Cancer Metabolism, Methotrexate and Oxidative Stress · to its antimetabolite properties....

Cancer Metabolism, Methotrexate and Oxidative Stress

Joshua A. Hess, MD Marshall University Joan C. Edwards School of Medicine 1600 Medical Center Drive Huntington, WV 25701

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The Open Hydrant © 2014

Cancer Metabolism, Methotrexate and Oxidative Stress Joshua A. Hess Dept. of Internal Medicine and Pediatrics, Joan C. Edwards School of Medicine, Huntington, WV, 25701 Published to www.TheOpenHydrant.com on April 18th, 2014 http://dx.doi.org/10.6084/m9.figshare.1007690

Abstract Purpose- Methotrexate has been an anti-cancer agent for over 60 years and inhibition of dihydrofolate reductase is its best known mechanism of action. Interactions have been studied involving multiple pathways of cellular metabolism and signaling. Metabolic dysfunction is a hallmark of many cancer cells, its role in carcinogenesis and cancer therapy remain of great debate. This review aims to explore, in part, this metabolic dysfunction in cancer cells and how a more complete understanding of methotrexate function provides significant insight into additional mechanisms of existing chemotherapeutic agents. Methods - Online literature searches of PubMed and Google Scholar were performed using the following terms and related biochemical pathways: autophagy, aerobic glycolysis, cancer metabolism, hydrogenosomes, glutamine metabolism, methotrexate, mitophagy, oxidative stress and cellular redox balance.

Results - Non-dihydrofolate reductase dependent methotrexate mechanisms have an intimate involvement in the disruption of mainly mitochondrial, but also cytosolic, metabolic pathways. The resulting metabolic interference leads to the depletion of NAD(P)H, an increase in cellular oxidative stress, and, ultimately, cell death. Conclusions - The additional methotrexate mechanisms reveal a novel dependence of cancer cells on their metabolic abnormalities to buffer oxidative stress. Mitochondria appear to have a significant role in buffering oxidative stress and in both the development and growth of cancer cells. The alterations in glycolytic metabolism seen in cancer cells may aid this mitochondrial ability. Further research is needed to better understand the involvement of other chemotherapeutic agents on these pathways.

Contents:

I. INTRODUCTION --------------------------------------------- 2 II. METHODS ---------------------------------------------------- 3 III. METABOLIC DYSFUNCTION OF THE CANCER CELL --------------------------------------------------------------- 4

I. THE WARBURG EFFECT -------------------------------------- 4 II. ENERGY PRODUCTION -------------------------------------- 4 III. HYPOXIA ---------------------------------------------------- 4

IV. MITOCHONDRIAL ROLES IN OXIDATIVE STRESS AND CARCINOGENESIS -------------------------------------- 5

I. THE OXIDANT BUFFERING ORIGINS OF MITOCHONDRIA -- 5 II. INFLAMMATION --------------------------------------------- 6 III. ELECTROCHEMICAL METABOLISM ------------------------- 6

IV. GLUTATHIONE ---------------------------------------------- 6 V. AUTOPHAGY AND MTOR ----------------------------------- 7 VI. MITOCHONDRIAL PROLIFERATION AND THE CELL CYCLE 7 VII. DNA DAMAGE AND MALIGNANT TRANSFORMATION -- 8

V. METHOTREXATE PHARMACOKINETICS ------------- 8 I. METHOTREXATE POLYGLUTAMATES AND METABOLIC

SYNERGY -------------------------------------------------------- 8 II. METHOTREXATE AND RHEUMATOID ARTHRITIS ----------- 9 III. METHOTREXATE AND NAD(P)H-DEPENDENT

OXIDOREDUCTASES ------------------------------------------ 11 VI. CONCLUSION--------------------------------------------- 14

I. Introduction The genetics of the cancer cell is a fervently

studied area of medicine and has been for decades. In recent years, the area of cancer immunotherapy has grown tremendously and therapies with monoclonal antibodies have become an impressive addition to anti-cancer repertoire. Although genetic involvement was known of in the early twentieth century, the Otto Warburg was awarded the 1931 Nobel Prize for Physiology or Medicine as a result of his research regarding cellular respiration and hypothesized that cancerous transformation of tissue was a result of an injured respiration, or metabolism. As the popularity

of DNA grew with discoveries made in the mid-twentieth century, pursuits in understanding the metabolic peculiarities of cancer became less common. Today, cancer metabolism is a field of study quickly regaining momentum.

Methotrexate (MTX) is a structural analog of folic

acid. It is used in the therapy of various diseases due to its antimetabolite properties. Historically, aminopterin, was the antifolate initially successful in treating leukemia in the late 1940’s. Its classic mechanism of action is inhibition of dihydrofolate reductase (DHFR) to deplete cellular pools of tetrahydrofolate, stopping the production of

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thymidylate. Cells lacking adequate thymidine are unable to synthesize DNA, which results in the arrest of cellular proliferation. The combined effects lead to the demise of rapidly dividing cell populations either through apoptosis or autophagy. Sustained maximal DHFR inhibition is targeted and important for several reasons. First, it provides steady inhibition of DNA synthesis, and, second, it minimizes the risk of developing resistance to MTX[2]. With only 1% of the average cellular DHFR concentration is required to maintain a sufficient reserve of reduced folate coenzymes, high doses of MTX are required to achieve this effect are often accompanied by toxicities[1]. Major adverse effects of MTX therapy include dermatitis, elevated transaminases,

mucositis, and myelosuppression. Leucovorin is used to curb the incidence and severity of these effects and often found in high dose MTX protocols. (See Figure 1)

In vitro mechanics of the anti-folate MTX on DHFR are well known. Newer studies have explored the in vivo biochemical interactions of MTX with multiple enzymes. These studies reveal an intimate involvement of MTX with multiple metabolic pathways ranging from cellular energy production, anti-oxidant regeneration, nucleotide synthesis and buffering of oxidative stress [3,4,1]. This review investigates mechanisms of MTX and their interference with cytosolic and mitochondrial

abilities to buffer oxidative stress. This involvement reveals multiple pathways that are affected, directly or indirectly, by many other classes of currently

used chemotherapeutic

agents.

II. Methods Online

literature searches of PubMed and Google Scholar were performed using the following terms and related biochemical

pathways: autophagy, aerobic glycolysis, cancer

metabolism, hydrogenosomes,

glutamine metabolism,

methotrexate, mitophagy,

oxidative stress and cellular redox balance.

Figure 1. Effects of methotrexate on folate metabolism in relation to nucleotide metabolism. Via its inhibition of dihydrofolate reductase, methotrexate interferes with the generation of tetrahydrofolate and subsequently, DNA synthesis. Tetrahydrofolate is metabolized further for use as a cofactor in the synthesis of dTMP. Folinic acid, leuvovorin, is often used to rescue cells from methotrexate and restore cellular stores of tetrahydrofolate. In addition to interference with DNA synthesis, this diagram superficially shows the involvement of tetrahydrofolate in the metabolism of purines. Legend: ADP, adenosine diphosphate; AMP, adenosine monophosphate; CDP, cytidine diphosphate; CTP, cytidine triphosphate; dCDP, deoxycytidine diphosphate; DHFR, dihydrofolate reductase; dTMP, deoxythimidine monophosphate; dUDP, deoxyuridine diphosphate; dUMP, deoxyuridine monophosphate; GDP, guanine diphosphate; GMP, guanine monophosphate; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; IMP, inosine monophosphate; Methyl THF, methyltetrahydrofolate; MTX, methotrexate; PNP, purine nucleotide phosphorylase; PRPP, phosphoribosyl pyrophosphate; UDP, uracil diphosphate; UMP, uracil monophosphate; UTP, uracil triphosphate; XMP, xanthine monophosphate

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III. Metabolic Dysfunction of the Cancer

Cell

i. The Warburg Effect

“The Warburg Effect” describes a dysfunctional metabolic state present in a variety of cancers [5] [6]. This metabolic state is dominated by glycolysis ending in lactate production. Warburg postulated that this dysfunctional metabolism was the primary cause of cancer. The metabolic differences between normal and tumor cells are characterized as an increase in glycolytic flux, even in the presence of adequate oxygen [7]. This change in cellular metabolism is oft considered a result, not an instigator, of cancerous transformation. The idea of metabolic disarray as an initiator of cancer has gained additional support in recent years [7]. Some may argue against this theory as oxygen consumption in these transformed cells continues despite the production of ATP via aerobic glycolysis [8]. However, the perturbed metabolism of transformed cells does not fully exclude oxidative phosphorylation as a source of ATP production. The metabolic ratios of glycolysis and mitochondrial oxidative respiration may show similarities to proliferating non-transformed cells but the ability of transformed cells to return to non-Warburg, or non-stressed, metabolic profile is hindered.

Cancer cell research has characterized a very

complex state of metabolic disarray involving glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, the electron transport chain and amino acid metabolism [9,1,10,11,7]. While the initiating insult remains largely unidentified, it is likely to be as complicated as the resulting metabolic state. The derangements seen in cancer cells result from a combination of factors including hypoxia, poor total body and cellular antioxidant reserve, mitochondrial dysfunction and free radical production. Some research suggests that alterations in TCA associated enzymes, with resultant pseudohypoxic phenotypes, may lead to changes in epigenetic homeostasis with tumor promoting effects[7].

ii. Energy Production

Senescent thymocytes obtain 88% of ATP production occurs from oxidative glucose metabolism. However, when stimulated to proliferate, 86% of ATP production is obtained without oxidative phosphorylation, by the conversion of glucose to lactate [6]. This shift to non-oxidative

ATP production pathways results in significant increases, as much as 20-fold, in glucose utilization in proliferating cells [6,12]. This metabolic redirection to aerobic glycolysis and lactate production offers several benefits for the proliferating cell [6]. These include the production of numerous metabolic intermediates to serve as antioxidants, creation of an acidic environment to improve oxygenation and stimulation of angiogenesis via enzyme activation and the production of various inflammatory mediators- all of additional benefit to cancer cells in a hypoxic environment[6]. Outside of the process of cell division, this same metabolic redirection occurs during episodes of hypoxic stress.

iii. Hypoxia

Under normal oxygen tensions, hypoxia-inducible factor alpha (HIF-1α) is produced and degraded at an equal rate. The Von Hippel-Lindau tumor suppressor gene product, pVHL, has multiple functions, one of which is a recognition subunit of an E3 ubiquitin ligase complex. Prolyl hydoxylases (PHD) catalyze the hydroxylation of proline residues on the oxygen-dependent degradation site of HIF-1α creating a docking site for pVHL directed degradation [13]. Degradation of HIF-1α via pVHL is oxygen-dependant, converting oxygen and α –ketoglutarate to carbon dioxide, and appears to be a rate-limiting reaction dependent on the functional capacity of pVHL [14]. In the absence of sufficient reactants the HIF-1α protein stabilizes, binds HIF-b and is transported to the nucleus where it stimulates the transcription of genes containing a hypoxia response element.

