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Anti-Cancer Agents in Medicinal Chemistry, 2008 , 8, 305-312 305

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The Warburg Effect: Why and How do Cancer Cells Activate Glycolysis in thePresence of Oxygen?

Miguel López-Lázaro*

Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain

Abstract: Cells can obtain energy through the oxygen-dependent pathway of oxidative phosphorylation (OXPHOS) and through theoxygen-independent pathway of glycolysis. Since OXPHOS is more efficient in generating ATP than glycolysis, it is recognized that the

presence of oxygen results in the activation of OXPHOS and the inhibition of glycolysis (Pasteur effect). However, it has been known formany years that cancer cells and non-malignant proliferating cells can activate glycolysis in the presence of adequate oxygen levels

(aerobic glycolysis or Warburg effect). Accumulating evidence suggests that the persistent activation of aerobic glycolysis in tumor cells plays a crucial role in cancer development; the inhibition of the increased glycolytic capacity of malignant cells may therefore represent akey anticancer strategy. Although some important knowledge has been gained in the last few years on this growing field of research, the

basis of the Warburg effect still remains poorly understood. This communication analyzes why cancer cells switch from OXPHOS toglycolysis in the presence of adequate oxygen levels, and how these cells manage to avoid the inhibition of glycolysis induced by oxy-gen. Several strategies and drugs that may interfere with the glycolytic metabolism of cancer cells are also shown. This information may

help develop anticancer approaches that may have clinical relevance.

Key Words: Aerobic glycolysis, glycolysis inhibitors, metabolism, dysoxic metabolism, hypoxia-inducible factor 1, reactive oxygen species,hydrogen peroxide, superoxide anion.

1. INTRODUCTION

Most of the energy that cells need to live is produced in the

mitochondria through an oxygen-dependent process called oxida-tive phosphorylation (OXPHOS). This process couples the oxida-tion of NADH and FADH2 with the phosphorylation of ADP to

form ATP, the chemical energy currency of cells. OXPHOS is oxy-gen-dependent because the electrons resulting from the oxidation of NADH and FADH2 need to be ultimately accepted by oxygen (O2).

Cells can also produce ATP through glycolysis, which takes placein the cytosol and does not require O2. In the process of glycolysis,one molecule of glucose is broken down into two molecules of

pyruvate, resulting in the production of ATP. This process con-sumes NAD+, which can be regenerated by the conversion of pyru-

vate to lactate (Fig. 1). OXPHOS is more efficient in generatingATP than glycolysis; the oxidation of one molecule of glucosegives a net yield of 30 ATPs via OXPHOS and 2 ATPs via glycoly-

sis. It is comprehensible, therefore, that cells generate ATP throughOXPHOS when they have enough O2 levels. This was first

noted by

Pasteur in the late 19th

century, who observed that, as the O2 levels

decreased, the generation of ATP shifted from OXPHOS to glyco-lysis (Pasteur effect) [1-4].

In the first half of the 20th

century, Otto Warburg first observedthat cancer cells had increased rates of glycolysis despite the pres-ence of adequate O2 levels [5]. This phenomenon, called aerobicglycolysis or Warburg effect, has repeatedly been observed in can-cer cells [6-8]. Indeed, the widespread clinical use of the imagingtechnique positron-emission tomography using the glucose ana-

logue tracer 18fluorodeoxyglucose (FdG PET) has demonstrated thatthe glycolytic phenotype is observed in most human cancers [6]. Ithas also been known for several decades that the metabolic switch

from OXPHOS to aerobic glycolysis is not a unique feature of tu-mor cells, as it is also found in non-transformed proliferating cells[9-11]. These experimental observations raise two questions that

have long puzzled cancer biologists. First, if OXPHOS is moreefficient in generating ATP than glycolysis, why do cancer cellsand non-malignant proliferating cells activate glycolysis when the

O2 levels are adequate? Second, if O2 acts as a glycolysis inhibitor

*Address correspondence to this author at the Department of Pharmacology,

Faculty of Pharmacy, C/ Profesor García González, 41012 Sevilla, Spain;Fax: +34 954 23 37 65; E-mail: [email protected]

(Pasteur effect), how do these cells manage to activate glycolysis inthe presence of O2?

