PDF Publication of U of A Study in Cancer Cell Journal

Cancer Cell Article: A Normalization of the Mitochondria-K+ Channel Axis Promotes Apoptosis and Inhibits Cancer Growth Bonnet, Sebastien Alois Haromy, Christian Beaulieu, Richard Thompson, Christopher T. Lee, Gary D. Lopaschuk, Lakshmi Puttagunta, Joan Allalunis-Turner, Stephen L. Archer, 1 Gwyneth Harry, 7 Sandra Bonnet Bernard Thebaud,1,6, Miguel A. Andrade,1,8, Kyoko Hashimoto,1, Christopher J. Porter,8, Evangelos D. Michelakis,1, and * Vascular Biology Group and the Pulmonary Hypertension Program 1 Physiology Department 2 Oncology Department 3 Biomedical Engineering Department 4 Pharmacology Department 5 Division of Pediatrics Department of Pathology and Laboratory Medicine Edmonton, Alberta, Canada T6G 2B7, University of Alberta cellular and molecular medicine department at the University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada, and the Ontario Genomics Innovation Centre.

 Contact information is Reference:


 Apoptosis resistance may be conferred by the distinct metabolic profile of cancer (aerobic glycolysis), which may be therapeutically targeted. Numerous human malignancies differ from normal cells in having high mitochondrial membrane potential (DJm) and low expression of the K+ channel Kv1.5, both of which promote resistance to apoptosis. All cancer cells, but not normal cells, respond to dichloroacetate (DCA) by inhibiting mitochondrial pyruvate dehydrogenase kinase (PDK), switching metabolism from glycolysis to glucose oxidation, lowering DJm, raising mitochondrial H2O2, and activating Kv channels. DCA upregulates Kv1.5 through an NFAT1-dependent mechanism. Without exhibiting any visible harm, DCA causes apoptosis, lowers proliferation, and reduces tumor growth. The siRNA-induced molecular suppression of PDK2 imitates DCA. Cancer therapy should focus on the mitochondria-NFAT-Kv axis and PDK, and the orally accessible DCA is a promising selective anticancer drug. INTRODUCTION Suppression of apoptosis plays a role in both the development of cancer and its resistance to treatment, at least in part. Despite the fact that mitochondria are understood to be apoptosis regulators, their significance as targets for cancer therapy has not been sufficiently investigated or practically utilized. In 1930, Warburg proposed that the hallmark metabolic phenotype of cancer, aerobic glycolysis, is caused by mitochondrial malfunction (Warburg, 1930). It has recently been established through the use of positron emission tomography (PET) imaging that most malignant tumors have enhanced glucose absorption and metabolism. Although a useful cancer marker, this bioenergetic characteristic has not been used as a treatment target.


 Since many years ago, lactic acidosis and hereditary mitochondrial illnesses have been treated in humans using the tiny chemical DCA, a metabolic modulator. The metabolicelectrical remodeling that we describe in numerous cancer lines (hyperpolarized mitochondria, activated NFAT1, downregulated Kv1.5) is reversed by DCA without affecting normal cells, causing apoptosis and slowed tumor development. DCA inhibits and reverses tumor growth in vivo at therapeutically relevant doses for up to 3 months without causing any apparent toxicity or altering hemoglobin, transaminases, or creatinine levels. DCA is a desirable option for proapoptotic cancer therapy because of its simplicity of administration, selectivity, and potency, which may be quickly converted into phase II-III clinical trials. January 2007, Cancer Cell 11, 37-51 Elsevier Inc. 37 is believed to be a symptom, not a cause, of cancer, meaning that cells primarily produce energy by glycolysis due to chronic mitochondrial damage that prevents oxidative phosphorylation. However, it is still unclear if cancer patients’ mitochondria are actually harmed and whether this damage is irreversible.

 Recently, interest in the metabolic theory of cancer has returned. The altered cells initially need to rely on glycolysis for energy production since early carcinogenesis takes place in a hypoxic microenvironment, according to a recent theory by Gatenby and Gillies (Gatenby and Gillies, 2004). This early metabolic adaption does, however, seem to provide a proliferative benefit by inhibiting apoptosis. Additionally, the “byproducts” of glycolysis (lactate and acidosis, for example) aid in cell motility, break down the extracellular matrix, and raise the risk for metastatic disease. Thus, the glycolytic phenotype persists even when the tumors eventually vascularize and their oxygen levels rise, creating the “paradox” of glycolysis under aerobic settings (the Warburg effect). The convergent metabolic and apoptotic pathways in the mitochondria are interdependent, and it appears that the glycolytic phenotype is in fact linked to apoptosis resistance (Plas and Thompson, 2002). Numerous oncoproteins increase the production of glycolytic enzymes, and several glycolytic enzymes are known to regulate apoptosis (Kim and Dang, 2005). For instance, Akt activates hexokinase, an enzyme that catalyzes the first and irreversible step in glycolysis and promotes resistance to apoptosis (Elstrom et al., 2004).

 Hexokinase is translocated to the mitochondrial membrane by Akt, which is mediated via its downstream mediator glycogen synthase kinase 3 (GSK3). There, it binds to the voltage-dependent anion channel (VDAC) and inhibits apoptosis (Kim and Dang, 2005; Pastorino et al., 2005). Cancer cells that have GSK3 inhibition lose the ability to attach hexokinase to VDAC, undergo apoptosis, and become more sensitive to chemotherapy (Pastorino et al., 2005). This shows that the metabolic phenotype in cancer may originate from a potentially changeable remodeling of the mitochondria, which inhibits oxidative phosphorylation, increases glycolysis, and inhibits apoptosis. Pyruvate dehydrogenase (PDH), a gate-keeping mitochondrial enzyme, regulates whether glucose metabolism will conclude with glycolysis in the cytoplasm (converting pyruvate to lactate), or continue with glucose oxidation in the mitochondria (Figure 1). Acetyl-CoA is produced via PDH’s conversion of pyruvate to acetyl-CoA, which is then supplied to the Krebs cycle to produce the electron donors NADH and FADH2. The electron transport chain’s (ETC) complex I receives electrons from NADH (and FADH2 to complex III). The generation of reactive oxygen species (ROS) and the efflux of H+, which results in a negative mitochondrial membrane potential, are linked to the flux of electrons along the ETC (DJm). Since ATP is produced by the F1F0-ATP synthase using the energy that is stored in the DJm, the DJm is a good indicator of both mitochondrial and ETC activity. PDH kinase phosphorylates PDH to inhibit it (PDK). Unknown is the function of PDH and PDK in cancer. Because mitochondria govern various crucial activities like [Ca2+]i and ROS-redox regulation, mitochondrial remodeling has numerous downstream effects in addition to energy production. Both Ca2+-sensitive transcription factors and the opening of plasma-membrane ion channels are regulated by mitochondria through the release of ROS and [Ca2+]i, respectively. Some of these downstream pathways play a role in apoptosis as well and may be involved in cancer’s apoptosis resistance. For instance, by lowering the tonic outflow of K+ down its intracellular/extracellular gradient (145/5 mEq), blockage or downregulation of K+ channels increases [K+]i. K+ channel blockage or downregulation prevents apoptosis in a variety of cell types, including cancer cells, as [K+]i has a tonic inhibitory impact on caspases (Andersson et al., 2006; Remillard and Yuan, 2004; Wang et al., 2002; Yu et al., 1997). Due to its redox sensitivity, the voltage-gated K+ channel family (Kv) can be controlled by mitochondria. For instance, the comparatively stable ROS H2O2 produced from mitochondria can activate Kv1.5 (Caouette et al., 2003). Furthermore, the antiapoptotic protein bcl-2 blocks Kv channels whereas the mitochondria-derived proapoptotic mediator cytochrome c activates them (Remillard and Yuan, 2004). It is now understood that the mitochondria-ROS-Kv channel axis forms the foundation of a crucial O2-sensing mechanism in numerous tissues (Michelakis et al., 2004). In preliminary research, we examined a number of cancer cell lines with normal cell lines and discovered that cancer cells had more hyperpolarized mitochondria and lacked Kv channels on average. Reversing this metabolic-electrical remodeling could boost apoptosis and slow the spread of cancer if it is an adaptive response. 