The resulting HIF transcription cascade results in

increased transcription of proteins including glucose transporters GLUT1 and GLUT3, 6-phosphofructo-2-kinase, phosphoglycerate kinase 1, pyruvate kinase M2, monocarboxylate transporter MCT4 (a lactate efflux pump), and pyruvate dehydrogenase kinase (PDK1). When phosphorylated by PDK1, the pyruvate dehydrogenase enzyme is inactivated and inhibits the conversion of pyruvate into acetyl-CoA for the TCA cycle [15]. Despite a lack of glycolysis derived acetyl-CoA, TCA cycle activity continues. In oxygen concentrations as low at 1%, NADH production maintains a normal rate with mitochondrial ATP production significantly reduced [16].

Stabilization of HIF-1α outside of a true hypoxic

environment is referred to as pseudohypoxia and has

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been associated with, among many causes, defects in the mitochondrial enzymes succinate dehydrogenase and fumarate dehydrogenase[13]. Both of these enzymes possess tumor suppressing functions and loss of that function has been associated with many VHL-associated renal tumors [13]. The defects of these mitochondrial enzymes may be from inhibition exogenous to the enzyme, such as inactivation through reaction with a free radicals, or a functional defect resulting from genetic mutation.

GLUT1 and GLUT3 maintaining an insulin-

independent influx of glucose, activation of insulin receptors would be associated with a much larger volume of raw materials for its metabolic processes. The cell remains normoxic with aberrantly expressed PDK1 blocking delivery of glycolysis derived substrate to the TCA cycle. Mitochondria in the pseudohypoxic cell become starved for materials begin catabolizing amino acids, such as glutamine and asparagine, to supply the TCA cycle with intermediates, a-ketoglutarate and oxaloacetate respectively [17,18,11]. The changes in fuel sources and pathways may offer an explanation for the inverse relationship between the aggressiveness in tumor growth and proliferation and the drop in serum glutamine concentrations [19]. The conversion of amino acids to TCA intermediates has been seen as glutamine derived carbon exiting the TCA cycle as malic acid and being catalyzed by malic enzymes to produce NADPH; a reactant in the regeneration of free radical scavengers like glutathione [20]. The catabolism of glutamine and asparagine seen in highly metabolically active cancers is likely due to the ease of which they are transaminated to supply TCA intermediates. Additionally, high concentrations of pyruvate serve as a sink for nitrogen freed from amino acid transaminations [21].

IV. Mitochondrial Roles in Oxidative Stress

and Carcinogenesis

i. The Oxidant Buffering Origins of

Mitochondria Endosymbiotic Theory is currently the most

accepted route of mitochondrial origin. Two general assumptions accompany this theory. The first being that mitochondrial acquisition accompanied an accumulation of atmospheric oxygen. The second marks the separation of aerobic and anaerobic (assumed lack of mitochondria) eukaryotes. The

recent inclusion of various anaerobes to eukaryotic lineages is secondary to a much better understanding of “variant mitochondria”-like organelles- i.e. hydrogenosomes, mitosomes. Recent biological and geochemical theories propose the atmospheric oxygen increased long before a noticeable increase in oceanic waters. This atmospheric oxygenation led to oxidation and solubilization of land bound sulfide deposits making ocean waters increasingly anoxic and sulfidic. In the light of a Proterozoic ocean chemistry theory for mitochondrial endosymbiosis, one can rationalize that the benefit of mitochondrial acquisition was not solely ability to provide ATP via its utilization of oxygen but its ability to use electrons, as reduced molecules, to buffer oxidant stresses in a highly anoxic and sulfate rich environment. As a more electronegative atom than sulfur, increasing concentrations of dissolved oxygen would make it a much more efficient recipient of those free electrons.[22]

Mitochondria are very dynamic organelles, they

fuse, divide, power metabolic detoxification, generate stored energy and serve many important regulatory functions. Recent literature describes a localizing mechanism of mitochondria with definitive influence by the oxygen tension of the cell [23,24]. In hypoxic states, mitochondria in pulmonary artery endothelial cells undergo inward migration, via microtubules, and localize around the nucleus[23]. Mitochondrial respiration is a significant producer of free radicals with the electron transport chain being the primary source. This movement is associated with a local increase in production of free radicals and reactive oxygen species in the vicinity of the nucleus[23].

Mechanisms have evolved, alongside the

mitochondria, to mitigate damage from oxidative stresses produced within the cell. Superoxide dismutase (SOD), catalase, glutathione (GSH) and thioredoxin are well known scavengers of free radicals and their reactive metabolites[25]. Intermediates and products of glycolysis may also serve either antioxidant roles, such as pyruvate, or serve as a substrate for an antioxidant regenerating pathway as glucose-6-phosphate, glyceraldehyde-3-phosphate via NADH production[26,25]. The absence of adequate oxygen, presence of a dysfunctional electron transport chain, or a deficiency of cofactors place the mitochondria at risk of increased formation of free radicals.

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Despite the cell’s extensive ability to neutralize

developing free radicals, in a scenario of overwhelming oxidant stress the reserve capabilities of endogenous and exogenous antioxidants may be unable to defend the cell from the damaging effects of free radical. These stresses may be environmental or nutrition related or due to the genetic predispositions of the cell. Iron, copper, various forms of radiation, inflammation, smoke, vehicle exhaust, anti-neoplastic drugs and ischemia/reperfusion injuries are all known to increase cellular oxidant stress through various mechanisms [27]. Down syndrome is associated with increased oxidant stress secondary to over-expression of superoxide dismutase relative to catalase and glutathione peroxidase [28-30]. The SOD1 gene coding for superoxide dismutase is found on chromosome 21, 21q22.1 [30]. Individuals with Down Syndrome have lower plasma total antioxidant status measured as reduced glutathione [30]. This inborn dysfunctional ratio leads to an increased susceptibility to cellular oxidant stress. It is well known that patients with Down syndrome are at an increased risk of not only malignancy, but also Alzheimer's disease and adverse drug reactions to many chemotherapeutic agents, including MTX. In addition to Down syndrome, similar mitochondrial and metabolic dysfunctions are accompanied by sensitivity to oxidant stresses in Ataxia-Telangiectasia, Fanconi anemia, and Werner syndrome [29].

ii. Inflammation

Inflammation has a variety of effects in the body. Chronic inflammation is a significant factor in the development of many human diseases. A state of normal oxygen tension encourages mitochondrial localization in the cell periphery and a subsequent local increase in free radical concentrations. For a metabolically normal cell, injury induces the production of reactive oxygen species produced via NAD(P)H oxidase and then secretion into the extracellular matrix to initiate the process of healing. This peripheral increase in free radicals place cell membrane components at risk. The hydroxyl radical, regardless of its source, is one that may freely diffuse through cell membranes. One membrane component particularly vulnerable to hydroxyl radicals is arachidonic acid often esterified to membrane phospholipids. A class of eicosanoids called isoprostanes are formed upon reaction of arachidonic acid with free radicals; i.e. hydroxyl radicals [31].

Isoprostanes are isomers of the cyclo-oxygenase enzyme product prostaglandin F2 and will subsequently induce the same inflammatory cascade [32]. There is also some evidence to suggest the formation of thromboxane A2 via isoprostane endoperoxide rearrangement [33]. In a cell with poor antioxidant reserve, similar hypoxic events increase the likelihood of subsequent DNA damage from those free radicals.

iii. Electrochemical Metabolism

Via the Cytochrome P450 family of enzymes, mitochondria are arguably the most significant physiologic players in chemical metabolism. Cytochrome P450 enzymes make up a superfamily of heme-thiolate proteins involved in steroid synthesis and responsible for 75% of phase I drug metabolism [34-36]. The majority of these enzymes are associated with NAD(P)H-reductases and found in either the endoplasmic reticulum or the mitochondria[36]. An electron is obtained from NAD(P)H via the associated reductase enzyme and transferred to the substrate at the enzyme’s binding site via its heme group[34]. The overall efficiency of electron transfer from NAD(P)H to the cytochrome P450 enzyme and then to its substrate can vary. Although this reaction typically detoxifies a chemical, it may also transform a procarcinogen to its carcinogenic metabolite; i.e. 5-methylchrysene from tobacco smoke [37]. While some studies report an electron leakage flow of only 0.15%, in steroidogenic tissues the cytochrome P450 components outnumber electron transport chain components ten to one[36]. The “leaked” electrons can react with a variety of molecules to form free radicals, just as those from the electron transport chain.

iv. Glutathione

In vitro studies show that MTX therapy decreases intracellular concentrations of reduced glutathione[38]. While NAD(P)H is required for regeneration of reduced glutathione, it is not for required for de novo synthesis. In de novo synthesis, glutamate, cysteine, glycine and ATP are used in an enzyme catalyzed reaction. Depletion of glutathione in serine starved cells (a precursor to cysteine) has been associated with impaired glycolysis and an elevation in cellular ROS [39]. Tumor suppressor p53, in conjunction with the cyclin-dependent kinase inhibitor, p21, arrests cellular development and promoted cellular survival by rapid shunting of exogenous serine into the glutathione synthesis

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pathway [39]. In cells without p53, this shunt did not occur. The resulting serine depletion and oxidative stress lead to not only impaired proliferation, but also a reduction in cellular viability [39].

v. Autophagy and mTOR

A growing area of investigation into cancer metabolism centers on autophagy. Autophagy is the process in which cells degrade and recycle macromolecules and organelles via two distinct pathways; the ubiquitin-proteasome system (UP system) and the lysosome. Smaller, short-lived proteins are the typical degradation targets of the UP system. However, the size and stability large protein aggregates and organelles leave the lysosomal pathway as the most effective means of autophagy. In mammalian cells, lysosomal autophagy is directed by a chaperone system marking macromolecules and organelles for digestion. The most apparent benefits of autophagy are resultant decreases in damaged, unneeded or toxic cellular components while producing raw materials for cellular metabolism.