An understanding of the Warburg effect might be exploitedtherapeutically, as the sustained activation of glycolysis in tumorcells seems to play a crucial role in cancer development. For in-

stance, recent data suggest that cancer cells may depend on glycoly-sis for ATP generation and that cancer cells’ dependence on glyco-lytic energy progressively increases as malignant transformation

occurs [7, 12-14]. Furthermore, it has been demonstrated that theaccumulation of glucose metabolites caused by the constitutiveactivation of glycolysis results in the activation of hypoxia-

inducible factor 1 (HIF-1) [15, 16]. The activation of HIF-1 in-creases the transcription of many genes that code for proteins that

favor cancer development, including proteins involved in glucosemetabolism, apoptosis resistance, invasion, metastasis and angio-genesis [17-19]. Evidence suggests that the persistent activation of

glycolysis can also favor tumor growth [20, 21], as well as tumorinvasion and metastasis via acidification of the tumor microenvi-ronment [22, 23]; indeed, it has been proposed recently that the

glycolytic phenotype is necessary for evolution of invasive humancancers [6, 22]. The present communication analyzes why cancercells switch from OXPHOS to aerobic glycolysis and how they

avoid O2-induced glycolysis inhibition. In addition, possible strate-gies aimed at inhibiting glycolysis in cancer cells are discussed, andseveral drugs that may interfere with the glycolytic metabolism of

cancer cells are examined. This knowledge may help develop effec-tive antitumor strategies.

2. WHY DO CANCER CELLS ACTIVATE GLYCOLYSIS INTHE PRESENCE OF O2?

The first explanation for the phenomenon of aerobic glycolysiswas given by Otto Warburg several decades ago. He proposed thatcancer cells have increased glycolytic rates despite the presence ofO2 because these cells have irreversible damages to OXPHOS [5].Despite being rejected in the beginning, recent observations seem tosupport this hypothesis. For instance, it has been reported that cellsfrom the most common cancer types have decreased expression ofATP synthase [24, 25], a protein complex required for OXPHOS.Mitochondrial mutations, which may lead to malfunction in OX-PHOS, have also been observed in tumor cells [7]. It has also beenshown that inactivation of p53—one of the most commonly mu-tated genes in cancer—may trigger the Warburg effect; p53 is in-



306 Anti-Cancer Agents in Medicinal Chemistry, 2008 , Vol. 8, No. 3 Miguel López-Lázaro

volved in the activity of cytochrome c oxidase, a protein complexinvolved in OXPHOS [26]. However, recent data have shown thatglycolysis inhibition in cancer cells can enhance OXPHOS activity;this seems to indicate that the Warburg effect is not caused by irre-versible damages to OXPHOS [21, 27]. This last observation issupported by the fact that the Warburg effect has also been ob-served in non-transformed proliferating cells, which are not sup- posed to have irreversible damages to OXPHOS.

Based on mathematical models and empirical observations, ithas been proposed recently that cancer cells activate aerobic glyco-lysis because the persistent activation of glycolysis leads to envi-ronmental acidosis, which is toxic to normal cells but harmless to

cancer cells. The sustained activation of glycolysis would therefore provide cancer cells with a powerful growth advantage that would

prevail over an efficient ATP generation through OXPHOS [6].However, it is known that normal cells (e.g. lymphocytes) activateaerobic glycolysis when they are proliferating but come back to

OXPHOS when they are in a resting state [11]. If cells activateaerobic glycolysis in order to get a powerful growth advantage, it isnot clear why cancer cells keep this advantage while normal cells

come back to OXPHOS and give up this benefit. Although we can-not rule out that cancer cells and non-malignant proliferating cellsactivate aerobic glycolysis for different reasons, it seems sensible to

believe that there may be common reasons that explain the Warburgeffect in both cell types.

