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Scientists at MIT and Massachusetts General Hospital have discovered how cancer cells latch onto blood vessels and invade tissues to form new tumors — a finding that could help them develop drugs that inhibit this process and prevent cancers from metastasizing.
Cancer cells circulating in the bloodstream can stick to blood vessel walls and construct tiny “bridges” through which they inject genetic material that transforms the endothelial cells lining the blood vessels, making them much more hospitable to additional cancer cells, according to the new study.
The researchers also found that they could greatly reduce metastasis in mice by inhibiting the formation of these nanobridges. “Endothelial cells line every blood vessel and are the first cells in contact with any blood-borne element. They serve as the gateway into and out of tumors and have been the focus of intense research in vascular and cancer biology.
Building bridges
Metastasis is a multistep process that allows cancer to spread from its original site and form new tumors elsewhere in the body. Certain cancers tend to metastasize to specific locations; for example, lung tumors tend to spread to the brain, and breast tumors to the liver and bone.
To metastasize, tumor cells must first become mobile so they can detach from the initial tumor. Then they break into nearby blood vessels so they can flow through the body, where they become circulating tumor cells (CTCs). These CTCs must then find a spot where they can latch onto the blood vessel walls and penetrate into adjacent tissue to form a new tumor.
Blood vessels are lined with endothelial cells, which are typically resistant to intruders.
The researchers first spotted tiny bridges between cancer cells and endothelial cells while using electron microscopy to study the interactions between those cell types. They speculated that the cancer cells might be sending some kind of signal to the endothelial cells.
“Once we saw that these structures allowed for a ubiquitous transfer of a lot of different materials, microRNAs were an obvious interesting molecule because they’re able to very broadly control the genome of a cell in ways that we don’t really understand,” Connor says. “That became our focus.”
MicroRNA, discovered in the early 1990s, helps a cell to fine-tune its gene expression. These strands of RNA, about 22 base pairs long, can interfere with messenger RNA, preventing it from being translated into proteins.
In this case, the researchers found, the injected microRNA makes the endothelial cells “sticky.” That is, the cells begin to express proteins on their surfaces that attract other cells to adhere to them. This allows additional CTCs to bind to the same site and penetrate through the vessels into the adjacent tissue, forming a new tumor.
Non-metastatic cancer cells did not produce these invasive nanobridges when grown on endothelial cells.
Shutting down metastasis
The nanobridges are made from the proteins actin and tubulin (NB-the protein actin is abundant in all eukaryotic cells. It was first discovered in skeletal muscle, where actin filaments slide along filaments of another protein called myosin to make the cells contract. In non-muscle cells, actinfilaments are less organized and myosin is much less prominent, and a tubulin is a protein that is the main constituent of the micro-tubules of living cells, which also form the cytoskeleton that gives cells their structure). The researchers found that they could inhibit the formation of these nanobridges, which are about 300 microns long, by giving low doses of drugs that interfere with actin.
When the researchers gave these drugs to mice with tumors that normally metastasize, the tumors did not spread.
Sengupta’s lab is now trying to figure out the mechanism of nanobridge formation in more detail, with an eye toward developing drugs that act more specifically to inhibit the process.
The SEM picture below,
is a rounded cancer cell (top left) that sends out nanotubes connecting with endothelial cells. Genetic material can be injected via these nanotubes, transforming the endothelial cells and making them more hospitable to additional cancer cells. Image credit: Sengupta Lab. The second picture below,
is showing a cancer cell (bottom center) that creates a gap and enters the endothelial tube. Another cancer cell (middle right) sends out nanotubes to connect with endothelial cells. Both image are credited toSengupta Lab
An interesting comment on why plants do not develop cancer is given here, The article states that in plants, as in animals, most cells that constitute the organism limit their reproductive potential in order to provide collective support for the immortal germ line. And, as in animals, the mechanisms that restrict the proliferation of somatic cells in plants can fail, leading to tumors. There are intriguing similarities in tumorigenesis between plants and animals, including the involvement of the retinoblastoma pathway as well as overlap with mechanisms that are used for stem cell maintenance. However, plant tumors are less frequent and are not as lethal as those in animals. The authors of the article argue that fundamental differences between plant and animal development make it much more difficult for individual plant cells to escape communal controls.
The structure of the endothelium, the thin layer of cells that line our arteries and veins, is visible here. The endothelium is like a gatekeeper, controlling the movement of materials into and out of the bloodstream. Endothelial cells are held tightly together by specialized proteins that function like strong ropes (red) and others that act like cement (blue). In the picture here, the cell is preparing to divide. Two copies of each chromosome (blue) are lined
up next to each other in the center of the cell. Next, protein strands (red) will pull apart these paired
chromosomes and drag them to opposite sides of the cell. The cell will then split to form two daughter cells, each with a single, complete set of chromosomes. It is interesting that protein strands (red) in this picture are implicated on pulling apart the two copies of chromosomes in the same way the proteins actin and tubules do in the cancer cell interaction with the veins and arteries. It looks like the proteins shaped as strings, the nanobridges, due their shape to the tension development between cancer cells and the veins.
11.1.1 Electrically-driven modulation of surface-grafted RGD peptides for .. cell adhesion
11.1.2 The metabolic state of cancer stem cells—a target for cancer therapy
11.1.3 Regulation of tissue morphogenesis by endothelial cell-derived signals
11.1.4 Novel approach to bis(indolyl)methanes. De novo synthesis of 1-hydroxyimino-methyl derivatives with anti-cancer properties
11.1.5 Synthesis and Biological Evaluation of New 1,3-Thiazolidine-4-one Derivatives of 2-(4-Isobutylphenyl)propionic Acid molecules
11.1.6 Targeting pyruvate kinase M2 contributes to radiosensitivity of NSCLC cells
11.1.7 The tyrosine kinase inhibitor nilotinib has antineoplastic activity in prostate cancer cells but up-regulates the ERK survival signal—Implications for targeted therapies
11.1.8 PAF and EZH2 Induce Wnt.β-Catenin Signaling Hyperactivation
11.1.9 PAF Makes It EZ(H2) for β-Catenin Transactivation
11.1.10 PI3K.AKT.mTOR pathway as a therapeutic target in ovarian cancer
11.1.11 Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway
11.1.12 Curcumin-could-reduce-the-monomer-of-ttr-with-tyr114cys-mutationvia autophagy in cell model of familial amyloid polyneuropathy.
11.1.1 Electrically-driven modulation of surface-grafted RGD peptides for .. cell adhesion
Reported herein is a switchable surface that relies on electrically-induced conformational changes within surface-grafted arginine–glycine–aspartate (RGD) oligopeptides as the means of modulating cell adhesion
Stimuli-responsive surfaces that are capable of modulating their biological properties in response to an external stimuli, including temperature,1,2 light,3 magnetic field4 and electrical potential,5–9 are of growing interest for a variety of biological and medical applications.10,11 Switchable surfaces that can be controlled on-demand are playing an increasingly important part in the development of highly sensitive biosensors,12–15novel drug delivery systems16–18 and functional microfluidic, bioanalysis, and bioseparation systems.19–22Additionally, dynamic, synthetic surfaces that can control the presentation of regulatory signals to a cell are expected to have a significant impact in the field of tissue engineering and regenerative medicine, and to provide unprecedented opportunities in fundamental studies of cell biology.23,24 The availability of sophisticated and functional switchable surfaces is expected to emulate more complex in vivo like extracellular environments, and provide a powerful means to probe and control the dynamic interactions between the cell and its external environments.
The majority of studies on stimuli-responsive surfaces reported to date either rely25–29 on controlling non-specific interactions (i.e., hydrophobic/hydrophilic and electrostatic) of the biomolecules with the active surface, or have focused30–32 on demonstrating modulation of specific biomolecular interactions using relatively simple biological systems (e.g. biotin–streptavidin) and conditions (i.e. water or buffer solutions). For example, Zareie et al.30 fabricated a mixed self-assembled monolayer (SAM) on gold comprising oligo(ethylene glycol) (OEG) thiol molecules and shorter disulfides carrying biotin end-groups that regulated the interaction between biotin and streptavidin in water. The OEG thiols were able to switch in response to a change in temperature below and above their lower critical solution temperature (LCST = 37 °C). At 23 °C the structure of the OEG molecules was fully extended hindering the shorter biotin disulfide components. On the contrary, at 45 °C the OEG backbone collapsed, thus allowing the specific interaction between the biotin molecule on the surface and the protein streptavidin in solution. In our previous work,7–9 electrically controlled switching has been applied to regulate the conformational changes of modified positively charged oligolysine peptides tethered to a gold surface, such that biotin moieties incorporated into the oligolysines could be reversibly exposed or concealed on demand, as a function of surface potential. Switchable SAMs used to control biomolecular interactions via an electrical stimulus are particularly appealing because of their fast response times, ease of creating multiple individually addressable switchable regions on the same surface, as well as low-drive voltage and electric fields, which are compatible with biological systems.33 Our previous reported electrically switchable surface was able to control directly the biomolecular interactions between biotin and neutravidin in phosphate buffer saline (PBS) solution.
However, switchable surfaces have been scarcely used, thus far, to control biomolecular interactions on more complex systems such as those involving modulation of cell responsiveness.34–37 Jonkheijm and co-workers35 have reported a cucurbit[8]uril-based SAM system to electrochemically control the release of cells. Charged end groups on SAM surfaces have been exploited to electrically control the early stages of bacterial cell adhesion37 and form patterned surfaces with two independent dynamic functions for inducing cell migration.36 In spite of these efforts, given cellular complexity and diversity, such studies are very limited in number, as are the opportunities to further understand and control the complex interplay of events and interactions occurring within living cells.
Herein, we report on a stimuli-responsive surface that relies on electrically-induced conformational changes within surface-grafted arginine–glycine–aspartate (RGD) oligopeptides as the means of modulating cell adhesion. RGD, which is present in most of the adhesive ECM proteins (e.g. fibronectin, vitronectin, laminin and collagen), is specific for integrin-mediated cell adhesion.38 The RGD modified electrode is used here to dynamically regulate the adhesion of immune macrophage cells. The stimuli-responsive surface is fabricated on a gold surface and comprises a mixed SAM consisting of two components (Fig. 1): (i) an oligopeptide containing a terminal cysteine for attachment to the gold surface, three lysine residues as the main switching unit, and a glycine–arginine–glycine–aspartate–serine (GRGDS) as the recognition motif for cell adhesion –C3K-GRGDS, and (ii) an ethylene glycol-terminated thiol (C11TEG) to space out the oligopeptides. Since the charged backbone of the oligopeptide can be potentially harnessed7–9 to induce its folding on the surface upon an application of an electrical potential, we reasoned that such conformational changes can be employed to selectively expose under open circuit (OC) conditions (bio-active state) or conceal under negative potential (bio-inactive state) the RGD to the cell and dynamically regulate cell adhesion.
Fig. 1 Schematic of the dynamic RDG oligopeptide SAM utilised for controlling specific cellular interactions. The electrically switchable SAM exposes the RGD peptide and supports cell adhesion under open circuit (OC) conditions (no applied potential), while …
Mixed SAMs of C3K-GRGDS : C11TEG were formed from a solution ratio of 1 : 40 and characterised by X-ray photoelectron spectroscopy (XPS) (Fig. S2, ESI†). XPS analysis confirmed the formation of the C3K-GRGDS:C11TEG mixed monolayer and displayed signals from S, N, C and O. The chemical state of the sulphur atom was probed using the XPS spectra of the S 2p emission (Fig. S2, ESI†). The S 2p spectrum (Fig. S2a, ESI†) consists of two doublet peaks, with one doublet peak at 162.0 eV (S 2p3/2) and 163.2 eV (S 2p1/2), indicating that the sulphur is chemisorbed on the gold surface.39 A second small doublet peak can be observed at 163.8 eV and 165.0 eV, which can be attributed to the S–H bond, indicating a small presence of unbound sulphur. No sulphur peaks above 166 eV were observed, indicating that no oxidised sulphur is present at the surface. The N 1s spectrum (Fig. S2b, ESI†) can be de-convoluted into two peaks, which support the presence of the peptide on the surface. The first peak centred at 400.5 eV is attributed to amino (NH2) and amide (CONH) moieties. The second peak centred at 402.8 eV is ascribed to protonated amino groups.40 Note that no nitrogen peak was observed for pure C11TEG SAMs. The C 1s spectrum (Fig. S2c, ESI†) can be de-convoluted into three peaks, which are attributed to five different binding environments. The peak at 285.0 eV is attributed to C–C bonds,41 while the peak at 286.7 eV corresponds to C 1s of the three binding environments of C–S, C–N and C–O.41 The third and smaller peak (288.6 eV) is assigned to the C 1s photoelectron of the carbonyl moiety, C O.41 The O 1s spectrum (Fig. S2d, ESI†) is de-convoluted into two different peaks, corresponding to two different binding environments, arising from the C–O (533.3 eV) and C O (532.0 eV) bonds.41 From integrating the area of the S 2 p and N 1s peaks and taking into consideration that the C3K-GRGDS oligopeptide consists of 15 N atoms and 1 S atom and C11TEG has no N and 1 S atom only, it was possible to infer that the ratio of C3K-GRGDS:C11TEG on the surface is 1 : 10 ± 2. The presence of C11TEG was utilised not only to ensure sufficient spatial freedom for molecular reorientation of the surface bound oligopeptide, but also to stop non-specific binding to the surface.
The C3K-GRGDS:C11TEG mixed SAMs were shown to support adhesion of immune macrophage cells as determined by cell counting42,43 (Fig. 2). When RAW 264.7 mouse macrophages were cultured on theC3K-GRGDS:C11TEG mixed SAM in supplemented Dulbecco’s Modified Eagle Medium (DMEM), the number of cells adhered to the surface increased with incubation time, reaching 1792 ± 157 cells per mm2after 24 hours. This is in contrast with the weak cell adhesion observed in two control surfaces, pureC11TEG SAMs and clean gold, in which the number of cells that adhere was 60% and 50% lower, respectively, after 24 hours (Fig. 2).
Fig. 2Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAM, pure C11TEG SAM and bare gold surfaces that were normalized against the density of cells adherent onto the C3K-GRGDS:C11TEG mixed SAM. The surfaces were cultured in RAW 264.7 mouse macrophage cells under OC conditions for 24 hours.
In order to demonstrate that the C3K-GRGDS:C11TEG mixed SAMs can support or resist cell adhesion on demand, the macrophage cells were cultured on the C3K-GRGDS:C11TEG mixed SAM in DMEM medium under OC conditions and applied negative potential (–0.4 V) for a period of 1 h. Note that DMEM contains a mixture of inorganic salts, amino acids, glucose and vitamins. On application of the applied potential of –0.4 V the number of adherent cells was 70% less compared to the C3K-GRGDS:C11TEGmixed SAMs under OC conditions, Fig. 3. Similar switching efficiencies have been observed in another oligopeptide system using different DMEM solutions.44 These findings suggest that the negative potential induces the conformational changes in the C3K moiety of C3K-GRGDS in the SAM which in turn leads to the RGD moiety being concealed and hence reducing the binding of the cells.
Fig. 3 Density of adhered cells on C3K-GRGDS:C11TEG, C11TEG, C6EG-GRGDS:C11TEG mixed SAMs that were normalized against the density of cells adherent onto the C3K-GRGDS:C11TEG mixed SAM. The surfaces were cultured in RAW 264.7 for 1 h under OC conditions or while applying –0.4 V.
Previous studies have shown that small conformational and orientational changes in proteins and peptides modulate the availability and potency of the active sites for cell surface receptors.45–47 Thus, in a similar manner, small changes in the conformation/orientation of the RGD peptide on the surface induced by application of an electrical potential are able to affect the binding activity of the peptide. Recently, we have conducted detailed theoretical8 and experimental9 studies aimed at understanding the switching mechanism of oligopeptide-based switchable surfaces, that similarly as in the case of the C3K-GRGDS:C11TEG mixed SAMs, use lysine residues to act as the switching unit. These previous studies unraveled that the surface-appended oligolysines undergo conformational changes between fully extended, partially extended and collapsed conformer structures in response to an applied positive potential, open circuit conditions and negative electrical potential, respectively. Thus, these previous findings allow us to propose that when a negative potential is applied to the GRGDS:C11TEG mixed SAM surface, the oligopeptide chain adopts a collapsed conformation on the surface and the RGD binding motif is partially embedded on the C11TEGmatrix, thus showing no bioactivity (“OFF” state).
In order to verify that the changes in adhesion upon application of a negative surface potential occur due to changes in the conformational orientation of the RGD instead of cell repulsion or cell damage due to the presence of an electrical potential, control mixed SAMs were also prepared using C11TEG and a peptide where the 3 lysine residues as the switching unit were replaced by 6 non-switchable ethylene glycol units –C6EG-GRGDS (Fig. S1, ESI†). Fig. 3 demonstrates that cells adhered in similar numbers to the C11TEGand C6EG-GRGDS:C11TEG mixed SAMs under OC conditions and an applied negative potential. These results provide strong evidence that control over cell adhesion using the C3K-GRGDS:C11TEG mixed SAM is due to a conformational behaviour of the lysine-containing oligopeptide that can either expose or conceal the RGD moiety.
Cell viability was checked following application of –0.4 V for 1 h by performing a trypan blue assay. Cells that were dead were stained blue due to a break down in membrane integrity. Incubation of the cells under a negative potential had negligible effect on cell viability, which was greater than 98%. Cyclic voltammetric studies (outlined in detail in the Fig. S3, ESI†) were also performed to demonstrate that no significant faradaic process occur over the potential range studied, and thus ions are not participating in redox reactions and consequently redox chemistry is not being significantly affected by application of the potential used. In agreement with other studies,35,36,48 we conclude that the electrical modulation of the surface neither affected cell viability nor induced any redox process in the medium that could have had an effect on cells.
We then addressed the question of whether the C3K-GRGDS:C11TEG surfaces could be switched between different cell adhesive states (cell-resistant and cell-adhesive states). To begin with, we investigated the switching from a cell-adhesive state to a cell-resistant state, and the possibility to detach the cells from the substrate upon the application of a negative potential. Cells were incubated in the C3K-GRGDS:C11TEGmixed SAMs for 1 h under OC conditions, thereby exposing the RGD moiety and allowing for cell attachment. This step was followed by the application of a potential of –0.4 V for 1 h in order to detach the cells from the surface, by concealing the RGD moieties. Cell counts showed no significant differences between the pre and post application of the –0.4 V, suggesting that the electrostatic force generated by the applied negative electrical potential might not be sufficient to disrupt the RGD–integrin interaction. These results were to a certain extent expected since adherent cells are able to withstand strong detachment forces due to the adhesion being mediated by multiple RGD–integrin bonds in parallel.49
In contrast, a reversal of the switching sequence demonstrated that our surfaces can be dynamically switched from a non-adhesive to cell-adhesive state. Cells were incubated in the C3K-GRGDS:C11TEG mixed SAMs for 1 h while holding the potential at –0.4 V for 1 h making the RGD peptide inaccessible for recognition by the corresponding integrin. As above, the number of adherent cells when a negative potential of –0.4 V was applied was 70% of the number that adhered to the C3K-GRGDS:C11TEG mixed SAMs under OC conditions, Fig. 4. The potential was then shifted to open circuit conditions for 1 h on those exposed to a potential of –0.4 V, which resulted in a significant increase in the number of cells as a result of the exposure of the RGD moiety to the cells (Fig. 4). These values were similar to those obtained for the samples that were only incubated for 1 hour under OC conditions (Fig. 4), indicating that the surfaces were highly effective at switching from a non-adhesive to cell-adhesive state.
Fig. 4Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAMs that were incubated with cells for 1 h while applying –0.4 V and subsequently in OC conditions for 1 h. The density was normalized against the density of cells adherent onto C3K-GRGDS:C11TEG mixed SAMs that were incubated with cells in OC conditions for 1 h.
In summary, an electrically switchable surface has been devised and fabricated that is capable of efficiently exposing and concealing the RGD cell adhesion motif and dynamically regulate the adhesion of immune macrophage cells. This study will no doubt be useful in developing more realistic dynamic extracellular matrix models and is certainly applicable in a wide variety of biological and medical applications. For instance, macrophage cell adhesion to surfaces plays a key role in mediating immune response to foreign materials.50 Thus, development of such dynamic in vitro model systems that can control macrophage cell adhesion on demand are likely to provide new opportunities to understand adhesion signaling in macrophages51 and develop effective approaches for prolonging the life-span of implantable medical devices and other biomaterials.52
11.1.2 The metabolic state of cancer stem cells—a target for cancer therapy
Bulk tumor cell populations rely on aerobic glycolysis.
Cancer stem cells are in a specific metabolic state.
Cancer stem cells in breast cancer, glioblastoma, and leukemia rely on oxidative phosphorylation of glucose.
In the 1920s Otto Warburg first described high glucose uptake, aerobic glycolysis, and high lactate production in tumors. Since then high glucose uptake has been utilized in the development of PET imaging for cancer. However, despite a deepened understanding of the molecular underpinnings of glucose metabolism in cancer, this fundamental difference between normal and malignant tissue has yet to be employed in targeted cancer therapy in the clinic. In this review, we highlight attempts in the recent literature to target cancer cell metabolism and elaborate on the challenges and controversies of these strategies in general and in the context of tumor cell heterogeneity in cancer.
11.1.3 Regulation of tissue morphogenesis by endothelial cell-derived signals
Endothelial cells lining blood vessels induce organ formation and other morphogenetic processes in the embryo.
Blood vessels are also an important source of paracrine (angiocrine) signals acting on other cell types in organ regeneration.
Vascular niches and endothelial cell-derived signals generate microenvironments for stem and progenitor cells.
Endothelial cells (ECs) form an extensive network of blood vessels that has numerous essential functions in the vertebrate body. In addition to their well-established role as a versatile transport network, blood vessels can induce organ formation or direct growth and differentiation processes by providing signals in a paracrine (angiocrine) fashion. Tissue repair also requires the local restoration of vasculature. ECs are emerging as important signaling centers that coordinate regeneration and help to prevent deregulated, disease-promoting processes. Vascular cells are also part of stem cell niches and have key roles in hematopoiesis, bone formation, and neurogenesis. Here, we review these newly identified roles of ECs in the regulation of organ morphogenesis, maintenance, and regeneration.
The examples provided in this review highlight the important roles of ECs in tissue development, patterning, homeostasis, and regeneration. The endothelium often takes a central position in these processes and there are many reasons why ECs are ideally positioned as the source of important instructive, angiocrine signals. The vascular transport network extends into every organ system and needs to be embedded in those tissues in a certain spacing or pattern, which places ECs in central and, therefore, strategic positions for the regulation of morphogenesis and organ homeostasis.
Given that ECs and other cell types frequently form functional units, such as kidney glomeruli, liver lobules, or lung alveoli, the assembly, differentiation, and function of the different cellular components needs to be tightly coordinated. In addition, because circulating blood cells extensively rely on the vascular conduit system and frequently interact with the endothelium, it is perhaps not surprising that ECs contribute to niche microenvironments. During tissue repair, proliferative cell expansion processes are sometimes temporally separated from cell differentiation and tissue patterning events. The latter has to involve the restoration of a fully functional vascular network so that ECs appear ideally suited as the source of molecular signals that can trigger or suppress processes in the surrounding tissue.
11.1.4 Novel approach to bis(indolyl)methanes. De novo synthesis of 1-hydroxyimino-methyl derivatives with anti-cancer properties
A versatile and broad range approach to previously unknown bis(indolyl)methane oximes based on two consecutive hetero Diels-Alder cycloaddition reactions of electrophilic conjugated nitrosoalkenes with indoles is disclosed. The cytotoxic properties and selectivity of some adducts against several human cancer cell lines pointing to a promising role in the development of anti-tumoural drugs, in particular for leukemia and lymphoma.
The one-pot synthetic strategy to bis(indolyl)methanes is outlined in Scheme 3. The starting a,a 0-dihalogenooximes 3 were efficiently prepared from the respective ketones by known procedures [58,61]. These compounds, in the presence of base, were converted, in situ, into the corresponding transient and reactive nitrosoalkenes 4, which were intercepted bya first molecule of the appropriate indole 5 originating the intermediate indole oximes 6. The initially formed tetrahydroxazines undergo ring-opening to the corresponding oximes, under the driving force of the energy gain on rearomatisation. Subsequent dehydro-halogenation of 6 produces nitrosoalkenes 7 which reacted with a second molecule of indole, producing the target bis(indolyl)methanes 8. The results obtained are summarised in Table 1.
The reaction yields may be considered generally good, taking into account that the synthetic process involves a sequence of reactions. On the other hand, no other products could be obtained, which indicates that the reactions were regioselective. The results have shown also that both alkyl and aryl oximes can be used in the synthesis of bis(indolyl)methanes. Starting from aryl oximes 3aef the expected (E) oximes 9 were obtained as single or major products (Entries 1e11) whereas alkyl oxime 3g reacted with indole to give the (Z)-oxime 10g as the major product (Entries 12e13). The stereochemistry assignment of oximes 9 and 10 was confirmed by analysis of the NOESY spectra of 9d, 9g, 10d and 10g. In the spectra of 10d and 10g, connectivity was observed between the hydroxyl proton and the phenyl protons and the methyl protons, respectively, whereas in the case of 9d and 9g no connectivity was observed. Moreover, oximes 9 and 10 are also characterized by 1H NMR spectra with different features. The chemical shift of the methylenic proton appears at higher value for (E)-oximes 9 (9b: δ 6.81 ppm; 9d: δ = 6.82 ppm; 9g: δ = 6.39 ppm) than for the corresponding (Z) oximes 10 (10b: δ = 5.74 ppm; 10d: δ = 5.77 ppm; 10g: δ = 5.41 ppm).
The synthesis of two isomeric oximes from the reaction of arylnitrosoethylenes with pyrrole and dipyrromethanes has been previously observed [62]. The process was rationalized considering the conjugate addition of the heterocycle to the nitrosoalkene, at the s-cis or s-trans conformation, followed by rearomatization of the pyrrole unit leading to (E)- and (Z)-oxime, respectively. Thus, the synthesis of the BIM oximes via 1,4-conjugate addition of indole to the nitrosoelkene cannot be ruled out.
The use of water as solvent in Diels- Alder reactions has been shown to be advantageous, not only in environmental terms but also inducing critical improvements in reaction times, yields and selectivity [51,63]. We observed that carrying out the synthesis of bis(indolyl)methanes in water using dichloromethane as co-solvent is a valuable alternative to the use of dichloromethane as the only solvent. Generally the yields were better or comparable to those obtained in dichloromethane and reaction time significantly shorter (the reaction time was reduced from 36 h to 3 h). Clearly the efficiency of the reaction, using H2O/CH2Cl2 system, amongst the nitrosoalkenes bearing halogenated aryl substituents increases in the order F > Cl > Br > H the order of electron withdrawing ability and consequently the order of the expected effectiveness for an inverse electron demand Diels-Alder reaction (entries 2, 5, 7 and 9). However, the isolated yields from the reaction carried out in CH2Cl2 do not reflect the expected reactivity, which can be explained considering differences in the efficiency of the purification process.