While its pathways are not fully understood, the

regulation of these processes are intimately linked with their function. Cellular starvation is a powerful inducer of autophagy. A fall in intracellular amino acid concentrations is another stimulant of autophagy. Constructing a spectrum of starvation signals, nitrogen would be an excellent marker of cellular nutrition. To clarify, in a very well supplied cell, amino acids would activate cellular anabolic pathways via conjugated tRNA activation of the Gcn protein kinases [40]. As amino acid concentrations drop, the number of transamination and deamination reactions in the cell likely increase to attenuate this lack amino acids. Cellular concentrations of ammonia, especially in concert with glutaminolysis, serve as a powerful regulator in the basal cellular autophagy rates[41]. Further along the spectrum, however, literature has described total nitrogen starvation as more potent activator than either of the previous nitrogen-related autophagy stimulants [40].

Reactive oxygen species are also potent

activators of autophagy. Superoxide, in particular, has been found to be a major player in the activation of reactive oxygen species induced autophagy. Superoxide is a product of a dysfunctional mitochondrial electron transport chain as well. In properly functioning cells, it is reduced to hydrogen peroxide and then to water via SOD1, SOD2 and

catalase or scavenged by one of many different antioxidants present in the cell or in circulation.

When antioxidant systems are overwhelmed,

mitochondrial components are at a very distinct geographical disadvantage to the effects of free radicals and reactive oxygen species. Although the GSH regenerating abilities of mitochondria are present, mitochondrial autophagy, or mitophagy, may ensue if sufficiently injured damaged. This has been seen in association with significant decreases in the ratio of GSH/GSSG inducing mitophagy, while ample concentrations of GSH are inhibitory [42].

2-deoxyglucose (2DG), and glucose starvation,

have been observed to induce apoptosis in some cancer cells. Recent research has reveals a dichotomy of effect depending on cellular conditions. In hypoxic environments, 2DG and glucose starvation inhibit autophagic processes, possibly through significant decreases in ATP through PI3K III-Beclin1 complex blocking the effects of autophagy activating genes[43]. These same conditions will activate autophagy in a normoxic environment through separate pathways initiated by ROS formation [44,43]. This dual effect could be explained by the utilization of glycolysis intermediates and end-products in mitochondrial redox buffering roles.

vi. Mitochondrial Proliferation and the Cell

Cycle

As previously discussed, cellular ATP production shifts from mitochondria as the primary source to glycolysis during cell division. However, prior to cell division, mitochondrial mass expands largely for two reasons. The first is in preparation for increased metabolic demands of replication. The second being the preparation for splitting of the parent cell’s mitochondrial population between to two daughter cells. As with mitochondrial localizations induced by HIF, microtubules are involved in mitochondrial changes seen in preparation for cell division [45].

Mitochondrial inner membrane production in

HeLa cells, with functional enzymatic capabilities, is completed by mid-S-Phase [46]. At this point in the cell cycle, there is no increase in mitochondrial membrane potential. Prior to entering M-Phase, there is a significant increase in mitochondrial mass and membrane potential, as much as 168% and 228% respectively, with less than a 20% increase in ATP [46]. There is a noted discrepancy, and delay,

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between the cessation of inner mitochondrial membrane accumulation, in mid-S phase, and the increase of mitochondrial membrane potential prior to M-phase. Metabolism during these transitional stages is highly dependent on glutamine for cellular metabolic demands. When starved for glutamine, synchronized HeLa do not progress from G1- to S-phase and S- to G2/M-phase [47]. When those same cells are placed on media with glutamine without glucose, the cell cycle does progress at these points, although it does so at a slower rate [47].

Cellular progression of G1- to S-phase is

dependent on a favorable redox status in the cell and is a significant signaling pathway [48]. If we assume that the TCA cycle progresses at a constant rate and increased inner membrane mitochondrial volume correlates with total TCA cycle enzyme then the G1/S phase would have a high [NADH]/[NAD+] redox status [46]. In addition to mitochondrial morphologic changes, there is an associated decrease in ATP production due to decreased NADH flow through the electron transport change. High concentrations of NAD(P)H have been shown to have multiple effects on regulatory proteins, gene expression, directly or epigenetically, and cell cycle progression.

vii. DNA Damage and Malignant

Transformation

Cellular hypoxia induces a peri-nuclear mitochondrial migration. Applying the same cellular state of oxidative stress and free radical production in the vicinity of the nucleus would place the nuclear DNA at increased risk of damage. Beyond the risk for genetic damage and mutation during chromosomal replication, the current genome profile being expressed in a senescent cell would expose many regulatory and functional sites to an even higher risk of damage. Many commonly mutated tumor suppressor genes and onocogenes, P53, MYC and BCL as examples, induce metabolic changes in non-transformed cells in periods of high cellular stress [49-55]. Affecting amino acid metabolism, mitochondrial function, glycolysis and autophagy, the end function of these genes is to temporarily halt cellular self-destruction while processes attempt to overcome that stress. A Pyrrhic victory of sorts, these genes may prolong the life of the cell but at the cost of potential mutation when recovery fails; thus leading to cancerous transformation.

Mitochondrial DNA would be at a much higher risk for damage via this mechanism. Two important differences are present between nuclear and mitochondrial DNA. The first being that mitochondrial chromosomes are not protected via histones[56]. In nuclear DNA, genetic material is wrapped around histones and physically protected, to some degree, from the damaging effects of free radicals. The second factor is that the status of cellular oxygen tension would likely have no significant effect on the proximity of free radicals produced by mitochondria. Low level reactive oxygen species could be severe enough to induce mtDNA damage if not neutralized.

Combining the two factors in a state of cellular

hypoxia and increased free radical production would put the mitochondrial DNA at a much higher risk for damage than nuclear DNA for damage. Periods of chronic reactive oxygen species exposure has been shown to damage mtDNA and other macromolecules more extensively and with longer lasting effect, than damage to nuclear DNA[57]. Whether it’s resultant acute damage or epigenetic changes through chronic FR exposure of nuclear or mitochondrial DNA, this process could be responsible for “locking” cancer cells into this abnormal metabolism.

V. Methotrexate Pharmacokinetics

i. Methotrexate Polyglutamates and

Metabolic Synergy

In vivo MTX metabolism follows a similar pathway to natural folates with polyglutamation via the enzyme folylpolyglutamyl synthetase [58,59]. MTX polyglutamates, especially with three or more glutamate residues, are retained in cells longer, and with higher enzyme affinity, than unmodified MTX [60]. Cellular concentrations of polyglutamated methotrexate rise even in the absence of circulating drug, until cellular efflux begins [58]. Interestingly enough, animals fed a diet rich in supplemented glutamine showed not only a decreased incidence of MTX adverse effects, but also an enhanced anti-tumor effect in cancer cells[1]. This effect was also observed in the treatment of human tumors, and is likely secondary to MTX polyglutamylation [61].

Intracellular polyglutamated MTX concentrations

are an important factor in achieving maximum pharmacologic effects in the treatment of malignancy [60]. Rodent studies have shown that administration of oral glutamine in conjunction with high dose MTX

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therapy was not only associated decreases in enteric mucositis, but also a 300% increase in polyglutamated MTX concentrations within malignant cells when compared to normal cells[61]. In a phase I trial based on these rodent studies, nine patients with inflammatory breast cancer were placed on a similar regimen. In this study, one case of grade I mucositis was reported and eight of the nine patients responded to the chemotherapy regimen with a median survival of 35 months[61].

This process of polyglutamylation appears

responsible for the schedule dependent response to combination therapy with L-asparaginase. Pre-treatment of cancer cells with L-asparaginase leads to significant interference with MTX polyglutamation[62]. Conversely, in post-MTX treatment with L-asparaginase, the deamination of glutamine has a synergistic effect on the activity of L-asparaginase[63,62]. Asparaginases are frequently used in combination with MTX. Efficacy of this combination therapy has been found to be very schedule dependent[62]. Pretreatment with asparaginase decreases tumor cell retention, and lowers the tumoricidal effects, of MTX. Current literature suggests that this effect is largely due to a massive deamination of glutamine being required for optimal asparagine deamination[63]. While its primary enzymatic activity is focused on asparagine, L-asparaginases have varying levels of glutaminase activity that is depending on the source of the enzyme.

