Is the Warburg effect an effect of deregulated space occupancy of methylome?
Larry H. Bernstein and Radoslav Bozov, co-curation
LPBI
It would appear that pyruvate is directly used by cancer cell machinery for sustaining genome independence, and that CRISP-Cas9 system is essentially a modified CAD protein for making small bases.
13C-labeled biochemical probes for the study of cancer metabolism with dynamic nuclear polarization-enhanced magnetic resonance imaging
Lucia Salamanca-Cardona and Kayvan R. Keshari
Cancer & Metabolism 2015; 3:9 http://dx.doi.org:/10.1186/s40170-015-0136-2
In recent years, advances in metabolic imaging have become dependable tools for the diagnosis and treatment assessment in cancer. Dynamic nuclear polarization (DNP) has recently emerged as a promising technology in hyperpolarized (HP) magnetic resonance imaging (MRI) and has reached clinical relevance with the successful visualization of [1-13C] pyruvate as a molecular imaging probe in human prostate cancer. This review focuses on introducing representative compounds relevant to metabolism that are characteristic of cancer tissue: aerobic glycolysis and pyruvate metabolism, glutamine addiction and glutamine/glutamate metabolism, and the redox state and ascorbate/dehydroascorbate metabolism. In addition, a brief introduction of probes that can be used to trace necrosis, pH changes, and other pathways relevant to cancer is presented to demonstrate the potential that HP MRI has to revolutionize the use of molecular imaging for diagnosis and assessment of treatments in cancer.
Since the hallmark discovery of the Warburg effect in cancer cells in the 1920s, it has been widely accepted that the metabolic properties of cancer cells are vastly different from those of normal cells [1]. Starting from the observation that many cancerous (neoplastic) cells have higher rates of glucose utilization and lactate production, the development of tools and methods to correlate specific cellular metabolic processes to different types of cancer cells has received increased research focus [2, 3]. Several imaging techniques are currently in use for this purpose, including radiography, scintigraphy, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance (MR) [4, 5].
For more than 30 years, MR has been a revolutionary diagnostic tool, used in a wide range of settings from the central nervous system to cardiomyopathies and cancers. MR imaging (MRI) can outline molecular and cellular processes with high spatial resolution. Typically, MRI of body tissues is achieved via contrast visualization of the protons (1H) of water, which are present in high abundance in living systems. This can be extended to MR spectroscopy (MRS), which can further differentiate between less abundant, carbon-bearing, biological metabolites in vivo utilizing 1Hs of these compounds [6, 7]. However, despite its usefulness in imaging whole body tissues, 1H MRS has low spectral resolution and poor sensitivity for these less abundant metabolites. In addition,13C MRS is increasingly difficult, in comparison to 1H MRS, in that both the gyromagnetic ratio (approximately 25 % of 1H) and natural abundance (1.1 % of 1H) are significantly lower, making the detection of carbon-bearing compounds difficult [8, 9]. The low spectral resolution of 1H MRS for metabolites can be addressed by using 13C-enriched compounds, and with this direct 13C MRS, metabolic processes can be traced, utilizing enriched tags on specific carbons in a given metabolite [10]. While enrichment of molecules in 13C can also moderately address the sensitivity limitation of MRS, recent work in hyperpolarization (HP) provides a means of dramatically increasing sensitivity and enhancing signals, well beyond that of the equilibrium state obtained via MRS. [11, 12]. The focus of this review will be the introduction of this approach in the setting of cancer metabolism, delineating probes of interest, which have been applied to study metabolic processes in vivo.
Obtaining a hyperpolarized probe
In MR, a desired target is placed in a magnetic field where the nuclear spins of molecules are aligned with or against the direction of the magnetic field. The nuclear spins have thus different energies, and an MR signal is detected upon relaxation of nuclear spins of higher energy. At thermal equilibrium, the number of spins aligned with the magnetic field nearly equals the number of spins opposing the direction of the magnetic field. Thus, at thermal equilibrium, spin polarization is in the order of >0.0005 % resulting in a limited signal. Signal increases on the order of 100,000-fold can be achieved by hyperpolarizing the system via the redistribution of the spin population levels found at equilibrium [10, 13]. There are several techniques that have been used to achieve hyperpolarization of various nuclei: spin-exchange optical pumping of 3He and 129Xe, parahydrogen-induced polarization (PHIP), and dissolution dynamic nuclear polarization (DNP) [11,14, 15]. Both PHIP and DNP techniques can polarize biologically relevant nuclei like 13C and 15N, although there is a wider range of molecules that can be targeted for hyperpolarization using dissolution DNP [14, 16–18].