The cytotoxicity of compounds 9a, 9e and 9d was evaluated in different tumorl cell lines, namely HepG2 (hepatocellular carcinoma), MDA-MB-468 (human breast carcinoma), RAW 264.7 (murine leukemic monocyte macrophages), THP1 (human acute monocytic leukaemia), U937 (human leukaemic monocytic lymphoma) and EL4 cells (murine T-lymphoma). The compounds’ selectivity towards tumoural cells was assessed determining their cytotoxicity with respect to two non-tumoural derived cell lines S17 (murine bone marrow) and N9 cells (murine microglial). Results of the half maximal concentrations (IC50) are shown in Table 2 together with the toxicity of etoposide, a known antitumoural drug. Compound 9e was considerably less cytotoxic on tumor cell lines than the other two compounds, with IC50 values ranging from 35.7 (HepG2) to 124 mM (THP1) and was not selective. Compounds 9a and 9d, however, were considerably cytotoxic to all cells tested, with IC50 values ranging from 1.62 (THP1) to 23.9 mM (RAW) and from 10.7 (MDA) to 34.1 mM (U937), respectively. Compound 9a was particularly active against non-adherent cell lines with IC50 values ranging from 1.62 in THP1 to 1.65 mM in EL4.
Some conclusions regarding structure activity relationships can be redrawn based on the biological evaluation of these bis(indolyl)methanes. There is a dramatic difference in anticancer activitybetweenN-unsubstituted bis(indolyl)methanes 9a and the Nmethyl substituted derivative 9e, the latter characterized by high IC50 values. On the other hand, the significantly lower IC50 values observed for 9a for non-adherent cell lines in comparisonwith the ones obtained for 9d demonstrates that the presence of the bromo substituent leads to higher cytotoxic activity.
The observed high cytotoxicity of compound 9a against THP1, EL4 and U937 cell lines led us to extend the study to BIMs 9c, 9g and 10g (Table 3). Compound 9c, bearing a 4-fluorophenyl substituent, showed moderate anti-cancer activity which reinforces the observation that the 4-bromophenyl group is crucial to ensure low IC50 values. On the other hand, alkyl oximes 9g and 10g were even less cytotoxic against THP1, EL4 and U937 cell lines. None of these compounds were selective towards the tumor cell lines (selectivity index calculated for non-tumour cell line S17). In addition to having displayed higher toxicity towards the nontumor cell lines than all the studied compounds, compound 9a demonstrated the highest selectivity indexes: 9.86-14.2. Further studies using 9a as scaffold in the development of anti-tumoural drugs for leukaemia and lymphoma is worth pursuing since it presents lower IC50 and higher selectivity than etoposide.
Conclusions
The reliable preparation of a variety of unknown BIMs bearing different oxime substituents at the methylene bridge was presented. This strategy, supported on the robust and proved methodology of Diels-Alder cyclo addition reactions of electrophilic nitrosoalkenes with electron rich indoles, may pave the way for the synthesis of a vast library of new compounds.
Table 1 Preparation of bis(indolyl)methane oxime
Scheme 1. Selected biological active bis(indolyl)methanes.
Scheme 2. Common methods for BIMs’ preparation [27e44].
Scheme 3. Synthetic strategy towards BIM oximes.
Synthesis of a new bis(indolyl)methane that inhibits growth and induces apoptosis in human prostate cancer cells
The synthesis and the antiproliferative activity against the human breast MCF-7, SkBr3 and the prostate LNCaP cancer cell lines of a series of bis(indolyl)methane derivatives are reported. The synthesis of new compounds was first accomplished by the reaction of different indoles with trimethoxyacetophenone in the presence of catalytic amounts of hydrochloric acid. A second procedure involving the use of oxalic acid dihydrate [(CO2H)2·2H2O] and N-cetyl-N,N,N-trimethylammonium bromide in water was carried out and led to better yields. Compound 5b significantly reduced LNCaP prostate cancer cell viability in a dose-dependent manner, with an IC50 of 0.64 ± 0.09 μM. To determine whether the growth inhibition was associated with the induction of apoptosis, treated cells were stained using DAPI. LNCaP cells treated with 1 μM of 5b showed the morphological changes characteristic of apoptosis after 24 h of incubation.
11.1.5 Synthesis and Biological Evaluation of New 1,3-Thiazolidine-4-one Derivatives of 2-(4-Isobutylphenyl)propionic Acid molecules
New thiazolidine-4-one derivatives of 2-(4-isobutylphenyl)propionic acid (ibuprofen) have been synthesized as potential anti-inflammatory drugs. The structure of the new compounds was proved using spectral methods (FR-IR, 1H-NMR, 13C-NMR, MS). The in vitro antioxidant potential of the synthesized compounds was evaluated according to the total antioxidant activity, the DPPH and ABTS radical scavenging assays. Reactive oxygen species (ROS) and free radicals are considered to be involved in many pathological events like diabetes mellitus, neurodegenerative diseases, cancer, infections and more recently, in inflammation. It is known that overproduction of free radicals may initiate and amplify the inflammatory process via upregulation of genes involved in the production of proinflammatory cytokines and adhesion molecules. The chemical modulation of acyl hydrazones of ibuprofen 3a–l through cyclization to the corresponding thiazolidine-4-ones 4a–n led to increased antioxidant potential, as all thiazolidine-4-ones were more active than their parent acyl hydrazones and also ibuprofen. The most active compounds are the thiazolidine-4-ones 4e, m, which showed the highest DPPH radical scavenging ability, their activity being comparable with vitamin E.
In order to improve the anti-inflammatory effect and safety profile of representative NSAIDs, one research strategy is derivatization of the carboxylic acid group with various heterocyclic systems (oxazole, izoxazole, pyrazole, oxadiazole, thiazole, thiadiazole, triazole, etc.) [9,10]. In the past two decades there has been considerable interest in the role of reactive oxygen species (ROS) in inflammation [11]. ROS mediate the oxidative degradation of cellular components and alteration of protease/antiprotease balance with damage to the corresponding tissue. In the early stages of the inflammatory process, ROS exert their actions through activation of nuclear factors, such as NFkB or AP-1, that induce the synthesis of cytokines. In later stages, endothelial cells are activated due to the synergy between free radicals and cytokines, promoting the synthesis of inflammatory mediators and adhesion of molecules. In the last step free radicals react with different cellular components (trypsin, collagen, LDL, DNA, lipids) inducing the death of cells [12,13].
The thiazolidine-4-one moiety is a heterocycle that has received more attention in the last years due its important biological properties [14]. Many effects have been found, including anti-inflammatory and analgesic [15], antitubercular [16], antimicrobial and antifungal [17], antiviral, especially as anti-HIV agents [18], anticancer, antioxidants [19], anticonvulsants [20] and antidiabetic activity [21]. In the present study, some new derivatives of ibuprofen that contain thiazolidine-4-one scaffolds were synthesized in order to obtain compounds with double effect—antioxidant and anti-inflammatory properties. The structures of the compounds were assigned based on their spectral data (FT-IR, 1H-NMR, 13C-NMR, MS) and the compounds were screened for their in vitro antioxidant potential.
The 1,3-thiazolidine-4-one derivatives 4a–m were synthesized in several steps using the method summarized in Scheme 1 and Table 1. First 2-(4-isobutylphenyl)propionic acid (ibuprofen, 1) was reacted with thionyl chloride, followed by treatment with dry ethanol to get 2-(4-isobutylphenyl)propionic acid ethyl ester, which was turned in 2-(4-isobutylphenyl)propionic acid hydrazide (2) by reaction with 66% hydrazine hydrate [22]. The condensation of compound 2 with various aromatic aldehydes allowed the preparation of the corresponding hydrazone derivatives 3a–l in satisfactory yields. Finally, the hydrazone derivatives of ibuprofen upon reaction with mercaptoacetic acid led to the thiazolidine-4-one derivatives 4a–l in moderate to good yields. By reduction of compound 4g in presence of tin chloride and few drops of acetic acid in ethanol, the thiazolidine-4-one 4m was obtained in 90% yield. Acetylation of 4m with acetyl chloride gave thiazolidine-4-one 4n in moderate yield.
In the acyl hydrazone series most of the the tested compounds showed a radical scavenging ability comparable with ibuprofen (Table 4). The most active compounds were 3e and 3f which are about three times and two times more active than their parent compound, respectively. The scavenging ability of the acyl hydrazones was improved by cyclization to the corresponding thiazolidine-4-one derivatives, these compounds all being more active than ibuprofen, except for compound 4j which contains a CF3 group in the metaposition of phenyl ring (Table 5). The most active compounds were 4e and 4m which contain NO2 and NH2 groups in ortho and paraposition of the phenyl ring, respectively. For these compounds the radical scavenging ability (%) was 94.42 ± 0.43 and 94.88 ± 0.57, which means that the compounds are about 23 times more active than ibuprofen (4.15 ± 0.22). The activity of these compounds is comparable with that of vitamin E used as positive control. Important radical scavenging ability was also shown by compound 4b(81.31 ± 0.55), which contains a Cl group in the para position of the phenyl ring, the compound being 20 times more active than ibuprofen.
The acyl hydrazone derivatives showed an antioxidant activity comparable with ibuprofen. The most active compound in this series was 3h, with radical scavenging activity of 13.31 ± 0.81, which means that this compound is three times more active than ibuprofen (4.42 ± 0.18). In the thiazolidine-4-one series the most active compounds were 4b, 4e and 4k, which contain Cl(4), NO2(2) and CN(4), respectively, as substituents on the phenyl ring. These compounds, which showed a scavenging ability of around 50%, are 12 times more active than ibuprofen. In comparison with the corresponding acyl hydrazones 3b, 3e and 3k the thiazolidine-4-ones were 10 times (4b), seven times (4e) and 13 times (3k) more active. The improved antiradical activity of acyl hydrazones by cyclization to form thiazolidine-4-ones was also observed for compounds 3d, 3f and 3g. The most favorable influence was observed for acyl hydrazone 4g, which contains a NO2 in the para position of the phenyl ring. The corresponding thiazolidine-4-one (4g, 37.14 ± 1.10) is 22 times more active than 3g (1.67 ± 0.35). These data strongly support the favorable influence of the thiazolidine-4-one ring on the antioxidant potential of these compounds. The tested compounds were less active than vitamin E.
In this study new heterocyclic compounds that combine the thiazolidine-4-one structure with the arylpropionic acid one have been synthesized. The structure of the new compounds was proved using spectral methods (IR, 1H-NMR, 13C-NMR, MS). The compounds were evaluated for their antioxidant effects using in vitro assays: total antioxidant activity, DPPH and ABTS radical scavenging ability. All thiazolidin-4-one derivatives 4a–n showed improved antioxidant effects in comparison with the corresponding acyl hydrazones 3a–l and ibuprofen, the parent compound. The encouraging preliminary results illustrate the antioxidant potential of the synthesized compounds and motivate our next research focused on their anti-inflammatory effects in chronic and acute inflammation models.
11.1.6 Targeting pyruvate kinase M2 contributes to radiosensitivity of NSCLC cells
Aerobic glycolysis, a metabolic hallmark of cancer, is associated with radioresistance in non-small cell lung cancer (NSCLC). Pyruvate kinase M2 isoform (PKM2), a key regulator of glycolysis, is expressed exclusively in cancers. However, the impact of PKM2 silencing on the radiosensitivity of NSCLC has not been explored. Here, we show a plasmid of shRNA-PKM2 for expressing a short hairpin RNA targeting PKM2 (pshRNA-PKM2) and demonstrate that treatment with pshRNA-PKM2 effectively inhibits PKM2 expression in NSCLC cell lines and xenografts. Silencing of PKM2 expression enhanced ionizing radiation (IR)-induced apoptosis and autophagy in vitro and in vivo, accompanied by inhibiting AKT and PDK1 phosphorylation, but enhanced ERK and GSK3β phosphorylation. These results demonstrated that knockdown of PKM2 expression enhances the radiosensitivity of NSCLC cell lines and xenografts as well as may aid in the design of new therapies for the treatment of NSCLC.
Knockdown of PKM2 expression increases the sensitivity of NSCLC cells to radiotherapy in vitro
To examine PKM2 expressions levels in the normal lung epithelial cell and the NSCLC cell lines, we evaluated the expression levels of PKM2 in normal lung bronchial epithelial cell BEAS-2B and five NSCLC cell lines including A549, H460, H1299, H292, and H520 by Western blotting assays, and our results demonstrated that PKM2 expression was elevated in almost five NSCLC cell lines examined compared to autologous normal lung bronchial epithelial cell, although the expression levels fluctuated slightly depending on the different cell lines (Fig.1A). To test the role of PKM2 in the sensitivity of NSCLC to radiotherapy, we generated plasmids of pshRNA-PKM2 and control pshRNA-Con by inserting the DNA fragment for a pshRNA specifically targeting the PKM2 or control into the pGenesil2 vector. After demonstrating the authenticity, A549 and H460 cells were transfected with the plasmid for 48h and the levels of PKM2 expression were tested by Western blot assays. Obviously, transfection with control plasmid did not significantly modulate PKM2 expression; while transfection with pshRNA-PKM2 reduced the levels of PKM2 expression (Fig.1B and Appendix: Supplementary Fig.S1A). Quantitative analysis revealed that transfection with pshRNA-PKM2 significantly reduced PKM2 expressions as compared with that in the mock-treated and control pshRNA-Con plasmid-transfected cells, respectively (p<0.05, Fig.1C). Mock-treated and pshRNA-PKM2-trasnfected A549 and H460 cells were subjected to IR (4Gy), and 12 and 24h after IR, these cells, together with un-irradiated mock-treated, pshRNA-Con-transfected, and pshRNA-PKM2-trasnfected cells, were tested for cell viability by trypan blue staining. Knockdown of PKM2 reduced the percentage of A549 viable cells by 12.6–20% and IR treatment decreased the frequency of viable cells by 17.1–28.2%. However, the percentages of viable cells in the PKM2-silencing and irradiated cells were reduced by 27.7–48.7%, which were significantly lower than that in other groups (Fig.1D, p<0.05). Furthermore, it was consistent with the above results of A549 cells that knockdown of PKM2 significantly reduced the percentage of H460 viable cells (Appendix: Supplementary Fig.S1B). In addition, to further validate PKM2 silencing on their radiosensitivity,unirradiated control, mock-treated, and pshRNA-PKM2 transfected A549 cells were subjected to IR (0, 2, 4, 6, and 8Gy), and two weeks after IR, these cells were tested for the capacity for colony formation. The results showed that the numbers of colonies formed by pshRNA-PKM2 cells were significantly decreased compared with that of mock-treated and control cells; however, there was no significant change in mock-treated cells compared with control cells. These results suggested that pshRNA-PKM2 cells were more sensitive to IR than mock-treated and control cells (Fig.1E and F). Given that IR usually causes DNA double-strand breaks [28], we characterized the frequency of γ-H2AX nuclear foci positive cells by immunofluorescent assays. While IR treatment dramatically increased the frequency of γ-H2AX+ cells, the same dose of IR further significantly increased the percentages of γ-H2AX+ cells when combined with PKM2 silencing at 12 and 24h after IR, and there was a significant difference in γ-H2AX+ cells between these two groups at 12 and 24 h after IR (Fig. 1G and H, p < 0.05).
Fig. 1. The PKM2 expression levels in the normal lung epithelial cell and the NSCLC cell lines and knockdown of PKM2 expression enhance the radiosensitivity of A549 cells in vitro. The expression levels of PKM2 in normal lung bronchial epithelial cell BEAS-2B and five NSCLC cell lines including A549, H460, H1299, H292, and H520 were determined by Western blotting assay (A). A549 cells were transfected with pshRNA-PKM2 or pshRNA-Con plasmid for 48h, and the levels of PKM2 expression were determined by Western blot assays using a PKM2-specific antibody and β-actin as an internal control (B and C). Data are representative images or expressed as mean±SD of the relative levels of PKM2 to control β-actin in individual groups of cells from three separate experiments. # p
Knockdown of PKM2 enhances IR-induced apoptosis in NSCLC cells
Next, we tested the impact of PKM2-silencing on IR-induced cell death types. One day after IR, the apoptotic cells in the irradiatedmock-treated,pshRNA-PKM2-trasnfected cells, and one group of cells that had been pre-treated with 30μM Z-VAD for 1h prior to IR, together with mock-treated, unirradiated pshRNA-Contransfected, and pshRNA-PKM2-trasnfected groups of cells were characterized by TUNEL assays and/or FACS analysis (Fig.2A and C). In comparison with that in mock-treated and control plasmid transfected cells, the frequency of apoptotic cells in the PKM2 silencing or IR-treated cells increased moderately, while the percentages of apoptotic cells in the cells receiving combined treatment with IR and PKM2-silencing were significantly greater. However, the frequency of apoptotic cells in the Z-VAD-pretreated cells was partially reduced. Apparently, knockdown of PKM2 and IR induced apoptosis in NSCLC cells in vitro (Fig. 2B and D, and Appendix: Supplementary Fig.S1C).
Fig. 2. Knockdown of PKM2 expression enhances IR-induced apoptosis in A549 cells. A549 cells were transfected with, or without, pshRNA-Con or pshRNA-PKM2 for 48h and treated with, or without, Z-VAD for 1h. Subsequently, the cells were subjected to IR, and 24h later, the frequency of apoptotic cells was determined by TUNEL assays and FACS. (A and C) TUNEL and FACS analyses of apoptotic cells. (B and D) Quantitative analysis of the percentage of apoptotic cells. Data are representative images or expressed as mean%±SD of individual groups of cells from three independent experiments. * p
Knockdown of PKM2 enhances IR-induced autophagy in NSCLC cells
The cell autophagy is characterized by the formation of numerous autophagic vacuoles, autophagosome, in the cytoplasm and elevated levels of the microtubule-associated protein 1 light chain 3 (LC3)-II [29]. To test the impact of PKM2 silencing on IR-induced autophagy, the presence of autophagosome in mock-treated, pshRNACon-transfected, pshRNA-PKM2-transfected, IR-treated alone, IR + pshRNA-PKM2-transfected, and 1 mM 3-MA-pretreated IR + pshRNA-PKM2-transfected cells was characterized by electronic microphotography (EM). Intriguingly and importantly, numerous autophagosomes were detected in the IR + pshRNAPKM2-transfected cells, and only a few were detected in the sensitivity of the NSCLC cells to radiotherapy in vitro. It was noted that pshRNA-Con had almost no effect on A549 cells, therefore, some subsequently experiments did not set this group.
Fig. 3. Knockdown of PKM2 and IR induce A549 cell autophagy. A549 cells were transfected with, or without, pshRNA-Con or pshRNA-PKM2 for 48h and treated with, or without, 3-MA for 1h. Subsequently, the cells were subjected to IR, and 2h later, the presence of autophagic vacuoles and autolysosomes in A549 cells was determined by EM and the relative levels of LC3-I, LC3-II, AKT, ERK1/2, and control β-actin expression and AKT, ERK1/2, GSK3β, PDK1 phosphorylation were determined by Western blot assays using specific antibodies. Data are representative images and expressed as mean values of the relative levels of target protein to control in individual groups of cells from three separate experiments. The relative levels of target protein to control in mock-treated cells were designated as 1. (A) EM analysis of autophagic vacuoles and autophagosomes. Black arrows point to autophagic vacuoles and autophagosomes in the cytoplasma of A549 cells. (B) Western blot analysis of LC3-I and LC3-II expression. The values indicate the ratios of the relative levels of LC3-II to LC3-I in individual groups. (C) Western blotting analysis of individual signal events. The values indicate the relative levels of target protein to control β-actin in individual groups of cell
Fig. 4. The impact of 3-MA or/and V-ZAD on cell viability, colony formation, apoptosis and autophagy in A549 cells. A549 cells were transfected with, or without, pshRNACon or pshRNA-PKM2 for 48h and pre-treated with, or without, 3-MA or V-ZAD for 1h, respectively. Subsequently, the cells were subjected to IR. Twenty-four hours later and two weeks, the viability, apoptosis, and colony formation were determined. Two hours after treatment, autophagy and the relative levels of LC3-I and LC3-II expression in different groups of cells were determined. Data are representative images and expressed as mean%±SD of individual groups of cells from three separate experiments. (A) The percentages of viable cells. (B) The capacity of cell colony formation. (C) Quantitative analysis of apoptotic cells. (D) Western blot analysis of LC3-I and LC3-II expression. The values indicate the ratios of LC3-II to LC3-I in individual groups of cells. * p
Fig. 5. Treatment with pshRNA-PKM2 enhances the IR-inhibited growth of implanted tumors in mice. The nude mice were inoculated with A549 cells and when the tumor grew at 50mm3 in one dimension, the mice were randomized and treated with vehicle (PS), plasmid of pshRNA-Con or pshRNA-PKM2 alone or IR (4Gy×7f) alone or in combination with pshRNA-PKM2 and IR, respectively. The body weights and tumor growths of individual mice were monitored longitudinally. At the end of the in vivo experiment, the tumor tissues were dissected out and the frequency of apoptotic cells, the presence of autophagosomes and the expression of PKM2 were determined by TUNEL, EM and immunohistochemistry, respectively. Data are representative images or expressed as mean±SD of individual groups of mice (n=6 per group). (A) The body weights of mice. (B and C) The tumor growth curve of implanted tumors and the log-transformed tumor growth curve of implanted tumors in mice. (D) Quantitative analysis of the frequency of apoptotic cells.(E) EM analysis of autophagy. (F)The expression of PKM2.(G) Quantitative analysis of PKM2 expression.The cells with brown cytoplasma were considered as positive anti-PKM2 staining and the percentage of PKM2-positive cells was obtained by dividing the numbers of the PKM2-positive cells by the total number of cancer cells in the same field.
11.1.7 The tyrosine kinase inhibitor nilotinib has antineoplastic activity in prostate cancer cells but up-regulates the ERK survival signal—Implications for targeted therapies
Background: Novel therapeutic options beyond hormone ablation and chemotherapy are urgently needed for patients with advanced prostate cancer. Tyrosine kinase inhibitors (TKIs) are an attractive option as advanced prostate cancers show a highly altered phosphotyrosine proteome. However, despite favorable initial clinical results, the combination of the TKI dasatinib with docetaxel did not result in improved patient survival for reasons that are not known in detail. Methods: The National Cancer Institute-Approved Oncology Drug Set II was used in a phenotypic drug screen to identify novel compounds with antineoplastic activity in prostate cancer cells. Validation experiments were carried out in vitro and in vivo. Results: We identified the TKI nilotinib as a novel compound with antineoplastic activity in hormone-refractory prostate cancer cells. However, further analyses revealed that treatment with nilotinib was associated with a significant up-regulation of the phospho-extracellular-signal-regulated kinases (ERK) survival signal. ERK blockade alone led to a significant antitumoral effect and enhanced the cytotoxicity of nilotinib when used in combination. Conclusions: Our findings underscore that TKIs, such as nilotinib, have antitumoral activity in prostate cancer cells but that survival signals, such as ERK up-regulation, may mitigate their effectiveness. ERK blockade alone or in combination with TKIs may represent a promising therapeutic strategy in advanced prostate cancer.
Identification of nilotinib as a novel antineoplastic compound in prostate cancer cells
Using the NCI-Approved Oncology Drug Panel II for a phenotypic drug screen of normal prostate epithelial cells and prostate cancer cell lines (Fig. 1) [7], we identified the TKI nilotinib as a positive hit in hormone-refractory DU-145 prostate cancer cells.
Fig. 1. Discovery of nilotinib as a novel antineoplastic agent in prostate cancer cells using a phenotypic drug screen. Overview of the drug screen procedure (see text for details).
Results were confirmed using annexin V staining, which showed a significant induction of apoptosis beginning at 24 hours (Fig. 2A). The IC50 of nilotinib against DU-145 cells was determined at 10 μM using an MTT cell viability assay (Fig. 2B). Immunoblot experiments confirmed an induction of apoptosis using PARP cleavage in DU-145 cells and in hormonerefractory PC-3 prostate cancer cells at this drug concentration (Fig. 2C). An onset of apoptosis at 24 hours was likewise confirmed using PARP cleavage at a nilotinib concentration of 10 μM(Fig. 2D). PWR-1E prostate epithelial cells and hormone-sensitive prostate LNCaP prostate cancer cells were not found to undergo enhanced apoptosis when treated with nilotinib (not shown).
Fig. 2. Antitumoral effects of nilotinib in prostate cancer cells: (A) flow cytometric analysis of DU-145 prostate cancer cells for annexin V to detect apoptotic cells after treatment with 10 μM of nilotinib for the indicated intervals; (B) cell viability (MTT) assay to determine the IC50 of nilotinib in DU-145 cells (24-h treatment); (C and D) immunoblot analysis of DU-145 and PC-3 prostate cancer cells for PARP cleavage (arrow) at nilotinib concentrations and time intervals as indicated. GAPDH is shown for protein loading; and (E) colony growth assay of DU-145 cells after drug treatment and washout as shown. Cells grown in 60-mm dishes were stained with crystal violet to visualize viable cells at the time points indicated. (Color version of figure is available online.
To further confirm the effect of nilotinib on prostate cancer cell growth, we performed a colony growth assay in which DU-145 cells were treated with nilotinib for 72 hours followed by a washout of the drug and continued culture for additional 9 days (Fig. 2E). We found that nilotinib induced significant cytotoxicity after 72 hours and that a minor regrowth of cancer cells did not occur until 6 to 9 days after the washout, which is comparable to other TKIs [8]. Next, we sought to identify the targets of nilotinib in DU-145 prostate cancer cells. Overall, 5 well-established targets, including ABL1, KIT, PDGFRA, DDR1, and NQO2, were analyzed for their role in the drug response. We found that protein expression of 3 of these targets (ABL1, KIT, and PDGFRA) was not detectable in DU-145 cells and that small interfering RNA–mediated knockdown of the remaining 2 targets, DDR1 and NQO2, did not result in apoptosis (not shown). Collectively, these results show a significant antitumoral activity of nilotinib in prostate cancer cells. However, this effect was associated with a relatively high IC50 and was independent of known nilotinib targets.
