delicate
mechanisms of conformational interaction of proteins. Further-
more, this system also shows on the opposite side of our
reasoning scheme, how covalent changes are adequate for
more stable changes than those mediated by Van der Wall’s
forces between proteins. Yet, these delicate forces are the
same involved when Sc-Prion transfers its rogue
conformation to c-Prion proteins and other similar events.
The Jacob-Monod Model

A combination of genetic and biochemical experiments in
bacteria led to the initial recognition of

  1. protein-binding regulatory sequences associated with genes and
  2. proteins whose binding to a gene’s regulatory sequences
    either activate or repress its transcription.

These key components underlie the ability of both prokaryotic and
eukaryotic cells to turn genes on and off. The  experimental findings lead to a general model of bacterial transcription control.

Gene control serves to allow a single cell to adjust to changes in its
nutritional environment so that its growth and division can be optimized.
Thus, the prime focus of research has been on genes that encode
inducible proteins whose production varies depending on the nutritional
status of the cells. Its most characteristic and biologically far-reaching
purpose in eukaryotes, distinctive from single cell organisms is the
regulation of a genetic program that underlies embryological
development and tissue differentiation.

The principles of transcription have already been described in this
series under the translation of the genetic code into amino acids
that are the building blocks for proteins.

E.coli can use either glucose or other sugars such as the
disaccharide lactose as the sole source of carbon and energy.
When E. coli cells are grown in a glucose-containing medium,
the activity of the enzymes needed to metabolize lactose is
very low. When these cells are switched to a medium
containing lactose but no glucose, the activities of the lactose-metabolizing enzymes increase. Early studies showed that the
increase in the activity of these enzymes resulted from the
synthesis of new enzyme molecules, a phenomenon termed
induction. The enzymes induced in the presence of lactose
are encoded by the lac operon, which includes two genes, Z
and Y, that are required for metabolism of lactose and a third
gene. The lac Y gene encodes lactose permease, which spans the E. coli cell membrane and uses the energy available from
the electrochemical gradient across the membrane to pump
lactose into the cell. The lac Z gene encodes β-galactosidase,
which splits the disaccharide lactose into the monosaccharides
glucose and galactose, which are further metabolized through
the action of enzymes encoded in other operons. The third
gene encodes thiogalactoside transacetylase.

Synthesis of all three enzymes encoded in the lac operon is rapidly
induced when E. coli cells are placed in a medium containing lactose
as the only carbon source and repressed when the cells are switched
to a medium without lactose. Thus all three genes of the lac operon
are coordinately regulated. The lac operon in E. coli provides one
of the earliest and still best-understood examples of gene control.
Much of the pioneering research on the lac operon was conducted by
Francois Jacob, Jacques Monod, and their colleagues in the 1960s.

Some molecules similar in structure to lactose can induce expression
of the lacoperon genes even though they cannot be hydrolyzed by β-galactosidase. Such small molecules (i.e., smaller than proteins) are
called inducers. One of these, isopropyl-β-D-thiogalactoside,
abbreviated IPTG,is particularly useful in genetic studies of the lac
operon, because it can diffuse into cells and, it is not metabolized.
Insight into the mechanisms controlling synthesis of β-galactosidase
and lactose permease came from the study of mutants in which control
of β-galactosidase expression was abnormal and used a colorimetric
assay for β-galactosidase.

When the cells are exposed to chemical mutagens before plating on
X-gal/glucose plates, rare blue colonies appear, but when cells
from these blue colonies are recovered and grown in media containing
glucose, they overexpress all the genes of the lac operon. These cells
are called constitutive mutants because they fail to repress the lac
operon in media lacking lactose and instead continuously express the
enzymes, and the genes were mapped to a region on the E. coli
chromosome. This led to the conclusion that these cells had a defect
in a protein that normally repressed expression of the lac operon in
the absence of lactose, and that it blocks transcription by binding to
a site on the E. coli genome where transcription of the lac operon is
initiated. In addition, it binds to the lac repressor in the lactose
medium and decreases its affinity for the repressor-binding site
on the DNA causing the repressor to unbind the DNA. Thereby,
transcription of the lac operon is initiated, leading to synthesis of
β-galactosidase, lactose permease, and thiogalactoside
transacetylase.

 regulation of the lac operon by lac repressor

Jacob and Monod model of transcriptional regulation of the lac operon

Next, Jacob and Monod isolated mutants that expressed the lac operon
constitutively even when two copies of the wild-type lacI gene
encoding the lac repressor were present in the same cell, and the
constitutive mutations mapped to one end of the lac operon, as the
model predicted.  Further, there are rare cells that carry a mutation
located at the region, promoter, that block initiation of transcription by
RNA polymerase.

lac I+ gene is trans-acting, & encodes a protein, which binds to a lac operator

 lac I+ gene is trans-acting, & encodes a protein, which
binds to a lac operator

They further demonstrated that the two types of mutations lac I and
lac I+, were cis- and trans-acting, the latter encoding a protein that
binds to the lac operator. The cis-acting Oc mutations prevent
binding of the lac repressor to the operator, and  mutations in the
lac promoter are cis-acting, since they alter the binding site for RNA
polymerase. In general, trans-acting genes that regulate expression
of genes on other DNA molecules encode diffusible products. In
most cases these are proteins, but in some cases RNA molecules
can act in trans to regulate gene expression.

According to the Jacob and Monod model of transcriptional control,
transcription of the lac operon, which encodes three inducible
proteins, is repressed by binding of lac repressor protein to the
operator sequence.

 (Section 10.1Bacterial Gene Control: The Jacob-Monod Model.)
This book is accessible by the search feature.

Comment: This seminal work was done a half century ago. It was a
decade after the Watson-Crick model for DNA. The model is
elaborated for the Eukaryote in the examples that follow.

(The next two articles were called to my attention by R. Bosov at
University of Virginia).

An acetate switch regulates stress erythropoiesis

M Xu,  JS Nagati, Ji Xie, J Li, H Walters, Young-Ah Moon, et al.
Nature Medicine 10 Aug 2014(20): 1018–1026.
http://dx.doi.org:/10.1038/nm.3587

message: 1- ( -CH3 ) = Ln ( (1/sqrt(1-Acetate^2) –
sqrt oxalate))/ Ln(oxygen) – K(o)
rsb5n@virginia.edu

The hormone erythropoietin (EPO), synthesized in the kidney or liver
of adult mammals, controls erythrocyte production and is regulated by
the stress-responsive transcription factor hypoxia-inducible factor-2
(HIF-2).
 HIFα acetylation and efficient HIF-2–dependent EPO
induction during hypoxia requires  the lysine acetyltransferase CREB-binding protein (CBP) . These processes require acetate-dependent
acetyl CoA synthetase 2 (ACSS2) as follows.Acetate levels rise and
ACSS2 is required for HIF-2α acetylation, CBP–HIF-2α complex
formation, CBP–HIF-2α recruitment to the EPO enhancer and induction
of EPO gene expression
 in human Hep3B hepatoma cells and in EPO-generating organs of hypoxic or acutely anemic mice. In acutely anemic
mice, acetate supplementation augments stress erythropoiesis in an
ACSS2-dependent manner. Moreover, in acquired and inherited
chronic anemia mouse models, acetate supplementation increases
EPO expression
 and the resting hematocrit. Thus, a mammalian
stress-responsive acetate switch controls HIF-2 signaling and EPO
induction during pathophysiological states marked by tissue hypoxia.

Figure 1: Acss2 controls HIF-2 signaling in hypoxic cells.
Time course of endogenous HIF-2α acetylation during hypoxia following
immunoprecipitation (IP) of HIF-2α from whole-cell extracts and detection
of acetylated lysines by immunoblotting (IB).
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F1.jpg

Figure 2: Acss2 regulates hypoxia-induced renal Epo expression in mice.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F2.jpg

Figure 3: Acute anemia induces Acss2-dependent HIF-2 signaling in mice.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F3.jpg

Figure 4: An acetate switch regulates Cbp–HIF-2 interactions in cells.
(a) HIF-2α acetylation following immunoprecipitation of endogenous
HIF-2α and detection by immunoblotting with antibodies to acetylated
lysine or HIF-2α.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F4.jpg

Figure 5: Acss2 signaling in cells requires intact HIF-2 acetylation.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F5.jpg

Figure 6: Acetate facilitates recovery from anemia.

Acetate facilitates recovery from anemia

Acetate facilitates recovery from anemia

(a) Serial hematocrits of CD1 wild-type female mice after PHZ treatment, followed
by once daily per os (p.o.) supplementation with water vehicle (Veh; n = 7 mice),
GTA (n = 6 mice), GTB (n = 8 mice) or GTP (n = 7 mice) (single measurem…

http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F6.jpg

see also-.
1. Bunn, H.F. & Poyton, R.O. Oxygen sensing and molecular adaptation to
hypoxia. Physiol. Rev. 76, 839–885 (1996).

  1. .Richalet, J.P. Oxygen sensors in the organism: examples of regulation
    under altitude hypoxia in mammals. Comp. Biochem. Physiol. A Physiol.
    118, 9–14 (1997).
  2. .Koury, M.J. Erythropoietin: the story of hypoxia and a finely regulated
    hematopoietic hormone. Exp. Hematol. 33, 1263–1270 (2005).
  3. Wang, G.L., Jiang, B.H., Rue, E.A. & Semenza, G.L. Hypoxia-inducible
    factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated
    by cellular O2 tension. Proc. Natl. Acad. Sci. USA92, 5510–5514 (1995).
  4. Chen, R. et al. The acetylase/deacetylase couple CREB-binding
    protein/sirtuin 1 controls hypoxia-inducible factor 2 signaling. J. Biol.
    Chem. 287, 30800–30811 (2012).
  5. .Papandreou, I., Cairns, R.A., Fontana, L., Lim, A.L. & Denko, N.C.
    HIF-1 mediates adaptation to hypoxia by actively down-regulating
    mitochondrial oxygen consumption. Cell Metab. 3,187–197 (2006).

14. Kim, J.W., Tchernyshyov, I., Semenza, G.L. & Dang, C.V. HIF-1-
mediated expression of pyruvate dehydrogenase kinase: a metabolic
switch required for cellular adaptation to hypoxia. Cell Metab. 3,
177–185 (2006).

16. Fujino, T., Kondo, J., Ishikawa, M., Morikawa, K. & Yamamoto, T.T.
Acetyl-CoA synthetase 2, a mitochondrial matrix enzyme involved in the
oxidation of acetate. J. Biol. Chem. 276,11420–11426 (2001).

17..Luong, A., Hannah, V.C., Brown, M.S. & Goldstein, J.L. Molecular
characterization of human acetyl-CoA synthetase, an enzyme regulated
by sterol regulatory element-binding proteins. J. Biol. Chem. 275,
26458–26466 (2000).

20 .Wellen, K.E. et al. ATP-citrate lyase links cellular metabolism to
histone acetylation. Science324, 1076–1080 (2009).

24. McBrian, M.A. et al. Histone acetylation regulates intracellular pH.
Mol. Cell 49, 310–321(2013).

Asymmetric mRNA localization contributes to fidelity and sensitivity
of spatially localized systems

Robert J Weatheritt, Toby J Gibson & M Madan Babu
Nature Structural & Molecular Biology 21, 833–839 (2014)
http://www.nature.com/nsmb/journal/v21/n9/abs/nsmb.2876.html 

Although many proteins are localized after translation, asymmetric
protein distribution is also achieved by translation after mRNA localization.
Why are certain mRNA transported to a distal location and translated
on-site? Here we undertake a systematic, genome-scale study of
asymmetrically distributed protein and mRNA in mammalian cells.
Our findings suggest that asymmetric protein distribution by mRNA
localization enhances interaction fidelity and signaling sensitivity
.
Proteins synthesized at distal locations frequently contain intrinsically
disordered segments. These regions are generally rich in assembly-
promoting modules and are often regulated by post-translational
modifications. Such proteins are tightly regulated but display distinct
temporal dynamics upon stimulation with growth factors. Thus, proteins
synthesized on-site may rapidly alter proteome composition and
act as dynamically regulated scaffolds to promote the formation
of reversible cellular assemblies. 
Our observations are consistent
across multiple mammalian species, cell types and developmental stages,
suggesting that localized translation is a recurring feature of cell
signaling and regulation.

Figure 1: Classification and characterization of TAS and DSS proteins.

The two major mechanisms for localizing proteins to distal sites in the cell

The two major mechanisms for localizing proteins to distal sites in the cell

(a)The two major mechanisms for localizing proteins to distal sites in the cell.
(b) Data sets used to identify groups of DSS and TAS transcripts, as well as
DSS and TAS proteins in mouse neuroblastoma cells

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F1.jpg

Figure 2: Structural analysis of DSS proteins reveals an enrichment
in disordered regions.

Distributions of the various structural properties of the DSS and TAS proteins of the mouse neuroblastoma data sets

Distributions of the various structural properties of the DSS and TAS proteins of the mouse neuroblastoma data sets

(a,b) Distributions of the various structural properties of the DSS and TAS
proteins of the mouse neuroblastoma data sets (a), the mouse pseudopodia,
the rat embryonic sensory neuron data set and the adult sensory neuron data set (b).…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F2.jpg

Figure 3: Analysis of DSS proteins reveals an enrichment for linear motifs, phase-
transition (i.e., higher-order assembly) promoting segments and PTM sites that act
as molecular switches.

(a,b) Distributions of the various regulatory and structural properties of the DSS
and TAS proteins of the mouse neuroblastoma data sets
http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F3.jpg

Figure 4: Dynamic regulation of DSS transcripts and proteins.

Dynamic regulation of DSS transcripts and proteins

Dynamic regulation of DSS transcripts and proteins

Genome-wide quantitative measurements of gene expression of DSS (n = 289)
and TAS (n = 1,292) proteins in mouse fibroblast cells. DSS transcripts and
proteins have a lower abundance and shorter half-lives

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F4.jpg

Figure 5: An overview of the potential advantages conferred by distal-site protein
synthesis, inferred from our analysis.

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis

Turquoise and red filled circle represents off-target and correct interaction partners,
respectively. Wavy lines – a disordered region within a distal site synthesis protein.

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F5.jpg

The identification of asymmetrically localized proteins and transcripts.

The identification of asymmetrically localized proteins and transcripts

The identification of asymmetrically localized proteins and transcripts

An illustrative explanation of the resolution of the study and the concept of asymmetric
localization of proteins and mRNA. In this example, on the left a neuron is divided into
its cell body and axon terminal, and transcriptome/proteo…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-SF1.jpg

Graphs and boxplots of functional and structural properties for distal site synthesis
(DSS) proteins (red) and transport after synthesis (TAS) proteins (gray).
See Online Methods for details and legend of Figure 2 for a description of boxplots
and statistical tests.
http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-SF2.jpg

See also –
1. Martin, K.C. & Ephrussi, A. mRNA localization: gene expression in the spatial
dimension. Cell136, 719–730 (2009).

  1. Scott, J.D. & Pawson, T. Cell signaling in space and time: where proteins come
    together and when they’re apart. Science 326, 1220–1224 (2009).

4..Holt, C.E. & Bullock, S.L. Subcellular mRNA localization in animal cells
and why it matters.Science 326, 1212–1216 (2009).

  1. Jung, H., Gkogkas, C.G., Sonenberg, N. & Holt, C.E. Remote control of
    gene function by local translation. Cell 157, 26–40 (2014). 

Regulation of metabolism by hypoxia-inducible factor 1.   
Semenza GL.    Author information
Cold Spring Harb Symp Quant Biol. 2011;76:347-53.
http://dx.doi.org:/10.1101/sqb.2011.76.010678.

The maintenance of oxygen homeostasis is critical for survival, and the
master regulator of this process in metazoan species is hypoxia-inducible
factor 1 (HIF-1), which

  • controls both O(2) delivery and utilization.

Under conditions of reduced O(2) availability,

  • HIF-1 activates the transcription of genes, whose protein products
  • mediate a switch from oxidative to glycolytic metabolism.

HIF-1 is activated in cancer cells as a result of intratumoral hypoxia
and/or genetic alterations.

In cancer cells, metabolism is reprogrammed to

  • favor glycolysis even under aerobic conditions.

Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and
metabolism, although the mechanism by which it exerts these effects is
unclear. Recent studies indicate that

PKM2 interacts with HIF-1α physically and functionally to

  1. stimulate the binding of HIF-1 at target genes,
  2. the recruitment of coactivators,
  3. histone acetylation, and
  4. gene transcription.

Interaction with HIF-1α is facilitated by

  • hydroxylation of PKM2 at proline-403 and -408 by PHD3.

Knockdown of PHD3

  • decreases glucose transporter 1, lactate dehydrogenase A, and
    pyruvate dehydrogenase kinase 1 expression;
  • decreases glucose uptake and lactate production; and
  • increases O(2) consumption.

The effect of PKM2/PHD3 is not limited to genes encoding metabolic
enzymes because VEGF is similarly regulated.

These results provide a mechanism by which PKM2

  • promotes metabolic reprogramming and

suggest that it plays a broader role in cancer progression than has
previously been appreciated.   PMID: 21785006   

Cadherins

Cadherins are thought to be the primary mediators of adhesion
between the cells
 of vertebrate animals, and also function in cell
adhesion in many invertebrates. The expression of numerous cadherins
during development is highly regulated, and the precise pattern of
cadherin expression plays a pivotal role in the morphogenesis of tissues
and organs. The cadherins are also important in the continued maintenance
of tissue structure and integrity. The loss of cadherin expression appears
to be highly correlated with the invasiveness of some types of tumors. Cadherin adhesion is also dependent on the presence of calcium ions
in the extracellular milieu.

The cadherin protein superfamily, defined as proteins containing a
cadherin-like domain, can be divided into several sub-groups. These include

  • the classical (type I) cadherins, which mediate adhesion at adherens junctions;
  • the highly-related type II cadherins;
  • the desmosomal cadherins found in desmosome junctions;
  • protocadherins, expressed only in the nervous system; and
  • atypical cadherin-like domain containing proteins.

Members of all but the atypical group have been shown to play a role
in intercellular adhesion.

Part II.  PKM2 and regulation of glycolysis

PKM2 regulates the Warburg effect and promotes ​HMGB1
release in sepsis

L Yang, M Xie, M Yang, Y Yu, S Zhu, W Hou, R Kang, …, & D Tang
Nature Communic 14 July 2014; 5(4436)
http://dx.doi.org/doi:10.1038/ncomms5436

Increasing evidence suggests the important role of metabolic reprogramming

  • in the regulation of the innate inflammatory response,

We provide evidence to support a novel role for the

  • ​pyruvate kinase M2 (​PKM2)-mediated Warburg effect,

namely aerobic glycolysis,

  • in the regulation of ​high-mobility group box 1 (​HMGB1) release. ​
  1. PKM2 interacts with ​hypoxia-inducible factor 1α (​HIF1α) and
  2. activates the ​HIF-1α-dependent transcription of enzymes necessary
    for aerobic glycolysis in macrophages.

Knockdown of ​PKM2, ​HIF1α and glycolysis-related genes

  • uniformly decreases ​lactate production and ​HMGB1 release.

Similarly, a potential ​PKM2 inhibitor, ​shikonin,

  1. reduces serum ​lactate and ​HMGB1 levels, and
  2. protects mice from lethal endotoxemia and sepsis.

Collectively, these findings shed light on a novel mechanism for

  • metabolic control of inflammation by
  • regulating ​HMGB1 release and

highlight the importance of targeting aerobic glycolysis in the treatment
of sepsis and other inflammatory diseases.

  1. Glycolytic inhibitor ​2-D G attenuates ​HMGB1 release by activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f1.jpg
  2. Figure 2: Upregulated ​PKM2 promotes aerobic glycolysis and ​HMGB1
    release in activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f2.jpg
  3. Figure 3: ​PKM2-mediated ​HIF1α activation is required for ​HMGB1
    release in activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f3.jpg

 

ERK1/2-dependent phosphorylation and nuclear translocation of
PKM2 promotes the Warburg effect  

W Yang, Y Zheng, Y Xia, Ha Ji, X Chen, F Guo, CA Lyssiotis, & Zhimin Lu
Nature Cell Biology  2012 (27 June 2014); 14: 1295–1304
Corrigendum (January, 2013)  http://dx.doi.org:/10.1038/ncb2629

Pyruvate kinase M2 (PKM2) is upregulated in multiple cancer types and
contributes to the Warburg. We demonstrate that

  • EGFR-activated ERK2 binds directly to PKM2 Ile 429/Leu 431
  • through the ERK2 docking groove
  • and phosphorylates PKM2 at Ser 37, but
  • does not phosphorylate PKM1.

Phosphorylated PKM2 Ser 37

  1. recruits PIN1 for cis–trans isomerization of PKM2, which
  2. promotes PKM2 binding to importin α5
  3. and PKM2 translocates to the nucleus.

Nuclear PKM2 acts as

  • a coactivator of β-catenin to
  • induce c-Myc expression,

This is followed by

  1. the upregulation of GLUT1, LDHA and,
  2. in a positive feedback loop,
  • PTB-dependent PKM2 expression.

Replacement of wild-type PKM2 with

  • a nuclear translocation-deficient mutant (S37A)
  • blocks the EGFR-promoted Warburg effect
    and brain tumour development in mice.

In addition, levels of PKM2 Ser 37 phosphorylation

  • correlate with EGFR and ERK1/2 activity
    in human glioblastoma specimens.

Our findings highlight the importance of

  • nuclear functions of PKM2 in the Warburg effect
    and tumorigenesis.
  1. ERK is required for PKM2 nucleus translocation.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f1.jpg
  2. ERK2 phosphorylates PKM2 Ser 37.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f2.jpg
  3. Figure 3: PKM2 Ser 37 phosphorylation recruits PIN1.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f3.jpg

 Pyruvate kinase M2 activators promote tetramer formation
and suppress tumorigenesis

D Anastasiou, Y Yu, WJ Israelsen, Jian-Kang Jiang, MB Boxer, B Hong, et al.
Nature Chemical Biology  11 Oct 2012; 8: 839–847

Cancer cells engage in a metabolic program to

  • enhance biosynthesis and support cell proliferation.

The regulatory properties of pyruvate kinase M2 (PKM2)

  • influence altered glucose metabolism in cancer.

The interaction of PKM2 with phosphotyrosine-containing proteins

  • inhibits PTM2 enzyme activity and
  • increases the availability of glycolytic metabolites
  • supporting cell proliferation.

This suggests that high pyruvate kinase activity may suppress
tumor growth
.

  1. expression of PKM1,  the pyruvate kinase isoform with high
    constitutive activity, or
  2. exposure to published small-molecule PKM2 activators
  • inhibits the growth of xenograft tumors.

Structural studies reveal that

  • small-molecule activators bind PKM2
  • at the subunit interaction interface,
  • a site that is distinct from that of the
    • endogenous activator fructose-1,6-bisphosphate (FBP).

However, unlike FBP,

  • binding of activators to PKM2 promotes
  • a constitutively active enzyme state that is resistant to inhibition
  • by tyrosine-phosphorylated proteins.

These data support the notion that small-molecule activation of PKM2
can interfere with anabolic metabolism

  1. PKM1 expression in cancer cells impairs xenograft tumor growth.
    http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F1.jpg
  2. TEPP-46 and DASA-58 isoform specificity in vitro and in cells.
    TEPP-46 and DASA-58 isoform specificity in vitro and in cells.

    TEPP-46 and DASA-58 isoform specificity in vitro and in cells.

    (a) Structures of the PKM2 activators TEPP-46 and DASA-58. (b) Pyruvate kinase (PK) activity in purified recombinant human
    PKM1 or PKM2 expressed in bacteria in the presence of increasing
    concentrations of TEPP-46 or DASA-58. M1, PKM1;…
    http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F2.jpg

  3. Activators promote PKM2 tetramer formation and prevent
    inhibition by phosphotyrosine signaling.
Activators promote PKM2 tetramer formation and prevent inhibition by phosphotyrosine signaling.

Activators promote PKM2 tetramer formation and prevent inhibition by phosphotyrosine signaling.

Sucrose gradient ultracentrifugation profiles of purified recombinant
PKM2 (rPKM2) and the effects of FBP and TEPP-46 on PKM2 subunit stoichiometry.
http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F3.jpg

Figure 5: Metabolic effects of cell treatment with PKM2 activators.
(a) Effects of TEPP-46, DASA-58 (both used at 30 μM) or PKM1
expression on the doubling time of H1299 cells under normoxia
(21% O2) or hypoxia (1% O2). (b) Effects of DASA-58 on lactate
production from glucose. The P value shown was ca…
http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F5.jpg

EGFR has a tumour-promoting role in liver macrophages during
hepatocellular carcinoma formation

H Lanaya, A Natarajan, K Komposch, L Li, N Amberg, …, & Maria Sibilia
Nature Cell Biology 31 Aug 2014   http://dx.doi.org:/10.1038/ncb3031

Tumorigenesis has been linked with macrophage-mediated chronic
inflammation and diverse signaling pathways, including the ​epidermal
growth factor receptor (​EGFR) pathway. ​EGFR is expressed in liver
macrophages in both human HCC and in a mouse HCC model. Mice
lacking ​EGFR in macrophages show impaired hepatocarcinogenesis,
Mice lacking ​EGFR in hepatocytes develop HCC owing to increased
hepatocyte damage and compensatory proliferation. EGFR is required
in liver macrophages to transcriptionally induce ​interleukin-6 following
interleukin-1 stimulation, which triggers hepatocyte proliferation and HCC.
Importantly, the presence of ​EGFR-positive liver macrophages in HCC
patients is associated with poor survival. This study demonstrates a

  • tumour-promoting mechanism for ​EGFR in non-tumour cells,
  • which could lead to more effective precision medicine strategies.
  1. HCC formation in mice lacking ​EGFRin hepatocytes or all liver cells.
    http://www.nature.com/ncb/journal/vaop/ncurrent/carousel/ncb3031-f1.jpg

2. EGFR expression in Kupffer cells/liver macrophages promotes HCC development.

EGFR c2a expression in Kupffer cells.liver macrophages promotes HCC development.

EGFR c2a expression in Kupffer cells.liver macrophages promotes HCC development.

http://www.nature.com/ncb/journal/vaop/ncurrent/carousel/ncb3031-f2.jpg

Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates
the Warburg effect in carcinogenesis
.

Lu H1, Forbes RA, Verma A.
J Biol Chem. 2002 Jun 28;277(26):23111-5. Epub 2002 Apr 9

Cancer cells display high rates of aerobic glycolysis, a phenomenon
known historically as the Warburg effect. Lactate and pyruvate, the end
products of glycolysis, are highly produced by cancer cells even in the
presence of oxygen
.

Hypoxia-induced gene expression in cancer cells

  • has been linked to malignant transformation.

Here we provide evidence that lactate and pyruvate

  • regulate hypoxia-inducible gene expression
  • independently of hypoxia
  • by stimulating the accumulation of hypoxia-inducible Factor 1alpha
    (HIF-1alpha).

In human gliomas and other cancer cell lines,

  • the accumulation of HIF-1alpha protein under aerobic conditions
  • requires the metabolism of glucose to pyruvate that
  1. prevents the aerobic degradation of HIF-1alpha protein,
  2. activates HIF-1 DNA binding activity, and
  3. enhances the expression of several HIF-1-activated genes
  4. erythropoietin,
  5. vascular endothelial growth factor,
  6. glucose transporter 3, and
  7. aldolase A.

Our findings support a novel role for pyruvate in metabolic signaling
and suggest a mechanism by which

  • high rates of aerobic glycolysis
  • can promote the malignant transformation and
  • survival of cancer cells.PMID: 11943784

Part IV. Transcription control and innate immunity

 c-Myc-induced transcription factor AP4 is required for
host protection mediated by CD8+ T cells

C Chou, AK Pinto, JD Curtis, SP Persaud, M Cella, Chih-Chung Lin, … & T Egawa Nature Immunology 17 Jun 2014;   http://dx.doi.org:/10.1038/ni.2943

The transcription factor c-Myc is essential for

  • the establishment of a metabolically active and proliferative state
  • in T cells after priming,

We identified AP4 as the transcription factor

  • that was induced by c-Myc and
  • sustained activation of antigen-specific CD8+ T cells.

Despite normal priming,

  • AP4-deficient CD8+ T cells
  • failed to continue transcription of a broad range of
    c-Myc-dependent targets.

Mice lacking AP4 specifically in CD8+ T cells showed

  • enhanced susceptibility to infection with West Nile virus.

Genome-wide analysis suggested that

  • many activation-induced genes encoding molecules
  • involved in metabolism were shared targets of
  • c-Myc and AP4.

Thus, AP4 maintains c-Myc-initiated cellular activation programs

  • in CD8+ T cells to control microbial infection.
  1. AP4 is regulated post-transcriptionally in CD8+ T cells.

Microarray analysis of transcription factor–encoding genes with a difference
in expression of >1.8-fold in activated CD8+ T cells treated for 12 h with
IL-2 (100 U/ml; + IL-2) relative to their expression in activated CD8+ T cells…
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F1.jpg

2. AP4 is required for the population expansion of antigen specific
CD8+ T cells following infection with LCMV-Arm.

Expression of CD4, CD8α and KLRG1 (a) and binding of an
H-2Db–gp(33–41) tetramer and expression of CD8α, KLRG1 and
CD62L (b) in splenocytes from wild-type (WT) and Tfap4−/− mice,
assessed by flow cytometry 8 d after infection
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F2.jpg

3. AP4 is required for the sustained clonal expansion of CD8+ T cells
but  not for their initial proliferation.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F3.jpg

  1. AP4 is essential for host protection against infection with WNV, in
    a CD8+ T cell–intrinsic manner.
AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.

AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.

  •  Survival of Tfap4F/FCre− control mice (Cre−; n = 16) and
  • Tfap4F/FCD8-Cre+ mice (CD8-Cre+; n = 22) following infection with WNV.
    (b,c) Viral titers in the brain (b) and spleen (c) of Tfap4F/F Cre− and Tfap4F/F
    CD8-Cre+ mice  on day 9…
    http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F4.jpg

AP4 is essential for the sustained expression of genes that are targets of c-Myc.

Normalized signal intensity (NSI) of endogenous transcripts in
Tfap4+/+ and Tfap4−/− OT-I donor T cells adoptively transferred into
host mice and assessed on day 4 after infection of the host with LM-OVA
(top), and that of ERCC controls
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F6.jpg

Sustained c-Myc expression ‘rescues’ defects of Tfap4−/− CD8+ T cells.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F7.jpg

AP4 and c-Myc have distinct biological functions.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-SF7.jpg

Mucosal memory CD8+ T cells are selected in the periphery
by an MHC class I molecule

Y Huang, Y Park, Y Wang-Zhu, …A Larange, R Arens, & H Cheroutre

Nature Immunology 2 Oct 2011; 12: 1086–1095
http://dx.doi.org:/10.1038/ni.2106

The presence of immune memory at pathogen-entry sites is a prerequisite
for protection. We show that the non-classical major histocompatibility
complex (MHC) class I molecule

  • thymus leukemia antigen (TL),
  • induced on dendritic cells interacting with CD8αα on activated CD8αβ+ T cells,
  • mediated affinity-based selection of memory precursor cells.

Furthermore, constitutive expression of TL on epithelial cells

  • led to continued selection of mature CD8αβ+ memory T cells.

The memory process driven by TL and CD8αα

  • was essential for the generation of CD8αβ+ memory T cells in the intestine and
  • the accumulation of highly antigen-sensitive CD8αβ+ memory T cells
  • that form the first line of defense at the largest entry port for pathogens.

The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.

Marçais A, Cherfils-Vicini J, Viant C, Degouve S, Viel S, Fenis A, Rabilloud J,
Mayol K, Tavares A, Bienvenu J, Gangloff YG, Gilson E, Vivier E,Walzer T.
Nat Immunol. 2014 Aug; 15(8):749-757. Epub 2014 Jun 29
http://dx.doi.org:/10.1038/ni.2936  .    PMID: 24973821

Interleukin 15 (IL-15) controls

  • both the homeostasis and the peripheral activation of natural killer (NK) cells.

We found that the metabolic checkpoint kinase

  • mTOR was activated and boosted bioenergetic metabolism
  • after exposure of NK cells to high concentrations of IL-15,

whereas low doses of IL-15 triggered

  • only phosphorylation of the transcription factor STAT5.

mTOR

  • stimulated the growth and nutrient uptake of NK cells and
  • positively fed back on the receptor for IL-15.

This process was essential for

  • sustaining NK cell proliferation during development and
  • the acquisition of cytolytic potential during inflammation
    or viral infection.

The mTORC1 inhibitor rapamycin 

  • inhibited NK cell cytotoxicity both in mice and humans;
    • this probably contributes to the immunosuppressive
      activity of this drug in different clinical settings.

The Critical Role of IL-15-PI3K-mTOR Pathway in Natural Killer Cell
Effector Functions.
Nandagopal NAli AKKomal AKLee SH.   Author information
Front Immunol. 2014 Apr 23; 5:187. eCollection 2014.
http://dx.doi.org:/10.3389/fimmu.2014.00187

Natural killer (NK) cells were so named for their uniqueness in killing
certain tumor and virus-infected cells without prior sensitization.
Their functions are modulated in vivo by several soluble immune mediators;

  • interleukin-15 (IL-15) being the most potent among them in
    enabling NK cell homeostasis, maturation, and activation.

During microbial infections,

  • NK cells stimulated with IL-15 display enhanced cytokine responses.