We employed dichloroacetate (DCA), a well-known PDK inhibitor that is a tiny, orally accessible small chemical (BowkerKinley et al., 1998; Knoechel et al., 2006; Stacpoole, 1989). As seen in Figure 1, pyruvate metabolism changes from glycolysis and lactate generation to glucose oxidation in the mitochondria when PDK is inhibited. Lactic acidosis, which exacerbates hereditary mitochondrial illnesses in humans, is treated for more than 30 years using DCA’s capacity to lower lactate generation (Stacpoole et al., 1988, 2006). We predicted that the subsequent impacts of the DCA-induced change in metabolism would be advantageous for the treatment of cancer (Figure 1). 

We demonstrate that, as expected, DCA switches cancer cells’ metabolism from cytoplasm-based glycolysis to mitochondria-based glucose oxidation. This is related to the release of proapoptotic mediators from the mitochondria, induction of mitochondria-dependent apoptosis, and increased production of ROS and decreased DJm in all cancer cells but not in normal cells. In addition, DCA reverses Kv1.5 inhibition and downregulation in all cancer cells, but not in normal cells. The resulting K+ efflux and drop in intracellular K+ intensifies DCA’s proapoptotic effects. In vitro, DCA successfully slows the growth of tumors. 38 Cancer Cell 11, 37-51, January 2007, published by Elsevier Inc. In cancer and in vivo, there is a metabolic-electrical remodeling. We demonstrate that apoptosis resistance in cancer is regulated by metabolic-electrical remodeling. Furthermore, a straightforward medicine that is already prescribed to humans can quickly reverse this anomaly. RESULTS Cancer DCA Reverses Hyperpolarized Mitochondria and Suppressed Oxidative Metabolism in Mitochondria A549 (non-small-cell lung cancer), M059K (glioblastoma), and MCF-7 (breast cancer) are three human cancer cell lines that we examined for DJm and contrasted with SAEC, fibroblasts, and pulmonary artery smooth muscle cells, three normal, non-cancerous human cell lines (PASMC). Compared to normal cells, all cancer cell lines displayed a considerable increase in DJm hyperpolarization (increased fluorescence of the DJm-sensitive positive dye tetramethyl rhodamine methyl ester; TMRM). All three types of cancer cells were incubated with DCA for 48 hours, which reversed the hyperpolarization and brought the DJm back to that of normal cells (n = 80 cells; n = 10 plates/cell line/group). In contrast, DCA did not change the DJm of the PASMC, fibroblasts, or SAEC (Figure 2A) (not shown). 

The effects of DCA on mitochondrial DJm may be seen in as little as 5 minutes, and they were dose-dependent (Figure 2B). We used a protonophore (carbonylcyanide-ptrifluoromethoxyphenylhydrazone; FCCP) and demonstrated that it decreased DJm in all cancer cell lines in a dose-dependent manner, eventually depolarizing untreated and treated cancer cells, as the intracellular distribution of TMRM can theoretically be affected by variations in the plasma-membrane potential (Em). Image 1. A Cancerous Metabolic-Electrical Remodeling That Is Reversible Several Potential Therapeutic Targets are Revealed and Contributes to Resistance to Apoptosis Cancer cells rely on cytoplasmic glycolysis for energy production since mitochondrial glucose oxidation is hindered. This mitochondrial “inactivity” most likely causes a state of apoptosis resistance. By encouraging the inflow of acetylCoA into the mitochondria and the Krebs cycle, activating PDH by DCA increases glucose oxidation. This increases NADH delivery to complex I of the electron transport chain, increasing the production of superoxide, which is dismutated into the more stable H2O2 in the presence of MnSOD. Continuously producing more ROS can harm the redoxsensitive complex I, preventing H+ efflux and lowering DJm. Apoptosis-inducing factor and cytochrome c can escape when the DJm-sensitive mitochondrial transition pore (MTP) is opened (AIF). Both cytochrome c and H2O2 hyperpolarize the cell (increased Em) and open the plasma membrane’s redox-sensitive K+ channel Kv1.5, which prevents a voltage-dependent Ca2+ entry. The removal of NFAT from the nucleus as a result of the decreased [Ca2+]i causes an increase in Kv1.5 expression.

 Apoptosis is further boosted by the decreased tonic inhibition of [K+]i on caspases caused by the enhanced outflow of K+ from the cell. DCA’s efficiency is explained by its dual mode of apoptosis induction, both via depolarizing mitochondria (proximal pathway) and activating/upregulating Kv1.5. DCA’s selectivity is predicated on its ability to target the distinct metabolic profile that characterizes most malignancies (distal pathway). a2007 Elsevier Inc., Cancer Cell 11, 37–51, January tumor cell Cancer cells treated with DCA undergo a metabolic-electrical remodeling at the same low DJm (see Figure S1 in the Supplemental Data available with this article online). This shows that the TMRM signal was caused by actual changes in the DJm rather than being confused by DCA effects on the Em. We assessed glycolysis (Gl), glucose oxidation (GO), and fatty acid oxidation (FAO) in A549 cells (10 plates/experiment, n = 3), in order to ascertain the impact of DCA on metabolism. With a simultaneous decline in Gl and FAO, DCA considerably raised GO rates (+23%). (Figure 2C). Lactic-acid levels in the culture medium of the DCA-treated cells decreased (Figure 2D, n = 10 plates/group) and the intracellular pH rose (Figure 2E, n = 5 plates, 60 cells/group), as anticipated from the DCA-induced shift of pyruvate metabolism away from lactate and toward acetylCoA and the Krebs cycle.