Nilotinib up-regulates the ERK survival signal in prostate cancer cells
To further investigate why relatively high concentrations of nilotinib were required to induce cytotoxicity, we analyzed 40,6-diamidino-2-phenylindole–stained DU-145 cells treated with 10 μM of nilotinib for 24 hours using fluorescence microscopy (Fig. 3A).
Fig. 3. Nilotinib up-regulates the ERK survival signal in prostate cancer cells. (A) Fluorescence microscopic analysis of DAPI-stained DU-145 cells. (B and C) Immunoblot analyses of DU-145 cells (B) or DU-145 cells in comparison with LNCaP and PC-3 cells (C) treated with nilotinib for the expression of phospho-ERK1/2 T202/Y204 and total ERK. Immunoblot for GAPDH is shown as a loading control. (D) Immunohistochemical staining of xenografted DU-145 cells after 21 days of treatment with 75 mg/kg/d of nilotinib for phospho-ERK1/2 T202/Y204 expression. It can be noted that tumors explanted from vehicle-treated mice showed mostly positivity at the tumor periphery, whereas tumors explanted from nilotinib-treated mice showed a more evenly distributed phospho-ERK immunostaining (left panels). Quantification of phospho-ERK–positive DU-145 xenografts explanted after 21 days of treatment. Mean and standard errors of positive cells per high-power field (HPF; [1]40) from at least 3 tumors are given (right panel). (E) Immunoblot analysis of DU-145 cells treated with U0126 alone or in combination with nilotinib shows abrogation of phospho-ERK1/2 T202/Y204 expression by U0126. (F) Quantification of viable cells compared with that of controls using the MTT assay after treatment with U0126 (10 μM) or nilotinib (10 μM) or both and after either pretreatment (24 h) or simultaneous treatment (72 h). DAPI ¼ 40,6-diamidino-2-phenylindole. (Color version of figure is available online.)
We found that, despite the presence of apoptotic cells, there was also a population of actively dividing tumor cells in the presence of nilotinib as well as a population of viable but multinucleated cells (Fig. 3A). We interpreted these results as evidence that a subset of tumor cells has the ability to resist TKI treatment. To reconcile these results, we analyzed the activation of ERK1/2, which is known to function as a prosurvival signal in TKI-treated tumor cells [9,10]. We detected a robust overexpression of phospho-ERK1/2 T202/Y204 in nilotinib-treated DU-145 cells (Fig. 3B). An up-regulation of phospho-ERK1/2 T202/Y204 was also detectable in nilotinib-treated LNCaP cells, albeit at a lower level, and was not found in PC-3 cells (Fig. 3C). To further corroborate the evidence of phospho-ERK upregulation in vivo, we analyzed explanted DU-145 xenografts from a representative experiment in which nilotinib was used at a 75-mg/kg/d concentration. This initial dosage was based on published animal experiments [11] but yielded no or incomplete tumor control in our experiment (data not shown).
In vivo antitumoral activity of nilotinib and ERK blockade
Our results raised 2 important questions First, can a higher dose of nilotinib induce improved tumor control, and second, is a combination of nilotinib with the MEK inhibitor U0126 to block ERK activity superior to nilotinib alone?
Fig. 4. In vivo antitumoral activity of nilotinib and ERK blockade in prostate cancer cells: (A) tumor growth curves of DU-145 xenografts in NMRI-nude mice and (B) analysis of tumor volumes on day 21. Asterisks indicate statistical significance (**P r 0.01 and ***P r 0.001). (Color version of figure is available online.)
11.1.8 PAF and EZH2 Induce Wnt.β-Catenin Signaling Hyperactivation
Fine-control of Wnt signaling is essential for various cellular and developmental decision making processes. However, deregulation of Wnt signaling leads to pathological consequences including cancer. Here, we identify a novel function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells, and required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing secondary axis with β-catenin target gene upregulation. Upon Wnt signaling activation, PAF is dissociated from PCNA, and directly binds to β-catenin. Then, PAF recruits EZH2 to β-catenin transcriptional complex, and specifically enhances Wnt target gene transactivation, independently of EZH2’s methyltransferase activity. In mouse, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling, and propose a novel regulatory mechanism of Wnt signaling during tumorigenesis. Fine-control of Wnt signaling is essential for various cellular and developmental decision making processes. However, deregulation of Wnt signaling leads to pathological consequences including cancer. Here, we identify a novel function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells, and required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing secondary axis with β-catenin target gene upregulation. Upon Wnt signaling activation, PAF is dissociated from PCNA, and directly binds to β-catenin. Then, PAF recruits EZH2 to β-catenin transcriptional complex, and specifically enhances Wnt target gene transactivation, independently of EZH2’s methyltransferase activity. In mouse, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling, and propose a novel regulatory mechanism of Wnt signaling during tumorigenesis.
Keywords: Wnt, β-catenin, PAF, KIAA0101, EZH2
Strict regulation of stem cell proliferation and differentiation is required for mammalian tissue homeostasis, and its repair in the setting of tissue damage. These processes are precisely orchestrated by various developmental signaling pathways, with dysregulation contributing to disease and genetic disorders, including cancer (Beachy et al., 2004). Cancer is initiated by the inactivation of tumor suppressor genes and activation of oncogenes. For instance, colon cancer cells harbor genetic mutations in Wnt/β-catenin pathway constituents such as adenomatous polyposis coli (APC), Axin, and β-catenin (Polakis, 2007). In mouse models, inactivation of APC or activation of β-catenin results in the development of intestinal hyperplasia and adenocarcinoma (Moser et al., 1990), indicating that hyperactivation of Wnt signaling promotes intestinal tumorigenesis.
In canonical Wnt signaling, Wnt ligand induces stabilization of β-catenin protein via inhibition of the protein destruction complex (glycogen synthase kinase 3, APC, casein kinase I, and Axin). Then, activated β-catenin is translocated into the nucleus and binds to its nuclear interacting partners, TCF/LEF. Finally, β-catenin-TCF/LEF transactivates the expression of its target genes (Clevers and Nusse, 2012).
Although various Wnt/β-catenin modulators have been identified (Wnt homepage; wnt.stanford.edu), the pathological relevance of these modulators to tumorigenesis remains elusive. Also, many reports have suggested that mutation-driven Wnt signaling activation can be enhanced further (Goentoro and Kirschner, 2009; He et al., 2005; Suzuki et al., 2004; Vermeulen et al., 2010), which implies the presence of an additional layer of Wnt-signaling regulation in cancer beyond genetic mutations in APC or β-catenin. Here, we unraveled a novel function of the DNA repair gene, PAF (PCNA-associated factor) /KIAA0101). PAF was shown to be involved in translesion DNA synthesis (TLS), an error-prone DNA repair process that permits DNA replication machinery to replicate DNA lesions with specialized translesion DNA polymerase (Emanuele et al., 2011; Povlsen et al., 2012; Sale et al., 2012). Our comprehensive approaches uncover that cancer-specifically expressed PAF hyperactivates Wnt/β-catenin signaling and induces intestinal tumorigenesis.
Mitogenic role of PAF via Wnt signaling
To identify colon cancer-specific Wnt signaling regulators, we analyzed multiple sets of human colon cancer tissue samples using the publicly available database (www.oncomine.org), and selected genes that are highly expressed in colon cancer cells (fold change > 2; P < 0.0001; top 10% ranked). Among several genes, we investigated the biological role of PAF, based on its significant overexpression in human colon adenocarcinoma with correlated expression of Axin2, a well-established specific target gene of β-catenin (Jho et al., 2002; Lustig et al., 2002)(Figure 1A). To validate our in silico analysis, we performed immunostaining of colon cancer tissue microarray, and confirmed that PAF was highly expressed in colon cancer cells, whereas its expression was barely detectable in normal intestine (Figure 1B). Consistently, PAF was strongly expressed and mainly localized in the nucleus of colon cancer cell lines (Figure 1C). Additionally, we found that PAF was not expressed in non-transformed cells such as NIH3T3, mouse embryonic fibroblasts, and mammary epithelial cells (data not shown). Next, to assess the relevance of PAF upregulation in colon cancer cell proliferation, we depleted endogenous PAF using short hairpin RNAs (shRNAs) in these cell lines. Intriguingly, PAF knockdown (sh-PAF) inhibited colon cancer cell proliferation (Figures 1D and 1E). Given that PAF was shown to interact with PCNA via PIP box (Yu et al., 2001), we also examined whether PAF-PCNA interaction is required for mitogenic effects of PAF. In reconstitution experiments, sh-PAF-induced cell growth inhibition was rescued by ectopic expression of both shRNA non-targetable wild-type PAF (nt-PAF) and PIP mutant PAF (mutPIP-PAF) (Figure 1F), indicating that the PAF-PCNA interaction is not necessary for PAF-mediated colon cancer cell proliferation. Interestingly, PAF knockdown downregulated cell proliferation–related genes (Cyclin D1 and c-Myc) (Figure 1G). Given that Cyclin D1 and c-Myc are β-catenin direct target genes (He et al., 1998; Tetsu and McCormick, 1999), PAF likely participates in regulating Wnt/β-catenin signaling. Interestingly, PAF depletion-induced downregulation of Cyclin D1 andc-Myc was only observed in SW620 colon cancer cells, but not in Panc-1 and MDA-MB-231 cells (Figure 1G), indicating the specific effects of PAF on Cyclin D1 and c-Myc expression in colon cancer cells. We also assessed the effects of PAF knockdown on Axin2. Indeed, PAF knockdown suppressed Axin2transcription in colon cancer cells (Figure 1H). Moreover, as nt-PAF did, β-catenin ectopic expression reverted sh-PAF–induced cell growth arrest (Figure 1I), implying that PAF might be functionally associated with Wnt/β-catenin. We also examined whether other mitogenic signaling pathways mediate PAF’s mitogenic role. Of note, except HT29, other colon cancer cell lines (SW620, HCT116, HCC2998, and HCT15) harbor oncogenic mutations in K-Ras gene. Nonetheless, PAF depletion induced the suppression of cell growth on all five colon cancer cells (Figure 1D), indicating that PAF’s mitogenic function is independent of Ras/MAPK signaling activation. Additionally, overexpression of wild-type Akt or constitutively active form of Akt (myristoylated form of Akt [Myr-Akt]) did not rescue sh-PAF-induced inhibition of cell proliferation (Figure 1I). Moreover, β-catenin activation did not revert cell proliferation suppression resulted from MAPK or PI3K inhibition (Figure 1J), indicating that β-catenin-mediated mitogenic function is independent of MAPK and PI3K signaling pathways. These results suggest that PAF contributes to colon cancer cell proliferation, possibly via Wnt/β-catenin signaling.
Figure 1Mitogenic role of PAF in colon cancer cells
PAF positively modulates Wnt signaling
Given that many cancers develop as a result of deregulation of developmental signalings (Beachy et al., 2004), analyzing PAF expression during development may provide insights into the mechanisms of PAF-mediated signaling regulation. Whole mount immunostaining of mouse embryos, showed that PAF was specifically enriched in the apical ectodermal ridge (AER) of the limb bud, midbrain, hindbrain, and somites (Figure 2A and data not shown). During limb development, AER induction is specifically coordinated by active Wnt signaling (Figure 2B)(Kengaku et al., 1998). Using, Axin2-LacZ, a β-catenin reporter (Lustig et al., 2002), mouse embryos, we confirmed the specific activation of Wnt signaling in AER (Figure 2C). Intriguingly, Wnt signaling activity as exhibited in the AER, overlapped with the pattern of PAF expression (Figures 2A and 2C). Given that (1) Wnt signaling is deregulated in most colon cancer, (2) PAF is highly overexpressed in colon cancer cells, (3) PAF is required for colon cancer cell proliferation (Figure 1D), and (4) PAF is enriched in AER where Wnt signaling is active (Figure 2A), we hypothesized that PAF modulates the Wnt signaling pathway. To test this, we first examined the impact of PAF on β-catenin transcriptional activity using TOPFLASH reporter assays. In HeLa cells, PAF knockdown decreased β-catenin reporter activation by 6-bromoindirubin-3′-oxime, a GSK3 inhibitor (Figure 2D). Similarly, Wnt3A-induced transcriptional activation of Axin2 was also inhibited by PAF depletion (Figure 2E). These data suggest that PAF might be required for Wnt target gene expression.
To gain better insight of PAF’s role in Wnt signaling regulation, we utilized Xenopus laevis embryos for axis duplication assays (Funayama et al., 1995), as previously performed (Park et al., 2009). Because of Wnt signaling’s pivotal role in vertebrate anterior-posterior axis development, the effects of Xenopus PAF (xPAF) on Wnt signaling can be monitored and quantified on the basis of secondary axis formation following injection of in vitro transcribed mRNAs. xβ-catenin mRNA, titrated to a subphenotypic level when expressed in isolation, was co-injected with xPAF mRNA into ventrovegetal blastomeres. Unlike the controls (β-catenin and β-galactosidase mRNA), the experimental group (β-catenin and xPAF mRNA) displayed axis-duplications (Figures 2F-H). Of note, the ventrovegetal injection of xPAF mRNA alone failed to induce secondary axes (data not shown), showing that PAF hyperactivates Wnt/β-catenin signaling only in the presence of active β-catenin. Consistent with the results of axis duplication assays, qRT-PCR assays showed that xPAF expression upregulated expression of Siamois and Xnr3, β-catenin targets in frogs (Figure 2I). Furthermore, we examined the specificity of PAF on Wnt/β-catenin signaling activity, using various luciferase assays. Ectopic expression of PAF hyperactivates Wnt3A or LiCl, a GSK3 inhibitor, -induced activation of β-catenin target gene reporter activity (MegaTOPFLASH, Siamois, c-Myc, and Cyclin D1). Of note, BMP/Smad pathway also plays an essential role in the developing limb AER (Ahn et al., 2001). However, PAF knockdown or overexpression did not affect BMP/Smad or FoxO signalings, respectively, (Figure 2J) indicating the specificity of PAF in regulating Wnt signaling. These results suggest that PAF positively modulates Wnt/β-catenin signaling in vitro and in vivo.
Next, we investigated the molecular mechanism underlying PAF hyperactivation of Wnt signaling. Given that stabilization of β-catenin protein is a key process in transducing Wnt signaling, we asked whether PAF affects β-catenin protein level. However, we found that the level of β-catenin protein was not altered by PAF knockdown or overexpression (Figures 2E and and3A),3A), leading us to test whether PAF controls the β-catenin/TCF transcriptional complex activity. Owing to the nuclear specific localization of PAF in colon cancer cells (Figure 1C), we tested whether PAF interacts with β-catenin transcriptional complex. Using a glutathione S-transferase (GST) pull-down assay, we found that PAF bound to β-catenin and TCF proteins (Figure 3B). Also, endogenous PAF interacted with β-catenin and TCF3 in SW620 cells that display constitutive hyperactivation of Wnt signaling by APC mutation (Figure 3C). Moreover, binding domain mapping assays showed that the Armadillo repeat domain of β-catenin was essential for its interaction with PAF (Figure 3D). Although PAF is a cell cycle-regulated anaphase-promoting complex substrate (Emanuele et al., 2011), PAF-β-catenin interaction was not affected (Figure S1). These data suggest that PAF directly binds to β-catenin transcriptional complex and this interaction is independent of cell cycle. Next, due to interaction of PAF with β-catenin and TCF, we tested whether PAF is also associated with Wnt/β-catenin target genes. First, we analyzed the subnuclear localization of PAF by chromatin fractionation. We found that PAF was only detected in the chromatin fraction of HCT116 cells (Figure 3E). Additionally, chromatin immunoprecipitation (ChIP) assays showed that both ectopically expressed and endogenous PAF occupied the TCF-binding element (TBE)-containing proximal promoter of the β-catenin targets (c-Myc and Cyclin D1) in HCT116 cells (Figures 3F and 3G). These data show that PAF is specifically associated with the promoters of Wnt/β-catenin target genes.
Figure 3PAF-EZH2-β-catenin transcriptional complex at target gene promoters
In intestine, Wnt/β-catenin signaling constitutively activates intestinal stem cells (ISCs) to give rise to progenitor cells, which replenishes intestinal epithelium (Figure 3H). Given the involvement of PAF on Wnt/β-catenin signaling regulation (Figure 2), we analyzed the spatial expression of PAF in intestinal epithelium. Immunostaining showed that PAF was specifically expressed in B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) positive intestinal stem cells (ISCs)(Figures 3I and 3J). Bmi1 and its associated components in Polycomb-repressive complex 1 (PRC1) and 2 (PRC2) are shown to epigenetically regulate gene expression (Sparmann and van Lohuizen, 2006). Due to (1) specific association of PAF with TBEs of β-catenin target promoters (Figures 3F and 3G) and (2) co-localization with Bmi1 positive ISCs (Figure 3J), we asked whether PAF is associated with components of PRC1 and PRC2, using co-immunoprecipitation (co-IP) assays. Intriguingly, PAF interacted with both Bmi1 and enhancer of zeste homolog 2 (EZH2) in SW620 cells (Figure 3K), which led us to test whether either Bmi1 or EZH2 is functionally associated with PAF-mediated Wnt signaling hyperactivation. To do this, we assessed the effects of Bmi1 and EZH2 on β-catenin transcriptional activity, using β-catenin reporter assays. We observed that ectopic expression of EZH2 upregulated β-catenin transcriptional activity, but Bmi1 overexpression did not (data not shown), implying that EZH2 might be associated with Wnt signaling activation. Binding domain mapping analysis showed that EZH2 bound to PAF via the middle region of EZH2 including the CXC cysteine-rich domain (Figure 3L). In conjunction with the Bmi1-containing PRC1, EZH2-containing PRC2 catalyzes histone H3 lysine 27 trimethylation (H3K27me3) via histone methyltransferase domain. Despite the crucial role of EZH2 in H3K27me3-meidated gene regulation, we found that other core components of PRC2, EED, and Suz12 were not associated with PAF (Figure 3K). Moreover, although EZH2 overexpression in cancer induces PRC4 formation in association with the NAD+-dependent histone deacetylase Sirt1 (Kuzmichev et al., 2005), the PAF-EZH2 complex did not contain Sirt1 (Figure 3K). These data indicate that PAF-EZH2 complex is distinct from the conventional PRCs in cancer cells. Also, we questioned whether PCNA is required for PAF’s interaction with either PAF or β-catenin. Interestingly, β-catenin-PAF and EZH2-PAF complexes existed only in PCNA-free fractions (Figure 3M, compare lanes 1 and 2), which is consistent with PCNA-independent mitogenic role of PAF in colon cancer cell proliferation (Figure 1I). Due to exclusive interaction of PAF with either PCNA or β-catenin, we asked whether Wnt signaling activation affects either PAF-β-catenin or PAF-PCNA interaction. Co-IP assays showed that, in HeLa cells, PAF-β-catenin interaction was only detected upon LiCl treatment, while PAF-EZH2 interaction remained constant. Moreover, PAF-PCNA association was decreased by LiCl or Wnt3A treatment (Figures 3N and 3O, compare lanes 3 and 4). These data suggest that Wnt signaling activation is required for PAF-β-catenin interaction. Due to absence of endogenous Wnt signaling activity in HeLa cells, we also assessed the effects of active Wnt/β-catenin signaling on PAF-PCNA binding in colon cancer cell lines that exhibit hyperactivation of Wnt signaling by genetic mutations in APC or β-catenin alleles. Surprisingly, PAF-PCNA interaction was barely detectable in colon cancer cell lines, whereas 293T and HeLa cells displayed strong PAF-PCNA association (Figure 3P), implying that active β-catenin may sequester PAF from PCNA. In binding domain mapping analysis, we also found that N-terminal and PIP regions are required for β-catenin interaction (Figure S2), suggesting that β-catenin competes with PCNA for PAF interaction. These results suggest that, upon Wnt signaling activation, PAF is conditionally associated with β-catenin transcriptional complex.
PAF activates β-catenin target genes by recruiting EZH2 to promoters
Previous studies showed that EZH2 interacts with β-catenin (Li et al., 2009; Shi et al., 2007). Also, we found that PAF is physically associated with EZH2, independently of PRC2 complex (Figure 3). These evidences prompted us to ask whether EZH2 mediates PAF-induced Wnt signaling hyperactivation. Given PAF-EZH2-β-catenin complex formation, we tested whether EZH2 is also associated with the promoters of β-catenin target genes. Intriguingly, PAF, EZH2, and β-catenin steadily co-occupied the promoters of c-Myc,Cyclin D1, and Axin2 in HCT116 cells carrying β-catenin mutation, whereas PCNA, EED, and Suz12 did not (Figure 4A), which recapitulates PRC2 complex-independent association of EZH2 with PAF (see Figures 3K and 3N). Next, we asked whether PAF, EZH2, and β-catenin are recruited to β-catenin target gene promoter upon Wnt signaling activation, as PAF-β-catenin interaction was dependent of Wnt signaling activation (Figure 3N). In HeLa cells, we found that PAF, EZH2, and β-catenin conditionally bound to TBEs in the c-Myc and Axin2 promoters, only upon LiCl treatment (Figure 4B), indicating that Wnt signaling activation is a prerequisite for PAF-β-catenin-EZH2’s promoter association. To further confirm the specificity of PAF-EZH2-β-catenin’s recruitment to β-catenin target promoters, we performed ChIP promoter scanning of 10 kb of the c-Myc promoter, and found that PAF, EZH2, and β-catenin specifically co-occupied the proximal promoter containing TBEs of the c-Myc gene (at -1037 and -459 bp) (He et al., 1998) in HCT116 cells (Figure 4C). Also, the analysis of EZH2 ChIP-sequencing data from mouse embryonic stem cells showed that EZH2 was specifically enriched in the proximal promoters of β-catenin targets (Lef1, Lgr5, c-Myc, and Axin2) (Figure 4D).
Next, we asked whether EZH2 promoter recruitment is necessary for activation of β-catenin target gene transcription. Previously, depletion of EZH2 was shown to inhibit c-Myc expression in DLD-1 colon cancer cells (Fussbroich et al., 2011). Consistently, EZH2 knockdown downregulated β-catenin target genes, Axin2and Cyclin D1 in HCT116 cells (Figure 4E), and decreased LiCl-induced β-catenin reporter activation (Figure 4F), suggesting that EZH2 is required for PAF-mediated Wnt target gene hyperactivation. These results are also supported by previous finding that EZH2 enhances β-catenin transcriptional activity by connecting β-catenin with the Med1/RNA polymerase II (Pol II) complex (Shi et al., 2007). Indeed, Med1/TRAAP220 and Pol II conditionally binds to c-Myc and Axin2 promoters in LiCl-treated HeLa cells (Figure 4G). Given that PRC2-indepednent interaction between EZH2 and PAF (Figures 3K and 3N), we asked whether EZH2’s histone methyltransferase activity is dispensable in β-catenin regulation. We utilized an EZH2 point mutant (F681I) that disrupts the contact between the EZH2 hydrophobic pocket and histone lysine residue H3K27 (Joshi et al., 2008). Ectopic expression of either EZH2 or EZH2-F681I enhanced β-catenin reporter activity (Figure 4H). Also, PAF knockdown did not change the H3K27 methylation status (H3K27me3) of proximal promoters of c-Myc, Axin2, Cyclin D1, and DCC in HCT116 cells (Figure 4I). These results indicate a methyltransferase-independent role of EZH2 in transactivating β-catenin targets.
Due to PAF’s (1) small size (111 amino acids, one α-helix), (2) lack of a specific catalytic domain, and (3) binding to both β-catenin and EZH2, PAF may facilitate the interaction between EZH2 and β-catenin through recruiting EZH2 to the promoter. We tested this using ChIP assays for EZH2 in the setting of PAF depletion. Indeed, PAF-depleted HCT116 cells displayed decreased EZH2-association at the c-Myc promoter (Figure 4J), suggesting that PAF assists or is needed to recruit EZH2 to β-catenin transcriptional complex. Also, β-catenin knockdown decreased recruitment of PAF and EZH2 to promoters (Figure 4K), showing that PAF and EZH2 occupy target promoters via β-catenin. We then asked whether PAF promotes β-catenin-EZH2 binding. In vitro binding assays showed that the addition of GST-PAF protein increased EZH2-β-catenin association (Figure 4L). Moreover, ectopic expression of PAF promoted the EZH2-β-catenin interaction in HeLa cells treated with LiCl (Figure 4M). Additionally, we tested whether Wnt signaling-induced post-translational modification of either β-catenin or PAF is required for EZH2 interaction. However, in GST pull-down assays, we found that bacterially expressed either GST-β-catenin or –PAF bound to EZH2 (Figure S3). Due to the lack of post-translational modification in GST protein expression system, these data indicate that post-translation modification of either β-catenin or PAF is not necessary for EZH2 interaction. Together, these results suggest that PAF acts as a molecular adaptor to facilitate EZH2-β-catenin complex, and subsequently enhances the transcriptional activity of the β-catenin transcriptional complex at Wnt target promoters (Figure 4N).
Intestinal tumorigenesis following PAF conditional expression
Having determined that PAF is overexpressed in colon cancer cells and hyperactivates Wnt/β-catenin signaling, we aimed to determine whether mimicking PAF overexpression drives intestinal tumorigenesis, using genetically engineered mouse models. To conditionally express PAF, we generated doxycycline (doxy)-inducible PAF transgenic mice (TetO-PAF-IRES-emGFP [iPAF]). For intestine-specific expression of PAF, we used iPAF:Villin-Cre:Rosa26-LSL-rtTA mouse strains. Villin-Cre is specifically expressed in intestinal epithelial cells (IECs), including ISCs and progenitor cells. Cre removes a floxed stop cassette (loxP-STOP-loxP [LSL]) from the Rosa26 allele and induces rtTA expression. Upon doxy treatment, rtTA drives the transcriptional activation of the tetracycline-responsive element promoter, resulting in conditional transactivation of PAF selectively in IECs. We also utilized the Rosa26-rtTA strain for ubiquitous expression of PAF (Figure 5A and Figure S4). First, we examined the effects of PAF induction on IEC proliferation using a crypt organoid culture system (Figure S5A). Intriguingly, PAF conditional expression (2 weeks) induced expansion of the crypt organoids (Figures 5B and 5C), which recapitulates the mitogenic function of PAF (Figure 1). In mouse, IEC-specific PAF expression (iPAF:Villin-Cre:Rosa26-LSL-rtTA; 2 months) developed adenoma in both small intestine and colon (Figure 5D). Also, microscopic analysis using hematoxylin and eosin (H&E) staining showed aberrant IEC growth and crypt foci formation (Figures 5E and 5F), with disorganized epithelial cell arrangements (Figure S5B). Consistently, PAF-induced IEC hyperproliferation was manifested by increased Ki67 expression, a mitotic marker (Figure 5G). Importantly, these lesions exhibited the upregulation of CD44, a β-catenin target gene, whereas CD44 was expressed strictly in the crypts of normal intestine (Figure 5H). Next, we examined whether PAF directly hyperactivates Wnt/β-catenin in vivo using BAT-gal, a β-catenin reporter transgenic mouse carrying multiple TBEs followed by a LacZ reporter. To quantify the early effects of PAF on β-catenin activity, we treated mice with doxy for 1 week, and found that short-term induction of PAF increased β-catenin transcriptional activity as represented by enhanced X-gal staining (Figure 5I). Moreover, conditional PAF expression upregulated the β-catenin target genes, Axin2, Lgr5, CD44, Cyclin D1, and c-Myc in crypt organoids (Figure 5J). Additionally, mice ubiquitously expressing PAF exhibited intestinal hypertrophy (Figure S5C), which is similar to that induced by R-Spondin1, a secreted Wnt agonist (Kim et al., 2005). These data strongly suggest that PAF expression is sufficient to initiate intestinal tumorigenesis via Wnt signaling hyperactivation.
