The Warburg Effect and Mitochondrial Stability in Cancer Cells

Molecular Aspects of Medicine 31 (2010) 60–74

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Molecular Aspects of Medicine

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Review The Warburg effect and mitochondrial stability in cancer cells

Vladimir Gogvadze, Boris Zhivotovsky, Sten Orrenius *

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, Stockholm SE-171 77, Sweden article info abstract

Article history: The last decade has witnessed a renaissance of Otto Warburg’s fundamental hypothesis, Received 22 June 2009 which he put forward more than 80 years ago, that mitochondrial malfunction and subse- Received in revised form 31 July 2009 quent stimulation of cellular glucose utilization lead to the development of cancer. Since Accepted 2 December 2009 most tumor cells demonstrate a remarkable resistance to drugs that kill non-malignant cells, the question has arisen whether such resistance might be a consequence of the abnormalities in tumor mitochondria predicted by Warburg. The present review discusses Keywords: potential mechanisms underlying the upregulation of glycolysis and silencing of mitochon- The Warburg effect drial activity in cancer cells, and how pharmaceutical intervention in cellular energy Apoptosis Mitochondria metabolism might make tumor cells more susceptible to anti-cancer treatment. Cancer Ó 2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction: The Warburg effect vs. the Pasteur effect – historical background and current views...... 61 2. Possible mechanisms of mitochondrial silencing in cancer cells ...... 62 2.1. The Crabtree effect ...... 62 2.2. HIF and suppression of mitochondrial activity ...... 62 2.3. Importance of ROS in cancer ...... 63 3. Consequences of the glycolytic switch in tumors ...... 64 4. Mitochondrial stabilization in glycolytic tumor cells...... 65 4.1. The role of Bcl-2 proteins in OMM permeabilization ...... 65 4.2. Hexokinase and OMM stability ...... 67 4.3. ANT and OMM stability ...... 67 4.4. Akt-mediated OMM stability ...... 68 4.5. Contribution of p53 to OMM stability ...... 68 5. Stimulation of cancer cell death via intervention with cellular ATP-generating pathways...... 69 5.1. Suppression of the glycolytic pathway ...... 69 5.2. Shifting metabolism from glycolysis to glucose oxidation ...... 70 5.3. Alteration of mitochondrial functions ...... 70 6. Concluding remarks ...... 71

Abbreviations: ACL, ATP citrate lyase; ANT, adenine nucleotide translocase; ATRA, All-Trans retinoic acid; 3-BrPA, 3-bromopyruvate; COX, cytochrome oxidase; CsA, cyclosporin A; CyPD, cyclophilin D; DCA, dichloroacetate; HIF-1, hypoxia-inducible factor-1; IMM, inner mitochondrial membrane; LDH, lactate dehydrogenase; mtDNA, mitochondrial DNA; OMM, outer mitochondrial membrane; PDH, pyruvate dehydrogenase; PDK1, pyruvate dehydrogenase kinase 1; PPIase, peptidyl-prolyl cis–trans isomerases; MPT, mitochondrial permeability transition; PI3-K, phosphoinositide 3-kinase; ROS, reactive oxygen species; SCO2, Synthesis of cytochrome c oxidase 2; TIGAR, TP53-Induced glycolysis and apoptosis regulator; a-TOS, a-tocopheryl succinate; VDAC, voltage- dependent anion channel. * Corresponding author. E-mail address: [email protected] (S. Orrenius).

Acknowledgements ...... 71 References ...... 71

1. Introduction: The Warburg effect vs. the Pasteur effect – historical background and current views

In 1926, Otto Warburg reported that cancer cells produce most of their ATP via glycolysis, also under aerobic conditions (Warburg, 1926). This finding contradicted the Pasteur effect, named after Louis Pasteur, who found that in most mammalian cells the rate of glycolysis decreases significantly in the presence of oxygen. Glycolytic production of ATP under aerobic conditions, the Warburg effect, was found to be a characteristic of most cancer cells, and this finding was confirmed in various laboratories. Many cancer cell lines were also shown to rely on glycolysis for ATP production, and typically, the most glycolytic tumor cells were also found to be the most aggressive ones (Simonnet et al., 2002). Warburg originally assumed that aerobic glycolysis was a universal property of malignant cells due to defects in mitochondrial respiration, and he suggested that cancer might in fact be caused by impaired mitochondrial metabolism. In his lecture at the meeting of Nobel Laureates at Lindau, Lake Constance in June 1966, he stated: ‘‘Cancer, above all other diseases, has countless secondary causes. But, even for cancer, there is only one prime cause. Summarized in a few words, the prime cause of cancer is the replacement of the respiration of oxygen in normal body cells by a fermentation of sugar” (Warburg, 1967). He further hypothesized that moderate doses of ionizing radiation might reduce the activity of mitochondria in tumor cells below a threshold level essential for cell survival, whereas mitochondria in normal cells would still be able to produce ATP, and that inhibition of oxidative phosphorylation could be a tool in cancer cell elimination. Indeed, in some forms of cancer, analysis of possible alterations in the oxidative phosphorylation machinery revealed down-regulation of the catalytic subunit of the mitochondrial ATP synthase (b-F1-ATPase) (Isidoro et al., 2005). Remarkably, the expression level of the b-F1-ATPase protein was found to inversely correlate with the rate of aerobic glycolysis. A link between mitochondrial dysfunction and upregulation of glycolysis could also be demonstrated in an in vitro model, when inhibition of oxidative phosphorylation by oligomycin in lung carcinoma cells was found to trigger a rapid increase in aerobic glycolysis. This observation suggests that cells might become glycolytic as a result of suppression of mitochondrial energy production (Lopez-Rios et al., 2007). Similarly, inhibition of respiration in two human lung cancer cell lines, H460 and A549, significantly upregulated glycolysis. In contrast, when glycolysis was suppressed, the cells were unable to sufficiently upregulate mitochondrial oxidative phosphorylation, which might have been a sign of mitochondrial dysfunction in these tumor cells (Wu et al., 2007). In contrast to these findings, other studies have demonstrated that tumor mitochondria are fairly functional with regards to respiration and ATP synthesis, exhibiting normal P/O and respiratory control ratios, and normal capabilities for the oxidation of respiratory substrates (Eakin et al., 1972). In fact, in some tumors, oxygen consumption was found to be similar, or even higher than in the normal tissue. Hence, in a critical analysis of Warburg’s theory, Sidney Weinhouse concluded: ‘‘no substantial evidence has been found that would indicate a respiratory defect, either in the machinery of electron transport, or in the coupling of respiration with ATP formation, or in the unique presence or absence of mitochondrial enzymes or cofactors involved in electron transport” (Weinhouse, 1976). In accordance with this, a comprehensive study of mitochondrial function in slowly growing hepatomas showed almost no differences in respiratory parameters as compared to normal liver mitochondria (Pedersen et al., 1970). Mitochondria isolated from the ‘‘minimal deviation” hepatomas were also very similar in their electron transport activity. On the other hand, mitochondria isolated from rapidly growing hepatomas had diluted-appearing matrices and were incapable of oxidizing b-hydroxybutyrate, although oxidation of succinate oc- curred at rates comparable to, or even higher than in liver mitochondria. Mitochondria from these rapidly growing hepatomas were able to phosphorylate added ADP, but they exhibited lower respiratory control ratio, indicative of increased proton permeability of the inner mitochondrial membrane (IMM). Thus, mitochondrial dysfunction can, indeed, be observed in tumors, particularly in the more aggressive and rapidly growing forms. When fast-growing tumors shift their ATP production towards glycolysis, mitochondrial activity slows down. Is this because hypoxic conditions trigger destructive processes in the mitochondria, or do they remain functional after normalization of oxygen supply? In other words, is the Pasteur effect rather than the Warburg effect responsible for mitochondrial silencing (Zu and Guppy, 2004)? As mentioned above, Warburg originally suggested that aerobic glycolysis in cancer cells might re- flect defects in what we now know is mitochondrial oxidative phosphorylation; however, the severity of the anticipated mitochondrial damage was probably rather exaggerated. Nowadays, the Warburg effect is generally explained by alterations in signaling pathways that govern glucose uptake and utilization, which are also involved in the regulation of mitochondrial activity, rather than by mitochondrial defects, although some aggressive tumors do display mitochondrial deterioration. However, independently of mitochondrial damage, most tumor cells do display high rates of glycolysis, aerobic or anaerobic, and this property is now widely used for their visualization by positron emission tomography. Mitochondrial function in cell metabolism is, however, not restricted to ATP production for cellular demands. Mitochon- dria also generate reactive oxygen species (ROS), which normally participate in the regulation of multiple physiological processes, but might be harmful if produced excessively. For example, ROS are critically involved in the regulation of cell death pathways, apoptosis as well as necrosis. In fact, cell death signaling has emerged as a second major function of the mitochon- 62 V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 dria, and the resistance of tumor cells to apoptotic cell death can often be related to deficiencies in the ‘‘internal” (mitochondrial) pathway of apoptosis signaling. Thus, characterization of mitochondrial alterations in tumor cells is also important for the development of more efficient chemotherapy.