 Proapoptotic factors are effluxed from mitochondria by DCA, which also increases the production of ROS. When cancer cells (A549) were exposed to DCA, cytochrome c was diffusely present in the cytoplasm and apoptosis-inducing factor (AIF) was translocated to the nucleus, both of which indicated induction of apoptosis. In contrast, the untreated cancer cells (A549) showed cytochrome c and apoptosis-inducing factor (AIF) restricted to the mitochondria (colocalized with mit Rotenone blocked the dose-dependent rise in H2O2 generation caused by DCA, indicating that complex I of the ETC provided the basis for this increase (Figure 3B, n = 5 plates/group). In isolated mitochondria, we also assessed NADH concentrations, and the results demonstrated that DCA enhanced intramitochondrial NADH (Figure 3C, 5 plates/experiment, n = 5). The inhibitor of VDAC, a vital part of the mitochondrial transition pore (MTP), 40 -diisothiocyano-2,20 -disulfonic acid stilbene (DIDS; 0.5 mM), prevented the DCA from causing a drop in DJm (Granville and Gottlieb, 2003) (N = 5, 60 cells per group, Figure 3D). We investigated the effects of low-dose cyanide to ascertain whether cancer cells are less reliant on the ETC and oxidative phosphorylation (a complex-IV inhibitor and a well-known poison for normal cells). Figure 2 shows the impact of cyanide on mitochondria (as determined by DJm). In cancer cells but not in healthy cells, DCA reverses the glycolytic phenotype and depolarizes the mitochondria (A) A549, MO59K, and MCF-7 cancer cells were noticeably depolarized after 48 hours of DCA (0.5 mM), although healthy SAEC were unaffected. (B) DCA immediately (5–10 min) and dose-dependently depolarizes mitochondria. In A549 cells, DCA inhibits glycolysis and fatty acid oxidation while boosting glucose oxidation. (D and E) DCA (0.5 mM) treatment for 48 hours reduced extracellular lactate levels and raised pH in A549 cells as determined by the SNARF-1 ratiometric dye. *p 0.05 compared to control. 40 Cancer Cell 11 (January 2007), 37–51 ª2007 The Cancer Cell, Elsevier Inc. Compared to the DCA-treated cells, the cancer cells’ metabolic-electrical remodeling was substantially less dramatic (Figure 3E, n = 5, 60 cells/group). Kv Channels in Cancer Cells are Activated by DCA Through an H2O2-Dependent Mechanism. We demonstrated, using whole-cell patch clamping, that the outward K+ current (Ik) was modest and essentially voltage independent in all untreated cancer cell lines. The Ik was not changed in the non-cancerous SAEC (Figure 4A), PASMC, or fibroblasts (not shown) (Figure 4A, n = 7-8/group), but DCA dramatically elevated the Ik in all cancer cell lines. After 48 hours of DCA treatment, the increase in Ik maintained, starting as early as 5 minutes. The majority of the elevated Ik was voltage-dependent and was stopped by the particular Kv channel inhibitor 4-aminopyridine.

 DCA also decreased cell capacitance, an electrophysiologic proxy for cell size/volume, similar with the cell shrinkage that marks early apoptosis. The elevated Ik resulted in hyperpolarization of the plasmamembrane Em (Figure 4A). Intracellular catalase supplied via patch pipette (i.e., due to H2O2) and rotenone (i.e., due to H2O2 created by complex I) both prevented the DCA from increasing Ik, but not thenoyltrifluoroacetone. Image 3. In A549 Cells, DCA Increases Intramitochondrial NADH and H2O2 Production, Opens the MTP, and Induces Mitochondrial Apoptosis (A) In untreated cancer cells, cytochrome c in green colocalizes with the mitotracker red labeling, however after 48 hours of treatment with DCA (0.5 mM), cytochrome c escapes from the mitochondria into the cytosol. In contrast to untreated A549 cells, DCA-treated cells primarily locate apoptosis-inducing factor (AIF, red) in the nucleus (i.e., are activated). (B) Rotenone (5 mM), an inhibitor of mitochondrial electron transport chain complex I, almost totally inhibits DCA’s dose-dependent rise in H2O2 generation (*p 0.05) (#p 0.05). In mitochondria extracted from untreated control and DCA-treated A549 cells (48 hr, 0.5 mM), DCA increases intramitochondrial NADH (C) (*p 0.05). (D) DIDS, an inhibitor of the mitochondrial VDAC, a crucial part of MTP, prevents the DCA-induced mitochondrial depolarization (*p 0.05). (E) The relative independence of cancer cells from the mitochondrial ETC is supported by the finding that cyanide (5 mM) had less effect on mitochondrial function (DJm) in cancer cells compared to DCA-treated cells (*p 0.05). January 2007, Cancer Cell 11, 37-51 41 Elsevier Inc. tumor cell (Figure 4B, n = 5) is a Metabolic-Electrical Remodeling in Cancer (TTFA; an inhibitor of complex II of the ETC). The human ether-a-go-gorelated gene (HERG) inhibitor E4031 (50 nM) (Wang et al., 2002) did not also prevent it from occurring (not shown). Due to efflux of K+ down its concentration gradient, the activation of Kv channels by DCA led to a decrease in intracellular K+. The effects of DCA on reducing intracellular K+ were blocked when KCl was added to this gradient (Figure 4C, n = 20). Through NFAT1 Inhibition, DCA Reduces [Ca2+]i and Increases Kv1.5 Expression. Recently, we demonstrated that DCA enhances Kv1.5 expression in PASMC (McMurtry et al., 2004). Kv1.5 expression (but not other Kv channels) correlates with tumor grade in human gliomas, meaning that the greater the grade, the lower the Kv1.5 expression. This is the only study to link Kv1.5 with cancer (Preussat et al., 2003). In contrast to Kir2.1, a K+ channel from a different family, we performed quantitative real-time polymerase chain reaction (n = 5) and immunoblots to demonstrate that Kv1.5 is dramatically enhanced in DCA-treated A549 cancer cells (Figure 4D) (not shown). We used archived tumors from a cohort of 30 consecutive patients with non-small-cell lung cancer to investigate whether Kv1.5 expression correlates with human tumor grade. Kv1.5 and survivin expression levels were assessed in 


Blinded readers correlated each tumor sample’s values with its histologic tumor grade. Survivin, a tumor aggressiveness and resistance to apoptosis marker, has recently been demonstrated to control mitochondria-dependent Image 4. In cancer cells but not in healthy cells, DCA activates a Kv current and induces the expression of Kv1.5. (A) In all cancer cell lines (A549, MO59K, and MCF-7) 48 hours of DCA (0.5 mM) increases K+ current density (current amplitude/cell capacitance, pA/pF), but has no effect on non-cancerous SAEC. Original K+ current traces for both untreated and DCA-treated cells are seen on the left. Given that the majority of the increased current is 4-aminopyridine sensitive, the rise in K+ current density is primarily caused by an increase in Kv current. The DCA-induced Kv current is displayed in the insets after current subtraction has been done. Additionally, DCA administration results in a significant rise in the plasma-membrane resting potential and a decrease in membrane capacitance (*p 0.05 versus control). (B) Acute exposure of cancer (A549) cells to DCA (0.5 mM) was used to examine the mechanism by which DCA raises K+ current (5–10 min). 4-Aminopyridine (5 mM), catalase (10,000 units, administered intracellularly via pipette), and rotenone (5 mM) all inhibited the effects of DCA on K+ current, however a particular complex II blocker (TTFA, 1 mM) had no effect on the effects of DCA (*p 0.05 against control). (C) DCA reduces [K+]i, and this is prevented when extracellular KCl is added to weaken the K+ gradient. (D) Kv1.5 mRNA and protein levels were higher in DCA-treated cells (48 hr) compared to untreated cells (*p 0.05 versus untreated cells). 42 January 2007, Cancer Cell 11, 37-51 The Cancer Cell, Elsevier Inc.

