The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (ΔΨm) and low expression of the K + channel Kv1.5, both contributing to apoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases ΔΨm, increases mitochondrial H 2 O 2 , and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

The small molecule DCA is a metabolic modulator that has been used in humans for decades in the treatment of lactic acidosis and inherited mitochondrial diseases. Without affecting normal cells, DCA reverses the metabolic-electrical remodeling that we describe in several cancer lines (hyperpolarized mitochondria, activated NFAT1, downregulated Kv1.5), inducing apoptosis and decreasing tumor growth. DCA in the drinking water at clinically relevant doses for up to 3 months prevents and reverses tumor growth in vivo, without apparent toxicity and without affecting hemoglobin, transaminases, or creatinine levels. The ease of delivery, selectivity, and effectiveness make DCA an attractive candidate for proapoptotic cancer therapy which can be rapidly translated into phase II–III clinical trials.

Introduction

Warburg, 1930 Warburg O. Ueber den stoffwechsel der tumoren. Cancer progression and its resistance to treatment depend, at least in part, on suppression of apoptosis. Although mitochondria are recognized as regulators of apoptosis, their importance as targets for cancer therapy has not been adequately explored or clinically exploited. In 1930, Warburg suggested that mitochondrial dysfunction in cancer results in a characteristic metabolic phenotype, that is, aerobic glycolysis (). Positron emission tomography (PET) imaging has now confirmed that most malignant tumors have increased glucose uptake and metabolism. This bioenergetic feature is a good marker of cancer but has not been therapeutically pursued, as it is thought to be a result and not a cause of cancer; that is, the cells rely mostly on glycolysis for energy production because of permanent mitochondrial damage, preventing oxidative phosphorylation. However, whether the mitochondria in cancer are indeed damaged and whether this is reversible remain unknown.

Gatenby and Gillies, 2004 Gatenby R.A.

Gillies R.J. Why do cancers have high aerobic glycolysis?. 2 levels increase, the glycolytic phenotype persists, resulting in the “paradox” of glycolysis during aerobic conditions (the Warburg effect). Metabolic and apoptotic pathways that converge in the mitochondria are not independent from each other, and it appears that glycolytic phenotype is indeed associated with a state of apoptosis resistance ( Plas and Thompson, 2002 Plas D.R.

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Thompson C.B. Akt stimulates aerobic glycolysis in cancer cells. Kim and Dang, 2005 Kim J.W.

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Shulga N. Activation of glycogen synthase kinase 3β disrupts the binding of hexokinase II to mitochondria by phosphorylating voltage-dependent anion channel and potentiates chemotherapy-induced cytotoxicity. The metabolic hypothesis of cancer has recently been rekindled. Gatenby and Gillies recently proposed that because early carcinogenesis occurs in a hypoxic microenvironment, the transformed cells initially have to rely on glycolysis for energy production (). However, this early metabolic adaptation appears to also offer a proliferative advantage, suppressing apoptosis. Furthermore, the “byproducts” of glycolysis (i.e., lactate and acidosis) contribute to the breakdown of the extracellular matrix, facilitate cell mobility, and increase the metastatic potential. Therefore, even though the tumors eventually become vascularized and Olevels increase, the glycolytic phenotype persists, resulting in the “paradox” of glycolysis during aerobic conditions (the Warburg effect). Metabolic and apoptotic pathways that converge in the mitochondria are not independent from each other, and it appears that glycolytic phenotype is indeed associated with a state of apoptosis resistance (). Many glycolytic enzymes have been recognized to also regulate apoptosis, and several oncoproteins induce the expression of glycolytic enzymes (). For example, Akt, which stimulates glycolysis and induces resistance to apoptosis (), activates hexokinase, an enzyme catalyzing the first and irreversible step in glycolysis. Via its downstream mediator glycogen synthase kinase 3 (GSK3), Akt induces the translocation of hexokinase to the mitochondrial membrane where it binds to the voltage-dependent anion channel (VDAC), suppressing apoptosis (). Inhibition of GSK3 in cancer cells causes unbinding of hexokinase from VDAC, induces apoptosis, and increases sensitivity to chemotherapy (). This suggests that perhaps the metabolic phenotype in cancer is due to a potentially plastic mitochondrial remodeling that results in suppressed oxidative phosphorylation, enhanced glycolysis, and suppressed apoptosis.