2. Possible mechanisms of mitochondrial silencing in cancer cells

2.1. The Crabtree effect

Low mitochondrial contribution to cellular ATP production is not a characteristic of tumor cells only, but is also observed in a variety of fast-growing normal cells (Wang et al., 1976). One of the ﬁrst demonstrations of inhibition of mitochondrial respiration by stimulated glycolysis, a phenomenon known as the Crabtree effect (reviewed in (Ibsen, 1961)), was made in cells with approximately equal glycolytic and respiratory capacities for ATP synthesis. Thus, the Crabtree effect is absent in thymocytes in the resting state, whereas thymocytes in the proliferative state demonstrate a signiﬁcantly higher rate of glycolysis and a strong glucose-induced inhibition of oxygen consumption (Guppy et al., 1993). Various mechanisms have been suggested to explain the Crabtree effect in tumor cells: (a) competition between oxidative phosphorylation and glycolysis for ADP and inorganic phosphate (Sussman et al., 1980); (b) a decrease in the cytosolic pH, as a consequence of lactic acid formation, that diminishes the activity of oxidative enzymes (Heinz et al., 1981); (c) damage of the mitochondrial membranes by free radicals, produced as a consequence of glucose catabolism (Yang et al., 1997b); and (d) glucose-induced release of Ca2+ from the endoplasmic reticulum followed by enhanced uptake of Ca2+ by the mitochondria, which would inhibit ATP synthase (Wojtczak et al., 1999). The latter explanation would be in agreement with the observed elevation of the Ca2+ content in tumor mitochondria. Apparently, all four mechanisms could be involved in the Crabtree effect, but their relative contribution to mitochondrial suppression might vary between different tumors.

2.2. HIF and suppression of mitochondrial activity

Fast proliferation is one of the main characteristics of cancer cells. This leads to hypoxia in tumors due to inability of the local vascular system to provide adequate oxygenation. Thus, the tumor cells must adapt to these unfavorable conditions until new blood vessels are formed, and the oxygen tension is normalized. Ischemic conditions are usually lethal to non- malignant cells due to hypoxia-mediated, p53-dependent cell death. However, tumor cells can survive under hypoxic conditions because of mutations in p53, or its low expression (Moll and Schramm, 1998). Due to the inability of the mitochondria to provide enough ATP for cell survival under hypoxic conditions, tumor cells must upregulate the glycolytic pathway. This occurs via induction of hypoxia-inducible factor-1 (HIF-1) (Wang and Semenza, 1993). HIF-1 is a heterodimer that consists of a constitutively expressed HIF-1b subunit and a HIF-1a subunit, the expression of the latter is highly regulated and is determined by the relative rates of its synthesis and degradation. Synthesis of HIF-1a is regulated via oxygen-independent mechanisms, whereas its degradation is oxygen-dependent (Semenza, 2003). Specifically, in the presence of oxygen, the HIF- 1a chains are polyhydroxylated on conserved prolyl and arginyl residues by oxygen-dependent prolyl and arginyl hydroxylases. Once hydroxylated, HIF-1a protein binds to von Hippel–Lindau tumor suppressor protein (VHL) – the recognition component of E3 ubiquitin-protein ligases. Ubiquitinated HIF-1a is rapidly degraded by the proteasomes. Under conditions of low oxygen tension (hypoxia, ischemia), hydroxylation is suppressed which causes stabilization of HIF-1a and its accumulation in the nucleus, where it forms a complex with the constitutively expressed HIF-1b (Semenza, 2004). HIF-1 induces genes that control crucial features of cancer biology, including angiogenesis, glucose metabolism and cell proliferation and invasion. In particular, HIF-1 stimulates key steps of glycolysis, such as glucose transporters and hexokinases, which catalyze the first reaction of glycolysis – glucose phosphorylation. It should be mentioned, however, that in certain tumors, high levels of HIF-1 are observed also in well-oxygenated medium, indicating that there are other factors than hypoxia that can cause HIF-1 stabilization (Hagg and Wennstrom, 2005). Thus, in vascular smooth muscle cells, stabilization of HIF-1 was observed after treatment with several hormones and growth factors, such as fetal calf serum, angiotensin II, thrombin, platelet-derived growth factor, etc., to levels that were substantially higher than those observed during hypoxia (Richard et al., 2000). HIF-1 does not only stimulate glycolysis in tumor cells. In addition, it was shown to suppress the respiratory activity of mitochondria, suggesting that HIF-1 can function as a switch between glycolysis and oxidative phosphorylation. There are several possible mechanisms for such regulation. The mode of cellular ATP production depends on the fate of the end-product of glycolysis – pyruvate. The fate of pyruvate depends, in turn, on the relative activities of two enzymes, pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH). The ‘‘gate-keeping” mitochondrial enzyme, PDH converts pyruvate into acetyl CoA, which enters the Krebs cycle where it undergoes stepwise transformation. The activity of PDH is under control of pyruvate dehydrogenase kinase 1 (PDK1), the enzyme that phosphorylates and inactivates PDH. HIF-1 was shown to induce PDK1 and thereby to inactivate PDH and, as a result, to suppress the Krebs cycle and mitochondrial respiration (Kim et al., 2006; Papandreou et al., 2006). Under these circumstances, instead of supplying reduced equivalents to mitochondria, pyruvate undergoes conversion into lactate, reoxidizing cytosolic NADH and facilitating continued glycolysis. In addition to the inhibition of mitochondrially-directed pyruvate oxidation, HIF-1 was shown to stimulate expression of the LDH-A gene (Semenza et al., 1996), whose product converts pyruvate into lactate. LDH is a tetrameric enzyme with five isoforms composed of combinations of two subunits, LDH-A and LDH-B. The LDH-A subunit converts pyruvate to lactate V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 63 under anaerobic conditions in normal cells. The other isoenzyme, LDH-B, kinetically favors the conversion of lactate to pyruvate and is expressed at high levels in aerobic tissues, such as the heart. Taken together, these factors suppress the delivery of acetyl-CoA to the Krebs cycle and mitochondrial respiration in tumors. Suppression of pyruvate oxidation through HIF-1- mediated PDK1 upregulation can, in turn, serve as a mechanism that protects cells from toxic production of ROS. Hence, in HIF-1-deficient mouse embryonic fibroblasts the level of ROS increased drastically, leading to cell death. ROS levels and cell death were markedly reduced in subclones transfected with an expression vector encoding PDK1 (reviewed in (Semenza, 2007)). These results clearly indicate that stimulation of the mitochondrial pathway of energy production could be an efficient tool in tumor cell killing. Another possible route, by which HIF-1 can modulate mitochondrial functioning in tumors, in particular the activity of the mitochondrial respiratory chain, is by its involvement in the regulation of cytochrome oxidase (COX). COX, located in the IMM, represents a dimer, in which each monomer consists of 13 subunits (Tsukihara et al., 1996). Mammalian cells ex- press a predominant COX4–1 isoform. An alternative COX4–2 isoform is also expressed, but only in some tissues. Thus, Northern analysis and quantitative PCR of human and rat tissues revealed high COX4–2 expression in the adult lung and lower expression in all other tissues investigated, including the fetal lung (Huttemann et al., 2001). Under hypoxic conditions, the expression of the COX4–2 subunit is increased, optimizing COX activity, whereas the COX4–1 subunit, which is responsible for COX activity under aerobic conditions, is downregulated (Fukuda et al., 2007). Degradation of COX4–1 occurs via activation of LON, a mitochondrial protease. Apparently, switching between COX4 subunits provides a mechanism to maintain the efficiency of respiration under conditions of reduced oxygen availability and may be involved in the adaptive response to hypoxia. A signal for stimulation of glycolysis via HIF-1 induction can arise from mitochondria as well. Thus, when mitochondrial activity it tumor cells is downregulated, due to either hypoxic conditions or malfunction of respiratory chain complexes, accumulation of Krebs cycle substrates, such as fumarate and succinate, might serve as a signal for the upregulation of glycolysis (Gottlieb and Tomlinson, 2005). Succinate was shown to inhibit HIF-1a prolyl hydroxylases in the cytosol, leading to stabilization and activation of HIF-1a. Succinate accumulation in mitochondria results from inhibition of succinate dehydrogenase, which catalyses the oxidative dehydrogenation of succinate coupled to the reduction of ubiquinone. Mutations in this enzyme are principally involved in familial predisposition to benign tumors, therefore, succinate dehydrogenase can be considered as a classical tumor suppressor (Baysal et al., 2000; Lu et al., 2002). A similar stabilizing effect was described for lactate and pyruvate (Lu et al., 2002). Although both lactate and pyruvate were found to accumulate in cancer cells and to increase HIF-1 protein level, lactate appeared to require conversion to pyruvate for its effect.

