Glycolytic enzyme inhibitors in cancer treatment

Review

10.1517/13543780802411053 © 2008 Informa UK Ltd ISSN 1354-3784 1533All rights reserved: reproduction in whole or in part not permitted

Oncologic

Glycolytic enzyme inhibitors in cancer treatment Roberto Scatena † , Patrizia Bottoni , Alessandro Pontoglio , Lucia Mastrototaro & Bruno Giardina Catholic University, Department of Laboratory Medicine, Rome, Italy

Background : The radio- and chemotherapeutics currently used for the treatment of cancer are widely known to be characterized by a low therapeutic index. An interesting approach to overcoming some of the limits of these techniques is the exploitation of the so-called Warburg effect, which typically characterizes neoplastic cells. Interestingly, this feature has already been utilized with good results, but only for diagnostic purposes (PET and SPECT). From a pharmacological point of view, drugs able to perturb cancer cell metabolism, specifically at the level of glycolysis, may display interesting therapeutic activities in cancer. Objective : The pharmacological actions of these glycolytic enzyme inhibitors, based primarily on ATP depletion, could include: i) amelioration of drug selectivity by exploiting the particular glycolysis addiction of cancer cell; ii) inhibition of energetic and anabolic processes; iii) reduction of hypoxia-linked cancer-cell resistance; iv) reduction of ATP-dependent multi-drug resistance; and v) cytotoxic synergism with conventional cancer treatments. Conclusion : Several glycolytic inhibitors are currently in preclinical and clinical development. Their clinical value as anticancer agents, above all in terms of therapeutic index, strictly depends on a careful reevaluation of the pathophyiological role of the unique metabolism of cancer cells in general and of Warburg effect in particular.

Keywords: cancer stem cell , cancer therapeutics , cancer-cell metabolism , chemotherapy , glycolysis , mitochondria , Warburg effect

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1. Introduction

Current chemotherapy permits curative intervention in patients with several types of solid tumor and hematologic neoplasms. These results are further ameliorated by integration of pharmacotherapy with surgery and radiation therapy with positive results [1,2] . However, some important obstacles are typically encountered in the use of chemotherapy, such as its intrinsic toxicity towards normal tissues/organs, and the resistance of cancer cells towards chemotherapeutic agents. In addition to these serious drawbacks, a significant percentage of patients with cancer still develop metastatic disease during their lifetime, despite local control of their neoplasia. Hence, for most patients, cancer has to be considered as a systemic and evolutionary disease, requiring progressively more aggressive systemic treatment. For this purpose, the typical narrow therapeutic index of chemotherapy and radiotherapy (due mainly to intrinsic low target selectivity) can seriously jeopardize the health status of the cancer patient, exacerbating the evolution of a cancer cachexia [1,2] .

Cancer-cell target selectivity is considered to be one of the main aims of oncopharmacology. Current molecular oncology studies are significantly expanding the knowledge of cellular mechanisms at the basis of transformation of a normal cell into a tumor cell. Specifically, many researchers have worked hard to elucidate

1. Introduction

2. Glycolysis as target for

anticancer treatment

3. Conclusions

4. Expert opinion

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the role of various oncogenes and tumor-suppressor genes. Mainly on the basis of these studies, the inactivation of tumor-suppressor genes by mutation, deletion, or whole-chromosome loss, and/or activation of oncogenes by chromosomal translocation or amplification of point mutation, have both been related to disease progression, poor prognosis, and/or resistance to therapy. These findings have contributed to the development of pharmacologically active compounds capable of interfering with specific molecules participating in signal transduction pathways involved in carcinogenesis and/or cancer progression. Moreover, drugs interfering with different stromal factors and angiogenesis mechanisms in cancer have become available [1-4] .

This new level of understanding of the molecular pathways through which chemotherapy works, and by which genetic change can result in resistance to drug therapy, has facilitated the implementation of new therapeutic strategies. Molecular, genetic, and biologic therapies can be used in combination to directly attack new and specific targets, increasing the chemosensitivity of malignant cells to treatment and protecting the normal tissues of the body from therapy-induced side effects [3,4] .

Furthermore, a new approach to therapeutic strategy is emerging, based on the peculiar metabolism of the cancer cell. Specifically, glycolysis has long been considered the main source of energy for the cancer cell [5] . This particular metabolic status was defined as the ‘Warburg effect’ by Otto Warburg, winner of the Nobel Prize in Medicine in 1931 [6] . Cancer cells tend to produce ATP mainly by ‘aerobic glycolysis,’ a metabolic shift characterized by high glucose uptake and increased production of lactate. Originally, this different metabolic state was considered to be the result of adaptation of some cancer cells – via Hypoxia-inducible factor (HIF) and/or Akt – to the new, potentially anoxic, environment of the neoplastic lesion, implicating a reduction/damage of mitochondrial metabolism. In fact, it is becoming clear that this strongly induced aerobic glycolysis of cancer is an epiphenomenon that results from a more complex metabolic rearrangement. Thus not only glycolysis but also the Krebs cycle, beta oxidation, and anabolic metabolism in general are readdressed to respond to the new primary function of this cell (i.e., uncontrolled proliferation) by providing not only energy but also building blocks for the synthesis of nucleotides and amino and fatty acids [7-10] .

Some glycolytic enzymes, strongly induced in the cancer cell (i.e., hexokinase II, lactate dehydrogenase A, glucose-6-phosphate isomerase) may possess different biological functions, acting as both facilitators and gatekeepers of malignancy [11,12] . Moreover, the recent evidence linking cellular metabolism (both in terms of glycolysis and the Krebs cycle), growth factors and apoptosis (primarily in terms of cross-talk between Bcl-2 family members and overall glucose metabolism) further stresses the interrelationship between cancer-cell growth, cancer-cell metabolism, and cancer-cell apoptotic resistance [7] . In this sense, a dramatic indirect proof of the

interrelationship between glycolysis and neoplastic transforma-tion is the strong ability of imatinib to normalize glucose metabolism in leukemic cells [13] . These data, together with other new evidence relating glycolysis to the pentose phosphate pathway and to mitochondrial metabolism, seem to indicate the existence of a peculiar metabolic status typical of proliferating cells, in which glycolysis represents the central metabolic signal transduction pathway that finely regulates cell proliferation, adapting cancer-cell metabolism to the selection pressures of the cancer milieu [14-18] .