In settings of glutamine depletion, increased

expression of asparagine synthetase has been observed in hamster cell lines[64]. With pretreatment of cancer cells with methotrexate significantly handicapping the regeneration of NAD(P)H, glutamine and glutamate shift from supplying intermediates of the TCA cycle into a significant resource sink, de novo synthesis of glutathione. As intracellular levels of glutamine decrease, expression of asparagine synthetase likely increases with subsequent glutamine depletion and increased ammonia production. With an evolving depletion of glutamine and cellular shift toward increased asparagine synthesis, the addition of L-asparaginase induces a starvation of asparagine and glutamine and produces significant concentrations of aspartate, glutamate, and ammonia. The restriction of both asparagine and glutamine have been observed to induce expression of asparagine synthetase,

glutamine synthetase, IGF-I and –II, IGFBP-1, CHOP, Ornithine decarboxylase, oncogenes c-JUN and c-FOS and ribosomal proteins L17 and L25 [65]. In asparaginase-resistant cells, inhibition of glutamine synthetase has been shown to both block proliferation and decrease cellular viability [66]. Additionally, as expression of asparagine and glutamine synthetase increases there is concomitant stabilization and up-regulation of the Myc and BCL-2 oncoproteins[65].

ii. Methotrexate and Rheumatoid Arthritis

Methotrexate is well-established in the treatment of rheumatoid arthritis (RA). Its role in this disease has suggested additional mechanisms of action[38]. Aside from DHFR inhibition, MTX inhibits the production of spermine and spermidine as well as indirectly increasing extracellular concentrations of adenosine. Although spermine and spermidine are found in high concentrations within chronically inflamed tissues, there is little evidence supporting it as the effective mechanism in RA management. Its effect on increasing extracellular adenosine, however, has increasing support as the major mechanism of action in the treatment of RA[38]. Extracellular concentrations of adenosine increase with MTX inhibition of AICAR transformylase, an enzyme involved in purine metabolism [67,38]. With MTX blocking its action, increased AICAR is followed by increases in a dephosphorylated metabolite, AICARiboside. (See Figure 2) These directly inhibit AMP deaminase and adenosine deaminase, respectively, and effectively handicaps purine metabolism [68,38].

Increased extracellular ATP is a normal response

to stress, cell injury and is secreted by prokaryotes. Membrane proteins CD39 and CD73, both ectonucleosidases, catalyze the hydrolysis of ATP and ADP to adenosine. Physiologic concentrations of interstitial adenosine can range from 30-300nM. In periods of hypoxic stress, adenosine concentrations may increase 100-fold. MTX induces increased intracellular concentrations of AMP and adenosine leading to increased extracellular concentrations via efflux and enzymatic dephosphorylation[68].

The presence of increased concentrations of

adenosine, both intra and extra-cellularly, induce a wide range of cascading effects. Cellular energy status can be measured as a ratio of nucleoside triphosphate to its dephosphorylated and free nucleotide forms. In

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a healthy cell with a normal metabolic state, increases in AMP and free adenosine indicate cellular starvation and activate various cellular pathways. Contrasting the two ends of this spectrum, extracellular ATP has pro-inflammatory effects on immune response while adenosine has displayed anti-inflammatory effects, as seen in the treatment of RA[69].

Cell membrane receptors for the molecules of

ATP/ADP and adenosine are referred to as purinergic signaling, and is a conserved pathway in eukaryotes. These receptors are subdivided into two general categories P1 and P2. The P1 receptor group, selective for adenosine, has four subtypes; A1, A2a, A2b, and A3. While the P2 receptor group, selective for ATP and ADP, has been broken down into the two broad categories of P2X and P2Y, each with several variations. [69]

P1 Purinergic receptors A1 and A3 are Gi-coupled receptors that are actived at physiologic concentrations of extracellular adenosine. Their activation induces MAP kinase, phosphatidylinositol-3-kinase and protein kinase C pathways while inhibiting adenylyl cyclase and protein kinase A pathways. The difference between the two being that

desensitization of the A3 receptor occurs following chronic stimulation. Purinergic receptor A2A, a Gs-coupled receptor, is also active at physiologic adenosine concentrations and its stimulation induces adenylyl cyclase and protein kinase A pathways. The A2B receptor has a lower affinity for adenosine than the other subtypes is and only activated in the presence of pathologic concentrations of extracellular adenosine. Analagous to the A2A receptor, the A2B receptor is a Gs-coupled receptor and activates the adenylyl cyclase and protein kinase A pathway.

P2X group are trimeric

cation channels permeable to Na+, K+, and Ca+. P2X receptors have been observed in 2 general groups with multiple forms, six homotrimeric

(P2X1-5 and P2X7) and six heterotrimeric receptors. The P2X7 receptor is an atypical adenosinergic receptor with highly regulated activity. In low concentrations of ATP, the P2X7 receptor activates the NLRP3 inflammasome via K+ efflux and pannexin-1 membrane pore. In high ATP concentrations, the P2X7 channel undergoes significant permeability changes in the pore that allows for the passage of very large (up to 900kDa) molecules leading to cell death. [69] The P2Y purinergic receptor group is divided into two subtypes with five Gq/G11-coupled receptors and three Gi/o-coupled receptors

Figure 2. The effects of methotrexate on pathways of adenosine metabolism. By inhibiting the regeneration of THF, MTX inhibits the enzyme AICAR transformylase. A subsequent build-up of this enzyme’s substrates lead to an accumulation of AMP and adenosine in the cell via inhibition of AMPDA and ADA. This intracellular accumulation leads to an increase in extracellular concentrations of AMP and adenosine via efflux pumps and enzymatic cleavage of phosphate bonds. This extracellular accumulation of AMP and adenosine is currently thought to be the effective mechanism of action for MTX in the treatment of rheumatoid arthritis. Legend: ADP, Adenosine diphosphate; ADA, Adenosine deaminase; AICAR, 5-aminoimidazole-4- carboxamide ribonucleotide; AICARiboside, 5-aminoimidazole-4-carboxamide ribonucleoside; AMP, Adenosine monophosphate; AMPDA, Adenosine monophosphate deaminase; ATP, Adenosine triphosphate; DHF, dihydrofolate; DHFR, dihydrofolate reductase; Ecto-5'NT, exto-5'-nucleotidase; FAICAR, 5-Formamidoimidazole-4-carboxamide ribotide; IMP, Inosine monophosphate; MTX, Methotrexate; NTPDase, Nucleotide triphosphate dephosphorylase

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Signaling from ATP/adenosine flux has multiple effects in both normal and pathologic states. In the presence of a normal metabolic state, increases in interstitial ATP concentrations have inflammatory effects on the immune system. Conversely, increased interstitial adenosine induces a significant anti-inflammatory effect with down-regulation of immune system activity. It is thought that tumor cells evade the immune response through this mechanism. (See Table 1)

In addition to metabolic dysfunction, the cancer

cell has a perturbed expression of purinergic signaling proteins. Cancer cells show an up-regulation in P1 receptors A1 and A3, both of which support cellular proliferation when activated. Likely due to induced down-regulation of the receptor, chronic therapies with A3 receptor agonists were reported to have an inhibitory effect on tumor cell survival and proliferation. Expression of A2A receptors in lung cancer cells was found to be similar to expression in normal lung tissue with the exception of a marked A2A increase in early stage cancer[69]. Stimulation of the A2A receptor was found to not only be a significant promoter of cancer cell survival, but also a powerful suppressor of tumor immuno-surveillance. Mice deficient in the A2A receptor have been observed to mount spontaneous anti-tumor immune responses inducing T-cell dependent tumor rejection[69].

While definitive literature is lacking, A2B

receptor expression varies in different malignant cells. Although the A2B receptor is consistently up-regulated in colon adenocarcinoma cells, one study of these cells found that expression of this receptor was further inducible by hypoxia [70]. The increased expression of these receptors offered the cells an increased survival and pro-angiogenic advantage. After eight hours of hypoxic incubation of these cells, antagonism of the A2B adenosine receptor led to significant dose-dependant inhibition of colonic carcinoma cell proliferation[70]. P2Y2 receptor agonism induces a pathway leading to cellular release of EGFR ligand, TGF-a, and transactivation of EGFR secondary to increased ROS production from mitochondria[69]. The P2X7 receptor is expressed in higher concentrations in many cancers and offers significant survival advantages in serum free environment with antagonism of the receptor on leukemic cells suppressing proliferation[69].

iii. Methotrexate and NAD(P)H-Dependent

Oxidoreductases

MTX is an inhibitor of dihydrofolate reductase and, more generally speaking, many NAD(P)H-dependent oxidoreductases. Unfortunately, its clinical use is associated with various toxicities. High doses of MTX are required to induce inhibition of the entire cellular pool of DHFR and are often associated with severe side effects [1]. Folinic acid, or leucovorin, has long been used to rescue cells from MTX toxicity. Beyond very high dose leucovorin rescues, few reports can be found of markedly reduced MTX efficacy with low dose leucovorin rescues or concurrent folate supplementation. This suggests methotrexate toxicity and pharmacokinetics in chemotherapy may be secondary to its effects on cellular abilities to buffer oxidative flux. Multiple studies report various “antioxidant” therapies using L-carnitine, lipoic acid, melatonin, and curcumin are effective in reducing MTX toxicity [71,9,72-74]. Quercetin has been shown to have multiple effects dependent on the cell. In studies of nontumor cells, quercetin has been shown to decrease the incidence of MTX-induced hepatotoxicity [71]. Folic acid is often taken in MTX treatment of rheumatoid arthritis and has been shown inhibit superoxide anion production via the NADPH oxidase enzyme [75].

Dihydrofolate reductase is an NADPH-dependent

oxidoreductase that oxidizes NADPH to NADP+ while reducing dihydrofolic acid to tetrahydrofolic acid. Reversing the reaction, this same enzyme can be referred to as tetrahydrofolate dehydrogenase, an NADP+-dependent enzyme. In addition to DHFR, MTX inhibits many NAD(P)-dehydrogenase enzymes; glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), pyruvate dehydrogenase,

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isocitrate dehydrogenase, alpha-ketoglutarate

dehydrogenase, and malate dehydrogenase

[9,76,3,4,1,74]. (See Figure 3) There is an additional

inhibitory effect on several enzymes more directly

involved with maintenance of the cellular redox state

via the regeneration of glutathione. Glutathione

Table 1. Effects of Purinergic Signalling on Immune Cell Function Increased Extracellular Purines

Action Receptor

B-Cells Adenosine stimulates increases in protein kinase A- shown to inhibit B-Cell receptor activation of NF-kB. Increased CD73 and adenosine pathway genes expression on memory B-Cells suggests a role in adenosine and in controlling re-stimulation.