2.3. Importance of ROS in cancer

ROS accumulation is one of the factors leading to mitochondrial dysfunction. ROS are tumorigenic by their ability to increase cell proliferation and migration, but also by inducing genetic lesions that can initiate tumorigenicity and sustain subsequent tumor progression (Storz, 2005). A multitude of cancer cells are characterized by elevated ROS level. What are then the mechanisms of stimulated ROS production in tumors? In most cells, the majority of ROS are byproducts of mitochondrial respiration. Approximately 2% of the molecular oxygen consumed during respiration are converted into superoxide radical – the precursor of most ROS. The mitochondrial electron transport chain contains several redox centers that may leak electrons to molecular oxygen, serving as the primary source of superoxide production in most tissues. The one-electron reduction of oxygen is thermodynamically favorable for most mitochondrial oxido-reductases (Turrens, 2003). Superoxide-producing sites and enzymes were recently analyzed in detail in a comprehensive review (Andreyev et al., 2005). ROS, if not detoxified, oxidize cellular proteins, lipids, and nucleic acids and, by doing so, cause cellular dysfunction or death. The harmful ROS activity is suppressed by a cascade of water- and lipid-sol- uble antioxidants and antioxidant enzymes. An imbalance that favors the production of ROS over antioxidant defense is generally defined as ‘‘oxidative stress” and is implicated in a wide variety of pathologies, including malignant disease. It should be kept in mind, however, that mitochondria are not only a major source of ROS, they are also a sensitive target for the damaging effects of oxygen radicals. ROS produced by mitochondria oxidize proteins and induce lipid peroxidation, compromising the barrier properties of mitochondrial membrane. One of the targets of ROS is mitochondrial DNA (mtDNA), which encodes several proteins essential for mitochondrial respiratory chain function and ATP synthesis by oxidative phosphorylation (Anderson et al., 1981). Therefore, mtDNA represents a critical cellular target for oxidative damage that could lead to lethal cell injury through the loss of mitochondrial electron transport, membrane potential and ATP generation. mtDNA is especially susceptible to attack by ROS owing to its close proximity to the electron transport chain, the major locus for free radical production, and the lack of protective histones. For example, mitochondrially generated ROS can trigger the formation of 8-hydroxydeoxy-guanosine as a result of oxidative DNA damage; the level of oxidatively modified bases in mtDNA is almost 15-fold higher than that in nuclear DNA (Richter et al., 1988). Oxidative damage induced by ROS is probably a major source of mitochondrial genomic instability leading to respiratory dysfunction. The hypoxic environment of proliferating tumor tissue facilitates ROS production. Cellular hypoxia and reoxygenation are two essential elements of ischemia/reperfusion injury. Massive production of ROS is normally observed in reoxygenation of hypoxic tissue. However, the ROS level also may increase by hypoxia, when electron transport complexes are in the reduced state (Guzy and Schumacker, 2006). Therefore, especially after normalization of oxygen supply, the production of ROS in tumor cells maintained under hypoxic conditions can be enhanced and cause damage to vital cell components, including 64 V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 mitochondrial DNA. This might trigger a vicious cycle: hypoxia – ROS production – mtDNA mutations – malfunction of the mitochondrial respiratory chain – further stimulation of ROS production, etc. Even though enhanced formation of ROS in cancer cells has been documented in numerous publications, there is still some uncertainty about the extent of this process in tumors. A majority of malignant cells do indeed produce considerable amounts of ROS when cultured under conditions, which can be regarded as hyperoxic (about 159 mmHg) (Halliwell, 2007). However, the absence of antioxidants in the culture medium, and the presence of iron in some media, might contribute to the excessive rate of ROS formation seen under in vitro conditions. On the other hand, the elevated level of ROS in tumors can be a consequence of suppressed activity of antioxidant enzymes responsible for detoxication of ROS. Thus, the level of MnSOD, catalyzing the conversion of superoxide to hydrogen peroxide in mitochondria, seems to be lowered in certain cancer cells, and stimulated expression of Mn-SOD appears to suppress malignant phenotypes in some experimental models (Behrend et al., 2003). Alterations of glutathione S-transferases in tumor cells have also has been reported, suggesting an association between cancer incidence and various disorders of GSH-related enzyme functions (Pastore et al., 2003). Another uncertainty about massive ROS production in tumors is related to the observation that cellular production of ROS was shown to be inversely correlated to the state of proliferation of the cell and dependent on the mode of ATP supply. Thus, in proliferating thymocytes, producing 86% of their ATP by glycolysis, PMA-induced ROS production was nearly abolished. This was in contrast to the finding with resting thymocytes, producing ATP (88%) mainly by oxidative phosphorylation. Sim- ilarly, no ROS formation was observed in proliferating human promyelocytic HL-60 cells, whereas differentiated, non-proliferating HL-60 cells showed a marked response upon priming with PMA (Brand and Hermfisse, 1997). The observed reduction of ROS formation by resting thymocytes incubated with pyruvate suggested that pyruvate might function as a H2O2 scaven- ger, indicating that when the mitochondrial pathway is suppressed, accumulation of pyruvate can control the level of ROS in tumor cells. In accordance with this hypothesis, pyruvate protected mitochondria from oxidative stress in neuroblastoma cells (Wang et al., 2007) and prevented DNA damage by hypoxia/reoxygenation in hepatocellular carcinoma cells (Roudier et al., 2007). Stimulation of ROS production as a result of glucose deprivation caused inhibition of cell proliferation (Aulwurm and Brand, 2000). Conversely, enhanced glycolysis was reported to protect cells from oxidative stress, which might be a potential mechanism of cellular immortalization (reviewed in (Kondoh et al., 2007). Immortalized mouse embryonic fibroblasts (MEFs) suffered less oxidative damage than control cells, as estimated by cytosolic ROS staining or quantification of 8- hydroxydeoxy-guanosine, a hallmark of oxidative DNA damage (Kondoh et al., 2005). Therefore, extrapolation from cell cultures to tumors in vivo must be made with caution. Tumor cells certainly can respond to hypoxic conditions by production of ROS, but subsequent activation of glucose metabolism may represent a mechanism of protection that rescues them from oxidative stress-induced cell death and allows them to continue to proliferate.