Established biochemical evidence indicates that, in addition togenerating ATP, the activation of glycolysis serves the key purpose

of providing carbon skeletons for biosyntheses. This implies thatthe activation of glycolysis is essential for cell proliferation, as cell

proliferation requires the synthesis of new molecules (e.g. nucleicacids, lipids, proteins) and as glycolysis provides most of the build-ing blocks required for the synthesis of these molecules [2-4]. Forinstance, as represented in Fig. (2), cells need to synthesize nucleicacids in order to proliferate (S-phase of the cell cycle). The starting point for nucleotide biosynthesis is the sugar ribose-5-phosphate,which is derived from the glycolytic metabolite glucose-6-phosphate. Likewise, the glycolyt ic metabolites glucose 6-phosphate, 3- phosphoglycerate, phosphoenolpyruvate and pyruvate are key pre-cursors in the biosynthesis of several amino acids (i.e. His, Ser,Cys, Gly, Tyr, Phe, Trp, Ala, Val, Leu) [4] required for the synthe-sis of proteins. In addition, the glycolytic metabolite dihydroxyace-tone phosphate is the key precursor of glycerol, a necessary me-

tabolite for the synthesis of lipids. Lipid formation also requiresfatty acids, and experimental data support that glucose-inducedcytosolic synthesis of acetyl CoA is required for the synthesis offatty acids [27-29].

The essential role of glycolysis in cell proliferation is supported by experimental observations that have revealed that cell prolifera-tion is inhibited in normal and cancer cells placed in glucose defi-

cient media containing high levels of alternative energy sources, orin cells treated with the non-metabolizable glucose analogue 2-deoxyglucose (3) [11, 30-32]. These observations support that can-

cer cells and non-malignant proliferating cells may activate glyco-lysis in the presence of O2 in order to proliferate (Fig. 2). In other

words, if glycolysis was always inhibited in the presence of O2,cells could not keep sustained proliferative rates under aerobic con-ditions. It is important to note that, despite being a powerful reason,

there is evidence that suggests that non-malignant proliferating cells

Fig. (1). Simplified diagram of the processes of glycolysis and oxidative phosphorylation (OXPHOS). Cells can produce ATP in the cytosol through glycolysis

by converting glucose into pyruvate. This process consumes NAD+, which can be regenerated by reducing pyruvate to lactate in a reaction catalyzed by the

enzyme lactate dehydrogenase (LDH). Pyruvate can also be used to produce ATP in the mitochondria through OXPHOS. Pyruvate passes the outer mitochon-drial membrane (OMM) through voltage dependent anion channels (VDAC). Pyruvate transport into the mitochondrial matrix across the inner mitochondrial

membrane (IMM) is driven by an electrochemical H+ gradient across this membrane. Once in the matrix, pyruvate dehydrogenase (PDH) catalyzes the conver-

sion of pyruvate to acetyl-CoA; this enzyme is inhibited by pyruvate dehydrogenase kinase (PDK). Acetyl-CoA is oxidized in the tricarboxylic acid cycle(TCA) to CO2, producing NADH and FADH2. The electrons of NADH and FADH2 are transported along the electron transport chain (ETC) to O 2. This elec-

tron transport drives the pumping of H

+

from the mitochondrial matrix to the intermembrane space. This generates an electrochemical H

+

gradient across theinner mitochondrial membrane, which is used by ATP synthase to phosphorylate ADP to form ATP.