The goal of DNP is the transfer of polarization from highly polarized unpaired electron spins to the nuclear spins of a desired target compound. This is achieved by applying an external magnetic field to a free-radical agent in order to polarize electron spins, followed by saturating the electron spin resonance via microwave irradiation in order to obtain polarization transfer. The free-radical agent is generally a stable organic compound that is compatible with aqueous buffers, which are used as solvent in order to obtain a homogeneous distribution of the radical [13]. Nearly 100 % of the electrons on the free-radical agent are polarized when the free-radical/solvent mixture is subjected to high magnetic fields (≥3.3 T) followed by rapid freezing to 1 K using liquid helium in order to obtain a sample frozen to an amorphous state, which is necessary for retention and transfer of polarization [18]. For biological applications, after transfer of electron spin polarization to the nuclei of interest has occurred, the preparation must exist in solution, which can be achieved utilizing a dissolution process in which the solid sample is rapidly melted via injection of a hot solvent, typically a biologically compatible buffer, into the frozen sample [13]. The dissolution process results in a liquid sample at room temperature, while still preserving the enhanced polarization obtained by the microwave irradiation of the frozen sample [8]. Additionally, the use of chelating agents (e.g., EDTA) with the solvent to eliminate trace metals and more recently the use of gadolinium (Gd) chelates with the DNP sample have been used to further enhance and retain polarization in the liquid sample, albeit with caution over potential toxic effects when applied in vivo and the potential for loss of hyperpolarization due to T 1 shortening [11, 19, 20]. More in-depth exploration of the technical aspects of probe development has been previously reviewed [8, 11].
Considerations in probe selection and current research
The usefulness of a molecule for hyperpolarized MRS is dependent on the polarization lifetime of the nucleus of interest, and this property is determined by the spin-lattice relaxation constant (T1) [21]. Dipolar coupling, the magnetic field range, and molecular size can also affect the T1 of a given nucleus. In general, high magnetic fields and large molecular weights decrease the T1. Dipole-dipole coupling of 13C with 1H is common in biologically relevant molecules, and it shortens relaxation times; therefore, carbon atoms directly bound to 1H are generally not useful as probes for HP. For example, all carbons present in glucose (an important substrate in cancer cells) have relaxation times shorter than 2 s [22]. On the other hand, carbonyl carbons of biologically relevant molecules generally have T1’s above 20 s even at high magnetic fields like [1-13C] pyruvic acid, which has relaxation times of 67, 48, and 44 s at 3, 11.7, and 14.1 T, respectively [23–25]. Even carbons that are less oxidized than carbonyls, like the hemi-ketal in [2-13C] fructose have T1’s one order of magnitude higher than glucose carbons. Short spin-lattice relaxation times can sometimes be increased by deuterium enrichment of the sample. With this technique, protons that are directly bound to carbons are exchanged for deuterium atoms which results in the reduction of dipole-dipole relaxation, further preserving the hyperpolarized state [26]. This has resulted in increased T1’s of 13C nuclei in molecules such as glucose (T1 increased from 2 s to 10–14 s), providing the possibility of utilizing them in future metabolic studies [27–29]. Despite the effect of deuterium enrichment, research efforts have largely focused on developing carbonyl-bearing molecules as molecular imaging probes. The focus of this review is to introduce representative compounds relevant to metabolism that are characteristic of cancer tissue and have been applied in the work of multiple groups: aerobic glycolysis, glutamine addiction, and the redox state.