3. Consequences of the glycolytic switch in tumors

The main reason for the glycolytic shift in cancer cells is assumed to be an inadequate supply of oxygen (Gatenby and Gillies, 2007; Lopez-Lazaro, 2008). Surprisingly, however, tumor cells remain glycolytic also after restoration of the oxygen supply. In fact, the amount of glucose taken up by cancer cells exceeds their bioenergetic demand. It has been suggested that the excessive glycolysis in tumors is required to support cell growth (Vander Heiden et al., 2009). Thus, the end-product of glycolysis, pyruvate, is directed toward lipid synthesis, which is necessary for membrane assembly. A key enzyme linking glucose metabolism to lipid synthesis is ATP citrate lyase (ACL), which catalyzes the conversion of citrate to acetyl-CoA in the cytosol. Inhibition of ACL can suppress tumor cell survival and proliferation in vitro and can also reduce in vivo tumor growth and induce differentiation (Hatzivassiliou et al., 2005). In addition, shifting to the glycolytic pathway and to lactate production creates an acidic environment, which gives the cancer cells a competitive advantage for invasion (Gillies and Gatenby, 2007). Another important consequence of the glycolytic shift in tumor cells is their acquired resistance to apoptotic cell death. Apoptosis is an evolutionarily conserved and genetically regulated process of critical importance for embryonic development and maintenance of tissue homeostasis in the adult organism, which also plays a signiﬁcant role in tumor cell biology (Vik- torsson et al., 2005). Hence, apoptosis might be involved in mediating spontaneous regression, one of the unique features of tumors, whereas defects in apoptosis programs may contribute to tumor progression and resistance to treatment. Two major pathways of apoptosis signaling are known. Firstly, the extrinsic (receptor-mediated) pathway engages pro-caspase-8, a key initiator caspase that subsequently activates pro-caspase-3, which is primarily responsible for manifestation of the various biochemical and morphological features of apoptosis. Secondly, the intrinsic apoptotic pathway involves permeabilization of the outer mitochondrial membrane (OMM) followed by the release of cytochrome c and other proteins from the intermembrane space of mitochondria. Once in the cytosol, cytochrome c interacts with its adaptor molecule, Apaf-1, resulting in the recruitment, processing and activation of pro-caspase-9. Active caspase-9, in turn, cleaves and activates pro-caspase-3 and - 7; these effector caspases are responsible for the cleavage of cellular proteins leading to the characteristic features of apoptosis. Therefore, permeabilization of the OMM is considered a crucial event during the early phase of the apoptotic process (Gogvadze et al., 2006). However, the glycolytic shift in tumor cells makes the OMM less susceptible to permeabilization and thereby the cells more resistant to mitochondrially-mediated cell death. What are then the mechanisms of OMM permeabilization, and how could stimulation of the glycolytic pathway make mitochondria resistant to permeabilization? V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 65

4. Mitochondrial stabilization in glycolytic tumor cells

4.1. The role of Bcl-2 proteins in OMM permeabilization

OMM permeabilization engages pro-apoptotic members of the Bcl-2 family of proteins. The first indication that genes and proteins, which play a role in tumorigenesis, might be involved in the negative regulation of cell death came from the finding that the Bcl-2 protein is overexpressed as a result of a chromosomal translocation in B cell lymphomas (Tsujimoto et al., 1987). Overexpression of this protein was shown to inhibit cell death induced by different treatments, such as IL-3 deprivation, chemotherapeutic agents and heat shock (reviewed in (Tsujimoto and Shimizu, 2000)). In an early publication, the Bcl-2 protein, which blocks cell death rather than affecting proliferation, was shown to be localized to the mitochondria (Hocken- bery et al., 1990). Accumulation of Bcl-2 in mitochondria triggered a set of metabolic changes leading to the stabilization of this organelle. Cells overexpressing Bcl-2 had a higher mitochondrial membrane potential than wild-type cells, which was suggested to be responsible for the enhanced survival of cells after TNF challenge (Hennet et al., 1993). More than thirty Bcl-2 family members, or related proteins, have been identified today. They can be divided into three subgroups: Bcl-2-like survival factors, Bax-like, and BH3-only death factors (Festjens et al., 2004). The Bcl-2 family proteins contain between one and four Bcl-2 homology (BH) domains. The anti-apoptotic subfamily contains proteins with four BH domains. Most members of this subfamily also bear transmembrane domains that allow insertion into membranes. The pro- apoptotic Bax-like subfamily lacks the BH4 domain and promotes apoptosis by forming pores in the mitochondrial outer membrane. The BH3-only subfamily is a structurally diverse group of proteins that share homology within the small BH3 motif. Early indications of the importance of these proteins for the release of cytochrome c were obtained in 1997, when two groups independently showed that overexpression of Bcl-2 prevented the efflux of cytochrome c from mitochondria in cells treated with apoptotic stimuli as well as the initiation of apoptosis (Kluck et al., 1997; Yang et al., 1997a). It was concluded that one possible mechanism by which Bcl-2 can prevent apoptosis is to block cytochrome c release from mitochondria. Permeabilization of the outer mitochondrial membrane was shown to be a prerogative of the oligomeric form of Bax, whereas monomeric Bax was ineffective (Antonsson et al., 2000). Oligomerization of Bax is mediated by the truncated form of the BH3-only, pro-apoptotic protein Bid (tBid), cleaved by caspase-8 or several other proteases (Fig. 1(1)). Cells deficient in both Bax and Bak, but not cells lacking only one of these proteins, have been found to be resistant to tBid-induced cytochrome c release and apoptosis. Moreover, Bax- and Bak-deficient cells were also resistant to a variety of apoptotic stimuli that act through the mitochondrial pathway, such as staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimulus, thapsigargin (Wei et al., 2001). Thus, activation of a ‘‘multidomain”, pro-apoptotic Bcl-2 family member, Bax or Bak, and subsequent OMM permeabilization appear to be a principal gateway to the mitochondrial release of pro-apoptotic proteins in response to diverse cell death stimuli. Permeabilization of OMM is averted when anti-apoptotic proteins, e.g. Bcl-2, Bcl-XL, Mcl-1, and Bcl-w, interact with the pro-apoptotic proteins, Bax and Bak, to prevent their oligomerization (Fig. 1(2)). Thus, the balance between pro- and anti-apoptotic proteins in the OMM is a critical factor that regulates apoptosis induction. Disturbance of the balance between anti- and pro-apoptotic Bcl-2 family members in favor of the latter can proceed by mechanisms involving BH3-only proteins that sequester the anti-apoptotic proteins, thereby liberating Bax and Bak. For example, the BH3-only proteins PUMA and NOXA, which are expressed in a p53-dependent manner upon DNA damage, were shown to cause outer membrane permeabilization (Nakano