Figure 5Induction of intestinal neoplasia by PAF expression
Herein we reveal the unexpected role of PAF in modulating Wnt/β-catenin signaling. PAF enhances the transcription of Wnt targets by recruiting EZH2 to the β-catenin transcriptional complex. This is similar to the mechanism by which Lgl/BCL9 binds to β-catenin and thereby recruits the PHD-finger protein Pygopus, to bridge the β-catenin/TCF complex to Med12 and Med13 (Carrera et al., 2008). Importantly, due to specific overexpression of PAF in cancer cells, our studies identified an additional layer of the regulatory mechanism of β-catenin target gene transactivation.
In cancer cells, the upregulation of EZH2 contributes to tumorigenesis through the epigenetic repression of various genes including tumor suppressor genes, Wnt antagonists, and DNA repair genes (Chang et al., 2011; Cheng et al., 2011; Kondo et al., 2008). Our results propose a noncanonical function of EZH2 in activating β-catenin target genes in conjunction with PAF. Consistently, recent study also suggests methyltransferase activity-independent function of EZH2 in gene activation (Xu et al., 2012). Moreover, this non-canonical role of EZH2 is supported by several lines of evidence: (a) EZH2 transactivates β-catenin target genes (Li et al., 2009; Shi et al., 2007) (Figures 4E and 4F); (b) EZH2 overexpression in murine mammary epithelium induces ductal hyperplasia (Li et al., 2009), which phenocopies that in a ∆Nβ-catenin (constitutively active form of β-catenin) mouse model (Imbert et al., 2001); (c) EZH2 occupies β-catenin target promoters (Figures 4A-D); and (d) EZH2’s methyltransferase activity is dispensable for β-catenin target activation (Figures 4H and 4I). Moreover, similar to PAF expression in the AER (Figure 2A), EZH2 is also specifically expressed there to maintain of Hox cluster gene transcription (Wyngaarden et al., 2011). Thus, it is plausible that EZH2 and PAF cooperatively control Hox gene activation in the developing limb. Interestingly, despite the presence of a physical and functional connection between Bmi1 and EZH2 in H3K27me3-mediated gene repression, EZH2 is expressed only in crypt IECs including ISCs (Figure S6), whereas Bmi1 is expressed in ISCs at position 4 (Figure 3J), implying a Bmi1-independent role for EZH2 in gene regulation. These results demonstrate the novel function of EZH2 in β-catenin target gene activation, independent of the histone methyltransferase activity of EZH2.
Previously, we found that TERT, a catalytic subunit of telomerase, positively modulates Wnt signaling (Park et al., 2009), elucidating a non-telomeric function of telomerase in development and cancer. Here our results propose that one component of DNA damage bypass process also functions in regulating Wnt signaling, dependent of context. In cancer, PAF overexpression may play a dual role in inducing (a) cell hyperproliferation (via Wnt signaling hyperactivation) and (b) the accumulation of mutations arising from DNA lesion bypass (by PAF-mediated TLS) (Povlsen et al., 2012). Importantly, PAF is only expressed in cancer cells, but not in normal epithelial cells. Thus, upon DNA damage, instead of cell growth arrest to permit high-fidelity DNA repair, the PAF overexpression in cancer cells is likely to induce DNA lesion bypass by facilitating TLS. However, in the setting of Wnt signaling deregulation, nuclear β-catenin sequesters PAF from PCNA and utilize PAF as a co-factor of transcriptional complex, which induces Wnt signaling hyperactivation and possibly lead to increased mutagenesis.
We observed that PAF marked the stemness of ISCs and mouse embryonic stem cells (Figure S7), implicating its roles in stem cell regulation under physiological conditions. In a previous study, a PAFgermline knockout mouse model displayed defects in hematopoietic stem cell self-renewal (Amrani et al., 2011), suggesting a crucial role of PAF in stem cell maintenance and activation. In the intestine, β-catenin activation in Lgr5-positive or Bmi1-positive cells is sufficient to develop intestinal adenoma (Barker et al., 2009; Sangiorgi and Capecchi, 2008), suggesting an essential role of tissue stem cells in tumor initiation. Considering PAF expression in Bmi1-positive ISCs, PAF upregulation in ISCs likely hyperactivates the Wnt/β-catenin signaling and contributes to intestinal tumor initiation.
Despite the critical role of Wnt signaling in early vertebrate, development PAF germline knockout mice are viable (Amrani et al., 2011). It is noteworthy that, whereas deletion of any core component in the Wnt signaling pathway causes embryonic lethality, mice with germline knockout of Wnt signaling modulators, including Nkd1/2, Pygo1/2, and BCL9/9-2, exhibit no lethal phenotypes (Deka et al., 2010; Schwab et al., 2007; Zhang et al., 2007). This may result from the robustness of Wnt signaling during embryogenesis because of functional compensation not only via the presence of multiple Wnt signaling regulators per se but also via other types of signaling crosstalk. Therefore, as described previously in pRb studies (Sage et al., 2003), acute deletion of PAF in a conditional knockout mouse model may disrupt the developmental balance or tissue homeostasis, and then reveal the full spectrum of the physiological and pathological roles of PAF in tumorigenesis. Taken together, our findings reveal unexpected function of PAF and EZH2 in modulating Wnt signaling, and highlight the impacts of PAF-induced Wnt signaling deregulation on tumorigenesis.
11.1.9 PAF Makes It EZ(H2) for β-Catenin Transactivation
In this issue of Molecular Cell, Park and colleagues (Jung et al., 2013) show that PAF (PCNA-associatedfactor) binds to and hyperactivates transcriptional function of β-catenin in colon cancer cells by recruiting EZH2 to the coactivator complex. PAF-β-catenin and PAF-PCNA interactions are competitive, raising the question of whether β-catenin might regulate PCNA-dependent DNA replication and repair.
Wnt signaling through stabilization of transcription co-activator β-catenin plays critical roles in animal development and tissue homeostasis, and its deregulation is involved in myriad human diseases including cancer (Clevers and Nusse, 2012). Notably, most colorectal cancers (CRCs) have elevated β-catenin signaling caused by mutations of Wnt pathway components such as the tumor suppressor APC (Adenomatosis polyposis coli) and β-catenin itself (Clevers and Nusse, 2012). Much effort has focused on studying β-catenin-dependent transactivation in CRCs, including the current study by Park and colleagues that identifies PAF as an unexpected β-catenin co-activator (Jung et al., 2013).
PAF, for PCNA (proliferating cell nuclear antigen)-associated factor (also known as KIAA0101 or p15PAF), is an interacting partner of PCNA (Yu et al., 2001). PCNA has a key role in DNA replication and repair by assembling various DNA polymerase and repair complexes at the replication fork (Mailand et al., 2013). Dynamic regulation of PAF abundance and/or interaction with PCNA appears to be important for engaging DNA damage repair and bypass pathways (Emanuele et al., 2011; Povlsen et al., 2012). PAF is overexpressed in many types of cancers and required for cell proliferation (e.g., Yu et al., 2001).
In the current study (Jung et al., 2013), Jung et al. show that PAF is overexpressed in CRCs in a manner that parallels expression of Axin2, an established Wnt/β-catenin target gene. PAF knockdown inhibits CRC proliferation, and this effect is independent of PAF-PCNA interaction and can be rescued by a PAF mutant that does not binds to PCNA or by β-catenin overexpression. PAF knockdown downregulates the expression of Wnt/β-catenin target genes Cyclin D1, c-Myc, and Axin2 in a CRC line, leading the authors to hypothesize that PAF participates in Wnt/β-catenin signaling. Indeed PAF knockdown reduces, and its overexpression augments, Wnt/β-catenin responsive TOPFLASH reporter and target gene expression induced by Wnt3a or by pharmacological agents that stabilize β-catenin. In Xenopus embryos, PAF synergizes with β-catenin to induce Wnt target gene expression and axis duplication (a hallmark of Wnt/β-catenin activation). In mouse embryos, PAF is highly expressed in regions known for Wnt/β-catenin signaling such as the apical ectodermal ridge of the limb bud. Therefore PAF appears to be a positive regulator of Wnt/β-catenin signaling in CRCs and vertebrate embryos.
PAF does not affect β-catenin protein levels and is localized in the nucleus. Protein binding assays show that PAF interacts, directly or indirectly, with β-catenin (via the Armadillo-repeat domain) and its DNA-bound partner TCF (T Cell factor). Indeed PAF is associated with promoters of Wnt/β-catenin target genes in chromatin in CRC cells. Interestingly in the mouse intestine, the PAF protein is enriched in Bmi (B lymphoma Mo-MLV insertion region 1 homolog)-positive stem cells (at the “+4” position) (Sangiorgi and Capecchi, 2008). Bmi1 is a component of Polycomb Repressive Complex 1 (PRC1), which, together with the PRC2 complex that modifies Histone H3, has critical functions in transcriptional epigenetic silencing. Previous studies have suggested that a core PRC2 component, EZH2 (enhancer of zeste homolog 2), is a partner and paradoxically a co-activator of β-catenin, acting in a manner that is independent of EZH2’s methyltransferase activity (Li et al., 2009; Shi et al., 2007). Jung et al. found that PAF indeed interacts with both Bmi1 and EZH2, but not other PRC2 components, and EZH2 overexpression augments β-catenin transcriptional activity. PAF, EZH2, and β-catenin are found to co-occupy promoters of several Wnt/β-catenin target genes in CRC and mouse ES cells, and PAF depletion decreases EZH2 association with the c-Myc promoter, and β-catenin depletion decreases the association of both PAF and EZH2 with the promoter. Thus the β-catenin-PAF-EZH2 complex appears to constitute a chain of co-activators (Figure 1), and indeed PAF, which binds to both β-catenin and EZH2, enhances β-catenin-EZH2 co-immunoprecipitation. Together with an earlier study (Shi et al., 2007), these results suggest a model that PAF brings EZH2 and the associated RNA polymerase II Mediator complex to β-catenin target genes for transactivation in CRCs (Figure 1). Consistent with this model, transgenic overexpression of PAF in the mouse intestine induces β-catenin-dependent target and reporter gene expression, intestinal overgrowth, and adenoma formation in vivo and crypt organoid expansion in vitro, resembling Wnt/β-catenin signaling activation in the gastrointestinal tract.
Figure 1 β-catenin transactivation mediated by PAF and EZH2 in the G1 phase and a speculative role of β-catenin in modulating PAF-PCNA-dependent DNA replication and repair/bypass pathways in the S phase.
PAF and EZH2 represent newer additions to β-catenin’s plethora of co-activators (Mosimann et al., 2009), which offer multiple routes to engage the basal transcription apparatus. These co-activators may have partially redundant and/or context-dependent functions for numerous Wnt/β-catenin-dependent gene programs. Mouse mutants that lack an individual β-catenin co-activator are often viable (MacDonald et al., 2009; Mosimann et al., 2009). Paf−/− mice are viable but exhibit defects in hematopoietic stem cell properties (Amrani et al., 2011). PAF is also expressed in self-renewing mouse ES cells but the expression is downregulated upon ES cell differentiation (Jung et al., 2013). Whether PAF has a general role in self-renewal of embryonic and adult stem cells through its role in β-catenin signaling or DNA replication and repair pathways remains to be investigated.
PAF-β-catenin interaction is observed under Wnt stimulation, likely as a consequence of β-catenin accumulation (Jung et al., 2013). In some cell types PAF is ubiquitinated and degraded by the anaphase promoting complex and thus exhibits the lowest level in the G1 phase of the cell cycle (Emanuele et al., 2011). In these cells PAF may have a limited role as a co-activator for β-catenin-dependent transcription, which primarily occurs in G1. But in CRC and other cancers where PAF is overexpressed, PAF may have a prominent role as a β-catenin co-activator.
PAF-PCNA interaction is well documented (e.g., Yu et al., 2001). Surprisingly however, in CRCs with high levels of β-catenin, PAF-PCNA interaction is barely detectable (Jung et al., 2013). Conversely, in cells where the basal level of Wnt/β-catenin signaling is low, PAF-PCNA interaction is detected but is diminished by Wnt3a or pharmacological agents that stabilize β-catenin (Jung et al., 2013). PAF seems to interact with β-catenin and PCNA via an overlapping domain (although this remains to be better defined), offering a possible explanation why PAF-β-catenin and PAF-PCNA complexes appear to be mutually exclusive (Jung et al., 2013). This may simply reflect the fact that PAF-β-catenin (for RNA transcription) and PAF-PCNA (for DNA replication/repair) complexes act in G1 and S, respectively (Figure 1). However, when β-catenin levels are high in Wnt-stimulated cells or in CRCs, one may speculate that β-catenin accumulation could inhibit PAF-PCNA complex formation in the S phase, thereby enabling Wnt/β-catenin signaling to modulate PAF-PCNA-dependent DNA replication and repair/bypass pathways (Figure 1). This would constitute an unsuspected role for Wnt/β-catenin signaling in genomic stability beyond its established transcriptional function and could have implications to tumorigenesis.
Amrani YM, Gill J, Matevossian A, Alonzo ES, Yang C, Shieh JH, Moore MA, Park CY, Sant’Angelo DB, Denzin LK. J Exp Med. 2011;208:1757–1765. [PMC free article] [PubMed]
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Background: Ovarian cancer is one of the major causes of death in women worldwide. Despite improvements in conventional treatment approaches, such as surgery and chemotherapy, a majority of patients with advanced ovarian cancer experience relapse and eventually succumb to the disease; the outcome of patients remains poor. Hence, new therapeutic strategies are urgently required. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) is activated in approximately 70 % of ovarian cancers, resulting in hyperactive signaling cascades that relate to cellular growth, proliferation, survival, metabolism, and angiogenesis. Consistent with this, a number of clinical studies are focusing on PI3K pathway as an attractive target in the treatment of ovarian cancer. In this review, we present an overview of PI3K pathway as well as its pathological aberrations reported in ovarian cancer. We also discuss inhibitors of PI3K pathway that are currently under clinical investigations and the challenges these inhibitors face in future clinical utility.Methods: PubMed was searched for articles of relevance to ovarian cancer and the PI3K pathway. In addition, the ClinicalTrials.gov was also scanned for data on novel therapeutic inhibitors targeting the PI3K pathway. Results: Genetic aberrations at different levels of PI3K pathway are frequently observed in ovarian cancer, resulting in hyperactivation of this pathway. The alterations of this pathway make the PI3K pathway an attractive therapeutic target in ovarian cancer. Currently, several inhibitors of PI3K pathway, such as PI3K/AKT inhibitors, rapamycin analogs for mTOR inhibition, and dual PI3K/mTOR inhibitors are in clinical testing in patients with ovarian cancer. Conclusions: PI3K pathway inhibitors have shown great promise in the treatment of ovarian cancer. However, further researches on selection patients that respond to PI3K inhibitors and exploration of effective combinatorial therapies are required to improve the management of ovarian cancer.
Fig.1. Inputs from receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCR) to class I PI3K.
Fig. 2. Schematic representation of the PI3K/AKT/mTOR signaling pathway.
Fig.3. PI3K/AKT/mTOR inhibitors.
AKT inhibitors
AKT inhibitors can be grouped into three classes including lipid based phosphatidylinositol (PI) analogs, ATP-competitive inhibitors, and allosteric inhibitors. Perifosine, which is the most clinically studied AKT inhibitor, is a lipid-based PIanalog that targets the pleckstrin homology domain of AKT, preventing its translocation to the cell membrane. Amongthe three classes of AKT inhibitors, allosteric AKT inhibitors display highly specific selectivity for AKT isoforms. Considering the genetic background of ovarian cancer, allosteric AKT inhibitors such as MK2206 that can target both AKT1 and AKT2 might be the best agents for treating ovarian cancer.In clinical trials, AKT inhibitors have shown similar toxicities to those caused by PI3K inhibitors, such as hyperglycemia, rashes, stomatitis, and gastrointestinal side effects [25].
mTOR inhibitors
Rapamycin and its analogs Rapamycin (sirolimus), a potent inhibitor of mTORC1, was first isolated in 1975 from the bacterium Streptomyces hygroscopicus. Rapamycin inhibits mTORC1 by first binding to the intracellular protein FK506 binding protein 12 (FKBP12). The resultant rapamycin–FKBP12 complex then binds to the FKBP12–rapamycin-binding domain (FRB) of mTORC1 and inhibits the serine/threonine kinase activity of mTORC1 via an allosteric mechanism. In contrast to mTORC1, the rapamycin–FKBP12 complex cannot interact with the FRB domain of mTORC2, and thus,mTORC2 is generally resistant to rapamycin treatment [12]. As rapamycin displays very poor water solubility, which limits its clinical use, several soluble ester analogs of rapamycin (rapalogs) have been developed [12]. Currently, these analogs include temsirolimus, everolimus, and ridaforolimus. Temsirolimus and everolimus are formulated for intravenous and oral administration, respectively. Ridaforolimus was initially developed as an intravenous formulation, but an oral formulation was subsequently produced [12,28]. Clinically, rapalogs are generally well tolerated, with the most common side effects including stomatitis, rashes, fatigue, hyperglycemia, hyperlipidemia, hypercholesterolemia, and myelosuppression [3,12,25].
ATP-competitive inhibitors
Different from rapalogs, ATP-competitive inhibitors do not require co-factors such as FKBP12 to bind to mTOR. By competingwith ATP for theATP-binding sites of mTOR, this class of mTOR inhibitors can inhibit the kinase activity of both mTORC1 and mTORC2. Although there is a concern that the simultaneous inhibition of mTORC1 and mTORC2 might result in greater toxicities in normal tissues, ATP-competitive mTOR inhibitors have been shown to display stronger anti-proliferative activity than rapalogs across a broad range of cancers includingovarian cancer [12,15].
Metformin
Metformin,the most commonly prescribed oral anti-diabetic agent, has been shown to reduce the incidence of malignancies in patients with diabetes. The activation of 5′ adenosine monophosphateactivated protein kinase (AMPK) by metformin plays an important role in mediating the drug’s effects. AMPK activation results in the phosphorylation and activation of TSC2, which exerts inhibitory effects on mTORC1. Metformin-induced AMPK activation also reduces AKT activity by inhibiting insulin receptor substrate 1 (IRS-1). Ultimately, AMPK activation results in the inhibition of the PI3K/AKT/mTOR signaling pathway, making metformin an effective treatment for cancer [28].
Pan-class I PI3K inhibitors Pan-class IPI3K inhibitors can inhibit the kinase activity ofall 4 isoforms of classI PI3K.The main advantage of pan-class IPI3K inhibitors is that most cancer cells express multiple PI3K isoforms with redundant oncogenic signaling functions. Early clinical trials have suggested that the most common toxicitiesof pan-class IPI3K inhibitors are hyperglycemia, skin toxicities, stomatitis, and gastrointestinal side effects. Of these, hyperglycemia is likely to be a mechanism-based toxicity given the well described role of PI3K in insulin receptor signaling [3,25].
Isoform-selective PI3K inhibitors
This class of agents target the specific PI3K p110 isoforms involved in particular types of cancer. The p110α isoform (which is encoded by the PIK3CA gene) is a frequent genetic driver (PIK3CA mutations) of ovarian cancer, whereas p110β activity is known to be essential in cancer cells lacking PTEN. As for the p110δ isoform, it plays a fundamental role in the survival of normal B cells and is implicated in malignancies affecting this lineage. Thus, the main theoretical advantage of these inhibitors is that they have the potential to completely block the relevant target whilst causing limited toxicities compared with pan-PI3K inhibitors. Consistent withthese findings, preclinical studies have detected significant activities of PI3Kα inhibitor in tumors exhibiting PIK3CA mutations, PI3Kβ inhibitors in tumors with PTEN loss, and PI3Kδ inhibitors in hematologic malignancies. In addition, PI3Kδ inhibitors have already shown very promising activity in patients with chronic lymphocytic leukemia [26].
Dual PI3K/mTOR inhibitors
Structural similarities between the ATP-binding domain of p110 and the catalytic domain of mTOR have led to the development of a class of agents that inhibit both class I PI3K and mTORC1/2. Theoretically, dual mTOR/PI3K inhibitors should lead to more complete suppression of the PI3K/AKT/mTOR pathway than targeting either component independently.In agreement with this, in preclinical studies of ovarian cancer dual PI3K/mTOR inhibitors were found to exhibit greater in vitro and in vivo anti-tumor activity than mTOR inhibitors alone [27]. The safety profile of these inhibitors is similar to that of pan-PI3K inhibitors, with common adverse events including nausea, diarrhea, fatigue, and vomiting [3,25].
11.1.11 Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway
Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3-Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.
Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.
Rac proteins are members of the Rho GTPase family and act as molecular switches in regulating a variety of biological response pathways, including cell motility, gene transcription, cell transformation, and cell-cycle progression (1). The Rac family includes Rac1, the myeloid-lineage-specific Rac2, and the recently cloned Rac3 proteins (2). Rac3 differs from Rac1 and Rac2 in two domains, the insert region and the C terminus, which influence transformation (3, 4), interaction with guanine nucleotide exchange factors (GEFs) (5, 6), and subcellular localization (7, 8). Small GTPases, including Rac, cycle between an inactive GDP-bound state and an active GTP-bound state. Two classes of regulatory factors, GTPase-activating proteins (GAPs) and GEFs, determine by their opposing effects the ratio of GDP versus GTP, which is bound to the GTPase (1). GAP proteins increase the intrinsic rate of GTP hydrolysis, rendering the GTPase inactive, whereas GEFs enhance the exchange of bound GDP for GTP, thereby activating the protein. Active Rac regulates distinct downstream signaling pathways by interacting with specific effector proteins, including a family of serine-threonine protein kinases termed Paks (p21-activated kinases) (9–11).
Apart from its well documented role in cytoskeletal rearrangements in growth factor-stimulated cells (12), Rac1 is required for Ras-induced malignant transformation and is involved in transcription and growth control (1, 13, 14). Recently, the importance of the Rac effector Pak in cell transformation has been highlighted by inhibiting RasV12- and Rac1V12-induced transformation of Rat-1 fibroblasts with a catalytically inactive form of Pak (15, 16). The involvement of Rac1 in driving cell-cycle progression through the G1 phase and stimulating DNA synthesis has been shown by introducing dominant-active and -negative Rac1 mutants into fibroblasts (17, 18). However, the signaling pathways used by Rac to control mitogenesis and proliferation still remain poorly understood. Overexpression of constitutively active Rac-effector-domain mutants in fibroblasts indicated that although Rac1 mediated cyclin D1 transcription by Pak in NIH 3T3 cells (19), Pak was not involved in the DNA synthesis of Swiss 3T3 cells (20). Accumulating evidence, however, suggests higher complexity where Pak-binding proteins, such as the GEF Pix, contribute to the Rac–Pak interaction in vivo and influence subsequent cellular functions (21–23).
All biological functions listed above have been attributed to Rac1 in experimental cell systems using overexpression or microinjection of mutant forms. Endogenously active Rho GTPases, including Rac, have not yet been observed. In this paper, we describe a consistently active Rac3 GTPase leading to hyperactivity of its effector protein kinase, Pak, in human breast cancer-derived epithelial cell lines. Analysis of growth properties and DNA synthesis revealed that both proteins are required to convey the highly proliferative phenotype displayed by these cells.
Highly Proliferating Cancer Cells Contain Hyperactive Rac3.
Comparison of growth rates among several breast cancer cell lines showed that three lines (MDA-MB 435, T47D, and MCF 7) grew faster under normal and low-serum conditions (Fig. (Fig.1).1). Interestingly, in contrast to MDA-MD 231 and Hs578T cells, these three highly proliferative cell lines do not possess mutated Ras (28, 29). To assess whether Rho GTPases drive this cellular phenotype, we determined whether these cell lines contained active GTP-bound Rac or Cdc42. We used a recently described assay, the PBD-pulldown assay (24), which is based on the specific binding of the GTP-bound forms of Rac and Cdc42 to the PBD of Pak (10). Neither active Rac1 (Fig. (Fig.22A) nor active Cdc42 (data not shown) could be detected in any of the cell lysates obtained from serum-starved cells. However, both proteins were detected if the PBD-pulldown assay was performed with in vitro guanosine 5′-[γ-thio]triphosphate (GTP[γS])-loaded cell lysates, confirming that Rac1 and Cdc42 were present in their inactive GDP-bound forms in these cells (Fig. (Fig.22A for Rac1). Next we wanted to determine whether active Rac3 was present in breast cancer cell lines. Because Rac3 effectors have not yet been characterized, we demonstrated by overlay binding and kinase assays that Rac3 bound to and activated Pak as efficiently as Rac1 (data not shown). We verified that the PBD-pulldown assay specifically detected the active GTP-bound form of Rac3 (GTP[γS]-loaded Rac3wt or Rac3V12, Fig. Fig.22B) and not the inactive form. To probe for Rac3 protein in breast cell lysates, a Rac3-specific antibody was used. GST-PBD-pulldown experiments from cell lysates revealed the presence of hyperactive Rac3 in highly proliferative cell lines (MDA-MB 435, T47D, and MCF 7), but not in normal breast cell lines or in less proliferative breast cancer cells (Fig. (Fig.22C). Additionally, as indicated by the virtual absence of Rac3 in the supernatant of the PBD pulldown, all the Rac3 protein present in these cell lines was active (Fig. (Fig.22C). To demonstrate that consistent Rac3 activation is not limited to cell lines, we performed an initial screening of human metastatic breast cancer tissues and found active Rac3 in one of three samples, underlining the potential clinical relevance of the cellular findings (Fig. (Fig.22D).