 A Metabolic-Electrical Remodeling in Cancer apoptosis in both vascular tissues and cancer (Dohi et al., 2004) (McMurtry et al., 2005). There was no change in the expression of Kir2.1, and Survivin correlated positively with tumor grade while Kv1.5 negatively correlated (the higher the Kv1.5, the lower the grade) (Figure S2). We hypothesized that this might also happen in cancer because NFAT (nuclear factor of activated T lymphocytes) decreases both apoptosis (Pu et al., 2003) and the production of Kv1.5 in cardiac cells (Rossow et al., 2004). When [Ca2+]i rises, calcineurin is activated and dephosphorylates NFAT, allowing it to go to the nucleus where it controls gene transcription (Macian, 2005). According to our hypothesis, DCA-induced activation of Kv1.5 causes plasmalemmal hyperpolarization, which inhibits voltage-gated Ca2+ channels (which are active even in nonexcitable cells; Dietl et al., 1995), lowers [Ca2+]i, and inhibits NFAT while increasing Kv1.5 expression (Figure 1). As expected, compared to untreated cells, DCA-treated A549 cells had decreased [Ca2+]i (Figure 5A). After 48 hours of exposure to DCA, the drop in [Ca2+]i is persistent and happens within 5 minutes. At both time points, 4-aminopyridine and rotenone block DCA’s effects on [Ca2+]i, and t-butyryl-H2O2 mimics those effects, indicating that they involve opening of Kv channels by complex I-derived H2O2 (n 20 plates/group). Lanthanum (n = 6), a blocker of Ca2+ entrance into the cell, inhibits the 4-aminopyridine-induced increase in [Ca2+]i in the DCA-treated cells (Figure 5A), demonstrating the existence of a functional voltage-dependent Ca2+ entry route. 

Most untreated A549 cells exhibit NFAT1 activation (characterized by its translocation to the nucleus). Image 5. By lowering [Ca2+]i and inhibiting the Ca2+-Dependent Transcription Factor, DCA increases Kv1.5. Using FLUO-3, the free cytosolic calcium ([Ca2+]i) concentration of NFAT1 was determined. Untreated A549 cells had greater [Ca2+]i than cells that have been treated with DCA or t-butyrylH2O2. 4-Aminopyridine (5 mM) inhibits the decrease in [Ca2+]i caused by DCA and H2O2, indicating that it involves the opening of Kv channels. Rotenone (5 mM) also inhibits it, indicating that complex I-produced ROS are involved. In contrast to untreated controls, lanthanum (10 mM) blocks the effects of 4-aminopyridine (*p 0.01, **p 0.05). (B) Confocal imaging and triple-staining of A549 cells demonstrated that NFAT1 (green) is activated because it is mostly located in the nucleus (stained blue by DAPI) of the majority of the untreated cells. The expression of Kv1.5 in these cells is extremely low (red). Since NFAT1 is primarily found in the cytoplasm, both DCA and H2O2 prevent its activation. Cells that contain more cytoplasmic NFAT1 also express Kv1.5 more frequently. 

The NFAT inhibitor VIVIT (4 mM) generates a substantial increase of Kv1.5 while displacing NFAT1 from the nucleus. (C) NFAT1 activation is linked to lower Kv1.5 expression and a higher histologic grade in representative clinical samples of non-small-cell lung cancer. In each sample, cells with activated NFAT1 exhibit the lowest levels of Kv1.5 expression (arrows). (D) Upregulating Kv1.5 causes VIVIT to lower [Ca2+]i, indicating that a downregulation of Kv1.5 is the cause of the elevated [Ca2+]i levels in cancer cells. *p 0.05 versus control. Elsevier Inc., January 2007, Cancer Cell 11, 37–51 tumor cell Cancer cells undergo metabolic-electrical remodeling, and the expression of Kv1.5 is low in these cells (Figure 5B). In contrast, NFAT1 is primarily found in the cytoplasm of DCA-treated cells, and Kv1.5 expression is correspondingly elevated. Similar to Ik (Figure 4B) and [Ca2+]i (Figure 5A), t-butyrylH2O2 mirrored the effects of DCA on NFAT1 (Figure 5B). Then, using VIVIT, a competitive peptide that selectively inhibits NFAT-regulated pathways by preventing calcineurin from docking on NFAT and reducing its activation without impairing calcineurin’s catalytic site, we treated A549 cells (Aramburu et al., 1999).

 The VIVIT-treated cells exhibit enhanced Kv1.5 expression and NFAT1 displacement from the nucleus (Figure 5B). The same was valid for cells exposed to cyclosporine, a general calcineurin inhibitor (Figure S3A). Low-grade histology was connected to high Kv1.5 expression and low NFAT1 expression, most of which was cytoplasmic, in human non-small-cell lung cancers costained with anti-Kv1.5, anti-NFAT1, and 40,6-diamidino-2-phenylindole (DAPI); the opposite pattern was observed in high-grade tumors (Figure 5C). A positive feedback loop consisting of low Kv1.5, depolarized plasma membrane, Ca2+ inflow, high [Ca2+]i, activated NFAT, and low Kv1.5 may enhance the effects of NFAT1. Because VIVIT lowers [Ca2+]i (n = 5 plates/group, Figure 5D), NFAT inhibition successfully breaks this loop.

DCA Reduces Proliferation and Induces Mitochondria-Dependent Apoptosis In vitro DCA upregulates annexin expression, upregulates the proportion of TUNEL-positive nuclei by six times, and activates caspases 3 and 9 in A549 cells (Figures 6A and 6D). DCA reduces markers of proliferation, such as BrdU incorporation and expression of proliferating cell nuclear antigen, by eliminating highly proliferative cells by inducing apoptosis and by lowering [Ca2+]i levels (Figures 6B and 6D) (PCNA). Image 6. Mitochondria and Kv1.5 Upregulation Contribute to the Dual Mechanism by Which DCA Induces Apoptosis and Reduces Proliferation in Cancer Cells (A) The expression of annexin and the higher percentage of TUNEL-positive cells (mean data in [D]) in A549 cells demonstrate that DCA enhances apoptosis as compared to the untreated controls. The annexin-positive cells’ lack of propidium iodide (PPI) staining indicates that the cells were apoptotic rather than necrotic. 

Caspases 3 and 9 are both activated by DCA, as shown by immunoblots, which show an active band for each caspase. (B) PCNA and BrdU measurements show that DCA therapy reduces cell proliferation (mean values in [D]). DCA also lowers survivin expression. (C and D) Comparatively to cells infected with Ad-GFP, Kv1.5 gene transfer using Ad-GFP-Kv1.5 adenovirus enhances the K+ current and causes apoptosis (for these tests, TUNEL is red in GFP-positive cells) (C). However, despite a bigger rise in the outward K+ current, this exogenous gene transfer of human Kv1.5 results in less apoptosis and a lower suppression of proliferation compared to the DCA-treated cells (D) (compare with Figure 4). By 32%, 4-aminopyridine reduces the apoptosis caused by DCA (also see Figure S4). *p 0.05 compared to controls. 44 January 2007, Cancer Cell 11, 37-51 The Cancer Cell, Elsevier Inc. A Cancerous Metabolic-Electrical Remodeling DCA also lowers the expression of survivin (Figure 6B). Two pathways are involved in the progression of DCA-induced apoptosis: one occurs in the mitochondria, where depolarization triggers mitochondria-dependent apoptosis, and the other occurs at the level of the plasmalemmal, where activation/upregulation of Kv1.5 channels reduces [K+]i and triggers the activation of caspases. 