This reevaluation of the Warburg effect in particular, and of cancer-cell metabolism in general, suggests new potential therapeutic strategies. Inhibition of glycolysis to curtail energy and the substrates necessary for cancer-cell proliferation may facilitate, or even induce, apoptosis. Interestingly, this peculiar drug-induced metabolic stress could have other significant therapeutic activities at both cell and tissue levels, such as:

• Reduction of hypoxia-linked cancer-cell resistance : An inhibition of glycolysis, hampering the molecular adaptive mechanism related to typical neoplastic-tissue low oxygen levels, should reduce the mass of cancer cells in general, and of the so-called dormant cancer cells in particular [19,20] . Moreover, the perturbation of this cancer-cell glycolysis addiction could alter the molecular pathways of hypoxia, hypoxia–reoxygenation cycling, free radical production, infl ammation/immune cell infi ltration. This in turn may decrease HIF1 activity, thereby reducing molecular mechanisms at the basis of promotion of angiogenesis and cancer cell survival [21-23] . • Cytotoxic synergism with conventional cancer treatments : An abrupt energetic collapse due to glycolysis block could synergize with other physical and chemical anti-cancer treatments. In the context of certain cancers, this could cause neoplastic cells to reutilize mitochondria, strengthening the therapeutic activity of antineoplastic treatments dependent on the induction of free radicals (i.e., doxorubicin) [10,23-25] . • Amelioration of drug selectivity : Glycolysis inhibition in cells strongly addicted to this metabolic pathway could also ameliorate selectivity of other conventional and less selective treatments [24,25] . • Reduction of ATP-dependent multi-drug resistance : The energetic deprivation as a result of glycolytic inhibition could perturb the function of certain ATP-binding cassette (ABC) transporter proteins that are responsible for the multi-drug resistance of cancer cells towards structurally diverse chemotherapeutic compounds. Intriguingly, this functional inhibition of multi-drug-resistance (MDR) proteins could also favorably infl uence the pharmacokinetic properties of various antineoplastic drugs [26,27] .

A major drawback for the development of this class of anticancer agent is the potential for metabolic disruption of normal cells. For example, neurons need copious glucose for

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energetic purposes and have been found to take up more fluorodeoxyglucose than invasive solid malignancies in ∼ 30% of cancer patients studied. Other vital organs, such as the liver and heart, take up substantial glucose and an acknowledgement of this potential drawback to antimetabolite development in cancer needs careful evaluation [28-30] . In this sense, it could be also useful to consider potential glycolytic inhibitors with low blood–brain-barrier penetration properties.

These considerations justify a careful evaluation by pharmacological and/or clinical studies of the role of glycolytic enzyme drug inhibitors in cancer therapeutic protocols. Specifically, some glycolytic inhibitors are already in preclinical and clinical Phase trials and a host of new molecules are under development.

2. Glycolysis as target for anticancer treatment

A better understanding of the biochemical and pharmaco-logical properties of this new class of drug could benefit from describing them according to their enzyme target.

2.1 Hexokinase This enzyme is one of the main targets of glycolysis inhibitors, probably due to its peculiar structural and functional properties. Hexokinase (HK) is composed of two domains. The binding sites for glucose and ATP lie in a cleft between these domains, and the domains move toward each other to narrow the cleft when glucose binds. The hexose binding greatly increases the affinity of the enzyme for ATP. HK phosphorylates a six-carbon sugar (a hexose), generally glucose, to a hexose phosphate (i.e., glucose-6-phosphate), and is responsible for the first metabolic step of glycolysis, during the so-called priming phase of this metabolic pathway, when phosphorylated glucose is trapped in the cytoplasm, ready to be utilized for catabolic and/or anabolic purposes. There are four hexokinase isozymes (EC 2.7.1.1) in mammals, with molecular weights of approximately 100 kD. These isozymes vary somewhat in their subcellular locations, kinetic characteristics, and physiological and pathological functions. They are classically defined as hexokinases I (tissue specificity: common), II (tissue specificity: erythrocytes), III, and IV (testis specific). Hexokinases I, II, and III have a low K m for glucose (< 1 mM), which results in a high affinity for glucose even at low concentrations. Importantly, these specific isoenzymes are strongly inhibited by their product, glucose-6-phosphate. Each isoenzyme consists of two similar 50-kD halves but only in hexokinase II do both halves have functional active sites. Hexokinase I is found in all mammalian tissues and is considered to be a ‘housekeeping enzyme,’ unaffected by most physiological, hormonal, and metabolic changes. Hexokinase III is inhibited by excessive glucose (substrate inhibition). Hexokinase IV, better known as gluco-kinase, has unique characteristics and functions compared with other hexokinases. In fact, glucokinase can phosphorylate

glucose only if the concentration of this substrate is high enough; its K m for glucose is about 200 times higher than those of hexokinases I, II, and III. It is monomeric, about 50 kD, displays positive cooperativity with glucose, and is not allosterically inhibited by its product, glucose-6-phosphate [31,32] .