A2A

T-Cells Adenosine stimulation inhibits t-cell receptor activation of NF-kB, cytotoxicity and cytokine production. This inhibited phenotype is present even after removal of A2A agonist. In the presence of A2A agonists, activated T-Cells fail to both proliferate and produce IFN-y. A2A activation induces CD4+ T-Cell differentiation toward Foxp3+ Treg Cells with increase production of TGF-B and IL-6. A2A activation also promotes generation of LAG-3+ Tregs

A2A

T-helper type 17 cells

ATP constitutively released by commensal gut bacteria activates these cells. Excess and prolonged exposure can exacerbate colitis and increases susceptibility to Crohn’s disease.

Foxp3+ T-Cells Recent studies have shown that ATP may be an activating molecule for Foxp3+ T-Cells with adenosine having a suppressing effect in vitro.

NK T-Cells NK T-Cells lacking A2A receptor produce less IL-4, IL-10, and TGF-B but more IFN-y. A2A

Neutrophils Interplay between the two receptors induced neutrophils to secrete ATP at their leading edge, possibly to promote macrophage migration.

A3/P2Y2

Adenosine inhibits degranulation, adhesion to endothelial cells, and superoxide production.

Monocytes/ Macrophages

Extrinsic ATP is a chemo-attractant and cause migrations towards the source of the gradient

P2Y2

Adenosine inhibits phagocytosis, superoxide production, nitric oxide production, TNF-a release, MHC-II expression, and IL-12 production.

NK Cells Adenosine inhibits IFN-y production secondary to decreased ability to increase intracellular cAMP levels.

A2A

Possible link between adenosine and enhanced NK function (Unknown if due to chronic exposure to A3 agonists and desensitization)

A3

Dendtritic Cells ATP is chemotactic and acutely promotes migration towards areas of high concentration while prolonged ATP exposure induces production of less inflammatory cytokines (IL-10 and IFN-Y.)

P2Y

Adenosine increases chemotaxis of immature DC’s but inhibits production of IL-12 when these cells are activated.

Adenosine induces DC differentiation toward cell lines with increase production of VEGF, IL-6, IL-8, IL-10, COX-2, TGF-B and indoleamine-2,3-dioxygenase.

A2B

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reductase, gamma-glutamylcysteine synthetase,

glutathione peroxidase and lactate dehydrogenase

are also inhibited on a concentration dependent

manner by MTX with no affect from co-administered

leucovorin [1].

These NAD-dependent dehydrogenase enzymes have conserved super-secondary structure with varying subunits [77]. While all are related in regards to this structure, differences lie in individual enzyme subunits. These subunits may be responsible for the

variable responses to methotrexate and its polyglutamates. (See Table 2) Function of the enzymes in this study was measured by the rate of NAD(P)H oxidation at increasing MTX concentrations. Direct comparison of MTX’s inhibitory effects on the activity of DHFR is difficult as studies using the same result measure could not be found. The serum concentrations of methotrexate seen in the table, however, are frequently reached in high dose methotrexate administrations. Both G6PD and 6PGD enzymes catalyze the reduction of NAD(P)+ to NAD(P)H in the pentose phosphate pathway for

Figure 3. An overview of carbon metabolism with enzymes affected by methotrexate. MTX can be seen inhibiting many enzymes involved in glucose metabolism. The enzymes inhibited in this figure are all dehydrogenase enzymes and involved in reduction of NAD+/NADP+ to NADH/NADPH. These reduced compounds are involved in multiple cellular reactions, the regeneration of reduced glutathione being one of them. Also seen in this figure is the incorporation of asparagine and glutamine as carbon sources for the production of TCA cycle intermediates. Legend: NAD+, nicotinamide adenine dinucleotide; NADP+, nicotinamide adenine dinucleotide phosphate; NADH, reduced nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; Star-shape indicates reaction of multiple reactants and products with various intermediates Dashed lines indicate a metabolite is present in two locations along the metabolic pathway. Bold lines indicate an irreversible reaction.

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eventual use in either the reduction of oxidized glutathione or the production of a free radicals via NADPH oxidase, cholesterol production,

steroidogenesis, etc. The remaining enzymes are largely involved in TCA cycle reactions resulting in the reduction of NAD+ to NADH within the mitochondria. In proliferating cells, the pentose phosphate pathway and actions of malic enzyme are currently considered the most significant source of cellular NAD(P)+ regeneration[1]. With the blockage of these key enzymes, the cellular ability to regenerate glutathione, and deal with oxidative stress, is inhibited. Highlighting the dependence of cells on

glutamine in dealing with oxidative stress, loss of glutaminase-2 production leads to decreased NAD(P)H production, decreased glutathione

production and sensitizes cancer cells to oxidative stresses like those induced by ionizing radiation [78]. Higher doses impose a greater oxidant buffering handicap, proving fatal for both normal and abnormal cells. Cellular depletion of NAD(P)H leading to cell death is also seen in the treatment of infectious diseases. Antimicrobial therapy of both gram negative and gram positive organisms with bactericidal, but not bacteriostatic, antibiotics leads to a transient depletion of NADH, increased oxidative stress and ultimately cell death [79].

VI. Conclusion

Genetic predispositions aside, the literature supports the postulate that genetic mutations in most cancers are secondary to severe metabolic and mitochondrial dysfunction leading to dysfunctional buffering of oxidant stresses. Better understanding of the complex interactions of cellular metabolism, mitochondrial function, and oxidative stress appears to have very widespread implications in the treatment of disease. Immediate application of this review falls on the current target of chemotherapeutics, but applications may exist for the treatment or prevention of many pathologies.

While exact environmental parameters

of Preterozoic oceans are of some debate, an increased level of electrochemical redox stress from dissolving land bound sulfide deposits prior to oceanic oxygenation is

understood. Acquisition of an organelle with the sole purpose buffering changes in redox stress would undoubtedly increase cellular fitness, expand the number of tolerable environments and allow for the evolution of more complex cellular processes. Coincidentally, the acquisition of these organelles, and subsequent stabilization of environmental redox status, is followed by a massive increase in biodiversity known as the Cambrian Explosion that marks the end of the Proterozoic Era.

Table 2: Inhibitory Effect of Methotrexate on NADPH-Dependent Oxidoreductases[1]

Enzyme [MTX] µM Activity*

2-Oxaloglutarate Dehydrogenase

15 50%

100 0%

Isocitrate Dehydrogenase

100 50%

500 0%

Malate Dehydrogenase

150 50%

400 0%

Succinate Dehydrogenase

500 80%

Glutathione Reductase

CFE-HeLa Cell Enzyme 80 50%

250 10%

Purified enzyme 110 50%

200 20%

6-Phosphogluconate Dehydrogenase

CFE-Hela Cell Enzyme 50 50%

200 10%

Purified Enzyme 100 50%

Glucose-6-Phosphate Dehydrogenase


250 10%


250 10%

Gamma-glutamylcysteine synthetase

100 46%

Glutathione Peroxidase

100 50%

Values in this table were obtained directly from the text or via interpretation of figures in the reference.

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Current understandings describe the primary

mitochondrial role as an oxygen dependent power plant with the primary role of ATP production via oxidative phosphorylation. Recent research into mitochondrial origins and the environmental redox status of early eukaryotic cells may further expose early roles of the ancestral organelle. Investigations of mitosomes and hydrogenosomes reveal organelles very similar to mitochondria, with reduced or non-existent ATP-producing functions. Interestingly, all three organelles have in common the ability to create and store reduced molecules for later use by the cell. Some research suggests that molecular hydrogen, one product of hydrogenosomes, may serve as an effective antioxidant and has been shown to decrease area of infarction in rat brains affected by ischemia-reperfusion injuries [80]. The mitochondrial currency of stored reduced molecules, NAD(P)H, and its ratio to NAD+ has been shown to have intimate interactions in the growth and metastasis of tumors. Excessive concentrations of NAD(P)H in relation to NAD+ have been shown to have a positive effect on the growth and metastasis of breast cancers[81].

Many cancers originate in hypoxic

microenvironments. Detailed studies have documented how characteristics of the tumor microenvironment can lead to significant changes in tumor phenotypes. Survival of these cells appears to be dependent upon mitochondrial abilities to regenerate free radical scavengers to buffer oxidant stress. Experimental studies have shown a dependence on mitochondrial and cytolosolic properties to perpetuate tumors. By placing normal and tumor nuclei in tumor and normal cells respectively, normal daughter cells were produced upon division of the tumor nuclei and normal cell cytoplasm [82]. In the second population of cells, with normal nuclei and tumor cytoplasmic contents, division resulted in the production of more tumor cells or cell death [82].

The resultant metabolism of these cells becomes

highly dependent on the catabolism of amino acids, particularly glutamine, to preserve the cell via regeneration of free radical scavengers. Although it The Warburg Effect seemingly covers only part of the metabolic derangements seen in cancer. The increase in glycolysis and lactate production may serve as a depository for nitrogen freed from transamination reactions needed to fuel enzyme catalyzed reduction

of NAD(P)+. Methotrexate interferes with many enzymes involved in reduction of NAD(P)+ to NAD(P)H and, more significantly, cellular abilities to buffer oxidative stress. This may be a clue to the paradox seen in Down syndrome with increased risk of leukemia yet an increased sensitivity to chemotherapy.

Interference with cellular abilities to maintain

supply of glutamine, glutamate and glutathione is detrimental, and often fatal, to the cell. Glutaminase 2, a phosphate-activated mitochondrial enzyme, catalyzes the hydrolysis of glutamine to glutamate. Loss of this enzyme results in an effect similar to methotrexate therapy; decreased NADH and glutathione with increased sensitivity to oxidant stress. Combination chemotherapy with metabolically active agents, i.e asparaginase, effectively starves the cancer cell of two sources of fuel used to maintain functional output of the TCA cycle.