We used adenoviral gene transfer to compare the apoptosis caused by DCA to the apoptosis caused by a primary increase in Kv1.5 expression in order to assess the relative relevance of the two processes (Figures 6C and 6D). The percentage of TUNEL-positive cells was much higher in the adenovirus carrying GFP plus cloned human Kv1.5 than in the adenovirus carrying only green fluorescent protein (GFP) (Pozeg et al., 2003). Though the increase in Ik brought about by the gene transfer was greater than the increase brought about by DCA (Figure 6C; compare with Figure 4A), the increase in apoptosis brought about by the gene transfer was noticeably lower than that brought about by DCA (Figure 6D). Then, using the Kv family blocker 4-aminopyridine (5 mM), we evaluated the DCA-induced apoptosis. In addition to A549 cells, we also looked at glioblastoma, a type of excitable cell in which Kv channels may play a more significant role in regulating apoptosis than they do in epithelial A549 cells. By itself, DCA alone caused 68% of the apoptosis (% TUNEL-positive cells) that was caused by 4-aminopyridine (Figure 6D). 

Similar to this, DCA Plus 4-aminopyridine caused 62% more apoptosis in glioblastoma cells than DCA alone did (Figure S4). Furthermore, DCA’s ability to release cytochrome c from mitochondria and start mitochondria-based apoptosis was unaffected by 4-aminopyridine (Figure S4). These findings support the notion that the mitochondrial element of DCA’s proapoptotic effects is overwhelmingly significant. The fact that VIVIT induced apoptosis and reduced proliferation similarly to DCA supported the hypothesis that NFAT1 is a distal mediator in DCA’s anticancer actions (Figure S3B). 

For imaging experiments, we used 30 slides per group and four random fields per slide, whereas for patch clamping, we used six to eight cells per group. DCA Mimicked by Molecular Inhibition of PDK2 We examined whether molecular inhibition of PDK2 by siRNA mimics DCA in order to confirm that PDK inhibition is the primary mechanism underlying the effects of DCA. Since PDK1 and 3 are exclusively found in the heart and testis, respectively, while PDK4 is mostly expressed in skeletal muscle and the heart, we chose PDK2 as the only isoenzyme that is expressed throughout the body. The lowest Ki for DCA (0.2 mM) and most active of all is PDK2 (Bowker-Kinley et al., 1998). siRNA for PDK2 decreased protein expression (measured by both immunoblots and immunohistochemistry) by 70% and inhibited mRNA by up to 80% in a dose-dependent manner (Figure S5).

 We examined three commercially available PDK2 siRNAs, all of which similarly suppressed the gene. Simultaneously scrambled PDK2 and PDK1 siRNA did not reduce PDK2 expression (Figure S5). DCA given to siRNA-treated cells had no further effects, although the scrambled siRNA had no effect on A549 cells, whereas the PDK2 siRNA lowered DJm and elevated mitochondrial ROS in a manner similar to DCA (Figures 7A and 7B, n 20 plates/group) (data not shown). Cancer cells’ proliferation and apoptosis were both increased when PDK2 was inhibited by siRNA (Figure 7C, n 30 plates/group). We immunoprecipitated PDH to demonstrate that DCA increased the nonphosphorylated fraction (i.e., active) of the catalytic subunit (E1a), further demonstrating that DCA activates PDH via inhibiting PDK (Figure 7D). Inducing Apoptosis and Reducing Tumor Growth in Vivo by Drinking Water DCA We examined naked athymic rats that had 3 3 106 A549 cells subcutaneously implanted. The rats had unrestricted access to either DCA (75 mg/l) or water. In the initial set of tests (protocol a), 21 animals were split into three groups: untreated controls (n = 5); rats receiving DCA soon after cell injection for 5 weeks; and rats receiving DCA 2 weeks after cell injection for 3 additional weeks. The untreated rats developed tumors quickly, growing exponentially at a constant rate (Figure 8A). Tumor weight at sacrifice and maximal diameter were used to gauge tumor size in both DCA-treated groups. In certain rats, in vivo magnetic resonance imaging allowed us to see the tumors in vivo and determine their volume. 

An increase in apoptosis (TUNEL) and a decrease in proliferation (PCNA) were linked to DCA’s ability to slow tumor growth (Figure 8B). Apoptosis and tumor growth in the treated rats showed an inverse relationship (Figure 8B). In the DCA-treated rats, Kv1.5 was increased and survivin was downregulated, supporting our in vitro findings (Figure 8C) (Figures 4 and 6). We investigated if the effects of DCA were sustained over longer periods of time and whether DCA would have a comparable effect in more advanced tumors in a second set of tests (protocol b). An untreated control group, a preventative group (rats given DCA at the time of tumor cell injection), and a reversal group (rats given DCA at week 10 for 2 weeks) were all monitored for 12 weeks. Each group of rats (n = 6) received DCA. Similar to protocol A, rats in the preventive group consistently had smaller tumors than the untreated controls. At week 10, DCA quickly prevented tumor growth, with a significant reduction occurring even after just one week of therapy. According to various blood tests, DCA therapy had no harmful side effects (Figure 8D; also see McMurtry et al., 2004). DISCUSSION Here, we demonstrate that the apoptosis resistance that underlies a number of human malignancies is mediated by metabolic-electrical remodeling (hyperpolarized mitochondria, downregulated Kv channels). 

This remodeling is reversed by the tiny chemical DCA, which targets the mitochondria. This results in Cancer Cell 11, 37-51, January 2007. 45 Elsevier Inc. Cancer cell A metabolic-electrical remodeling in cancer cell death and reducing cancer growth in vitro and in vivo These advantageous outcomes take place without harming non-cancerous cells or causing systemic toxicity. The well-known metabolic cancer signature of aerobic glycolysis (which only occurs in functional mitochondria) is reversible, rather than the result of long-term mitochondrial damage, according to DCA treatment, which significantly increases glucose oxidation (which only occurs in functional mitochondria). Both of the avenues via which DCA exerts its positive effects—mitochondrial depolarization and the outflow of proapoptotic mediators and an increase in Kv channel expression/function—induce apoptosis. By blocking NFAT1, a calcium-sensitive transcription factor that controls cell-differentiation processes in a variety of cell types but has not yet been studied in cancer, DCA promotes Kv channel expression. 