This priming effect has previously been shown with respect to increased
IFN-γ production in NK cells

  • upon IL-12 and IL-15/IL-2 co-stimulation.
  • we explored if this effect of IL-15 priming 
  • can be extended to various other cytokines and
  • observed enhanced NK cell responses to stimulation
    • with IL-4, IL-21, IFN-α, and IL-2 in addition to IL-12.
  • we also observed elevated IFN-γ production in primed NK cells

Currently, the fundamental processes required for priming and

  • whether these signaling pathways work collaboratively or
    independently 

    • for NK cell functions are poorly understood.

We examined IL-15 effects on NK cells in which

  • the pathways emanating from IL-15 receptor activation
    • were blocked with specific inhibitors
    • To identify the key signaling events for NK cell priming,

Our results demonstrate that

the PI3K-AKT-mTOR pathway is critical for cytokine responses
in IL-15 primed NK cells. 

This pathway is also implicated in a broad range of

  • IL-15-induced NK cell effector functions such as
    • proliferation and cytotoxicity.

Likewise, NK cells from mice

  • treated with rapamycin to block the mTOR pathway
  • displayed defects in proliferation, and IFN-γ and granzyme B productions
  • resulting in elevated viral burdens upon murine cytomegalovirus infection.

Taken together, our data demonstrate

  • the requirement of PI3K-mTOR pathway
    • for enhanced NK cell functions by IL-15, thereby
  • coupling the metabolic sensor mTOR to NK cell anti-viral responses.

KEYWORDS: IL-15; JAK–STAT pathway; mTOR pathway; natural killer cells; signal transduction

Part V. Predicting Therapeutic Targets 

New discovery approach accelerates identification of potential cancer treatments
 Laura Williams, Univ. of Michigan   09/30/2014
http://www.rdmag.com/news/2014/09/new-discovery-approach-accelerates-identification-potential-cancer-treatments

Researchers at the Univ. of Michigan have described a new approach to
discovering potential cancer treatments that

  • requires a fraction of the time needed for more traditional methods.

They used the platform to identify

  • a novel antibody that is undergoing further investigation as a potential
    treatment for breast, ovarian and other cancers.

In research published online in the Proceedings of the National Academy
of Sciences
, researchers in the laboratory of Stephen Weiss at the U-M Life
Sciences Institute detail an approach

  • that replicates the native environment of cancer cells and
  • increases the likelihood that drugs effective against the growth of
    tumor cells in test tube models
  • will also stop cancer from growing in humans.

The researchers have used their method

  • to identify an antibody that stops breast cancer tumor growth in animal models, and
  • they are investigating the antibody as a potential treatment in humans.

“Discovering new targets for cancer therapeutics is a long and tedious undertaking, and

  • identifying and developing a potential drug to specifically hit that
    target without harming healthy cells is a daunting task,” Weiss said.
  • “Our approach allows us to identify potential therapeutics
    • in a fraction of the time that traditional methods require.”

The researchers began by

  • creating a 3-D “matrix” of collagen, a connective tissue molecule very similar to that found
    • surrounding breast cancer cells in human patients.
  • They then embedded breast cancer cells into the collagen matrix,
    • where the cells grew as they would in human tissue.

The investigators then injected the cancer-collagen tissue composites into mice that then

  • recognize the human cancer cells as foreign tissue.
    • Much in the way that our immune system generates antibodies
      to fight infection,
  • the mice began to generate thousands of antibodies directed against
    the human cancer cells.
  • These antibodies were then tested for the ability to stop the growth
    of the human tumor cells.

“We create an environment in which cells cultured in the laboratory ‘think’
they are growing in the body and then

  • rapidly screen large numbers of antibodies to see if any exert
    anti-cancer effects,” Weiss said.
  • “This allows us to select promising antibodies very quickly and then

They discovered a particular antibody, 4C3, which was able to

  • almost completely stop the proliferation of the breast cancer cells.

They then identified the molecule on the cancer cells that the antibody targets.

The antibody can be further engineered to generate

  • humanized monoclonal antibodies for use in patients

“We still need to do a lot more work to determine how effective 4C3 might be as a
treatment for breast and other cancers, on its own or in conjunction with other
therapies,” Weiss said. “But we have enough data to warrant further pursuit,
and are expanding our efforts to use this discovery platform to find similarly promising antibodies.”

Source: Univ. of Michigan

  1. Jose Eduardo de Salles Roselino

    Larry,
    I think you have made a great effort in order to connect basic ideas of metabolic regulation with those of gene expression control “modern” mechanisms.
    Yet, I do not think that at this stage it will be clear for all readers. At least, for the great majority of the readers. The most important factor I my opinion, is derived from the fact that modern readers considers that metabolic regulation deals with so called “housekeeping activities” of the cell. Something that is of secondary, tertiary or even less level of relevance.
    My idea, that you have mentioned in the text when you write at the beginning, the word biochemistry, in order to resume it, derives from the reading of What is life together with Prof. Leloir . For me and also, for him, biochemistry comprises a set of techniques and also a framework of reasoning about scientific results. As a set of techniques, Schrodinger has considered that it will lead to better understanding of genetics and of physiology as a two legs structure supporting the future progress related to his time (mid-forties). For Leloir, the key was the understanding of chemical reactivity and I agree with him. However, as I was able to talk and discuss it with him in detail, we should also take into account levels of stabilities of macromolecules and above all, regulation of activities and function (this is where) Pasteur effect that I was studying in Leloir´s lab at that time, 1970-72, gets into the general picture.
    Regulation for complex living beings , that also have cancer cell as a great topic of research problem can be understood through the understanding of two quite different results when opposition with lack of regulation is taken into account or experimentally elicited. The most clearly line of experiments can follow the Pasteur Effect as the intracellular result best seen when aerobiosis is compared with anaerobiosis as conditions in which maintenance of ATP levels and required metabolic regulation (Energy charge D.E, Atkinson etc) is studied. Another line of experiments is one that takes into account the extracellular result or for instance the homeostatic regulation of blood glucose levels. The blood glucose level is the most conspicuous and related to Pasteur Effect regulatory event that can be studied in the liver taking into account both final results tested or compared regarding its regulation, ATP levels maintenance (intracellular) and blood glucose maintenance (extracellular).
    My key idea is to consider that the same factors that elicits fast regulatory responses also elicits the slow energetic expensive regulatory responses. The biologic logic behind this common root is the ATP economy. In case, the regulatory stimulus fades out quickly the fast regulatory responses are good enough to maintain life and the time requiring, energetic costly responses will soon be stopped cutting short the ATP expenditure. In case, the stimulus last for long periods of time the fast responses are replaced by adaptive responses that in general will follow the line of cell differentiation mechanisms with changes in gene expression etc.
    The change from fast response mechanisms to long lasting developmentally linked ones is not sharp. Therefore, somehow, cancer cells becomes trapped into a metastable regulatory mechanism that prevents cell differentiation and reinforces those mechanisms linked to its internal regulatory goals. This metastable mechanism takes advantage from the fact that other cells, tissues and organs will take good care of homeostatic mechanisms that provide for their nutritional needs. In the case of my Hepatology work you will see a Piruvate kinase that does not responds to homeostatic signals .

Read Full Post »


Metabolomic analysis of two leukemia cell lines. I.

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

Leaders in Pharmaceutical Intelligence

 

I have just posted a review of metabolomics.  In the last few weeks, the Human Metabolome was published.  I am hopeful that my decision has taken the right path to prepare my readers adequately if they will have read the articles that preceded this.  I pondered how I would present this massive piece of work, a study using two leukemia cell lines and mapping the features and differences that drive the carcinogenesis pathways, and identify key metabolic signatures in these differentiated cell types and subtypes.  It is a culmination of a large collaborative effort that required cell culture, enzymatic assays, mass spectrometry, the full measure of which I need not present here, and a very superb validation of the model with a description of method limitations or conflicts.  This is a beautiful piece of work carried out by a small group by today’s standards.

I shall begin this by asking a few questions that will be addressed in the article, which I need to beak up into parts, to draw the readers in more effectively.

Q 1. What metabolic pathways do you expect to have the largest role in the study about to be presented?

Q2. What are the largest metabolic differences that one expects to see in compairing the two lymphoblastic cell lines?

Q3. What methods would be used to extract the information based on external metabolites, enzymes, substrates, etc., to create the model for the cell internal metabolome?

 

 

Abstract

Metabolic models can provide a mechanistic framework to analyze information-rich omics data sets, and are increasingly being used

  • to investigate metabolic alternations in human diseases.

An expression of the altered metabolic pathway utilization is

  • the selection of metabolites consumed and released by cells.

However, methods for the inference of intracellular metabolic states from extracellular measurements in the context of metabolic models

  • remain underdeveloped compared to methods for other omics data.

Herein, we describe a workflow for such an integrative analysis

  • extracting the information from extracellular metabolomics data.

We demonstrate, using the lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM, how

  • our methods can reveal differences in cell metabolism.

Our models explain metabolite uptake and secretion by

  • predicting a more glycolytic phenotype for the CCRF-CEM model and
  • a more oxidative phenotype for the Molt-4 model, which
  • was supported by our experimental data.

Gene expression analysis revealed altered expression of gene products at

  • key regulatory steps in those central metabolic pathways,

and literature query emphasized

  • the role of these genes in cancer metabolism.

Moreover, in silico gene knock-outs identified

  • unique control points for each cell line model, e.g., phosphoglycerate dehydrogenase for the Molt-4 model.

Thus, our workflow is well suited to the characterization of cellular metabolic traits based on

  • extracellular metabolomic data, and
  • it allows the integration of multiple omics data sets into a cohesive picture based on a defined model context.

Keywords Constraint-based modeling _ Metabolomics _Multi-omics _ Metabolic network _ Transcriptomics

 

Reviewer Summary:

  1. A model is introduced to demonstrate a lymphocytic integrated data set using to cell lines.
  2. The method is required to integrate extracted data sets from extracellular metabolites to an intracellular picture of cellular metabolism for each cell line.
  3. The method predicts a more glycolytic or a more oxidative metabolic framework for one or the othe cell line.
  4. The genetic phenotypes differ with a unique control point for each cell line.
  5. The model presents an integration of omics data sets into a cohesive picture based on the model context.

Without having seen the full presentation –

  1. Is the method a snapshot of the neoplastic processes described?
  2. Does the model give insight into the cellular metabolism of an initial cell state for either one or both cell lines?
  3. Would one be able to predict a therapeutic strategy based on the model for either or both cell lines?

Before proceeding further into the study, I would conjecture that there is no way of knowing the initial state ( consistent with what is described by Ilya Prigogine for a self-organizing system) because the model is based on the study of cultured cells that had an unknown metabolic control profile in a host proliferating bone marrow that is likely B-cell origin.  So this is a snapshot of a stable state of two incubated cell lines.  Then the question that is raised is whether there is not only a genetic-phenotypic relationship between the cells in culture and the external metabolites produced, but also whether differences can be discerned between the  internal metabolic constructions that would fit into a family tree.

 

Introduction

Modern high-throughput techniques

  • have increased the pace of biological data generation.

Also referred to as the ‘‘omics avalanche’’, this wealth of data

  • provides great opportunities for metabolic discovery.

Omics data sets contain a snapshot of almost the entire repertoire of

  • mRNA, protein, or metabolites at a given time point or
  • under a particular set of experimental conditions.

Because of the high complexity of the data sets,

  • computational modeling is essential for their integrative analysis.

Currently, such data analysis

  • is a bottleneck in the research process and
  • methods are needed to facilitate the use of these data sets, e.g.,
  1. through meta-analysis of data available in public databases
    [e.g., the human protein atlas (Uhlen et al. 2010)
  2. or the gene expression omnibus (Barrett  et al.  2011)], and
  3. to increase the accessibility of valuable information
    for the biomedical research community.

Constraint-based modeling and analysis (COBRA) is

  • a computational approach that has been successfully used
  • to investigate and engineer microbial metabolism through
    the prediction of steady-states (Durot et al.2009).

The basis of COBRA is network reconstruction: networks are assembled

  1. in a bottom-up fashion based on genomic data and
  2. extensive organism-specific information from the literature.

Metabolic reconstructions

  1. capture information on the known biochemical transformations
    taking place in a target organism
  2. to generate a biochemical, genetic and genomic knowledge base
    (Reed et al. 2006).

Once assembled, a metabolic reconstruction

  • can be converted into a mathematical model
    (Thiele and Palsson 2010), and
  • model properties can be interrogated using a great variety of methods
    (Schellenberger et al. 2011).

The ability of COBRA models to represent

  • genotype–phenotype and environment–phenotype relationships
  • arises through the imposition of constraints,
  • which limit the system to a subset of possible network states
    (Lewis et al. 2012).

Currently, COBRA models exist for more than 100 organisms, including humans
(Duarte et al. 2007; Thiele et al. 2013).

Since the first human metabolic reconstruction was described
[Recon 1 (Duarte et al. 2007)],

  • biomedical applications of COBRA have increased
    (Bordbar and Palsson 2012).

One way to contextualize networks is to

  • define their system boundaries
  • according to the metabolic states of the system,
    e.g., disease or dietary regimes.

The consequences of the applied constraints

  • can then be assessed for the entire network
    (Sahoo and Thiele 2013).

Additionally, omics data sets have frequently been used

  • to generate cell-type or condition-specific metabolic models.

Models exist for specific cell types, such as

  • enterocytes (Sahoo and Thiele2013),
  • macrophages (Bordbar et al. 2010), and
  • adipocytes (Mardinoglu et al. 2013), and
  • even multi-cell assemblies that represent
    the interactions of brain cells (Lewis et al. 2010).

All of these cell type specific models,

  • except the enterocyte reconstruction
  • were generated based on omics data sets.

Cell-type-specific models have been used

  • to study diverse human disease conditions.

For example, an adipocyte model was generated using

  • transcriptomic,
  • proteomic, and
  • metabolomics data.

This model was subsequently used to investigate

  • metabolic alternations in adipocytes
  • that would allow for the stratification of obese patients
    (Mardinoglu et al. 2013).

One highly active field within the biomedical applications of COBRA is

  • cancer metabolism (Jerby and Ruppin, 2012).

Omics-driven large-scale models have been used

  • to predict drug targets (Folger et al. 2011; Jerby et al. 2012).

A cancer model was generated using

  • multiple gene expression data sets and
  • subsequently used to predict synthetic lethal gene pairs
  • as potential drug targets selective for the cancer model,
  • but non-toxic to the global model (Recon 1),
  • a consequence of the reduced redundancy in the
    cancer specific model (Folger et al. 2011).

In a follow up study, lethal synergy between

  • FH and enzymes of the heme metabolic pathway
    were experimentally validated and
  • resolved the mechanism by which FH deficient cells,
    e.g., in renal-cell cancer cells
  • survive a non-functional TCA cycle (Frezza et al. 2011).

Contextualized models, which contain only 

  • the subset of reactions active in 
  • a particular tissue (or cell-) type,
  • can be generated in different ways
    (Becker and Palsson, 2008; Jerby et al. 2010).

However, the existing algorithms mainly consider

  • gene expression and proteomic data to define the reaction sets
  • that comprise the contextualized metabolic models.

These subset of reactions are usually defined based on

  • the expression or absence of expression of the genes or proteins
    (present and absent calls), or
  • inferred from expression values or differential gene expression.

Comprehensive reviews of the methods are available
(Blazier and Papin, 2012; Hyduke et al. 2013).

Only the compilation of a large set of omics data sets

  • can result in a tissue (or cell-type) specific metabolic model, whereas

the representation of one particular experimental condition is achieved through

  • the integration of omics data set generated from one experiment only
    (condition-specific cell line model).

Recently, metabolomic data sets

  • have become more comprehensive and using these data sets allow
  • direct determination of the metabolic network components (the metabolites).

Additionally, metabolomics has proven to be

  1. stable,
  2. relatively inexpensive, and
  3. highly reproducible
    (Antonucci et al. 2012).

These factors make metabolomic data sets

  •  particularly valuable for interrogation of metabolic phenotypes. 

Thus, the integration of these data sets is now an active field of research
(Li et al. 2013; Mo et al. 2009; Paglia et al. 2012b; Schmidt et al. 2013).

Generally, metabolomic data can be incorporated into metabolic networks as

  1. qualitative,
  2. quantitative, and
  3. thermodynamic constraints
    (Fleming et al. 2009; Mo et al. 2009).

Mo et al. used metabolites detected in the spent medium
of yeast cells to determine

  • intracellular flux states through a sampling analysis (Mo et al. 2009),
  • which allowed unbiased interrogation of the possible network states
    (Schellenberger and Palsson 2009)
  • and prediction of internal pathway use.

Such analyses have also been used

  • to reveal the effects of enzymopathies on red blood cells (Price et al. 2004),
  • to study effects of diet on diabetes (Thiele et al. 2005) and
  • to define macrophage metabolic states (Bordbar et al. 2010).

This type of analysis is available as a function in the COBRA toolbox
(Schellenberger et al. 2011).

 

 

 

In this study, we established a workflow for the generation and analysis of

  • condition-specific metabolic cell line models that
  • can facilitate the interpretation of metabolomic data.

Our modeling yields meaningful predictions regarding

  • metabolic differences between two lymphoblastic leukemia cell lines
    (Fig. 1A).
Differences in the use of the TCA cycle by the CCRF-CEM

Differences in the use of the TCA cycle by the CCRF-CEM

 

 

 

http://link.springer.com/static-content/images/404/art%253A10.1007%252
Fs11306-014-0721-3/MediaObjects/11306_2014_721_Fig1_HTML.gif

Fig. 1

A  Combined experimental and computational pipeline to study human metabolism.
Experimental work and omics data analysis steps precede computational modeling. Model

  • predictions are validated based on targeted experimental data.

Metabolomic and transcriptomic data are used for

  • model refinement and submodel extraction.

Functional analysis methods are used to characterize

  • the metabolism of the cell-line models and compare it to additional experimental
    data.

The validated models are subsequently 

  • used for the prediction of drug targets.

B Uptake and secretion pattern of model.
All metabolite uptakes and secretions that were mapped during model
generation are shown.
Metabolite uptakes are depicted on the left, and

  • secreted metabolites are shown on the right.

A number of metabolite exchanges mapped to the model

  • were unique to one cell line.

Differences between cell lines were used to set

  • quantitative constraints for the sampling analysis.

C Statistics about the cell line-specific network generation.

 Quantitative constraints.
For the sampling analysis, an additional

  • set of constraints was imposed on the cell line specific models,
  • emphasizing the differences in metabolite uptake and secretion between cell lines.

Higher uptake of a metabolite was allowed in the model of the cell line

  • that consumed more of the metabolite in vitro, whereas
  • the supply was restricted for the model with lower in vitro uptake.

This was done by establishing the same ratio between the models bounds as detected in vitro.
X denotes the factor(slope ratio) that

  1. distinguishes the bounds, and
  2. which was individual for each metabolite.
  • (a) The uptake of a metabolite could be x times higher in CCRF-CEM cells,
    (b) the metabolite uptake could be x times higher in Molt-4,
    (c) metabolite secretion could be x times higher in CCRF-CEM, or
    (d) metabolite secretion could be x times higher in Molt-4 cells. LOD limit of detection.

The consequence of the adjustment was, in case of uptake, that  one model

  1. was constrained to a lower metabolite uptake (A, B), and the difference
  2. depended on the ratio detected in vitro.

In case of secretion,

  • one model had to secrete more of the metabolite, and again

the difference depended on

  • the experimental difference detected between the cell lines.

Q5. What is your expectation that this type of integrative approach could be used for facilitating medical data interpretations?

The most inventive approach was made years ago by using data constructions from the medical literature by a pioneer in the medical record development, but the technology was  not what it is today, and the cost of data input was high.  Nevertheless, the data acquisition would not be uniform across institutions, except for those that belong to a consolidated network with all of the data in the cloud, and the calculations would be carried out with a separate engine.  However, whether the uniform capture of the massive amount of data needed is not possible in the near foreseeable future.  There is no accurate way of assessing the system cost, and predicting the benefits.  In carrying this model forward there has to be a minimal amount of insufficient data.  The developments in the regulatory sphere have created a high barrier.

This concludes a first portion of this presentation.

 

Read Full Post »

Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics


Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer and Curator: Larry H. Bernstein, MD, FCAP 

 

The human genome is estimated to encode over 30,000 genes, and to be responsible for generating more than 100,000 functionally distinct proteins. Understanding the interrelationships among

  1. genes,
  2. gene products, and
  3. dietary habits

is fundamental to identifying those who will benefit most from or be placed at risk by intervention strategies.

Unraveling the multitude of

  • nutrigenomic,
  • proteomic, and
  • metabolomic patterns

that arise from the ingestion of foods or their

  • bioactive food components

will not be simple but is likely to provide insights into a tailored approach to diet and health. The use of new and innovative technologies, such as

  • microarrays,
  • RNA interference, and
  • nanotechnologies,

will provide needed insights into molecular targets for specific bioactive food components and

  • how they harmonize to influence individual phenotypes(1).

Nutrigenetics asks the question how individual genetic disposition, manifesting as

  • single nucleotide polymorphisms,
  • copy-number polymorphisms and
  • epigenetic phenomena,

affects susceptibility to diet.

Nutrigenomics addresses the inverse relationship, that is how diet influences

  • gene transcription,
  • protein expression and
  • metabolism.

A major methodological challenge and first pre-requisite of nutrigenomics is integrating

  • genomics (gene analysis),
  • transcriptomics (gene expression analysis),
  • proteomics (protein expression analysis) and
  • metabonomics (metabolite profiling)

to define a “healthy” phenotype. The long-term deliverable of nutrigenomics is personalised nutrition (2).

Science is beginning to understand how genetic variation and epigenetic events

  • alter requirements for, and responses to, nutrients (nutrigenomics).

At the same time, methods for profiling almost all of the products of metabolism in a single sample of blood or urine are being developed (metabolomics). Relations between

  • diet and nutrigenomic and metabolomic profiles and
  • between those profiles and health

have become important components of research that could change clinical practice in nutrition.

Most nutrition studies assume that all persons have average dietary requirements, and the studies often

  • do not plan for a large subset of subjects who differ in requirements for a nutrient.

Large variances in responses that occur when such a population exists

  • can result in statistical analyses that argue for a null effect.

If nutrition studies could better identify responders and differentiate them from nonresponders on the basis of nutrigenomic or metabolomic profiles,

  • the sensitivity to detect differences between groups could be greatly increased, and
  • the resulting dietary recommendations could be appropriately targeted (3).

In recent years, nutrition research has moved from classical epidemiology and physiology to molecular biology and genetics. Following this trend,

  • Nutrigenomics has emerged as a novel and multidisciplinary research field in nutritional science that
  • aims to elucidate how diet can influence human health.

It is already well known that bioactive food compounds can interact with genes affecting

  • transcription factors,
  • protein expression and
  • metabolite production.

The study of these complex interactions requires the development of

  • advanced analytical approaches combined with bioinformatics.

Thus, to carry out these studies

  • Transcriptomics,
  • Proteomics and
  • Metabolomics

approaches are employed together with an adequate integration of the information that they provide(4).

Metabonomics is a diagnostic tool for metabolic classification of individuals with the asset of quantitative, non-invasive analysis of easily accessible human body fluids such as urine, blood and saliva. This feature also applies to some extent to Proteomics, with the constraint that

  • the latter discipline is more complex in terms of composition and dynamic range of the sample.

Apart from addressing the most complex “Ome”, Proteomics represents

  • the only platform that delivers not only markers for disposition and efficacy
  • but also targets of intervention.

Application of integrated Omic technologies will drive the understanding of

  • interrelated pathways in healthy and pathological conditions and
  • will help to define molecular ‘switchboards’,
  • necessary to develop disease related biomarkers.

This will contribute to the development of new preventive and therapeutic strategies for both pharmacological and nutritional interventions (5).

Human health is affected by many factors. Diet and inherited genes play an important role. Food constituents,

  • including secondary metabolites of fruits and vegetables, may
  • interact directly with DNA via methylation and changes in expression profiles (mRNA, proteins)
  • which results in metabolite content changes.

Many studies have shown that

  • food constituents may affect human health and
  • the exact knowledge of genotypes and food constituent interactions with
  • both genes and proteins may delay or prevent the onset of diseases.

Many high throughput methods have been employed to get some insight into the whole process and several examples of successful research, namely in the field of genomics and transcriptomics, exist. Studies on epigenetics and RNome significance have been launched. Proteomics and metabolomics need to encompass large numbers of experiments and linked data. Due to the nature of the proteins, as well as due to the properties of various metabolites, experimental approaches require the use of

  • comprehensive high throughput methods and a sufficiency of analysed tissue or body fluids (6).

New experimental tools that investigate gene function at the subcellular, cellular, organ, organismal, and ecosystem level need to be developed. New bioinformatics tools to analyze and extract meaning

  • from increasingly systems-based datasets will need to be developed.

These will require, in part, creation of entirely new tools. An important and revolutionary aspect of “The 2010 Project”  is that it implicitly endorses

  • the allocation of resources to attempts to assign function to genes that have no known function.

This represents a significant departure from the common practice of defining and justifying a scientific goal based on the biological phenomena. The rationale for endorsing this radical change is that

  • for the first time it is feasible to envision a whole-systems approach to gene and protein function.

This whole-systems approach promises to be orders of magnitude more efficient than the conventional approach (7).

The Institute of Medicine recently convened a workshop to review the state of the various domains of nutritional genomics research and policy and to provide guidance for further development and translation of this knowledge into nutrition practice and policy (8). Nutritional genomics holds the promise to revolutionize both clinical and public health nutrition practice and facilitate the establishment of

(a) genome-informed nutrient and food-based dietary guidelines for disease prevention and healthful aging,

(b) individualized medical nutrition therapy for disease management, and

(c) better targeted public health nutrition interventions (including micronutrient fortification and supplementation) that

  • maximize benefit and minimize adverse outcomes within genetically diverse human populations.

As the field of nutritional genomics matures, which will include filling fundamental gaps in

  • knowledge of nutrient-genome interactions in health and disease and
  • demonstrating the potential benefits of customizing nutrition prescriptions based on genetics,
  • registered dietitians will be faced with the opportunity of making genetically driven dietary recommendations aimed at improving human health.

The new era of nutrition research translates empirical knowledge to evidence-based molecular science (9). Modern nutrition research focuses on

  • promoting health,
  • preventing or delaying the onset of disease,
  • optimizing performance, and
  • assessing risk.

Personalized nutrition is a conceptual analogue to personalized medicine and means adapting food to individual needs. Nutrigenomics and nutrigenetics

  • build the science foundation for understanding human variability in
  • preferences, requirements, and responses to diet and
  • may become the future tools for consumer assessment

motivated by personalized nutritional counseling for health maintenance and disease prevention.

The primary aim of ―omic‖ technologies is

  • the non-targeted identification of all gene products (transcripts, proteins, and metabolites) present in a specific biological sample.

By their nature, these technologies reveal unexpected properties of biological systems.

A second and more challenging aspect of ―omic‖ technologies is

  • the refined analysis of quantitative dynamics in biological systems (10).

For metabolomics, gas and liquid chromatography coupled to mass spectrometry are well suited for coping with

  • high sample numbers in reliable measurement times with respect to
  • both technical accuracy and the identification and quantitation of small-molecular-weight metabolites.

This potential is a prerequisite for the analysis of dynamic systems. Thus, metabolomics is a key technology for systems biology.

In modern nutrition research, mass spectrometry has developed into a tool

  • to assess health, sensory as well as quality and safety aspects of food.

In this review, we focus on health-related benefits of food components and, accordingly,

  • on biomarkers of exposure (bioavailability) and bioefficacy.

Current nutrition research focuses on unraveling the link between

  • dietary patterns,
  • individual foods or
  • food constituents and

the physiological effects at cellular, tissue and whole body level

  • after acute and chronic uptake.

The bioavailability of bioactive food constituents as well as dose-effect correlations are key information to understand

  • the impact of food on defined health outcomes.

Both strongly depend on appropriate analytical tools

  • to identify and quantify minute amounts of individual compounds in highly complex matrices–food or biological fluids–and
  • to monitor molecular changes in the body in a highly specific and sensitive manner.

Based on these requirements,

  • mass spectrometry has become the analytical method of choice
  • with broad applications throughout all areas of nutrition research (11).

Recent advances in high data-density analytical techniques offer unrivaled promise for improved medical diagnostics in the coming decade. Genomics, proteomics and metabonomics (as well as a whole slew of less well known ―omics‖ technologies) provide a detailed descriptor of each individual. Relating the large quantity of data on many different individuals to their current (and possibly even future) phenotype is a task not well suited to classical multivariate statistics. The datasets generated by ―omics‖ techniques very often violate the requirements for multiple regression. However, another statistical approach exists, which is already well established in areas such as medicinal chemistry and process control, but which is new to medical diagnostics, that can overcome these problems. This approach, called megavariate analysis (MVA),

  • has the potential to revolutionise medical diagnostics in a broad range of diseases.

It opens up the possibility of expert systems that can diagnose the presence of many different diseases simultaneously, and

  • even make exacting predictions about the future diseases an individual is likely to suffer from (12).

Cardiovascular diseases

Cardiovascular diseases are the leading cause of morbidity and mortality in Western countries. Although coronary thrombosis is the final event in acute coronary syndromes,

  • there is increasing evidence that inflammation also plays a role in development of atherosclerosis and its clinical manifestations, such as
  • myocardial infarction, stroke, and peripheral vascular disease.

The beneficial cardiovascular health effects of

  • diets rich in fruits and vegetables are in part mediated by their flavanol content.

This concept is supported by findings from small-scale intervention studies with surrogate endpoints including

  1. endothelium-dependent vasodilation,
  2. blood pressure,
  3. platelet function, and
  4. glucose tolerance.

Mechanistically, short term effects on endothelium-dependent vasodilation

  • following the consumption of flavanol-rich foods, as well as purified flavanols,
  • have been linked to an increased nitric oxide bioactivity.

The critical biological target(s) for flavanols have yet to be identified (13), but we are beginning to see over the horizon.

Nutritional sciences

Nutrition sciences apply

  1. transcriptomics,
  2. proteomics and
  3. metabolomics

to molecularly assess nutritional adaptations.

Transcriptomics can generate a

  • holistic overview on molecular changes to dietary interventions.

Proteomics is most challenging because of the higher complexity of proteomes as compared to transcriptomes and metabolomes. However, it delivers

  • not only markers but also
  • targets of intervention, such as
  • enzymes or transporters, and
  • it is the platform of choice for discovering bioactive food proteins and peptides.

Metabolomics is a tool for metabolic characterization of individuals and

  • can deliver metabolic endpoints possibly related to health or disease.

Omics in nutrition should be deployed in an integrated fashion to elucidate biomarkers

  • for defining an individual’s susceptibility to diet in nutritional interventions and
  • for assessing food ingredient efficacy (14).

The more elaborate tools offered by metabolomics opened the door to exploring an active role played by adipose tissue that is affected by diet, race, sex, and probably age and activity. When the multifactorial is brought into play, and the effect of changes in diet and activities studied we leave the study of metabolomics and enter the world of ―metabonomics‖. Adiponectin and adipokines arrive (15-22). We shall discuss ―adiposity‖ later.

Potential Applications of Metabolomics

Either individually or grouped as a profile, metabolites are detected by either

  • nuclear magnetic resonance spectroscopy or mass spectrometry.

There is potential for a multitude of uses of metabolome research, including

  1. the early detection and diagnosis of cancer and as
  2. both a predictive and pharmacodynamic marker of drug effect.

However, the knowledge regarding metabolomics, its technical challenges, and clinical applications is unappreciated

  • even though when used as a translational research tool,
  • it can provide a link between the laboratory and clinic.

Precise numbers of human metabolites is unknown, with estimates ranging from the thousands to tens of thousands. Metabolomics is a term that encompasses several types of analyses, including

(a) metabolic fingerprinting, which measures a subset of the whole profile with little differentiation or quantitation of metabolites;

(b) metabolic profiling, the quantitative study of a group of metabolites, known or unknown, within or associated with a particular metabolic pathway; and

(c) target isotope-based analysis, which focuses on a particular segment of the metabolome by analyzing

  • only a few selected metabolites that comprise a specific biochemical pathway.

 

Dynamic Construct of the –Omics

Dynamic Construct of the –Omics

 

Dynamic Construct of the –Omics

 

 

Iron metabolism – Anemia

Hepcidin is a key hormone governing mammalian iron homeostasis and may be directly or indirectly involved in the development of most iron deficiency/overload and inflammation-induced anemia. The anemia of chronic disease (ACD) is characterized by macrophage iron retention induced by cytokines and hepcidin regulation. Hepcidin controls cellular iron efflux on binding to the iron export protein ferroportin. While patients present with both ACD and iron deficiency anemia (ACD/IDA), the latter results from chronic blood loss. Iron retention during inflammation occurs in macrophages and the spleen, but not in the liver. In ACD, serum hepcidin concentrations are elevated, which is related to reduced duodenal and macrophage expression of ferroportin. Individuals with ACD/IDA have significantly lower hepcidin levels than ACD subjects. ACD/IDA patients, in contrast to ACD subjects, were able to absorb dietary iron from the gut and to mobilize iron from macrophages. Hepcidin elevation may affect iron transport in ACD and ACD/IDA and it is more responsive to iron demand with IDA than to inflammation. Hepcidin determination may aid in selecting appropriate therapy for these patients (23).