The Warburg Effect Anti-Cancer Agents in Medicinal Chemistry, 2008 , Vol. 8, No. 3 307

and cancer cells do not activate aerobic glycolysis with the sole

purpose of proliferating [33]. For instance, the activation of aerobicglycolysis seems to play an important role in the protection againstcell death induced by reactive oxygen species (ROS) such as hy-

drogen peroxide [10, 34-37]. Likewise, it has been proposed that,although OXPHOS produces more energy per molecule of glucosethan glycolysis, glycolysis is capable of producing ATP considera- bly faster than OXPHOS as long as glucose supplies are unlimited.Growing cells have an enormous demand for ATP to fuel theirgrowth, and glycolysis seems much better suited to meeting thisdemand [27, 38].

3. HOW DO CANCER CELLS ACTIVATE GLYCOLYSIS IN

THE PRESENCE OF O2?

Recent reports have shown evidence that suggests that cancercells have an alteration in O2 metabolism (dysoxia or dysoxic me-tabolism), which may drive tumor growth, invasion and metastasis[20, 23, 39]. This section discusses that this alteration in O2 metabo-

lism may also play a key role in the Warburg effect. In the processof OXPHOS, ATP generation is coupled with a reaction in whichO2 is reduced to H2O. But under certain conditions, O2 can also be

reduced to H2O via the ROS superoxide anion (O2•–

) and hydrogen

peroxide (H2O2). Fig. (3) represents that a deviation of O2 metabo-lism from the route that generates ATP to the route that produces

O2•–

and H2O2 can activate aerobic glycolysis.

A decrease in O2 metabolism via OXPHOS can activate aerobicglycolysis. It is recognized that high ATP levels can repress glyco-lysis via allosteric inhibition of phosphofructokinase (PFK), a keyenzyme in the regulation of glycolysis [2]. The possible basis of the

Pasteur effect is that O2 allows ATP synthesis through OXPHOS;

this produces an allosteric inhibition of PFK resulting in glycolysis

inhibition [2, 39]. This implies that glycolysis is not directly inhib-ited by O2, but by ATP. It makes sense to think, therefore, that the presence of O2 will not cause the inhibition of glycolysis when O2

is not used to generate ATP. Accordingly, Fig. (3) proposes thatcells manage to activate glycolysis in the presence of O2 by reduc-ing O2 metabolism through OXPHOS. A decrease in O2 metabolismthrough OXPHOS would reduce ATP generation; this would re-lease PFK inhibition by ATP and would activate glycolysis. Theactivation of glycolysis would compensate the decreased ATP gen-eration through OXPHOS.

An increase in O2 metabolism through the ROS O2•–

and H2O2

may also produce aerobic glycolysis. It is known that O2•

can pro-duce intracellular alkalinization ( pH

i) [40, 41] and that intracellu-

lar alkalinization can activate glycolysis by increasing the activity

of PFK; the activity of this enzyme is extremely sensitive to smallchanges in pH in the physiological range, a high pH increasing theactivity of this enzyme) [39, 42, 43]. Although the activation of the

enzyme PFK is fundamental in the activation of glycolysis, cellsneed to increase the expression of glucose transporters (e.g.GLUTs) and glycolytic enzymes (e.g. hexokinase, PFK, pyruvatekinase) to keep sustained glycolytic rates. It is now well acceptedthat an increase in the cellular levels of H2O2 activates HIF-1 [39,44-46]. HIF-1 activation plays a key role in the transcription ofgenes that code for glucose transporters and glycolytic enzymes[17, 47-49]; indeed, it has been observed that cells lacking HIF-1exhibit decreased glycolytic capacity [48]. H2O2 can also activateAkt [50-52] and the oncogenes ras, src, and myc [53, 54], which areinvolved in the synthesis of glucose transporters and glycolytic

enzymes [55, 56]. Although Akt, ras and src are known to activate

Fig. (2). Key role of glycolysis in cell proliferation. Cell proliferation requires the presence of mitogenic signals and the synthesis of new macromolecules (e.g.nucleic acids, lipids, proteins). Glycolysis provides building blocks (e.g. glucose 6-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, phosphoe-

nolpyruvate, pyruvate) that participate in the synthesis of these macromolecules. If glycolysis was always inhibited in the presence of O 2 (Pasteur effect), cell

proliferation would be restricted under aerobic conditions. This may contribute to explain why normal proliferating cells and cancer cells activate glycolysis inthe presence of O2.