The importance and function of the

mitochondria and its changes during the cell cycle also offer insight into peculiarities of other chemotherapeutic agents. In regards to the antimitotic chemotherapeutics, such as paclitaxel, a proliferation rate paradox is described by these agents selectively targeting of cells with a high rates of division yet these same agents will induce regression in tumors with low rates of proliferation [83]. Beyond cell division, microtubules are highly utilized in the intracellular localization of mitochondria. Agents interfering with the proper function of microtubules likely interfere with the purposeful migration and localization of mitochondria throughout the cell cycle.

The early mitochondrial organelle seemingly led

to increased cellular fitness due to its ability to buffer redox stresses secondary to changes in the cells’ immediate environment. This buffering capabilities may have increase cellular hardiness allowing survival during environmental changes. Although part of a whole, individual cells throughout the body are subject to changes and stresses in their own microenvironments. Cellular metabolites serve as intermediate signals of the extracellular environment to affect genetic expression. New understandings on the pharmacologic mechanisms of methotrexate may suggest cellular redox buffering, metabolism and mitochondrial function is a more susceptible target in

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cancer therapy than genomic attack. Better understanding metabolic pathways and the effects of other anti-cancer agents on them may offers significant insight into paradoxical effects seen with many chemotherapeutic drugs.

For millennia, living cells have evolved

mechanisms to protect its genome, and function, at all costs. Despite overwhelming stress, severe starvation or massive free radical attack, the cell persists. At the cost of growth and detriment to its host, the cancer cell persists. When its metabolism has "damaged" its cellular blueprints enough to thrive in its new environment, the cancer cell proliferates, escapes, settles elsewhere in its new world and, again, persists.

Acknowledgements:

Special thanks to Dr. Mark Mogul, Dr. Darshana Shah, Dr. Gary Rankin and Dr. Pier Paulo Cluadio for their feedback and encouragement on this project, as well as Dr. Richard Niles and Dr. Lynne Goebel for their feedback, proofreading and editing of the manuscript.

REFERENCES

1. Babiak RM, Campello AP, Carnieri EG, Oliveira MB (1998) Methotrexate: pentose cycle and oxidative stress. Cell biochemistry and function 16 (4):283-293. doi:10.1002/(SICI)1099-0844(1998120)16:4<283::AID-CBF801>3.0.CO;2-E 2. Sirotnak FM, Donsbach RC (1974) The intracellular concentration dependence of antifolate inhibition of DNA synthesis in L1210 leukemia cells. Cancer research 34 (12):332-340 3. Akkemik E, Budak H, Ciftci M (2010) Effects of some drugs on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro study. Journal of enzyme inhibition and medicinal chemistry 25 (4):476-479. doi:10.3109/14756360903257900 4. Akkemik E, Budak H, Ciftci M (2010) Effects of some drugs on human erythrocyte glucose 6-phosphate dehydrogenase: an in vitro study. Journal of enzyme inhibition and medicinal chemistry 25 (6):871-875. doi:10.3109/14756360903489581 5. Warburg O (1956) On the origin of cancer cells. Science 123 (3191):309-314 6. Brand KA, Hermfisse U (1997) Aerobic glycolysis by proliferating cells: a protective strategy against reactive oxygen species. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 11 (5):388-395 7. Cardaci S, Ciriolo MR (2012) TCA Cycle Defects and Cancer: When Metabolism Tunes Redox State. International journal of cell biology 2012:161837. doi:10.1155/2012/161837 8. Guppy M, Leedman P, Zu X, Russell V (2002) Contribution by different fuels and metabolic pathways to the total ATP turnover of proliferating MCF-7 breast cancer cells. The Biochemical journal 364 (Pt 1):309-315 9. Uzar E, Koyuncuoglu HR, Uz E, Yilmaz HR, Kutluhan S, Kilbas S, Gultekin F (2006) The activities of antioxidant enzymes and the level of malondialdehyde in cerebellum of rats subjected to methotrexate: protective effect of caffeic acid phenethyl ester. Molecular and cellular biochemistry 291 (1-2):63-68. doi:10.1007/s11010-006-9196-5

10. Sullivan SG, Stern A (1984) Glucose metabolism of oxidatively stressed human red blood cells incubated in plasma or medium containing physiologic concentrations of lactate, pyruvate and ascorbate. Biochemical pharmacology 33 (9):1417-1421 11. Medina MA (2001) Glutamine and cancer. The Journal of nutrition 131 (9 Suppl):2539S-2542S; discussion 2550S-2531S 12. Pelicano H, Martin DS, Xu RH, Huang P (2006) Glycolysis inhibition for anticancer treatment. Oncogene 25 (34):4633-4646. doi:10.1038/sj.onc.1209597 13. MacKenzie ED, Selak MA, Tennant DA, Payne LJ, Crosby S, Frederiksen CM, Watson DG, Gottlieb E (2007) Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells. Molecular and cellular biology 27 (9):3282-3289. doi:10.1128/MCB.01927-06 14. Snell CE, Turley H, McIntyre A, Li D, Masiero M, Schofield CJ, Gatter KC, Harris AL, Pezzella F (2014) Proline-Hydroxylated Hypoxia-Inducible Factor 1alpha (HIF-1alpha) Upregulation in Human Tumours. PloS one 9 (2):e88955. doi:10.1371/journal.pone.0088955 15. Liu W, Shen SM, Zhao XY, Chen GQ (2012) Targeted genes and interacting proteins of hypoxia inducible factor-1. International journal of biochemistry and molecular biology 3 (2):165-178 16. Frezza C, Zheng L, Tennant DA, Papkovsky DB, Hedley BA, Kalna G, Watson DG, Gottlieb E (2011) Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival. PloS one 6 (9):e24411. doi:10.1371/journal.pone.0024411 17. Sookoian S, Pirola CJ (2012) Alanine and aspartate aminotransferase and glutamine-cycling pathway: their roles in pathogenesis of metabolic syndrome. World journal of gastroenterology : WJG 18 (29):3775-3781. doi:10.3748/wjg.v18.i29.3775 18. Anastasiou D, Cantley LC (2012) Breathless cancer cells get fat on glutamine. Cell research 22 (3):443-446. doi:10.1038/cr.2012.5 19. Mohamed A, Deng X, Khuri FR, Owonikoko TK (2014) Altered glutamine metabolism and therapeutic opportunities for lung cancer. Clinical lung cancer 15 (1):7-15. doi:10.1016/j.cllc.2013.09.001 20. Des Rosiers C, Di Donato L, Comte B, Laplante A, Marcoux C, David F, Fernandez CA, Brunengraber H (1995) Isotopomer analysis of citric acid cycle and gluconeogenesis in rat liver. Reversibility of isocitrate dehydrogenase and involvement of ATP-citrate lyase in gluconeogenesis. The Journal of biological chemistry 270 (17):10027-10036 21. Le A, Lane AN, Hamaker M, Bose S, Gouw A, Barbi J, Tsukamoto T, Rojas CJ, Slusher BS, Zhang H, Zimmerman LJ, Liebler DC, Slebos RJ, Lorkiewicz PK, Higashi RM, Fan TW, Dang CV (2012) Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell metabolism 15 (1):110-121. doi:10.1016/j.cmet.2011.12.009 22. Mentel M, Martin W (2008) Energy metabolism among eukaryotic anaerobes in light of Proterozoic ocean chemistry. Philosophical transactions of the Royal Society of London Series B, Biological sciences 363 (1504):2717-2729. doi:10.1098/rstb.2008.0031 23. Murphy MP (2012) Modulating mitochondrial intracellular location as a redox signal. Science signaling 5 (242):pe39. doi:10.1126/scisignal.2003386 24. Al-Mehdi AB, Pastukh VM, Swiger BM, Reed DJ, Patel MR, Bardwell GC, Pastukh VV, Alexeyev MF, Gillespie MN (2012) Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription. Science signaling 5 (231):ra47. doi:10.1126/scisignal.2002712 25. Berridge Michael J (2012) Cell Signalling Biology: Module 2 - Cell signalling pathways. Biochemical Journal. doi:10.1042/csb0001002