There is correlation between serum hepcidin, iron and inflammatory indicators associated with anemia of chronic disease (ACD), ACD, ACD concomitant iron-deficiency anemia (ACD/IDA), pure IDA and acute inflammation (AcI) patients. Hepcidin levels in anemia types were statistically different, from high to low: ACD, AcI > ACD/IDA > the control > IDA. Serum ferritin levels were significantly increased in ACD and AcI patients but were decreased significantly in ACD/IDA and IDA. Elevated serum EPO concentrations were found in ACD, ACD/IDA and IDA patients but not in AcI patients and the controls. A positive correlation exists between hepcidin and IL-6 levels only in ACD/IDA, AcI and the control groups. A positive correlation between hepcidin and ferritin was marked in the control group, while a negative correlation between hepcidin and ferritin was noted in IDA. The significant negative correlation between hepcidin expression and reticulocyte count was marked in both ACD/IDA and IDA groups. If the hepcidin role in pathogenesis of ACD, ACD/IDA and IDA, it could be a potential marker for detection and differentiation of these anemias (24).

Cancer

Because cancer cells are known to possess a highly unique metabolic phenotype, development of specific biomarkers in oncology is possible and might be used in identifying fingerprints, profiles, or signatures to detect the presence of cancer, determine prognosis, and/or assess the pharmacodynamic effects of therapy (25).

HDM2, a negative regulator of the tumor suppressor p53, is over-expressed in many cancers that retain wild-type p53. Consequently, the effectiveness of chemotherapies that induce p53 might be limited, and inhibitors of the HDM2–p53 interaction are being sought as tumor-selective drugs. A binding site within HDM2 has been dentified which can be blocked with peptides inducing p53 transcriptional activity. A recent report demonstrates the principle using drug-like small molecules that target HDM2 (26).

Obesity, CRP, interleukins, and chronic inflammatory disease

Elevated CRP levels and clinically raised CRP levels were present in 27.6% and 6.7% of the population, respectively. Both overweight (body mass index [BMI], 25-29.9 kg/m2) and obese (BMI, 30 kg/m2) persons were more likely to have elevated CRP levels than their normal-weight counterparts (BMI, <25 kg/m2). After adjusting for potential confounders, the odds ratio (OR) for elevated CRP was 2.13 for obese men and 6.21 for obese women. In addition, BMI was associated with clinically raised CRP levels in women, with an OR of 4.76 (95% CI, 3.42-6.61) for obese women. Waist-to-hip ratio was positively associated with both elevated and clinically raised CRP levels, independent of BMI. Restricting the analyses to young adults (aged 17-39 years) and excluding smokers, persons with inflammatory disease, cardiovascular disease, or diabetes mellitus and estrogen users did not change the main findings (27).

A study of C-reactive protein and interleukin-6 with measures of obesity and of chronic infection as their putative determinants related levels of C-reactive protein and interleukin-6 to markers of the insulin resistance syndrome and of endothelial dysfunction. Levels of C-reactive protein were significantly related to those of interleukin-6 (r=0.37, P<0.0005) and tumor necrosis factor-a (r=0.46, P<0.0001), and concentrations of C-reactive protein were related to insulin resistance as calculated from the homoeostasis model and to markers of endothelial dysfunction (plasma levels of von Willebrand factor, tissue plasminogen activator, and cellular fibronectin). A mean standard deviation score of levels of acute phase markers correlated closely with a similar score of insulin resistance syndrome variables (r=0.59, P<0.00005) and the data suggested that adipose tissue is an important determinant of a low level, chronic inflammatory state as reflected by levels of interleukin-6, tumor necrosis factor-a, and C-reactive protein (28).

A number of other studies have indicated the inflammatory ties of visceral obesity to adipose tissue metabolic profiles, suggesting a role in ―metabolic syndrome‖. There is now a concept of altered liver metabolism in ―non-alcoholic‖ fatty liver disease (NAFLD) progressing from steatosis to steatohepatitis (NASH) (31,32).

These unifying concepts were incomprehensible 50 years ago. It was only known that insulin is anabolic and that insulin deficiency (or resistance) would have consequences in the point of entry into the citric acid cycle, which generates 16 ATPs. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In the case of this cycle there is a tie in with both catabolism and anabolism.

 

TCA_reactions

TCA_reactions

 http://www.newworldencyclopedia.org/entry/Image:TCA_reactions.gif

 

For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative G of phosphate hydrolysis than ATP.

The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following:

(a) Pyruvate Carboxylase (Gluconeogenesis) catalyzes:

pyruvate + HCO3 + ATP — oxaloacetate + ADP + Pi

(b) PEP Carboxykinase (Gluconeogenesis) catalyzes:

oxaloacetate + GTP — phosphoenolpyruvate + GDP + CO2

The concept of anomalies in the pathways with respect to diabetes was sketchy then, and there was much to be filled in. This has been substantially done, and is by no means complete. However, one can see how this comes into play with diabetic ketoacidosis accompanied by gluconeogenesis and in severe injury or sepsis with peripheral proteolysis to provide gluconeogenic precursors. The reprioritization of liver synthetic processes is also brought into play with the conundrum of protein-energy malnutrition.

The picture began to be filled in with the improvements in technology that emerged at the end of the 1980s with the ability to profile tissue and body fluids by NMR and by MS. There was already a good inkling of a relationship of type 2 diabetes to major indicators of CVD (29,30). And a long suspected relationship between obesity and type 2 diabetes was evident. But how did it tie together?

End Stage Renal Disease and Cardiovascular Risk

Mortality is markedly elevated in patients with end-stage renal disease. The leading cause of death is cardiovascular disease.

As renal function declines,

  • the prevalence of both malnutrition and cardiovascular disease increase.

Malnutrition and vascular disease correlate with the levels of

  • markers of inflammation in patients treated with dialysis and in those not yet on dialysis.

The causes of inflammation are likely to be multifactorial. CRP levels are associated with cardio-vascular risk in the general population.

The changes in endothelial cell function,

  • in plasma proteins, and
  • in lpiids in inflammation

are likely to be atherogenic.

That cardiovascular risk is inversely correlated with serum cholesterol in dialysis patients, suggests that

  • hyperlipidemia plays a minor role in the incidence of cardiovascular disease.

Hypoalbuminemia, ascribed to malnutrition, has been one of the most powerful risk factors that predict all-cause and cardiovascular mortality in dialysis patients. The presence of inflammation, as evidenced by increased levels of specific cytokines (interleukin-6 and tumor necrosis factor a) or acute-phase proteins (C-reactive protein and serum amyloid A), however, has been found to be associated with vascular disease in the general population as well as in dialysis patients. Patients have

  • loss of muscle mass and changes in plasma composition—decreases in serum albumin, prealbumin, and transferrin levels, also associated with malnutrition.

Inflammation alters

  • lipoprotein structure and function as well as
  • endothelial structure and function

to favor atherogenesis and increases

  • the concentration of atherogenic proteins in serum.

In addition, proinflammatory compounds, such as

  • advanced glycation end products, accumulate in renal failure, and
  • defense mechanisms against oxidative injury are reduced,

contributing to inflammation and to its effect on the vascular endothelium (33,34).

Endogenous copper can play an important role in postischemic reperfusion injury, a condition associated with endothelial cell activation and increased interleukin 8 (IL-8) production. Excessive endothelial IL-8 secreted during trauma, major surgery, and sepsis may contribute to the development of systemic inflammatory response syndrome (SIRS), adult respiratory distress syndrome (ARDS), and multiple organ failure (MOF). No previous reports have indicated that copper has a direct role in stimulating human endothelial IL-8 secretion. Copper did not stimulate secretion of other cytokines. Cu(II) appeared to be the primary copper ion responsible for the observed increase in IL-8 because a specific high-affinity Cu(II)-binding peptide, d-Asp-d-Ala-d-Hisd-Lys (d-DAHK), completely abolished this effect in a dose-dependent manner. These results suggest that Cu(II) may induce endothelial IL-8 by a mechanism independent of known Cu(I) generation of reactive oxygen species (35).

Blood coagulation plays a key role among numerous mediating systems that are activated in inflammation. Receptors of the PAR family serve as sensors of serine proteinases of the blood clotting system in the target cells involved in inflammation. Activation of PAR_1 by thrombin and of PAR_2 by factor Xa leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade. Other receptors that can modulate responses of the cells activated by proteinases through PAR receptors are also involved in the association of coagulation and inflammation together with the receptors of the PAR family. The presence of PAR receptors on mast cells is responsible for their reactivity to thrombin and factor Xa , essential to the inflammation and blood clotting processes (36).

The understanding of regulation of the inflammatory process in chronic inflammatory diseases is advancing.

Evidence consistently indicates that T-cells play a key role in initiating and perpetuating inflammation, not only via the production of soluble mediators but also via cell/cell contact interactions with a variety of cell types through membrane receptors and their ligands. Signalling through CD40 and CD40 ligand is a versatile pathway that is potently involved in all these processes. Many inflammatory genes relevant to atherosclerosis are influenced by the transcriptional regulator nuclear factor κ B (NFκB). In these events T-cells become activated by dendritic cells or inflammatory cytokines, and these T-cells activate, in turn, monocytes / macrophages, endothelial cells, smooth muscle cells and fibroblasts to produce pro-inflammatory cytokines, chemokines, the coagulation cascade in vivo, and finally matrix metalloproteinases, responsible for tissue destruction. Moreover, CD40 ligand at inflammatory sites stimulates fibroblasts and tissue monocyte/macrophage production of VEGF, leading to angiogenesis, which promotes and maintains the chronic inflammatory process.

NFκB plays a pivotal role in co-ordinating the expression of genes involved in the immune and inflammatory response, evoking tumor necrosis factor α (TNFα), chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-8, matrix metalloproteinase enzymes (MMP), and genes involved in cell survival. A complex array of mechanisms, including T cell activation, leukocyte extravasation, tissue factor expression, MMP expression and activation, as well induction of cytokines and chemokines, implicated in atherosclerosis, are regulated by NFκB.

Expression of NFκB in the atherosclerotic milieu may have a number of potentially harmful consequences. IL-1 activates NFκB upregulating expression of MMP-1, -3, and -9. Oxidized LDL increases macrophage MMP-9, associated with increased nuclear binding of NFκB and AP-1. Expression of tissue factor, initiating the coagulation cascade, is regulated by NFκB. In atherosclerotic plaque cells, tissue factor antigen and activity were inhibited following over-expression of IκBα and dominant-negative IKK-2, but not by dominant negative IKK-1 or NIK. Tis supports the concept that activation of the ―canonical‖ pathway upregulates pro-thrombotic mediators involved in disease. Many of the cytokines and chemokines which have been detected in human atherosclerotic plaques are also regulated by NFκB. Over-expression of IκBα inhibits release of TNFα, IL-1, IL-6, and IL-8 in macrophages stimulated with LPS and CD40 ligand (CD40L). This report describes how NFκB activation upregulates major pro-inflammatory and pro-thrombotic mediators of atherosclerosis (37-41).

This review is both focused and comprehensive. The details of evolving methods are avoided in order to build the argument that a very rapid expansion of discovery has been evolving depicting disease, disease mechanisms, disease associations, metabolic biomarkers, study of effects of diet and diet modification, and opportunities for targeted drug development. The extent of future success will depend on the duration and strength of the developed interventions, and possibly the avoidance of dead end interventions that are unexpectedly bypassed. I anticipate the prospects for the interplay between genomics, metabolomics, metabonomics, and personalized medicine may be realized for several of the most common conditions worldwide within a few decades (42-44).

References

  1. Trujillo E, Davis C, Milner J. Nutrigenomics, proteomics, metabolomics, and the practice of dietetics. J Am Diet Assoc. 2006;106(3):403-13.
  2. Kussmann M, Raymond F, Affolter M. OMICS-driven biomarker discovery in nutrition and health. J Biotechnol. 2006;124(4):758-87.
  3. (Zeisel SH. Nutrigenomics and metabolomics will change clinical nutrition and public health practice: insights from studies on dietary requirements for choline. Am J Clin Nutr. 2007;86(3):542-8.
  4. García-Cañas V, Simó C, León C, Cifuentes A. Advances in Nutrigenomics research: novel and future analytical approaches to investigate the biological activity of natural compounds and food functions. J Pharm Biomed Anal. 2010;51(2):290-304.
  5. Kussmann M, Blum S. OMICS-derived targets for inflammatory gut disorders: opportunities for the development of nutrition related biomarkers. Endocr Metab Immune Disord Drug Targets. 2007;7(4):271-87.
  6. Ovesná J, Slabý O, Toussaint O, Kodícek M, et al. High throughput ‘omics’ approaches to assess the effects of phytochemicals in human health studies. Br J Nutr. 2008;99 E Suppl 1:ES127-34.
  7. Workshop Report: ―The 2010 Project‖. Chory J, Ecker JR, Briggs S, et al. A Blueprint for Understanding How Plants Are Built and How to Improve Them. Plant Physiology 2000;123:423–425, http://www.plantphysiol.org.
  8. Stover PJ, Caudill MA. Genetic and epigenetic contributions to human nutrition and health: managing genome-diet interactions. J Am Diet Assoc. 2008 Sep;108(9):1480-7.
  9. Kussmann M, Panchaud A, Affolter M.. Proteomics in nutrition: status quo and outlook for biomarkers and bioactives. J Proteome Res. 201;9(10):4876-87.
  10. Wolfram Weckwerth. Metabolomics in Systems Biology. Annual Review of Plant Biology 2003; 54: 669-689.
  11. Kussmann M, Affolter M, Nagy K, Holst B, Fay LB. Mass spectrometry in nutrition: understanding dietary health effects at the molecular level. Mass Spectrom Rev. 2007;26(6):727-50.
  12. Grainger DJ. Megavariate Statistics meets High Data-density Analytical Methods: The Future of Medical Diagnostics? IRTL Reviews 2003;1:1-6.
  13. Heiss; C, Keen CL, Kelm M. Flavanols and Cardiovascular Disease Prevention. European Heart Journal 2010;31(21):2583-2592.
  14. Kussmann M, Rezzi S, Daniel H. Profiling techniques in nutrition and health research. Curr Opin Biotechnol. 2008;19 (2):83-99.
  15. Ohashi N, Ito C, Fujikawa R, Yamamoto H, et al. The impact of visceral adipose tissue and high-molecular weight adiponectin on cardia-ankle vascular index in asymptomatic Japanese subjects. Metabolism 2009; 58:1023-9. [CAVI and VAT and HMW adiponectin levels];
  1. Zha JM, Di WJ, Zhu T, Xie T, et al. Comparison of gene transcription between subcutaneous and visceral adipose tissue in chinese adults. Endocr J 2009;56:934-44. [TLR4 signaling, 11 beta-HSD1 and GR levels in VAT];
  2. Albert L, Girola A, Gilardini L, Conti A, et al. Type 2 diabetes and metabolic syndrome are associated with increased expression of 11 beta-hydroxysteroid dehydrogenase 1 in obese subjects. Int J Obesity (Lond) 2007;31:1826-31;
  3. Fabbrini E, Markos F, Mohammed BS, Pietka T, et al. Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity. PNAS 2009;106:15430-5;
  4. Tong J, Boyko EJ, Utzschneider KM, McNeely MJ, et al. Intraabdominal fat accumulation predicts the development of the metabolic syndrome in non-diabetic Japanese-Americans. Diabetologia 2007;50:1156-60;
  5. Kim K, Valentine RJ, Shin Y, Gong K. Association of visceral adiposity and exercise participation with C- reactive protein, insulin resistance, and endothelial dysfunction in Korean healthy adults. Metabolism 2008;57:1181-9. [(VAT-EC exhibits a marked angiogenic and proinflammatory state];
  6. Villaret A, Galitzky J, Decaunes P, Exteve D, et al. Adipose tisue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence. Diabetes 2010;59:2755-63;
  7. van Dijk -, Feskens EJ, Bos MB, Hoelen DW, et al. A saturated fatty acid-rich diet induces an obesity-linked proinflammatory gene expression profile in adipose tissue of subjects at risk of metabolic syndrome. Am J Clin Nutr 2009;90:1656-64.[MUFA in LDL lowering].
  8. Theurl I, Aigner E, Theurl M, Nairz M, et al. Regulation of iron homeostasis in anemia of chronic disease and iron deficiency anemia: diagnostic and therapeutic implications. Blood. 2009;113(21):5277-86
  9. Cheng PP, Jiao XY, Wang XH, Lin JH, Cai YM. Hepcidin expression in anemia of chronic disease and concomitant iron-deficiency anemia. Clin Exp Med. 2010 May 25. [Epub ahead of print].
  10. Spratlin JL, Serkova NJ, and Eckhardt SG. Clinical Applications of Metabolomics in Oncology: A Review. Clin Cancer Res. 2009 ;15; 15(2): 431–440.
  11. Fischer PM, Lane DP. Small molecule inhibitors of thep53 suppressor HDM2: have protein-protein interactions come of age as drug targets? Trends in Pharm Sci 2004;25(7):343-346.
  12. Visser M, Bouter LM, McQuillan GM, Wener HM. Elevated C-Reactive Protein Levels in Overweight and Obese Adults. JAMA. 1999;282:2131-2135.
  13. Yudkin JS, Stehouwer CDA, Emeis JJ, Coppack SW. C-Reactive Protein in Healthy Subjects: Associations With Obesity, Insulin Resistance, and Endothelial Dysfunction : A Potential Role for Cytokines Originating From Adipose Tissue? Arterioscler. Thromb. Vasc. Biol. 1999; 19:972-978.
  14. Visvikis-Siest S, Siest G. The STANISLAS cohort: a 10-year followup of supposed healthy families. Gene-environment interactions, reference values and evaluation of biomarkers in prevention of cardiovascular diseases. Clin Chem Lab Med 2008;46:733-47.
  15. Schmidt MI, Duncan BB. Diabesity: an inflammatory metabolic condition. Clin Chem Lab Med 2003;41:1120-1130.
  16. Fenkci S, Rota S, Sabir N, Akdag B. Ultrasonographic and biochemical evaluation of visceral obesity in obese women with non-alcoholic fatty liver disease. Eur J Med Res 2007;12:68-73. (VAT, HOMA)
  17. Lee JW, Lee HR, Shim JY, Im JA, et al. Viscerally obese women with normal body weight have greater brachial-ankle pulse wave velocity than non viscerally obese women with excessive body weight. Clin Endocrinol (Oxf) 2007;66:572-8. [visceral obesity – high trigly, high baPWV, greater SFA and thigh SFA].
  18. Kaysen GE. The Microinflammatory State in Uremia: Causes and Potential Consequences. J Am Soc Nephrol 2001;12:1549–1557.
  19. Kaysen GE. Role of Inflammation and Its Treatment in ESRD Patients. Blood Purif 2002;20:70–80.
  20. Bar-Or D, Thomas GW, Yukl RL, Rael LT, et al. Copper stimulates the synthesis and release of interleukin-8 in human endothelial cells: a possible early role in systemic inflammatory responses. Shock 2003;20(2):154–158.
  21. Dugina TN, Kiseleva EV, Chistov IV, Umarova BA, and Strukova SM. Receptors of the PAR Family as a Link between Blood Coagulation and Inflammation. Biochemistry (Moscow), 2002; 67(1):65-74. [Translated from Biokhimiya 2002;67(1):77-87].
  22. Monaco C, Andreakos E, Kiriakidis S, Feldmann M, and and Ewa Paleolog. T-Cell-Mediated Signalling in Immune, Inflammatory and Angiogenic Processes: The Cascade of Events Leading to Inflammatory Diseases. Current Drug Targets – Inflammation & Allergy, 2004, 3, 35-42.
  23. Monaco C, Grosjean J, and Paleolog E. The role of the NFκB pathway in atherosclerosis. [E-mail: e.paleolog@imperial.ac.uk]
  24. Libby P, Ridker PM, and Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135-43.
  25. Karin M, Yamamoto Y, Wang QM. The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov 2004;3:17-26.
  26. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol 2000;18:621-63.
  27. Lee DY, Bowen BP, and Northen TR. Mass spectrometry–based metabolomics, analysis of metabolite-protein interactions, and imaging. BioTechniques 2010;49:557-565.
  28. Faca V, Krasnoselsky A, and Hanash S. Innovative proteomic approaches for cancer biomarker discove.
  29. Sharp, P, and MIT faculty. ‘Convergence’ offers potential for revolutionary advance in biomedicine. The Third Revolution: Convergence of the Life Sciences, Physical Sciences and Engineering. White paper. Reported in Biotechnology Jan 5, 2011. [Convergence is a new paradigm that can yield critical advances in a broad array of sectors]

 

Read Full Post »


Epilogue: Envisioning New Insights in Cancer Translational Biology

Author and Curator: Larry H Bernstein, MD, FCAP

 

The foregoing  summary leads to a beginning as it is a conclusion.  It concludes a body of work in the e-book series,

Series C: e-Books on Cancer & Oncology

Series C Content Consultant: Larry H. Bernstein, MD, FCAP

 

VOLUME ONE 

Cancer Biology and Genomics for Disease Diagnosis

2014

Stephen J. Williams, PhD, Senior Editor

sjwilliamspa@comcast.net

Tilda Barliya, PhD, Editor

tildabarliya@gmail.com

Ritu Saxena, PhD, Editor

ritu.uab@gmail.com

Leaders in Pharmaceutical Business Intelligence 

that has been presented by the cancer team of professional experts, e-Book concept was conceived by Aviva Lev-Ari, PhD, RN, e-Series Editor-in-Chief and Founder of Leaders in Pharmaceutical Business Intelligence 

and the Open Access Online Scientific Journal

http://pharmaceuticalintelligence.com

Stephen J. Williams, PhD, Senior Editor, and other notable contributors in  various aspects of cancer research in the emerging fields of targeted  pharmacology,  nanotechnology, cancer imaging, molecular pathology, transcriptional and regulatory ‘OMICS’, metabolism, medical and allied health related sciences, synthetic biology, pharmaceutical discovery, and translational medicine.

This  volume and its content have been conceived and organized to capture the organized events that emerge in embryological development, leading to the major organ systems that we recognize anatomically and physiologically as an integrated being.  We capture the dynamic interactions between the systems under stress  that are elicited by cytokine-driven hormonal responses, long thought to be circulatory and multisystem, that affect the major compartments of  fat and lean body mass, and are as much the drivers of metabolic pathway changes that emerge as epigenetics, without disregarding primary genetic diseases.

The greatest difficulty in organizing such a work is in whether it is to be merely a compilation of cancer expression organized by organ systems, or whether it is to capture developing concepts of underlying stem cell expressed changes that were once referred to as “dedifferentiation”.  In proceeding through the stages of neoplastic transformation, there occur adaptive local changes in cellular utilization of anabolic and catabolic pathways, and a retention or partial retention of functional specificities.

This  effectively results in the same cancer types not all fitting into the same “shoe”. There is a sequential loss of identity associated with cell migration, cell-cell interactions with underlying stroma, and metastasis., but cells may still retain identifying “signatures” in microRNA combinatorial patterns.  The story is still incomplete, with gaps in our knowledge that challenge the imagination.

What we have laid out is a map with substructural ordered concepts forming subsets within the structural maps.  There are the traditional energy pathways with terms aerobic and anaerobic glycolysis, gluconeogenesis, triose phosphate branch chains, pentose shunt, and TCA cycle vs the Lynen cycle, the Cori cycle, glycogenolysis, lipid peroxidation, oxidative stress, autosomy and mitosomy, and genetic transcription, cell degradation and repair, muscle contraction, nerve transmission, and their involved anatomic structures (cytoskeleton, cytoplasm, mitochondria, liposomes and phagosomes, contractile apparatus, synapse.

Then there is beneath this macro-domain the order of signaling pathways that regulate these domains and through mechanisms of cellular regulatory control have pleiotropic inhibitory or activation effects, that are driven by extracellular and intracellular energy modulating conditions through three recognized structures: the mitochondrial inner membrane, the intercellular matrix, and the ion-channels.

What remains to be done?

  1. There is still to be elucidated the differences in patterns within cancer types the distinct phenotypic and genotypic features  that mitigate anaplastic behavior. One leg of this problem lies in the density of mitochondria, that varies between organ types, but might vary also within cell type of a common function.  Another leg of this problem has also appeared to lie in the cell death mechanism that relates to the proeosomal activity acting on both the ribosome and mitochondrion in a coordinated manner.  This is an unsolved mystery of molecular biology.

 

  1. Then there is a need to elucidate the major differences between tumors of endocrine, sexual, and structural organs, which are distinguished by primarily a synthetic or primarily a catabolic function, and organs that are neither primarily one or the other.  For example, tumors of the thyroid and paratnhyroids, islet cells of pancreas, adrenal cortex, and pituitary glands have the longest 5 year survivals.  They and the sexual organs are in the visceral compartment.  The rest of the visceral compartment would be the liver, pancreas, salivary glands, gastrointestinal tract, and lungs (which are embryologically an outpouching of the gastrointestinal tract), kidneys and lower urinary tract.  Cancers of these organs have a much less favorable survival (brain, breast and prostate, lymphatic, blood forming organ, skin).  The case  is intermediate for breast and prostate between the endocrine organs and GI tract, based on natural history, irrespective of the available treatments.  Just consider the dilemma over what we do about screening for prostate cancer in men over the age of 60 years age who have a 70 percent incident silent carcinoma of the prostate that could be associated with unrelated cause of death.  The very rapid turnover of the gastric and colonic GI epithelium, and of the  subepithelial  B cell mucosal lymphocytic structures  is associated  with a greater aggressiveness of the tumor.

 

  1. However, we  have to reconsider the observation by NO Kaplan than the synthetic and catabolic functions are highlighted by differences in the expressions of the balance of  the two major pyridine nucleotides – DPN (NAD) and TPN (NADP) – which also might be related to the density of mitochondria  which is associated with both NADP and synthetic activity, and  with efficient aerobic function.  These are in an equilibrium through the “transhydrogenase reaction” co-discovered by Kaplan, in Fritz Lipmann’s laboratory. There does  arise a conundrum involving the regulation of mitochondria in these high turnover epithelial tissues  that rely on aerobic energy, and generate ATP through TPN linked activity, when they undergo carcinogenesis. The cells  replicate and they become utilizers of glycolysis, while at the same time, the cell death pathway is quiescent. The result becomes the introduction of peripheral muscle and liver synthesized protein cannabolization (cancer cachexia) to provide glucose  from proteolytic amino acid sources.

 

  1. There is also the structural compartment of the lean body mass. This is the heart, skeletal  structures (includes smooth muscle of GI tract, uterus, urinary bladder, brain, bone, bone marrow).  The contractile component is associated with sarcomas.  What is most striking is that the heart, skeletal muscle, and inflammatory cells are highly catabolic, not anabolic.  NO Kaplan referred tp them as DPN (NAD) tissues. This compartment requires high oxygen supply, and has a high mechanical function. But again, we return to the original observations of enrgy requirements at rest being different than at high demand.  At work, skeletal muscle generates lactic acid, but the heart can use lactic acid as fuel,.

 

  1. The liver is supplied by both the portal vein and the hepatic artery, so it is not prone to local ischemic injury (Zahn infarct). It is exceptional in that it carries out synthesis of all the circulating transport proteins, has a major function in lipid synthesis and in glycogenesis and glycogenolysis, with the added role of drug detoxification through the P450 system.  It is not only the largest organ (except for brain), but is highly active both anabolically and catabolically (by ubiquitilation).
  2. The expected cellular turnover rates for these tissues and their balance of catabolic and anabolic function would have to be taken into account to account for the occurrence and the activities of oncogenesis. This is by no means a static picture, but a dynamic organism constantly in flux imposed by internal and external challenges.  It is also important to note the the organs have a concentration of mitochondria, associated with energy synthetic and catabolic requirements provided by oxygen supply and the electron transport mechanism for oxidative phosphorylation.  For example, tissues that are primarily synthetic do not have intermitent states of resting and high demand, as seen in skeletal muscle, or perhaps myocardium (which is syncytial and uses lactic acid generated from skeletal muscle when there is high demand).
  3. The existence of  lncDNA has been discovered only as a result of the human genome project (HGP). This was previously known only as “dark DNA”.  It has become clear that lncDNA has an important role in cellular regulatory activities centered in the chromatin modeling.  Moreover, just as proteins exhibit functionality in their folding, related to tertiary structure and highly influenced by location of –S-S- bridges and amino acid residue distances (allosteric effects), there is a less studied effect as the chromatin becomes more compressed within the nucleus, that should have a bearing on cellular expression.

According to Jose Eduardo de Salles Roselino , when the Na/Glucose transport system (for a review Silvermann, M. in Annu. Rev. Biochem.60: 757-794(1991)) was  found in kidneys as well as in key absorptive cells of digestive tract, it should be stressed its functional relationship with “internal milieu” and real meaning, homeostasis. It is easy to understand how the major topic was presented as how to prevent diarrheal deaths in infants, while detected in early stages. However, from a biochemical point of view, as presented in Schrödinger´s What is life?, (biochemistry offering a molecular view for two legs of biology, physiology and genetics). Why should it be driven to the sole target of understanding genetics? Why the understanding of physiology in molecular terms should be so neglected?

From a biochemical point of view, here in a single protein. It is found the transport of the cation most directly related to water maintenance, the internal solvent that bath our cells and the hydrocarbon whose concentration is kept under homeostatic control on that solvent. Completely at variance with what is presented in microorganisms as previously mentioned in Moyed and Umbarger revision (Ann. Rev42: 444(1962)) that does not regulates the environment where they live and appears to influence it only as an incidental result of their metabolism.

In case any attempt is made in order to explain why the best leg that supports scientific reasoning from biology for medical purposes was led to atrophy, several possibilities can be raised. However, none of them could be placed strictly in scientific terms. Factors that bare little relationship with scientific progress in general terms must also be taken into account.

One simple possibility of explanation can be found in one review (G. Scatchard – Solutions of Electrolytes Ann. Rev. Physical Chemistry 14: 161-176 (1963)).  A simple reading of it and the sophisticated differences among researchers will discourage one hundred per cent of biologists to keep in touch with this line of research. Biochemists may keep on reading.  However, consider that first: Complexity is not amenable to reductionist vision in all cases. Second, as coupling between scalar flows such as chemical reactions and vector flows such as diffusion flows, heat flows, and electrical current can occur only in anisotropic system…let them with their problems of solvents, ions and etc. and let our biochemical reactions on another basket. At the interface, for instance, at membrane level, we will agree that ATP is converted to ADP because it is far from equilibrium and the continuous replenishment of ATP that maintain relatively constant ATP levels inside the cell and this requires some non-stationary flow.

Our major point must be to understand that our biological limits are far clearer present in our limited ability to regulate the information stored in the DNA than in the amount of information we have in the DNA as the master regulator of the cells.

The amazing revelation that Masahiro Chiga   (discovery of liver adenylate kinase  distinct from that of muscle) taught  me (LHB) is – draw 2 circles  that intersect, one of which represents what we know, the other – what we don’t know.  We don’t teach how much we don’t know!  Even today, as much as 40 years ago, there is a lot we need to get on top of this.

 

The observation is rather similar to the presentations I  (Jose Eduardo de Salles Rosalino) was previously allowed to make of the conformational energy as made by R Marcus in his Nobel lecture revised (J. of  Electroanalytical Chemistry 438:(1997) p251-259. His description of the energetic coordinates of a landscape of a chemical reaction is only a two-dimensional cut of what in fact is a volcano crater (in three dimensions) ( each one varie but the sum of the two is constant. Solvational+vibrational=100% in ordinate) nuclear coordinates in abcissa. In case we could represent it by research methods that allow us to discriminate in one by one degree of different pairs of energy, we would most likely have 360 other similar representations of the same phenomenon. The real representation would take into account all those 360 representation together. In case our methodology was not that fine, for instance it discriminate only differences of minimal 10 degrees in 360 possible, will have 36 partial representations of something that to be perfectly represented will require all 36 being taken together. Can you reconcile it with ATGC? Yet, when complete genome sequences were presented they were described as we will know everything about this living being. The most important problems in biology will be viewed by limited vision always and the awareness of this limited is something we should acknowledge and teach it. Therefore, our knowledge is made up of partial representations.

 

Even though we may have complete genome data for the most intricate biological problems, they are not so amenable to this level of reductionism. However, from general views of signals and symptoms we could get to the most detailed molecular view and in this case the genome provides an anchor. This is somehow, what Houssay was saying to me and to Leloir when he pointed out that only in very rare occasions biological phenomena could be described in three terms: Pacco, the dog and the anesthetic (previous e-mail). The non-coding region, to me will be important guiding places for protein interactions.

Read Full Post »

A Synthesis of the Beauty and Complexity of How We View Cancer


A Synthesis of the Beauty and Complexity of How We View Cancer

Author: Larry H. Bernstein, MD, FCAP

Cancer Volume One – Summary

A Synthesis of the Beauty and Complexity of How We View Cancer

 

This document has covered a broad spectrum of the research, translational biology, diagnostics (both laboratory and imaging methodologies), and treatments for a variety of cancers, mainly by organs, and selectively by the most common cancers seen in human populations. A number of observations stand out on review of all the material presented. 1. The most common cancers affecting humans is spread worldwide, with some variation by region. 2. Cancers within geographic regions may be expressed differently in relationship to population migrations, the incidence of specific environmental pollutants, occurrence of insect transmitted and sexually transmitted diseases (HIV, HCV, HPV), and possibly according to age, or relationship to ultraviolet or high dose radiation exposure. 3. Cancers are expressed within generally recognized age timelines. For example, acute lymphocytic leukemia and neuroblastoma in children under 10 years age; malignant giant cell tumor and osteosarcoma in the third and fourth decade; prostate cancer and breast cancer over age 40, and are more aggressive at an earlier age, both having a strong sex hormone dependence. 4. There is dispute about the effectiveness of screening for cancer with respect to what age, excessive risk in treatment modality, and the duration of progression free survival. Despite the evidence of several years potential life extension, a long term survival of 10 years is not the expected outcome. However, the quality of life in the remaining years is a valid point in favor of progress. 5. There has been a significant reduction in toxicity of treatment, but attention has been focused on a patient-centric decision process. 6. There has been a dramatic improvement in surgical approaches, post-surgical surveillance, and in diagnosis by invasive and noninvasive methods, especially in the combination of needle biopsy and imaging techniques. 7. There is significant variation within cancer cell types with respect to disease-free survival.