HIF-1 [17, 55], the possibility that H2O2-induced transcription ofglucose transporters and glycolytic enzymes may occur independ-ently of HIF-1 cannot be excluded [57].

Experimental observations support that many situations thathave been linked to the Warburg effect may be integrated in the

model represented in Fig. (3). As mentioned before, the first expla-nation to the Warburg effect was given by Otto Warburg, who proposed that this phenomenon was caused by irreversible damages toOXPHOS [5]. While some observations support that cancer cells

have alterations in proteins involved in OXPHOS [7, 24-26], otherssuggest that the Warburg effect is not caused by irreversible dam-ages to OXPHOS [21, 27]. All these observations can be integrated

in the model shown in Fig. (3), which proposes any situation that produces a decrease in OXPHOS activity in the presence of O2 can

produce the Warburg effect.

It has been discussed recently that cancer cells have increasedrates of aerobic glycolysis because of an adaptation to intermittent

hypoxia [6]. It is known that intermittent hypoxia increases O2•

generation [58, 59] and, according to the model represented in Fig.(3), an increased O2

• generation would lead to the activation of

aerobic glycolysis. The present model also agrees with the proposalthat intracellular alkalinization is involved in the Warburg effect[60-62], as intracellular alkalinization may cause OXPHOS repres-

sion [39] and glycolysis activation [42, 60, 61]. Accordingly, experimental data suggest that intracellular alkalinization may inacti-

vate p53 [63], and p53 inactivation may cause the Warburg effect[26]. Although it has been shown that IL-3 can increase glycolysisthrough activation of the Akt/Pim kinases [33, 64], IL-3-induced

activation of glycolysis might also be integrated in Fig. (3), as IL-3can increase O2

• generation [65] and the intracellular pH [66].

Fig. (3). A deviation of O2 metabolism from the route that generates ATP(OXPHOS) to the route that produces O2

•– and H2O2 may cause the activa-

tion of glycolysis in the presence of O2 (aerobic glycolysis or Warburgeffect). This deviation of O2 metabolism would avoid the inhibition of gly-colysis induced by the presence of O2 (Pasteur effect).

The increased generation of H2O2 caused by the alteration in O2 metabolism shown in Fig. (3) might explain the activation of aerobic glycolysis induced by several apparently unrelated factors. Be-cause the activation of HIF-1, akt, ras, src and myc is involved inthe synthesis of glucose transporters and glycolytic enzymes, these

factors have been linked to the activation of aerobic glycolysis [15,17, 55, 56]. Interestingly, it has been shown that H2O2 can activateHIF-1 [44-46], akt [50-52], ras, src, and myc [53, 54]. The activityof the Na

+/K

+-ATPase pump has also been associated with the acti-

vation of aerobic glycolysis [67, 68], and it has been observed that

H2O2 can activate Na

+

/K

+

-ATPase [69] and that catalase preventsthe activation of this pump [70]. In brief, it seems that many of thesituations that have been linked to the activation of aerobic glycoly-sis may be integrated in the model represented in Fig. (3).