Hess


26. Krasnov GS, Dmitriev AA, Snezhkina AV, Kudryavtseva AV (2013) Deregulation of glycolysis in cancer: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a therapeutic target. Expert opinion on therapeutic targets. doi:10.1517/14728222.2013.775253 27. Toyokuni S (1998) Oxidative stress and cancer: the role of redox regulation. Biotherapy 11 (2-3):147-154 28. Campos C, Guzman R, Lopez-Fernandez E, Casado A (2011) Evaluation of urinary biomarkers of oxidative/nitrosative stress in children with Down syndrome. Life sciences 89 (17-18):655-661. doi:10.1016/j.lfs.2011.08.006 29. Pallardo FV, Lloret A, Lebel M, d'Ischia M, Cogger VC, Le Couteur DG, Gadaleta MN, Castello G, Pagano G (2010) Mitochondrial dysfunction in some oxidative stress-related genetic diseases: Ataxia-Telangiectasia, Down Syndrome, Fanconi Anaemia and Werner Syndrome. Biogerontology 11 (4):401-419. doi:10.1007/s10522-010-9269-4 30. Sulthana SM, Kumar SN, Sridhar MG, Bhat BV, Rao KR (2012) Levels of non enzymatic antioxidants in Down syndrome. Indian journal of pediatrics 79 (11):1473-1476. doi:10.1007/s12098-012-0795-8 31. Yin H, Zhou Y, Zhu M, Hou S, Li Z, Zhong H, Lu J, Meng T, Wang J, Xia L, Xu Y, Wu Y (2012) Role of mitochondria in programmed cell death mediated by arachidonic acid-derived eicosanoids. Mitochondrion. doi:10.1016/j.mito.2012.10.003 32. Strydom A, Dickinson MJ, Shende S, Pratico D, Walker Z (2009) Oxidative stress and cognitive ability in adults with Down syndrome. Progress in neuro-psychopharmacology & biological psychiatry 33 (1):76-80. doi:10.1016/j.pnpbp.2008.10.006 33. Karamouzis I, Pervanidou P, Berardelli R, Iliadis S, Papassotiriou I, Karamouzis M, Chrousos GP, Kanaka-Gantenbein C (2011) Enhanced oxidative stress and platelet activation combined with reduced antioxidant capacity in obese prepubertal and adolescent girls with full or partial metabolic syndrome. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme 43 (9):607-613. doi:10.1055/s-0031-1284355 34. Danielson PB (2002) The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Current drug metabolism 3 (6):561-597 35. Ingelman-Sundberg M (2004) Human drug metabolising cytochrome P450 enzymes: properties and polymorphisms. Naunyn-Schmiedeberg's archives of pharmacology 369 (1):89-104. doi:10.1007/s00210-003-0819-z 36. Hanukoglu I (2006) Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. Drug metabolism reviews 38 (1-2):171-196. doi:10.1080/03602530600570040 37. Shimada T, Hayes CL, Yamazaki H, Amin S, Hecht SS, Guengerich FP, Sutter TR (1996) Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer research 56 (13):2979-2984 38. Cronstein BN (2005) Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis. Pharmacological reviews 57 (2):163-172. doi:10.1124/pr.57.2.3 39. Maddocks OD, Berkers CR, Mason SM, Zheng L, Blyth K, Gottlieb E, Vousden KH (2013) Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells. Nature 493 (7433):542-546. doi:10.1038/nature11743 40. Ecker N, Mor A, Journo D, Abeliovich H (2010) Induction of autophagic flux by amino acid deprivation is distinct from nitrogen starvation-induced macroautophagy. Autophagy 6 (7):879-890. doi:10.4161/auto.6.7.12753

41. Eng CH, Yu K, Lucas J, White E, Abraham RT (2010) Ammonia derived from glutaminolysis is a diffusible regulator of autophagy. Science signaling 3 (119):ra31. doi:10.1126/scisignal.2000911 42. Deffieu M, Bhatia-Kissova I, Salin B, Galinier A, Manon S, Camougrand N (2009) Glutathione participates in the regulation of mitophagy in yeast. The Journal of biological chemistry 284 (22):14828-14837. doi:10.1074/jbc.M109.005181 43. Xi H, Barredo JC, Merchan JR, Lampidis TJ (2013) Endoplasmic reticulum stress induced by 2-deoxyglucose but not glucose starvation activates AMPK through CaMKKbeta leading to autophagy. Biochemical pharmacology. doi:10.1016/j.bcp.2013.02.037 44. Wang Q, Liang B, Shirwany NA, Zou MH (2011) 2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase. PloS one 6 (2):e17234. doi:10.1371/journal.pone.0017234 45. Karbowski M, Spodnik JH, Teranishi M, Wozniak M, Nishizawa Y, Usukura J, Wakabayashi T (2001) Opposite effects of microtubule-stabilizing and microtubule-destabilizing drugs on biogenesis of mitochondria in mammalian cells. Journal of cell science 114 (Pt 2):281-291 46. Sweet S, Singh G (1999) Changes in mitochondrial mass, membrane potential, and cellular adenosine triphosphate content during the cell cycle of human leukemic (HL-60) cells. Journal of cellular physiology 180 (1):91-96. doi:10.1002/(SICI)1097-4652(199907)180:1<91::AID-JCP10>3.0.CO;2-6 47. Colombo SL, Palacios-Callender M, Frakich N, Carcamo S, Kovacs I, Tudzarova S, Moncada S (2011) Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells. Proceedings of the National Academy of Sciences of the United States of America 108 (52):21069-21074. doi:10.1073/pnas.1117500108 48. Menon SG, Sarsour EH, Spitz DR, Higashikubo R, Sturm M, Zhang H, Goswami PC (2003) Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle. Cancer research 63 (9):2109-2117 49. Zhang C, Lin M, Wu R, Wang X, Yang B, Levine AJ, Hu W, Feng Z (2011) Parkin, a p53 target gene, mediates the role of p53 in glucose metabolism and the Warburg effect. Proceedings of the National Academy of Sciences of the United States of America 108 (39):16259-16264. doi:10.1073/pnas.1113884108 50. Zhang XD, Qin ZH, Wang J (2010) The role of p53 in cell metabolism. Acta pharmacologica Sinica 31 (9):1208-1212. doi:10.1038/aps.2010.151 51. Yamada E, Singh R (2012) Mapping autophagy on to your metabolic radar. Diabetes 61 (2):272-280. doi:10.2337/db11-1199 52. Cimmino A, Capasso R, Muller F, Sambri I, Masella L, Raimo M, De Bonis ML, D'Angelo S, Zappia V, Galletti P, Ingrosso D (2008) Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation. PloS one 3 (9):e3258. doi:10.1371/journal.pone.0003258 53. Dang CV (2010) Rethinking the Warburg effect with Myc micromanaging glutamine metabolism. Cancer research 70 (3):859-862. doi:10.1158/0008-5472.CAN-09-3556 54. Cook SA, Sugden PH, Clerk A (1999) Regulation of Bcl-2 Family Proteins During Development and in Response to Oxidative Stress in Cardiac Myocytes : Association With Changes in Mitochondrial Membrane Potential. Circulation research 85 (10):940-949. doi:10.1161/01.res.85.10.940 55. Zirath H, Frenzel A, Oliynyk G, Segerstrom L, Westermark UK, Larsson K, Munksgaard Persson M, Hultenby K, Lehtio J, Einvik C, Pahlman S, Kogner P, Jakobsson PJ, Henriksson MA (2013) MYC inhibition induces metabolic changes leading to accumulation of

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lipid droplets in tumor cells. Proceedings of the National Academy of Sciences of the United States of America 110 (25):10258-10263. doi:10.1073/pnas.1222404110 56. Pieczenik SR, Neustadt J (2007) Mitochondrial dysfunction and molecular pathways of disease. Experimental and molecular pathology 83 (1):84-92. doi:10.1016/j.yexmp.2006.09.008 57. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress (1997). 58. Chabner BA, Allegra CJ, Curt GA, Clendeninn NJ, Baram J, Koizumi S, Drake JC, Jolivet J (1985) Polyglutamation of methotrexate. Is methotrexate a prodrug? The Journal of clinical investigation 76 (3):907-912. doi:10.1172/JCI112088 59. Weigand M, Frei E, Graf N, Wiessler M (2000) Comparative analysis of methotrexate polyglutamates in lymphoblast preparations from bone marrow and blood, and the contribution of residual red blood cells. Journal of cancer research and clinical oncology 126 (7):407-411 60. Synold TW, Relling MV, Boyett JM, Rivera GK, Sandlund JT, Mahmoud H, Crist WM, Pui CH, Evans WE (1994) Blast cell methotrexate-polyglutamate accumulation in vivo differs by lineage, ploidy, and methotrexate dose in acute lymphoblastic leukemia. The Journal of clinical investigation 94 (5):1996-2001. doi:10.1172/JCI117552 61. Rubio IT, Cao Y, Hutchins LF, Westbrook KC, Klimberg VS (1998) Effect of glutamine on methotrexate efficacy and toxicity. Annals of surgery 227 (5):772-778; discussion 778-780 62. Sur P, Fernandes DJ, Kute TE, Capizzi RL (1987) L-asparaginase-induced modulation of methotrexate polyglutamylation in murine leukemia L5178Y. Cancer research 47 (5):1313-1318 63. Panosyan EH, Grigoryan RS, Avramis IA, Seibel NL, Gaynon PS, Siegel SE, Fingert HJ, Avramis VI (2004) Deamination of glutamine is a prerequisite for optimal asparagine deamination by asparaginases in vivo (CCG-1961). Anticancer research 24 (2C):1121-1125 64. Gong SS, Guerrini L, Basilico C (1991) Regulation of asparagine synthetase gene expression by amino acid starvation. Molecular and cellular biology 11 (12):6059-6066 65. Avramis VI (2012) Asparaginases: biochemical pharmacology and modes of drug resistance. Anticancer research 32 (7):2423-2437 66. Rotoli BM, Uggeri J, Dall'Asta V, Visigalli R, Barilli A, Gatti R, Orlandini G, Gazzola GC, Bussolati O (2005) Inhibition of glutamine synthetase triggers apoptosis in asparaginase-resistant cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 15 (6):281-292. doi:10.1159/000087238 67. Stamp LK, Hazlett J, Roberts RL, Frampton C, Highton J, Hessian PA (2012) Adenosine receptor expression in rheumatoid synovium: a basis for methotrexate action. Arthritis research & therapy 14 (3):R138. doi:10.1186/ar3871 68. Hasko G, Cronstein B (2013) Regulation of inflammation by adenosine. Frontiers in immunology 4:85. doi:10.3389/fimmu.2013.00085 69. Stagg J, Smyth MJ (2010) Extracellular adenosine triphosphate and adenosine in cancer. Oncogene 29 (39):5346-5358. doi:10.1038/onc.2010.292 70. Ma DF, Kondo T, Nakazawa T, Niu DF, Mochizuki K, Kawasaki T, Yamane T, Katoh R (2010) Hypoxia-inducible adenosine A2B receptor modulates proliferation of colon carcinoma cells. Human pathology 41 (11):1550-1557. doi:10.1016/j.humpath.2010.04.008