The work presented has several main components: First, there is the biology and mechanisms involved in carcinogenesis related to (1) mutations; (2) carcinogenesis; (3) cell regulatory mechanisms; (4) cell signaling pathways; (5) apoptosis (6) ubitination (7) mitochondrial dysfunction; (8) cell-cell interactions; (9) cell migration; (10) metastasis. Then there are large portions covering (1) imaging; (2) specific targeted therapy; (3) nanotechology-based therapy; (4) specific organ-type cancers; (5) genomics-based testing; (6) circulating cancer cells; (7) miRNAs; (8) siRNAs; (9) cancer immunology and (10) immunotherapy.

Classically, we refer to cancer development in terms of the germ cell layers – ectoderm, mesoderm, and endoderm. These are formative in embryonic development. The most active development occurs during embryonic development, with a high growth rate of cells and also a high utilization of energy. The cells utilize oxidation for energy in this period characterized by movement of cells in differentiation and organogenesis. This was observed to be unlike the cell metabolism in carcinogenesis, which is characterized by impaired mitochondrial function and reliance on lactate production for energy – termed anaerobic glycolysis, as investigated by Meyerhof, Embden, Warburg, Szent-Gyorgy, H. Krebs, Theorell, AV Hill, B Chance, P Mitchell, P Boyer, F Lippman, and others.

In addition, the body economy has been divided into two major metabolic compartments: fat and lean body mass (LBM), which is further denoted as visceral and structural. This denotes the gut, kidneys, liver, lung, pancreas, sexual organs, endocrines, brain and fat cells in one compartment, and skeletal muscle, bone and cardiovascular in another. LBM is calculated as fat free mass. Further, brown fat is distinguished from white fat. But this was a first layer of construction of the human body. One peels away this layer to find a second layer. For example, the gut viscera have an inner (outer) epithelial layer, a muscularis, and a deep epithelium, which has circulation and fat. There is also an interstitium between the gut epithelium and muscularis. The lung has an epithelium exposed to the airspaces, then capillaries, and then epithelium, designed for exchange of O2 and CO2, the source of heat generation. The pancreas has an endocrine portion in the islets that are embedded in an exocrine secretory organ. The sexual organs have a combination of glandular structures embedded in a mesothelium.

The structural compartment is entirely accounted for by the force of contraction. If this is purely anatomical, that is not really the case when one goes into the functioning substructures of these tissues – cytoplasm, endoplasmic reticulum (ribosomal), mitochondria, liposomes, chromatin apparatus, cell membrane and vesicles. Within and between these structures are the working and interacting mechanisms of the cell in its unique role. What ties these together was first thought to be found in the dogma following the discovery of the genetic code in 1953 that begat DNA to RNA to protein.

This led to many other discoveries that made it clear that it was only a first approximation. It did not account for noncoding DNA, which became unmasked with the culmination of the Human Genome Project and concurrent advances in genomics (mtDNA, mtRNA, siRNA, exosomes, proteomics, synthetic biology, predictive analytics, and regulatory pathways directed by signaling molecules. Here is a list of signaling pathways: 1. JAK-STAT 2. GPCR 3. Endocrine 4. Cytochemical 5. RTK 6. P13K 7. NF-KB 8. MAPK 9. Ubiquitin 10. TGF-beta 11. Stem cell These signaling pathways have become the basis for the discovery of inhibitors of signaling pathways (suppressors), as well as activators, as these have been considered as specific targets for selective therapy. (.See Figure below) Of course, extensive examination of these pathways has required that all such findings are validated based on the STRENGTH of their effect on the target and in the impact of suppression.

inhibitors of signaling pathways-1

http://www.SelleckChem.com

 

Let us continue this discussion elucidating several major points.  While the early observations that drove the interest in biochemical behavior of cancer cells has been displaced, it has not faded from view.

Bioenergetics of Cancer cells

Michael J. Gonzalez (Bioenergetic_Theory_of_Carcinigenesis. http://www.academia.edu/2224071/ Bioenergetic_Theory_of_Carcinigenesis) maintains that the altered energy metabolism of tumor cells provides a viable target for a non-toxic chemotherapeutic approach.  An increased glucose consumption rate  has been observed in malignant cells. Warburg (NobelLaureate in medicine) postulated that the respiratory process of malignant cells was impaired in the malignant transformation. Szent-Györgyi (Nobel in medicine) also viewed cancer as originating from insufficient oxygen utilization. Oxygen inhibits anaerobic  metabolism (fermentation and lactic acid production). Interestingly, during cell differentiation (where cell energy level is high) there is an increased cellular production of oxidation products that appear to provide physiological stimulation for changes in gene expression that may lead to a terminal differentiated state. The failure to maintain high ATP production (high cell energy levels) may be a consequence of inactivation of key enzymes, especially those related to the Krebs cycle and the electron transport system. A distorted mitochondrial function (transmembrane potential) may result.  This  aspect could be suggestive of an important mitochondrial involvement in the carcinogenic process in addition to presenting it as a possible therapeutic target for cancer. Intermediate metabolic correction of the mitochondria is postulated as a possible non-toxic therapeutic approach for cancer.

Fermentation is the anaerobic metabolic breakdown of glucose without net oxidation. Fermentation does not release all the available energy of glucose or need oxygen as part of its biochemical reactions ;  it merely allows glycolysis  (a process that yields two ATP per mole of glucose) to continue by replenishing reduced coenzymes and yields lactate as its final product. The first step in aerobic and anaerobic energy producing pathways, it occurs in the cytoplasm of cells, not in specialized organelles, and is found in all living organisms.  Cancer cells have a fundamentally different energy metabolism compared to normal cells, that  are obligate aerobes (oxygen-requiring cells)  meeting their energy needs with oxidative metabolic processes., while cancer cells do not  require oxygen for their survival. This increase in glycolytic  flux is a metabolic strategy of tumor cells to ensure growth and    survival  in  environments  with  low   oxygen concentrations.

Radoslav Bozov has commented that the process of genomic evolution cannot be fully revealed through comparative genomicsHe states that DNA would be entropic- favorable stable state going towards absolute ZERO temp. Themodynamics measurement in subnano discrete space would go negative towards negativity. DNA is like a cold melting/growing crystal, quite stable as it appears not due to hydrogen bonding , but due to interference of C-N-O. That force is contradicted via proteins onto which we now know large amount of negative quantum redox state carbon attaches. The more locally one attempts to observe, the more hidden variables would emerge as a consequence of discrete energy spaces opposing continuity of matter/time. But stability emerges out of non-stable states, and never reaches absolute stability, for there would be neither feelings nor freedom.

Membrane potential(Vm)

Membrane potential (Vm), the voltage across the plasma membrane, arises because of the presence of differention channels/transporters with specific ion selectivity and permeability. Vm is a key biophysical signal in non-excitable cells, modulating important cellular activities, such as proliferation and differentiation. Therefore, the multiplicities of various ion channels/transporters expressed on different cells are finely tuned in order to regulate the Vm. (M Yang and WJ Brackenbury.

Membrane potential and cancer progression. Frontiers in Physiol.  2013(4); 185: 1.  http://dx.doi.org/10.3389/fphys.2013.00185)

It is well-established that cancer cells possess distinct bioelectrical properties. Notably, electrophysiological analyses in many cancer cell types have revealed a depolarized Vm that favors cell proliferation. Ion channels/transporters control cell volume and migration, and emerging data also suggest that the level of Vm has functional roles in cancer cell migration. In addition, yperpolarization is necessary for stem cell differentiation. For example, both osteogenesis and adipogenesis are hindered in human mesenchymal stem cells (hMSCs) under depolarizing conditions. Therefore, in the context of cancer, membrane depolarization might be important for the emergence and maintenance of cancer stem cells (CSCs), giving rise to sustained tumor growth. This review aims to provide a broad understanding of the Vm as a bioelectrical signal in cancer cells by examining several key types of ion channels that contribute to its regulation. The mechanisms by which Vm regulates cancer cell proliferation, migration, and differentiation will be discussed. In the long term, Vm might be avaluable clinical marker for tumor detection with prognostic value, and could even be artificially modified in order to inhibit tumor growth and metastasis.

Perspective beyond Cancer Genomics: Bioenergetics of Cancer Stem Cells

Hideshi Ishii, Yuichiro Doki, and Masaki Mori
Yonsei Med J 2010; 51(5):617-621.  http://dx.doi.org/10.3349/ymj.2010.51.5.617   pISSN: 0513-5796, eISSN: 1976-2437

Although the notion that cancer is a disease caused by genetic and epigenetic alterations is now widely accepted, perhaps more emphasis has been given to the fact that cancr is a genetic disease. It should be noted that in the post-genome sequencing project period of the 21st century, the underlined phenomenon nevertheless could not be discarded towards the complete control of cancer disaster as the whole strategy, and in depth investigation of the factors associated with tumorigenesis is required for achieving it. Otto Warburg has won a Nobel Prize in 1931 for the discovery of tumor bioenergetics, which is now commonly used as the basis of positron emission tomography (PET), a highly sensitive noninvasive technique used in cancer diagnosis. Furthermore, the importance of the cancer stem cell (CSC) hypothesis in therapy-related resistance and metastasis has been recognized during the past 2 decades. Accumulating evidence suggests that tumor bioenergetics plays a critical role in CSC regulation; this finding has opened up a new era of cancer medicine, which goes beyond cancer genomics.

Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H+-ATP synthase.

Gema Santamaría1,#, Marta Martínez-Diez1,#, Isabel Fabregat2 and José M. Cuezva1,*
Carcinogenesis 2006 27(5):925-935      http://dx.doi.org/10.1093/carcin/bgi315

There is a large body of clinical data documenting that most human carcinomas contain reduced levels of the catalytic subunit of the mitochondrial H+-ATP synthase. In colon and lung cancer this alteration correlates with a poor patient prognosis. Furthermore, recent findings in colon cancer cells indicate that down-regulation of the H+-ATP synthase is linked to the resistance of the cells to chemotherapy. However, the mechanism by which the H+-ATP synthase participates in cancer progression is unknown. In this work, we show that inhibitors of the H+-ATP synthase delay

staurosporine-induced cell death in liver cells that are dependent on oxidative phosphorylation for energy provision whereas it has no effect on glycolytic cells. Efficient execution of cell death requires the generation of reactive oxygen species (ROS) controlled by the activity of the H+-ATP synthase in a process that is concurrent with the rapid disorganization of the cellular mitochondrial network. The generation of ROS after staurosporine treatment is highly dependent on the mitochondrial membrane potential and most likely caused by reverse electron flow to Complex I. The generated ROS promote the carbonylation and covalent modification of cellular and mitochondrial proteins. Inhibition of the activity of the H+-ATP synthase blunted ROS production, prevented the oxidation of cellular proteins and the modification of mitochondrial proteins, delaying the release of cyt c and the execution of cell death. The results in this work establish the down-regulation of the H+-ATP synthase, and thus of oxidative phosphorylation, as part of the molecular strategy adapted by cancer cells to avoid reactive oxygen species-mediated cell death. Furthermore, the results provide a mechanistic explanation to understand chemotherapeutic resistance of cancer cells that rely on glycolysis as main energy provision pathway.

see also –

The tumor suppressor function of mitochondria: Translation into the clinics

José M. CuezvaÁlvaro D. OrtegaImke Willers, et al.  
Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease  Dec 2009;  1792(12): 1145–1158  http://dx.doi.org/10.1016/j.bbadis.2009.01.006

Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life–death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial β-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.

Self-Destructive Behavior in Cells May Hold Key to a Longer Life

Carl Zimmer, MY Times  October 5, 2009

In recent years, scientists have found evidence of autophagy in preventing a much wider range of diseases. Many disorders, like Alzheimer’s disease, are the result of certain kinds of proteins forming clumps. Lysosomes can devour these clumps before they cause damage, slowing the onset of diseases.

Lysosomes may also protect against cancer. As mitochondria get old, they cast off charged molecules that can wreak havoc in a cell and lead to potentially cancerous mutations. By gobbling up defective mitochondria, lysosomes may make cells less likely to damage their DNA. Many scientists suspect it is no coincidence that breast cancer cells are often missing autophagy-related genes. The genes may have been deleted by mistake as a breast cell divided. Unable to clear away defective mitochondria, the cell’s descendants become more vulnerable to mutations.

Unfortunately, as we get older, our cells lose their cannibalistic prowess. The decline of autophagy may be an important factor in the rise of cancer, Alzheimer’s disease and other disorders that become common in old age. Unable to clear away the cellular garbage, our bodies start to fail.

If this hypothesis turns out to be right, then it may be possible to slow the aging process by raising autophagy. It has long been known, for example, that animals that are put on a strict low-calorie diet can live much longer than animals that eat all they can. Recent research has shown that caloric restriction raises autophagy in animals and keeps it high. The animals seem to be responding to their low-calorie diet by feeding on their own cells, as they do during famines. In the process, their cells may also be clearing away more defective molecules, so that the animals age more slowly.

Some scientists are investigating how to manipulate autophagy directly. Dr. Cuervo and her colleagues, for example, have observed that in the livers of old mice, lysosomes produce fewer portals on their surface for taking in defective proteins. So they engineered mice to produce lysosomes with more portals. They found that the altered lysosomes of the old experimental mice could clear away more defective proteins. This change allowed the livers to work better.

 

Essentiality of pyruvate kinase, oxidation, and phosphorylation

We can move to the next level with greater clarity. Yu et al. reported an important relationship between Pyruvate kinase M2 (PKM2) and the Warburg effect of cancer cells ( M Yu, et al. PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells. J Biol Chem (PMID: 24142698) http://dx.doi.org10.1074/jbc.M113.508226 ).  They found that PIM2 could directly phosphorylate PKM2 on the Thr454 residue, which resulted in an increase of PKM2 protein levels. PKM2 with a phosphorylation-defective mutation displayed a reduced effect on glycolysis compared to the wild-type, thereby co-activating HIF-1α and β-catenin, and enhanced mitochondria respiration and chemotherapeutic sensitivity of cancer cells. This indicated that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target.

In another study of the effect of 3 homoplastic mtDNA mutations on oxidative metabolism of osteosarcoma cells, there was a difference proportional to the magnitude of the defect. (Iommarini L, et al. Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment. Hum Mol Genet. 2013 Nov 11. [Epub ahead of print]; PMID: 24163135 ).   Osteosarcoma cells carrying the most marked impairment of the gene encoding mitochondrial complex I  (CI) of oxidative phosphorylation displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction. The severe CI dysfunction was an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation.  The result suggested that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia-inducible factor 1α stabilization, indicating that structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.

An unrelated finding shares some agreement with what has been identified (Systematic isolation of context-dependent vulnerabilities in NSCLC. Cell, 24 Oct 2013; 155 (3): 552-566, http://dx.doi.org/10.1016/ j.cell.2013.09.041). They report  three distinct target/response-indicator pairings that are represented with significant frequencies (6%–16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature.   This is depicted in the Figure below.  The authors noted a frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets.

Print

 

 

The forging of a cancer-metabolism link and twists in the chain (Biome 19th April 2013)

Ten years ago, Grahame Hardie and Dario Alessi discovered that the elusive upstream kinase required for the activation of AMP-activated protein kinase (AMPK) by metabolic stress that the Hardie lab had been pursuing in their research on the metabolic regulator AMPK was the tumor suppressor, LKB1, that the neighbouring Alessi lab was working on at the time. This finding represented the first clear link between AMPK and cancer.

The resulting paper [1], published in 2003 in what was then Journal of Biology (now BMC Biology), was one [1] of three [2, 3] connecting these two kinases and that helped to swell of a surge of interest in the metabolism of tumor cells that was just beginning at about that time and is still growing. (LKB1 and AMPK and the cancer-metabolism link – ten years after.  D Grahame Hardie, and Dario R Alessi.  BMC Biology 2013, 11:36.   http://dx doi.org.10.1186/1741-7007-11-36.)

 

In September 2003, both groups published a joint paper [1] in Journal of Biology (now BMC Biology) that identified the long-sought and elusive upstream kinase acting on AMP-activated protein kinase (AMPK) as a complex containing LKB1, a known tumor suppressor. Similar findings were reported at about the same time by David Carling and Marian Carlson [2] and by Reuben Shaw and Lew Cantley [3]; at the time of writing these three papers have received between them a total of over 2,000 citations. These findings provided a direct link between a protein kinase, AMPK, which at the time was mainly associated with regulation of metabolism, and another protein kinase, LKB1, which was known from genetic studies to be a tumor suppressor. While the idea that cancer is in part a metabolic disorder (first suggested by Warburg in the 1920s [4]) is well recognized today [5], this was not the case in 2003, and our paper perhaps contributed towards its renaissance.

The distinctive metabolic feature of tumor cells that enables them to meet the demands of unrestrained growth is the switch from oxidative generation of ATP to aerobic glycolysis – a phenomenon now well known as the Warburg effect. Operating this switch is one of the central functions of the AMP-activated protein kinase (AMPK) that has long been the focus of research in the Hardie lab. AMPK is an energy sensor that is allosterically tuned by competitive binding of ATP, ADP and AMP to sites on its g regulatory subunit (its portrait here, with AMP bound at two sites, was kindly provided by Bing Xiao and Stephen Gamblin). When phosphorylated by LKB1, AMPK responds to depletion of ATP by turning off anabolic reactions required for growth, and turning on catabolic reactions and oxidative phosphorylation – the reverse of the Warburg effect. In this light, it is not surprising that LKB1  is inactivated in some proportion of many different types of tumors.

AMPK as an energy sensor and metabolic switch

AMPK was discovered as a protein kinase activity that phosphorylated and inactivated two key enzymes of fatty acid and sterol biosynthesis: acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The ACC kinase activity was reported to be activated by 5’-AMP, and the HMGR kinase activity by reversible phosphorylation, but for many years the two activities were thought to be due to distinct enzymes. However, in 1987 the DGH laboratory showed that both were functions of a single protein kinase, which we renamed AMPK after its allosteric activator, 5’-AMP. It was subsequently found that AMPK regulated not only lipid biosynthesis, but also many other metabolic pathways, both by direct phosphorylation of metabolic enzymes, and through longer-term effects mediated by phosphorylation of transcription factors and co-activators. In general, AMPK switches off anabolic pathways that consume ATP and NADPH, while switching on catabolic pathways that generate ATP (Figure 1).

 

target proteins and metabolic pathways regulated by AMPK 1741-7007-11-36-1_1

 

Summary of a selection of target proteins and metabolic pathways regulated by AMPK. Anabolic pathways switched off by AMPK are shown in the top half of the ‘wheel’ and catabolic pathways switched on by AMPK in the bottom half. Where a protein target for AMPK responsible for the effect is known, it is shown in the inner wheel; a question mark indicates that it is not yet certain that the protein is directly phosphorylated. For original references see [54].

Key to acronyms: ACC1/ACC2, acetyl-CoA carboxylases-1/-2; HMGR, HMG-CoA reductase; SREBP1c, sterol response element binding protein-1c; CHREBP, carbohydrate response element binding protein; TIF-1A, transcription initiation factor-1A; mTORC1, mechanistic target-of-rapamycin complex-1; PFKFB2/3, 6-phosphofructo-2-kinase, cardiac and inducible isoforms; TBC1D1, TBC1 domain protein-1; SIRT1, sirtuin-1; PGC-1α, PPAR-γ coactivator-1α; ULK1, Unc51-like kinase-1.

Regulation of AMPK  1741-7007-11-36-3

 

Regulation of AMPK. AMPK can be activated by increases in cellular AMP:ATP or ADP:ATP ratio, or Ca2+ concentration. AMPK is activated >100-fold on conversion from a dephosphorylated form (AMPK) to a form phosphorylated at Thr172 (AMPK-P) catalyzed by at least two upstream kinases: LKB1, which appears to be constitutively active, and CaMKKβ, which is only active when intracellular Ca2+ increases. Increases in AMP or ADP activate AMPK by three mechanisms: (1) binding of AMP or ADP to AMPK, causing a conformational change that promotes phosphorylation by upstream kinases (usually this will be LKB1, unless [Ca2+] is elevated); (2) binding of AMP or ADP, causing a conformational change that inhibits dephosphorylation by protein phosphatases; (3) binding of AMP (and not ADP), causing allosteric activation of AMPK-P. All three effects are antagonized by ATP, allowing AMPK to act as an energy sensor.

AMPK and AMPK-related kinase (ARK) family  1741-7007-11-36-4

 

Members of the AMPK and AMPK-related kinase (ARK) family. All the kinases named in the figure are phosphorylated and activated by LKB1, although what regulates this phosphorylation is known only for AMPK. Alternative names are shown, where applicable.

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

 

 

Three possible mechanisms to explain how the AMPK-activating drugs metformin or phenformin might provide protection against cancer. (a) Metformin acts on the liver and other insulin target tissues by activating AMPK (and probably via other targets), normalizing blood glucose; this reduces insulin secretion from pancreatic β cells, reducing the growth-promoting effects of insulin (and high glucose) on tumor cells. Since metformin does not reduce glucose levels in normoglycemic individuals, this mechanism would only operate in insulin-resistant subjects. (b) Metformin or phenformin activates AMPK in pre-neoplastic cells, restraining their growth and proliferation and thus delaying the onset of tumorigenesis; this mechanism would only operate in cells where the LKB1-AMPK pathway was intact. (c) Metformin or phenformin inhibits mitochondrial ATP synthesis in tumor cells, promoting cell death. If the LKB1-AMPK pathway was down-regulated in the tumor cells, they would be more sensitive to cell death induced by the biguanides than surrounding normal cells.

Metformin and phenformin are biguanides that inhibit mitochondrial function and so deplete ATP by inhibiting its production . AMPK is activated by any metabolic stress that depletes ATP, either by inhibiting its production (as do hypoxia, glucose deprivation, and treatment with biguanides) or by accelerating its consumption (as does muscle contraction). By switching off anabolism and other ATP-consuming processes and switching on alternative ATP-producing catabolic pathways, AMPK acts to restore cellular energy homeostasis.

Findings that AMPK is activated in skeletal muscle during exercise and that it increases muscle glucose uptake and fatty acid oxidation led to the suggestion that AMPK-activating drugs might be useful for treating type 2 diabetes. Indeed, it turned out that AMPK is activated by metformin, a drug that had at that time been used to treat type 2 diabetes for over 40 years, and by phenformin , a closely related drug that had been withdrawn for treatment of diabetes due to side effects of lactic acidosis.

If only it were so simple. Effects of metformin on cancer in type 2 diabetics could be secondary to reduction in insulin levels, and although there is evidence for direct effects of AMPK activation on the development of tumors in mice, there is also recent evidence that tumors that become established without down-regulating LKB1 survive metformin better than those that have lost it – probably because metformin poisons the mitochondrial respiratory chain, depressing ATP levels, and cells in which AMPK can still be activated in response to the challenge do better than those in which it can’t.

In their review, Hardie and Alessi chart these  twists and turns, and point to the explosion of further possibilities opened up by the discovery, since their 2003 publication, of at least one other class of kinase upstream of AMPK (the CaM kinases), and at least a dozen other downstream targets of LKB1 (AMPK-related kinases, or ARKs) – not to mention the innumerable downstream targets of AMPK; all which make half their schematic illustrations look like hedgehogs.

Analysis of respiration  in human cancer

Bioenergetic profiling of cancer cells is of great potential because it can bring forward new and effective

Therapeutic  strategies along with early diagnosis. Metabolic Control Analysis (MCA) is a methodology that enables quantification of the flux control exerted by different enzymatic steps in a metabolic network thus assessing their contribution to the system‘s function.

(T Kaambre,V Chekulayev, I Shevchuk, et al. Metabolic control analysis of respiration  in human cancer tissue.  Frontiers Physiol 2013 (4); 151:  1. http://dx.doi.org/10.3389/fphys.2013.00151)

Our main goal is to demonstrate the applicability of MCA for in situ studies of energy

Metabolism in human breast and colorectal cancer cells as well as in normal tissues .We seek to determine the metabolic conditions leading to energy flux redirection in cancer cells. A main result obtained is that the adenine nucleotide translocator exhibits the highest control of respiration in human breast cancer thus becoming a prospective therapeutic target. Additionally, we present evidence suggesting the existence of mitochondrial respiratory supercomplexes that may represent a way by which cancer cells avoid apoptosis. The data obtained show that MCA applied in situ can be insightful in cancer cell energetic research.

Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001

Metabolic control analysis of respiration in human cancer tissue.

Representative traces of change in the rate of oxygen consumption by permeabilized human colorectal cancer (HCC) fibers after their titration with increasing concentrations of mersalyl, an inhibitor of inorganic phosphate carrier (panel A). The values of respiration rate obtained were plotted vs. mersalyl concentration (panel B) and from the plot the corresponding flux control coefficient was calculated. Bars are ±SEM.

Oncologic diseases such as breast and colorectal cancers are still one of the main causes of premature death. The low efficiency of contemporary medicine in the treatment of these malignancies is largely mediated by a poor understanding of the processes involved in metastatic dissemination of cancer cells as well as the unique energetic properties of mitochondria from tumors. Current knowledge supports the idea that human breast and colorectal cancer cells exhibit increased rates of glucose consumption displaying Warburg phenotype,i.e.,elevated glycolysis even in the presence of oxygen (Warburg and Dickens, 1930; Warburg, 1956 ;Izuishietal., 2012). Notwithstanding,  there are some evidences that in these malignancies mitochondrial oxidative phosphorylation (OXPHOS) is the main source of ATP rather than glycolysis. Cancer cells have been classified according to their pattern of metabolic remodeling depending of the relative balance between aerobic glycolysis and OXPHOS (Bellanceetal.,2012). The first type of tumor cells is highly glycolytic, the second OXPHOS deficient and the third type of tumors dislay enhanced OXPHOS. Recent studies strongly sug gest  that cancer cells can utilize lactate, free fatty acids, ketone bodies, butyrate and glutamine as key respiratory substrate selic iting metabolic remodeling of normal surrounding cells toward aerobic glycolysis—“reverse Warburg”effect (Whitaker-Menezes et al.,2011;Salem et al.,2012;Sotgia et al.,2012;Witkiewicz et al., 2012).

In normal cells,the OXPHOS system is usually closely linked to phosphotransfer systems, including various creatine kinase(CK) isotypes,which ensure a safe operation of energetics over a broad functional range of cellular activities (Dzejaand Terzic,2003).  However, our current knowledge about the function of CK/creatine (Cr) system in human breast and colorectal cancer is insufficient. In some malignancies, for example sarcomas the CK/Cr system was shown to be strongly downregulated (Beraetal.,2008;Patraetal.,2008).  Our previous studies showed  that the mitochondrial-bound CK (MtCK) activity was significantly decreased in HL-1 tumor cells (Mongeetal.,2009), as compared to normal parent cardiac cells where the OXPHOS is the main ATP source of and the CK system is a main energy carrier. In the present study,we estimated the role of MtCK in maintaining energy homeostasis in human colorectal cancer cells. Understanding the control and regulation of energy metabolism requires analytical tools that take into account  the existing interactions between individual network components and their impact on systemic network function. Metabolic Control Analysis(MCA) is a theoretical framework relating the properties of metabolic systems to the kinetic characteristics of their individual enzymatic components (Fell,2005). An experimental approach of MCA has been already successfully applied to the studies of OXPHOS in isolated mitochondria (Tageretal.,1983; Kunzetal.,1999; Rossignoletal.,2000)  and in skinned muscle fibers (Kuznetsovetal.,1997;Teppetal.,2010).

Metabolic control analysis of respiration in human cancer tissue

Values of basal (Vo) and maximal respiration rate (Vmax, in the presence of 2 mM ADP) and apparent Michaelis Menten constant (Km) for ADP in permeabilized human breast and colorectal cancer samples as well as health tissue. – See more at: http://journal.frontiersin.org/Journal/10.3389/fphys.2013.00151/full#sthash.VBXPdodj.dpuf

Role of Uncoupling Proteins in Cancer

Adamo Valle, Jordi Oliver and Pilar Roca *
Cancers 2010; 2: 567-591;   http://dx.doi.org/10.3390/cancers2020567

Since Otto Warburg discovered that most cancer cells predominantly produce energy by glycolysis rather than by oxidative phosphorylation in mitochondria, much interest has been focused on the alterations of these organelles in cancer cells. Mitochondria have been shown to be key players in numerous cellular events tightly related with the biology of cancer. Although energy production relies on the glycolytic pathway in cancer cells, these organelles also participate in many other processes essential for cell survival and proliferation such as ROS production, apoptotic and necrotic cell death, modulation of oxygen concentration, calcium and iron homeostasis, and certain metabolic and biosynthetic pathways. Many of these mitochondrial-dependent processes are altered in cancer cells, leading to a phenotype characterized, among others, by higher oxidative stress, inhibition of apoptosis, enhanced cell proliferation, chemoresistance, induction of angiogenic genes and aggressive fatty acid oxidation. Uncoupling proteins, a family of inner mitochondrial membrane proteins specialized in energy-dissipation, has aroused enormous interest in cancer due to their relevant impact on such processes and their potential for the development of novel therapeutic strategies.

Uncoupling proteins (UCPs) are a family of inner mitochondrial membrane proteins whose function is to allow the re-entry of protons to the mitochondrial matrix, by dissipating the proton gradient and, subsequently, decreasing membrane potential and production of reactive oxygen species (ROS). Due to their pivotal role in the intersection between energy efficiency and oxidative stress UCPs are being investigated for a potential role in cancer. In this review we compile the latest evidence showing a link between uncoupling and the carcinogenic process, paying special attention to their involvement in cancer initiation, progression and drug chemoresistance.

The Warburg Effect

Uncoupling the Warburg effect from cancer

A Najafov and DR Alessi
Proc Nat Acad Sci                                      www.pnas.org/cgi/doi/10.1073/pnas.1014047107
A remarkable trademark of most tumors is their ability to break down glucose by glycolysis at a vastly higher rate than in normal tissues, even when oxygen is copious. This phenomenon, known as the Warburg effect, enables rapidly dividing tumor cells to generate essential biosynthetic building blocks such as nucleic acids, amino acids, and lipids from glycolytic intermediates to permit growth and duplication of cellular components during  division (1). An assumption dominating research in this area is that the Warburg effect is specific to cancer. Thus, much of the focus has been on uncovering mechanisms by which cancer-causing mutations influence metabolism to stimulate glycolysis.

This has lead to many exciting discoveries. For example, the p53 tumor suppressor can suppress glycolysis through its ability to control expression of key metabolic genes, such as phosphoglycerate mutase (2), synthesis of cytochrome C oxidase-2 (3), and TP53-induced glycolysis and apoptosis regulator (TIGAR) (4). Many cancer-causing mutations lead to activation of the Akt and mammalian target of rapamycin (mTOR) pathway that profoundly influences metabolism and expression of metabolic enzymes to promoteglycolysis (5).

Strikingly, all cancer cells but not nontransformed cells express a specific splice variant of pyruvate kinase, termed M2-PK, that is less active, leading to the build up of phosphoenolpyruvate (6). Recent work has revealed that reduced activity of M2-PK promotes a unique glycolytic pathway in which phosphoenolpyruvate is converted to pyruvate by a histidine-dependent phosphorylation of phosphoglycerate mutase, promoting assimilation of glycolytic products into biomass (7). However, despite these observations, one might imagine that the Warburg effect need not be specific for cancer and that any normal cell would need to stimulate glycolysis to generate sufficient biosynthetic materials to fuel expansion and division.

Recent work by Salvador Moncada’s group published in PNAS (8) and other recent work from the same group (9, 10) provides exciting evidence supporting the idea that the Warburg effect is also required for the proliferation of noncancer cells.

The key discovery was that the anaphase promoting complex/cyclosome-Cdh1(APC/C-Cdh1), a master regulator of the transition of G1 to S phase of the cell cycle, inhibits glycolysis in proliferating noncancer cells by mediating the degradation of two key metabolic enzymes, namely 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase isoform3 (PFKFB3) (9, 10) and glutaminase-(Fig. 1) (8).

Fig. 1. Mechanism by which APC_C-Cdh1 inhibits glycolysis and glutaminolysis to suppress cell proliferation

 

Fig.  Mechanism by which APC/C-Cdh1 inhibits glycolysis and glutaminolysis to suppress cell proliferation.

APC/C-Cdh1 E3 ligase recognizes KEN-box–containing metabolic enzymes, such as PFKFB3 and glutaminase-1 (GLS1), and ubiquitinates and targets them for proteasomal degradation. This inhibits glycolysis and glutaminolysis, leading to decrease in metabolites that can be assimilated into biomass, thereby suppressing proliferation.

PFKFB3 potently stimulates glycolysis by catalyzing the formation of fructose-2,6-bisphosphate, the allosteric activatorof 6-phosphofructo-1-kinase (11). Glutaminase-1 is the first enzyme in glutaminolysis, converting glutamine to lactate, yielding biosyntheticintermediates required for cell proliferation (12).