Pyruvate and aerobic glycolysis

Early work that utilized HP pyruvate to assess the response of tumors to treatment was conducted in mice xenografted with EL-4 lymphoma cells and treated with etoposide, a topoisomerase inhibitor that causes rapid cell death [38, 39]. In this study, tumor necrosis was correlated to a decrease in the flux of hyperpolarized lactate which was suggested to be due to a decrease in NAD+ and NADH in the intracellular pool as well as loss of LDH function. More recently, HP [1-13C] pyruvate has been used as a biomarker to evaluate early response to radiation therapy in glioma tumors by observing a decrease in hyperpolarized lactate suggested to be a result of changes in tumor perfusion which can be detected between 24 and 96 h following treatment [40, 41]. HP [1-13C] pyruvate has also been used to detect early response to temozolomide (TMZ) treatment on human glioblastoma rat models [42]. The study successfully showed for the first time detection of response to TMZ therapy 1 day after TMZ administration. The continued reports on using HP pyruvate as an imaging probe for assessing treatment response indicate the potential of the compound to become a standard in the field. Moreover, these studies demonstrate the usefulness of HP [1-13C] pyruvate as a tool for early assessment of therapy response, which can improve treatment selection at the clinical level. Pyruvate has also been validated as a metabolic imaging marker for use in humans [43]. In a two-phase study, patients with biopsy-proven prostate cancer of various histological grades were injected with HP [1-13C] pyruvate. In the first phase, a maximum dose level was determined to establish pharmacological safety of the HP probe while still injecting enough pyruvate to allow visualization. This addressed one of the major challenges faced in translating HP MRI to clinical applications: the potential toxicity of compounds that must be injected into patients. In the second phase, metabolism of pyruvate was visualized in real time and differences in the ratio of [1-13C] lactate to [1-13C] pyruvate between identified cancerous regions and normal tissue regions were successfully observed (Fig. 1a–d). [1-13C] lactate in regions that did not contain tumor was not detected, confirming previous biopsy and preclinical studies that demonstrated low flux of [1-13C] pyruvate to lactate and low concentrations of lactate in benign prostate tissues [44, 45]. Preliminary results indicated the possibility of detecting previously unobserved cancerous regions by HP [1-13C] pyruvate, later confirmed to be Gleason 4+3 cancer by biopsy, though further investigation into the relationship between grade and metabolism in prostate cancer patients is needed. While there are challenges associated with translation to clinical use for HP [1-13C] pyruvate, the first in human study demonstrated the feasibility of hyperpolarization technology as a safe diagnostic tool and provides the potential for utilizing this approach in preclinical models with direct translation to the clinic.
Glutamine metabolism

Dehydroascorbate as a redox sensor

Other metabolic imaging probes
While the three probes discussed earlier are the most well studied in metabolic events that are characteristic of cancer cells in general, other molecules have been evaluated in their potential to be used as biomarkers. Hyperpolarized bicarbonate (H13CO3−) has been successfully used to determine the pH in extracellular matrix of lymphoma tumors in mice, and a correlation between acidic environments and cancer was established [67]. The relaxation times for bicarbonate compounds at 3 T are between 34 and 50 s, which is enough time to detect the rapid conversion of H13CO3− and 13CO2 catalyzed by carbonic anhydrase [23]. The attractive feature of this probe is based on how ubiquitous acidic extracellular environments are to a wide variety of diseases; thus, HP bicarbonate has the potential for clinical translation beyond cancer research, though extensive work will be necessary to generate a preparation which will result in an adequate dose for the clinic [68, 69]. More recently, the potential of α-ketoisocaproate (KIC) as a molecular probe for in vivo detection of branched chain amino acid transaminase (BCAT) has been explored. BCAT catalyzes the conversion of KIC to leucine, and its expression has been suggested to correlate to genetic characterization of certain tumors. In a pilot study, HP α-keto-[1-13C]-isocaproate was shown to have a T1 of 100 s so its metabolism can be sensitively traced for over a minute after injection [70]. In the same study, metabolism of HP [1-13C] KIC to [1-13C] leucine by BCAT was observed in murine lymphoma tumor tissue but was absent in rat mammary adenocarcinoma with a correlation between BCAT expression and [1-13C] leucine signal detection [70]. Additionally, in the same models, [1-13C] pyruvate conversion to [1-13C] lactate and [1-13C] alanine was detected in both types of tumors. These findings show the promise of using [1-13C] KIC as a discriminative probe in addition to pyruvate in order to diagnose different types of cancer [71, 72]. Furthermore, the correlation between BCAT expression and [1-13C] leucine detection was also shown in rat brain tissue, confirming