Fig. 1. (1) Oligomerization of Bax is mediated by the truncated form of the BH3-only, pro-apoptotic protein Bid (tBid); (2) Bcl-2, Bcl-XL, Mcl-1, and Bcl-w, interact with the pro-apoptotic proteins, Bax and Bak, to prevent their oligomerization; (3) The anti-apoptotic protein Bcl-XL prevents tBid-induced closure of VDAC and apoptosis by maintaining VDAC in open configuration allowing ADT/ATP exchange and normal mitochondrial functioning; (4) MPT pore is a multimeric complex, composed of VDAC located in the OMM, ANT, an integral protein of the IMM, and a matrix protein, CyPD; (5) Interaction with VDAC allows hexokinase to use exclusively intramitochondrial ATP to phosphorylate glucose, thereby maintaining high rate of glycolysis. 66 V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 and Vousden, 2001; Oda et al., 2000). In many tumors the mitochondrial pathway of apoptosis signaling is suppressed due to a disproportion between anti-apoptotic and pro-apoptotic mediators in favor of the former (Abel et al., 2005). This overexpression of Bcl-2 and Bcl-XL in tumor cells, in particular neuroblastomas, contributes to the drug resistance characteristic of high-risk neuroblastomas. Hence, an imbalance between Bcl-2 proteins in favor of the anti-apoptotic family members is a phenomenon that occurs frequently in cancer cells. Could this imbalance then contribute to the alteration of mitochondrial respiration and ATP production in tumors? Bcl-2 proteins can not pass through the OMM and, accordingly, would not be expected to directly affect the respiratory complexes in the inner membrane. Indeed, assessment of mitochondrial respiration in wild- type and Bcl-2 overexpressing PC12 and GT1–7 neural cells did not reveal any differences in oxygen consumption (Kowaltowski et al., 2002). However, Bcl-2 overexpressing cells demonstrated an increased mitochondrial volume and structural complexity, which might be associated with the anti-apoptotic properties of this protein at the mitochondrial level. Prevention of apoptosis via modulation of mitochondrial function may also occur by the interaction of anti-apoptotic proteins, in particular Bcl-XL, with voltage-dependent anion channel (VDAC) (Shimizu et al., 2000), a protein located in the OMM and responsible for most of the metabolite fluxes across the mitochondrial outer membrane (Colombini, 1983). Closure of VDAC upon growth factor withdrawal triggered apoptosis via prevention of ADP/ATP exchange and the concomitant decrease in metabolite fluxes over the mitochondrial membranes and led to OMM permeabilization and cytochrome c release

(Vander Heiden et al., 2001). The anti-apoptotic protein, Bcl-XL, prevented apoptosis by maintaining VDAC in the open configuration, allowing ADP/ATP exchange and normal mitochondrial functioning (Fig. 1(3)). It is not clear, however, how the closure of VDAC would cause OMM permeabilization. Recently, it was suggested that VDAC shows a higher permeability to Ca2+ in the closed, than in the open state (Tan and Colombini, 2007). Thus, VDAC closure might favor Ca2+ uptake into mitochondria, which could lead to induction of mitochondrial permeability transition (MPT) – another mechanism of OMM permeabilization. MPT was described some thirty years ago by Haworth and Hunter, who showed that upon accumulation of Ca2+ by mitochondria drastic changes in mitochondrial morphology and functional activity are induced (Haworth and Hunter, 1979). These changes were explained by opening of a non-specific pore in the mitochondrial inner membrane, commonly known as the MPT pore. Traditionally, the MPT pore was regarded as a multimeric complex, composed of VDAC located in the OMM, adenine nucleotide translocase (ANT), an integral protein of the IMM, and a matrix protein, cyclophilin D (CyPD). This complex is located at contact sites between the mitochondrial inner and outer membranes (Fig. 1(4)). Other proteins may bind to the pore complex, in particular kinases (hexokinase, creatine kinase) (Crompton, 2000). Pore opening is Ca2+-dependent and can be facilitated by inorganic phosphate, oxidation of pyridine nucleotides, ATP depletion, low pH and ROS (reviewed in (Crompton, 1999). MPT was shown to be a key event in both necrotic and apoptotic cell death. Opening of Ca2+-dependent, non-specific pores in the IMM is followed by the influx of water and ions into the matrix, which results in mitochondrial swelling, rupture of the OMM and the release of proteins, including cytochrome c, from the intermembrane space. Tumor cell mitochondria demonstrate some resistance towards MPT induction. Comparison of Ca2+ accumulation by mitochondria from liver and hepatoma revealed that 2–5-fold more Ca2+ was needed for MPT induction in hepatoma mitochondria. Such resistance might be due to higher expression of the Bcl-2 protein in hepatoma cells as compared to hepatocytes (Evtodienko et al., 1999). Hence, the increased stability of mitochondria in tumor cells towards MPT might be one factor that contributes to their resistance to treatment. The role of CyPD, a putative component of the MPT pore, in apoptotic cell death is still controversial and needs further investigation. Cyclophilins constitute a group of peptidyl-prolyl cis–trans isomerases (PPIase) with highly conserved protein sequences, which are important for protein folding (Gothel and Marahiel, 1999). For many years, CyPD was considered critical for the opening of the MPT pore. This view was based on the observation that cyclosporin A (CsA), a potent inhibitor of some forms of necrosis and mitochondrially-mediated apoptosis (Lemasters et al., 1999), blocks the opening of the MPT pore at concentrations similar to those needed to inhibit the enzymatic activity of CyPD. Based on these properties, CyPD was thought to promote the opening of the mitochondrial permeability transition pore and thereby to facilitate cell death. In- deed, CyPD overexpression sensitizes the ANT to agents (Ca2+ and oxidants) that transform it into the MPT pore. CyPD overexpression was further found to promote MPT pore formation in both stressed and unstressed B50 cells, as well as in isolated mitochondria (Li et al., 2004). In addition, CyPD was shown to activate the formation of permeability transition pores from purified ANT in black lipid membranes (Brustovetsky et al., 2002). Moreover, it has been reported that CypD-deficient mitochondria do not undergo cyclosporin A-sensitive MPT. However, in knock-out experiments CypD-deficient cells died normally in response to various apoptotic stimuli, but showed resistance to necrotic cell death induced by reactive oxygen species and Ca2+ overload (Nakagawa et al., 2005). In contrast to the observations reported above, overexpression of CyPD was found to augment the resistance of HEK293 and rat glioma C6 cells to apoptotic stimuli (Lin and Lechleiter, 2002). Protection from apoptosis required PPIase activity, whereas CyPD binding to ANT was not affected by the loss of enzyme activity. Thus, it seems unlikely that the protective effect of CyPD on apoptotic cell death is due to binding of CyPD to ANT. More recently, CyPD was shown to be specifically upregulated in human tumors of the breast, ovary, and uterus (Schubert and Grimm, 2004). The authors suggested that CyPD is a new type of apoptosis inhibitor, which is effective at a functional level different from that of the previously known inhibitors of the Bcl-2 family. V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 67

Although the molecular mechanism by which CyPD prevents apoptosis is unclear, it has been reported that suppression of apoptosis by CyPD correlated with the amount of hexokinase II bound to mitochondria (Machida et al., 2006). Amazingly, inactivation of endogenous CyPD by small interference RNA, or by a cyclophilin inhibitor caused detachment of hexokinase II from the mitochondria and stimulation of Bax-mediated apoptosis. Interestingly, mitochondrial binding of hexokinase II was essential for apoptosis suppression by CyPD, since its anti-apoptotic effect was lost upon detachment of hexokinase II from the mitochondria. Furthermore, CypD dysfunction appears to abrogate hexokinase II-mediated apoptosis suppression, indicating that it is in fact required for the anti-apoptotic effect of hexokinase II. A recent study claims that the anti-apoptotic effect of CypD is MPT-independent, but requires the interaction with some key apoptosis regulator, such as Bcl-2 (Eliseev et al., 2009). Although Bcl-2 resides in the OMM and is spatially separated from CypD, which is located in the matrix, the presence of Bcl-2 at mitochondrial contact sites could make such interaction possible, considering that MPT complexes, including CypD, are also localized to mitochondrial contact sites. Based on their data, the authors propose that targeting CypD to disrupt its interaction with Bcl-2 might increase the sensitivity of cells to apoptosis. Apparently, overexpression of CyPD might be important for tumor cell protection against apoptosis, although the molecular mechanism of this protection remains elusive. Both Bax/Bak- and MPT-mediated release of cytochrome c is facilitated by ROS. Cytochrome c is normally bound to the outer surface of the IMM by both electrostatic and hydrophobic interactions with the unique mitochondrial phospholipid, cardiolipin. Stimulation of ROS production resulting in oxidation of cardiolipin leads to the dissociation of cytochrome c from its binding to the IMM and subsequent release into the cytosol through pores in OMM formed by Bax/Bak (Ott et al., 2002). In addition, ROS can stimulate Bax activation. Thus it has been shown that oxidative dimerization of Bax promotes its translocation to the mitochondria (D’Alessio et al., 2005), and that cysteine 62 of Bax is critical for its conformational activation and pro-apoptotic activity in response to ROS (Nie et al., 2008). Mitochondria can also trigger the release of pro-apoptotic proteins through excessive ROS generation and self-directed induction of MPT. A critical involvement of ROS in apoptosis provides a plausible explanation for the anti-apoptotic effects reported for multiple mitochondrial antioxidant enzymes and anti-tumor compounds with antioxidant properties (Orrenius et al., 2007). As discussed above, overexpression of anti-apoptotic Bcl-2 family proteins is typical for a variety of malignant cells. Localization of these proteins to the OMM makes this membrane stable towards permeabilization and abrogates the release of cytochrome c and other pro-apoptotic proteins from the mitochondria; it also enhances the capacity of tumor cells to proliferate.