APC/C is a cell cycle-regulated E3 ubiquitin ligase that promotes ubiquitination of a distinct set of cell cycle proteins containing either a D-box (destruction box) or a KEN-box, named after the essential Lys-Glu-Asn motif required for APC recognition (13). Among its well-known substrates are crucial cell cycle proteins, such as cyclin B1, securin, and Plk1. By ubiquitinating and targeting its substrates to 26S proteasome-mediated degradation, APC/C regulates processes in late mitotic stage, exit  from mitosis, and several events in G1 (14). The Cdh1 subunit is the KENbox binding adaptor of the APC/C ligase and is essential for G1/S transition.

Importantly, APC/C-Cdh1 is inactivated at the initiation of the S-phase of the cell cycle when DNA and cellular organelles are replicated at the time of the greatest need for generation of biosynthetic materials. APC/C-Cdh1 is reactivated later at the mitosis/G1 phase of the cell cycle when there is a lower requirement for biomassgeneration.

Both PFKFB3 (9, 10) and glutaminase-1 (8) possess a KEN-box and are rapidly degraded in nonneoplastic lymphocytes during the cell cycle when APC/C-Cdh1 is active. Consistent with destruction being mediated by APC-C-Cdh1, ablation of the KEN-box prevents degradation of PFKFB3 (9, 10) and glutaminase-1 (8). Inhibiting the proteasomal-dependent degradation with the MG132 inhibitor

markedly increases levels of ubiquitinated PFKFB3 and glutaminase-1 (8). Moreover, overexpression of Cdh1 to activate APC/C-Cdh1 decreases levels of PFKFB3 as well as glutmaninase-1 and concomitantly inhibited glycolysis, as judged by decrease in lactate production. This effect is also observed when cells were treated with a glutaminase-1 inhibitor (6-diazo-5- oxo-L-norleucine) (8). The final evidence supporting the authors’ hypothesis is that proliferation and glycolysis is inhibited after shRNA-mediated silencing of either PFKFB3 or glutaminase-1 (8).

These results are interesting, because unlike most recent work in this area, Colombo et al. (8) link the Warburg effect to the machinery of the cell cycle that is present in all cells rather than to cancer driving mutations. Further work is required to properly define the overall importance of this pathway, which has thus far only been studied in a limited number of cells. It would also be of value to undertake a more detailed analysis of how the rate of glycolysis and other metabolic pathways vary during the cell cycle of normal and cancer cells…(see full 2 page article) at PNAS.

 

The Warburg Effect Suppresses Oxidative Stress Induced Apoptosis in a Yeast Model for Cancer

C Ruckenstuhl, S Buttner, D Carmona-Gutierre, et al.
PLoS ONE 2009; 4(2): e4592.  http://dx.doi.org/10.1371/journal.pone.0004592

Colonies of Saccharomyces cerevisiae, suitable for manipulation of mitochondrial respiration and shows mitochondria-mediated cell death, were used as a model. Repression of respiration as well as ROS-scavenging via glutathione inhibited apoptosis, conferred a survival advantage during seeding and early development of this fast proliferating solid cell population. In contrast, enhancement of respiration triggered cell death.

Conclusion/Significance: The Warburg effect might directly contribute to the initiation of cancer formation – not only by enhanced glycolysis – but also via decreased respiration in the presence of oxygen, which suppresses apoptosis.

 

PIM2 phosphorylates PKM2 and promotes Glycolysis in Cancer Cells
Z Yu, L Huang, T Zhang, et al.
J Biol Chem 2013;                               http://dx.doi.org/10.1074/jbc.M113.508226

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.508226

Serine/threonine protein kinase PIM2, a known oncogene is a binding partner of pyruvate kinase M2 (PKM2), a key player in the Warburg effect of cancer cells.   PIM2 interacts with PKM2 and phosphorylates PKM2 on the Thr454 residue.

The phosphorylation of PKM2 increases glycolysis and proliferation in cancer cells.

The PIM2-dependent phosphoirylation of ZPKM2 is critical for regulating the Warburg effect in cancer.

 

Genome-Scale Metabolic Modeling Elucidates the Role of Proliferative Adaptation in Causing the Warburg Effect

Shlomi T, Benyamini T, Gottlieb E, Sharan R, Ruppin E
PLoS Comput Biol 2011; 7(3): e1002018.    http://dx.doi.org/10.1371/journal.pcbi.1002018
The Warburg effect – a classical hallmark of cancer metabolism – is a counter-intuitive phenomenon in which rapidly proliferating cancer cells resort to inefficient ATP production via glycolysis leading to lactate secretion, instead of relying primarily on more efficient energy production through mitochondrial oxidative phosphorylation, as most normal cells do.

The causes for the Warburg effect have remained a subject of considerable controversy since its discovery over 80 years ago, with several competing hypotheses. Here, utilizing a genome-scale human metabolic network model accounting for stoichiometric and enzyme solvent capacity considerations, we show that the Warburg effect is a direct consequence of the metabolic adaptation of cancer cells to increase biomass production rate. The analysis is shown to accurately capture a three phase metabolic behavior that is observed experimentally during oncogenic progression, as well as a prominent characteristic of cancer cells involving their preference for glutamine uptake over other amino acids.

 

The metabolic advantage of tumor cells

Maurice Israël and Laurent Schwartz

Additional article information

Abstract

1- Oncogenes express proteins of “Tyrosine kinase receptor pathways”, a receptor family including insulin or IGF-Growth Hormone receptors. Other oncogenes alter the PP2A phosphatase brake over these kinases.

2- Experiments on pancreatectomized animals; treated with pure insulin or total pancreatic extracts, showed that choline in the extract, preserved them from hepatomas.

Since choline is a methyle donor, and since methylation regulates PP2A, the choline protection may result from PP2A methylation, which then attenuates kinases.

3- Moreover, kinases activated by the boosted signaling pathway inactivate pyruvate kinase and pyruvate dehydrogenase. In addition, demethylated PP2A would no longer dephosphorylate these enzymes. A “bottleneck” between glycolysis and the oxidative-citrate cycle interrupts the glycolytic pyruvate supply now provided via proteolysis and alanine transamination. This pyruvate forms lactate (Warburg effect) and NAD+ for glycolysis. Lipolysis and fatty acids provide acetyl CoA; the citrate condensation increases, unusual oxaloacetate sources are available. ATP citrate lyase follows, supporting aberrant transaminations with glutaminolysis and tumor lipogenesis. Truncated urea cycles, increased polyamine synthesis, consume the methyl donor SAM favoring carcinogenesis.

4- The decrease of butyrate, a histone deacetylase inhibitor, elicits epigenic changes (PETEN, P53, IGFBP decrease; hexokinase, fetal-genes-M2, increase)

5- IGFBP stops binding the IGF – IGFR complex, it is perhaps no longer inherited by a single mitotic daughter cell; leading to two daughter cells with a mitotic capability.

6- An excess of IGF induces a decrease of the major histocompatibility complex MHC1, Natural killer lymphocytes should eliminate such cells that start the tumor, unless the fever prostaglandin PGE2 or inflammation, inhibit them…

Introduction

The metabolic network of biochemical pathways forms a system controlled by a few switches, changing the finality of this system. Specific substrates and hormones control such switches. If for example, glycemia is elevated, the pancreas releases insulin, activating anabolism and oxidative glycolysis, energy being required to form new substance or refill stores. If starvation decreases glycemia, glucagon and epinephrine activate gluconeogenesis and ketogenesis to form nutriments, mobilizing body stores. The different finalities of the system are or oriented by switches sensing the NADH/NAD+, the ATP/AMP, the cAMP/AMP ratios or the O2 supply… We will not describe here these metabolic finalities and their controls found in biochemistry books.

Many of the switches depend of the phosphorylation of key enzymes that are active or not. Evidently, there is some coordination closing or opening the different pathways. Take for example gluconeogenesis, the citrate condensation slows down, sparing OAA, which starts the gluconeogenic pathway. In parallel, one also has to close pyruvate kinase (PK); if not, phosphoenolpyruvate would give back pyruvate, interrupting the pathway. Hence, the properties of key enzymes acting like switches on the pathway specify the finality of the system. Our aim is to show that tumor cells invent a new specific finality, with mixed glycolysis and gluconeogenesis features. This very special metabolism gives to tumor cells a selective advantage over normal cells, helping the tumor to develop at the detriment of the rest of the body.

I Abnormal metabolism of tumors, a selective advantage

The initial observation of Warburg 1956 on tumor glycolysis with lactate production is still a crucial observation [1]. Two fundamental findings complete the metabolic picture: the discovery of the M2 pyruvate kinase (PK) typical of tumors [2] and the implication of tyrosine kinase signals and subsequent phosphorylations in the M2 PK blockade [35].

A typical feature of tumor cells is a glycolysis associated to an inhibition of apoptosis. Tumors over-express the high affinity hexokinase 2, which strongly interacts with the mitochondrial ANT-VDAC-PTP complex. In this position, close to the ATP/ADP exchanger (ANT), the hexokinase receives efficiently its ATP substrate [6,7]. As long as hexokinase occupies this mitochondria site, glycolysis is efficient. However, this has another consequence, hexokinase pushes away from the mitochondria site the permeability transition pore (PTP), which inhibits the release of cytochrome C, the apoptotic trigger [8]. The site also contains a voltage dependent anion channel (VDAC) and other proteins. The repulsion of PTP by hexokinase would reduce the pore size and the release of cytochrome C. Thus, the apoptosome-caspase proteolytic structure does not assemble in the cytoplasm. The liver hexokinase or glucokinase, is different it has less interaction with the site, has a lower affinity for glucose; because of this difference, glucose goes preferentially to the brain.

Further, phosphofructokinase gives fructose 1-6 bis phosphate; glycolysis is stimulated if an allosteric analogue, fructose 2-6 bis phosphate increases in response to a decrease of cAMP. The activation of insulin receptors in tumors has multiple effects, among them; a decrease of cAMP, which will stimulate glycolysis.

Another control point is glyceraldehyde P dehydrogenase that requires NAD+ in the glycolytic direction. If the oxygen supply is normal, the mitochondria malate/aspartate (MAL/ASP) shuttle forms the required NAD+ in the cytosol and NADH in the mitochondria. In hypoxic conditions, the NAD+ will essentially come via lactate dehydrogenase converting pyruvate into lactate. This reaction is prominent in tumor cells; it is the first discovery of Warburg on cancer.

At the last step of glycolysis, pyruvate kinase (PK) converts phosphoenolpyruvate (PEP) into pyruvate, which enters in the mitochondria as acetyl CoA, starting the citric acid cycle and oxidative metabolism. To explain the PK situation in tumors we must recall that PK only works in the glycolytic direction, from PEP to pyruvate, which implies that gluconeogenesis uses other enzymes for converting pyruvate into PEP. In starvation, when cells need glucose, one switches from glycolysis to gluconeogenesis and ketogenesis; PK and pyruvate dehydrogenase (PDH) are off, in a phosphorylated form, presumably following a cAMP-glucagon-adrenergic signal. In parallel, pyruvate carboxylase (Pcarb) becomes active. Moreover, in starvation, much alanine comes from muscle protein proteolysis, and is transaminated into pyruvate. Pyruvate carboxylase first converts pyruvate to OAA and then, PEP carboxykinase converts OAA to PEP etc…, until glucose. The inhibition of PK is necessary, if not one would go back to pyruvate. Phosphorylation of PK, and alanine, inhibit the enzyme.

Well, tumors have a PK and a PDH inhibited by phosphorylation and alanine, like for gluconeogenesis, in spite of an increased glycolysis! Moreover, in tumors, one finds a particular PK, the M2 embryonic enzyme [2,9,10] the dimeric, phosphorylated form is inactive, leading to a “bottleneck “. The M2 PK has to be activated by fructose 1-6 bis P its allosteric activator, whereas the M1 adult enzyme is a constitutive active form. The M2 PK bottleneck between glycolysis and the citric acid cycle is a typical feature of tumor cell glycolysis.

We also know that starvation mobilizes lipid stores from adipocyte to form ketone bodies, they are like glucose, nutriments for cells. Growth hormone, cAMP, AMP, activate a lipase, which provides fatty acids; their β oxidation cuts them into acetyl CoA in mitochondria and in peroxisomes for very long fatty acids; forming ketone bodies. Normally, citrate synthase slows down, to spare acetyl CoA for the ketogenic route, and OAA for the gluconeogenic pathway. Like for starvation, tumors mobilize lipid stores. But here, citrate synthase activity is elevated, condensing acetyl CoA and OAA [1113]; citrate increases, ketone bodies decrease. Consequently, ketone bodies will stop stimulating Pcarb. In tumors, the OAA needed for citrate synthase will presumably come from PEP, via reversible PEP carboxykinase or other sources. The quiescent Pcarb will not process the pyruvate produced by alanine transamination after proteolysis, leaving even more pyruvate to lactate dehydrogenase, increasing the lactate released by the tumor, and the NAD+ required for glycolysis.

Above the bottleneck, the massive entry of glucose accumulates PEP, which converts to OAA via mitochondria PEP carboxykinase, an enzyme requiring biotine-CO2-GDP. This source of OAA is abnormal, since Pcarb, another biotin-requiring enzyme, should have provided OAA. Tumors may indeed contain “morule inclusions” of biotin-enzyme [14] suggesting an inhibition of Pcarb, presumably a consequence of the maintained citrate synthase activity, and decrease of ketone bodies that normally stimulate Pcarb. The OAA coming via PEP carboxykinase and OAA coming from aspartate transamination or via malate dehydrogenase condenses with acetyl CoA, feeding the elevated tumoral citric acid condensation starting the Krebs cycle. Thus, tumors have to find large amounts of acetyl CoA for their condensation reaction; it comes essentially from lipolysis and β oxidation of fatty acids, and enters in the mitochondria via the carnitine transporter. This is the major source of acetyl CoA; since PDH that might have provided acetyl CoA remains in tumors, like PK, in the inactive phosphorylated form. The blockade of PDH [15] was recently reversed by inhibiting its kinase [16,17].

The key question is then to find out why NADH, a natural citrate synthase inhibitor did not switch off the enzyme in tumor cells. Probably, the synthesis of NADH by the dehydrogenases of the Krebs cycle and malate/aspartate shuttle, was too low, or the oxidation of NADH via the respiratory electron transport chain and mitochondrial complex1 (NADH dehydrogenase) was abnormally elevated. Another important point concerns PDH and α ketoglutarate dehydrogenase that are homologous enzymes, they might be regulated in a concerted way; when PDH is off, α ketoglutarate dehydrogenase might be also be slowed. Moreover, this could be associated to an upstream inhibition of aconinase by NO, or more probably to a blockade of isocitrate dehydrogenase, which favors in tumor cells, the citrate efflux from mitochondria, and the ATP citrate lyase route.

Normally, an increase of NADH inhibits the citrate condensation, favoring the ketogenic route associated to gluconeogenesis, which turns off glycolysis. Apparently, this regulation does not occur in tumors, since citrate synthase remains active. Moreover, in tumor cells, the α ketoglutarate not processed by
α ketoglutarate dehydrogenase converts to glutamate, via glutamate dehydrogenase, in this direction the reaction forms NAD+, backing up the LDH production. Other sources of glutamate are glutaminolysis, which increases in tumors [2].

The Figure Figure11 shows how tumors bypass the PK and PDH bottlenecks and evidently, the increase of glucose influx above the bottleneck, favors the supply of substrates to the pentose shunt, as pentose is needed for synthesizing ribonucleotides, RNA and DNA. The Figure Figure11 represents the stop below the citrate condensation. Hence, citrate quits the mitochondria to give via ATP citrate lyase, acetyl CoA and OAA in the cytosol of tumor cells. Acetyl CoA supports the synthesis of fatty acids and the formation of triglycerides. The other product of the ATP citrate lyase reaction, OAA, drives the transaminase cascade (ALAT and GOT transaminases) in a direction that consumes GLU and glutamine and converts in fine alanine into pyruvate and lactate plus NAD+. This consumes protein body stores that provide amino acids and much alanine (like in starvation).

The Figure Figure11 indicates that malate dehydrogenase is a source of NAD+ converting OAA into malate, which backs-up LDH. Part of the malate converts to pyruvate (malic enzyme) and processed by LDH. Moreover, malate enters in mitochondria via the shuttle and gives back OAA to feed the citrate condensation. Glutamine will also provide amino groups for the “de novo” synthesis of purine and pyrimidine bases particularly needed by tumor cells. The Figure Figure11 indicates that ASP shuttled out of the mitochondrial, joins the ASP formed by cytosolic transaminases, to feed the synthesis of pyrimidine bases via ASP transcarbamylase, a process also enhanced in tumor cells. In tumors, this silences the argininosuccinate synthetase step of the urea cycle [1820].

This blockade also limits the supply of fumarate to the Krebs cycle. The latter, utilizes the α ketoglutarate provided by the transaminase reaction, since α ketoglutarate coming via aconitase slows down. Indeed, NO and peroxynitrite increase in tumors and probably block aconitase. The Figure Figure11 indicates the cleavage of arginine into urea and ornithine. In tumors, the ornithine production increases, following the polyamine pathway. Ornithine is decarboxylated into putrescine by ornithine decarboxylase, then it captures the backbone of S adenosyl methionine (SAM) to form polyamines spermine then spermidine, the enzyme controlling the process is SAM decarboxylase. The other reaction product, 5-methlthioribose is then decomposed into methylthioribose and adenine, providing purine bases to the tumor. We shall analyze below the role of SAM in the carcinogenic mechanism, its destruction aggravates the process.

metabolic pathways 1476-4598-10-70-1
Cancer metabolism. Glycolysis is elevated in tumors, but a pyruvate kinase (PK) “bottleneck” interrupts phosphoenol pyruvate (PEP) to pyruvate conversion. Thus, alanine following muscle proteolysis transaminates to pyruvate, feeding lactate dehydrogenase,

In summary, it is like if the mechanism switching from gluconeogenesis to glycolysis was jammed in tumors, PK and PDH are at rest, like for gluconeogenesis, but citrate synthase is on. Thus, citric acid condensation pulls the glucose flux in the glycolytic direction, which needs NAD+; it will come from the pyruvate to lactate conversion by lactate dehydrogenase (LDH) no longer in competition with a quiescent Pcarb. Since the citrate condensation consumes acetyl CoA, ketone bodies do not form; while citrate will support the synthesis of triglycerides via ATP citrate lyase and fatty acid synthesis… The cytosolic OAA drives the transaminases in a direction consuming amino acid. The result of these metabolic changes is that tumors burn glucose while consuming muscle protein and lipid stores of the organism. In a normal physiological situation, one mobilizes stores for making glucose or ketone bodies, but not while burning glucose! Tumor cell metabolism gives them a selective advantage over normal cells. However, one may attack some vulnerable points.

Cancer metabolism. Glycolysis is elevated in tumors, but a pyruvate kinase (PK) “bottleneck” interrupts phosphoenol pyruvate (PEP) to pyruvate conversion. Thus, alanine following muscle proteolysis transaminates to pyruvate, feeding lactate dehydrogenase, converting pyruvate to lactate, (Warburg effect) and NAD+ required for glycolysis. Cytosolic malate dehydrogenase also provides NAD+ (in OAA to MAL direction). Malate moves through the shuttle giving back OAA in the mitochondria. Below the PK-bottleneck, pyruvate dehydrogenase (PDH) is phosphorylated (second bottleneck). However, citrate condensation increases: acetyl-CoA, will thus come from fatty acids β-oxydation and lipolysis, while OAA sources are via PEP carboxy kinase, and malate dehydrogenase, (pyruvate carboxylase is inactive). Citrate quits the mitochondria, (note interrupted Krebs cycle). In the cytosol, ATPcitrate lyase cleaves citrate into acetyl CoA and OAA. Acetyl CoA will make fatty acids-triglycerides. Above all, OAA pushes transaminases in a direction usually associated to gluconeogenesis! This consumes protein stores, providing alanine (ALA); like glutamine, it is essential for tumors. The transaminases output is aspartate (ASP) it joins with ASP from the shuttle and feeds ASP transcarbamylase, starting pyrimidine synthesis. ASP in not processed by argininosuccinate synthetase, which is blocked, interrupting the urea cycle. Arginine gives ornithine via arginase, ornithine is decarboxylated into putrescine by ornithine decarboxylase. Putrescine and SAM form polyamines (spermine spermidine) via SAM decarboxylase. The other product 5-methylthioadenosine provides adenine. Arginine deprivation should affect tumors. The SAM destruction impairs methylations, particularly of PP2A, removing the “signaling kinase brake”, PP2A also fails to dephosphorylate PK and PDH, forming the “bottlenecks”. (Black arrows = interrupted pathways).

 II Starters for cancer metabolic anomaly

1. Lessons from oncogenes

Following the discovery of Rous sarcoma virus transmitting cancer [21], we have to wait the work of Stehelin [22] to realize that this retrovirus only transmitted a gene captured from a previous host. When one finds that the transmitted gene encodes the Src tyrosine kinase, we are back again to the tyrosine kinase signals, similar to those activated by insulin or IGF, which control carbohydrate metabolism, anabolism and mitosis.

An up regulation of the gene product, now under viral control causes tumors. However, the captured viral oncogene (v-oncogene) derives from a normal host gene the proto-oncogene. The virus only perturbs the expression of a cellular gene the proto-oncogene. It may modify its expression, or its regulation, or transmit a mutated form of the proto-oncogene. Independently of any viral infection, a similar tumorigenic process takes place, if the proto-oncogene is translocated in another chromosome; and transcribed under the control of stronger promoters. In this case, the proto-oncogene becomes an oncogene of cellular origin (c-oncogene). The third mode for converting a prot-oncogene into an oncogene occurs if a retrovirus simply inserts its strong promoters in front of the proto-oncogene enhancing its expression.

It is impressive to find that retroviral oncogenes and cellular oncogenes disturb this major signaling pathway: the MAP kinases mitogenic pathways. At the ligand level we find tumors such Wilm’s kidney cancer, resulting from an increased expression of insulin like growth factor; we have also the erbB or V-int-2 oncogenes expressing respectively NGF and FGF growth factor receptors. The receptors for these ligands activate tyrosine kinase signals, similarly to insulin receptors. The Rous sarcoma virus transmits the src tyrosine kinase, which activates these signals, leading to a chicken leukemia. Similarly, in murine leukemia, a virus captures and retransmits the tyrosine kinase abl. Moreover, abl is also stimulated if translocated and expressed with the bcr gene of chromosome 22, as a fusion protein (Philadelphia chromosome). Further, ahead Ras exchanging protein for GTP/GDP, and then the Raf serine-threonine kinases proto-oncogenes are known targets for oncogenes. Finally, at the level of transcription factors activated by MAP kinases, one finds cjun, cfos or cmyc. An avian leucosis virus stimulates cmyc, by inserting its strong viral promoter. The retroviral attacks boost the mitogenic MAP kinases similarly to inflammatory cytokins, or to insulin signals, that control glucose transport and gycolysis.

In addition to the MAP kinase mitogenic pathway, tyrosine kinase receptors activate PI3 kinase pathways; PTEN phosphatase counteracts this effect, thus acting as a tumor suppressor. Recall that a DNA virus, the Epstein-Barr virus of infectious mononucleose, gives also the Burkitt lymphoma; the effect of the virus is to enhance PI3 kinase. Down stream, we find mTOR (the target of rapamycine, an immune-suppressor) mTOR, inhibits PP2A phosphatase, which is also a target for the simian SV40 and Polyoma viruses. Schematically, one may consider that the different steps of MAP kinase pathways are targets for retroviruses, while the different steps of PI3 kinase pathway are targets for DNA viruses. The viral-driven enhanced function of these pathways mimics the effects of their prolonged activation by their usual triggers, such as insulin or IGF; one then expects to find an associated increase of glycolysis. The insulin or IGF actions boost the cellular influx of glucose and glycolysis. However, if the signaling pathway gets out of control, the tyrosine kinase phosphorylations may lead to a parallel PK blockade [35] explaining the tumor bottleneck at the end of glycolysis. Since an activation of enyme kinases may indeed block essential enzymes (PK, PDH and others); in principle, the inactivation of phosphatases may also keep these enzymes in a phosphorylated form and lead to a similar bottleneck and we do know that oncogenes bind and affect PP2A phosphatase. In sum, a perturbed MAP kinase pathway, elicits metabolic features that would give to tumor cells their metabolic advantage.

2. The methylation hypothesis and the role of PP2A phosphatase

In a remarkable comment, Newberne [23] highlights interesting observations on the carcinogenicity of diethanolamine [24] showing that diethanolamine decreased choline derivatives and methyl donors in the liver, like does a choline deficient diet. Such conditions trigger tumors in mice, particularly in the B6C3F1 strain. Again, the historical perspective recalled by Newberne’s comment brings us back to insulin. Indeed, after the discovery of insulin in 1922, Banting and Best were able to keep alive for several months depancreatized dogs, treated with pure insulin. However, these dogs developed a fatty liver and died. Unlike pure insulin, the total pancreatic extract contained a substance that prevented fatty liver: a lipotropic substance identified later as being choline [25]. Like other lipotropes, (methionine, folate, B12) choline supports transmethylation reactions, of a variety of substrates, that would change their cellular fate, or action, after methylation. In the particular case concerned here, the removal of triglycerides from the liver, as very low-density lipoprotein particles (VLDL), requires the synthesis of lecithin, which might decrease if choline and S-adenosyl methionine (SAM) are missing. Hence, a choline deficient diet decreases the removal of triglycerides from the liver; a fatty liver and tumors may then form. In sum, we have seen that pathways exemplified by the insulin-tyrosine kinase signaling pathway, which control anabolic processes, mitosis, growth and cell death, are at each step targets for oncogenes; we now find that insulin may also provoke fatty liver and cancer, when choline is not associated to insulin.

We must now find how the lipotropic methyl donor controls the signaling pathway. We know that after the tyrosine kinase reaction, serine-threonine kinases take over along the signaling route. It is thus highly probable that serine-threonine phosphatases will counteract the kinases and limit the intensity of the insulin or insulin like signals. One of the phosphatases involved is PP2A, itself the target of DNA viral oncogenes (Polyoma or SV40 antigens react with PP2A subunits and cause tumors). We found a possible link between the PP2A phosphatase brake and choline in works on Alzheimer’s disease [26]. Indeed, the catalytic C subunit of PP2A is associated to a structural subunit A. When C receives a methyle, the dimer recruits a regulatory subunit B. The trimer then targets specific proteins that are dephosphorylated [27].

In Alzheimer’s disease, the poor methylation of PP2A is associated to an increase of homocysteine in the blood [26]. The result of the PP2A methylation failure is a hyperphosphorylation of Tau protein and the formation of tangles in the brain. Tau protein is involved in tubulin polymerization, controlling axonal flow but also the mitotic spindle. It is thus possible that choline, via SAM, methylates PP2A, which is targeted toward the serine-threonine kinases that are counteracted along the insulin-signaling pathway. The choline dependent methylation of PP2A is the brake, the “antidote”, which limits “the poison” resulting from an excess of insulin signaling. Moreover, it seems that choline deficiency is involved in the L to M2 transition of PK isoenzymes [28].

3. Cellular distribution of PP2A

In fact, the negative regulation of Ras/MAP kinase signals mediated by PP2A phosphatase seems to be complex. The serine-threonine phosphatase does more than simply counteracting kinases; it binds to the intermediate Shc protein on the signaling cascade, which is inhibited [29]. The targeting of PP2A towards proteins of the signaling pathway depends of the assembly of the different holoenzymes. The carboxyl methylation of C-terminal leucine 309 of the catalytic C unit, permits to a dimeric form made of C and a structural unit A, to recruit one of the many regulatory units B, giving a great diversity of possible enzymes and effects. The different methylated ABC trimers would then find specific targets. It is consequently essential to have more information on methyl transferases and methyl esterases that control the assembly or disassembly of PP2A trimeric forms.

A specific carboxyl methyltransferase for PP2A [30] was purified and shown to be essential for normal progression through mitosis [31]. In addition, a specific methylesterase that demethylates PP2A has been purified [32]. Is seems that the methyl esterase cancels the action of PP2A, on signaling kinases that increase in glioma [33]. Evidently, the cellular localization of the methyl transferase (LCMT-1) and the phosphatase methyl esterase (PME-1) are crucial for controlling PP2A methylation and targeting. Apparently, LCMT-1 mainly localizes to the cytoplasm and not in the nucleus, where PME-1 is present, and the latter harbors a nuclear localization signal [34]. From these observations, one may suggest that PP2A gets its methyles in the cytoplasm and regulates the tyrosine kinase-signaling pathway, attenuating its effects.

A methylation deficit should then decrease the methylation of PP2A and boost the mitotic insulin signals as discussed above for choline deficiency, steatosis and hepatoma. At the nucleus, where PME-1 is present, it will remove the methyl, from PP2A, favoring the formation of dimeric AC species that have different targets, presumably proteins involved in the cell cycle. It is interesting to quote here the structural mechanism associated to the demethylation of PP2A. The crystal structures of PME-1 alone or in complex with PP2A dimeric core was reported [35] PME-1 binds directly to the active site of PP2A and this rearranges the catalytic triad of PME-1 into an active conformation that should demethylate PP2A, but this also seems to evict a manganese required for the phosphatase activity. Hence, demethylation and inactivation would take place in parallel, blocking mitotic actions.

However, another player is here involved, the so-called PTPA protein, which is a PP2A phosphatase activator. Apparently, this activator is a new type of cis/trans of prolyl isomerase, acting on Pro190 of the catalytic C unit isomerized in presence of Mg-ATP [36], which would then cancel the inactivation mediated by PME-1. Following the PTPA action, the demethylated phosphatase would become active again in the nucleus, and stimulate cell cycle proteins [37,38] inducing mitosis. Unfortunately, the ligand of this new prolyl isomerase is still unknown. Moreover, we have to consider that other enzymes such as cytochrome P450 have also demethylation properties.

In spite of deficient methylations and choline dehydrogenase pathway, tumor cells display an enhanced choline kinase activity, associated to a parallel synthesis of lecithin and triglycerides.

The hypothesis to consider is that triglycerides change the fate of methylated PP2A, by targeting it to the nucleus, there a methylesterase demethylates it; the phosphatase attacks new targets such as cell cycle proteins, inducing mitosis. Moreover, the phosphatase action on nuclear membrane proteins may render the nuclear membrane permeable to SAM the general methyl donor; promoters get methylated inducing epigenetic changes.

The relative decrease of methylated PP2A in the cytosol, not only cancels the brake over the signaling kinases, but also favors the inactivation of PK and PDH, which remain phosphorylated, contributing to the metabolic anomaly of tumor cells.

In order to prevent tumors, one should then favor the methylation route rather than the phosphorylation route for choline metabolism. This would decrease triglycerides, promote the methylation of PP2A and keep it in the cytosol, reestablishing the brake over signaling kinases.

Hypoxia is an essential issue to discuss

Many adequate “adult proteins” replace their fetal isoform: muscle proteins utrophine, switches to dystrophine; enzymes such as embryonic M2 PK [39] is replaced by M1. Hypoxic conditions seem to trigger back the expression of the fetal gene packet via HIF1-Von-Hippel signals. The mechanism would depend of a double switch since not all fetal genes become active after hypoxia. First, the histones have to be in an acetylated form, opening the way to transcription factors, this depends either of histone deacetylase (HDAC) inhibition or of histone acetyltransferase (HAT) activation, and represents the main switch. Second, a more specific switch must be open, indicating the adult/fetal gene couple concerned, or more generally the isoform of a given gene that is more adapted to the specific situation. When the adult gene mutates, an unbound ligand may indeed indicate, directly or indirectly, the particular fetal copy gene to reactivate [40]. In anoxia, lactate is more difficult to release against its external gradient, leading to a cytosolic increase of up-stream glycolytic products, 3P glycerate or others. These products may then be a second signal controlling the specific switch for triggering the expression of fetal genes, such as fetal hemoglobin or the embryonic M2 PK; this takes place if histones (main switch) are in an acetylated form.

Growth hormone-IGF actions, the control of asymmetrical mitosis

When IGF – Growth hormone operate, the fatty acid source of acetyl CoA takes over. Indeed, GH stimulates a triglyceride lipase in adipocytes, increasing the release of fatty acids and their β oxidation. In parallel, GH would close the glycolytic source of acetyl CoA, perhaps inhibiting the hexokinase interaction with the mitochondrial ANT site. This effect, which renders apoptosis possible, does not occur in tumor cells. GH mobilizes the fatty acid source of acetyl CoA from adipocytes, which should help the formation of ketone bodies, but since citrate synthase activity is elevated in tumors, ketone bodies do not form.

Compounds for correcting tumor metabolism

The figure figure1 indicates interrupted and enhanced metabolic pathways in tumor cells.

In table table1,1, the numbered pathways represent possible therapeutic targets; they cover several enzymes. When the activity of the pathway is increased, one may give inhibitors; when the activity of the pathway decreases, we propose possible activators

Table - metabolic  targets

Table 1 Mol Cancer. 2011; 10 70. Published online Jun 7, 2011. doi  10.1186_1476-4598-10-70

The origin of Cancers by means of metabolic selection

The disruption of cells by internal or external compounds, releases substrates stimulating the tyrosine kinase signals for anabolism proliferation and stem cell repair, like for most oncogenes. If such signals are not limited, there is a parallel blockade of key metabolic enzymes by activated kinases or inhibited phosphatases. The result is a metabolism typical of tumor cells, which gives them a selective advantage; stabilized by epigenetic changes. A proliferation process, in which the two daughter cells divide, increases the tumor mass at the detriment of the body. Inevitable mutations follow.

Maurice Israël, et al. Mol Cancer. 2011;10:70-70.
Transcriptomics and Regulatory Processes

What are lncRNAs?