the usefulness of HP [1-13C] KIC in assessing BCAT activity in vivo [73]. Choline is another compound that has been evaluated as a molecular imaging probe since elevated choline and choline-derived metabolites have been correlated by 1H-MRS imaging to cancer in the brain, breast, colon, cervix, and prostate [74–76]. Despite its potential as a global marker for cancer because of the long T1 relaxation times that can be achieved with deuterium and 15N enrichment [77, 78], HP applications of 13C enriched choline are limited due to the small change in chemical shifts of choline and choline-derived metabolites as well as its potential toxicity [16, 79, 80]. It has been shown that choline toxicity occurs at doses of 53 mg/kg in mice, although a recent study successfully detected HP 13C choline in vivo without adverse effects in rats at doses of 50 mg/kg by using atropine to prevent cholinergic intoxication [81, 82] though metabolic products have been difficult to visualize in vivo. As mentioned earlier, the usefulness of glucose as a probe is limited due to the short relaxation times of all the carbons present in the molecule and although the T1’s can be increased through deuterium enrichment, the lifetime of the probe remains a hurdle for clinical applications [27, 28]. Thus, fructose (a pentose analog of glucose) has been successfully used as an alternative to probe glycolytic pathways [83] in TRAMP models where differences in HP [2-13C] fructose uptake and metabolism was visualized in tumor regions compared to surrounding normal tissues. Like choline, the limiting factor in the usefulness of [2-13C] fructose for in vivo studies is in small chemical shifts between the metabolite and its phosphorylated product. Finally, tumor necrosis can be used as a measure of treatment response, particularly early necrosis. HP [1,4-,13C] malate has been visualized in lymphoma mice models after injection of HP [1,4-13C] fumarate [84]. In normal cells, fumarate has a slow rate of transport into the mitochondria; however, in necrotic cells where the mitochondrial membrane is degraded, fumarase has access to the HP fumarate and its ubiquitous cofactor, water, thus facilitating rapid conversion to malate. Preliminary studies have shown the potential for its use in animal models though further work is required to determine the necessary density of necrotic cells for detection and the timings required for adequate visualization in patients.
Conclusions

Abbreviations
- ALT: alanine transaminase; BCAT: branched chain amino acid transaminase; DHA: dehydroascorbate; DNP: dynamic nuclear polarization; EDTA: ethylenediaminetetraacetic acid; GSH: glutathione; GSSG: glutathione sulfide; HCC: hepatocellular carcinoma; HIF: hypoxia-inducible factor; HP: hyperpolarized/hyperpolarization; IDH: isocitrate dehydrogenase; KIC: ketoisocaproate; LDH: lactate dehydrogenase; MR: magnetic resonance; MRI: Magnetic resonance imaging; MRS: magnetic resonance spectroscopy; NAD(H): nicotinamide adenine dinucleotide; NADP(H): nicotinamide adenine dinucleotide phosphate; PET: positron emission tomography; ROS: reactive oxygen species; SPECT: single-photon emission computed tomography; TRAMP: transgenic adenocarcinoma of mouse prostate
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sjwilliamspa commented on Is the Warburg effect an effect of deregulated space occupancy of methylome?
Is the Warburg effect an effect of deregulated space occupancy of methylome? Larry H. Bernstein and Radoslav Bozov, …
It would be an interesting figure, although I am not sure anyone has been able to measure it, is the spatial distribution of lactate and pyruvate over the tumor as a function of diffusion distance such as a heat map to see if pyruvate and lactate levels have a gradiant over a solid tumor. I am not sure it would but interesting to see where tumor cells, which undergo Warburg type metabolic phenotype actually exist, if it is a function of angiogenesis or a function of the proliferative capacity of cells in situ.
Response by LHB…
Radoslav Bozov has repeatedly referred to the real problem of space/time in the required experimental view that is intractable, as seen by Erwin Schroedinger. It is confounded by
the restrictions imposed by research, and to an extent also the dilemma of location and velocity.
I think it is to an extent also inherent in the modern revelations of autophagy and apoptosis that were not part of the view in the mid 20th century. However, the work of B. Chance led to a substantially better understanding of the hydride transfer in the NAD/NADH. What is overlooked is the important role cited by NO Kaplan of NADPH/NADP vs NADH/NAD associated with synthetic and, alternatively, catabolic processes in the cell. What role the pyridine nucleotide transhydrogenase would play is anyones guess. In any case the proliferation of malignant cells is dependent on NADPH. This would limit the NAD/NADH related reactions. The effect in the cytoplasm is PYR –> LAC, with generation of NAD from NADH. In addition, the type of isoenzyme favored should be consequential. For instance, the M-type LDH does not form an abortive ternary complex LDH*NAD+*PYR. In addition, Bernstein, Everse and Grisham showed that in cancer there is an aberrant cytoplasmic MDH.