Numerous new prospective targets for proapoptotic therapy with great therapeutic selectivity are provided by the mitochondria-NFAT-Kv pathway in cancer. Cancer and Glycolysis: More Than an Epiphenomenon The majority of malignancies have a glycolytic character, which is now widely acknowledged. Warburg proposed—without providing evidence—that this was caused by “abnormal mitochondria” (Warburg, 1930); in other words, cancer cells are compelled to employ ineffective, nonmitochondrial methods of ATP production. Our findings imply that the purported mitochondrial “dysfunction” is actually reversible. According to Plas and Thompson (2002), who provide Figure 7, DCA Activates PDH by Inhibiting PDK2; Molecular Inhibition of PDK2 by siRNA, Oxidative Metabolism in Cancer Could Be Actively Suppressed; The Resultant Shift to Glycolysis May Lead to Apoptosis Resistance. imitates DCA (A) While scrambled siRNA has little effect, siRNA that effectively inhibits PDK2 expression (see Figure S1) depolarizes A549 mitochondria in a manner similar to DCA. (B) Mitosox, a fluorescent dye, was used to quantify the increase in mitochondrial-derived ROS caused by siRNA suppression of PDK2, which likewise resembles DCA. Similar to DCA, (C) siRNA-based PDK2 inhibition induces apoptosis (% TUNEL-positive cells). 

(D) A549 cells were used to immunoprecipitate human PDH, which was then subjected to a cocktail of monoclonal antibodies directed against various PDH subunits. The E1a subunit, or catalytic subunit, is fully phosphorylated in untreated cancer cells, indicating that PDH is being maximally inhibited by PDK. The nonphosphorylated proportion of the E1a subunit is considerably higher in cells that have been exposed to DCA, which suggests that PDH activity has risen. *p 0.05 compared to controls. 46 Cancer Cell 11, 37-51, January 2007, published by Elsevier Inc. Cancer cells with metabolic-electrical remodeling have a survival advantage (Gatenby and Gillies, 2004). This implies that undoing this metabolic/mitochondrial remodeling might be a unique strategy to overcome apoptosis resistance. We demonstrate that encouraging oxidative phosphorylation can quickly change the glycolytic profile of cancer (Figure 2C). This is connected to mitochondrial depolarization, which promotes apoptosis and slows the growth of tumors. In comparison to many noncancerous cell lines, all of the human cancer cell lines tested had greater negative DJm (Figure 2A), which may be a sign of malignancy. 

Although mitochondrial depolarization is not necessarily related with apoptosis, our observations support the finding that cationic lipophilic medicines preferentially accumulate to tumor mitochondria (Don and Hogg, 2004). Additionally, positively charged rhodamine-based dyes (such TMRM) have been tested as “carriers” for the targeted delivery of medications to treat cancer. More than 200 carcinomas were screened, and it was discovered that they accumulated rhodamine significantly more than noncarcinoma cells. Although the mechanism was unclear at the time, these findings were first reviewed in 1988 (Chen, 1988), and they most likely reflect the more unfavorable DJm of cancerous cells in comparison to noncancerous cells. Our research clearly demonstrates that this relative rise in DJm is linked to an increase in apoptosis resistance, and that its “normalization” would boost apoptosis and slow the progression of cancer. In line with our hypothesis, it has recently been demonstrated that the DJm of colon cancer cells predicts the aggressiveness of the tumor cells, i.e., the more hyperpolarized the DJm, the more aggressive and metastatic the tumor (Heerdt et al., 2005). 

Studying DJm in fresh tumor samples may be a practical way to foretell chemotherapeutic proapoptotic resistance, which has significant clinical decision-making implications. Figure 8 shows that an increase in apoptosis and a decrease in cell proliferation are the causes of the decreased tumor size in DCA-treated naked rats. (A) Within a week of injecting A549 cells into the flank of naked rats, detectable tumors appear. The tumors are smaller in DCA-treated rats in both the preventive and reversal groups of protocols a and b (see Results). The tumors’ dimensions were measured in-vivo and at the moment of death using weight, calipers, or magnetic resonance imaging, as shown. (B) Triple staining revealed that tumors in the DCA-treated rats were much smaller as a result of an increase in apoptosis (TUNEL) and a decrease in proliferation (PCNA). The percentage of TUNEL-positive cells and tumor diameter and weight were found to be significantly correlated; the higher the percentage of TUNEL-positive cells, the smaller the tumor. (C) Both immunohistochemistry and immunoblotting results demonstrate that DCA enhances Kv1.5 and decreases survivin expression, which is consistent with our in vitro observations. (D) DCA-treated rats showed no signs of liver damage.

Both immunohistochemistry and immunoblotting demonstrate that DCA enhances Kv1.5 and lowers survivin expression. (D) No liver (AST), kidney (creatinine), or blood (hemoglobin) damage was present in the DCA-treated rats. *p 0.05 compared to the untreated controls. January 2007, Cancer Cell 11, 37–51 a2007 Elsevier Inc. tumor cell A Cancerous Metabolic-Electrical Remodeling What Mechanisms Does DCA Use to Modify Metabolism, Depolarize Mitochondria, and Initiate Apoptosis? By increasing the intramitochondrial production of the electron-donor NADH (Figure 3C), a substrate of the ETC complex I, and shifting the metabolism of pyruvate away from lactate and toward acetyl-CoA and the Krebs cycle (Figures 2C and 2D), caused by DCA or molecular inhibition of PDK2 (Figure 7), more complex I-based ROS are produced (Figures 3B and 7B) (Kushnareva et al., 2002). The ETC, especially complex I, can suffer oxidative damage from an extended increase in ROS production. Due to its size (46 subunits), the presence of at least nine ROS-sensitive iron-sulfur centers, and the presence of seven mitochondrial DNA-encoded subunits, this megacomplex is the most vulnerable of all ETC complexes to oxidative damage. 

Complex-I dysfunction brought on by ROS can restrict H+ efflux and lower DJm. The voltage-sensitive MTP opens in response to a continuous and large decline in DJm (Zamzami and Kroemer, 2001), allowing the efflux of several proapoptotic substances and the beginning of apoptosis (Figures 3A, 6, and 8B). As a result, mitochondrial ROS production rises even more, possibly perpetuating a positive feedback loop that promotes apoptosis (Zamzami and Kroemer, 2001). Congenital mitochondrial disorders and neurodegenerative illnesses are examples of this “complex I-centered” suggested mechanism. Patients with congenital complex-I deficiency produce more ROS and less DJm (Pitkanen and Robinson, 1996). 

Complex I inhibition in cell lines is dose-dependently correlated with reduced DJm and increased ROS generation; in other words, the higher the percentage of complex-I inhibition, the higher the ROS and the lower the DJm (Barrientos and Moraes, 1999). In the pathogenesis of neurodegenerative diseases like Parkinson’s, where complex-I dysfunction and ROS-mediated oxidative damage are well described, a similar mechanism is proposed where dose-dependent inhibition of complex I leads to dose-dependent efflux of cytochrome c and apoptosis (Clayton et al., 2005). (Bao et al., 2005; Schon and Manfredi, 2003). The reduction in DJm caused by DCA was constrained by VDAC inhibition (Figure 3D). ADP, a substrate for the F1F0-ATPase, is transported from the cytoplasm into the mitochondria by VDAC and the adenine nucleotide translocase. 