It was traditionally thought that the transcriptome would be mostly comprised of mRNAs, however advances in high-throughput RNA sequencing technologies have revealed the complexity of our genome. Non-coding RNA is now known to make up the majority of transcribed RNAs and in addition to those that carry out well-known housekeeping functions (e.g. tRNA, rRNA etc), many different types of regulatory RNAs have been and continue to be discovered.

Long noncoding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins. Their expression is developmentally regulated and lncRNAs can be tissue- and cell-type specific. A significant proportion of lncRNAs are located exclusively in the nucleus. They are comprised of many types of transcripts that can structurally resemble mRNAs, and are sometimes transcribed as whole or partial antisense transcripts to coding genes. LncRNAs are thought to carry out important regulatory functions, adding yet another layer of complexity to our understanding of genomic regulation.

lncRNA-s   A summary of the various functions described for lncRNA

 

The evolution of genome-scale models of cancer metabolism
The importance of metabolism in cancer is becoming increasingly apparent with the identification of metabolic enzyme mutations and the growing awareness of the influence of metabolism on signaling, epigenetic markers, and transcription. However, the complexity of these processes has challenged our ability to make sense of the metabolic changes in cancer. Fortunately, constraint-based modeling, a systems biology approach, now enables one to study the entirety of cancer metabolism and simulate basic phenotypes. With the newness of this field, there has been a rapid evolution of both the scope of these models and their applications. (NE Lewis and AM.Abdel-Haleem. frontiers physiol  2013;4(237): 1   http://dx.doi.org/10.3389/fphys.2013.00237)

Here we review the various constraint-based models built for cancer metabolism and how their predictions are shedding new light on basic cancer phenotypes, elucidating pathway differences between tumors, and discovering putative anti-cancer targets. As the field continues to evolve, the scope of these genome-scale cancer models must expand beyond central metabolism to address questions related to the diverse processes contributing to tumor development and metastasis.

“One of the goals of cancer research is to ascertain the mechanisms of cancer.”These words, penned by Dulbecco (1986), began a treatise on how a mechanistic understanding of cancer requires a sequenced human genome. Now with the abundance of sequence data, we are finding diverse genetic changes among different cancers (Vogelstein et al.,2013). While we are cataloging these mutations, the associated mechanisms leading to phenotypic changes are often unclear since mutations occur in the context of complex biological networks. For example, mutations to isocitrate dehydrogenase lead to oncometabolite synthesis, which alters DNA methylation and ultimately changes gene expression and the balance of normal cell processes (Sasakietal.,2012). Furthermore, many different combinations of mutations can lead to cancer. Since the genetic heterogeneity between tumors can be large, the biomolecular mechanisms underlying tumor physiology can vary substantially.

This is apparent in metabolism, where tumors can differ in serine metabolism  dependence (Possematoetal., 2011) or TCA cycle function (Frezzaetal., 2011b). In addition, diverse mutations can alter NADPH synthesis by differentially regulat ing  signaling pathways, such as the AMPK pathway (Cairnsetal., 2011; Jeonetal., 2012). The challenges regarding complexity and heterogeneity in cancer metabolism are beginning to be addressed with the COnstraint-Based Reconstruction and Analysis (COBRA) approach (Hernández Patiñoetal., 2012; Sharma and König,  2013), an emerging field in systems biology.Specifically, it accounts for the complexity of the perturbed biochemical processes by using genome-scale metabolic network reconstructions (Duarteetal., 2007; Maetal., 2007;Thieleetal., 2013).

In a reconstruction, the stoichiometric chemical reactions in a cell are carefully annotated and stitched together into a large network, often containing thousands of reactions. Genes and enzymes associated with each reaction are also delineated. The networks are converted into computational models and analyzed using many algorithms (Lewisetal., 2012). COBRA approaches are also beginning to address heterogeneity in cancer by integrating experimental data with the reconstructions (Blazier and Papin, 2012; Hydukeetal., 2013)  to tailor the models to the unique gene expression profiles of general cancer tissue, and even individual cell lines and tumors. Here we describe the recent conceptual evolution that has occurred for constraint-based cancer modeling.

Targeting of  gene expression

Tumor Suppressor Genes and its Implications in Human Cancer

Gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes (TSG) lead to cancer. In most human cancers, these mutations occur in somatic tissues. However, hereditary forms of cancer exist for which individuals are heterozygous for a germline mutation in a TSG locus at birth. The second allele is frequently inactivated by gene deletion, point mutation, or promoter methylation in classical TSGs that meet Knudson’s two-hit hypothesis. Conversely, the second allele remains as wild-type, even in tumors in which the gene is haplo-insufficient for tumor suppression. (K Inoue, EA Fry and Pj Taneja. Recent Progress in Mouse Models for Tumor Suppressor Genes and its Implications in Human Cancer. Clinical Medicine Insights: Oncology2013:7 103–122). This article highlights the importance of PTEN, APC, and other tumor suppressors for counteracting aberrant PI3K, β-catenin, and other oncogenic signaling pathways. We discuss the use of gene-engineered mouse models (GEMM) of human cancer focusing on Pten and Apc knockout mice that recapitulate key genetic events involved in initiation and progression of human neoplasia.

Targeting cancer metabolism – aiming at a tumour’s sweet-spot
Neil P. Jones and Almut Schulze
Drug Discovery Today   January 2012

Targeting cancer metabolism has emerged as a hot topic for drug discovery. Most cancers have a high demand for metabolic inputs (i.e. glucose/glutamine), which aid proliferation and survival. Interest in targeting cancer metabolism has been renewed in recent years with the discovery that many cancer related (e.g. oncogenic and tumor suppressor) pathways have a profound effect on metabolism and that many tumors become dependent on specific metabolic processes. Considering the recent increase in our understanding of cancer metabolism and the increasing knowledge of the enzymes and pathways involved, the question arises: could metabolism be cancer’s Achilles heel?
During recent years, interest into the possible therapeutic benefit of targeting metabolic pathways in cancer has increased dramatically with academic and pharmaceutical groups actively pursuing this aspect of tumor physiology. Therefore, what has fuelled this revived interest in targeting cancer metabolism and what are the major advances and potential challenges faced in the race to develop new therapeutics in this area? This review will attempt to answer these questions and illustrate why we, and others, believe that targeting metabolism in cancer presents such a promising therapeutic rationale.

Oncogenes and cancer metabolism
Glycolysis  TCA cycle  Pentose phosphate pathway

 FIGURE 1

Schematic representation of the regulation of cancer metabolism pathways. Metabolic enzymes are regulated by signaling pathways involving oncogenes and tumor suppressors. Complex regulatory mechanisms, key pathway interactions and enzymes are shown along with key metabolic endpoints (shown in purple) necessary for proliferation and survival (biosynthetic intermediates and NADPH). Key oncogenic pathways are shown in green and key tumor suppressor pathways are shown in red. Mutant IDH (mIDH) pathway is listed but is only functional in cancers containing mIDH.

FIGURE 2

Schematic representation of key components of the pentose phosphate pathway (PPP). Key enzymes are shown in blue boxes and key intermediates in purple text/box outline. DNA damage can activate ATM which in turn activates G6PDH to upregulate nucleotide synthesis for DNA repair and NAPDH to combat reactive oxygen species. PPP is also regulated by the tumour suppressor p53. The PPP can function as two separate branches (oxidative and non-oxidative) or be coupled into a recycling pathway – the pentose phosphate shunt – for maximum NADPH production.

Serine biosynthesis

Another branch diverting from glycolysis recently implicated in cancer is the serine biosynthesis pathway which converts the glycolytic intermediate 3-phosphoglycerate into serine (Fig. 3). Serine is an amino acid and an important neurotransmitter but can also provide fuel for the synthesis of other amino acids and nucleotides. The serine biosynthesis pathway also provides another key metabolic intermediate, a-KG, from glutamate breakdown via the action of phosphoserine aminotransferase (PSAT1). This pathway couples glycolysis (via 3-phosphoglycerate) with glutaminolysis (via glutamate), thereby linking two metabolic pathways known to be activated in many cancers.

FIGURE 3

Schematic representation of the serine biosynthesis pathway. Synthesis of serine involves integration of metabolites from glycolysis and  glutaminolysis pathways  and generates a-ketoglutarate, a key biosynthetic intermediate, and serine. Serine has many essential uses in the cell including amino acid, phospholipid and nucleotide synthesis.

 

Silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1)

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressorgenes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely,demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action.  (Mayada Achour, et al. Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1.  Biochemical and Biophysical Research Communications 430 (2013) 208–212.    http://dx.doi.org/10.1016/j.bbrc.2012.11.087)

Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16INK4A  is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.

Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and –active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored.  (Citation: Richard M. Gallo, et al. (2013) Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant. J Cancer Res Therap Oncol 1: 1-10)

Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavage by gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

EGF Receptor

 Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction.

(CM Ardito,BM Gruner. ,EGF Receptor Is Required for KRAS-Induced Pancreatic Tumorigenesis.  http://dx.doi.org/10.1016/j.ccr.2012.07.024)

Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation

The almost universal lethality of PDA has led to the intense study of genetic mutations responsible for its formation and progression. The most common oncogenic mutations associated with all PDA stages are found in the KRAS gene, suggesting it as the primary initiator of pancreatic neoplasia. However, mutant Kras expression throughout the mouse pancreatic parenchyma shows that the oncogene remains largely indolent until secondary events, such as pancreatitis, unlock its transforming potential. We find KRAS requires an inside-outside-in signaling axis that involves ligand-dependent EGFR activation to initiate the signal transduction and cell biological changes that link PDA and pancreatitis. (Cancer Cell (2012); 22: 304–317).

HER4 (EGFR/ErbB, HER2/Neu, HER3)

 ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and –active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored.

ErbB4 Possesses Multiple Functional Motifs and Mutations Have Been Engineered to Target These Motifs.

The organization of ErbB4 is as indicated in this schematic. The extracellular ligand-binding motifs reside in the amino-terminal region upstream of amino acid residue 651. The singlepass transmembrane domain consists of amino acid residues 652-675. The cytoplasmic tyrosine kinase domain consists of amino acid residues 713-989. The majority of cytoplasmic sites of tyrosine phosphorylation reside in amino acid residues 990-1308, most notably Tyr1056. Additional putative functional motifs include a TACE cleavage site, a gamma-secretase cleavage site, two LXXLL (steroid hormone receptor binding) motifs, a BH3 domain, three WW domain binding motifs, and a PDZ domain binding motif. Mutations that disrupt these motifs are noted. Finally, note the two locations of alternative transcriptional splicing, resulting in a total of four different splicing isoforms.

 

 

 

Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavageby gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

(Richard M. Gallo, et al. (2013) Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant. J Cancer Res Therap Oncol 1: 1-10)

Resistance to Receptor Tyrosine Kinase Inhibition

Receptor tyrosine kinases (RTKs) are activated by somatic genetic alterations in a subset of cancers, and such cancers are often sensitive to specific inhibitors of the activated kinase. Two well-established examples of this paradigm include lung cancers with either EGFR mutations or ALK translocations. In these cancers, inhibition of the corresponding RTK leads to suppression of key downstream signaling pathways, such as the PI3K (phosphatidylinositol 3-kinase)/AKT and MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal–regulated kinase) pathways, resulting in cell growth arrest and death. Despite the initial clinical efficacy of ALK (anaplastic lymphoma kinase) and EGFR (epidermal growth factor receptor) inhibitors in these cancers, resistance invariably develops, typically within 1 to 2 years. (MJ Niederst and JA Engelman. Sci Signal, 24 Sep 2013; 6(294), p. re6 .  http://dx.doi.org/10.1126/scisignal.2004652)

Over the past several years, multiple molecular mechanisms of resistance have been identified, and some common themes have emerged. One is the development of resistance mutations in the drug target that prevent the drug from effectively inhibiting the respective RTK. A second is activation of alternative RTKs that maintain the signaling of key downstream pathways despite sustained inhibition of the original drug target. Indeed, several different RTKs have been implicated in promoting resistance to EGFR and ALK inhibitors in both laboratory studies and patient samples. In this mini-review, we summarize the concepts underlying RTK-mediated resistance, the specific examples known to date, and the challenges of applying this knowledge to develop improved therapeutic strategies to prevent or overcome resistance.

The TGF-β Pathway

Aberrations in the enzymes that modify ubiquitin moieties have been observed to cause a myriad of diseases, including cancer. Therefore a better understanding of these enzymes and their substrates will lead to the identification of prospective druggable targets. Here we discuss the role of ubiquitin modifying enzymes in the canonical TGF-β pathway highlighting the ubiquitin regulating enzymes, which may potentially be targeted by small molecule inhibitors. (Pieter Eichhorn. (DE) -Ubiquitination in The TGF-β Pathway. J Cancer Res Therap Oncol 2013; 1: 1-6).

TGF-β is a multifunctional cytokine that plays a key role in embryogenesis and adult tissue homoeostasis. TGF-β is secreted by a myriad of cell types triggering a varied array of cellular functions including apoptosis, proliferation, migration, endothelial and mesenchymal transition, and extracellular matrix production. Downstream TGFβ responses can also be modulated by other signalling pathways (i.e. PI3K, ERK, WNT, etc.) resulting in a complex web of TGF-β pathway activation or repression depending on the nature of the signal and cellular context. Apart from TGF-β mediated cell autonomous effects TGF-β can further play an important function in regulating tumour microenvironments effecting the interaction between stromal fibroblasts and tumour cells.
Due to the central role of TGF-β in cellular processes it is therefore unsurprising that loss of TGF-β pathway integrity is frequently observed in a variety of human diseases, including cancer. However, the TGF-β pathway plays a complex dual role in cancer. In normal epithelial cells and premalignant cells TGF-β acts a potent tumor suppressor eliciting a cytostatic response inhibiting tumor progression. Supporting this notion, inactivating mutations in members of the TGF-βpathway have been observed in a variety of cancers including pancreatic, colorectal, and head and neck cancer.

In contrast, during tumor progression the TGF-β antiproliferative function is lost, and in certain advanced cancers TGF-β becomes an oncogenic factor inducing cellular proliferation, invasion, angiogenesis, and immune suppression. As a consequence, the TGFβ pathway is currently considered a therapeutic target in advanced cancers and several anti- TGF-β agents in clinical trials have shown promising results. However, due to the complex dichotomous role of TGF-β in oncogenesis a detailed understanding of TGF-β biology is required in order to design successful therapeutic strategies to identify patient populations that will benefit most from these compounds.

G protein receptor

 G protein-coupled receptors (GPCRs) modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers.

(D Leyva-Illades, S DeMorrow . Orphan G protein receptor GPR55 as an emerging target in cancer therapy and management.  Cancer Management and Research 2013:5 147–155)

Signal transduction is essential for maintaining cellular homeostasis and to coordinate the activity of cells in all organisms. Proteins localized in the cell membrane serve as the interface between the outside and inside of the cell. G protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and are encoded by at least 800 genes in the human genome. GPCRs are also known as seven-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors. GPCRs can detect an expansive array of extracellular signals or ligands that include photons, ions, odors, pheromones, hormones, and neurotransmitters. Nonsensory GPCRs (excluding light, odor, and taste receptors) have been classified into four families: class A rhodopsin-like, class B secretin-like, class C metabotropic glutamate/pheromone, and frizzled receptors. They have a peculiar structure that has been highly conserved over the course of evolution and are made up of an amino acid chain, the N-terminal of which is localized outside of the cellular membrane and the C-terminal in the cytoplasm. The amino acid chain spans the cellular membrane seven times and has three intracellular and three extracellular loops.

GPCRs are called that because they exert their actions by associating with a family of heterotrimeric proteins (made up of α, β, and γ subunits) that are capable of binding and hydrolyzing guanosine triphosphate (GTP).To date, 16 different α subunits, five β subunits, and 11 γ subunits have been described in mammalian tissues. When activated, these receptors undergo conformational changes that are mechanically transduced to the G proteins, which then initiate a cycle of activation and inactivationassociated with the binding and hydrolysis of GTP. Activated G proteins can then positively or negatively modulate ion channels (mainly potassium and calcium) or the second messenger generating enzymes (ie, adenylate cyclase and phospholipase C [PLC]) that allow the signal to be propagated to the interior of the cell to ultimately affect cell function.

 Matrix Metalloproteinases

Degradation of extracellular matrix is crucial for malignant tumour growth, invasion, metastasis and angiogenesis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all  components of the ECM. Elevated levels of distinct MMPs can be detected in tumour tissue or serumof patients with advanced cancer and their role as prognostic indicators in cancer is studied. In addition, therapeutic intervention of tumour growth and invasion based on inhibition of MMP activity is under intensive investigation and several MMP inhibitors are in clinical trials in cancer. In this review, we discuss the current view on the feasibility of MMPs as prognostic markers and as targets for therapeutic intervention in cancer.

(MATRIX METALLOPROTEINASES IN CANCER: PROGNOSTIC MARKERS AND THERAPEUTIC TARGETS.

Pia Vihinen and Veli-Matti Kahari.  Int. J. Cancer 2002;99: 157–166. http://dx.doi.org/10.1002/ijc.10329

Common properties of the MMPs include the requirement of zinc in their catalytic site for activity and their synthesis as inactive zymogens that generally need to be proteolytically cleaved to be active. Normally the MMPs are expressed only when and where needed for tissue remodeling accompanies various processes such as during embryonic development, wound healing, uterine and mammary involution, cartilage-to-bone transition during ossification, and trophoblast invasion into the endometrial stoma during placenta development. However, aberrant expression of various MMPs has been correlated with pathological conditions, such as periodontitis, rheumatoid arthritis, and tumor cell invasion and metastasis .

There are now over 20 members of the MMP family, and they can be subgrouped based on their structures. The minimal domain structure consists of a signal peptide, prodomain, and catalytic domain. The propeptide domain contains a conserved cysteine residue (the “cysteine switch”) that coordinates to the catalytic zinc to maintain inactivity. MMPs with only the minimal domain are referred to as matrilysins (MMP-7 and -26). The most common structures for secreted MMPs, including collagenases and stromelysins, have an additional hemopexin-like domain connected by a hinge region to the catalytic domain (MMP-1, -3, -8, -10, -12, -13, -19, and -20).

Terms: 1FN, fibronectin; 2M, 2-macroglobulin; 1PI, 1-proteinase inhibitor; COMP, cartilage oligomeric matrix protein; ND, not determined; TACE, TNF-converting enzyme; OP, osteopontin

FIGURE 1 – Structure of human matrix metalloproteinases

 

FIGURE 1 – Structure of human matrix metalloproteinases. The signal peptide directs the proenzyme for secretion. The propeptide contains a conserved sequence (PRCGxPD), in which the cysteine forms a covalent bond (cysteine switch), with the catalytic zinc (Zn2_) to maintain the latency of proMMPs. Catalytic domain contains the highly conserved zinc binding site (HExGHxxGxxHS) in which Zn2_is coordinated by 3 histidines. The proline-rich hinge region links the catalytic domain to the hemopexin domain, which determines the substrate specificity of specific MMPs. The hemopexin domain is absent in matrilysin (MMP-7) and matrilysin-2 (endometase, MMP-26). Gelatinases  A and B (MMP-2 and MMP-9, respectively) contain 3 repeats of the fibronectin-type II domain inserted in the catalytic domain. MT1-, MT2-, MT3- and MT5-MMP contain a transmembrane domain and MT4- and MT6-MMPs contain a glycosylphosphatidylinositol (GPI) anchor in the C-terminus of the molecule, which attach these MMPs to the cell surface. MT-MMPs, MMP-11, MMP-23 and MMP-28 contain a furin cleavage site (RxKR) between the propeptide and catalytic domain, making these proenzymes susceptible to activation by intracellular furin convertases. MMP-23 contains an N-terminal signal anchor, which anchors proMMP-23 to the Golgi complex and has a different C-terminal domain instead of hemopexin-like domain.

The physiologic expression of MMP-13 in vivo is limited to situations, such as fetal bone development and fetal wound repair, in which rapid remodeling of collagenous ECM is required. MMP-13 is expressed in pathologic conditions, such as arthritis, chronic dermal and intestinal ulcers, chronic periodontal inflammation and atherosclerotic plaques. The expression of MMP-13 is detected in vivo in invasive malignant tumours, breast carcinomas, squamous cell carcinomas (SCCs) of the head and neck and vulva, malignant melanomas, chondrosarcomas and urinary bladder carcinomas.

Table I. Human MMPS, their chromosomal localization, substrates, exogenous activators, and activating capacity1
Enzyme Chromosomal location Substrates Activated by Activator of
  • FN, fibronectin; 2M, 2-macroglobulin; 1PI, 1-proteinase inhibitor; COMP, cartilage oligomeric matrix protein; ND, not determined; TACE, TNF-converting enzyme; OP, osteopontin.

    …………..

Collagenases
 Collagenase-1 (MMP-1) 11q22.2-22.3 Collagen I, II, III, VII, VIII, X, aggregan, serpins, 2M MMP-3, -7, -10, plasmin kallikrein, chymase MMP-2
 Collagenase-2 (MMP-8) 11q22.2-22.3 Collagen I, II, III, aggregan, serpins, 2M MMP-3, -10, plasmin ND
 Collagenase-3 (MMP-13) 11q22.2-22.3 Collagen I, II, III, IV, IX, X, XIV, gelatin, FN, laminin, large tenascin aggrecan, fibrillin, osteonectin, serpins MMP-2, -3, -10, -14, -15, plasmin MMP-2, -9
Stromelysins
 Stromelysin-1 (MMP-3) 11q22.2-22.3 Collagen IV, V, IX, X, FN, elastin, gelatin, laminin, aggrecan, nidoge fibrillin*, osteonectin*, 1PI*, myelin basic protein*, OP, E-cadherin Plasmin, kallikrein, chymas tryptase MMP-1, -8, -9, -13
 Stromelysin-2 (MMP-10) 11q22.2-3 As MMP-3, except * Elastase, cathepsin G MMP-1, -7, -8, -9, -13
Stromelysin-like MMPs
 Stromelysin-3 (MMP-11) 22q11.2 Serine proteinase inhibitors, 1PI Furin ND
 Metalloelastase (MMP-12) 11q22.2-22.3 Collagen IV, gelatin, FN, laminin, vitronectin, elastin, fibrillin, 1-PI, myelin basic protein, apolipoprotein A ND ND
Matrilysins
 Matrilysin (MMP-7) 11q22.2-22.3 Elastin, FN, laminin, nidogen, collagen IV, tenascin, versican, 1PI, O E-cadherin, TNF- MMP-3, plasmin MMP-9
 Matrilysin-2 (MMP-26) 11q22.2 Gelatin, 1PI, synthetic MMP-substrates, TACE-substrate ND ND
Gelatinases
 Gelatinase A (MMP-2) 16q13 Gelatin, collagen I, IV, V, VII, X, FN, tenascin, fibrillin, osteonectin, Monocyte chemoattractant protein 3 MMP-1, -13, -14, -15, -16, -tryptase? MMP-9, -13
 Gelatinase B (MMP-9) 20q12-13 Gelatin, collagen IV, V, VII, XI, XIV, elastin, fibrillin, osteonectin 2 MMP-2, -3, 7, -13, plasmin, trypsin, chymotrypsin, cathepsin G ND
Membrane-type MMPs
 MT1-MMP (MMP-14) 14q12.2 Collagen I, II, III, gelatin, FN, laminin, vitronectin, aggrecan, tenasci nidogen, perlecan, fibrillin, 1PI, 2M, fibrin Plasmin, furin MMP-2, -13
 MT2-MMP (MMP-15) 16q12.2 FN, laminin, aggrecan, tenascin, nidogen, perlecan ND MMP-2, -13

 

MMP expression and activity are regulated at several levels. In most cases, MMPs are not synthesized until needed. Transcription can be induced by various signals including cytokines, growth factors, and mechanical stress. In certain cases, regulation of mRNA stability and translational efficiencyhave been reported. Because most MMPs are secreted as inactive zymogens, they need to be activated, usually by proteolytic cleavage of their NH2-terminal prodomains. Some MMPs are activated by other serine proteases such as plasmin and furin, whereas some of the MMPs can activate other members of their family. The most well characterized is the activation of pro-MMP-2 by MT1-MMP.

A number of MMPs have been strongly implicated in multiple stages of cancer progression including the acquisition of invasive and metastatic properties. Thus, efforts have been made for the past 20 years to develop MMPIs that can be used to halt the spread of cancer, which is what ultimately kills the person. However, initial clinical trials using first generation MMPIs proved to be disappointing . In the ensuing years, much has been learned about the roles of specific MMPs in the different processes of carcinogenesis and more specific MMPIs are being developed and brought to clinical trials.

However, the dosing and scheduling for optimal efficacy is not the same as required for conventional cytotoxic drugs because the MMPIs do not directly kill cancer cells, but instead target such processes as angiogenesis (the development of new blood vessels), invasion, and metastatic spread. (Matrix Metalloproteinases, Angiogenesis, and Cancer. Joyce E. Rundhaug.  Commentary re: A. C. Lockhart et al., Reduction of Wound Angiogenesis in Patients Treated with BMS-275291, a Broad Spectrum Matrix Metalloproteinase Inhibitor. Clin. Cancer Res., 2003; 9551–554).

 Role of p38 MAP Kinase Signal Transduction in Solid Tumors

HK Koul, M Pal, and S Koul. Genes & Cancer  2013 ; 4(9-10) 342–359.  http://dx.doi.org/10.1177/ 1947601913507951

Mitogen-activated protein kinases (MAPKs) mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the main subgroups, the p38 MAP kinases, has been implicated in a wide range of complex biologic processes, such as cell proliferation, cell differentiation, cell death, cell migration, and invasion. Dysregulation of p38 MAPK levels in patients are associated with advanced stages and short survival in cancer patients (e.g., prostate, breast, bladder, liver, and lung cancer). p38 MAPK plays a dual role as a regulator of cell death, and it can either mediate cell survival or cell death depending not only on the type of stimulus but also in a cell type specific manner. In addition to modulating cell survival, an essential role of p38 MAPK in modulation of cell migration and invasion offers a distinct opportunity to target this pathway with respect to tumor metastasis. The specific function of p38 MAPK appears to depend not only on the cell type but also on the stimuli and/or the isoform that is activated.

Mitogen-activated protein kinase (MAPK) signal transduction pathways are evolutionarily conserved among eukaryotes and have been implicated to play key roles in a number of biological processes, including cell growth, differentiation, apoptosis, inflammation, and responses to environmental stresses.

They are typically organized in 3-tiered architecture consisting of a MAPK, a MAPK activator (MAPK kinase), and a MAPKK activator (MAPKK kinase). The MAPK pathways can be regulated at multiple levels as well as via multiple mechanisms, of which the regulation of mitogen-activated protein kinase kinase kinase (MAPKKK/MAP3K) has been proved to be the most challenging due to the great diversity and versatility between different modules at this level. The complex array of growth factors and other ligands that can initiate intracellular cell signaling requires a very high level of coordination among the different proteins involved.

GTP cyclohydrolase (GCH1)

GTP cyclohydrolase (GCH1) is the key-enzyme to produce the essential enzyme cofactor, tetrahydrobiopterin. The byproduct, neopterin is increased in advanced human cancer and used as cancer-biomarker, suggesting that pathologically increased GCH1 activity may promote tumor growth.

(G Picker, Hee-Young Lim, et al. Inhibition of GTP cyclohydrolase attenuates tumor growth by reducing angiogenesis and M2-like polarization of tumor associated macrophages. Int. J. Cancer 2003; 132: 591–604 (2013)  http://dx.doi.org/10.1002/ijc.27706 )

We found that inhibition or silencing of GCH1 reduced tumor cell proliferation and survival and the tube formation of human umbilical vein endothelial cells, which upon hypoxia increased GCH1 and

endothelial NOS expression, the latter prevented by inhibition of GCH1. In nude mice xenografted with HT29-Luc colon cancer cells GCH1 inhibition reduced tumor growth and angiogenesis, determined by in vivo luciferase and near-infrared imaging of newly formed blood vessels. The treatment with the GCH1 inhibitor shifted the phenotype of tumor associated macrophages from the proangiogenic M2 towards M1, accompanied with a shift of plasma chemokine profiles towards tumor-attacking chemokines including CXCL10 and RANTES. GCH1 expression was increased in mouse AOM/DSS-induced colon tumors and in high grade human colon and skin cancer and oppositely, the growth of GCH1-deficient HT29-Luc tumor cells in mice was strongly reduced. The data suggest that GCH1 inhibition reduces tumor growth by (i) direct killing of tumor cells, (ii) by inhibiting angiogenesis, and (iii) by enhancing the antitumoral immune response.

The Role of Stroma in Tumour-Host Co-Existence

Molnár et al.,  The Role of Stroma in Tumour-Host Co-Existence: Some Perspectives in Stroma-Targeted Therapy of Cancer   Biochem Pharmacol 2013, 2:1    http://dx.doi.org/10.4172/2167-0501.1000107

 Cancer grows at the expense of the host as a parasite or superparasite following the second law of thermodynamics (conservation of energy). When the cancer cell progresses via replication to the special state called “spheroid”, a new phase begins with its intimate interaction and development of responses from the stroma which together assist in the formation of a full blown cancer. Among the processes involved are the development of blood vessels and lymphatic channels which are essential for maintenance and further growth of the cancer mass. In this way the condition of “parasitism” is completed with simultaneous suppression of the immune response of the host to the histo-incompatability of the tumor mass. Stroma/parenchyma promotes cancer invasion by feeding cancer cells and inducing immune tolerance. The dynamic changes in composition of stroma and biological consequences as feeder of cancer cells and immune tolerance can give a perspective for rational drug design in anti-stromal therapy. There are differences between normal and cancer cells at subcellular level such as compartmentalzation and structure of cytoskeleton and energy distribution (that is low generally, but locally high in normal cells). In cancer cannibalism of normal cells, the growing cancer mass is a factor for progression and invasion.

Cancer cells have been shown to kill normal cells and the products of cell death used for progression of growth of the cancer cell. Serum and growth factors produced by tumor stroma also provide the needed nutrients and conditions for further tumor growth. Cancer cannot feed off other cancer cells and therefore grow poorly. Probably, although not yet proven, the inability of cancer to “parasitise” other cancer cell types is probably due to some kind of competition or interference. The tumor is in charge of its own development due to its induction proteinases, lipid mobilization factors and angiogenetic factors as well as its ability to negate immune responses of the host response to what is in essence a foreign body.

In our review co-existence of normal and cancer cells in tumor with the growth promoting factors, and the immune tolerance mediating factors produced in the stromal and cancer cells/tissues will be discussed with perspective of stroma targeted therapy.

The clinical significance of cell cannibalism is well defined and described in a large number of publications. The direction of process of cancer development is defined as the tumor invades the normal tissue which never occurs in the reverse direction. This suggests that the cancer cell strives to achieve the lowest energy level possible. Therefore the first of the development of a full blown cancer can be considered as the 2nd Thermodynamic principle  that explains, describes and drives the invading cancer into normal surrounding tissue.

From the normal living state, under particular conditions such as hypoxia, where ATP synthesis is decreased resulting in a switch to glycolytic pathways, cancer cells are selected from a fraction of the population [4]. Energetically, in the presence of electron transfer, by using high energy from respiration, the proliferating state is more stable than resting cells where a higher degree of protein stabilization occurs such as that needed for maintainance of the cytoskeleton of the cell. It was proposed that tumor-promotion might be controlled or modulated by small electronic currents originating from reactive oxygen species and transported through the cytoskeletal microfilament network of the cancer cell.

Aerobic glycolysis is the main energy producing process in cancer cells. Among many other aspects, recently the mitochondria have also been regarded as potential targets in the therapy of cancer. Several small molecules have been tested to restore their dysfunctional functions either by direct or indirect effects. Because of poorly functioning mitochondria, the electron transfer component of the respiration cycle is inefficient; therefore, cancer cells have smaller Gibbs energy than healthy cells. This means, that these cancer cells exists in a metastable state and are not able maintain normal cell structure.

Therefore, the cytoskeleton system is collapsed and dielectric bilayers are formed as a lower grade of cellular structure with decreased electron conductivity. Consequently, to halt cancer growth, one has to evaluate the process of cancer cell development in situ, where the primary tumor is growing as well as that of the metastatic cell that is invading surrounding or distal tissues. This affords one to suggest that the stroma is formed first during long term repeated oxidative stress, a process that is initially accompanied with inflammation due to an active immune response to the histoincompatability antigens present on the surface of the cancer cell. If the cancer cell evades the activity of killer T cells (Treg cells) by either secreting agents that reduce the response of the Treg cells or the immune system for whatever reason is ineffective (immunosuppressed states such as HIV/AIDS, pregnancy, transplantation  therapy, etc.), the formed cancer cells have the opportunity to initiate tumor development. Because of the limited capacity of its electron transfer cycle, cancer cells are essentially starving cells that require glycolytically useful substrates. These substrates are obtained from the killing of normal cells by agents secreted by the cancer cell and the products yielded from dead normal cells “eaten” (phagocytosed) by the starving cancer cell which is digested by the cancer cells lysosomal system. This autophagic process of cannibalism keeps the cancer cell alive and thriving and is known as cytophagy, i.e., cannibalism of normal cells. This type of autophagocytosis  results in a parasitic co-existence of tumor cells with normal cells and will determine the main pathway of interaction between the growing cancer tissue (tumor) and normal tissue where the cancer tissue gradually destroys normal tissues. This process obeys the second law of thermodynamics-conservation of energy within a defined system.

Treatments for Cancer

 Bosutinib: a SRC–ABL tyrosine kinase inhibitor for treatment of chronic myeloid leukemia. 