4.2. Hexokinase and OMM stability

Several recent observations have revealed that the stimulation of glycolysis in cancer cells can trigger a chain of events, which increase the resistance of mitochondria to OMM permeabilization and hence abrogate mitochondrial pathways in apoptosis. Hexokinase plays a leading role in this process. Four isoforms of hexokinase are known: hexokinase I, II, III, and IV, or glucokinase. Isoenzymes differ by their tissue speciﬁc expression and subcellular localization. Hexokinase-I is the predominant form in brain, kidney and retina, whereas hexokinase-II is expressed in muscle and adipose tissue. Type IV hexokinase (glucokinase), has long been known to be of key functional signiﬁcance in hepatocytes and pancreatic b-cells (Postic et al., 2001). In hepatomas, however, glucokinase is silenced, and hexokinase-II is activated. Among glycolytic enzymes, hexokinases, in particular hexokinase-I and -II, are exceptional in their ability to directly bind to mitochondria (Bustamente et al., 1977). In contrast, the type III isozyme lacks the hydrophobic N-terminal sequence known to be critical for the binding of the type I and type II isozymes to mitochondria (Wilson, 2003). Tumors are characterized by upregulation of hexokinase-I (in brain tumors) and -II (in almost all other tumors). The interaction with VDAC allows hexokinase to use exclusively intramitochondrial ATP to phosphorylate glucose, thereby maintaining a high rate of glycolysis (for recent review see (Mathupala et al., 2006)). Interestingly, glucose-6-phosphate is produced faster when ATP is provided by the ATP synthase, located in the IMM, than when cytosolic ATP is used. The interaction of VDAC with hexokinase not only facilitates glucose phosphorylation, using mitochondrially-generated ATP, but also keeps VDAC in the open state, which counteracts OMM permeabilization (Vander Heiden et al., 2001)(Fig. 1(5)). The interaction with VDAC also allows hexokinase to block binding sites for pro-apoptotic Bcl-2 family proteins on the OMM, thereby inter- fering with apoptosis induction (Pastorino et al., 2002).

4.3. ANT and OMM stability

In tumor cells, alterations of the expression proﬁle of ANT, a key player in MPT induction, can also contribute to OMM stability. In mammals, three isoforms of ANT are expressed in a tissue-speciﬁc manner. ANT1 is expressed predominantly in the heart, skeletal muscle and brain, ANT2 is expressed in liver and in proliferating cells, whereas ANT3 is expressed uni- versally (Stepien et al., 1992). Analysis of ANT isoform expression in several transformed human cell lines revealed prevailing expression of ANT2 (Chevrollier et al., 2005), which lacks the pro-apoptotic activity of ANT1 (Bauer et al., 1999). Transient overexpression of ANT3, or of ANT1, was shown to induce apoptosis as assessed by an increase in the sub-G1 fraction, an- nexin V staining, a decrease in W and activation of caspases-9 and -3 (Zamora et al., 2004). Thus, similar to binding of hexokinase to VDAC, the upregulation of ANT2 in tumors might stabilize mitochondria towards OMM permeabilization. 68 V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74

4.4. Akt-mediated OMM stability

An additional mechanism that can evoke glycolytic ATP production is activation of the serine/threonine kinase, Akt (also known as protein kinase B) (Elstrom et al., 2004), which has emerged as one of the most frequently activated protein kinases in human cancer (Robey and Hay, 2009). Phosphoinositide 3-kinase (PI3-K) and its downstream target, Akt, are en- gaged in a pathway that transmits survival signals from various cell surface receptors. In a variety of tumors, activation of Akt correlates with poor prognosis in vivo and with apoptosis resistance in vitro. Activation of the Akt/PKB protein kinase family triggers increases in cell size, glycolytic activity and cell survival (Plas and Thompson, 2005). Akt was found to regulate multiple steps in glycolysis via posttranscriptional mechanisms that include localization of the glucose transporter to the cell surface and maintenance of hexokinase function in the absence of extrinsic factors. Akt can be rendered constitutively active by targeting to the plasma membrane by myristoylation (mAkt), where it is phosphorylated by endogenous PI3-K-dependent kinases. When activated, Akt can phosphorylate target proteins which regulate glucose uptake and metabolism. Activation of Akt can also result from mitochondrial dysfunction. Defects in mitochondrial respiration may lead to an increase in NADH level, which can inactivate PTEN (a lipid phosphatase that antagonizes PI3-K function and thereby inhibits downstream signaling through Akt) through a redox modification mechanism, subsequently leading to Akt activation (Pel- icano et al., 2006). Activation of the Akt/PKB pathway is known to protect cells from apoptosis (Franke et al., 1997), although the precise mechanisms of this protection are still undetermined. However, Akt activation was early found to inhibit cytochrome c release from mitochondria and thereby to prevent initiation of the caspase cascade (Kennedy et al., 1999). Accordingly, when cytochrome c was injected into the cytoplasm, Akt was not able to suppress apoptosis. These findings indicate that the anti-apoptotic effect of Akt occurs upstream of OMM permeabilization, and that it might involve a stabilization of the OMM. How could then Akt influence OMM stability? Several mechanisms were suggested. Thus, Akt inhibits p53-mediated expression of Bax and could thereby decrease the probability of OMM permeabilization (Yamaguchi et al., 2001). The active, but not the inactive form of Akt was shown to phosphorylate the pro-apoptotic protein Bad, preventing its interaction with the OMM and its pro-apoptotic functions (Datta et al., 1997; del Peso et al., 1997). Activation of PI3-K leads to rapid accumulation of Akt in mitochondria, where it was found to reside not only in the OMM but also in the inner membrane and the matrix (Bijur and Jope, 2003). Although the functions of Akt in mitochondria are not yet fully understood, it has been reported that active Akt can reduce Bax oligomerization in the mitochondrial membrane, thereby preventing OMM permeabilization (Mookherjee et al., 2007). In addition, Akt was shown to promote the translocation of hexokinase to mitochondria, where it can interact with VDAC to inhibit OMM permeabilization (Majewski et al., 2004).