HIF-1 is a heterodimeric transcription factor that consists of aconstitutively expressed HIF-1 subunit and a HIF-1 subunit, theexpression of which is highly regulated. HIF-1 overexpression has

been observed in the most common cancer types, and is thereforeconsidered a potential target for cancer therapy [17, 71]. The activa-

tion of HIF-1 seems to play an important role in the metabolicswitch from OXPHOS to aerobic glycolysis (Warburg effect), asthis transcription factor not only can activate aerobic glycolysis but

also repress OXPHOS [72, 73]. Indeed, experimental data haveshown that the activation of HIF-1 mediates the expression of pyru-vate dehydrogenase kinase (PDK); this results in pyruvate dehydro-

genase (PDH) inhibition, decreased conversion of pyruvate to ace-tyl-CoA, reduced activity of the tricarboxylic acid cycle and subse-quent oxphos repression [72, 73] (Fig. 1). A recent article has re-

viewed that, in clear cell renal carcinoma (an perhaps in other hu-man cancers), HIF-1 mediates increased glucose uptake, increasedlactate production, and decreased OXPHOS, thus delineating for

the first time the molecular mechanisms underlying the switch fromoxidative to glycolytic metabolism in human cancer [49]. Recentevidence also suggests that an alteration in O2 metabolism (dysoxia)may be the main mechanism responsible for HIF-1 activation underhypoxic and aerobic conditions [39]; this supports the idea that analteration in O2 metabolism may cause the Warburg effect.

According to the model shown in Fig. (3), cancer cells wouldactivate glycolysis in the presence of O2 because of a deviation of

O2 metabolism from the route that generates ATP to the route that

produces O2

•–

and H2O2. This alteration in O2 metabolism wouldactivate aerobic glycolysis by producing OXPHOS repression, in-

creased generation of O2•–

and H2O2, intracellular alkalinization,and HIF-1 activation (see Fig. 3). This model is supported by experimental data that have revealed that most cancers have HIF-1overexpression [17, 18], alkaline intracellular pH values (7.12–7.65compared with 6.99–7.20 in normal tissues) [60, 62, 74], excessivegeneration of O2

• and H2O2 [75-78] and structural alterations in

OXPHOS that may cause OXPHOS repression [7, 24-26].

4. GLYCOLYSIS INHIBITION: STRATEGIES AND DRUGS

The attenuation or inhibition of glycolysis in tumor cells may be exploited for the development of cancer chemopreventive andchemotherapeutic strategies. On the one hand, as illustrated in Fig.(2) and discussed elsewhere [20, 22, 23], evidence suggests thatglycolysis is essential for cell proliferation, tumor invasion andmetastasis. The reduction of the glycolytic capacity of tumor cellswould restrict their ability to proliferate, invade adjacent tissues andmigrate to distant organs. This suggests that the attenuation of gly-colysis in tumor cells may represent a useful strategy for preventingor stopping the development of cancer (i.e. cancer chemopreven-tion). On the other hand, evidence supports that the activation ofglycolysis protects cells from H2O2-induced cell death [10, 34-37],and that cancer cells are more susceptible to H2O2-induced cell

death than normal cells [78, 79]. In addition, cancer cells are moredependent on glycolytic ATP than normal cells [7, 8, 12-14]. Thissuggests that the inhibition of glycolysis may produce selective

killing of cancer cells and be a practical strategy for cancer chemo-therapy (see ref. [105]).

Several strategies and drugs can be used to inhibit the glycolyticmetabolism of cancer cells. The fist and perhaps most straightfor-

ward way of inhibiting glycolysis is reducing the blood and intersti-




tial glucose levels by using insulin therapy. Mice can survive dosesof human insulin that results in a decrease of blood glucose by oneorder of magnitude (approximately, from 240 mg/dL to 24 mg/dL).The effect of insulin on xenograft tumors is currently under investi-gation [80].