FE Rassi, HJ Khoury. Pharmacogenomics and Personalized Medicine  2013:6 57–62.

Bosutinib is one of five tyrosine kinase inhibitors commercially available in the United States for the treatment of chronic myeloid leukemia. This review of bosutinib summarizes the mode of action, pharmacokinetics, efficacy and safety data, as well as the patient-focused perspective through quality-of-life data. Bosutinib has shown considerable and sustained efficacy in chronic myeloid leukemia, especially in the chronic phase, with resistance or intolerance to prior tyrosine kinase inhibitors. Bosutinib has distinct but manageable adverse events. In the absence of T315I and V299L mutations, there are no absolute contraindications for the use of bosutinib in this patient population

Chronic myeloid leukemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the presence of a signature hybrid oncogene, the BCR–ABL. The Philadelphia chromosome (Ph+) results from a reciprocal translocation between chromosome 9 and chromosome 22 that juxtaposes the two genes BCR and ABL and drives the leukemogenesis in CML. The ABL gene encodes for a nonreceptor tyrosine kinase that becomes deregulated and constitutively active after the juxtaposition of BCR. BCR–ABL is central in controlling downstream pathways involved in cell proliferation, regulation of cellular adhesion, and apoptosis.The understanding of the importance of this kinase activity in the pathophysiology of CML led to the development of tyrosine kinase inhibitors (TKI) that specifically target BCR–ABL. These agents became the mainstay of modern therapy in CML. CML has a triphasic clinical course, and the majority of patients (∼80%) are diagnosed during the early phase or the chronic phase (CP). However, and without effective treatment, CML invariably progresses to the advanced phases of the disease – the accelerated phase (AP) and the blast phase (BP). BP CML is a lethal refractory secondary leukemia with a short predicted survival.

Comprehensive molecular portraits of human breast tumors

 The Cancer Genome Atlas Network

Nature. 2012 October 4; 490(7418): 61–70. http://dx.doi.org/10.1038/nature11412.

We analyzed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. Our ability to integrate information across platforms provided key insights into previously-defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity.

Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at  > 10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the Luminal A subtype. We identified two novel protein expression-defined subgroups, possibly contributed by stromal/microenvironmental elements, and integrated analyses identified specific signaling pathways dominant in each molecular subtype including a HER2/p-HER2/HER1/p-HER1 signature within the HER2-Enriched expression subtype. Comparison of Basal-like breast tumors with high-grade Serous Ovarian tumors showed many molecular commonalities, suggesting a related etiology and similar therapeutic opportunities. The biologic finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biologic subtypes of breast cancer.

Most molecular studies of breast cancer have focused on just one or two high information content platforms, most frequently mRNA expression profiling or DNA copy number analysis, and more recently massively parallel sequencing. Supervised clustering of mRNA expression data has reproducibly established that breast cancers encompass several distinct disease entities, often referred to as the intrinsic subtypes of breast cancer. The recent development of additional high information content assays focused on abnormalities in DNA methylation, microRNA expression and protein expression, provide further opportunities to more completely characterize the molecular architecture of breast cancer.

Synbiology contribution and Nanotechnology

Synthetic RNAs Designed to Fight Cancer

Xiaowei Wang and his colleagues at  Washington University School of Medicine in St. Louis have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer.  They have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer.  RNA plays an important role in how genes are turned on and off in the body. Both siRNAs and microRNAs are snippets of RNA known to modulate a gene’s signal or shut it down entirely. Separately, siRNA and microRNA treatment strategies are in early clinical trials against cancer, but few groups have attempted to marry the two.

“We are trying to merge two largely separate fields of RNA research and harness the advantages of both,” said Xiaowei Wang, assistant professor of radiation oncology and a research member of the Siteman Cancer Center.  The study appears in the December issue of the journal RNA.

“We designed an artificial RNA that is a combination of siRNA and microRNA,” Wang said “our artificial RNA simultaneously inhibits both cell migration and proliferation.”  For therapeutic purposes, “small interfering” RNAs, or siRNAs, are designed and assembled in a lab and can be made to shut down– or interfere with– a single specific gene that drives cancer.  The siRNA molecules work extremely well at silencing a gene target because the siRNA sequence is made to perfectly complement the target sequence, thereby silencing a gene’s expression.

Though siRNAs are great at turning off the gene target, they also have potentially dangerous side effects: siRNAs inadvertently can shut down other genes that need to be expressed to carry out tasks that keep the body healthy.  The siRNAs interfere with off-target genesthat closely complement their “seed region,” a section of the siRNA  that governs binding to a gene target. “In the past, we tried to block the seed region in an attempt to reduce the side effects. Until now, we never tried to replace the seed region completely.”

Wang and his colleagues asked whether they could replace the siRNA’s seed region with the seed region from microRNA. Unlike siRNA, microRNA is a natural part of the body’s gene expression. And it can also shut down genes. As such, the microRNA seed region (with its natural targets) might reduce the toxic side effects caused by the artificial siRNA seed region. Plus, the microRNA seed region would add a new tool to shut down other genes that also may be driving cancer.

Wang’s group started with a bioinformatics approach, using a computer algorithm to design siRNA sequences against a common driver of cancer, a gene called AKT1 that encourages uncontrolled cell division. The program also selected siRNAs against AKT1 that had a seed region highly similar to the seed region of a microRNA known to inhibit a cell’s ability to move, thus potentially reducing the cancer’s ability to spread.

A Neutralizing RNA Aptamer

 Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity.

(CL Esposito, D Passaro, et al. A Neutralizing RNA Aptamer against EGFR Causes Selective Apoptotic Cell Death. PLoS ONE 6(9): e24071. http://dx.doi.org/10.1371/journal.pone.0024071)

Here we generated, by a cell-Systematic Evolution of  Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors.

In-Silico Molecular Docking Analysis of Cancer Biomarkers

Currently, in the research scenario for cancer, the identification of anti-cancer drugs using immuno-modulatory proteins and other molecular agents to initiate apoptosis in cancer cells and to inhibit the signaling pathways of cancer biomarkers as a drug targeted therapy, for cancer cell proliferation assays by the researchers. In-Silico analysis is used to recognize anticancer compounds as a future prospective for In-Vitro and In-Vivo analysis. A large number of herbal remedies (e.g. garlic, mistletoe) are used by cancer patients for treating the cancer and/or reducing the toxicities of chemotherapeutic drugs. Some herbal medicines have shown potentially beneficial effects on cancer progression and may ameliorate chemotherapy-induced toxicities.  (K. Gowri Shankar et al., In-Silico Molecular Docking Analysis of Cancer Biomarkers with Bioactive Compounds of Tribulus terrestris. Intl J NOVEL TRENDS PHARMAL SCI. 2013; 3(4).

Tribulus terrestris is mentioned in ancient Indian Ayurvedic medical texts dating back thousands of years. Tribulus terrestris has been widely used in the Ayurvedic system of medicine for the treatment of sexualdysfunction and various urinary disorders. The aim of the present study is to evaluate the interactions of some bioactive compounds of Tribulus terrestris for In-Silico anticancer analysis with cancer biomarkers as targets. The targeted biomarkers for analysis include NSE-Lung cancer, Follistatin-Prostrate cancer, GGT Hepatocellular carcinoma, Human Prostasin-Ovarian cancer.

GC-MS analysis of Tribulus terrestris whole plant methanol extract revealed the existence of the major compound like 3,7,11,15-tetramethylhexadec-2-en-1-ol, 1,2-Benzenedicarboxylic acid, disooctyl ester, 9,12,15-Octadecatrienoic acid, (z,z,z)-, 9,12-Octadecadienoic acid (z,z)-, Hexadecadienoic acid, ethyl ester, n-Hexadecadienoic acid, Octadecanoic acid, Phytol, α-Amyrin are chosen as ligands. Hence, by analyzing the minimum binding energy of the ligand binding complex with the receptors by dockinganalysis using AutoDock tools will show effective nature of inhibition of these receptors by the unique ligands. Based on the results low minimum binding energy ligands are identified and used as a future studies can be done for specific receptors  docking.

Anti-Cancerous Effect of4,4′-Dihydroxychalcone ((2E,2′E)-3,3′-(1,4-Phenylene) Bis (1-(4-hydroxyphenyl) Prop-2-en-1-one)) on T47D Breast Cancer Cell Line

Narges Mahmoodi, T Besharati-Seidani, N Motamed, and NO Mahmoodi*
Annual Research & Review in Biology 2014; 4(12): 2045-2052
SCIENCEDOMAIN international    www.sciencedomain.org

Aims: The majority of human breast tumors are estrogen receptor α (ERα) positive. However, not all of the ERα+ breast cancers respond to anti-estrogens drugs for those women who do respond, initial positive responses can be of short duration. Thus, more effective drugs are needed to enhance the efficacy of anti-estrogens drugs or to be used separately in a period of time. In view of potential cytotoxicity associated with silybin as polyhydroxy compounds a synthetic 4-hydroxychalcones (bis-phenol) was considered to explore its anti-carcinogenic effects in comparison to silybin on ERα+ breast cancer cell line.

Methodology: We have studied the inhibitory effect of 4,4′-dihydroxychalcone on the T47D breast cancer cell line by MTT test and the IC50s were estimated using Pharm PCS.

Results: The 4,4′-dihydroxychalcone showed significant dose- and time-dependent cell growth inhibitory effects on T47D breast cancer cells. The IC50 of 4,4′-dihydroxychalcone on T47D cells after 24 and 48 hours was 160.88+/1 μM, 62.20+/1 μM and for silybin was 373.42+/-1 μM,176.98+/1 μM respectively.

Conclusion: Our results strongly suggests that this premade synthetic 4,4′-dihydroxychalcone can promote anti carcinogenic actions on T47D cell line. All 4,4′-dihydroxychalcone doses had a much larger inhibitory effect on cell viability than silybin doses in T47D cells. The ratio of the IC50 of 4,4′-dihydroxychalcone to silybin after 24 and 48 hours was 1: 2.3 and 1: 2.8 respectively.

Anticancer and multidrug resistance-reversal effects of solanidine analogs synthetized from pregnadienolone acetate.

István Zupkó, Judit Molnár, Borbála Réthy, Renáta Minorics, Eva Frank, et al.
Molecules (Impact Factor: 2.43). 01/2014; 19(2):2061-76.  http://dx.doi.org/10.3390/molecules19022061
Source: PubMed

ABSTRACT A set of solanidine analogs  with antiproliferative properties were recently synthetized from pregnadienolone acetate, which occurs in Nature. The aim of the present study was an in vitro characterization of their antiproliferative action and an investigation of their multidrug resistance-reversal activity on cancer cells. Six of the compounds elicited the accumulation of a hypodiploid population of HeLa cells, indicating their apoptosis-inducing character, and another one caused cell cycle arrest at the G2/M phase. The most effective agents inhibited the activity of topoisomerase I, as evidenced by plasmid supercoil relaxation assays. One of the most potent analogs down-regulated the expression of cell-cycle related genes at the mRNA level, including tumor necrosis factor alpha and S-phase kinase-associated protein 2, and induced growth arrest and DNA damage protein 45 alpha. Some of the investigated compounds inhibited the ABCB1 transporter and caused rhodamine-123 accumulation in murine lymphoma cells transfected by human MDR1 gene, expressing the efflux pump (L5178). One of the most active agents in this aspect potentiated the antiproliferative action of doxorubicin without substantial intrinsic cytostatic capacity. The current results indicate that the modified solanidine skeleton is a suitable substrate for the rational design and synthesis of further innovative drug candidates with anticancer activities.

Nutrition and Cancer

 Ascorbic Acid and Selenium Interaction: Its Relevance in Carcinogenesis

 Michael J. Gonzalez
Journal of Orthomolecular Medicine 1990; 5(2)

Ascorbic acid and selenium are two nutrients that seem to have a preventive potential in the process of carcinogenesis; because of a possible synergistic action that may produce an enhanced anticarcinogenic effect. Interaction between these nutrients have been reported. Results indicate that the protective effect of the inorganic form of selenium (Na Selenite) was nullified by ascorbic acid, whereas the chemopreventive action of the organic form (seleno-DL-methionine) was not affected.

A possibility exists that Selenite is reduced by ascorbic acid to elemental selenium and is therefore not available for tissue uptake. In experiments using Selenite; plasma and erythrocyte glutathione peroxidase enzyme activity was directly related to the level of ascorbic acid fed.

Complementary RNA and Protein Profiling Identifies Iron as a Key Regulator of Mitochondrial Biogenesis

J W. Rensvold, Shao-En On, A Jeevananthan, et al.
Cell Rep. 2013 January 31; 3(1): .   http://dx.doi.org/10.1016/j.celrep.2012.11.029

Mitochondria are centers of metabolism and signaling whose content and function must adapt to
changing cellular environments. The biological signals that initiate mitochondrial restructuring
and the cellular processes that drive this adaptive response are largely obscure. To better define
these systems, we performed matched quantitative genomic and proteomic analyses of mouse
muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in
cellular iron homeostasis are highly coordinated with this process and that depletion of cellular
iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and
oxidative capacity. We further show that this process is universal across a broad range of cell
types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron
is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be
leveraged to explore posttranscriptional and posttranslational processes that are essential for
mitochondrial adaptation.

Avemar outshines new cancer ‘breakthrough’ drug

by Michael Traub
Townsend Letter / Oct, 2010

Many of us in the cancer research community were happy to hear about progress against metastatic melanoma reported this June at the annual meeting of the American Society of Clinical
Oncology (ASCO). since there has not been an improvement in overall survival from chemotherapy in over three decades.
Data from a phase III clinical trial of the experimental monoclonal antibody ipilimumab (pronounced “ep-eh-lim-uemab”) showed that patients with melanoma survived longer if they were taking ipilimumab than if they were not, regardless of whether they also were taking the other drug in the study, an experimental cancer vaccine. (1)

A Closer Look: How Big an Improvement, at What Cost to Patients?

Overall Survival: the ‘Gold Standard’ for Judging Cancer Therapies

Overall survival (OS) is the length of time that a patient actuallysurvives a cancer after treatment. It can also be measured as the percentage of patients surviving a specific time. It is the gold
standard by which the usefulness of a cancer treatment should be determined. Many things can help a patient, but the most important goal of doctors and patients is for the cancer patient to live longer, with a decent quality of life (QOL).

Among patients taking ipilimumab with or without the experimental vaccine, median overall survival was about 10 months. That is compared with 6.4 months’ overall survival among patients receiving the vaccine by itself. About 45.6% of patients taking ipilimumab survived one year, an improvement of some 7% over the 38% seen in some earlier studies. This very modest improvement in survival comes at quite a price.

Severe Side Effects in More Than One in Four Ipilimumab Patients Ipilimumab has some side effects that can be “both severe and long-lasting,” according to the study report. Among patients taking ipilimumab by itself (without the vaccine), 19.1% had side effects requiring hospitalization or invasive intervention, 3.8% died from the effects of the drug, and another 33.8% had life-threatening or disabling side effects. All totaled, 26.7% of the patients taking ipilimumab by itself– more than 1 in 4-had side effects that were severe, very severe, or fatal. Severe side effects included diarrhea, nausea, constipation, vomiting, abdominal pain, fatigue, cough, and headache. Vernon Sondak, MD, of the H. Lee Moffitt Cancer and Research Institute, said that “using the drug requires the medical team to be on guard to manage toxicity at all times.” But even with its severe side effects, the researchers said that the drug should be welcomed because it can increase median survival from 6.4 months to 10.1 months. That is because any lengthening of lives is welcome in a disease that hasn’t seen a new drug that can do that in many years.

Fermented Wheat Germ (Avemar) Improves Melanoma Survival Without Harsh Side Effects

But what if there already were such a treatment available-not a drug, but a safe, natural substance shown in clinical trials to have a remarkably similar ability to lengthen the lives of melanoma patients, without the severe side effects of the new drug?
What if the other substance had no significant side effects at all?
What if, instead of causing severe and sometimes fatal side effects, that other substance actually helped prevent and reduce serious side effects caused by chemotherapy and radiotherapy?
In fact, there is just such a treatment available. It is known as fermented wheat germ extract (FWGE) and by its trade name Avemar. It has been approved as a medical nutriment for cancer
patients in Europe for years and is available in the US as a dietary supplement. It has been compared to dacarbazine (DTIC), standard melanoma therapy, in a clinical trial with longer
follow-up than the ipilimumab trial. And with better results.

In 2008, data were published in the research journal Cancer Biotherapy and Radiopharmaceuticals from seven years’ follow-up on a trial at the N. N. Blokhin Cancer Center in Moscow,
Russia, involving 52 patients who had taken or not taken Avemar while taking dacarbazine for the year following surgical removal of their stage III melanoma tumors. (2) Patients who got only dacarbazine survived 44.7 months. Those who got Avemar along with their dacarbazine survived 66.2 months. This is an improvement in overall survival time of over 48%. In the Russian study,
just as it has in other studies, Avemar reduced side effects of the chemotherapy. Among those taking only dacarbazine, 11 % experienced severe (grade 3 or grade 4) side effects that required hospitalization or invasive intervention. None of the Avemar patients had grade 3 or 4 side effects. Since it is difficult to compare length of survival between the recent ipilimumab study and the Avemar melanoma study, because the ipilimumab study tested mostly stage 4 melanoma patients and the Avemar study tested mostly stage 3 melanoma patients, it is most instructive to look at
the percentage improvement in overall survival from adding either treatment to the regimen. Ipilimumab and Avemar both produced very similar improvements in OS (56% vs. 48%, respectively),

Avemar Ameliorates Conventional Treatment Side Effects

The improvement of survival and the amelioration of chemotherapy side effects by Avemar seen in the Russian melanoma study is typical of Avemar’s effects when used in treating other cancers, including in combination with chemotherapy or radiotherapy. Among 170 colorectal cancer patients in a 2003 study published in the British journal of Cancer, Avemar improved overall survival
and reduced metastasis and recurrences after surgery, chemotherapy, and radiotherapy. (3) Taking Avemar for six months during and after those conventional treatments resulted in a 61.8% reduction in the death rate among those patients, compared with those who received only the conventional treatment. Those taking Avemar experienced lower rates of recurrences and metastases
as well, even though most patients in the Avemar group came into the study with more advanced disease, had more radiation earlier, and had been diagnosed longer. Side effects of Avemar, as in
other Avemar trials., were rare, mild, and transient, with no serious adverse events occurring.

In a 2004 study published in the journal of Pediatric Hematology and Oncology, childhood cancer patients taking Avemar during and after conventional therapies had a 42.8% reduction in the
low white blood cell counts and high fever known as febrile neutropenia, which can be a life-threatening consequence of chemotherapy and radiation. (4) This and similar results with
Avemar in other cancers are consistent with animal studies showing that Avemar helps the immune system recover a full white blood cell count after chemotherapy and radiation faster
than would otherwise happen. This study also demonstrated the safety of Avemar for children.

Why Avemar Works in Many Different Kinds of Cancer

Extensive studies in cells and animals have shown how Avemar works. Perhaps its most important action is to restrict cancer cells’ use of glucose. (5) Cancer cells use up to 50 times more glucose
than normal cells, a phenomenon known as the Warburg effect. (6) They use those enormous amounts of glucose to make ribose, the backbone sugar of DNA, much faster than normal cells can. To
do this, they must use a different series of biochemical reactions (“pathway”) than normal cells. Avemar makes this very difficult for cancer cells to do, because it inhibits the activity of the key enzyme in that pathway, transketolase (TK). (7) With the TK pathway blocked, cancer cells cannot use large amounts of glucose to make DNA fast enough to support the proliferation that makes them so dangerous.(8-10)

In experiments in the US and abroad, scientists have learned that Avemar has these additional effects. It:

* lowers the levels of a DNA repair enzyme known as poly (ADPribose) polymerase (PARP).” With this effect, cancer cells are forced to self-destruct, preventing them from proliferating and
producing a synergistic cancer-cell killing effect when given with chemotherapy, which also works to damage cancer cells’ DNA;
* reduces the number of molecules on cancer cells that identify them as originating within the body (MHC-1 molecules). (12) With cancer cells stripped of that protection, the immune system,
which recognizes the cancer cells as abnormal, no longer gives them the pass given to cells originating in the body. The cancer cells are attacked by the immune system’s natural killer (NK)
cells and destroyed;
* increases levels of molecules called intercellular adhesion molecule-1 (ICAM-1) on the blood vessels of cancer tumors. (13). The increase helps immune system cells pass through the walls of the blood vessels supplying the tumor blood flow, moving directly into the tumor to attack its cancer cells; increases the activity of the primary anticancer cytokine, tumor necrosis factor alpha (TNF-a), and produces a synergistic effect in interaction with other anticancer cytokines. (14) Cytokines are substances produced by cells to act directly on other cells. TNF-a helps force cancer cells into the programmed death known as apoptosis and inhibits tumorigenesis, the process through which new tumors are formed;
* inhibits the activity of ribonucleotide reductase (RR), a key enzyme that cells must have to make new DNA so that each cancer cell can divide to make two more like it. (15) With DNA
production slowed, increases in cancer cell growth and replication are inhibited.

Antimetastatic and Immune-Boosting Effects Are Key to Survival

Because the biochemical changes listed above have consistently been shown in both animal and human studies to be directly linked to reducing cancer’s ability to metastasize and to
improving the immune system’s ability to fight cancer, scientists count them as among the most likely main causes of improved survival seen in cancer patients when Avemar is used alone or,
more often, as an adjuvant in addition to standard-of-care therapies such as chemotherapy, radiotherapy, or the combination of the two. (16-23)

Extending Life: How Long, Exactly, and At What Cost in Quality of Life?

Any improvement in advanced melanoma survival, no matter how small, is certainly an achievement. But ipilimumab had severe side effects requiring hospitalization or invasive intervention in
over one-quarter of patients treated with it. And it increased median survival only by 3-plus months. On the other hand, Avemar added to dacarbazine improved survival very markedly, with no severe side effects. If actually improving overall survival substantially without significant side effects means that a drug should be considered as the new standard of care for first-line therapy, then there is no need to wait for further results. Avemar has already demonstrated very significant improvement in survival over chemotherapy alone and has a safety profile unmatched by
conventional therapies.

Michael Traub, ND, FABNO, is in private practice and serves as a member of Oncology Association of Naturopathic Physicians board of examiners.
Notes
(1.) Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010 Jun 14.
(2.) Demidov LV. Manziuk LV, Kharkevitch GY, Pirogova NA,  Artamonova EV. Adjuvant fermented wheat germ extract (Avemar) nutraceutical improves survival of high-risk skin
melanoma patients; a randomized, pilot, phase ll clinical study with a 7-year follow-up. Cancer Biother Radiopharm. 2008 Aug. 23(4):477-482. Erratum in: Cancer Biother Radiopharm. 2008
Oct;2315):669.
(3.) Jakab F, Shoenfeld Y, Balogh A. et al. A medical nutriment has supportive value in the treatment of colorectal cancer. Br J Cancer. 2001 Aug 4;89(3):465-9.
(4.) Garami M, Schuler D, Babosa M, et al. Fermented wheat germ extract reduces chemotherapy-induced febrile neutropenia in pediatric cancer patients, J Pediatr Hematol Oncol. 2004
Oct;26(10):631-635.
(5.) Boros I.G, Lapis K, Szende B, et al. Wheat germ extract decreases glucose uptake and RNA ribose formation but increases fatty acid synthesis in MIA pancreatic adenocarcinoma
cells. Pancreas. 2001 Aug:23(2):141-147.
(6.) Warburg, O. On the origin of cancer cells. Science. 1956 Feb 24; 123(31 91):309-314.
(7.) Boros LG, Lee VVN, Go VL., A metabolic hypothesis of cell growth and death in pancreatic cancer, Pancreas. 2002 Jan;
24:(1):26 33.
(8.) Boros LG, Lapis K, Szende B, et al. Op cit.
(9.) Comin-Anduix B, Boros LG, Marin S, et al. Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase
activation in Jurkat T-cell leukemia tumor cells. J Biol Chem. 2002 Nov 29;277 (48):46408-46414. Epub 2002 Sep 25.
(23.) Garami M, Schuler D, Babosa M, et al. Fermented wheat germ extract reduces chemotherapy-induced febrile neutropenia in pediatric cancer patients. J Pediatr Hematol Oncol. 2004 Oct;
26(10):631-635.

by Michael Traub, ND, FABNO
COPYRIGHT 2010 The Townsend Letter Group
COPYRIGHT 2010 Gale, Cengage Learning

Nanotechnology in Cancer Drug Delivery and Selective Targeting

Nanoparticles are rapidly being developed and trialed to overcome several limitations of traditional drug delivery systems and are coming up as a distinct therapeutics for cancer treatment. Conventional chemotherapeutics possess some serious side effects including damage of the immune system and other organs with rapidly proliferating cells due to nonspecific targeting, lack of solubility, and inability to enter the core of the tumors resulting in impaired treatment with reduced dose and with low survival rate.

Nanotechnology has provided the opportunity to get direct access of the cancerous cells selectively with increased drug localization and cellular uptake. Nanoparticles can be programmed for recognizing the cancerous cells and giving selective and accurate drug delivery avoiding interaction with the healthy cells. This review focuses on cell recognizing ability of nanoparticles by various strategies having unique identifying properties that distinguish them from previous anticancer therapies. It also discusses specific drug delivery by nanoparticles inside the cells illustrating many successful researches and how nanoparticles remove the side effects of conventional therapies with tailored cancer treatment.

(Kumar Bishwajit Sutradhar and Md. Lutful Amin. Hindawi Publ. Corp.  2014, Article ID 939378, 12 pages

http://dx.doi.org/10.1155/2014/939378)

Cancer, the uncontrolled proliferation of cells where apoptosis is greatly disappeared, requires very complex process of treatment. Because of complexity in genetic and phenotypic levels, it shows clinical diversity and therapeutic resistance. A variety of approaches are being practiced for the treatment of cancer each of which has some significant limitations and side effects. Cancer treatment includes surgical removal, chemotherapy, radiation, and hormone therapy. Chemotherapy, a  very common treatment, delivers anticancer drugs systemically to patients for quenching the uncontrolled proliferation of cancerous cells. Unfortunately, due to nonspecific targeting by anticancer agents, many side effects occur and poor drug delivery of those agents cannot bring out the desired outcome in most of the cases. Cancer drug development involves a very complex procedure which is associated with advanced polymer chemistry and electronic engineering.

The main challenge of cancer therapeutics is to differentiate the cancerous cells and the normal body cells. That is why the main objective becomes engineering the drug in such a way as it can identify the cancer cells to diminish their growth and proliferation. Conventional chemotherapy fails to target the cancerous cells selectively without interacting with the normal body cells. Thus they cause serious side effects including organ damage resulting in impaired  treatment with lower dose and ultimately low survival rates.

Nanotechnology is the science that usually deals with the size range from a few nanometers (nm) to several hundrednm, depending on their intended use. It has been the area of interest over the last decade for developing precise drug delivery systems as it offers numerous benefits to overcome the limitations of conventional formulations . It is very promising both in cancer diagnosis and treatment since it can enter the tissues at molecular level.

Cisplatin-incorporated nanoparticles of poly(acrylic acid-co-methyl methacrylate) copolymer

K Dong Lee, Young-Il Jeong,  DH Kim,  Gyun-Taek Lim,  Ki-Choon Choi.  Intl J Nanomedicine 2013:8 2835–2845.

Although cisplatin is extensively used in the clinical field, its intrinsic toxicity limits its clinical use. We investigated nanoparticle formations of poly(acrylic acid-co-methyl methacrylate) (PAA-MMA) incorporating cisplatin and their antitumor activity in vitro and in vivo.

Methods: Cisplatin-incorporated nanoparticles were prepared through the ion-complex for­mation between acrylic acid and cisplatin. The anticancer activity of cisplatin-incorporated nanoparticles was assessed with CT26 colorectal carcinoma cells.

Results: Cisplatin-incorporated nanoparticles have small particle sizes of less than 200 nm with spherical shapes. Drug content was increased according to the increase of the feeding amount of cisplatin and acrylic acid content in the copolymer. The higher acrylic acid content in the copolymer induced increase of particle size and decrease of zeta potential. Cisplatin-incorporated nanoparticles showed a similar growth-inhibitory effect against CT26 tumor cells in vitro. However, cisplatin-incorporated nanoparticles showed improved antitumor activity against an animal tumor xenograft model.

Conclusion: We suggest that PAA-MMA nanoparticles incorporating cisplatin are promising carriers for an antitumor drug-delivery system.

Researchers Say Molecule May Help Overcome Cancer Drug Resistance
By Estel Grace Masangkay

A group of researchers from the University of Delaware has discovered that a deubiquitinase (DUB) complex, USP1-UAF1, may present a key target in helping fight resistance to platinum-based anticancer drugs. The research team’s findings were published online in Nature Chemical Biology.

Zhihao Zhuang, associate professor in the Department of Chemistry and Biochemistry at UD, and his team studied a DNA damage tolerance mechanism called translesion synthesis (TLS). Enzymes known as TLS polymerases synthesize DNA over damaged nucleotide bases, followed by replication after lesion. The enzymes have been linked with building cancer cell resistance to certain cancer drugs including cisplatin. Cisplatin is used in treatment of ovarian, bladder, and testicular cancers which have spread.

“Cancer drugs like cisplatin work by damaging DNA and thereby preventing cancer cells from replicating the genomic DNA and dividing. However, cancer cells quickly develop resistance to cisplatin, and we and other researchers suspect that a polymerase known as Pol η is involved in overcoming cisplatin-induced lesions,” Professor Zhuang said.

The team found that USP1-UAF1 may play a crucial role in regulating DNA damage response. A new molecule ML323 can be used to inhibit processes such as translesion synthesis. Zhuang said, “Using ML323, we studied the cellular response to DNA damage and revealed new insights into the role of deubiquitination in both the TLS pathway and another one called the Fanconi anemia, or FA, pathway. We’re very encouraged by the fact that a single molecule is effective at inhibiting the USP1-UAF1 DUB complex and disrupting two essential DNA damage tolerance pathways.”

A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

Cui-yan Han,  Li-ling Yue, Ling-yu Tai,  Li Zhou  et al.  Intl J Nanomedicine 2013:8 1541–1549

The discovery of suitable ligands that bind to cancer cells is important for drug delivery specifically targeted to tumors. Monoclonal antibodies and fragments that serve as ligands have specific targets. Natural ligands have strong mitogenic and neoangiogenic activities. Currently, small pep­tides are pursued as targeting moieties because of their small size, low immunogenicity, and their ability to be incorporated into certain delivery vectors.

The epidermal growth factor receptor (EGFR) serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. The epidermal growth factor receptor (EGFR) is a transmembrane protein on the cell surface that is overexpressed in a wide variety of human cancers. EGFR is an effective tumor-specific target because of its significant functions in tumor cell growth, differentiation, and migration. EGFR-targeted small molecule peptides such as YHWYGYTPQNVI have been successfully identified using phage display library screening; by contrast, the peptide LARLLT has been generated using computer-assisted design (CAD).

These peptides can be conjugated to the surfaces of liposomes that are then delivered selectively to tumors by the specific and efficient binding of these peptides to cancer cells that express high levels of EGFR.

In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD) These small peptides were labeled with fluorescein isothiocyanate (FITC) and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control.

Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors.

We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.

Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity

SR Sarker  Y Aoshima,   R Hokama  T Inoue  et al. Intl J Nanomedicine 2013:8 1361–1375.

Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group.

 Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000.

 We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular.

Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity.

The gene delivery efficiency of amino acid-based cationic assemblies is influ­enced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.

Molecularly targeted approaches herald a new era of non-small-cell lung cancer treatment

H Kaneda, T Yoshida,  I Okamoto.   Cancer Management and Research 2013:5 91–101.

The discovery of activating mutations in the epidermal growth-factor receptor (EGFR) gene in 2004 opened a new era of personalized treatment for non-small-cell lung cancer (NSCLC). EGFR mutations are associated with a high sensitivity to EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. Treatment with these agents in EGFR-mutant NSCLC patients results in dramatically high response rates and prolonged progression-free survival compared with conventional standard chemotherapy. Subsequently, echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK), a novel driver oncogene, has been found in 2007. Crizotinib, the first clinically available ALK tyrosine kinase inhibitor, appeared more effective compared with standard chemotherapy in NSCLC patients harboring EML4-ALK. The identification of EGFR mutations and ALK rearrangement in NSCLC has further accelerated the shift to personalized treatmentbased on the appropriate patient selection according to detailed molecular genetic characterization. This review summarizes these genetic biomarker-based approaches to NSCLC, which allow the instigation of individualized therapy to provide the desired clinical outcome.

Non-small-cell lung cancer (NSCLC) has a poor prognosis and remains the leading cause of death related to cancer worldwide. For most individuals with advanced, metastatic NSCLC, cytotoxic chemotherapy is the mainstay of treatment on the basis of the associated moderate improvement in survival and quality of life. However, the outcome of chemotherapy in such patients has reached a plateau in terms of overall response rate (25%–35%) and overall survival (OS; 8–10 months). This poor outcome, even for patients with advanced NSCLC who respond to such chemotherapy, has motivated a search for new therapeutic approaches.

Recent years have seen rapid progress in the development of new treatment strat­egies for advanced NSCLC, in particular the introduction of molecularly targeted therapiesand appropriate patient selection. First, the most important change has been customization of treatment according to patient selection based on the genetic profile of the tumor. Small-molecule tyrosine kinase inhibitors (TKIs) that target the epidermal growth-factor receptor (EGFR), such as gefitinib and erlotinib, are especially effective in the treatment of NSCLC patients who harbor activating EGFR mutations.