2 . NADH donates electrons to complex I of the electron transport chain (ETC) (and FADH 2 to complex III). The flux of electrons down the ETC is associated with production of reactive oxygen species (ROS) and with the efflux of H+, which causes a negative mitochondrial membrane potential (ΔΨm). The F1F0-ATP synthase uses the stored energy of the ΔΨm to synthesize ATP; thus the ΔΨm reflects ETC activity and mitochondrial function. PDH is inhibited by phosphorylation by PDH kinase (PDK). The role of PDH and PDK in cancer is unknown. Figure 1 A Reversible Metabolic-Electrical Remodeling in Cancer Contributes to Resistance to Apoptosis and Reveals Several Potential Therapeutic Targets Show full caption In cancer, mitochondrial glucose oxidation is inhibited and energy production relies on the cytoplasmic glycolysis. This “inactivity” of the mitochondria likely induces a state of apoptosis resistance. Activation of PDH by DCA increases glucose oxidation by promoting the influx of acetyl-CoA into the mitochondria and the Krebs cycle, thus increasing NADH delivery to complex I of the electron transport chain, increasing the production of superoxide, which in the presence of MnSOD is dismutated to the more stable H 2 O 2 . Sustained increase in ROS generation can damage the redox-sensitive complex I, inhibiting H+ efflux and decreasing ΔΨm. Opening of the ΔΨm-sensitive mitochondrial transition pore (MTP) allows the efflux of cytochrome c and apoptosis inducing factor (AIF). Both cytochrome c and H 2 O 2 open the redox-sensitive K+ channel Kv1.5 in the plasma membrane and hyperpolarize the cell (increased Em), inhibiting a voltage-dependent Ca2+ entry. The decreased [Ca2+] i suppresses a tonic activation of NFAT, resulting in its removal from the nucleus, thus increasing Kv1.5 expression. The increased efflux of K+ from the cell decreases the tonic inhibition of [K+] i on caspases, further enhancing apoptosis. DCA's selectivity is based on its ability to target the unique metabolic profile that characterizes most cancers, and its effectiveness is explained by its dual mechanism of apoptosis induction, both by depolarizing mitochondria (proximal pathway) and activating/upregulating Kv1.5 (distal pathway). Whether the metabolism of glucose will end with glycolysis in the cytoplasm (converting pyruvate to lactate) or continue with glucose oxidation in the mitochondria is controlled by a gate-keeping mitochondrial enzyme, pyruvate dehydrogenase (PDH) ( Figure 1 ). PDH converts pyruvate to acetyl-CoA which, along with the acetyl-CoA from the fatty acid β-oxidation, is fed to the Krebs cycle, producing the electron donors NADH and FADH. NADH donates electrons to complex I of the electron transport chain (ETC) (and FADHto complex III). The flux of electrons down the ETC is associated with production of reactive oxygen species (ROS) and with the efflux of H, which causes a negative mitochondrial membrane potential (ΔΨm). The F1F0-ATP synthase uses the stored energy of the ΔΨm to synthesize ATP; thus the ΔΨm reflects ETC activity and mitochondrial function. PDH is inhibited by phosphorylation by PDH kinase (PDK). The role of PDH and PDK in cancer is unknown.

2+] i and ROS-redox control. Through the release of ROS, mitochondria regulate the opening of plasma-membrane ion channels and through the control of [Ca2+] i , regulate Ca2+-sensitive transcription factors. Some of these downstream pathways are also important in apoptosis and might contribute to the apoptosis resistance in cancer. For example, inhibition or downregulation of K+ channels results in increased [K+] i , by decreasing the tonic efflux of K+ down its intracellular/extracellular gradient (145/5 mEq). Because [K+] i exerts a tonic inhibitory effect on caspases, K+ channel inhibition or downregulation suppresses apoptosis in several cell types, including cancer ( Andersson et al., 2006 Andersson B.

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Choi D.W. Mediation of neuronal apoptosis by enhancement of outward potassium current. + channels (Kv) is redox sensitive and therefore can be regulated by mitochondria. For example, mitochondria-derived H 2 O 2 (a relatively stable ROS) can activate Kv1.5 ( Caouette et al., 2003 Caouette D.

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Archer S.L. Hypoxic pulmonary vasoconstriction: redox regulation of O 2 -sensitive K+ channels by a mitochondrial O 2 -sensor in resistance artery smooth muscle cells. Mitochondrial remodeling has multiple downstream effects, beyond energy production, because mitochondria regulate several critical functions including [Caand ROS-redox control. Through the release of ROS, mitochondria regulate the opening of plasma-membrane ion channels and through the control of [Ca, regulate Ca-sensitive transcription factors. Some of these downstream pathways are also important in apoptosis and might contribute to the apoptosis resistance in cancer. For example, inhibition or downregulation of Kchannels results in increased [K, by decreasing the tonic efflux of Kdown its intracellular/extracellular gradient (145/5 mEq). Because [Kexerts a tonic inhibitory effect on caspases, Kchannel inhibition or downregulation suppresses apoptosis in several cell types, including cancer (). The voltage-gated family of Kchannels (Kv) is redox sensitive and therefore can be regulated by mitochondria. For example, mitochondria-derived H(a relatively stable ROS) can activate Kv1.5 (). Furthermore, the mitochondria-derived proapoptotic mediator cytochrome c activates, whereas the antiapoptotic bcl-2 inhibits, Kv channels (). This mitochondria-ROS-Kv channel axis is now recognized as a basis of an important O-sensing mechanism in many tissues ().

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et al. Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children. In preliminary experiments, we compared several cancer with normal cell lines and found that cancer cells had more hyperpolarized mitochondria and were relatively deficient in Kv channels. If this metabolic-electrical remodeling is an adaptive response, then its reversal might increase apoptosis and inhibit cancer growth. We used dichloroacetate (DCA), a small, orally available small molecule and a well-characterized inhibitor of PDK (). As seen in Figure 1 , inhibition of PDK shifts pyruvate metabolism from glycolysis and lactate production to glucose oxidation in the mitochondria. The ability of DCA to decrease lactate production has been used for more than 30 years in the treatment of lactic acidosis that complicates inherited mitochondrial diseases in humans ().