Inhibition of glycolytic enzymes is another strategy that mayresult in glycolysis inhibition in tumor cells. As illustrated in Fig.(2), hexokinase (HK), phosphofructokinase (PFK) and pyruvatekinase (PK) catalyze the three irreversible reactions of glycolysis;these enzymes are important control sites of glycolysis [2]. HK isthe fist and rate-limiting reaction in glycolysis and catalyses the phosphorylation of glucose to glucose-6-phosphate (G6P). This stepconverts a non-ionic molecule (glucose) to an anion (G6P) that istrapped in the cells. Lonidamine (1), a derivative of indazole-3-carboxilic acid that is currently undergoing clinical trials for thetreatment of benign prostatic hyperplasia, can inhibit the phosphorylation of glucose by HK. This drug is currently undergoingclinical trials in combination with other anticancer agents for thetreatment of different types of cancer [7, 80]. The pyruvate analog3-bromopyruvate (3-BrPA) (2) is a HK inhibitor that has shown

potent anticancer activity in preclinical studies [12, 81]. The glucoseanalog 2-deoxyglucose (2-DG) (3) is a competitive inhibitor ofglucose metabolism that competes with glucose for HK. Once 2-DG (3) is phosphorylated by HK, it cannot be metabolized further;

this leads to accumulation of 2-DG (3) within the cell and results inglycolysis inhibition [7]. This agent, however, seems to producetoxic effects when used as a primary therapy and it is currently

being explored in combination with other anticancer agents [82].Evidence suggests that HK and glucose-6-phosphate 1-dehydro-genase (G6PDH) are primary targets of imatinib (Gleevec) (4), an

anticancer drug already approved for clinical use [83]. As men-tioned before, the activity of PFK is extremely sensitive to smallchanges of pH in the physiological range, a high pH increasing the

activity of this enzyme [39, 42, 43]. Evidence suggests that cancercells have high intracellular pH values, which may activate PFKand contribute to explain the high glycolytic rates of these cells [60-

62]. Activation of H+ extruders such as the Na+/H+-exchanger NHE-1 has been shown to play a key role in the elevation of the intracel-

lular pH in cancer cells. Inhibitors of NHE-1, such as amiloride or5,5-dimethylamiloride (DMA) (5), may therefore reduce the intra-cellular pH, PFK activity and glycolysis, and produce anticancer

effects. Indeed, these two drugs have shown anticancer effects in

vivo [60, 84, 85]. In addition, it is well known that PFK is inhibited by citrate [3]. The enzyme ATP citrate lyase (ACL) catalyzes the

conversion of citrate to cytosolic acetyl-CoA. Inhibitors of ATPcitrate lyase (ACL), such as SB-204990 (6), may cause citrate to build up and inhibit glycolysis [14, 29, 86]. It has been shown that

SB-204990 (6) limits in vitro proliferation and survival of tumorcells displaying aerobic glycolysis and reduces in vivo tumorgrowth [29]. Inhibition of the enzyme pyruvate kinase (PK), whichcatalyzes the third irreversible reaction of glycolysis (Fig. 2), repre-

sents another strategy for inhibiting glycolysis; this enzyme seemsto play an important role in tumor growth and invasion, and itsinhibition may produce anticancer effects [87].

Fig. (1) shows that the enzymes lactate dehydrogenase (LDH),

pyruvate dehydrogenase (PDH) and pyruvate dehydrogenase kinase(PDK) play important roles in glycolysis and OXPHOS. The en-zyme LDH catalyzes a reaction by which pyruvate is reduced to

lactate; this reaction permits the regeneration of NAD+, needed for

glycolysis to continue (Fig. 1). Recent observations showed thatattenuation of LDH reduced the glycolytic metabolism of cancercells and produced antitumor effects in animals [21, 27]. Theauthors discussed that, because individuals with complete defi-ciency of LDH do not show any symptoms under ordinary circum-stances, the inhibition of LDH activity may represent a relativelynontoxic approach to interfere with tumor growth [21]. Deck et al .