From an oncologic point of view, it is interesting to point out that hexokinases I, III, and particularly II are physically associated with the outer surface of the external membrane of mitochondria through specific binding to a porin (voltage-dependent anion channel, VDAC). This association allows hexokinase direct access to mitochondrially generated ATP, one of its two substrates. This mitochondrial hexokinase is highly elevated in rapidly growing malignant tumor cells, with levels up to 200 times higher than found in normal tissues. Importantly, mitochondrially bound hexokinase has been demonstrated to be the driving force for the extremely high glycolytic rates that take place in different tumor cells [11,24,32,33] . Moreover, this mitochondrial localization affords the enzyme additional functions. It can inhibit programmed death in cancer cells by modulating interplay, directly and indirectly via Akt, between pro-and antiapoptotic factors related to the Bcl-2 family [11,24,33] .

Finally, it is significant that the rate-limiting enzyme in the utilization of glucose during glycolysis is classically considered to be phosphofructokinase, which catalyzes further metabolism of fructose 6-phosphate. However, if metabolism by this route is sufficiently rapid, as in proliferating cancer cells, the rate of formation of glucose 6-phosphate by hexokinase may become the real rate-limiting step.

These peculiar biological functions render HK in general, and mitochondrial HK specifically, an interesting target for glycolysis inhibition in cancer treatment. Hence, multiple HK inhibitors are in preclinical and clinical Phase trials, in particular the following.

2.1.1 2-Deoxyglucose 2-Deoxy- D -glucose (2-DG, Threshold Pharmaceuticals, Redwood City, CA, USA) is a glucose molecule in which the 2-hydroxyl group has been replaced by hydrogen. HKs phosphorylate this hexose to 2-DG-P, which cannot be metabolized by phosphohexose isomerase. Hence, its intracellular accumulation causes a competitive inhibition of the HKs types I, II, and III.

Notably, because HK IV is not inhibited by its products at the level of liver or pancreas, the physiological mechanisms of glucoregulation must be maintained. Interestingly, Le Goffe et al. [34] demonstrated that 2-deoxyglucose-6-phosphate, generated via the action of hexokinase, can also be metabolized via the pentose phosphate pathway in quantities sufficient to support the redox coupling critical for cell survival. That means that glucose metabolic block by 2-DG cannot be considered total. Finally, according to the classical competitive inhibition model, the action of 2-DG can be rapidly reversed by glucose [24] .

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However, it may be useful to cite some studies suggesting an alternative mechanism of action for 2-DG in cancer therapy. Specifically, Kurtoglu et al. [35] have shown that 2-DG elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N -linked glycosylation. This perturbed glycosylation can induce accumulation of misfolded proteins in the endoplasmic reticulum (ER), leading to induction of the ER stress response [36] . On the basis of these results, 2-DG could be used as a single agent to selectively kill both the aerobic (via interference with glycosylation) and hypoxic (via inhibition of glycolysis) cells of a solid tumor.

Confirmation of prevalent glycolysis inhibition is demonstrated by the observation that 2-DG leads to a decrease in the amount of hexokinase associated with mitochondria, due to its peculiar functional properties (i.e., glucose binding enhances the affinity for ATP). This interesting aspect should be further evaluated, above all in terms of apoptosis modulation and transcriptional regulation [11,24] .

Experimental studies have clearly shown that 2-DG exhibits cytotoxic effects in various cancer cells, especially those hypoxic for tissue conditions and/or displaying mitochondrial respiratory defects [24,37,38] . Moreover, 2-DG significantly increases the anticancer activity of adriamycin and paclitaxel in mice bearing human osteosarcoma, as well as in the MV522 non-small-cell lung cancer xenograft model [39] . Interestingly, a study by Ledoux et al. [40] and cited by Pelicano et al. [24] showed that 2-DG may induce the expression of P -glycoprotein encoded by the MDR1 gene; this effect was associated with a stimulation of [ 3 H]vinblastine efflux by P -glycoprotein despite impaired glycosylation, raising the possibility that this might help cancer cells to develop chemoresistance.

Interestingly, Phase I clinical studies showed that oral administration of 2-DG combined with large fractions of radiation (5 Gy/fraction/week) is safe and well-tolerated in patients with glioblastoma multiforme without significant acute toxicity and/or late radiation damage to the normal brain [41,42] .

At present, Threshold Pharmaceuticals is completing a Phase I trial with orally administered 2-DG to treat solid tumors, alone and in combination with other chemotherapy. This trial is a dose-escalation study to determine the safety, pharmacokinetics, and maximum tolerated dose of daily oral doses of 2DG given alone or in combination with docetaxel. The study should enroll about 40 patients with previously treated refractory advanced solid tumors. The study will also evaluate the effect of 2DG alone and in combination with docetaxel on tumor metabolism, and provide a preliminary assessment of efficacy, as assessed by computed tomography. Of note, 2DG has been already administered in clinical settings to approximately 700 people, principally to evaluate the hormonal and metabolic effects of glucose deprivation. These studies seem to show that single intravenous doses of 2DG as high as 200 mg/kg do not cause any serious adverse events.

The US National Institutes of Health website www.ClinicalTrials.gov reports other clinical studies in progress, such as:

A Phase I/II trial for the treatment of advanced cancer and • hormone refractory prostate cancer (HRPC) to determine both the safety and the optimal dose of 2-deoxyglucose administered daily to patients with advanced solid tumors and the biochemical response of this regimen in patients with HRPC. A safety study of 2-DG associated with stereotactic • radiosurgery for sensitizing cancer cells to ionizing radiation by inhibiting the use of glucose to detoxify reactive oxygen species produced by radiation.

2.1.2 3-Bromopyruvate Bromopyruvic acid, or bromopyruvate (3-BrPA), is a potent synthetic bromo-halogenated derivative of pyruvic acid. It is considered a classic inhibitor of hexokinase, capable of causing a severe depletion of ATP in cancer cells at µmolar concentration (about 100 µmol/l). However, as an aliphatic halogenated drug, it is also a strong alkylating agent toward thiol proteins and therefore cannot be considered an HK selective inhibitor [24,25] . In this sense, Robey et al. [43] showed that 3-BrPA inhibits HK activity, probably via selective alkylation of sulfhydryl groups important for enzymatic function but not involved in glucose or ATP binding. However, 3-BrPA glycolytic inhibitory potency correlates better with inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) than with inhibition of HK in non-tumor epithelial cells.