Differential growth rates of human breast cell lines. pq0104939001
Figure 1Differential growth rates of human breast cell lines. Human breast cell lines, including HMEC 184 (○), MDA-MB 231 (▵), Hs578T (□), MDA-MB 435 (●), T47D (▴), and MCF 7 (♦), were grown in 10% serum (A) or 0.5% serum (B) conditions. The cells were split in duplicate over 6-well plates at 5 × 105 cells per well and counted daily with a hemocytometer for 4 days. Data shown in A and B are representative of three independent experiments.
Figure 2 Active Rac3 is present in highly proliferative cell lines and in human breast cancer tissue. (A and C) Cell lysates from serum-starved breast cancer cell lines without (A and C) or after (+) GTP[γS] loading (A) were incubated with 10 μg of GST-PBD. Active Rac proteins (PBD pulldown) were detected by immunoblot with anti-Rac1 (A) or anti-Rac3 antibodies (C). Blotting of PBD supernatants revealed the GDP-bound form of Rac3 in lysates. Equal amounts of Rac3 protein were detected by immunoblot (IB) in all cell lines. (B) A PBD-pulldown assay of extracts from HeLa cells expressing Myc-Rac3wt or -Rac3 mutants, followed by an anti-Myc immunoblot, detected only active Rac3 (GTP[γS] loading or Rac3V12). (D) PBD pulldown of lysates obtained from three different human metastatic breast cancer tissues, followed by anti-Rac1 and anti-Rac3 immunoblots, revealed active Rac3 in tissue 1. (E) PBD pulldown of lysates derived from MDA-MB 435 and MDA-MB 231 cells expressing LacZ control or Myc-Rac3wt without or after in vitro GTP[γS] loading. Consistent activation of Myc-Rac3wt occurred only in MDA-MB 435 cells. (F) Subcellular localization of Rac1 and Rac3. Cytosol (c) and membranes (m) were obtained after nitrogen cavitation and fractionation of breast cancer cell lines and immunoblotted with anti-Rac1 and anti-Rac3 antibodies. All blots are representative of at least three experiments.
Subcellular Localization and GTPase-Regulatory Factors Influence Rac3 Activity.
Constitutive activation of Ras proteins in cancer cells is often caused by activating point mutations at the switch I or II regions (29). cDNA cloning and complete sequence analysis of full-length Rac3 did not reveal any mutations in the breast cell lines studied and did not explain the observed Rac3 activation. GTPase-regulatory proteins such as GEFs and GAPs, which are usually regulated by upstream stimuli, control cycling between the active and inactive forms of Rac. To confirm the presence of an altered regulatory mechanism involved in Rac3 activation, we used the PBD-pulldown assay to analyze the activation state of Myc-tagged Rac3wt transfected into either MDA-MB 231, a cell line harboring only GDP-Rac3, or MDA-MB 435, a cell line that contains endogenous, active GTP-Rac3. Fig. Fig.22E shows that activated Myc-Rac3 was detected only in the MDA-MB 435 cell line, confirming that the regulation of the GDP/GTP state of Rac3 was altered in these cells. We then investigated several upstream stimuli that have been shown to affect GTPase-regulatory proteins (28, 30–32). We excluded the possibility of an autocrine growth-stimulatory loop by culturing MDA-MB 231 cells with the conditioned medium from MDA-MB 435, which did not affect the Rac3 activation state (data not shown). Treatment of cell cultures with phosphatidylinositol 3-kinase or tyrosine kinase inhibitors, including wortmannin, LY294002, and genistein, did not decrease Rac3 activation (data not shown). At this point, we speculated that an oncogenic, Rac3-specific GEF is present in certain breast cancer cells. GEFs possess a pleckstrin homology domain that is essential for membrane localization and for their oncogenic properties (5, 33). Analysis of the subcellular localization of the Rac family members revealed that Rac3 is located in the membranes of breast epithelial cell lines, independently of its activation state (Fig. (Fig.22F). In contrast, endogenous Rac1 in its inactive GDP-bound state was essentially cytosolic (Fig. (Fig.22F). Thus, the distinct localization of Rac3 and Rac1 may contribute to their different activation states in certain breast cancer cell lines. It is conceivable that the highly proliferative cell lines (Fig. (Fig.1)1) express a constitutively active, membrane-bound Rho GEF that activates adjacent Rac3 protein. This hypothesis was further supported by using an hydroxymethylglutaryl-CoA reductase inhibitor, lovastatin, that interferes with isoprenoid synthesis and thereby with posttranslational processing of GTPases. Unprocessed Rac3 from lovastatin-treated MDA-MB 435 cells was predominantly cytosolic and inactive (GDP-Rac3) (data not shown). The requirement of membrane localization for consistent Rac3 activity was further supported by using a Rac3S189 mutant. Replacing cysteine-189 of the CAAX box with serine abolishes isoprenoid incorporation, rendering the GTPase cytosolic. This Rac3 mutant remained in its inactive GDP-bound state when transfected into MDA-MB 435 cells (data not shown).
Several Rho GTPase-regulating GEFs have been identified (5), including the Rac1-specific GEF Tiam-1, which has been linked to tumors such as invasive T-lymphomas (34). Although Tiam-1 is expressed in virtually all tissues, no evidence of oncogenic truncations or alternative splicing of Tiam-1 transcripts has been found (35). A variation of Tiam-1 transcript levels in certain cancer cell lines might lead to overexpression and possibly activation of Tiam-1 protein. However, the activation state of Rac3 protein in the cell lines used in this study does not seem to correlate with Tiam-1 expression levels as reported by Habets et al. (35). Hyperactivity of Rac3 in cancer cells could also result from an absent or dysfunctional Rac3-specific GAP protein. By accelerating the intrinsic GTP hydrolysis rate, GAPs render the GTPase inactive and act as tumor suppressors. Deletion or mutations in the RasGAP gene NF1 and the RhoGAP homologs bcr and DLC-1 have been reported in cancer cells (36, 37).
Active Rac3 Drives Epithelial Cell Proliferation.
To study whether active Rac3 could account for the high proliferation rate of certain breast cancer cells, we expressed a constitutively active Rac3 mutant (Rac3V12) in normal mammary epithelial cells (HMEC 184) that contain only GDP-Rac3 (Fig. (Fig.22C). Rac3V12 expression significantly increased the incorporation of BrdUrd into nascent DNA (Fig. (Fig.3),3), emphasizing that transfection of active Rac3 drives epithelial cell proliferation.
Rac3V12 induces DNA synthesis in human mammary epithelial cells pq0104939003
Rac3V12 induces DNA synthesis in human mammary epithelial cells
Figure 3Rac3V12 induces DNA synthesis in human mammary epithelial cells. HMEC 184 cells, infected with recombinant LacZ or Rac3V12 Semliki Forest virus, were allowed to express protein for 14 h in serum-free medium containing 10 μM BrdUrd. Cells were fixed and stained with anti-Myc antibody for Myc-Rac3V12 expression level (Upper) or with FITC-conjugated anti-BrdUrd antibody for BrdUrd incorporation (Lower). The presence of bright fluorescent nuclei indicates BrdUrd-positive cells. The percentage was calculated after counting 400 cells in each of three independent experiments.
Hyperactive Pak and c-Jun Kinases in Cancer Cells.
The signaling cascade utilized by Rac proteins to control cell proliferation still remains to be identified (1, 9), but might involve Paks. We analyzed Pak activity in cell lysates derived from serum-starved breast cancer cell lines by using in-gel kinase assays and by usingin vitro kinase assays after immunoprecipitation with Pak-specific antibodies. Pak activity was increased 4- to 6-fold in the three cell lines containing active Rac3 (Fig. (Fig.44A). This increased kinase activity was mainly associated with the Pak2 isoform, which can phosphorylate and positively regulate Raf-1 activity, another key component in cell proliferation (38–40).
Figure 4Rac3 activates Pak and JNK by two different pathways. (A) Breast cancer cell lysates from serum-starved cells were analyzed for Pak and JNK activities. Pak activities in cell lysates were analyzed by in-gel kinase assays. JNK activity was determined by …
Intracellular Rac-regulated signaling pathways impinge on distinct mitogen-activated protein kinase cascades. Constitutively active Rac has been shown to positively regulate the activity of the stress-activated kinases JNK and p38 (1). Moreover, ERK activity can be indirectly stimulated by Rac or mediated by crosstalk between the distinct mitogen-activated protein kinase cascades (1, 41). Determination of distinct mitogen-activated protein and stress-activated protein kinase activities in the breast cell lines studied here showed that consistent Rac3 and Pak kinase activities were associated with enhanced JNK activity (Fig. (Fig.44A). In contrast, no correlation existed between p38 or ERK kinase activities and active Rac3 or Pak (data not shown).
Rac3 Triggers Pak and JNK Activities by Separate Pathways.
To determine whether the highly proliferative phenotype of breast cancer cells depends directly on a consistently active Rac3-Pak-JNK cascade, we used virus-mediated protein expression in MDA-MB 435 cells to examine the ability of Rac3 and Paks to control JNK activation and cellular proliferation. The importance of Pak as an effector protein in Rac-mediated activation of JNK is still controversial and seems to be cell-type-dependent (42). Expression of the PBD domain, which controls the activity of both Rac and Pak (21), completely inhibited Pak and JNK stimulation (Fig. (Fig.44B). The mutation of leucine to phenylalanine at position 107 of the PBD domain suppresses the autoinhibitory function of the PBD (21). Thus, PBD F107 will act only to sequester active Rac3 and blocks its ability to bind and activate endogenous effectors. Expression of either dominant-negative Rac3N17 or PBD F107 almost completely blocked Pak and JNK activities, demonstrating that Rac3 is upstream of these proteins (Fig. (Fig.44B). Moreover, Pak kinase activity can be inhibited independently of Rac3 by overexpressing the kinase autoinhibitory domain, PID, which does not interact with Rac (21, 43). Transfection of PID into MDA-MB 435 cells dramatically inhibited Pak activity as expected, but did not decrease JNK activation (Fig. (Fig.44B). Our results indicate that in MDA-MB 435 cells, consistent stimulation of JNK by Rac3 is independent of PAK activity and that Rac3 initiates two different pathways involving Pak and JNK, respectively.
Rac3 and Pak Are Both Required for Breast Cancer Cell Proliferation.
We subsequently determined which of these two Rac3 pathways promoted the increased cell proliferation in breast cancer cell lines with hyperactive Rac3. We studied the consequence of expressing inhibitory Rac mutants or Pak fragments on DNA synthesis. LacZ-expressing MDA-MB 435 cells still proliferated in low-serum conditions and 35% incorporated BrdUrd (Fig. (Fig.5).5). This percentage increased to 50% when Rac3wt, which will be partially activated in these cells (Fig. (Fig.22E), is expressed (Fig. (Fig.55Bottom Right). In contrast, expression of inhibitory proteins, including Rac3N17 or the PBD that suppressed Pak and JNK activation (Fig. (Fig.44B), almost completely blocked S-phase entry, as indicated by the absence of BrdUrd incorporation (Fig. (Fig.5).5). Expression of the PID that inhibited Pak kinase activity without affecting JNK stimulation (Fig. (Fig.44B) also arrested proliferation in MDA-MB 435 cells (Fig. (Fig.5).5). These experiments emphasize the crucial role of active Rac3 for DNA synthesis in breast cancer cell lines and demonstrate that Pak kinase activity is necessary for Rac3-induced proliferation.
Rac3 mediates proliferation in MDA-MB 435 cells pq0104939005
Figure 5 Rac3 mediates proliferation in MDA-MB 435 cells by a Pak-dependent pathway. MDA-MB 435 cells growing in 0.5% FBS were infected with Semliki Forest virus encoding for LacZ, Rac3N17, Pak1-PBD, Pak1-PBD F107, Pak1-PID, or Rac3wt. After 12 to14 h of protein expression in serum-free medium, 20 μM BrdUrd was added for 20 min before the cells were fixed and stained with anti-Myc antibody and phalloidin for expression (Top) or with FITC-conjugated anti-BrdUrd antibody for BrdUrd incorporation (Lower five micrographs). The presence of bright fluorescent nuclei indicates BrdUrd-positive cells. The percentage was calculated after counting 400 cells in each of four independent experiments.
Our results establish the persistent activation of a small Rho GTPase, Rac3, and the effector kinase Pak in human breast cancer cells. In contrast to Rac1, endogenous Rac3 is localized at the plasma membrane in both guanine nucleotide states. It seems likely that a Rac3 regulatory protein is altered or deleted in highly proliferating cancer cells, and that its specificity toward Rac3 results from the adjacent location of both proteins at the membrane and/or from discrete Rac3 domains, which convey a specific interaction. The cytoskeletal phenotypes of serum-starved breast cancer cells, such as ruffles or lamellipodia typical of Rac1 protein activation, did not seem to correlate with the GDP versus GTP state of endogenous Rac3. This may suggest that Rac family members are specialized in certain cellular functions, as already reported for Rac2 in leukocyte phagocytosis (44) and now demonstrated by us for Rac3 in cancer cell proliferation. Our studies establish further that endogenous, active Rac3 is essential for breast cancer cell proliferation via a Pak-dependent pathway. Paks have been shown to directly phosphorylate Raf kinase, which binds to retinoblastoma protein and regulates its function (45), and to interact with cyclin-dependent kinases to up-regulate cyclin D1 expression (46). Initial screening of various human cancer-derived cell lines revealed the presence of hyperactive Rac3 and Pak kinase in other types of highly proliferating tumors (data not shown). Further investigations, primarily in animal models and clinical settings, will be necessary to assess whether loss of Rac3 and Pak regulation correlates with certain breast tumor stages and is accompanied by specific alterations in cell-cycle regulators. Approaches to inhibit Rac3 or Pak activity would then open a new avenue for cancer therapeutics.
11.1.12 Curcumin-could-reduce-the-monomer-of-ttr-with-tyr114cys-mutationvia autophagy in cell model of familial amyloid polyneuropathy.
Transthyretin (TTR) familial amyloid polyneuropathy (FAP) is an autosomal dominant inherited neurodegenerative disorder caused by various mutations in the transthyretin gene. We aimed to identify the mechanisms underlying TTR FAP with Tyr114Cys (Y114C) mutation. Our study showed that TTR Y114C mutation led to an increase in monomeric TTR and impaired autophagy. Treatment with curcumin resulted in a significant decrease of monomeric TTR by recovering autophagy. Our research suggests that impairment of autophagy might be involved in the pathogenesis of TTR FAP with Y114C mutation, and curcumin might be a potential therapeutic approach for TTR FAP.
Transthyretin (TTR) familial amyloid polyneuropathy (FAP) is an autosomal dominant inherited disease, characterized clinically by progressive sensory, motor, and autonomic impairment, which typically lead to death around a decade after diagnosis.1 Since the first identification of TTR with Val30Met mutation (TTR V30M), the most common gene mutation in FAP patients, more than 100 TTR mutations have been found to cause FAP.2 However, the detailed pathogenesis underlying TTR FAP remains undefined. Previous studies of the TTR V30M mutant have shown that misfolding and self-aggregation of TTR are implicated in the pathogenesis of TTR FAP involving abnormal endoplasmic reticulum (ER) stress.3
Corresponding to the various TTR gene mutations and a wide range of geographical distributions, FAP presents diverse characteristics in genotype-phenotype in different regions. We have recently published the first report of a TTR Tyr114Cys (TTR Y114C) mutation in a Chinese family with TTR FAP.4 Compared with TTR V30M, the TTR Y114C mutation showed different clinical manifestations, and was also observed in a Japanese family.5,6 This suggests that the pathogenesis of the TTR Y114C and TTR V30M mutations might be different. Studies focused on monomer generation and tetramer depolymerization have been performed.1,2 However, the mechanisms underlying the clearing of the abnormally increased monomer are unknown.
Autophagy is the major lysosomal pathway via which cells degrade intracytoplasmic protein. It is widely accepted that autophagy plays a key role in the process of amyloid deposition in certain neurodegenerative diseases, including alpha-synuclein, beta peptides, tau oligomers, and misfolded prion protein.7 Therefore, autophagy may be involved in degradation of the TTR monomer in TTR FAP.
Curcumin and its analogs have demonstrated a protective effect in many diseases involving antimicrobial, antitubercular,8 and anticancer mechanisms,9 and they can also modulate innate immunity.10 Of note, curcumin has been shown to promote autophagy.11 Therefore, we hypothesized that autophagy might be involved in the pathogenetic mechanism of the TTR Y114C mutation in TTR FAP and curcumin might have potential therapeutic role in this disease. In this study, we aimed to identify the role of autophagy in the pathogenetic mechanism of TTR FAP and to assess the therapeutic effect of curcumin in the disease.
TTR Y114C mutation led to increased monomeric TTR and impaired autophagy in vitro
To investigate the alteration of monomeric TTR with different mutations, we generated HEK293T cell lines with wild-type TTR, TTR Y114C, and stable overexpression of TTR V30M. Wild-type TTR represented the normal control and TTR V30M represented the positive control. Western blotting analysis of the TTR level in the cells when cultured for 24 hours showed that the monomer of TTR Y114C and TTR V30M was increased by approximately 2.3 times and 2.78 times, respectively, compared with wild-type TTR (Figure 1A and B). Mutation of TTR Y114C was related to the increase in monomeric TTR, as well as the mutation of TTR V30M.
Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C dddt-8-2121Fig1
Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C
Figure 1Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C.
Next we investigated the activation of several markers associated with ER stress, including ER-resident chaperone BiP and p-eIF2α. Our results showed the levels of BiP and p-eIF2α is higher in TTR V30M than those in wild-type TTR. In contrast, BiP and p-eIF2α levels in TTR Y114C were similar to those in wild-type TTR (Figure 1A and C), indicating ER stress might not be the main pathogenetic mechanism for the TTR Y114C mutation. We then investigated whether autophagy plays a role in the mechanism of TTR Y114C mutation. LC3-II is well known to be a robust marker of autophagosomes, and immunofluorescent staining of LC3-II can be used to assay for autophagosome formation. A high ratio of LC3-II to LC3-I would indicate induction of autophagy. Our results revealed that the ratio of LC3-II/I was markedly decreased for TTR Y114C, but less suppressed for TTR V30M (Figure 1A and D). Likewise, a significant decrease in LC3-II immunoreactivity was detected in TTR Y114C (Figure 1E). The results of Western blotting and immunofluorescence indicated that autophagy in TTR Y114C was significantly downregulated. Therefore, impaired autophagy might be responsible for the pathogenesis of TTR Y114C mutation.
Curcumin decreased monomeric TTR by promoting autophagy
The effects of curcumin were investigated in TTR Y114C and wild-type TTR stable overexpressed HEK293T cells. Curcumin did not show toxic effects in the stable overexpressed cell lines at curcumin concentrations below 10 µM (Figure 2A and B). We chose 5 µM as the experimental concentration, because it is the minimal effective concentration of curcumin in these cell lines. Further, we wanted to determine whether curcumin could decrease monomeric TTR by promoting autophagy at the minimal effective concentration. Therefore, we used curcumin (2.5 µM and 5 µM) as a protective agent to assess whether it could decrease monomeric TTR with mutation by promoting autophagy. Quantification of LC3-II and LC3-I indicated markedly higher activation of LC3 in TTR Y114C treated with curcumin 5 µM for 24 hours (Figure 2D). In contrast, treatment with curcumin at different concentrations could not activate LC3 in wild-type TTR (Figure 2C, E). We next examined the ratio of monomers to tetramers in TTR Y114C, which was significantly decreased after 24 hours of treatment with 5 µM curcumin compared with no treatment with curcumin (Figure 2D and F). However, for wild-type TTR, the ratio of monomers to tetramers was unchanged after treatment with curcumin (Figure 2C and E). These results indicate that treatment with curcumin 5 µM for 24 hours was able to decrease the monomer in the TTR Y114C mutation by promoting autophagy.
Curcumin decreased monomeric TTR by promoting autophagy dddt-8-2121Fig2
Curcumin decreased monomeric TTR by promoting autophagy
Figure 2Curcumin decreased monomeric TTR by promoting autophagy.
Protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA
To further validate whether the decrease in monomer by curcumin in our experiments was mediated by autophagy, 3-MA, an inhibitor of autophagosome formation, was implied to negatively regulate autophagy. 3-MA (1 mM) was added to the cell culture medium 2 hours before curcumin and incubated for 24 hours. Analysis of LC3, tetrameric TTR, and monomeric TTR from TTR Y114C revealed that 3-MA partly reversed the LC3 II activation induced by curcumin and increased the monomer of TTR Y114C (Figure 3). These results confirm that curcumin induced the decrease in the TTR Y114C monomer by promoting the autophagy pathway.
Figure 3 Protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA.
Discussion
TTR FAP is a severe autosomal dominant inherited disease, for which the treatment options are limited. Liver transplantation performed early in the course of the disease is the only therapeutic strategy known to stabilize this neuropathy.1,13 More recently, tafamidis meglumine, a potent inhibitor of misfolding and deposition of mutated TTR, has completed an 18-month, placebo-controlled Phase II/III clinical trial for the treatment of FAP.14 However, in June 2012, the US Food and Drug Administration Peripheral and Central Nervous System Drugs Advisory Committee rejected this drug, stating a lack of convincing data supporting its efficacy.15 Hence, it is important to identify the pathogenetic mechanism of FAP to find an alternative effective treatment strategy.
Accumulating studies focused on the TTR mutation gene and protein have provided insights into the pathogenesis of TTR FAP, including decreased stability of TTR tetramers, conformational change in the crystal structure of variant TTR, altered kinetics of denaturation, and disturbing endoplasmic ER quality control system.1,16–18 Previous studies have demonstrated that increased levels of ER stress are correlated with extracellular TTR deposition. Two ER stress markers, BiP and p-eIF2α, have been observed to be present and upregulated in the salivary gland tissue of FAP patients.3 However, the precise molecular mechanisms underlying TTR FAP and its phenotypic heterogeneity are not yet fully understood.
Our current study investigated whether the two mutations, TTR Y114C and TTR V30M, share the same pathogenesis and evaluated the effect of pathogenic mutations on the clearance of the monomer. Our results show that the ratio of LC3-II/I was markedly decreased, while BiP and p-eIF2α levels remained constant in TTR Y114C when compared with wild-type TTR and TTR 30M. The results of our research indicate the impaired autophagy contributed to the TTR Y114C mutation, but not ER stress. This observation indicates that abnormal accumulation of TTR caused by a different mutation might be cleared by different pathways, and more studies are necessary to confirm whether this difference applies to other TTR mutations.
Curcumin is known to have neuroprotective properties through a variety of mechanisms.8–11 Our research indicates that curcumin decreased the monomeric TTR by promoting autophagy, and without toxic effects. Moreover, this protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA. Pullakhandam et al showed that curcumin binds to wild-type TTR and prevents urea-induced perturbations in the tertiary structure of TTR in vitro.19 Recently, Ferreira et al reported that dietary curcumin modulated TTR amyloidogenicity.20 Therefore, curcumin might be an effective therapy for FAP involving multiple molecular pathways.
Overall, our findings show that abnormal accumulation of TTR caused by different mutations might be cleared in different ways, and curcumin might be an effective therapy for FAP by promoting autophagy. Further studies are necessary to determine whether this phenomenon exists in other TTR mutations.
Stephen Williams, PhD
For PI3K and related inhibitors of PI3K/AKT/mTOR i would refer you to two people who should be in the discussion of this signaling pathway and PI3K/AKT inhibitors used for chemotherapy. The first is Dr. Mien-Chie Hung and the second is Dr. Gordon Mills. They both had been at MD Anderson and developed some of the first inhibitors as well as the earliest discoveries of overactivity of PI3K/AKT in ovarian cancer.
Next the field had never progressed any inhibitors past Stage II as there has been some serious toxicities seen in preclinical phases (most long term tox studies are done after patients are enrolled in phase I).
These will show how inhibitors of certain isoforms of PI3K (namely delta) had to be developed to circumvent some of the severe toxicity seen with the earliest inhibitors (wortmanin and LY294002.
Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeuticshttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843585/ will explain about some of the toxicities and describes the one PI3K that has made it to phase II
Most of them have failed and I believe now are being thought as an adjuvant not front line therapy
Lab Director at Emergency County Hospital Targu Jiu
In experimental models, disrupting the MDM2–p53
interaction restored p53 function and sensitized tumors to
chemotherapy or radiotherapy. (Kojima et al., 2005). This
strategy could be particularly beneficial in treating
cancers that do not harbor TP53 mutations. For example
in hematologic malignancies, such as multiple myeloma,
chronic lymphocytic leukemia (CLL), acute lymphoblastic
leukemia (ALL), acute myeloid leukemia (AML), and
Hodgkin’s disease, the induction of p53 – using a small
MDM2-inhibitor molecule, nutlin-3 – can induce the
apoptosis of malignant cells. Nutlins are a group of cisimidazoline
analogs, first identified by Vassilev et al.
(2004), which have a high binding potency and selectivity
for MDM2. Crystallization data have shown that nutlin-3
mimics the three residues of the helical region of the
trans-activation domain of p53 (Phe19, Trp23 and
Leu26), which are conserved across species and critical
for binding to MDM2 (Wade et al., 2010). Nutlin-3
displaces p53 by competing for MDM2 binding. It has
also been found that nutlin-3 potently induces apoptosis
in cell lines derived from hematologic malignancies,
including AML, myeloma, ALL, and B-cell CLL (Secchiero
et al., 2010).
Stephen J Williams, PhD
Now as far as PKM2 you would want to look at a company called Synta Pharmaceuticals and their inhibitor Elesclomal. elesclomol binds copper ions causing a change in conformation that enables its uptake through membranes and into cells. Elesclomol binds copper in an oxidative, positively charged state called Cu(II). Once inside mitochondria, the elesclomol-Cu(II) complex interacts with the energy production mechanism of the cell, or the electron transport chain. This interaction reduces the copper from Cu(II) to Cu(I), resulting in a cascade of reduction-oxidation, or redox, reactions, that causes a rapid increase of oxidative stress, disruption of mitochondrial energy production, and ultimately, triggering of the mitochondrial apoptosis pathway.
The important part is that it seemed, to prefer tumors which had lower LDH activity, meaning that these tumor cells actually did have a more active electron transport chain than tumors with high LDH (Warburg) and therefore in clinical trials the tumors with lower LDH activity responded more favorably.
The following article is a review of the central relationship between the action of
metformin as a diabetic medication and its relationship to AMPK, the important and
essential regulator of glucose and lipid metabolism under normal activity, stress, with
its effects on skeletal muscle, the liver, the action of T3 and more.
We start with a case study and a publication in the J Can Med Assoc. Then we shall look
into key literature on these metabolic relationships.
Part I. Metformin , Diabetes Mellitus, and Thyroid Function
Hypothyroidism, Insulin resistance and Metformin
May 30, 2012 By Janie Bowthorpe
The following was written by a UK hypothyroid patient’s mother –
Sarah Wilson.