71. Aydin B (2011) Quercetin Prevents Methotrexate-induced Hepatotoxicity without Interfering with Methotrexate Metabolizing Enzymes in Liver of Mice. Journal of applied biological sciences 5 (2):75-80 72. Sener G, Eksioglu-Demiralp E, Cetiner M, Ercan F, Sirvanci S, Gedik N, Yegen BC (2006) L-Carnitine ameliorates methotrexate-induced oxidative organ injury and inhibits leukocyte death. Cell biology and toxicology 22 (1):47-60. doi:10.1007/s10565-006-0025-0 73. Abraham P, Kolli VK, Rabi S (2010) Melatonin attenuates methotrexate-induced oxidative stress and renal damage in rats. Cell biochemistry and function 28 (5):426-433. doi:10.1002/cbf.1676 74. Tabassum H, Parvez S, Pasha ST, Banerjee BD, Raisuddin S (2010) Protective effect of lipoic acid against methotrexate-induced oxidative stress in liver mitochondria. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 48 (7):1973-1979. doi:10.1016/j.fct.2010.04.047 75. Hwang SY, Siow YL, Au-Yeung KK, House J, O K (2011) Folic acid supplementation inhibits NADPH oxidase-mediated superoxide anion production in the kidney. American journal of physiology Renal physiology 300 (1):F189-198. doi:10.1152/ajprenal.00272.2010 76. Caetano NN, Campello AP, Carnieri EG, Kluppel ML, Oliveira MB (1997) Effect of methotrexate (MTX) on NAD(P)+ dehydrogenases of HeLa cells: malic enzyme, 2-oxoglutarate and isocitrate dehydrogenases. Cell biochemistry and function 15 (4):259-264. doi:10.1002/(SICI)1099-0844(199712)15:4<259::AID-CBF749>3.0.CO;2-D 77. Kutzenko AS, Lamzin VS, Popov VO (1998) Conserved supersecondary structural motif in NAD-dependent dehydrogenases. FEBS letters 423 (1):105-109 78. Xiang L, Xie G, Liu C, Zhou J, Chen J, Yu S, Li J, Pang X, Shi H, Liang H (2013) Knock-down of glutaminase 2 expression decreases glutathione, NADH, and sensitizes cervical cancer to ionizing radiation. Biochimica et biophysica acta 1833 (12):2996-3005. doi:10.1016/j.bbamcr.2013.08.003 79. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ (2007) A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130 (5):797-810. doi:10.1016/j.cell.2007.06.049 80. Ohta S (2012) Molecular hydrogen is a novel antioxidant to efficiently reduce oxidative stress with potential for the improvement of mitochondrial diseases. Biochimica et biophysica acta 1820 (5):586-594. doi:10.1016/j.bbagen.2011.05.006 81. Santidrian AF, Matsuno-Yagi A, Ritland M, Seo BB, LeBoeuf SE, Gay LJ, Yagi T, Felding-Habermann B (2013) Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression. The Journal of clinical investigation 123 (3):1068-1081. doi:10.1172/JCI64264 82. Seyfried TN, Flores R, Poff AM, D'Agostino DP (2013) Cancer as a Metabolic Disease: Implications for Novel Therapeutics. Carcinogenesis. doi:10.1093/carcin/bgt480 83. Mitchison TJ (2012) The proliferation rate paradox in antimitotic chemotherapy. Molecular biology of the cell 23 (1):1-6. doi:10.1091/mbc.E10-04-0335

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Figure Legends

Figure 1. Effects of methotrexate on folate metabolism in relation to nucleotide metabolism. Via its inhibition of dihydrofolate reductase, methotrexate interferes with the generation of tetrahydrofolate and subsequently, DNA synthesis. Tetrahydrofolate is metabolized further for use as a cofactor in the synthesis of dTMP. Folinic acid, leuvovorin, is often used to rescue cells from methotrexate and restore cellular stores of tetrahydrofolate. In addition to interference with DNA synthesis, this diagram superficially shows the involvement of tetrahydrofolate in the metabolism of purines. Legend: ADP, adenosine diphosphate; AMP, adenosine monophosphate; CDP, cytidine diphosphate; CTP, cytidine triphosphate; dCDP, deoxycytidine diphosphate; DHFR, dihydrofolate reductase; dTMP, deoxythimidine monophosphate; dUDP, deoxyuridine diphosphate; dUMP, deoxyuridine monophosphate; GDP, guanine diphosphate; GMP, guanine monophosphate; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; IMP, inosine monophosphate; Methyl THF, methyltetrahydrofolate; MTX, methotrexate; PNP, purine nucleotide phosphorylase; PRPP, phosphoribosyl pyrophosphate; UDP, uracil diphosphate; UMP, uracil monophosphate; UTP, uracil triphosphate; XMP, xanthine monophosphate Figure 2. The effects of methotrexate on pathways of adenosine metabolism. By inhibiting the regeneration of THF, MTX inhibits the enzyme AICAR transformylase. A subsequent build-up of this enzyme’s substrates lead to an accumulation of AMP and adenosine in the cell via inhibition of AMPDA and ADA. This intracellular accumulation leads to an increase in extracellular concentrations of AMP and adenosine via efflux pumps and enzymatic cleavage of phosphate bonds. This extracellular accumulation of AMP and adenosine is currently thought to be the effective mechanism of action for MTX in the treatment of rheumatoid arthritis. Legend: ADP, Adenosine diphosphate; ADA, Adenosine deaminase; AICAR, 5-aminoimidazole-4- carboxamide ribonucleotide; AICARiboside, 5-aminoimidazole-4-carboxamide ribonucleoside; AMP, Adenosine monophosphate; AMPDA, Adenosine monophosphate deaminase; ATP, Adenosine triphosphate; DHF, dihydrofolate; DHFR, dihydrofolate reductase; Ecto-5'NT, exto-5'-nucleotidase; FAICAR, 5-Formamidoimidazole-4-carboxamide ribotide; IMP, Inosine monophosphate; MTX, Methotrexate; NTPDase, Nucleotide triphosphate dephosphorylase Figure 3. An overview of carbon metabolism with enzymes affected by methotrexate. MTX can be seen inhibiting many enzymes involved in glucose metabolism. The enzymes inhibited in this figure are all dehydrogenase enzymes and involved in reduction of NAD+/NADP+ to NADH/NADPH. These reduced compounds are involved in multiple cellular reactions, the regeneration of reduced glutathione being one of them. Also seen in this figure is the incorporation of asparagine and glutamine as carbon sources for the production of TCA cycle intermediates. Legend: NAD+, nicotinamide adenine dinucleotide; NADP+, nicotinamide adenine dinucleotide phosphate; NADH, reduced nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; Star-shape indicates reaction of multiple reactants and products with various intermediates Dashed lines indicate a metabolite is present in two locations along the metabolic pathway. Bold lines indicate an irreversible reaction.

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TABLE 1. Effects of Purinergic Signalling on Immune Cell Function Increased Extracellular Purines

Action Receptor

B-Cells Adenosine stimulates increases in protein kinase A- shown to inhibit B-Cell receptor activation of NF-kB. Increased CD73 and adenosine pathway genes expression on memory B-Cells suggests a role in adenosine and in controlling re-stimulation.

A2A

T-Cells Adenosine stimulation inhibits t-cell receptor activation of NF-kB, cytotoxicity and cytokine production. This inhibited phenotype is present even after removal of A2A agonist. In the presence of A2A agonists, activated T-Cells fail to both proliferate and produce IFN-y. A2A activation induces CD4+ T-Cell differentiation toward Foxp3+ Treg Cells with increase production of TGF-B and IL-6. A2A activation also promotes generation of LAG-3+ Tregs

A2A

T-helper type 17 cells

ATP constitutively released by commensal gut bacteria activates these cells. Excess and prolonged exposure can exacerbate colitis and increases susceptibility to Crohn’s disease.

Foxp3+ T-Cells Recent studies have shown that ATP may be an activating molecule for Foxp3+ T-Cells with adenosine having a suppressing effect in vitro.

NK T-Cells NK T-Cells lacking A2A receptor produce less IL-4, IL-10, and TGF-B but more IFN-y.

A2A

Neutrophils Interplay between the two receptors induced neutrophils to secrete ATP at their leading edge, possibly to promote macrophage migration.

A3/P2Y2

Adenosine inhibits degranulation, adhesion to endothelial cells, and superoxide production.

Monocytes/ Macrophages

Extrinsic ATP is a chemo-attractant and cause migrations towards the source of the gradient

P2Y2

Adenosine inhibits phagocytosis, superoxide production, nitric oxide production, TNF-a release, MHC-II expression, and IL-12 production.

NK Cells Adenosine inhibits IFN-y production secondary to decreased ability to increase intracellular cAMP levels.

A2A

Possible link between adenosine and enhanced NK function (Unknown if due to chronic exposure to A3 agonists and desensitization)

A3

Dendtritic Cells ATP is chemotactic and acutely promotes migration towards areas of high concentration while prolonged ATP exposure induces production of less inflammatory cytokines (IL-10 and IFN-Y.)

P2Y

Adenosine increases chemotaxis of immature DC’s but inhibits production of IL-12 when these cells are activated.

Adenosine induces DC differentiation toward cell lines with increase production of VEGF, IL-6, IL-8, IL-10, COX-2, TGF-B and indoleamine-2,3-dioxygenase.

A2B

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Table 2. Inhibitory Effect of Methotrexate on NADPH-Dependent Oxidoreductases[1]

Values in this table were obtained directly from the text or via interpretation of figures in the reference.

Enzyme [MTX] µM Activity*

2-Oxaloglutarate Dehydrogenase

15 50%

100 0%

Isocitrate Dehydrogenase

100 50%

500 0%

Malate Dehydrogenase

150 50%

400 0%

Succinate Dehydrogenase

500 80%

Glutathione Reductase

CFE-HeLa Cell Enzyme 80 50%

250 10%

Purified enzyme 110 50%

200 20%

6-Phosphogluconate Dehydrogenase


200 10%


Glucose-6-Phosphate Dehydrogenase


250 10%


250 10%

Gamma-glutamylcysteine synthetase

100 46%

Glutathione Peroxidase

100 50%

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Figure 1

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Figure 2

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Figure 3

Cancer Metabolism, Methotrexate and Oxidative Stress · to its antimetabolite properties....

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