At the beginning, the anticancer/antiglycolytic activities of 3-BrPA were studied in different experimental models of hepatocarcinoma, and in particular on the rabbit VX2 tumor model [44-50] . Results showed that the drug was capable of strongly inhibiting HK, causing a serious inhibition of the glycolytic pathway in hypoxic tumors. Interestingly, some studies reported that selective intra-arterial administration of 3-BrPA can cause a significant toxicity not only in the liver but also in the gastrointestinal system [49,50] . By contrast, Shin et al. [51] adopted the same rabbit VX2 tumor model and did not observe therapeutic/toxic effects after a single session of intra-arterial injection of 3-BrPA.

An experimental study by Xu et al. [52] on human leukemia HL-60, human lymphoma cell line Raji, and human colon cancer HCT116 cells showed some molecular aspects related to the anticancer activity of 3-BrPA. Specifically, drug-induced glycolysis inhibition was associated with a rapid dephosphorylation of Bcl-2-associated death promoter (BAD) protein at the level of Ser 112 , relocalization of BCL-2-associated X (BAX) protein to mitochondria and activation of the apoptotic pathway.

Recent studies have evaluated the anticancer properties of 3-BrPA in a rat model of breast cancer [53] ; interestingly, the same compound was given in combination with a heat

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shock protein 90 (HSP90) inhibitor (geldanamycin) in an experimental model of chemotherapy-resistant pancreatic cancer in order to evaluate synergistic therapeutic efficacy and to reduce dose-limiting toxicity [54] . Results showed that 3-BrPA inhibited glycolysis and sensitized geldanamycin against pancreatic cancer cells through HSP90 client protein degradation. Importantly, the synergistic anticancer effect of reduced doses of geldanamycin and 3-BrPA was confirmed in xenograft models in vivo by more than 75% tumor growth inhibition.

Although preclinical studies seem to be promising, there are no human clinical trials yet. This lack of human studies could be the result of: i) a generally poor consideration of the potential for this class of drug; ii) a specific, prudent attitude towards a halogenated drug with potential multiple biological activities; iii) low economic interest in a non-patentable molecule; and iv) amelioration of the therapeutic index of other anticancer regimens.

Considering the high affinity for sulfhydryl groups of 3-BrPA, and that almost every enzyme and protein contains thiol groups, the cancer-cell selectivity of this sulfhydryl-reactive agent could be questionable. In fact, recently, Meng et al. [55] , comparing the activity of 3-BrPA directly to that of other glycolytic inhibitors, showed it to be neither specific nor very useful in targeting anaerobic or hypoxic cells. Based on these data, as well as some of what the review authors present, it appears that this drug will not be brought forward to the clinic as a useful glycolytic inhibitor. Moreover, the therapeutic index of this and other sulfhydryl-reactive compounds of pharmacological interest should be carefully investigated.

2.1.3 Lonidamine Lonidamine (TH-070; Threshold Pharmaceuticals, Inc.), also known as 1-(2,4-dichlorobenzyl)- 1 H-indazole-3-carboxylic acid, is an orally administered anticancer and antispermato-genic drug whose mechanism of action is still incompletely understood. Specifically, its biological activities seem to involve interference with cellular energy metabolism by disruption of the mitochondrial membrane and by inhibition of hexokinase, particularly mitochondrial hexokinase II [24,25] . Lonidamine antispermatogenic activity has been reported to inhibit germ-cell respiration. Mainly as a result of its inhibition of glycolysis, lonidamine has recently been indicated as a novel approach to the treatment of benign prostatic hyperplasia (BPH), in which prostatic cells typically show a high glycolytic flux for energetic purposes [56] .

The inhibition of hexokinase by lonidamine is well established, although the molecular mechanisms linking this inhibition of mitochondria-bound hexokinase, mitochondrial membrane depolarization, and induction of apoptosis (via release of cytochrome C, phosphatidylserine externalization, and DNA fragmentation) are still under debate [25,57] . In the context of cancer therapy, lonidamine seem to stop glycolysis in relatively hypoxic tumor cells, resulting in involution of

the cancer [56] . Because of this property, lonidamine (Doridamina, Angelini, Rome, Italy) has been approved for use in Europe in some cancer therapeutic protocols at doses of 450 – 900 mg daily in three divided doses [57-59] . In fact, this drug is considered helpful in various chemotherapeutic protocols, including head, neck, breast, and brain cancers. Myalgia is the most common side effect; other reported side effects are testicular pain, auditory dysfunction, gastrointestinal disturbances, drowsiness, weakness, and conjunctivitis with photophobia [58] . Recently, some positive clinical results have been obtained with lonidamine in association with: i) doxorubicine, in metastatic breast cancer patients [59] ; ii) paclitaxel and cisplatin, in advanced ovarian cancer [60] ; and iii) diazepam, in the treatment of recurrent glioblastoma multiforme [61] . These last data are particularly interesting considering that cellular metabolism in glioblastoma multiforme is characterized by a high rate of aerobic glycolysis that is dependent on mitochondria-bound hexokinase, and also given that recurrent glioblastoma multiforme (GBM) is resistant to most therapeutic endeavors, with low response rates and survival rarely exceeding 6 months [62,63] .

Other molecules acting as hexokinase inhibitors have been tested, including 5-thioglucose and mannoheptulose. At present, however, there are insufficient data to establish a biochemical and pharmacological profile for these molecules as glycolytic inhibitors. Interestingly, a series of derivatives of 2-DG, 5-thioglucose and 6-fluoro- D -glucose has been recently patented with claims of glycolytic inhibition in the context of cancer treatment.