By preventing the F1F0-ATPase from doing its job, inhibition of the VDAC would prevent H+ from building up in the intermembrane mitochondrial region, causing hyperpolarization of the DJm and reducing the depolarizing effects of DCA. This is supported by research from Thompson’s team (Vander Heiden et al., 1999), though some of these mechanisms may only be relevant under certain experimental conditions, like growth-factor withdrawal, and the function of VDAC in the regulation of DJm and the start of apoptosis is still debatable (Shimizu et al., 1999). (Vander Heiden et al., 1999). A further intriguing hypothesis is that DCA, which is an anion in and of itself (see structure in Figure 1), reaches the mitochondria via the VDAC, which would account, at least in part, for the limitation of DCA’s effects on DJm. Survivin is an apoptosis inhibitor, and DCA unexpectedly but in line with its therapeutic efficacy reduced its expression both in vitro and in vivo (Figures 6 and 8). 

Recently, survivalin has become a significant anti-apoptotic oncoprotein. Uncertainty surrounds the mechanism by which survivin is downregulated. Survivin may be involved in the mitochondrial remodeling of cancer, according to recent research demonstrating the direct role of a mitochondrial survivin pool in the regulation of apoptosis (Dohi et al., 2004; McMurtry et al., 2005). DCA Normalizes a Mitochondria-NFAT-Kv Channel Axis in Cancer, Associated with DCA’s Proapoptotic and Antiproliferative Effects There are probably several mechanisms involved in cancer’s resistance to apoptosis. The new research emphasizes how this resistance is influenced by Kv channel blockage or downregulation brought on by compromised mitochondrial signaling. The tonic inhibition that cytosolic K+ imposes on caspases is increased when K+ channels are closed or their expression is reduced.

 In A549 cells, Kv1.5 gene transfer directly induced apoptosis (Figures 6C and 6D). The DCA-induced apoptosis was reduced by 32% in A549 cells and by 38% in glioblastoma cells when all Kv channels were functionally inhibited (Figure 6D; Figure S4). This result suggests that although the majority of apoptosis in DCA-treated cells is a direct result of efflux of proapoptotic mediators from cancer cells, the secondary effects on Kv channels also play a significant role. Although K+ channel opening increases apoptosis in various tumors, the specific involvement of K+ channels in cancer is still unknown.

The opposite effect has also been observed (reviewed in Wang, 2004). The nature of the tumor or the well-known diversity of K+ channel families may be related to this. Different cell types’ apoptosis is now being significantly regulated by a subset of K+ channels. For instance, in different cancer cell lines, the Kv channel HERG mediates H2O2-dependent apoptosis (i.e., low HERG expression reduces death and increases proliferation) (Wang et al., 2002). Kv1.5 controls apoptosis in PASMC and is downregulated in proliferative and apoptosis-resistant vascular media in pulmonary hypertension, according to Remillard and Yuan (2004). (McMurtry et al., 2004, 2005; Pozeg et al., 2003). The very low turnover time of Kv1.5—less than 8 hours from transcription to functional expression—as a cancer apoptosis regulator and the reason for our focus on this channel (Levitan et al., 1995). We demonstrate that H2O2, a Kv1.5 channel opener produced by the ETC complex I, is insufficiently produced by cancer cells (Figure 3B). More significantly, Kv1.5 expression correlates inversely with histologic grade in a cohort of individuals with non-small-cell lung cancer (more aggressive tumors have less Kv1.5), and Kv1.5 is downregulated in cancer cell lines (Figure 4D) (Figure S2). As a key transcription factor for this Kv1.5 downregulation, NFAT1 has been found by us (more aggressive tumors have more activated NFAT1) (Figure 5C). 48 January 2007, Cancer Cell 11, 37-51 Company Elsevier Metabolic-electrical Remodeling in Cancer Cells In cancer, the cellular milieu is conducive to NFAT activation. 

Increased [Ca2+]i in A549 cells (Figure 5A), a direct activator of calcineurin and therefore NFAT (Macian, 2005). This rise in [Ca2+]i is caused, at least in part, by the increased Ca2+ influx brought on by the absence of Kv channels (Figure 4). Additionally, elevated ROS levels inhibit calcineurin (Namgaladze et al., 2005), hence cancer’s low mitochondrial ROS (Figures 3B and 7B) encourage NFAT activation. Additionally, Figure 2E’s acidotic environment in cancer (caused by aerobic glycolysis) would support NFAT activation even more (Komarova et al., 2005). Given that DCA raises ROS, raises pH, and lowers [Ca2+]i, it is interesting that all of these pathways are reversed by DCA, which accounts for its striking effects on NFAT (Figure 5B). The presence of the mitochondria-NFAT-Kv1.5 axis, which is repressed in cancer, is suggested by the elevation of Kv1.5 by a medication that directly impacts mitochondrial function. Our research implies that cancer treatments that target the mitochondria should take potential effects on Kv channels into account. 

Potential for Rapid Translation to Clinical Oncology of Metabolic Modulation in Cancer by DCA Our research demonstrates that metabolic modulators may act as apoptotic sensitizers in human cancer, either alone or in combination with conventional chemotherapies. This strategy may combine efficacy and selectivity by focusing on a fundamental and distinctive characteristic of cancer cells. In existing tumors, DCA (in clinically relevant doses; Stacpoole et al., 2006) was successful in delaying and suppressing tumor growth both early in their development (week 2) and late in it (week 10). (Figure 8A). In the reversal protocols, DCA had immediate effects that persisted even after one week of therapy. Microarray tests, which revealed a brief list of changed mitochondrial apoptosis, cell cycle, and ion channel genes, verified the relative specificity of DCA to target a metabolic (mitochondria) and electric (K+ channels) remodeling (Supplemental Results; Figure S6). The lack of any systemic toxicity in this (Figure 8D) and other recent animal (McMurtry et al., 2004) and human investigations demonstrates DCA’s selectivity, which is a very appealing trait (Stacpoole et al., 2006). Because of its capacity to “repair” DJm, DCA may be more effective in cells with very high DJm, such as cancer cells, and inactive in healthy cells (epithelial, fibroblasts, or PASMC). 

The selectivity of PDK may also be influenced by its preferential expression. Recent research on non-small-cell lung cancer specimens found that cancer cells expressed more PDK2 and less PDH (both of which are consistent with a glycolytic phenotype) in comparison to nearby non-malignant cells (Koukourakis et al., 2005). Our in vitro research on the hard-to-treat glioblastoma tumor revealed that DCA’s tiny size results in great tissue penetration following oral consumption, including the central nervous system (Stacpoole et al., 2003). Furthermore, DCA elevates intracellular pH and reduces tumor lactic-acid generation (Figure 2E); further research is required to explore the idea that these changes will lessen tumor invasiveness and metastatic potential (Gatenby and Gillies, 2004). Our research pinpoints the mitochondria-NFAT-Kv channel axis and PDK as essential elements of the metabolicelectrical reconfiguration that distinguishes many human malignancies and raises the intriguing possibility that DCA may be effective against specific types of cancer in patients. 