4.5. Contribution of p53 to OMM stability

Mutation of the p53 gene, one of the most frequently mutated genes in cancer, causes down-regulation of mitochondrial activity. In addition to its role as a central regulator of the cellular stress response, p53 can control the balance between the glycolytic pathway and mitochondrial oxidative phosphorylation (Matoba et al., 2006). One of the targets of p53 is the gene encoding Synthesis of Cytochrome c Oxidase 2 (SCO2), whose product, together with the SCO1 protein (Buchwald et al., 1991), is required for the assembly of COX, a multimeric protein complex required for oxidative phosphorylation. Mutation of p53 in tumors causes COX deficiency, impairment of the mitochondrial respiratory chain, and a shift of cellular energy metabolism toward glycolysis. Mutation or down-regulation of p53 does not only contribute to the suppression of mitochondrial respiratory activity, but can also play a role in maintenance of OMM intactness. Hence, p53 regulates the expression of the BH3-only proteins, PUMA and NOXA, which promote OMM permeabilization during apoptosis (Nakano and Vousden, 2001; Oda et al., 2000). PUMA and NOXA bind to and occupy the anti-apoptotic Bcl-2 proteins, thereby liberating Bax and Bak. Co-immunoprecipitation studies showed that NOXA binds to Bcl-2 and Bcl-XL, depending on a functional BH3-motif of NOXA, but not to Bax. Inter- estingly, p53 can also trigger OMM permeabilization via direct activation of Bax (Chipuk et al., 2004). Although the precise mechanism of this activation is still obscure, mutation of p53 in tumor cells resulted in stabilization of the OMM by down- regulation of pro-apoptotic proteins. Another target of p53 is TIGAR (TP53-Induced Glycolysis and Apoptosis Regulator). TIGAR inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. In addition, TIGAR stimulates NADPH generation via the pentose phosphate shunt, which results in increased intracellular NADPH and GSH levels (Bensaad et al., 2006). The authors suggested that the decreased intracellular ROS levels in response to TIGAR might be important for the ability of p53 to protect cells from accumulation of genomic damage. Recently, a new link between p53 and glucose metabolism has been demonstrated (Jones et al., 2005). Inhibition of glycolysis by glucose withdrawal was shown to serve as a signal for the phosphorylation and activation of p53. Therefore, one could expect that under stress conditions, such as glucose deprivation, activation of p53 might increase SCO2 expression and stimulate more efficient mitochondrial respiration and ATP production. Deficiencies in p53-mediated regulation of glycolysis and mitochondrial respiration are one reason for mitochondrial silencing and switch to aerobic glycolysis in tumors, while inadequate p53-modulated antioxidant defence mechanisms might contribute to mutagenesis and carcinogenesis (Olovnikov et al., 2009). V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 69

5. Stimulation of cancer cell death via intervention with cellular ATP-generating pathways

5.1. Suppression of the glycolytic pathway

The increased dependence of cancer cells on glycolysis offers a rationale for the design of therapeutic strategies to selectively kill cancer cells by inhibition of the glycolytic pathway. This strategy might be most useful in cells with mitochondrial defects, or under hypoxic conditions when glycolysis is the predominant source of ATP, and the mitochondrial contribution to cellular bioenergetics is minimal. Under such circumstances, inhibition of glycolysis would be expected to severely de- plete cellular ATP level (Pelicano et al., 2006). Indeed, in a variety of cancer cells inhibition of glycolysis was shown to cause marked ATP depletion, especially in clones with defective mitochondrial respiration (Xu et al., 2005). The administration of 2-deoxyglucose (Fig. 2(1)), a non-metabolizable glucose analogue, to human histiocytic lymphoma cells had an effect similar to that of reduction of the extracellular glucose concentration. The combined treatment with TNF and 2-deoxyglucose, led to apoptosis. Analysis of cell proliferation rates and viability revealed that TNF cytotoxicity was markedly potentiated by 2-deoxyglucose, whereas low concentrations of 2-deoxyglucose alone showed minimal cytotoxicity (Halicka et al., 1995). Glucose deprivation also promoted death receptor-mediated apoptosis in human tumor cells (Munoz- Pinedo et al., 2003). The suppression of intracellular ATP level by 2-deoxyglucose, alone or in combination with glucose-free medium, potentiated Fas-induced phosphatidylserine exposure, suggesting that intracellular ATP can inﬂuence the external- ization of PS during apoptosis (Gleiss et al., 2002). A lactic acid analogue, 3-bromopyruvate (3-BrPA) has been found to selectively kill hepatocellular carcinoma cells in vitro, leaving normal hepatocytes unharmed. Moreover, systemic delivery of 3-BrPA suppressed ‘‘metastatic” lung tumors with no apparent harm to other organs or to the animals (Geschwind et al., 2002). Analysis of cellular targets for 3-BrPA revealed that this compound markedly (by 80%) suppressed glycolytic capacity (Fig. 2), abolished the activity of mitochondrially-bound hexokinase and suppressed respiration in isolated mitochondria (Fig. 2(2))(Ko et al., 2001). Hence, cell death was associated with a dramatic decrease in the level of ATP (reviewed in (Pedersen, 2007)). Recently, it has been shown that in addition to causing ATP depletion in cancer cells, 3-BrPA stimulates the production of ROS and causes mitochondrial dysregulation, which contributes to cell death (Kim et al., 2008). Inhibition of glycolysis can markedly increase tumor sensitivity to common anti-cancer agents. In addition to compounds inhibiting key steps in the glycolytic pathway, suppression of glucose transporters may have a beneﬁcial effect in elimination of cancer cells. Thus, phloretin (Fig. 2(3)), a glucose transporter inhibitor, markedly enhanced the anti-cancer effects of daunorubicin (Cao et al., 2007). Combination of glycolysis inhibitors and conventional chemotherapeutic drugs may therefore provide a useful therapeutic strategy to overcome drug resistance in hypoxic tumor cells. Apparently, when the glycolytic

Fig. 2. Different sites of therapeutic intervention in cancer cell metabolism. (1) The non-metabolizable analog of glucose, 2-deoxyglucose, decreases ATP level in the cell; (2) 3-bromopyruvate suppresses the activity of hexokinase, and respiration in isolated mitochondria; (3) Phloretin a glucose transporter inhibitor, decreases ATP level in the cell and markedly enhances the anti-cancer effect of daunorubicin; (4) Dichloroacetate (DCA) shifts metabolism from glycolysis to glucose oxidation; (5) Apoptolidin, an inhibitor of mitochondrial ATP synthase, induces cell death in different malignant cell lines when applied together with the LDH inhibitor oxamate (6). 70 V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 pathway is suppressed, the fate of the cancer cell will depend entirely on how well mitochondrial oxidative phosphorylation can cope with cellular ATP demands.

5.2. Shifting metabolism from glycolysis to glucose oxidation

As discussed above, shifts in ATP production from glycolysis to oxidative phosphorylation can sensitize tumor cells to commonly used anti-cancer drugs and promote cell death. Attempts have been made to re-direct pyruvate towards oxidation in mitochondria. Thus, inhibition of PDK1 (hence activation of PDH) by dichloroacetate (DCA) was shown to shift metabolism from glycolysis to glucose oxidation (Fig. 2(4)). Treatment with DCA increased mitochondrial production of ROS in all tested cancer cells, but not in normal cells (Michelakis et al., 2008). Further, in cell lines with an apoptotic response, DCA treatment was shown to stimulate the expression of PUMA, suggesting the involvement of a p53-PUMA-mediated mechanism (Wong et al., 2008). DCA effectively sensitized human prostate cancer cells, both wild-type and Bcl-2 overexpressing cells, to radiation by modulating the expression of key members of the Bcl-2 family (Cao et al., 2008). A similar effect was achieved by inhibition of LDH and re-direction of pyruvate to mitochondria. Indeed, when the formation of lactate from pyruvate in malignant cells was suppressed by knocking down LDH-A, the ability of these tumor cells to proliferate under hypoxia was markedly compromised. Reduction of LDH-A activity resulted in stimulation of mitochondrial respiration and ATP synthesis. The ability to proliferate was restored by complementation with the human ortholog LDH-A protein (Fantin et al., 2006). Inhibition of LDH, or stimulation of PDH through the suppression of PDK1, could have a growth inhibitory effect in tumors with altered mitochondrial bioenergetics. However, in cancer cells with functionally active mitochondria this might not be enough, since the mitochondria would compensate for the inhibition of glycolysis. Therefore, under such circumstances sup- plementary suppression of mitochondrial activity might be needed to kill the cancer cells. Thus, non-toxic doses of apoptolidin (Fig. 2(5)), an inhibitor of mitochondrial ATP synthase, induced cell death in malignant cell lines when applied together with the LDH inhibitor, oxamate (Fig. 2(6))(Salomon et al., 2000). Similar results were obtained when 2-deoxyglucose was used instead of oxamate for glycolysis inhibition. Hence, combined strategies involving modulation of both glycolytic and mitochondrial pathways might be required for more efﬁcient elimination of malignant cells.