2.2 Glucose-6-phosphate isomerase The isomerization of glucose-6-phosphate into fructose-6-phosphate is the second step of glycolysis. This reversible reaction is catalyzed by glucose-6-phosphate isomerase (GPI), also know as phosphoglucose isomerase (EC 5.3.1.9) [31] . This essential enzyme of catabolic glycolysis and anabolic gluconeogenesis influences tumor cell growth. The two major isoforms are homodimers of subunits of 63.2 kDa (type A). Isoform 3 is a heterodimer composed of the type-A subunit and a previously unreported larger subunit of 69.8 kDa (type B). Isoform 4 is a BB-homodimer [64] . Interestingly, this protein has other biological activities. In fact, it is also known as autocrine motility factor (AMF), capable – in cancer cells – of promoting cell motility and proliferation in an autocrine manner [11,65] . Recently, Funasaka et al. [65,66] showed that GPI/AMF downregulation resulted in an increased sensitivity to oxidative stress and in oxidative-stress-induced cellular senescence in cancer cells. Pharmacological studies show that AMF consists of three domains, and that the substrate or inhibitors of GPI are located between the large and small domains, corresponding to approximately residues 117 – 288 [67,68] . Experimental drug inhibitors to be considered are D -fructose-6-phosphate, 6-phosphogluconic acid, and N -bromoacetyl-aminoethyl phosphate.

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2.3 Phosphofructokinase Phosphofructokinase (PFK) converts fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP. In human, there are three, tissue-specific, types of PFK isozymes: M (muscle), L (liver), and P (platelet). PFK is classically considered the most important regulatory enzyme (EC 2.7.1.11) of glycolysis. It is an allosteric enzyme comprising four subunits and controlled by several activators and inhibitors. The enzyme is activated by AMP, ADP, and inorganic phosphate; it is inhibited by ATP, citrate, and fatty acids. Importantly, PFK can also be considered a pacemaker of carbohydrate metabolism because it can regulate the metabolism of other monosaccharides, such as galactose, fructose, or mannose, which can feed into the glycolytic pathway upstream PFK enzymatic step [31] . Some authors [24] do not confer to PFK a significant physio-pathogenic role in cancer. However, others showed that Akt might phosphorylate and activate phosphofructokinase and release the inhibition of phosphofructokinase by ATP [24,69] . Moreover, at the level of cancer cells, it has been shown that genes coding for isoenzyme 3 (PFKFB3) of 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase) are expressed at high levels. This induces synthesis of the fructose-2,6-bisphosphate (F2,6BP) potent activator of phosphofructokinase [70] .

Hence, considering the particularly high metabolism of cancer cells, it could be hypothesized that PFK upregulation could be due to high levels of AMP, ADP, and F2,6BP, which probably override its inhibition with high amounts of hydrogen ions.

As a result of the direct (or indirect) role of PFK upregulation in cancer-cell metabolism, the modulation of this upregulation would be an interesting target for the development of new antineoplastic agents [71] . In this regard, an interesting study was recently published on a small-molecule inhibitor of PFKFB3 – 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) (Advanced Cancer Therapeutics, Louisville, KY, USA) – which has the capability of suppressing 6-phosphofructo-2-kinase activity, glycolytic flux, and tumor growth in several human malignant hematopoietic and adenocarcinoma cell lines, with an IC 50 ranging from 1.4 to 24 µmol/l. Interestingly, this molecule was shown to be selectively cytostatic to ras -transformed human bronchial epithelial cells relative to normal human bronchial epithelial cells [72] .

2.4 Aldolase Fructose-bisphosphate aldolase (EC 4.1.2.13) is responsible for the conversion of fructose 1,6-diphosphate into dihydroxy-acetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P). The three aldolase (ALDO) isozymes (A, B, and C) have a tetramer structure with identical molecular weights of about 160 kD. They are encoded by three different genes, differentially expressed during development. ALDOA is mainly produced by the developing embryo and in adult

muscle; ALDOB is produced by liver, kidney and intestine; and ALDOC is mainly produced by brain and other nervous tissue.

It is well known that cancer cells with a high glycolytic rate often exhibit an aberrant expression of all glycolytic enzymes, particularly type II hexokinase (HKII) and ALDOB. Analogs blocking aldolase-catalyzed cleavage are under development (i.e., 3-fluoro- D -glucose, 4-fluoro- D -glucose). These molecules should not be able to undergo aldolase-catalyzed cleavage [11,12] . The consequent fructose-1,6-bisphosphate (F1, 6BP) accumulation should stop glycolysis. Marin-Hernandez et al. [73] found that the greatest control of glycolysis (about 70 – 75%) in rapidly growing tumor cells is exerted by glucose transporters, HK, GPI, and PFK, whereas the remaining flux control is at the level of the so-called consuming block (from aldolase to lactate dehydrogenase).

Moreover, a recent study examining the aberrant expression of HKII and ALDOB in 203 surgically resected hepato-carcinomas showed a dramatic downregulation of ALDOB in 57% of samples. Interestingly, this downregulation was associated with advanced disease, early tumor recurrence and poor prognosis [74] . This intriguing data should be confirmed and carefully evaluated not only in terms of prognosis but also from a pathophysiological point of view.

2.5 Triosephosphate isomerase Triosephosphate isomerase (TPI; EC 5.3.1.1), a dimer of identical subunits, catalyzes the reversible conversion of dihydroxyacetone phosphate to glyceraldehydes-3-phosphate. Although equilibrium is in favor of dihydroxyacetone phosphate, glyceraldehyde-3-phosphate undergoes continued oxidation through the action of glyceraldehyde phosphate dehydrogenase and is thus removed from the equilibrium.

TPI is essential for the efficient energy production of glycolysis. Hence, in humans, deficiencies in TPI are associated with nonspherocytic hemolytic anemia and severe neurological disorders.