My daughter’s epilepsy is triggered by unstable blood sugars. And since taking
Metformin to control her blood sugar, she has significantly reduced the number of seizures. I have been doing research and read numerous academic medical journals,
which got me thinking about natural thyroid hormone and Hypothyroidism. My hunch
was that when patients develop hypothyroid symptoms, they are actually becoming
insulin resistant (IR). There are many symptoms in common between women with
polycystic ovaries and hypothyroidism–the hair loss, the weight gain, etc.
(http://insulinhub.hubpages.com/hub/PCOS-and-Hypothyroidism).
A hypothyroid person’s body behaves as if it’s going into starvation mode and so, to
preserve resources and prolong life, the metabolism changes. If hypothyroid is prolonged
or pronounced, then perhaps, chemical preservation mode becomes permanent even
with the reintroduction of thyroid hormones. To get back to normal, they need
a “jump-start” reinitiate a higher rate of metabolism. The kick start is initiated through
AMPK, which is known as the “master metabolic regulating enzyme.”
(http://en.wikipedia.org/wiki/AMP-activated protein kinase).
Note the following comments/partial statements:
“Hypothyroidism is characterized by decreased insulin responsiveness”;
“the pivotal regulatory role of T3 in major metabolic pathways”.
The community knows that T3/NTH (natural thyroid hormone [Armour]) makes
hypothyroid patients feel better – but the medical establishment is averse to T3/NTH
(treating subclinical hypoT (T3/T4 euthyroid) with natural dessicated thyroid (NDT).
The medical establishment might find an alternative view about impaired metabolism
more if shown real proof that the old NDT **was/is** having the right result –i.e., the
T3 is jump-starting the metabolism by re-activatingAMPK.
If NDT also can be used for hypothyroidism without the surmised “dangers” of NTH,
then they should consider it. [The reality in the choice is actually recombinant TH
(Synthroid)]. Metformin is cheap, stable and has very few serious side effects. I use the
car engine metaphor, and refer to glucose as our petrol, AMPK as the spark plug and
both T3 and Metformin as the ignition switches. Sometimes if you have flat batteries in
the car, it doesn’t matter how much you turn the ignition switch or pump the petrol
pedal, all it does is flatten the battery and flood the engine.
Dr. Skinner in the UK has been treating “pre-hypothyroidism” the way that some
doctors treat “pre-diabetes”. Those hypothyroid patients who get treated early
might not have had their AMPK pathways altered and the T4-T3 conversion still works.
There seems to be no reason why thyroid hormone replacement therapy shouldn’t
logically be given to ward off a greater problem down the line.
It’s my belief that there is clear and abundant academic evidence that the AMPK/
Metformin research should branch out to also look at thyroid disease.
Metformin Linked to Risk of Low Levels of Thyroid Hormone
CMAJ (Canadian Medical Association Journal) 09/22/2014
Metformin, the drug commonly for treating type 2 diabetes,
is linked to an increased risk of low thyroid-stimulating hormone (TSH) levels
in patients with underactive thyroids (hypothyroidism),
according to a study in CMAJ (Canadian Medical Association Journal).
Metformin is used to lower blood glucose levels
by reducing glucose production in the liver.
previous studies have raised concerns that
metformin may lower thyroid-stimulating hormone levels.
Study characteristics:
Retrospective long-term
74 300 patient who received metformin and sulfonylurea
25-year study period.
5689 had treated hypothyroidism
59 937 had normal thyroid function.
Metformin and low levels of thyroid-stimulating hormone in patients with type 2 diabetes mellitus
Jean-Pascal Fournier, Hui Yin, Oriana Hoi Yun Yu, Laurent Azoulay +
Centre for Clinical Epidemiology (Fournier, Yin, Yu, Azoulay), Lady Davis Institute,
Jewish General Hospital; Department of Epidemiology, Biostatistics and Occupational
Health (Fournier), McGill University; Division of Endocrinology (Yu), Jewish General
Hospital; Department of Oncology (Azoulay), McGill University, Montréal, Que., Cananda
metformin may lower thyroid-stimulating hormone (TSH) levels.
Objective:
determine whether the use of metformin monotherapy, when compared with
sulfonylurea monotherapy,
is associated with an increased risk of low TSH levels(< 0.4 mIU/L)
in patients with type 2 diabetes mellitus.
Methods:
Used the Clinical Practice Research Datalink,
identified patients who began receiving metformin or sulfonylurea monotherapy
between Jan. 1, 1988, and Dec. 31, 2012.
2 subcohorts of patients with treated hypothyroidism or euthyroidism,
followed them until Mar. 31, 2013.
Used Cox proportional hazards models to evaluate the association of low TSH
levels with metformin monotherapy, compared with sulfonylurea monotherapy,
in each subcohort.
Results:
5689 patients with treated hypothyroidism and 59 937 euthyroid patients were
included in the subcohorts.
For patients with treated hypothyroidism:
495 events of low TSH levels were observed (incidence rate 0.1197/person-years).
322 events of low TSH levels were observed (incidence rate 0.0045/person-years)
in the euthyroid group.
metformin monotherapy was associated with a 55% increased risk of low TSH
levels in patients with treated hypothyroidism (incidence rate 0.0795/person-years
vs.0.1252/ person-years, adjusted hazard ratio [HR] 1.55, 95% confidence
interval [CI] 1.09– 1.20), compared with sulfonylurea monotherapy,
the highest risk in the 90–180 days after initiation (adjusted HR 2.30, 95% CI
1.00–5.29).
No association was observed in euthyroid patients (adjusted HR 0.97, 95% CI 0.69–1.36).
Interpretation: The clinical consequences of this needs further investigation.
Crude and adjusted hazard ratios for suppressed thyroid-stimulating hormone
levels (< 0.1 mIU/L) associated with the use metformin monotherapy, compared
with sulfonylurea monotherapy, in patients with treated hypothyroidism or
euthyroidism and type 2 diabetes
Variable
No. events
suppressed
TSH levels
Person-years of
exposure
Incidence rate,
per 1000 person-years (95% CI)
Crude
HR
Adjusted HR*(95% CI)
Patients with treated hypothyroidism, n = 5689
Sulfonylure, n = 762
18
503
35.8
(21.2–56.6)
1.00
1.00
(reference)
Metformin, n = 4927
130
3 633
35.8
(29.9–42.5)
1.05
0.99
(0.57–1.72)
Euthyroid patients, n = 59 937
Sulfonylurea, n = 7980
12
8 576
1.4
(0.7–2.4)
1.00
1.00
(reference)
Metformin, n = 51 957
75
63 047
1.2
(0.9–1.5)
0.85
1.03
(0.52–2.03)
Part II. Metabolic Underpinning
(Source: Wikipedia, AMPK and thyroid)
5′ AMP-activated protein kinase or AMPK or 5′ adenosine monophosphate-activated protein kinase
is an enzyme that plays a role in cellular energy homeostasis.
It consists of three proteins (subunits) that
together make a functional enzyme, conserved from yeast to humans.
It is expressed in a number of tissues, including the liver, brain, and skeletal
muscle.
The net effect of AMPK activation is stimulation of
hepatic fatty acid oxidation and ketogenesis,
inhibition of cholesterol synthesis,
lipogenesis, and triglyceride synthesis,
inhibition of adipocyte lipolysis and lipogenesis,
stimulation of skeletal muscle fatty acid oxidation and muscle
glucose uptake, and
modulation of insulin secretion by pancreatic beta-cells.
The heterotrimeric protein AMPK is formed by α, β, and γ subunits. Each of these three
subunits takes on a specific role in both the stability and activity of AMPK.
the γ subunit includes four particular Cystathionine beta synthase (CBS) domains
giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio.
The four CBS domains create two binding sites for AMP commonly referred to as
Bateman domains. Binding of one AMP to a Bateman domain cooperatively
increases the binding affinity of the second AMP to the other Bateman domain.
As AMP binds both Bateman domains the γ subunit undergoes a conformational
change which exposes the catalytic domain found on the α subunit.
It is in this catalytic domain where AMPK becomes activated when
phosphorylation takes place at threonine-172by an upstream AMPK kinase
(AMPKK). The α, β, and γ subunits can also be found in different isoforms.
AMPK acts as a metabolic master switch regulating several intracellular systems
the cellular uptake of glucose,
the β-oxidation of fatty acids and
the biogenesis of glucose transporter 4 (GLUT4) and
mitochondria
The energy-sensing capability of AMPK can be attributed to
its ability to detect and react to fluctuations in the AMP:ATP ratio that take
place during rest and exercise (muscle stimulation).
During muscle stimulation,
AMP increases while ATP decreases, which changes AMPK into a good substrate
for activation.
AMPK activity increases while the muscle cell experiences metabolic stress
brought about by an extreme cellular demand for ATP.
Upon activation, AMPK increases cellular energy levels by
inhibiting anabolic energy consuming pathways (fatty acid synthesis,
protein synthesis, etc.) and
A recent JBC paper on mice at Johns Hopkins has shown that when the activity of brain
AMPK was pharmacologically inhibited,
the mice ate less and lost weight.
When AMPK activity was pharmacologically raised (AICAR see below)
the mice ate more and gained weight.
Research in Britain has shown that the appetite-stimulating hormone ghrelin also
affects AMPK levels.
The antidiabetic drug metformin (Glucophage) acts by stimulating AMPK, leading to
reduced glucose production in the liver and
reduced insulin resistance in the muscle.
(Metformin usually causes weight loss and reduced appetite, not weight gain and
increased appetite, ..opposite of expected from the Johns Hopkins mouse study results.)
Triggering the activation of AMPK can be carried out provided two conditions are met.
an AMP analogthat has been shown to activate AMPK.
Recent LKB1 knockout studies have shown that without LKB1,
electrical and AICAR stimulation of muscleresults in very little
phosphorylation of AMPK and of ACC, providing evidence that
LKB1-STRAD-MO25 is the major AMPKK in muscle.
Two particular adipokines, adiponectin and leptin, have even been demonstrated
to regulate AMPK. A main functions of leptin in skeletal muscle is
the upregulation of fatty acid oxidation.
Leptin works by way of the AMPK signaling pathway, and adiponectin also
stimulates the oxidation of fatty acids via the AMPK pathway, and
Adiponectin also stimulates the uptake of glucose in skeletal muscle.
An increase in enzymes which specialize in glucose uptake in cells such as GLUT4
and hexokinase II are thought to be mediated in part by AMPK when it is activated.
Increases in AMPK activity are brought about by increases in the AMP:ATP ratio
during single bouts of exercise and long-term training.
One of the key pathways in AMPK’s regulation of fatty acid oxidation is the
phosphorylation and inactivation of acetyl-CoA carboxylase.
Acetyl-CoA carboxylase (ACC) converts acetyl-CoA (ACA) to malonyl-CoA
(MCA), an inhibitor of carnitine palmitoyltransferase 1 (CPT-1).
CPT-1 transports fatty acids into the mitochondria for oxidation.
Inactivation of ACC results in increased fatty acid transport and oxidation.
the AMPK induced ACC inactivation and reduced conversion to MCA
may occur as a result of malonyl-CoA decarboxylase (MCD)
MCD as an antagonist to ACC, decarboxylatesmalonyl-CoA to acetyl-CoA
(reversal of ACC conversion of ACA to MCA)
This resultsin decreased malonyl-CoA and increased CPT-1 and fatty acid oxidation.
AMPK also plays an important role in lipid metabolism in the liver. It has long been
known that hepatic ACC has been regulated in the liver.
It phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)
acetyl-CoA(ACA) is converted to mevalonic acid (MVA) by ACC
with inhibition of CPT-1
HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from MVA
which then travels down several more metabolic steps to become cholesterol.
Insulin facilitates the uptake of glucose into cells via increased expression and
translocation of glucose transporter GLUT-4. In addition, glucose is phosphorylated
by hexokinase wheni iot enters the cell. The phosphorylated form keeps glucose from
leaving the cell,
The decreasedthe concentration of glucose molecules creates a gradient for more
glucose to be transported into the cell.
AMPK and thyroid hormone regulate some similar processes. Knowing these similarities,
Winder and Hardie et al. designed an experiment to see if AMPK was influenced by thyroid
hormone. They found that all of the subunits of AMPK were increased in skeletal muscle,
especially in the soleus and red quadriceps, with thyroid hormone treatment. There was
also an increase in phospho-ACC, a marker of AMPK activity.
Winder WW, Hardie DG (July 1999). “AMP-activated protein kinase,
a metabolic master switch: possible roles in type 2 diabetes”. J. Physiol.277
(1 Pt 1): E1–10. PMID10409121.
Winder WW, Hardie DG (February 1996). “Inactivation of acetyl-CoA
carboxylase and activation of AMP-activated protein kinase in muscle
during exercise”. J. Physiol.270 (2 Pt 1): E299–304. PMID8779952.
Hutber CA, Hardie DG, Winder WW (February 1997). “Electrical stimulation
inactivates muscle acetyl-CoA carboxylase and increases AMP-activated
protein kinase”. Am. J. Physiol. 272 (2 Pt 1): E262–6. PMID 9124333
Durante PE, Mustard KJ, Park SH, Winder WW, Hardie DG (July 2002).
“Effects of endurance training on activity and expression of AMP-activated
protein kinase isoforms in rat muscles”. Am. J. Physiol. Endocrinol.
Metab. 283 (1): E178–86. doi:10.1152/ajpendo.00404.2001. PMID 12067859
Corton JM, Gillespie JG, Hardie DG (April 1994). “Role of the AMP-activated
protein kinase in the cellular stress response”. Curr. Biol. 4 (4):
315–24. doi:10.1016/S0960-9822(00)00070-1. PMID 7922340
Winder WW (September 2001). “Energy-sensing and signaling by
AMP-activated protein kinase in skeletal muscle”. J. Appl. Physiol. 91 (3):
1017–28. PMID 11509493
Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D (October
2006). “Dissecting the role of 5′-AMP for allosteric stimulation, activation,
and deactivation of AMP-activated protein kinase”. J. Biol. Chem.
281 (43): 32207–6. doi:10.1074/jbc.M606357200. PMID 16943194
Part III. Pituitary-thyroid axis and diabetes mellitus The Interface Between Thyroid and Diabetes Mellitus
Leonidas H. Duntas, Jacques Orgiazzi, Georg Brabant Clin Endocrinol. 2011;75(1):1-9.
Interaction of Metformin and Thyroid Function
Metformin acts primarily by
suppressing hepatic gluconeogenesis via activation of AMPK
It has the opposite effects on hypothalamic AMPK,
inhibiting activity of the enzyme.
the metformin effects on hypothalamic AMPK activity will
counteractT3 effects at the hypothalamic level.
AMPK therefore represents a direct target for dual regulation
in the hypothalamic partitioning of energy homeostasis.
metformin crossesthe blood–brain barrier and
levels in the pituitary gland are substantially increased.
It convincinglysuppressesTSH
A recent study recruiting 66 patients with benign thyroid nodules furthermore
demonstrated that metformin significantly decreases nodule size in patients with
insulin resistance.[76] The effect of metformin, which was produced over a
6-month treatment period, parallelled a fall in TSH concentrations and achieved a
shrinkage amounting to 30% of the initial nodule size when metformin was
administered alone and up to 55% when it was added to ongoing LT4 treatment.
These studies reveal a
suppressive effect of metformin on TSH secretion patterns in
hypothyroid patients, an effect that is apparently
independent of T4 treatment and does not alter the TH profile.
A rebound of TSH secretion occurs at about 3 months following metformin
withdrawal.
It appears that recommendations for more frequent testing, on an annual to
biannual basis, seems justified in higher risk groups like patients over 50 or 55,
particularly with suggestive symptoms, raised antibody titres or dylipidaemia.
We thus would support the suggestion of an initial TSH and TPO antibody testing
which, as discussed, will help to predict the development of hypothyroidism in
patients with diabetes.
Hypothalamic AMPK and fatty acid metabolism mediate thyroid
regulation of energy balance
M López, L Varela, MJ Vázquez, S Rodríguez-Cuenca, CR González, …, & Vidal-Puig
Nature Medicine 29 Aug 2010; 16: 1001–1008 http://dx.doi.org:/10.1038/nm.2207
Thyroid hormones have widespread cellular effects; however it is unclear whether
their effects on the central nervous system (CNS) contribute to global energy balance.
Here we demonstrate that either
whole-body hyperthyroidism or central administration of triiodothyronine
(T3) decreases
the activity of hypothalamic AMP-activated protein kinase (AMPK),
increases sympathetic nervous system (SNS) activity and
upregulates thermogenic markers in brown adipose tissue (BAT).
Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus
(VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses
the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid
hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism.
This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this
enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and
increases expression of thermogenic markers in BAT. These effects are reversed by
pharmacological blockade of the SNS. Thus, thyroid hormone–induced modulation
of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of
whole-body energy homeostasis.
Metabolic Basis for Thyroid Hormone Liver Preconditioning:
Upregulation of AMP-Activated Protein Kinase Signaling
LA Videla,1 V Fernández, P Cornejo, and R Vargas
1Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
Faculty of Medicine, University of Chile, 2Faculty of Medicine, Diego Portales University,
Santiago, Chile
Academic Editors: H. M. Abu-Soud and D. Benke The Scientific World Journal 2012; 2012, ID 475675, 10 pp http://dx.doi.org/10.1100/2012/475675
The liver is a major organ responsible for most functions of cellular metabolism and
a mediator between dietary and endogenous sources of energy for extrahepatic tissues.
In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
constitutes an intrahepatic energy sensor
regulating physiological energy dynamics by limiting anabolism and stimulating
catabolism, thus increasing ATP availability.
This is achieved by mechanisms involving direct allosteric activation and
reversible phosphorylation of AMPK, in response to signals such as
energy status,
serum insulin/glucagon ratio,
nutritional stresses,
pharmacological and natural compounds, and
oxidative stress status.
Reactive oxygen species (ROS) lead to cellular AMPK activation and
downstream signaling under several experimental conditions.
Thyroid hormone (L-3,3′,5-triiodothyronine, T3) administration, a condition
that enhances liver ROS generation,
triggers the redox upregulation of cytoprotective proteins
affording preconditioning against ischemia-reperfusion (IR) liver injury.
Data discussed in this work suggest that T3-induced liver activation of AMPK
may be of importance in the promotion of metabolic processes
favouring energy supply for the induction and operation of preconditioning
mechanisms.
These include
antioxidant,
antiapoptotic, and
anti-inflammatory mechanisms,
repair or resynthesis of altered biomolecules,
induction of the homeostatic acute-phase response, and
stimulation of liver cell proliferation,
which are required to cope with the damaging processes set in by IR.
The liver functions as a mediator between dietary and endogenous sources
of energy and extrahepatic organs that continuously require energy, mainly
the brain and erythrocytes, under cycling conditions between fed and fasted states.
In the fed state, where insulin action predominates, digestion-derived glucose is
converted to pyruvate via glycolysis, which is oxidized to produce energy, whereas
fatty acid oxidation is suppressed. Excess glucose can be either stored as hepatic
glycogen or channelled into de novo lipogenesis.
In the fasted state, considerable liver fuel metabolism changes occur due to decreased
serum insulin/glucagon ratio, with higher glucose production as a consequence of
stimulated glycogenolysis and gluconeogenesis (from alanine, lactate, and glycerol).
Major enhancement in fatty acid oxidation also occurs to provide energy for liver
processes and ketogenesis to supply metabolic fuels for extrahepatic tissues. For these
reasons, the liver is considered as the metabolic processing organ of the body, and
alterations in liver functioning affect whole-body metabolism and energy homeostasis.
In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
is the downstream component of a protein kinase cascade acting as an
intracellular energy sensor regulating physiological energy dynamics by
limiting anabolic pathways, to prevent excessive adenosine triphosphate (ATP)
utilization, and
by stimulating catabolic processes, to increase ATP production.
Thus, the understanding of the mechanisms by which liver AMPK coordinates hepatic
energy metabolism represents a crucial point of convergence of regulatory signals
monitoring systemic and cellular energy status
Liver AMPK: Structure and Regulation
AMPK, a serine/threonine kinase, is a heterotrimeric complex comprising
a catalytic subunit α and
two regulatory subunits β and γ .
The α subunit has a threonine residue (Thr172) within the activation loop of the kinase
domain, with the C-terminal region being required for association with β and γ subunits.
The β subunit associates with α and γ by means of its C-terminal region , whereas
the γ subunit has four cystathionine β-synthase (CBS) motifs, which
bind AMP or ATP in a competitive manner.
75675.fig.001 (not shown)
Figure 1: Regulation of AMP-activated protein kinase (AMPK) by
(A) direct allosteric activation and
(B) reversible phosphorylation and downstream responses maintaining
intracellular energy balance.
Regulation of liver AMPK activity involves both direct allosteric activation and
reversible phosphorylation. AMPK is allosterically activated by AMP through
binding to the regulatory subunit-γ, which induces a conformational change in
the kinase domain of subunit α that protects AMPK from dephosphorylation
of Thr172, probably by protein phosphatase-2C.
Activation of AMPK requires phosphorylation of Thr172 in its α subunit, which can be
attained by either
(i) tumor suppressor LKB1 kinase following enhancement in the AMP/ATP ratio, a
kinase that plays a crucial role in AMPK-dependent control of liver glucose and
lipid metabolism;
(ii) Ca2+-calmodulin-dependent protein kinase kinase-β (CaMKKβ) that
phosphorylates AMPK in an AMP-independent, Ca2+-dependent manner;
(iii) transforming growth-factor-β-activated kinase-1 (TAK1), an important
kinase in hepatic Toll-like receptor 4 signaling in response to lipopolysaccharide.
Among these kinases, the relevance of CaMKKβ and TAK1 in liver AMPK activation
remains to be established in metabolic stress conditions. Both allosteric and
phosphorylation mechanisms are able to elicit
over 1000-fold increase in AMPK activity, thus allowing
the liver to respond to small changes in energy status in a highly sensitive fashion.
In addition to rapid AMPK regulation through allosterism and reversible phosphorylation
long-term effects of AMPK activation induce changes in hepatic gene expression.
This was demonstrated for
(i) the transcription factor carbohydrate-response element-binding protein (ChREBP),
whose Ser568 phosphorylation by activated AMPK
blocks its DNA binding capacity and glucose-induced gene transcription
under hyperlipidemic conditions;(ii) liver sterol regulatory element-binding
protein-1c (SREBP-1c), whose mRNA and protein expression and those of
its target gene for fatty acid synthase (FAS)
are reduced by metformin-induced AMPK activation,
decreasing lipogenesis and increasing fatty acid oxidation due to
malonyl-CoA depletion;
(iii) transcriptional coactivator transducer of regulated CREB activity-2 (TORC2),
a crucial component of the hepatic gluconeogenic program, was reported
to be phosphorylated by activated AMPK.
This modification leads to subsequent cytoplasmatic sequestration of TORC2 and
inhibition of gluconeogenic gene expression, a mechanism underlying
the plasma glucose-lowering effects of adiponectin and metformin
through AMPK activation by upstream LKB1.
Activation of AMPK in the liver is a key regulatory mechanism controlling glucose
and lipid metabolism,
inhibiting anabolic processes, and
enhancing catabolic pathways in response to different signals, including
energy status,
serum insulin/glucagon ratio,
nutritional stresses,
pharmacological and natural compounds, and
oxidative stress status
Reactive Oxygen Species (ROS) and AMPK Activation
The high energy demands required to cope with all the metabolic functions
of the liver are met by
fatty acid oxidation under conditions of both normal blood glucose levels and
hypoglycemia, whereas
glucose oxidation is favoured in hyperglycemic states, with consequent
generation of ROS.
Under normal conditions, ROS occur at relatively low levels due to their fast processing
by antioxidant mechanisms, whereas at acute or prolonged high ROS levels, severe
oxidation of biomolecules and dysregulation of signal transduction and gene expression
is achieved, with consequent cell death through necrotic and/or apoptotic-signaling
pathways.
Thyroid Hormone (L-3,3′,5-Triiodothyronine, T3), Metabolic Regulation,
and ROS Production
T3 is important for the normal function of most mammalian tissues, with major actions
on O2 consumption and metabolic rate, thus
determining enhancement in fuel consumption for oxidation processes
and ATP repletion.
T3 acts predominantly through nuclear receptors (TR) α and β, forming
functional complexes with retinoic X receptor that
bind to thyroid hormone response elements (TRE) to activate gene expression.
T3 calorigenesis is primarily due to the
induction of enzymes related to mitochondrial electron transport and ATP
synthesis, catabolism, and
some anabolic processes via upregulation of genomic mechanisms.
The net result of T3 action is the enhancement in the rate of O2 consumption of target
tissues such as liver, which may be effected by secondary processes induced by T3
(i) energy expenditure due to higher active cation transport,
(ii) energy loss due to futile cycles coupled to increase in catabolic and anabolic pathways, and
(iii) O2 equivalents used in hepatic ROS generation both in hepatocytes and Kupffer cells
In addition, T3-induced higher rates of mitochondrial oxidative phosphorylation are
likely to induce higher levels of ATP, which are partially balanced by intrinsic uncoupling
afforded by induction of uncoupling proteins by T3. In agreement with this view, the
cytosolic ATP/ADP ratio is decreased in hyperthyroid tissues, due to simultaneous
stimulation of ATP synthesis and consumption.
Regulation of fatty acid oxidation is mainly attained by carnitine palmitoyltransferase Iα (CPT-Iα),
catalyzing the transport of fatty acids from cytosol to mitochondria for β-oxidation,
and acyl-CoA oxidase (ACO),
catalyzing the first rate-limiting reaction of peroxisomal β-oxidation, enzymes that are
induced by both T3 and peroxisome proliferator-activated receptor α (PPAR-α).
Furthermore, PPAR-α-mediated upregulation of CPT-Iα mRNA is enhanced by PPAR-γ
coactivator 1α (PGC-1α), which in turn
augments T3 induction of CPT-Iα expression.
Interestingly, PGC-1α is induced by
T3,
AMPK activation, and
ROS,
thus establishing potential links between
T3 action, ROS generation, and AMPK activation
with the onset of mitochondrial biogenesis and fatty acid β-oxidation.
Liver ROS generation leads to activation of the transcription factors
nuclear factor-κB (NF-κB),
activating protein 1 (AP-1), and
signal transducer and activator of transcription 3 (STAT3)
at the Kupffer cell level, with upregulation of cytokine expression (TNF-α, IL-1, IL-6),
which upon interaction with specific receptors in hepatocytes trigger the expression of
cytoprotective proteins (Figure 3(A)).