synthesized several dihydroxynaphthoic acids that were potent in-

hibitors of LDH [88]; these drugs might inhibit glycolysis and dis-

play anticancer effects. On the other hand, recent evidence suggeststhat HIF-1 activation mediates the expression of PDK [72, 73]; thisenzyme repress OXPHOS by inhibiting PDH (Fig. 1) and maytherefore play a role in the activation of aerobic glycolysis. Inhibi-

tion of PDK may therefore attenuate glycolysis and produce anti-cancer effects. Several reports have shown that different groups ofcompounds are inhibitors of PDK [89-95]; these drugs may inhibitglycolysis and produce anticancer effects. Accordingly, a recentstudy have shown that the PDK inhibitor dichloroacetate (DCA) (7) produced marked anticancer effects [95]; DCA (7) is a known gly-colysis inhibitor that has been used in humans for decades in thetreatment of lactic acidosis and inherited mitochondrial diseases.The authors observed that DCA (7) in the drinking water at clini-cally relevant doses for up to 3 months prevented and reversed tu-mor growth in vivo, without apparent toxicity and without affectinghemoglobin, transaminases, or creatinine levels. They concludedthat the ease of delivery, selectivity, and effectiveness make DCA(7) an attractive candidate for cancer therapy which can be rapidlytranslated into phase II–III clinical trials.

Another strategy for reducing the increased glycolytic rates oftumor cells is the inhibition of the synthesis of glucose transportersand glycolytic enzymes. As discussed before, the activation of HIF-

1 plays a key role in the expression glucose transporters and glyco-lytic enzymes. Inhibition of HIF-1 may therefore reduce the persis-tent activation of aerobic glycolysis in tumor cells and produce

anticancer effects. Preclinical studies have already shown that inhibition of HIF-1 activity has marked effects on tumor growth [17].HIF-1 is considered a potential target for cancer chemoprevention[19] and therapy [17] and, recently, many efforts to develop newHIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories [17, 18, 96, 97]. As a result,several FDA-approved anticancer drugs (e.g. topotecan, imatinib(4), trastuzumab, NS398, celecoxib, ibuprofen) have been found toinhibit HIF-1 activity [18]. Several natural products (e.g. resvera-trol, genistein, apigenin, berberin) have also been found to inhibitthe activity of this transcription factor [18, 97].

Several other drugs have shown ability to inhibit glycolysis,including oxythiamine, genistein, 5-thioglucose, mannoheptulose,-chlorohydrin, ornidazole, glufosfamide, or arsenic compounds(see references [7, 8]). Oxamate (an analogue of pyruvate that blocks the step of glycolysis that converts pyruvate to lactic acid)and iodoacetate (an inhibitor of glyceraldehyde 3-phosphate dehy-drogenase) may also produce anticancer effects [98-100]. Bisphos- phonates [101] or tubercidin [102] may also inhibit glycolysis incancer cells. As mentioned before, the activity of the Na

+/K

+-

ATPase pump has also been associated with the activation of aerobic glycolysis [67, 68]. Cardiac glycosides, such as ouabain (8) ordigitoxin, are known inhibitors of the Na

+/K

+-ATPase pump that

have shown anticancer effects [103-105].

Finally, Fig. (3

) implies that the glycolytic capacity of cancercells may be restricted by preventing or reducing excessive cellularlevels of O2

• and H2O2. It has been demonstrated that the use of

antioxidants, such as the enzyme catalase, prevents the activation ofHIF-1 induced by hypoxia and non-hypoxic stimuli [44-46]. SinceHIF-1 plays a key role in the activation of glycolysis, the use of

antioxidants would prevent HIF-1 activation; this would attenuatethe glycolytic capacity of tumor cells and would prevent the devel-opment of invasive cancers [23].

5. CONCLUSIONS

Cells can obtain energy through the oxygen-dependent pathwayof OXPHOS and through the oxygen-independent pathway of gly-colysis. Since glycolysis is less efficient in generating ATP thanOXPHOS, it is comprehensible that glycolysis is inhibited in cellsunder aerobic conditions (Pasteur effect). It is not well understood,however, why cancer cells and non-malignant proliferating cells




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Received: 22 May 2007 Revised: 22 June 2007 Accepted: 25 June 2007

2008. ACA-MC. the Warburg Effect

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