Upregulation of TPI has been reported in various cancers (lung, liver, colon, breast, bone, prostate, etc.). Interestingly, a single primary isoform is observed in quiescent cells, while three primary isoforms of the dimeric glycolytic enzyme are detected in proliferating human cells. The electrophoretically separable isoforms result from the three possible combinations of constitutive subunits and subunits expressed only in proliferating cells. The pathophysiological role of these isoforms is still debated [75] .

At present, some TPI inhibitors are under study mainly as potential antiprotozoal drugs (2-carboxyethylphosphonic acid, N -hydroxy-4-phosphono-butanamide, 2-phosphoglyceric acid).

2.6 Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), by means of a redox reaction, converts glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate with concomitant reduction of

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nicotinamide adenine dinucleotide (NAD + ) to NADH. The enzyme structure consists of a homotetramer, each subunit of which may bind to NAD + [31,76] . Like other enzymes, GAPDH is a multifunctional protein with glycolytic and nonglycolytic functions, both of which can be of chemo-therapeutic value. Considering the extrametabolic activities, due to its ability to bind NAD + or NADH, the enzyme influences the pathway through which NADH can act as intracellular messenger [77] . Moreover, studies showed other activities for GAPDH [11] , including participation in DNA repair and telomeric DNA binding [78] , transcription regulation as a component of the OCA–S complex [24,25] , as well as a series of poorly understood functions in membrane fusion, RNA binding, tubulin bundling, apoptosis induction [11,12,24,25] , and, more recently, a significant role in preventing caspase-independent cell death (CICD). There is also intriguing evidence that GAPDH modulates and coordinates glycolysis and the autophagy pathway in cells [79,80] . Interestingly, Saunders et al. [81] showed six GAPDH isoforms in human cerebellar neurons. The more alkaline isoforms, 1 – 3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4 – 6, were the minor species. Levels of all six isoforms were increased after treatment with arabinoside C for 16 h; while a 4-h treatment increased levels of only isoforms 4 and 5. In opinion of these authors, it appears that various GAPDH isoforms are differentially regulated and might have distinct apoptotic roles.

Interestingly, some GAPDH inhibitors, in the form of adenosine analogs, are in preclinical phase as antiprotozoal drugs because glycolysis may provide virtually all of the energy in some phases of the vital cycle of these parasites (i.e., for the bloodstream form of Trypanosomas and Leishmanias) [82,83] . These data, together with the cited evidence for the role of GAPDH in preventing CICD, reinforce the potential role of the pharmacological inhibition of this particular enzyme in the context of cancer. Various molecules have already been considered for this therapeutic indication.

2.6.1 Iodoacetate Iodoacetate (IAA) is reported as a classical inhibitor of GAPDH, capable of reacting with the sulfhydryl group of the cysteine residue at the active site of the enzyme. Importantly, it is an inhibitor of glucose-6-phosphate dehydro-genase (G6PDH) and 6-phosphogluconate dehydrogenase as well [8,25] . Recently, Lai et al. [84] investigated the effects of 3-bromopyruvate (3-BrPA) and IAA on the survival of several different types of cancer cell, including glioblastoma, pancreatic, and oral cancer cells. Results seem to indicate that both 3-BrPA and IAA induce decreases in cell survival in all cancer cell types studied in a concentration- and time-related manner. Importantly, IAA was more potent than 3BP in inducing cell death in all the cancer cell types investigated. In the conclusion of these authors, their data prompt the hypothesis that glycolytic enzyme inhibitors,

such as IAA and 3BP, can be used as ‘proof-of-concept’ test drugs to derive a novel approach to inhibit cancer-cell proliferation and invasion. Partial opposite conclusions have been reported by Rodríguez-Enríquez et al. [85] showing that various mitochondrial inhibitors (i.e., rhodamines 123 and 6G, and casiopeina II-gly) inhibited tumor cell proliferation and oxidative phosphorylation but also glycolysis. By contrast, well-known GAPDH inhibitors (i.e., iodoacetate, gossypol, and arsenite) strongly blocked glycolysis without affecting tumor cell proliferation. In the authors’ opinion, fast-growing tumor cells have a predominantly oxidative type of metabolism, which might be a potential therapeutic target while GAPDH inhibitors have a minor therapeutic value.

Hence, for the alkylating agent iodoacetate can be asserted the same considerations of 3-BrPa in terms of cancer drug selectivity and toxicity and both molecules should be considered interesting proof-of-concept glycolytic inhibitors, rather than ready-to-use anticancer drugs.

2.6.2 Gossypol Gossypol (AT-101) is a well-known GAPDH inhibitor that Ascenta Therapeutics has in clinical development as an oral pan-Bcl-2 inhibitor. In fact, Phase I and Phase II clinical trials have already demonstrated that AT-101 shows cyto-reductive activity in chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, and prostate cancer [86] . Phase II combination trials are ongoing in hormone-refractory prostate cancer and non-small-cell and small-cell lung neoplasms, B-cell malignancies, glioblastoma multiforme, and esophageal cancer.

2.7 Phosphoglycerate kinase Phosphoglycerate kinase (PGK; E.C. 2.7.2.3) reversibly catalyzes the transfer of the phosphoryl group from the 1,3-bisphosphoglycerate to ADP with formation of ATP and 3-phosphoglycerate. By this reaction all the invested ATP is recovered. Importantly, at this level, the formation of ATP depends on substrate-level phosphorylation [31] . There are two isoforms of this enzyme, called 1 and 2. Testis-specific PGK-2 is considered critical to normal motility and fertility of mammalian spermatozoa [87] . Interestingly, the over-expression of the ubiquitous PGK-1 isoform seems to be associated to a multi-drug resistance phenotype in different cancers [88] .