These responses and the promotion of hepatocyte and Kupffer-cell proliferation
represent hormetic effects reestablishing
redox homeostasis,
promoting cell survival, and
protecting the liver against ischemia-reperfusion injury.
T3 liver preconditioning also involves the activation of the
Nrf2-Keap1 defense pathway
upregulating antioxidant proteins,
phase-2 detoxifying enzymes, and
multidrug resistance proteins, members of the ATP binding cassette (ABC)
superfamily of transporters (Figure 3(B))
In agreement with T3-induced liver preconditioning, T3 or L-thyroxin afford
preconditioning against IR injury in the heart, in association with
Figure 2: Calorigenic response of thyroid hormone (T3) and its relationship with O2
consumption, reactive oxygen species (ROS) generation, and antioxidant depletion in the liver.
Abbreviations: CYP2E1, cytochrome P450 isoform 2E1; GSH, reduced glutathione; QO2, rate
of O2 consumption; SOD, superoxide dismutase.
475675.fig.003
genomic signaling in T3 calorigenesis and ROS production 475675.fig.003
effects that are achieved through enhanced transcription of TRE-containing genes
Skeletal muscle AMPK activation is characterized by
(i) being a rapid and transient response,
(ii) upstream activation by Ca2+-induced mobilization and CaMKKβ activation,
(iii) upstream upregulation of LKB1 expression, which requires association with STRAD
and MO25 for optimal phosphorylation/activation of AMPK, and
(iv) stimulation of mitochondrial fatty acid β-oxidation.
T3-induced muscle AMPK activation was found to trigger two major downstream
signaling pathways, namely,
(i) peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA
expression and phosphorylation, a transcriptional regulator for genes related to
mitochondrial biogenesis,
fatty acid oxidation, and
gluconeogenesis and
(ii) cyclic AMP response element binding protein (CREB) phosphorylation, which
in turn induces PGC-1α expression in liver tissue, thus
reinforcing mechanism (i).
These data indicate that AMPK phosphorylation of PGC-1α initiates many of the
important gene regulatory functions of AMPK in skeletal muscle.
In heart, hyperthyroidism increased glycolysis and sarcolemmal GLUT4 levels by the
combined effects of AMPK activation and insulin stimulation, with concomitant increase
in fatty acid oxidation proportional to enhanced cardiac mass and contractile function.
Thyroid Hormone, AMPK Activation, and Liver Preconditioning
Recent studies by our group revealed that administration of a single dose of 0.1 mg T3/kg
to rats activates liver AMPK (Figure 4; unpublished work).
enhancement in phosphorylated AMPK/nonphosphorylated AMPK ratios in T3-
treated rats over control values thatis significant in the time period of 1 to 48
hours after hormone treatment
Administration of a substantially higher dose (0.4 mg T3/kg) resulted in
decreased liver AMPK activation at 4 h to return to control values at 6 h
after treatment
Activation of liver AMPK by T3 may be of relevance in terms of
promotion of fatty acid oxidation for ATP supply,
supporting hepatoprotection against IR injury (Figure 3(C)).
This proposal is based on the high energy demands underlying effective liver
preconditioning for full operation of hepatic
antioxidant, antiapoptotic, and anti-inflammatory mechanisms,
oxidized biomolecules repair or resynthesis,
induction of the homeostatic acute-phase response, and
promotion of hepatocyte and Kupffer cell proliferation,
mechanisms that are needed to cope with the damaging processes set in by IR.
T3 liver preconditioning , in addition to that afforded by
n-3 long-chain polyunsaturated fatty acids given alone or
combined with T3 at lower dosages, or
by iron supplementation,
constitutes protective strategies against hepatic IR injury.
Studies on the molecular mechanisms underlying T3-induced liver AMPK
activation (Figure 4) are currently under assessment in our laboratory.
References
Fernández and L. A. Videla, “Kupffer cell-dependent signaling in thyroid hormone
calorigenesis: possible applications for liver preconditioning,” Current Signal
Transduction Therapy 2009; 4(2): 144–151.
Viollet, B. Guigas, J. Leclerc et al., “AMP-activated protein kinase in the regulation
of hepatic energy metabolism: from physiology to therapeutic perspectives,” Acta
Physiologica 2009; 196(1): 81–98.
Carling, “The AMP-activated protein kinase cascade – A unifying system
for energy control,” Trends in Biochemical Sciences, 2004;. 29(1): 18–24.
E. Kemp, D. Stapleton, D. J. Campbell et al., “AMP-activated protein kinase,
super metabolic regulator,” Biochemical Society Transactions 2003; 31(1):
162–168.
G. Hardie, “AMP-activated protein kinase-an energy sensor that
regulates all ;aspects of cell function,” Genes and Development,
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Woods, P. C. F. Cheung, F. C. Smith et al., “Characterization of AMP-activated
protein kinase βandγ subunits Assembly of the heterotrimeric complex in vitro,”
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activated protein kinase,” Nature 2007; 449(7161): 496–500.
Although adenosine monophosphate activated protein kinase (AMPK) plays a crucial role
in energy metabolism, a direct effect of AMPK modulation on thyroid function has only
recently been reported, and much of its function in the thyroid is currently unknown.
The aim of this study was
to investigate the mechanism of AMPK modulation in iodide uptake.
to investigate the potential of the AMPK inhibitor compound C as an enhancer of
iodide uptake by thyrocytes.
Metformin reduced NIS promoter activity (0.6-fold of control), whereas compound C
stimulated its activity (3.4-fold) after 4 days. This largely coincides with
CRE activation (0.6- and 3.0-fold).
These experiments show that AMPK exerts its effects on iodide uptake, at least partly,
through the CRE element in the NIS promoter. Furthermore, we have used AMPK-alpha1
knockout mice to determine the long-term effects of AMPK inhibition without chemical compounds.
These mice have a less active thyroid, as shown by reduced colloid volume and reduced
responsiveness to thyrotropin.
NIS expression and iodine uptake in thyrocytes
can be modulated by metformin and compound C.
These compounds exert their effect by
modulation of AMPK, which, in turn, regulates
the activation of the CRE element in the NIS promoter.
Overall, this suggests that AMPK modulating compounds may be useful for the
enhancement of iodide uptake by thyrocytes, which could be useful for the
treatment of thyroid cancer patients with radioactive iodine.
AMPK induces a cascade of events within cells in response to the ever changing energy
charge of the cell. The role of AMPK in regulating cellular energy charge places this
enzyme at a central control point in maintaining energy homeostasis.
More recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients.
Once activated, AMPK-mediated phosphorylation events
How these latter two functions impact obesity and diabetes will be discussed below.
Regulation of AMPK
In the presence of AMP the activity of AMPK is increased approximately 5-fold.
However, more importantly is the role of AMP in regulating the level of phosphorylation
of AMPK. An increased AMP to ATP ratio leads to a conformational change in the γ-subunit
leading to increased phosphorylation and decreased dephosphorylation of AMPK.
The phosphorylation of AMPK results in activation by at least 100-fold. AMPK is
phosphorylated by at least three different upstream AMPK kinases (AMPKKs).
Phosphorylation of AMPK occurs in the α subunit at threonine 172 (T172) which
which phosphorylates and activates AMPK in response to increased calcium.
The distribution of CaMKKβ expression is primarily in the brain with detectable levels
also found in the testes, thymus, and T cells. As described for the Ca2+-mediated
regulation of glycogen metabolism,
increased release of intracellular stores of Ca2+ create a subsequent demand for
ATP.
Activation of AMPK in response to Ca fluxes
provides a mechanism for cells to anticipate the increased demand for ATP.
Evidence has also demonstrated that the serine-threonine kinase, LKB1 (also called
serine-threonine kinase 11, STK11) which is encoded by the Peutz-Jeghers syndrome
tumor suppressor gene, is required for activation of AMPK in response to stress.
The active LKB1 kinase is actually a complex of three proteins:
LKB1,
Ste20-related adaptor (STRAD) and
mouse protein 25 (MO25).
Thus, the enzyme complex is often referred to as LKB1-STRAD-MO25. Phosphorylation
of AMPK by LKB1 also occurs on T172. Unlike the limited distribution of CaMKKβ,
LKB1 is widely expressed, thus making it the primary AMPK-regulating kinase.
Loss of LKB1 activity in adult mouse liver leads to
near complete loss of AMPK activity and
is associated with hyperglycemia.
The hyperglycemia is, in part, due to an increase in the transcription of gluconeogenic
genes. Of particular significance is the increased expression of
the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator 1α
(PGC-1α), which drives gluconeogenesis.
Reduction in PGC-1α activity results in normalized blood glucose levels in
LKB1-deficient mice.
The third AMPK phosphorylating kinase is transforming growth factor-β-activated
kinase 1 (TAK1). However, the normal physiological conditions under which TAK1
phosphorylates AMPK are currently unclear.
The effects of AMP are two-fold:
a direct allosteric activation and making AMPK a poorer substrate for
dephosphorylation.
Because AMP affects both
the rate of AMPK phoshorylation in the positive direction and
dephosphorylation in the negative direction,
the cascade is ultrasensitive. This means that
a very small rise in AMP levels can induce a dramatic increase in the activity of
AMPK.
The activity of adenylate kinase, catalyzing the reaction shown below, ensures that
AMPK is highly sensitive to small changes in the intracellular [ATP]/[ADP] ratio.
2 ADP ——> ATP + AMP
Negative allosteric regulation of AMPK also occurs and this effect is exerted by phosphocreatine. As indicated above, the β subunits of AMPK have a glycogen-binding domain, GBD. In muscle, a high glycogen content
represses AMPK activity and
this is likely the result of interaction between the GBD and glycogen,
the GBD of AMPK allows association of the enzyme with the regulation of glycogen metabolism
by placing AMPK in close proximity to one of its substrates glycogen synthase.
AMPK has also been shown to be activated by receptors that are coupled to
phospholipase C-β (PLC-β) and by
hormones secreted by adipose tissue (termed adipokines) such as leptinand adiponectin (discussed below).
Targets of AMPK
The signaling cascades initiated by the activation of AMPK exert effects on
glucose and lipid metabolism,
gene expression and
protein synthesis.
These effects are most important for regulating metabolic events in the liver, skeletal
muscle, heart, adipose tissue, and pancreas.
Demonstration of the central role of AMPK in the regulation of metabolism in response
to events such as nutrient- or exercise-induced stress. Several of the known physiologic
targets for AMPK are included as well as several pathways whose flux is affected by
AMPK activation. Arrows indicate positive effects of AMPK, whereas, T-lines indicate
the resultant inhibitory effects of AMPK action.
The uptake, by skeletal muscle, accounts for >70% of the glucose removal from the
serum in humans. Therefore, it should be obvious that this event is extremely important
for overall glucose homeostasis, keeping in mind, of course, that glucose uptake by
cardiac muscle and adipocytes cannot be excluded from consideration. An important fact
related to skeletal muscle glucose uptake is that this process is markedly impaired in
individuals with type 2 diabetes.
The uptake of glucose increases dramatically in response to stress (such as ischemia) and
exercise and is stimulated by insulin-induced recruitment of glucose transporters
to the plasma membrane, primarily GLUT4. Insulin-independent recruitment of glucose
transporters also occurs in skeletal muscle in response to contraction (exercise).
The activation of AMPK plays an important, albeit not an exclusive, role in the induction of
GLUT4 recruitment to the plasma membrane. The ability of AMPK to stimulate
GLUT4 translocation to the plasma membrane in skeletal muscle is by a different mechanism
than that stimulated by insulin and insulin and AMPK effects are additive.
Under ischemic/hypoxic conditions in the heart the activation of AMPK leads to the
phosphorylation and activation of the kinase activity of phosphofructokinase-2, PFK-2
(6-phosphofructo-2-kinase). The product of the action of PFK-2 (fructose-2,6-bisphosphate,
F2,6BP) is one of the most potent regulators of the rate of flux through glycolysis and gluconeogenesis.
In liver the PKA-mediated phosphorylation of PFK-2 results in conversion of the
enzyme from a kinase that generates F2,6BP to a phosphatase that removes the
2-phosphate thus reducing the levels of the potent allosteric activator of the glycolytic
enzyme 6-phosphfructo-1-kinase, PFK-1 and the potent allosteric inhibitor
of the gluconeogenic enzyme fructose-1,6-bisphosphatase (F1,-6BPase).
It is important to note that like many enzymes, there are multiple isoforms of PFK-2
(at least 4) and neither the liver or the skeletal muscle isoforms contain the AMPK
phosphorylation sites found in the cardiac and inducible (iPFK2) isoforms of PFK-2.
Inducible PFK-2 is expressed in the monocyte/macrophage lineage in response to pro-
inflammatory stimuli. The ability to activate the kinase activity by phosphorylation of
PFK-2 in cardiac tissue and macrophages in response to ischemic conditions allows these
cells to continue to have a source of ATP via anaerobic glycolysis. This phenomenon is
recognized as the Pasteur effect: an increased rate of glycolysis in response to hypoxia.
Of pathological significance is the fact that the inducible form of PFK-2 is commonly
expressed in many tumor cells and this may allow AMPK to play an important role in
protecting tumor cells from hypoxic stress. Indeed, techniques for depleting AMPK in
tumor cells have shown that these cells become sensitized to nutritional stress upon loss
of AMPK activity.
Whereas, stress and exercise are powerful inducers of AMPK activity in skeletal muscle,
additional regulators of its activity have been identified.
Insulin-sensitizing drugs of the thiazolidinedione family (activators of PPAR-γ, see
below) as well as the hypoglycemia drug metformin exert a portion of their effects
through regulation of the activity of AMPK.
As indicated above, the activity of the AMPK activating kinase, LKB1, is critical for
regulating gluconeogenic flux and consequent glucose homeostasis. The action of
metformin in reducing blood glucose levels
requires the activity of LKB1 in the liver for this function.
AMPK-mediated phosphorylation of eNOS leads to increased activity and consequent
NO production and provides a link between metabolic stresses and cardiac function.
In platelets, insulin action leads to an increase in eNOS activity that is
due to its phosphorylation by AMPK.
Activation of NO production in platelets leads to
a decrease in thrombin-induced aggregation, thereby,
limiting the pro-coagulant effects of platelet activation.
The response of platelets to insulin function clearly indicates why disruption in insulin
action is a major contributing factor in the development of the metabolic syndrome
Activation of AMPK leads to a reduction in the level of SREBP
a transcription factor ®ulator of the expression of numerous
lipogenic enzymes
Another transcription factor reduced in response to AMPK activation is
hepatocyte nuclear factor 4α, HNF4α
a member of the steroid/thyroid hormone superfamily.
HNF4α is known to regulate the expression of several liver and
pancreatic β-cell genes such as GLUT2, L-PK and preproinsulin.
Of clinical significance is that mutations in HNF4α are responsible for
maturity-onset diabetes of the young, MODY-1.
Recent evidence indicates that the gene for the carbohydrate-response-element-
binding protein (ChREBP) is a target for AMPK-mediated transcriptional regulation
in the liver. ChREBP is rapidly being recognized as a master regulator of lipid
metabolism in liver, in particular in response to glucose uptake.
The transcription co-activator, p300, is phosphorylated by AMPK
which inhibits interaction of p300 with not only PPARγ but also
the retinoic acid receptor, retinoid X receptor, and
thyroid hormone receptor.
PPARγ is primarily expressed in adipose tissue and thus it was difficult to reconcile how
a drug that was apparently acting only in adipose tissue could lead to improved insulin
sensitivity of other tissues. The answer to this question came when it was discovered that the TZDs stimulated the expression and release of the adipocyte hormone (adipokine),
adiponectin. Adiponectin stimulates glucose uptake and fatty acid oxidation in skeletal
muscle. In addition, adiponectin stimulates fatty acid oxidation in liver while inhibiting
expression of gluconeogenic enzymes in this tissue.
These responses to adiponectin are exerted via activation of AMPK. Another
transcription factor target of AMPK is the forkhead protein, FKHR (now referred to as FoxO1). FoxO1 is involved in the activation of glucose-6-phosphatase expression and,
therefore, loss of FoxO1 activity in response to AMPK activation will lead to reduced
hepatic output of glucose.
This concludes a very complicated perspective that ties together the thyroid hormone
activity, the hypophysis, diabetes mellitus, and AMPK tegulation of metabolism in the
liver, skeletal muscle, adipose tissue, and heart. I also note at this time that there
nongenetic points to be made here:
The tissue specificity of isoenzymes
The modulatory role of AMP:ATP ratio in phosphorylation/dephosphorylation
effects on metabolism tied to AMPK
The tie in of stress or ROS with fast reactions to protect harm to tissues
The relationship of cytokine activation and release to the above metabolic events
The relationship of effective and commonly used diabetes medications to AMPK
mediated processes
The preceding presentation is notable for the importance of proteomic and
metabolomic invetigations in elucidation common chronic and nongenetic diseases
Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases
Author and Curator: Larry H Bernstein, MD, FCAP
We have covered a large amount of material that involves
the development,
application, and
validation of outcomes of medical and surgical procedures
that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade. Encouraging enabling developments have been:
1. The establishment of national and international outcomes databases for procedures by specialist medical societies
2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.
5. This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.
This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book. Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation. Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice. The time for this to occur in practice in the past has been through roughly a generation of physicians. That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased. This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.
However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care. This is not the place for or a matter for discussion here. It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes. The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.
I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.
Abstract:Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions
A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.
Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.
Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.
“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.
Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. : Hajjar & Mark Mercola. Nature (March 2014) http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13073.html
“Junk” DNA Tied to Heart Failure
Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support
The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.
Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.
The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.
In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.
“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”
‘Junk’ Genome Regions Linked to Heart Failure
In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.
“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.
In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.
“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”
Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.
Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.
Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis
In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.
Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.
ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.
Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.
Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.
A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.
2. Diagnostic Biomarker Status
A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.
ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.
Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.
ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.
ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.
ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.
Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.
Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies
Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.
Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair
Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.
Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.
Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1
Author and Curator: Larry H Bernstein, MD, FCAP
and
Curator: Aviva Lev-Ari, PhD, RN
Part 1 of Volume 4 in the e-series A: Cardiovascular Diseases and Translational Medicine, provides a foundation for grasping a rapidly developing surging scientific endeavor that is transcending laboratory hypothesis testing and providing guidelines to:
Target genomes and multiple nucleotide sequences involved in either coding or in regulation that might have an impact on complex diseases, not necessarily genetic in nature.
Target signaling pathways that are demonstrably maladjusted, activated or suppressed in many common and complex diseases, or in their progression.
Enable a reduction in failure due to toxicities in the later stages of clinical drug trials as a result of this science-based understanding.
Enable a reduction in complications from the improvement of machanical devices that have already had an impact on the practice of interventional procedures in cardiology, cardiac surgery, and radiological imaging, as well as improving laboratory diagnostics at the molecular level.
Enable the discovery of new drugs in the continuing emergence of drug resistance.
Enable the construction of critical pathways and better guidelines for patient management based on population outcomes data, that will be critically dependent on computational methods and large data-bases.
What has been presented can be essentially viewed in the following Table:
Summary Table for TM – Part 1
There are some developments that deserve additional development:
1. The importance of mitochondrial function in the activity state of the mitochondria in cellular work (combustion) is understood, and impairments of function are identified in diseases of muscle, cardiac contraction, nerve conduction, ion transport, water balance, and the cytoskeleton – beyond the disordered metabolism in cancer. A more detailed explanation of the energetics that was elucidated based on the electron transport chain might also be in order.
2. The processes that are enabling a more full application of technology to a host of problems in the environment we live in and in disease modification is growing rapidly, and will change the face of medicine and its allied health sciences.
Electron Transport and Bioenergetics
Deferred for metabolomics topic
Synthetic Biology
Introduction to Synthetic Biology and Metabolic Engineering
http://www.ibiology.org Lecturers generously donate their time to prepare these lectures. The project is funded by NSF and NIGMS, and is supported by the ASCB and HHMI.
Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”.
Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching. Learn more about how Kris became a scientist at
Prather 1: Synthetic Biology and Metabolic Engineering 2/6/14IntroductionLecture Overview In the first part of her lecture, Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”. The key material in building these machines is synthetic DNA. Synthetic DNA can be added in different combinations to biological hosts, such as bacteria, turning them into chemical factories that can produce small molecules of choice. In Part 2, Prather describes how her lab used design principles to engineer E. coli that produce glucaric acid from glucose. Glucaric acid is not naturally produced in bacteria, so Prather and her colleagues “bioprospected” enzymes from other organisms and expressed them in E. coli to build the needed enzymatic pathway. Prather walks us through the many steps of optimizing the timing, localization and levels of enzyme expression to produce the greatest yield. Speaker Bio: Kristala Jones Prather received her S.B. degree from the Massachusetts Institute of Technology and her PhD at the University of California, Berkeley both in chemical engineering. Upon graduation, Prather joined the Merck Research Labs for 4 years before returning to academia. Prather is now an Associate Professor of Chemical Engineering at MIT and an investigator with the multi-university Synthetic Biology Engineering Reseach Center (SynBERC). Her lab designs and constructs novel synthetic pathways in microorganisms converting them into tiny factories for the production of small molecules. Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.
Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells
in INTECH
Current Basic and Pathological Approaches to
the Function of Muscle Cells and Tissues – From Molecules to HumansLarissa Lipskaia, Isabelle Limon, Regis Bobe and Roger Hajjar
Additional information is available at the end of the chapter http://dx.doi.org/10.5772/48240
1. Introduction
Calcium ions (Ca ) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion messenger that carries information
as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca signal greatly differ from one cell type to another.
In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca signal. In each VSMC phenotype (synthetic/proliferating and contractile [1], tonic or phasic), the Ca signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca.
For instance, in contractile VSMCs, the initiation of contractile events is driven by mem- brane depolarization; and the principal entry-point for extracellular Ca is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca is the store-operated calcium (SOC) channel.
Whatever the cell type, the calcium signal consists of limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca ATPase (SERCA), has a critical role in determining the frequency of SR Ca release by upload into the sarcoplasmic
sensitivity of SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].
Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs
Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.
Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile re-sponse is initiated by extracellular Ca influx due to activation of Receptor Operated Ca (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca influx leads to large SR Ca IP3R or RyR clusters (“Ca -induced Ca SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca and setting the sensitivity of RyR or IP3R for the next spike.
Contraction of VSMCs occurs during oscillatory Ca transient.
Middle panel: schematic representa tion of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima.
Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca calcium pumps (only SERCA2b, having low turnover and low affinity to Ca depletion leads to translocation of SR Ca sensor STIM1 towards PM, resulting in extracellular Ca influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca transient is critical for activation of proliferation-related transcription factors ‘NFAT).
Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca /calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5- trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca sarcoplasmic reticulum.
Time for New DNA Synthesis and Sequencing Cost Curves
By Rob Carlson
I’ll start with the productivity plot, as this one isn’t new. For a discussion of the substantial performance increase in sequencing compared to Moore’s Law, as well as the difficulty of finding this data, please see this post. If nothing else, keep two features of the plot in mind: 1) the consistency of the pace of Moore’s Law and 2) the inconsistency and pace of sequencing productivity. Illumina appears to be the primary driver, and beneficiary, of improvements in productivity at the moment, especially if you are looking at share prices. It looks like the recently announced NextSeq and Hiseq instruments will provide substantially higher productivities (hand waving, I would say the next datum will come in another order of magnitude higher), but I think I need a bit more data before officially putting another point on the plot.
cost-of-oligo-and-gene-synthesis
Illumina’s instruments are now responsible for such a high percentage of sequencing output that the company is effectively setting prices for the entire industry. Illumina is being pushed by competition to increase performance, but this does not necessarily translate into lower prices. It doesn’t behoove Illumina to drop prices at this point, and we won’t see any substantial decrease until a serious competitor shows up and starts threatening Illumina’s market share. The absence of real competition is the primary reason sequencing prices have flattened out over the last couple of data points.
Note that the oligo prices above are for column-based synthesis, and that oligos synthesized on arrays are much less expensive. However, array synthesis comes with the usual caveat that the quality is generally lower, unless you are getting your DNA from Agilent, which probably means you are getting your dsDNA from Gen9.
Note also that the distinction between the price of oligos and the price of double-stranded sDNA is becoming less useful. Whether you are ordering from Life/Thermo or from your local academic facility, the cost of producing oligos is now, in most cases, independent of their length. That’s because the cost of capital (including rent, insurance, labor, etc) is now more significant than the cost of goods. Consequently, the price reflects the cost of capital rather than the cost of goods. Moreover, the cost of the columns, reagents, and shipping tubes is certainly more than the cost of the atoms in the sDNA you are ostensibly paying for. Once you get into longer oligos (substantially larger than 50-mers) this relationship breaks down and the sDNA is more expensive. But, at this point in time, most people aren’t going to use longer oligos to assemble genes unless they have a tricky job that doesn’t work using short oligos.
Looking forward, I suspect oligos aren’t going to get much cheaper unless someone sorts out how to either 1) replace the requisite human labor and thereby reduce the cost of capital, or 2) finally replace the phosphoramidite chemistry that the industry relies upon.
IDT’s gBlocks come at prices that are constant across quite substantial ranges in length. Moreover, part of the decrease in price for these products is embedded in the fact that you are buying smaller chunks of DNA that you then must assemble and integrate into your organism of choice.
Someone who has purchased and assembled an absolutely enormous amount of sDNA over the last decade, suggested that if prices fell by another order of magnitude, he could switch completely to outsourced assembly. This is a potentially interesting “tipping point”. However, what this person really needs is sDNA integrated in a particular way into a particular genome operating in a particular host. The integration and testing of the new genome in the host organism is where most of the cost is. Given the wide variety of emerging applications, and the growing array of hosts/chassis, it isn’t clear that any given technology or firm will be able to provide arbitrary synthetic sequences incorporated into arbitrary hosts.
Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine. Synthetic Biology combines engineering principles with basic science to build biological products, including regenerative medicines and cellular therapies. Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Regenerative therapies that are in clinical trials today may enable repair of damaged heart muscle following heart attack, replacement of skin for burn victims, restoration of movement after spinal cord injury, regeneration of pancreatic tissue for insulin production in diabetics and provide new treatments for Parkinson’s and Alzheimer’s diseases, to name just a few applications.
While the potential of the field is promising, the pace of development has been slow. One main reason for this is that the living cells required for these therapies are cost-prohibitive and not supplied at volumes that support many research and product development efforts. RoosterBio will manufacture large quantities of standardized primary cells at high quality and low cost, which will quicken the pace of scientific discovery and translation to the clinic. “Our goal is to accelerate the development of products that incorporate living cells by providing abundant, affordable and high quality materials to researchers that are developing and commercializing these regenerative technologies” says Dr. Rowley
NHMU Lecture featuring – J. Craig Venter, Ph.D. Founder, Chairman, and CEO – J. Craig Venter Institute; Co-Founder and CEO, Synthetic Genomics Inc.