5.3. Alteration of mitochondrial functions

As discussed above, the resistance of cancer cells to treatment is often associated with dysfunction of their apoptotic pro- gram. Successful elimination of tumor cells, therefore, largely depends on the ability of anti-cancer drugs to activate dormant apoptotic pathways. Mitochondria are promising candidates for such an approach. Indeed, this hypothesis was supported by numerous studies. Modulation of mitochondrial function by agents that suppress mitochondrial respiration, or uncouple oxidative phosphorylation, was found to provoke cell death. Thus, inhibitors of the mitochondrial respiratory chain, rotenone or antimycin A, and an inhibitor of mitochondrial ATP synthase, oligomycin, induced apoptosis in cultured human lymphoblas- toid and other mammalian cells. The same compounds failed to kill cells depleted of mitochondrial DNA and, hence, lacking an intact respiratory chain, indicating that induction of apoptosis was not caused by side-effects of these agents. Neither was apoptosis induced by the respiratory chain inhibitors suppressed by the anti-apoptotic protein, Bcl-2 (Wolvetang et al., 1994). Suppression of the activity of the mitochondrial respiratory chain also enhanced cellular susceptibility to common apoptotic stimuli. Thus, in HeLa cells treated with respiratory inhibitors, lower concentrations of Fas were sufficient to induce apoptosis as compared to untreated cells (Asoh et al., 1996). The importance of mitochondria as targets for anti-cancer treatment has also been documented in experiments with all- trans retinoic acid (ATRA), a natural derivative of vitamin A, which is used successfully in the treatment of acute promyelocytic leukemia (Schmidt-Mende et al., 2006). ATRA perturbed mitochondrial functions in the myeloid cell line P39 long be- fore any signs of apoptosis were detected. It suppressed mitochondrial respiration, decreased Dw and triggered opening of MPT pores. ATRA-induced mitochondrial dysfunction and activation of caspases were abolished by nifedipine, a calcium channel blocker, indicating the involvement of Ca2+ in mitochondrial deterioration. Cell death induced by inhibitors of the mitochondrial respiratory chain is often mediated by ROS. For example, benzyl isothiocyanate targets the mitochondrial respiratory chain to trigger ROS-dependent apoptosis in human breast cancer cells (Xiao et al., 2008). ROS production and apoptosis were inhibited by overexpression of catalase and Cu, Zn-superoxide dismu- tase, as well as by inhibition of the mitochondrial respiratory chain. Similarly, cells lacking mitochondrial DNA were resistant to benzyl isothiocyanate-mediated ROS generation and apoptosis. In 1982, Prasad and Edwards-Prasad reported for the first time that a-tocopheryl succinate (a-TOS), a redox-silent analogue of vitamin E, induced morphological changes and growth inhibition in mouse melanoma cells in culture (Prasad and Edwards-Prasad, 1982). a-TOS was shown to inhibit the proliferation of avian reticuloendotheliosis virus-transformed lym- phoblastoid cells in a dose-dependent manner, block the cells in the G2/M cell cycle phase, and induce apoptosis (Qian et al., 1996). a-TOS was also found to destabilize the mitochondria, stimulate the production of ROS and kill malignant cells at concentrations non-toxic to normal cells and tissues (Ottino and Duncan, 1997). a-TOS interacts with the CoQ binding site in complex II of the mitochondrial respiratory chain, causing leakage of electrons and formation of the superoxide radical (Dong et al., 2008). It facilitates translocation of Bax from the cytosol to the mitochondria and subsequent cytochrome c release (Yu et al., 2003). Although the precise mechanisms of a-TOS action remain to be elucidated, the results obtained so far makes it V. Gogvadze et al. / Molecular Aspects of Medicine 31 (2010) 60–74 71 an attractive candidate for anti-tumortherapy. Recently a range of compounds named mitocans (an abbreviation formed from mitochondria and cancer), were shown to cause cell death via targeting mitochondria (Ralph and Neuzil, 2009). A powerful tool to produce mitochondrial destabilization is oxidative stress. Modulation of the cellular redox balance via pharmacological stimulation of intracellular ROS production and/or depletion of protective reducing metabolites (such as glutathione and NADPH) may lead to oxidative stress and induction of apoptosis (Engel and Evens, 2006). Under such circumstances, a shift in the balance towards ROS formation can be expected to destabilize mitochondria and facilitate permeabilization of the OMM with release of cytochrome c and other intermembrane space proteins involved in apoptotic cell death. The anti-cancer effect of a number of conventional treatments (radiation, etoposide, arsenates) is based on their ability to stimulate ROS production. Thus, the anti-tumor effect of one such potent chemotherapeutic agent, arsenic trioxide, was shown to involve oxidative modification of thiol groups in ANT and, as a result thereof, MPT induction and release of cytochrome c. A direct effect of different arsenic compounds on the MPT pore was suggested to be the mechanism of apoptosis induction (Larochette et al., 1999; Nutt et al., 2005). Recent observations suggest that, in some experimental systems, stimulation rather than suppression of mitochondrial activity might be used as a tool against tumor growth. Accordingly, overexpression of frataxin, a protein associated with Friedreich ataxia, stimulated oxidative metabolism and enhanced mitochondrial Dw and ATP content in several colon cancer cell lines (Schulz et al., 2006). Friedreich ataxia is an inherited neurodegenerative disorder caused by the reduced expression of frataxin. It leads to impaired ATP synthesis and results in premature death due to cardiac failure. Frataxin was shown to promote mitochondrial oxidative metabolism, most probably by stimulation of the synthesis of Fe/S clusters in the mitochondria. In addition to stimulation of mitochondrial activity, frataxin inhibited colony formation and markedly suppressed tumor formation when injected into nude mice. Hence, depending on the conditions, either suppression or stimulation of mitochondrial metabolism might suppress the growth of tumor cells.

6. Concluding remarks

Despite the heterogeneity of tumors, which dictates an individual approach to anti-cancer treatment, almost all of them demonstrate enhanced uptake and utilization of glucose, a phenomenon known as the Warburg effect. One of the consequences of the upregulation of glycolysis in tumors is stabilization of the mitochondria and increased resistance to OMM permeabilization and apoptotic cell death. Successful elimination of cancer cells is therefore based on the ability of anti-cancer treatment to activate apoptotic pathways, which are suppressed in tumor cells. Targeting mitochondria might be a promising strategy to increase the sensitivity of tumor cells to apoptotic mechanisms. The existing interplay between apoptosis regulators and cellular energy producing systems should be considered in the search for novel anti-cancer drugs. The idea of selective activation of apoptosis in transformed cells, sparing normal cells, remains a primary strategic goal in fighting cancer, and extensive studies are being performed to find more efficient mechanisms of apoptosis induction in tumor cells. A successful outcome of this effort must include modulation of cellular energy metabolism to sensitize tumor cells to otherwise dormant apoptotic mechanisms. Targeting mitochondria is an essential component of this approach and may result in the development of new and more efficient therapeutic regiments for suppression of tumor growth.

Acknowledgements

Work in the authors’ laboratory was supported by grants from the Swedish and Stockholm Cancer Societies, the Swedish Childhood Cancer Foundation, the Swedish Research Council, the EC-FP-6 (Oncodeath and Chemores) and EC-FP-7 (APO-SYS) programs. We apologize to authors whose primary references could not be cited owing to space limitations.

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