Interestingly, the metabolic weight of this enzyme in glycolysis can be derived by the clinical picture of its deficiency. In fact, PGK deficiency is a rare X-linked disorder characterized by hemolytic anemia, seizures, muscle fatigue, and progressive neurological dysfunction [31] . The patho-genetic and prognostic role of PGK overexpression has been recently confirmed by interesting clinical proteomics studies on different cancers [89-91] . A series of novel, conformationally-restrained bisphosphonate analogs of 1,3-bisphosphoglyceric acid have been synthesized and evaluated as inhibitors of 3-PGK. Specifically, they are studied as competitive inhibitors

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of the human enzyme in erythrocytes. In this way, by diverting 1,3-diphosphoglycerate to 2,3-diphosphoglycerate via biphosphoglycerate mutase, power of the allosteric effector of hemoglobin is increased. The consequent increase of hemoglobin oxygen release could be therapeutically useful for different cardiovascular and respiratory disorders. Interestingly, PGK not only functions in glycolysis but is also secreted by tumor cells and participates in the angiogenic process as a disulfide reductase. The molecule facilitates cleavage of disulfide bonds in plasmin, which triggers proteolytic release of the angiogenesis inhibitor, angiostatin. However, its real role in tumor angiogenesis is still under debate [92,93] .

2.8 Phosphoglycerate mutase Phosphoglycerate mutase (PGM) catalyzes the internal transfer of a phosphate group from C-3 to C-2, which results in the conversion of 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) through a 2,3-bisphosphoglycerate intermediate. As it is a reversible reaction, it is not a site of major regulation for the glycolytic pathway. Phosphoglycerate mutase exists primarily as a dimer of two identical or closely related subunits of about 32 kDa. In mammals, the enzyme subunits appear to be either a muscle-derived form (m-type) or other tissue (b-type for brain where the b-isozyme was originally isolated); hence, the enzyme can be found in three isoforms, mm, bb or mb. The mm-type is found mainly in smooth muscle. The mb-isozyme is found in cardiac and skeletal muscle and the bb-type is ubiquitous [94] .

In humans, deficiency in the phosphoglycerate mutase function presents as a metabolic myopathy and is one of the many forms of syndromes formerly referred to as muscular dystrophy. From an oncologic point of view, PGM is found upregulated in many cancers including lung, colon, liver, and breast [24,95-97] . Interestingly, Kondoh et al. [97] recently demonstrated that a two-fold increase in this enzyme activity can reversibly immortalize mouse embryonic fibroblasts. However, at present, only some experimental inhibitors of PGM (benzene hexacarboxylic acid and 3-phosphoglyceric acid) are known.

2.9 Enolase Enolase (ENO) is a metalloenzyme that catalyzes the reversible dehydration of 2-phosphoglycerate to phosphoenol-pyruvate. Typically in adult human cells, three isoenzymes are more commonly found: enolase 1 ( α α or non-neuronal enolase [NNE], found in liver, kidney, spleen, adipose); enolase 2 ( γ γ or neuron-specific enolase [NSE]), and enolase 3 ( β β or muscle-specific enolase [MSE]).

ENO, and specifically ENO1, is a typical multifunctional enzyme that, as well as its role in glycolysis, has other different biological functions that influence growth control, hypoxia tolerance, and allergic responses. It also has a role in the intravascular and fibrinolytic system as receptor and activator of plasminogen on the cell surface of several cell

types. In cancer, an alternatively translated and truncated product of the ENO1 gene, also know as C-myc promoter-binding protein (MBP-1), is considered a tumor suppressor that binds to the c-myc promoter and acts as a transcriptional repressor [11,24,98] .

Moreover, ENO2 (NSE) is a classic tumor marker because increased serum levels may be measured in tumors of neuroendocrine origin (small cell lung cancer, melanoma, neuroblastoma etc.) [98] .

A classic experimental enolase inhibitor is sodium fluoride, while other molecules under evaluation are phosphonoacetohydroxamic acid and 2-phospho- D -glyceric acid.

2.10 Pyruvate kinase (PK) Pyruvate kinase (PK; 2.7.1.40) catalyzes the irreversible transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, producing one molecule of pyruvate and one molecule of ATP. Because the PK kinase reaction is essentially irreversible under intracellular conditions it is also an important site of regulation.

There are four isoezymes of PK (tetramers of 55 kD subunits) in mammals: L, R, M1, and M2. The L type predominates in liver; R is found in red cells; M1 is the main form in muscle, heart, and brain; and M2 is found in early fetal tissues. Each isoenzyme is differently and finely regulated. From a pharmacologic point of view, it is interesting to highlight that PK has an absolute require ment for a divalent metal ion (Mg 2+ ) and a monovalent metal ion (K + ), and that four sulfhydryl groups have a role at the active site [31] . Interestingly, tumor cells seem to express preferentially the pyruvate kinase isoenzyme type M2. Specifically, this isoenyme regulates the proportions of glucose carbons that are channeled to synthetic processes (inactive dimeric form) or used for glycolytic energy production (highly active tetrameric form). The switch between the tetrameric and dimeric form of M2-PK, regulated by direct interaction of M2-PK with certain oncoproteins, allows tumor cells to adapt to variation in environmental nutrient concentration [24,99] .

There are well-known experimental inhibitors, such as fluorophosphates, pyridoxal 5 ′ -phosphate, creatine phosphate, oxalate, and L -phospholactate (a specific inhibitor for M2-PK). Recently, synthesis of some phosphoenolpyruvate (PEP) analogs with modifications in the phosphate and the carboxylate function have been described [100,101] .

The most interesting PK inhibitor is CAP-232/TLN-232 (Thallion Pharmaceuticals, Montreal, Quebec, Canada), a synthetic cyclic heptapeptide with potential antineoplastic activity. In fact, TLN-232 targets pyruvate kinase M2, disrupting tumor-cell anaerobic glycolysis. This drug is already in clinical Phase II and a study of CAP-232 in patients with refractory metastatic renal cell carcinoma is active.

Thallion intends to initially develop TLN-232 for three primary applications: renal cell carcinoma, metastatic melanoma, and pancreatic cancer.