Surgical Nanorobotics using nanorobots made from advanced DNA origami and Synthetic Biology

Ido Bachelet’s moonshot to use nanorobotics for surgery has the potential to change lives globally. But who is the man behind the moonshot?

Ido graduated from the Hebrew University of Jerusalem with a PhD in pharmacology and experimental therapeutics. Afterwards he did two postdocs; one in engineering at MIT and one in synthetic biology in the lab of George Church at the Wyss Institute at Harvard.

Now, his group at Bar-Ilan University designs and studies diverse technologies inspired by nature.

They will deliver enzymes that break down cells via programmable nanoparticles.

Delivering insulin to tell cells to grow and regenerate tissue at the desired location.

Surgery would be performed by putting the programmable nanoparticles into saline and injecting them into the body to seek out remove bad cells and grow new cells and perform other medical work.

 

http://2.bp.blogspot.com/-bnAE6hL2RIE/Uy0wFB8pYPI/AAAAAAAAubM/BeSpFC4vLu0/s1600/screenshot-by-nimbus+(3).png

 

Robots killing and suppressing cancer cells

 

http://1.bp.blogspot.com/-LGsE1msGIrw/Uy0vKGoaQ3I/AAAAAAAAubE/2E1_lcAspao/s1600/screenshot-by-nimbus+(2).png

 

Robots delivering payload

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0

http://4.bp.blogspot.com/-kkfXlMyPRCI/Uy0wkYPMvBI/AAAAAAAAubU/0AQPpJpM5E4/s1600/screenshot-by-nimbus+(4).png

Molecular building blocks

 

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=236

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=283

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=287

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=292

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=333

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=397

http://2.bp.blogspot.com/-gCHiyZ2MBHg/Uy0ySRKw_II/AAAAAAAAubg/BeneEQ5bY-U/s1600/screenshot-by-nimbus+(5).png

 

Robot blocks neuron

http://4.bp.blogspot.com/-cbYNJnN_w7U/Uy0yrqyqebI/AAAAAAAAubo/b42r4WRMr8k/s1600/screenshot-by-nimbus+(6).png

 

automation of robotic surgery

 

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=470

Nanoparticles with computational logic has already been done

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=501

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=521

http://1.bp.blogspot.com/-rSyRzo7p50w/Uy0y5teQkDI/AAAAAAAAubw/8cxZ4t0WNHw/s1600/screenshot-by-nimbus+(7).png

 

 robotic algorithm

 

Load an ensemble of drugs into many particles for programmed release based on situation that is found in the body

http://1.bp.blogspot.com/-kc99CbOQYLs/Uy0zgUG13KI/AAAAAAAAub4/j6nM7hAVxUg/s1600/screenshot-by-nimbus+(8).png

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=572

http://www.youtube.com/watch?feature=player_embedded&v=aA-H0L3eEo0#t=577

 

robotic lung cancer Rx

 

chemotherapy regimen

 

Chemoprevention in Model Experiments

Effects of Two Disiloxanes ALIS-409 and ALIS-421 on Chemoprevention in Model Experiments

H TOKUDA,…. L AMARAL and J MOLNAR.ANTICANCER RESEARCH 33: 2021-2028 (2013).

ALIS

 

Figure 1. Chemical structures of ALIS-409 and ALIS-421.

Morpholino-disiloxane (ALIS-409) and piperazinodisiloxane (ALIS-421) compounds were developed as inhibitors of multidrug resistance of various types of cancer cells. In the present study, the effects of ALIS-409 and ALIS-421 compounds were investigated on cancer promotion and on co-existence of

tumor and normal cells. The two compounds were evaluated for their inhibitory effects on Epstein-Barr virus immediate early antigen (EBV-EA) expression induced by tetradecanoylphorbolacetate (TPA) in Raji cell cultures. The method is known as a primary screening test for antitumor effect, below the (IC50) concentration. ALIS-409 was more effective in inhibiting EBV-EA (100 μg/ml) and tumor promotion, than

ALIS-421, in the concentration range up to 1000 μg/ml. However, neither of the compounds were able to reduce tumor promotion significantly, expressed as inhibition of TPA-induced tumor antigen activation. Based on the in vitro results, the two disiloxanes were investigated in vivo for their effects on mouse skin tumors in a two-stage mouse skin carcinogenesis study.

 

 

 

 

 

 

Read Full Post »


Author: Ziv Raviv, PhD

Mitochondria are recognized as essential for both life and death fates of cells. Mitochondria are the site where oxidative phosphorylation happens, the process that is responsible for the majority of energy production of the cell in the form of adenosine triphosphate (ATP) synthesis. Therefore mitochondria are considered as the main power station of the cell. On the other hand, both apoptotic and necrotic cell death may result from mitochondrial perturbation [1]. In cancer cells, mitochondria are different from those of normal cells by several aspects: (i) In cancer cells the mitochondrial membrane potential is higher than that of normal cells, (ii) there is expression modulation of permeability transition pore complex (PTPC) components which include the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), and cyclophilin D, and (iii) there are enhanced rates of glycolysis even in the presence of oxygen, a phenomena that is known as the Warburg effect [2]. In fact, many chemotherapeutic drugs induce mitochondrial-mediated apoptotic cell death (intrinsic apoptosis pathway) and act via mitochondrial perturbation, causing mitochondrial membrane permeability transition (MPT), membrane depolarization, osmotic swelling, and release of cytochrome c leading to cancer cell death.

Hexokinase (HK) is the initial enzyme of glycolysis that catalyzes the phosphorylation of glucose to glucose-6-phosphate (G6P), which is also the rate-limiting step in glycolysis and sequesters glucose inside the cells.  In cancer cells, HK (mainly HK-II) is overexpressed and found mostly bound to mitochondria through VDAC1  [3]. An enhanced expression of HK is found in aggressive tumors such as gliomas [4], and hepatomas [5]. HK overexpression, along with its glucose phosphorylation activity, is suggested to play a pivotal role in cancer cell growth rate and survival [6]. Thus, mitochondrial-bound HK overexpression may contribute to the Warburg effect by facilitating the access  to ATP, the substrate of HK [7]. In addition, as one of the hallmarks of cancer [8] is evading apoptotic cell death, owing in part to overexpression of anti-apoptotic proteins of the Bcl-2 family and that of HK, the elevated levels of mitochondria-bound HK in cancer cells contribute to the protection against mitochondria-mediated cell death [6].  All together, these characteristics make HK attractive target for cancer therapy.

Anti-cancer agents targeting HK-mitochondria interactions:

Jasmonates

The jasmonates plant stress hormones aside from their natural function against microbial pathogens in plants were also discovered to have toxic activities towards mammalian cancer cells. These activities consist of two important characteristics for anti-cancer drugs: high selectivity towards cancer cells, and the ability to act against drug resistant cancer cells [9]. The main mechanism of action of jasmonates–induced cancer cell death is suggested to involve direct mitochondrial perturbation. Methyl jasmonate (MJ) is able to reduce intracellular levels of ATP in various cancer cells, preceding cell death induction. Thus, the impaired ability of cancer cell mitochondria to generate ATP renders them more sensitive to the rapid ATP depletion induced by MJ. In addition, MJ induces mitochondrial membrane depolarization and cytochrome c release in cancer cells, as well as swelling and cytochrome c release in isolated mitochondria derived from cancer cells in a PTPC-mediated manner, but not of normal cells [10]. These findings demonstrate that the jasmonates selective toxicity towards cancer cells relies on the differential mitochondrial status of cancer cells vs. non-cancerous cells. Most relevant, it was clearly demonstrated that MJ binds specifically to HK and disrupts its interaction with mitochondrial VDAC1, leading to detachment of HK from the mitochondria followed by cytochrome c release and subsequent cell death [11]. The direct interaction of MJ with HK was demonstrated using real-time surface plasmon resonance (SPR).  In addition, it was demonstrated that the susceptibility of cancer cells and mitochondria to MJ  depends on the expression of HK and its mitochondrial association [11]. Therefore, MJ-induced HK detachment from mitochondria perturbs mitochondrial permeability and induce overall cellular energy crises, leading to cell death (see figure). Jasmonates thus describe for the first time of a cytotoxic mechanism based on direct interaction between an anti-cancer agent and HK. This finding may stimulate the development of a novel class of small anticancer compounds that inhibit the HK-VDAC1 interaction [12].

Image

VDAC1-based peptides

As described above, the HK association to mitochondria in cancer cells mediated through VDAC1 [13]. It has been shown that HK–VDAC1 interaction prevents induction of apoptosis in tumor-derived cells. Thus interfering with HK binding to VDAC1, promoting detachment of HK would form the basis for novel cancer treatment. Two main classes of agents might affect the HK-VDAC1 association: inhibitors of HK activity, or compounds that compete with VDAC1 for HK binding.

Detailed studies were performed in order to elucidate the domains on VDAC1 sequence that are essential for its interactions with HK.  These studies were based upon VDAC1 biochemical/functional structural prediction and the recently elucidated VDAC1 3D structure. By mutagenesis and functional studies, suspected domains on VDAC1 were examined as for their role in VDAC1-HK interactions [14]. According to these studies selected cell-penetrable VDAC1-based peptides were designed and were demonstrated to directly interact with purified HK in vitro and to detach HK bound to mitochondria isolated from tumor cells. Not only that, it was clearly demonstrated that these peptides are capable to selectively kill cancer cells while spearing normal cells [15], all together supporting the notion that interfering with the binding of HK to mitochondria by VDAC1-based peptides indeed may offer a novel strategy by which to induce selective cancer cell death.

Further directions

In order to evaluate the potential of using the HK-mitochondrial interactions as valid targets for cancer therapy, more steps are needed to be taken on the road. The selectiveness of this therapy relays on the fact that cancer cells bare much more mitochondrial-bound HK than normal cells, which might serve as an Achilles heel of the cancer cell. As peptides could be easily degraded in the plasma, the VDAC1-based peptides efficacy against cancer should be evaluated in vivo as well as their plasma stability should be examined. New generation and formulations of VDAC1-based peptides should be developed based upon research progress. As for jasmonates, their main deficiency is the need of using relatively high concentration (at the range of millimolar) to exert their action. Therefore, in order to develop valid jasmonate-based therapies, there is an urgent need for the development of jasmonate analogs that actually work in much lower dosage, with increased solubility, yet still effective and potent against cancer. Furthermore, it is not clear yet what is the exact domain on HK that MJ is interacting with. Elucidating the plausible interaction site(s) of MJ with HK would give the opportunity to design other small molecules directed to that specific HK domain with the hope to achieve more effective anti-cancer agents.

References

1. Newmeyer DD, Ferguson-Miller S (2003) Mitochondria: releasing power for life and unleashing the machineries of death. Cell 112 (4):481-490

2. Warburg O (1956) On the origin of cancer cells. Science 123 (3191):309-314

3. Pedersen PL, Mathupala S, Rempel A, Geschwind JF, Ko YH (2002) Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention. Biochim Biophys Acta 1555 (1-3):14-20

4. Pastorino JG, Hoek JB (2003) Hexokinase II: the integration of energy metabolism and control of apoptosis. Curr Med Chem 10 (16):1535-1551

5. Gelb BD, Adams V, Jones SN, Griffin LD, MacGregor GR, McCabe ER (1992) Targeting of hexokinase 1 to liver and hepatoma mitochondria. Proc Natl Acad Sci U S A 89 (1):202-206

6. Mathupala SP, Ko YH, Pedersen PL (2006) Hexokinase II: cancer’s double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria. Oncogene 25 (34):4777-4786

7. Pedersen PL (2007) Warburg, me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancers’ most common phenotypes, the “Warburg Effect”, i.e., elevated glycolysis in the presence of oxygen. J Bioenerg Biomembr 39 (3):211-222

8. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144 (5):646-674

9. Raviv Z, Cohen S, Reischer-Pelech D (2013) The anti-cancer activities of jasmonates. Cancer Chemother Pharmacol 71 (2):275-285

10. Rotem R, Heyfets A, Fingrut O, Blickstein D, Shaklai M, Flescher E (2005) Jasmonates: novel anticancer agents acting directly and selectively on human cancer cell mitochondria. Cancer Res 65 (5):1984-1993

11. Goldin N, Arzoine L, Heyfets A, Israelson A, Zaslavsky Z, Bravman T, Bronner V, Notcovich A, Shoshan-Barmatz V, Flescher E (2008) Methyl jasmonate binds to and detaches mitochondria-bound hexokinase. Oncogene 27 (34):4636-4643

12. Galluzzi L, Kepp O, Tajeddine N, Kroemer G (2008) Disruption of the hexokinase-VDAC complex for tumor therapy. Oncogene 27 (34):4633-4635

13. Shoshan-Barmatz V, Zakar M, Rosenthal K, Abu-Hamad S (2009) Key regions of VDAC1 functioning in apoptosis induction and regulation by hexokinase. Biochim Biophys Acta 1787 (5):421-430

14. Abu-Hamad S, Zaid H, Israelson A, Nahon E, Shoshan-Barmatz V (2008) Hexokinase -I protection against apoptotic cell death is mediated via interaction with the voltage-dependent anion channel-1: Mapping the site of binding. J Biol Chem 19:13482-13490

15. Arzoine L, Zilberberg N, Ben-Romano R, Shoshan-Barmatz V (2009) Voltage-dependent anion channel 1-based peptides interact with hexokinase to prevent its anti-apoptotic activity. J Biol Chem 284 (6):3946-3955.

pharmaceuticalintelligence.com

https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view/

https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

Read Full Post »


Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis – with a Concomitant Influence on Mitochondrial Function

 

Reporter, Editor, and Topic Co-Leader: Larry H. Bernstein, MD, FACP, Clinical Pathologist and Biochemist

 

 

Apoptosis signaling pathways

Apoptosis signaling pathways (Photo credit: AJC1)

This discussion is a followup on a series of articles elucidating the importance of NO, eNOS, iNOS, cardiovascular and vascular endothelium effects, and therapeutic targets.

This mechanism of action and signaling actions have been introduced so that we identify endocrine, paracrine, and such effects in the normal, stressed, and dysfunctional state. The size and breadth of this vital adaptive process is now further explored.

The title is short, befitting a subtitle.  The full topic may be considered “Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer”.

Vascular endothelium

Vascular endothelium (Photo credit: Wikipedia)

Related articles

 

 

Nitric Oxide Synthase

Nitric Oxide Synthase (Photo credit: Wikipedia)

 

 

Nitric Oxide has a ubiquitous role in the regulation of glycolysis with a concomitant influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer.

Uncoupling of aerobic glycolysis
Potential cytotoxic mediators of endothelial cell (EC) apoptosis include increased formation of reactive oxygen and nitrogen species (ROSRNS) during the atherosclerotic process. Nitric oxide (NO) has a biphasic action on oxidative cell killing with low concentrations protecting against cell death, whereas higher concentrations are cytotoxic. High levels of NO can be produced by inducible nitric-oxide synthase in response to cytokine stimulation, primarily from macrophages, and elevated levels of NO is injurious to endothelium.Ccytochrome c release and caspase activation are involved in NO induced apoptosis. ROS also induces mitochondrial DNA damage in ECs, and this damage is accompanied by a decrease in mitochondrial RNA (mtRNA) transcripts, mitochondrial protein synthesis, and cellular ATP levels. Mitochondria have been recognized to play a pivotal role in the signaling cascade of apoptosis leading to atherosclerosis-induced damage in endothelial cells.
The processes involved in the signaling pathways leading to apoptosis are complex but have some degree of convergence between cell types including those in the vasculature. Release of cytochrome c from mitochondria is a proapoptotic signal, which activates several downstream signaling events including formation of the apoptosome and activation of caspases. Ubiquinol cytochrome c reductase (complex III) is a site for ROS formation, and cytochrome c oxidase (complex IV) is a target for the interaction of NO in mitochondria.
The impact of the inhibition of mitochondrial protein synthesis is particularly important in NO-dependent cytotoxicity, and depends also on other factors such as glycolysis. These authors examined whether the inhibition of mitochondrial protein synthesis by chloramphenicol increases the susceptibility of endothelial cells to undergo NO-dependent apoptosis in glucose-free media. Bovine aortic endothelial cells were treated with chloramphenicol, which resulted in a decreased ratio of mitochondrial complex IV to cytochrome c and increased oxidant production in the cell. Inhibition of mitochondrial protein synthesis was associated with a greater susceptibility of the cells to apoptosis induced by NO in glucose-free medium.
Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis. A Ramachandran, DR Moellering, E Ceaser, S Shiva, J Xu, and V Darley-Usmar. PNAS May 14, 2002: 99(10): 6643–6648 http://www.pnas.orgcgidoi10.1073pnas.102019899

Nitric oxide (NO) is a ubiquitous signaling molecule whose physiological roles mediated through the activation of the soluble guanylate cyclase are now clearly recognized. At physiological concentrations, NO also inhibits the mitochondrial enzyme cytochrome c oxidase (complex IV) in competition with oxygen, and recently we have suggested that the interplay between the two gases allows this enzyme to act as an oxygen sensor in cells. In addition, NO plays a variety of patho-physiological roles, some of which also may be the consequence of its action at a mitochondrial level. We have characterized the sequence of events that follow inhibition of complex IV by continuous exposure to NO.
The mitochondrion is a key organelle in the control of cell death. Nitric oxide (NO) inhibits complex IV in the respiratory chain and is reported to possess both proapoptotic and antiapoptotic actions. We investigated the effects of continuous inhibition of respiration by NO on mitochondrial energy status and cell viability. Serum-deprived human T cell leukemia (Jurkat) cells were exposed to NO at a concentration that caused continuous and complete (;85%) inhibition of respiration. Serum deprivation caused progressive loss of mitochondrial membrane potential (Dcm) and apoptotic cell death. In the presence of NO, Dcm was maintained compared to controls, and cells were protected from apoptosis. Similar results were obtained by using staurosporin as the apoptotic stimulus. As exposure of serum-deprived cells to NO progressed (>5 h), however, Dcm fell, correlating with the appearance of early apoptotic features and a decrease in cell viability. Glucose deprivation or iodoacetate treatment of cells in the presence of NO resulted in a collapse of Dcm, demonstrating involvement of glycolytic ATP in its maintenance. Under these conditions cell viability also was decreased. Treatment with oligomycin and or bongkrekic acid indicated that the maintenance of Dcm during exposure to NO is caused by reversal of the ATP synthase and other electrogenic pumps. Thus, blockade of complex IV by NO initiates a protective action in the mitochondrion to maintain Dcm; this results in prevention of apoptosis. It is likely that during cellular stress involving increased generation of NO this compound will trigger a similar sequence of events, depending on its concentration and duration of release. (mitochondrial membrane potential ; apoptosis ; necrosis)

The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death. B Beltra, A Mathur, MR Duchen, JD. Erusalimsky, and S Moncada. PNAS Dec 19, 2000; 97(26):4602–14607.

Another study by this group shows that inhibition of respiration by exogenous nitric oxide (NO) in Jurkat cells leads to mitochondrial membrane hyperpolarization dependent on the utilization of glycolytic ATP by the F1Fo-ATPase and other transporters acting in reverse mode. This process also occurs in astrocytes, which are highly glycolytic cells, but not in neurons , which do not invoke glycolysis to maintain ATP concentrations. In addition, this hyperpolarization correlates with protection against apoptotic cell death. Others found an early phase of mitochondrial hyperpolarization after treatment of a variety of cells with different pro-apoptotic stimuli, which precedes the generation of free. At present, no satisfactory explanation has been proposed to explain the mechanism of hyperpolarization, the reasons why free radicals are released from the mitochondrion, or the connection of these phenomena with apoptosis.
The authors surmise that a pro-apoptotic stimulus, anti-Fas Ab, leads to release of endogenous NO from Jurkat cells in sufficient amounts to inhibit cell respiration and cause a hyperpolarization dependent on the reversal of the F1Fo-ATPase. Moreover, the reduction of the mitochondrial electron transport chain, after inhibition of cytochrome oxidase by NO, leads to generation of superoxide anion (O2). They suggest the process is a cellular defense response that may be overcome by pro-apoptotic mechanisms that occur in parallel.

Inhibition of mitochondrial respiration by endogenous nitric oxide: A critical step in Fas signaling. B Beltran, M Quintero, E Garcıa-Zaragoza, E O’Connor, JV. Esplugues, and Salvador Moncada. PNAS June 25, 2002 99(13): 8892–8897. http://www.pnas.orgcgidoi10.1073pnas.092259799

Nitric oxide has been shown to render cells resistant to oxidative stress. Mechanisms proposed for the ability of nitric oxide to protect cells against oxidative stress include reactions of nitric oxide and the induction of adaptive responses that require protein synthesis. Nitric oxide forms iron complexes preventing the formation of strong oxidants. In addition, reactions of nitric oxide with lipid and or organic radicals protect against membrane peroxidation and peroxidative chemistry-induced cell injury. Exposure to low, nonlethal doses of nitric oxide induces adaptive responses that render cells resistant to lethal concentrations of nitric oxide and or peroxides, such as, the induction of hemoxygenase-1 (HO-1) and Mn superoxide dismutase. The up-regulation of HO-1 was accompanied by an increase in ferritin to account for the release of iron from HO-1, indicating a role of both iron heme and nonheme iron for peroxide-mediated cellular injury. Further, nitric oxide, by regulating critical mitochondrial functions such as respiration, membrane potential, and release of cytochrome c, is able to trigger defense mechanisms against cell death induced by pro-apoptotic stimuli.
This study investigates the potential contribution of nitric oxide’s ability to protect cells from oxidative stress, low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H2O2. Spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H2O2 (50–150 mM). Inhibition of nitric oxide synthesis abolished the protective effect against H2O2 exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria. ECV3041 eNOS cells with diminished mitochondria respiration are injured to the same extent as non-transfected ECV304 cells, and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H2O2-induced death. Nitric oxide had a profound effect in cell metabolism, because ECV3041eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H2O2 exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress. E Paxinou, M Weisse, Q Chen, JM Souza, et al. PNAS Sept 25, 2001; 98( 20): 11575–11580. http://www.pnas.orgycgiydoiy10.1073ypnas.201293198.

Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors of neurons. NO has also been implicated for such pathophysiological conditions as destruction of tumor cells by macrophages, rheumatoid arthritis, and focal brain ischemia. Some of these effects of NO are associated with hypoxic conditions. O2 radicals and ions that result from reactivity of NO are presumed to be involved in NO cytotoxicity. These investigators report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-Lglutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 mM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 5 6.6 mM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 5 2.5 mM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1a or HIF-1a-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1a to a DNA-binding form.
Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. K Sogawa, K Numayama-Tsuruta, M Ema, M Abe, et al. Proc. Natl. Acad. Sci. USA (Biochemistry) June 1998; 95:7368–7373. 1998. The National Academy of Sciences 0027-8424.98.957368-6. http:yywww.pnas.org.

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.
Glycolysis and the Tricarboxylic Acid Cycle Are Linked by Alanine Aminotransferase during Hypoxia Induced by Waterlogging of Lotus japonicus[W][OA]. M Rocha, F Licausi, WL Arau´ jo, A Nunes-Nesi, et al. Plant Physiology Mar 2010; 152: 1501–1513. http://www.plantphysiol.org 2010 Amer Soc Plant Biologists

DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP-ribose) polymerase-1 (PARP-1) cell death pathway. PARP-1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP-ribose) on acceptor proteins. The PARP-1 activation leads to cell death. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP-1 and the role of NAD+ depletion in this cell death program. PARP-1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near-complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD restored glycolytic function and prevented cell death. The addition of non-glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP-1 activation.
These findings suggest a sequence of events in which NAD+ depletion is a key event linking DNA damage to metabolic impairment and cell deathm. A similar scenario has been proposed by Zong et al. (2004), based on the finding that cell types that depend on aerobic glycolysis for ATP production exhibit a particularly high sensitivity to DNA damage and PARP-1 activation. In mature brain, glucose is normally the dominant metabolic substrate due to relatively slow transport of other metabolites across the blood– brain barrier. Oncein brain, glucose may be metabolized directly by neurons and glia or may be metabolized to lactate in glia and thelactate subsequently shuttled to neurons for oxidative metabolism (Dringen et al., 1993; Pellerin and Magistretti,1994; Wender et al., 2000; Dienel and Cruz, 2004). In either case, a block in glycolytic flux produced by NAD depletion will block energy metabolism in both neurons and glia in brain. Interestingly, the lactate shuttle hypothesis raises the possibility that activation of PARP-1 selectively in astroglia might also block energy metabolism in neurons.

These studies suggest PARP-1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD+ pool. Depletion of the cytosolic NAD+ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood–brain barrier. © 2004 Wiley-Liss, Inc.
Key words: mitochondria; permeability transition; poly(ADP-ribose) polymerase; ischemia; peroxynitrite
NAD+as a metabolic link between DNA damage and cell death. DNA damage induced by alkylating agents, oxidative stress, or other agents causes PARP-1 activation. PARP-1 activation leads to depletion in cytosolic NAD with, initially, a relative preservation of mitochondrial NAD and mitochondrial function. The depletion in cytosolic NAD+ blocks glycolysis, and in cells in which glucose is the primary energy substrate, this in turn leads to a block in substrate flux to mitochondria. The resulting mitochondrial dysfunction leads to mitochondrial permeability transition (MPT) and subsequent downstream events culminating in cell death.
NAD+ as a Metabolic Link Between DNA Damage and Cell Death. W Ying, CC Alano, P Garnier, and RA Swanson. Journal of Neuroscience Research 2005;79:216–223
Key words: glycolysis, mitochondrial energy production, nitric oxide
Abbreviations: NO, nitric oxide; SNAP, S-nitroso-N-acetylpenicyllamine; SNP, sodium nitroprusside.
The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.
Nitric oxide (NO) has been increasingly recognized as an important intra- and intercellular messenger molecule with a physiological role in vascular relaxation, platelet physiology, neurotransmission and immune responses (Moncada et al., 1991; Radomski et al., 1996; Szabó, 1996; Riedel et al., 1999; Titheradge 1999). In vitro NO is a strong inhibitor of platelet adhesion and aggregation (Radomski et al., 1996; Riedel et al., 1999;nSogo et al., 2000). In the blood stream, platelets remain in contact with NO that is permanently released from the endothelial cells and from activated macrophages (Moncada et al., 1991; Riedel et al., 1999; Titheradge 1999). It has been suggested that the activated platelet itself is able to produce NO (Lantoine et al., 1995; Zhou et al., 1995; Radomski et al., 1996). The mechanism responsible for the inhibitory effect of NO on platelet responses is not entirely clear. It is believed that the main intracellular target for NO in platelets is soluble cytosolic guanylate cyclase (Waldman & Walter 1989; Schmidt et al., 1993; Wang et al., 1998). NO activates the enzyme (Schmidt et al., 1993). Thus, elevated intracellular cGMP level inhibits platelet activation. There are suggestions, however, that elevated cGMP may not be the only intracellular factor directly involved in the inhibition of platelet activation (Gordge et al., 1998; Sogo et al., 2000; Beghetti et al., 2003).
Platelets are fairly active metabolically and have a total ATP turnover rate of about 3–8 times that of resting mammalian muscle (Akkerman, 1978; Akkerman et al., 1978; Holmsen, 1981; Niu et al., 1996). Platelets contain mitochondria which enable these cells to produce energy both in the oxidative and anaerobic way (Holmsen, 1981). Under aerobic conditions, ATP is produced by aerobic glycolysis using glucose or glycogen which can account for 30–50% of total ATP production, and by oxidative metabolism using glucose and glycogen (6–11%), amino-acids (7%) or free fatty acids (20–40%) (Holmsen 1981; Guppy et al., 1990; Niu et al., 1996).
The inhibition of mitochondrial respiration by removing oxygen or by respiratory chain blockers (antimycin A, cyanide, rotenone) results in the stimulation of glycolytic flux (Guppy et al., 1990). This phenomenon is known as Pasteur effect and indicates that in platelets glycolysis and mitochondrial respiration are tightly functionally connected (Akkerman, 1978; Holmsen, 1981; Guppy et al., 1995; Niu et al., 1996). It has been reported that the activation of human platelets by high concentration of thrombin is accompanied by an acceleration of lactate production and an increase in oxygen consumption (Akkerman & Holmsen, 1981; Niu et al., 1996).
The results presented here suggest that also porcine blood platelets stimulated by collagen produce more lactate. This indicates that both glycolytic and oxidativeATP production supports platelet responses. This also indicates that blocking of energy production in platelets may decrease their responses. It is well established that platelet responses have different metabolic energy (ATP) requirements increasing in the order: aggregation< dense and alfa granule secretion < acid hydrolase secretion (Holmsen et al., 1982; Verhoeven et al., 1984; Morimoto & Ogihara, 1996).
The present results indicate that exogenously added NO (in the form of NO donors)stimulates glycolysis in intact porcine platelets. Since in platelets glycolysis and mitochondrial respiration are tightly functionally connected, this can be interpreted to mean that the stimulatory effectof NO on glycolysis in intact platelets may be produced by non-functional mitochondria.This can be really the case since NO donors are able to inhibit both mitochondrial respiration and platelet cytochrome oxidase. Interestingly, the concentrations of NO donors inhibiting mitochondrial respiration and cytochrome oxidase were similar to those stimulating glycolysis in intact platelets.
Studies performed on intact J774 cells have shown that mitochondrial complex I is inhibited only after a prolonged (6–18 h) exposure to NO and that this inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex (Clementi et al., 1998). Further studies are needed to establish whether long term exposure of platelets to NO affects Mitochondrial complexes I and II.
Comparison of the concentrations of SNP and SNAP affecting cytochrome oxidase activityand mitochondrial respiration with those reducing the platelet responses indicates that NO cannot significantly reduce platelet aggregation through the inhibition of oxidative energy production. By contrast, the concentrations of the NO donors inhibiting platelet secretion, mitochondrial respiration and cytochrome oxidase were similar. This and the fact that the platelet release reaction strongly depends on the oxidative energy production may suggest that in porcine platelets NO can affect platelet secretion through the inhibition of mitochondrial energy production at the step of cytochrome oxidase.

Taking into account that platelets may contain NO synthase and are able to produce significant amounts of NO (Berkels et al., 1997)it seems possible that nitric oxide can function in these cells as a physiological regulator of mitochondrial energy production.
Nitric oxide and platelet energy metabolism. M Tomasiak, H Stelmach, T Rusak and J Wysocka. Acta Biochimica Polonica 2004; 51(3):789–803

These authors previously investigated the bioenergetic consequences of activating J774.A1 macrophages (MФ) with interferon (IFN)γ and lipopolysaccharide (LPS) and found that there is a nitric oxide (NO)-dependent mitochondrial impairment and stabilization of hypoxia inducible factor (HIF)-1α, which synergize to activate glycolysis and generate large
quantities of ATP. We now demonstrate, using TMRM fluorescence and time-lapse confocal microscopy, that these cells maintain a high mitochondrial membrane potential (ΔΨm) despite the complete inhibition of respiration. The maintenance of high ΔΨm is due to the utilization of a significant proportion of glycolytically generated ATP as a defence mechanism against cell death. This is achieved by the reverse functioning of FoF1-ATP synthase and adenine nucleotide translocase (ANT). Treatment of activated MФ with inhibitors of either of these enzymes, but not with inhibitors of the respiratory chain complexes I to IV, led to a collapse in ΔΨm and to an immediate increase in intracellular [ATP], due to the prevention of ATP hydrolysis by the FoF1-ATP synthase. This collapse in ΔΨm was followed by translocation of Bax from cytosol to the mitochondria, release of cytochrome c into the cytosol, activation of caspase 3 and 9 and subsequent apoptotic cell death. Our results indicate that during inflammatory activation “glycolytically competent cells” such as MФ utilize significant amounts of the glycolytically-generated ATP to maintain ΔΨm and thereby prevent apoptosis.

Activated macrophages utilize glycolytic ATP to maintain mitochondrial membranepotential and prevent apoptotic cell death. A Garedew, SO Henderson, S Moncada. Cell Death and Differentiation. 2010. DOI : 10.1038/cdd.2010.27
The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM). SNP decreased total and coupled, but increased uncoupled oxygen consumption. This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation. Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP. Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished. Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production. In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels. However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level). ATP/ADP ratio and adenylate energy charge level were significantly decreased. In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes. These alterations were accompanied with instability of energy status.

Effects of Exogenous Donor of Nitric Oxide – Sodium Nitroprusside on Energy Production of Rat Reticulocytes. SD MALETIĆ, L M DRAGIĆEVIĆ-DJOKOVIĆ, BI OGNJANOVIĆ, RV ŽIKIĆ, AŠ ŠTAJN, MB SPASIĆ.
Physiol. Res. 2004;53: 439-447.

Key points to take from this:
1. The role of NO in regulating cellular death is in many organs and central to this function is the stabilization of mitochondria through sufficient levels of NO. High levels of eNO leads to mitochondrial dysfunction that increases the dependence of ATP generated from glycolysis.
2. This is accompanied by inhibition of oxidative phosphorylation and stimulation of glycolysis, which brings the discussion to a different domain – cancer growth and Warburgh Effect.
3. This is accompanied by PPAR activation, cytoplasmic NAD+ depletion, and inhibition of glycolysis (critical in cells dependent on aerobic glycolysis), depletion of total energy production, and apoptosis.
4. Maintenance of high glycolytic generation of ATP is essential for cellular defense, but the oxygen consumption is uncoupled.
5. NO donors inhibiting mitochondrial respiration and cytochrome oxidase are similar to those stimulating glycolysis

More    (pharmaceuticalintelligence.com)

Read Full Post »