J. Craig Venter, Ph.D., is Founder, Chairman, and CEO of the J. Craig Venter Institute (JVCI), a not-for-profit, research organization dedicated to human, microbial, plant, synthetic and environmental research. He is also Co-Founder and CEO of Synthetic Genomics Inc. (SGI), a privately-held company dedicated to commercializing genomic-driven solutions to address global needs.
In 1998, Dr. Venter founded Celera Genomics to sequence the human genome using new tools and techniques he and his team developed. This research culminated with the February 2001 publication of the human genome in the journal, Science. Dr. Venter and his team at JVCI continue to blaze new trails in genomics. They have sequenced and a created a bacterial cell constructed with synthetic DNA, putting humankind at the threshold of a new phase of biological research. Whereas, we could previously read the genetic code (sequencing genomes), we can now write the genetic code for designing new species.
The science of synthetic genomics will have a profound impact on society, including new methods for chemical and energy production, human health and medical advances, clean water, and new food and nutritional products. One of the most prolific scientists of the 21st century for his numerous pioneering advances in genomics, he guides us through this emerging field, detailing its origins, current challenges, and the potential positive advances.
His work on synthetic biology truly embodies the theme of “pushing the boundaries of life.” Essentially, Venter is seeking to “write the software of life” to create microbes designed by humans rather than only through evolution. The potential benefits and risks of this new technology are enormous. It also requires us to examine, both scientifically and philosophically, the question of “What is life?”
J Craig Venter wants to digitize DNA and transmit the signal to teleport organisms
A technology trend analyst offers an overview of synthetic biology, its potential applications, obstacles to its development, and prospects for public approval.
In addition to boosting the economy, synthetic biology projects currently in development could have profound implications for the future of manufacturing, sustainability, and medicine.
Before society can fully reap the benefits of synthetic biology, however, the field requires development and faces a series of hurdles in the process. Do researchers have the scientific know-how and technical capabilities to develop the field?
Biology + Engineering = Synthetic Biology
Bioengineers aim to build synthetic biological systems using compatible standardized parts that behave predictably. Bioengineers synthesize DNA parts—oligonucleotides composed of 50–100 base pairs—which make specialized components that ultimately make a biological system. As biology becomes a true engineering discipline, bioengineers will create genomes using mass-produced modular units similar to the microelectronics and computer industries.
Currently, bioengineering projects cost millions of dollars and take years to develop products. For synthetic biology to become a Schumpeterian revolution, smaller companies will need to be able to afford to use bioengineering concepts for industrial applications. This will require standardized and automated processes.
A major challenge to developing synthetic biology is the complexity of biological systems. When bioengineers assemble synthetic parts, they must prevent cross talk between signals in other biological pathways. Until researchers better understand these undesired interactions that nature has already worked out, applications such as gene therapy will have unwanted side effects. Scientists do not fully understand the effects of environmental and developmental interaction on gene expression. Currently, bioengineers must repeatedly use trial and error to create predictable systems.
Similar to physics, synthetic biology requires the ability to model systems and quantify relationships between variables in biological systems at the molecular level.
The second major challenge to ensuring the success of synthetic biology is the development of enabling technologies. With genomes having billions of nucleotides, this requires fast, powerful, and cost-efficient computers. Moore’s law, named for Intel co-founder Gordon Moore, posits that computing power progresses at a predictable rate and that the number of components in integrated circuits doubles each year until its limits are reached. Since Moore’s prediction, computer power has increased at an exponential rate while pricing has declined.
DNA sequencers and synthesizers are necessary to identify genes and make synthetic DNA sequences. Bioengineer Robert Carlson calculated that the capabilities of DNA sequencers and synthesizers have followed a pattern similar to computing. This pattern, referred to as the Carlson Curve, projects that scientists are approaching the ability to sequence a human genome for $1,000, perhaps in 2020. Carlson calculated that the costs of reading and writing new genes and genomes are falling by a factor of two every 18–24 months. (see recent Carlson comment on requirement to read and write for a variety of limiting conditions).
Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products
Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging
Futurists have touted the twenty-first century as the century of biology based primarily on the promise of genomics. Medical researchers aim to use variations within genes as biomarkers for diseases, personalized treatments, and drug responses. Currently, we are experiencing a genomics bubble, but with advances in understanding biological complexity and the development of enabling technologies, synthetic biology is reviving optimism in many fields, particularly medicine.
Michael Brooks holds a PhD in quantum physics. He writes a weekly science column for the New Statesman,and his most recent book is The Secret Anarchy of Science.
The basic idea is that we take an organism – a bacterium, say – and re-engineer its genome so that it does something different. You might, for instance, make it ingest carbon dioxide from the atmosphere, process it and excrete crude oil.
That project is still under construction, but others, such as using synthesised DNA for data storage, have already been achieved. As evolution has proved, DNA is an extraordinarily stable medium that can preserve information for millions of years. In 2012, the Harvard geneticist George Church proved its potential by taking a book he had written, encoding it in a synthesised strand of DNA, and then making DNA sequencing machines read it back to him.
When we first started achieving such things it was costly and time-consuming and demanded extraordinary resources, such as those available to the millionaire biologist Craig Venter. Venter’s team spent most of the past two decades and tens of millions of dollars creating the first artificial organism, nicknamed “Synthia”. Using computer programs and robots that process the necessary chemicals, the team rebuilt the genome of the bacterium Mycoplasma mycoides from scratch. They also inserted a few watermarks and puzzles into the DNA sequence, partly as an identifying measure for safety’s sake, but mostly as a publicity stunt.
What they didn’t do was redesign the genome to do anything interesting. When the synthetic genome was inserted into an eviscerated bacterial cell, the new organism behaved exactly the same as its natural counterpart. Nevertheless, that Synthia, as Venter put it at the press conference to announce the research in 2010, was “the first self-replicating species we’ve had on the planet whose parent is a computer” made it a standout achievement.
Today, however, we have entered another era in synthetic biology and Venter faces stiff competition. The Steve Jobs to Venter’s Bill Gates is Jef Boeke, who researches yeast genetics at New York University.
Boeke wanted to redesign the yeast genome so that he could strip out various parts to see what they did. Because it took a private company a year to complete just a small part of the task, at a cost of $50,000, he realised he should go open-source. By teaching an undergraduate course on how to build a genome and teaming up with institutions all over the world, he has assembled a skilled workforce that, tinkering together, has made a synthetic chromosome for baker’s yeast.
Stepping into DIYbio and Synthetic Biology at ScienceHack
We got a crash course on genetics and protein pathways, and then set out to design and build our own pathways using both the “Genomikon: Violacein Factory” kit and Synbiota platform. With Synbiota’s software, we dragged and dropped the enzymes to create the sequence that we were then going to build out. After a process of sketching ideas, mocking up pathways, and writing hypotheses, we were ready to start building!
The night stretched long, and at midnight we were forced to vacate the school. Not quite finished, we loaded our delicate bacteria, incubator, and boxes of gloves onto the bus and headed back to complete our bacterial transformation in one of our hotel rooms. Jammed in between the beds and the mini-fridge, we heat-shocked our bacteria in the hotel ice bucket. It was a surreal moment.
While waiting for our bacteria, we held an “unconference” where we explored bioethics, security and risk related to synthetic biology, 3D printing on Mars, patterns in juggling (with live demonstration!), and even did a Google Hangout with Rob Carlson. Every few hours, we would excitedly check in on our bacteria, looking for bacterial colonies and the purple hue characteristic of violacein.
Most impressive was the wildly successful and seamless integration of a diverse set of people: in a matter of hours, we were transformed from individual experts and practitioners in assorted fields into cohesive and passionate teams of DIY biologists and science hackers. The ability of everyone to connect and learn was a powerful experience, and over the course of just one weekend we were able to challenge each other and grow.
Returning to work on Monday, we were hungry for more. We wanted to find a way to bring the excitement and energy from the weekend into the studio and into the projects we’re working on. It struck us that there are strong parallels between design and DIYbio, and we knew there was an opportunity to bring some of the scientific approaches and curiosity into our studio.
Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine
Author and Curator: Larry H Bernstein, MD, FCAP
and
Curator: Aviva Lev-Ari, PhD, RN
This document is entirely devoted to medical and surgical therapies that have made huge strides in
simplification of interventional procedures,
reduced complexity, resulting in procedures previously requiring surgery are now done, circumstances permitting, by medical intervention.
This revolution in cardiovascular interventional therapy is regenerative medicine. It is regenerative because it is largely driven by
the introduction into the impaired vasculature of an induced pleuripotent cell, called a stem cell, although
the level of differentiation may not be a most primitive cell line.
There is also a very closely aligned development in cell biology that extends beyond and including vascular regeneration that is called synthetic biology. These developments have occurred at an accelerated rate in the last 15 years. The methods of interventional cardiology were already well developed in the mid 1980s. This was at the peak of cardiothoracic bypass surgery.
Research on the endothelial cell,
endothelial cell proliferation,
shear flow in small arteries, especially at branch points, and
endothelial-platelet interactions
led to insights about plaque formation and vessel thrombosis.
Much was learned in biomechanics about the shear flow stresses on the luminal surface of the vasculature, and there was also
the concomitant discovery of nitric oxide,
oxidative stress, and
the isoenzymes of nitric oxide synthase (eNOS, iNOS, and nNOS).
It became a fundamental tenet of vascular biology that
atherogenesis is a maladjustment to oxidative stress not only through genetic, but also
non-genetic nutritional factors that could be related to the balance of omega (ω)-3 and omega (ω)-6 fatty acids,
a pro-inflammatory state that elicits inflammatory cytokines, such as, interleukin-6 (IL6) and c-reactive protein(CRP),
insulin resistance with excess carbohydrate associated with type 2 diabetes and beta (β) cell stress,
excess trans- and saturated fats, and perhaps
the now plausible colonic microbial population of the gastrointestinal tract (GIT).
There is also an association of abdominal adiposity,
including the visceral peritoneum, with both T2DM and with arteriosclerotic vessel disease,
which is presenting at a young age, and has ties to
the effects of an adipokine, adiponectin.
Much important work has already been discussed in the domain of cardiac catheterization and research done to
prevent atheroembolization.and beyond that,
research done to implant an endothelial growth matrix.
Even then, dramatic work had already been done on
the platelet structure and metabolism, and
this has transformed our knowledge of platelet biology.
The coagulation process has been discussed in detailed in a previous document. The result was the development of a
new class of platelet aggregation inhibitors designed to block the activation of protein on the platelet surface that
is critical in the coagulation cascade.
In addition, the term long used to describe atherosclerosis, atheroma notwithstanding, is “hardening of the arteries”. This is particularly notable with respect to mid-size arteries and arterioles that feed the heart and kidneys. Whether it is preceded by or develops concurrently with chronic renal insufficiency and lowered glomerular filtration rate is perhaps arguable. However, there is now a body of evidence that points to
a change in the vascular muscularis and vessel stiffness, in addition to the endothelial features already mentioned.
This has provided a basis for
targeted pharmaceutical intervention, and
reduction in salt intake.
So we have a group of metabolic disorders, which may alone or in combination,
lead to and be associated with the long term effects of cardiovascular disease, including
congestive heart failure.
This has been classically broken down into forward and backward failure,
depending on decrease outflow through the aorta (ejection fraction), or
decreased venous return through the vena cava,
which involves increased pulmonary vascular resistance and decreased return into the left atrium.
This also has ties to several causes, which may be cardiac or vascular. This document, as the previous, has four pats. They are broadly:
Stem Cells in Cardiovascular Diseases
Regenerative Cell and Molecular Biology
Therapeutics Levels In Molecular Cardiology
Research Proposals for Endogenous Augmentation of circulating Endothelial Progenitor Cells (cEPCs)
As in the previous section, we start with the biology of the stem cell and the degeneration in cardiovascular diseases, then proceed to regeneration, then therapeutics, and finally – proposals for augmenting therapy with circulating endogenous endothelial progenitor cells (cEPCs).
Circulating Endothelial Progenitors Cells (cEPCs) as Biomarkers
Article Curator: Larry H. Bernstein, MD, FCAP
and
Topic Curator: Aviva Lev-Ari, PhD, RN
Circulating progenitor cells have gained much interest rapidly in the past year primarily in identification of damaged tissue that has turnover of cells that are identifiable in the circulation. This has to require a sensitivity for identification at one or two logs lower than circulating hematopoietic cells. I mention this untested view only because cells of the circulation are detected routinely by automated hematology instruments like those of Beckman-Coulter and Siemens, with graphical presentation of results. The Sysmex also reports immature granulocytes that are a small percent of the neutrophil count. In the evaluation of leukemias, flow cytometry has been used for years, but require a preparative step. Cell types have been identified by acidic and basic dye stains to identify basophilic, acidophilic and neutrophilic granulocyte series, and by size of the cell population, and nuclear features, differentiating mature and nucleated red cells, the granulocyte series, monocytes and lymphocytes, as well as platelets (aggregation gives an underestimate of platelet count). But to detect cancer cells or damaged endothelial cells, the number of cells in the circulation requires and antibody to the surface with a visualizable ligand attached to an antibody for identification. Visualization could be by a fluorophor, or perhaps a luciferase reaction. Here are two articles that identify circulating endothelial cells, making them suitable for biomarkers of cardiovascular injury. Whether they can detect early predictive ischemia, or frank AMI needs investigation. The concept of piecemeal necrosis in the heart may be applicable to cardiomyocyte injury that is found unexpectedly at autopsy as “silent infarct”.
Circulating endothelial progenitors–cells as biomarkers
Rosenzweig, Anthony
N Engl J Med. 2005 Sep 8;353(10):1055-7
Comment on
Circulating endothelial progenitor cells and cardiovascular outcomes
Endothelial injury and dysfunction are thought to be critical events in the pathogenesis of atherosclerosis. Thus,
understanding the mechanisms that maintain and restore endothelial function
may have important clinical implications.
A series of clinical and basic studies prompted by the discovery
of bone marrow derived endothelial progenitor cells1 have
provided insights into these processes and
opened a door to the development of new therapeutic approaches.
Growing evidence suggests that bone marrow derived endothelial progenitor cells circulate in the blood and
play an important role in the formation of new blood vessels as well as
contribute to vascular homeostasis in the adult.
Circulating endothelial progenitor cells were initially identified
through their expression of CD34
(a surface marker common to hematopoietic stem cells and mature endothelial cells)
and vascular endothelial cell growth-factor receptor 2
(VEGFR2 or kinase-domain related [KDR] receptor),
but not of other markers seen on fully differentiated endothelial cells.1
Subsequent studies have also used other identifiers, such as
the stem-cell marker CD133, and
functional assays, including
the ability to form endothelial colonies.
Endothelial progenitor cells defined in these ways probably represent
a heterogeneous population, which,
in combination with the lack of a consensual definition,
complicates the interpretation of work in this field.
Nevertheless, numerous studies in animals have shown that endothelial progenitor cells can integrate into new and existing blood vessels.2,3,4
Intravenous injection of cytokine-mobilized human endothelial progenitor cells
improved myocardial neoangiogenesis and
the recovery of functioning in a rat model of infarction.3
Repeated injection of bone marrow derived cells in a mouse model of atherosclerosis
reduced the rate of plaque formation without altering serum lipids levels, and
donor endothelial progenitor cells could subsequently be identified in the recipient’s blood vessels.4
Previous clinical studies have shown that
traditional risk factors for coronary atherosclerosis
are associated with low levels of circulating endothelial progenitor cells,5 whereas
protective interventions, including statin therapy6 and exercise,7
appear to increase the supply of these cells.
Hill et al. found that even in healthy volunteers,
levels of endothelial progenitor cells were inversely correlated with the Framingham risk score and
actually appeared to predict vascular function better than the Framingham risk score.5
Together, these data suggest that circulating endothelial progenitor cells may participate
not only in forming new blood vessels
but also in maintaining the integrity and function of vascular endothelium,
thereby mitigating disease processes such as atherosclerosis.
In this issue of the Journal, Werner and colleagues have further advanced our understanding of the clinical implications of endothelial progenitor cells.8 Endothelial progenitor cells were quantitated in 519 patients with coronary artery disease who
were followed for one year after undergoing catheterization.
Patients with higher levels of endothelial progenitor cells had
a reduced risk of death from cardiovascular causes and of
the composite end point of major cardiovascular events.
These relationships were preserved even
after adjustment for traditional risk factors and prognostic variables.
A similar relationship was seen
whether endothelial progenitor cells were identified by virtue of expression either of CD34 and KDR or of CD133 or
because of their ability to form endothelial colonies,
further strengthening the authors’ conclusions. Repeated catheterization was not performed in this cohort, so
we do not know whether the reduction in clinical events reflected a slowed progression of atherosclerosis or some other clinical effect.
A dissociation between anatomical measures of atherosclerosis and clinical events has been well documented in other settings.
Although this study is consistent with prior work suggesting that circulating endothelial progenitor cells may play a protective role in vascular homeostasis, other explanations
for the association between endothelial progenitor number and outcome remain possible.
Changes in the number of endothelial progenitor cellsand
in clinical events might reflect a common underlying etiology,
rather than a causal relation.
For example, a defect in the production of nitric oxide, which plays an important role
in both the mobilization of endothelial progenitor cells9 and blood-vessel function, might account for both observations.
Similarly, the number of endothelial progenitor cells
may mirror a person’s regenerative capacity more broadly and
predict clinical events on that basis.
Even if endothelial progenitor cells are mechanistically linked to clinical cardiovascular events,
such clinical studies do not distinguish between the possibility
that the protection is mediated through the integration of endothelial progenitor cells into blood vessels and
its possible mediation by other mechanisms, such as the
paracrine benefits of endothelial progenitor cell secreted products.
Although such questions will undoubtedly continue to provide fertile ground for fundamental investigation,
the report by Werner and colleagues has more immediate clinical implications.
First, it suggests that circulating cell populations may represent a new class of biomarkers
that naturally integrate diverse genetic and environmental effects,
thereby providing robust physiological and prognostic insights.
Second, in the context of coronary disease, the study shows that
the number of endothelial progenitor cells is an independent predictor of hard clinical outcomes.
As with other biomarkers, a demonstration of clinical usefulness will ultimately require
the examination of other patient populations, as well as
a demonstration that clinical therapy can be guided and enhanced by this information.
Finally, the increased risk associated with reduced levels of endothelial progenitor cells
supports the growing interest in the therapeutic potential of enhancing the level of these cells.
The most dramatic extension of this line of reasoning involves
transferring bone marrow or peripheral blood cells that are likely to include endothelial progenitor cellsto patients with coronary artery disease. Although it would be premature to judge the clinical success of these strategies, early trials, including one randomized (though incompletely blinded) trial, have suggested
at least short-term functional benefits of intracoronary infusion of bone marrow cells after acute infarction.10
Trials are planned to address more definitively the potential benefits of such cells
in the settings of acute infarction and chronic ischemic cardiomyopathy.
Such efforts would be aided substantially by the identification of specific markers as well as
an improved understanding of the role of subtypes of endothelial progenitor cells and
of the mechanisms by which they work.
Ironically, the data presented by Werner and colleagues in combination with work showing
the impaired functioning of endothelial progenitor cells in high-risk patients5 suggest
that the patients most in need of endothelial progenitor cells may be
those who are least able to donate them for autologous transplantation.
Whether these limitations can be overcome through
ex vivo expansion or genetic modification of endothelial progenitor cells is unclear.
In addition to possible cell-based therapies, work on endothelial progenitor cells provides yet another rationale
for redoubling efforts to comply with established therapeutic guidelines,
including lifestyle modifications and the use of statin therapy,
both of which appear to enhance the number of circulating endothelial progenitor cells.
Whether there will be a downside to enhancing the number and function of endothelial progenitor cells remains unclear,
although obvious concerns include exacerbating conditions that are characterized by adverse vessel formation,
such as diabetic retinopathy and tumor angiogenesis.
Small studies have suggested an association between high levels of circulating endothelial progenitor cells and the risk of certain cancers, such as multiple myeloma.11 Moreover, studies in animals show that
bone marrow derived endothelial progenitors participate in tumor angiogenesis, thereby
enhancing tumor growth.12
In the study by Werner and colleagues,
the number of deaths from cardiovascular causes among patients with high levels of endothelial progenitor cells
was substantially lower than that among patients with lower levels of these cells,
without a reduction in the risk of death overall.8
Although this finding could raise the specter of a counterbalancing adverse effect of endothelial progenitor cells,
there was no apparent pattern in the deaths due to other causes,
and no deaths from cancer were noted in this population.
It is possible that as we learn more about the biology of endothelial progenitor cells, there may be opportunities
to target vessel formation more specifically.
In addition, therapeutic strategies
tailored to individualized risk will undoubtedly help in practice.
For example, in the study by Werner et al.,
patients in the group with the lowest baseline levels of endothelial progenitor cells
had a risk of death from cardiovascular causes of 8.3 percent during one year of follow-up,
suggesting that the benefits of enhancing the function and number of endothelial progenitor cells
may well outweigh the risks in such high-risk populations.
Additional studies will be necessary to address these questions definitively. Larger studies
of longer duration performed in different cohorts will be required to determine fully
the clinical usefulness of endothelial progenitor cells as a biomarker.
Rigorous interventional studies will indicate
whether levels of endothelial progenitor cells can be used to guide therapy and
whether cell transfer has a role in augmenting the levels of these cells.
Basic-science studies should help guide these clinical efforts by
further defining the desirable subpopulations of endothelial progenitor cells and
the mechanisms by which they mediate their effects.
By establishing a connection between circulating endothelial progenitor cells and hard clinical end points, Werner and colleagues
provide a potent stimulus for clinical and basic studies to address these important issues.
Source Information
From the Program in Cardiovascular Gene Therapy, Massachusetts General Hospital, and Harvard Medical School ― both in Boston.
References
Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964-967.
Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999;5:434-438.
Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7: 430-436.
Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 2003; 108: 457-463.
Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593-600.
Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation 2001; 103: 2885-2890.
Laufs U, Werner N, Link A, et al. Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis. Circulation 2004; 109: 220-226.
Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005; 353: 999-1007.
Aicher A, Heeschen C, Mildner-Rihm C, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med 2003; 9: 1370-1376.
Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004; 364: 141-148.
Zhang H, Vakil V, Braunstein M, et al. Circulating endothelial progenitor cells in multiple myeloma: implications and significance. Blood 2005; 105: 3286-3294.
Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001;7:1194-1201.
Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction.
Kelly Bethel, Madelyn S Luttgen, Samir Damani, Anand Kolatkar, Rachelle Lamy, Mohsen Sabouri-Ghomi, Sarah Topol, Eric J Topol, Peter Kuhn
Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted
a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay),
to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs.
Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined
by positive staining for DAPI, CD146 and von Willebrand Factor
and negative staining for CD45.
In addition, CECs exhibited distinct morphological features that
enable differentiation from surrounding white blood cells.
CECs were found both as individual cells and as aggregates.
CEC numbers were higher in MI patients compared with healthy controls.
VS patients had lower CEC counts when compared with MI patients
but were not different from healthy controls.
Both HD-CEC and CellSearch® assays could discriminate
MI patients from healthy controls with comparable accuracy
but the HD-CEC assay exhibited
higher specificity while maintaining high sensitivity.
Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.
MicroRNA function in endothelial cells
Solving the mystery of an unknown target gene using microRNA Target Site Blockers
Dr. Virginie Mattot
Dr. Virgine Mattot works in the team “Angiogenesis, endothelium activation and Cancer” directed by Dr. Fabrice Soncin at the Institut de Biologie de Lille in France where she studies the roles played by microRNAs in endothelial cells during physiological and pathological processes such as angiogenesis or endothelium activation. She has been using Target Site Blockers to investigate the role of microRNAs on putative targets which functions are yet unknown.
What is the main focus of the research conducted in your lab?
We are studying endothelial cell functions with a particular interest
in angiogenesis and endothelium activation during physiological and tumoral vascular development.
How did your research lead to the study of microRNAs?
A few years ago, we identified in my team
a new endothelial cell-specific gene which harbors a microRNA in its intronic sequence.
We have since been working on understanding
the functions of both this new gene and
its intronic microRNA in endothelial cells
What is the aim of your current project?
While we were searching for the functions of the intronic microRNA,
we identified an unknown gene as a putative target.
The aim of my project was to investigate if this unknown gene was actually a genuine targetand
if regulation of this gene by the microRNA was involved in endothelial cell function.
We had already characterized the endothelial cell phenotype associated with the inhibition of our intronic microRNA.
We then used miRCURY LNA™ Target Site Blockers to demonstrate
that the expression of this unknown gene is actually controlled by this microRNA.
Further, we also demonstrated that the microRNA regulates
specific endothelial cell properties through regulation of this unknown gene.
How did you perform the experiments and analyze the results?
LNA™ enhanced target site blockers (TSB) for our microRNA were designed by Exiqon. We transfected the TSBs into endothelial cells using our standard procedure and
analysed the induced phenotype.
As a control for these experiments, a mutated version of the TSB was designed by Exiqonand
transfected into endothelial cells.
We first verified that this TSB was functional by
analyzing the expression of the miRNA target
against which the TSB was directed in transfected cells.
Finally, we showed that the TSB induced similar phenotypes as those found when we inhibited the microRNA in the same cells.
What were some specific challenges in your experiments and how did you overcome them?
The fact that the target gene for our microRNA was unknown was a major challenge. Without specific available tools, like antibodies,
it becomes difficult to demonstrate the effect of the microRNA on the gene in question and
to show that the unknown gene is indeed responsible for the functions of the microRNA.
However through the use of specific target site blockers, we were able to demonstrate
that this unknown gene was associated with the phenotype observed
when the microRNA was inhibited in endothelial cells.
How do you feel about your results so far?
We are very pleased with the results of the TSB experiments and
altogether these results demonstrate that our miRNA of interest
is functional in endothelial cells
through the regulation of a target gene with a previously unknown role.
What do you find to be the main benefits/advantage of the LNA™ microRNA target site blockers from Exiqon?
Target Site Blockers are efficient tools to demonstrate the
specific involvement of putative microRNA targets
in the function played by this microRNA.
The use of LNA™ allows the design of short oligonucleotides that are very specific and easy to work with.
What would be your advice to colleagues about getting started with microRNA functional analysis?
In order to address the role played by a microRNA,
it is essential to perform both gain and loss of functions experiments.
What are the next steps in the current project and how do you plan to perform them?
We plan to use microRNA inhibitor libraries to identify
more microRNAs specifically involved in the processes that we currently study.
When and where will be hear /read more about your studies?
We are currently in the process of submitting a manuscript regarding the function of my microRNA of interest.
2012pharmaceutical
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