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2.11 Lactate dehydrogenase Lactate dehydrogenase (LDH; 1.1.1.27) permits glycolysis in human tissues with insufficient oxygen to support pyruvate and NADH oxidation by realizing the conversion of pyruvate to lactate coupled with oxidation of NADH to NAD + .

Mammalian lactate dehydrogenase (LDH) exists as five tetrameric isozymes composed of combinations of two different subunits, H and M, encoded by two different genes, A and B. The isozymes differ in catalytic, physical, and immunological properties. The polypeptide subunits combine to form two pure types of isozyme, H4 and M4, and three hybrids, H3M, H2M2, and HM3. Subunit ‘H’ predominates in heart muscle LDH, which is geared for aerobic oxidation of pyruvate. The ‘M’ subunit predominates in skeletal muscle and liver and is concerned more with aerobic metabolism and pyruvate reduction. LDH is of interest clinically in that the serum level of certain isozymes reflects pathological condition in particular tissues. Moreover, the serum level of LDH has independent prognostic significance in patients with advanced cancer and should be determined in all patients [102-105] . Increases in the serum concentration are often a reflection of tumor burden, growth rate, and cellular proliferation. Interestingly, LDH has recently been demonstrated to play a significant role in tumor maintenance [106] . Moreover, the LDH-A gene is controlled by hypoxia-inducible factor and its product binds DNA, thus influencing transcription [24] .

Inhibitors of LDHs are under development as antiprotozoal agents to specifically treat infections by the malarial parasite Plasmodium falciparum [107,108] .

3. Conclusions

Oncologic bioenergetics is progressively increasing its importance in pathophysiology, diagnosis, prognosis, and, above all, treatment of cancer. Warburg’s original observation of aerobic glycolysis that governs tumor-cell biology and the response to chemotherapeutics still retains some value. Glycolysis can still be considered the central metabolic pathway of cancer cells. Tumor cells bearing this metabolic change are uniquely sensitive to inhibition of glycolysis – unlike their normal counterparts – suggesting a potential therapeutic selectivity. The possibility of selectively killing cancer cells by deranging their peculiar bioenergetics seems an interesting approach, used alone and/or in combination with other systemic therapy.

Many molecules are in preclinical and clinical phase trials, acting as glycolysis enzyme inhibitors. These molecules, often acting as competitive inhibitors, are capable of reducing tumor burden alone or, preferably, in association with normal chemotherapy. In this sense, at present, the synergism with conventional antineoplastic drugs, by ameliorating therapeutic index of the latter, seems to be the main advantage of this therapeutic oncometabolic approach.

However, it should be stressed that glycolysis inhibition is not the only therapeutic approach. A series of old and new molecules are under development as modulators of tumor-cell energy metabolism. A few examples of these are oxythiamin, alpha-chlorohydrin, and ornidazole, which inhibit the pentose phosphate pathway (PPP), and clofazimine, F-16, rhodamine, and MKT-077, which target mitochondrial metabolism. Also for these molecules, it is fundamental to distinguish between drugs for proof-of-concept and molecules that for their pharmaco-toxicological profile could have a real clinical use.

All these drugs are likely to contribute to boosting the therapeutic potential of oncopharmacology, but to better utilize these metabolic bullets it will be fundamental to carefully evaluate the rationale for choosing specific drugs, combinations of drugs, or combinations of different types of treatment in various regimens (i.e., an inhibitor of PPP after a pro-oxidant pharmacological and/or radiotherapeutic treatment).

4. Expert opinion

The capability to selectively modulate cancer-cell metabolism is a fascinating approach, rich with therapeutic potentialities. The characteristic of cancer cells to carry active oncogenes (e.g., Ras , Her2 , Akt ) and/or lack single tumor suppressors (e.g., TSC1/2 , LKB1 , p53 ) is well-known to cause these cells to undergo apoptosis under low glucose conditions. Intriguingly, these same cells often display enhanced resistance to other forms of apoptotic stimuli (irradiation, chemotherapeutics).

The use of glycolytic inhibitors is therefore promising. Moreover the possibility of limiting the pitfalls of classic antineoplastic protocols (MDR, hypoxic regions, slow proliferation index) should push researchers to rapidly consider these molecules.

Importantly, the pharmacokinetic and pharmacodynamic properties of these drugs impose tailored protocols of administration strictly related to the particular associated antineoplastic treatment (e.g., boost administration just before or afterwards). Moreover, at present it is fundamental to distinguish between drugs for proof-of-concept as opposed to molecules that for their pharmaco-toxicological profile could have a real clinical use in this context. At last, it is also essential to understand the energy metabolism of the specific cancer in treatment.

Finally, it is becoming clear that the aerobic glycolysis of cancer is an epiphenomenon that results from a more complex metabolic rearrangement in which not only glycolysis but also the Krebs cycle, beta oxidation, and anabolic metabolism in general are readdressed to respond to the new primary function of this cell (i.e., uncontrolled proliferation) by providing not only energy but also building blocks for the synthesis of nucleotides and amino and fatty acids. Continuing to consider glycolysis

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as the main energetic metabolic pathway of neoplastic cells will never permit researchers to completely understand the mechanisms at the basis of the great ‘environmental’ adaptation of cancer. Understanding the real role of glycolysis as playmaker in cancer-cell metabolism in general, and in cancer-stem-cell bioenergetics in particular, could have significant implications not only for diagnosis and prognosis

but, above all, for the implementation of more selective cancer pharmacotherapy.

Declaration of interest

The authors declare no conflicts of interest and have received no payment for the preparation of this manuscript.

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Affi liation Roberto Scatena † , Patrizia Bottoni , Alessandro Pontoglio , Lucia Mastrototaro & Bruno Giardina † Author for correspondence Catholic University, Department of Laboratory Medicine, Largo A. Gemelli 8, 00168 Rome, Italy Tel: +39 0 630 154 222 ; Fax: +39 0 635 501 918 ; E-mail: [email protected]