Our research may be easily applied to clinical oncology given the very recent publication of the first randomized long-term clinical trial of oral DCA in children with congenital lactic acidosis (at doses comparable to those used in our in vivo experiments), which demonstrated that DCA was well tolerated and safe (Stacpoole et al., 2006). DEVELOPMENTAL METHODS The University of Alberta Human Ethics Committee approved the use of human tissues, while the University of Alberta Animal Ethics Committee approved all rodent experiments (Health Sciences Laboratory Animal Services). A Zeiss LSM 510 multiphoton confocal microscope (Carl Zeiss Canada, Toronto, ON) and multiple-staining methods were used to accomplish Confocal Microscopy Imaging, as previously described (McMurtry et al., 2004, 2005). See Supplemental Data for more information. Metabolic Research To assess the rates of glycolysis, fatty acid oxidation, and glucose oxidation in the presence or absence of DCA (0.5 mM, 48 hr), A549 cells were grown to confluency in T-175 flasks; for further information, see Supplemental Data. 

Measurements of Ca2+ FLUO-3AM was used to examine the intracellular free Ca2+ concentration ([Ca2+]i) in live A549 cancer cells (Invitrogen-Molecular Probes Canada, Burlington, ON). To allow for the cleavage of the acetoxymethyl esters, cells were loaded with FLUO-3AM (5 mmol/l) for 45 min (37C) in serum-free media and then washed for 30 min (37C) in PBS. Hoechst nuclear staining (1.0 mmol/l) was used in conjunction with FLUO-3 for 10 minutes (Molecular Probes). Excitation was at 488 nm, and fluorescence was recorded between 505 and 535 nm. Measurements of H2O2 On LabTek multiwell slides (Nalge Nunc, Rochester, NY, USA; VWR, Mississauga, ON, Canada), cancer cells were grown until confluent. Monolayers were pre-incubated with DCA for 1 hour either with or without the addition of 5 mM rotenone from Sigma-Aldrich Canada, Oakville, Ontario.

AmplexRed assay was used to evaluate the production of H2O2 (Molecular Probes). H2O2 levels were determined by comparison to a standard curve using fluorescence measured at 590 nm with illumination at 530 nm, as previously described (McMurtry et al., 2004). Electrophysiology Cells were voltage clamped at a holding potential of 70 mV using normal whole-cell patch-clamping procedures, and currents were induced by 200 ms test pulses from 70 to +70 mV with 20 mV steps, filtered at 1 kHz, and sampled at 2-4 kHz, as previously described (McMurtry et al., 2004, 2005). 

Quantitative Real-Time Polymerase Chain Reaction with Immunoblotting See Supplemental Data for more information, antibodies, and primers. Gene Transfer for Kv1.5 According to a previous report, replication-deficient serotype-5 adenoviruses were used to infect A549 cells with the genes for GFP and cloned human Kv1.5 (both listed under Cancer Cell 11, 37-51, January 2007 a2007 Elsevier Inc. 49 Cancer Cell A Metabolic-Electrical Remodeling in Cancer CMV promoters) (McMurtry et al., 2005; Pozeg et al., 2003). We were able to infect cells at a rate of 80%, and infected cells were chosen for analysis based on their green fluorescence. Assays for in vivo tumorigenicity Subcutaneous injections of A549 cell suspension in PBS (3 3 106 cells per injection) were given to naked, athymic rats. Two protocols were applied to three groups of rats: control (no treatment), prevention, and reversal. 

Rats were monitored for 5 weeks as part of protocol A; the prevention group received treatment for 5 weeks and the reversal group for 3 weeks (weeks 3–5). Rats were subjected to Protocol B for 12 weeks, with the prevention group receiving treatment for 12 weeks and the reversal group receiving treatment from week 10 to week 12. DCA (0.075 g/l) was added to the drinking water in both treatments. We estimated and modified the DCA concentration needed to get a daily dose that was comparable to that used therapeutically (50-100 mg/kg) based on the amount of water consumed. Stacpoole et al. (2006); McMurtry et al. (2004) The appearance of tumors at injection sites in rats was monitored weekly, and the size of each tumor was quantified weekly in each of the three groups using calipers. 

K+ intracellular Ratiometric measurements of the fluorescence of cells that were loaded with the acetoxymethyl ester form of PBFI were used to study intracellular K+ (PBFI-AM; 5 mM; Molecular Probes). See Supplemental Data for more information. NADH in mitochondria As previously published, mitochondria were extracted from both untreated and DCA-treated cells (500 mM, 48 hr) (Michelakis et al., 2002). The methods previously reported by Brandes and Bers were used to measure mitochondrial NADH ([NADH]m) (1996). Using a Photon Technology International Delta Scan 1 fluorescence spectrophotometer, light at a wavelength of 350 nm was used to stimulate the mitochondria. Fluorescence was then observed at a wavelength of 456 nm (London, ON, Canada). 

The primary source of the fluorescence signal at 456 nm is known to be [NADH]m (Eng et al., 1989). Activity PDH By counting the number of phosphorylated and unphosphorylated E1a subunits, PDH activity was calculated. Cells treated and untreated with DCA both produced three milligrams of protein. With the aid of the PDH complex immunocapture kit, PDH subunits were extracted (MitoSciences, Eugene, OR, USA). The human PDH subunits monoclonal antibody cocktail was then utilized to immunoblot the immunoprecipitated fraction (MitoSciences). siRNA Research In six-well culture dishes, A549 lung cancer cells were expanded to 80% confluence. In OptiMEM1 culture medium, the transfection agent siPORTamine (Ambion siRNA Transfection II kit 1631, Ambion, Austin, TX) was preincubated for 10 min at room temperature at a ratio of 1:12. (Invitrogen-GIBCO Canada, 31985-070, Burlington, ON). 

It had previously been proven that this level of transfection agent causes at least 60% gene knockdown and fewer than 15% cell death (data not shown). Following a further 10 minutes of incubation, the mixture was mixed with 75, 37.5, or 18.7 nmol of scrambled vs silencer RNA for human PDK2 and PDK1 (Ambion) in an equivalent volume of OptiMEM1. Additionally evaluated were the PDK2 silencer variants ID 264, 265 and 266. The transfection agent-RNA complex combination was allowed to spread over the monolayer before 1.5 ml of full F12K was added. The culture media was then aspirated from the cells. For 48 hours, plates were incubated at 37°C. Cultures of Cells See Supplemental Data for sources of culture media and cell lines. DNA microarrays, intracellular pH measurement, magnetic resonance imaging, and lactate measurement Review the Supplemental Data. 

Statistics The mean and SEM are used to express values. Intergroup differences were measured using Kruskal-Wallis or one-way ANOVA if necessary, and Fisher’s exact test was used for post hoc analysis (Statview 4.02, SAS Institute, Cary, NC, USA). Additional Information Six figures, Supplemental Results, and Supplemental Experimental Procedures are included in the Supplemental Data, which are available online with this publication at 37/DC1/. ACKNOWLEDGMENTS E.D.M. S.B. is supported by postdoctoral fellowships from both the CIHR and AHFMR, and this work was financed by funds from the Canadian Institutes for Health Research (CIHR), Alberta Heritage Foundation for Medical Research (AHFMR), and Canadian Foundation for Innovation. the 25th day of November 2005 12 July 2006, revised 18 October 2006 – Accepted 2015 January Published REFERENCES Along with Andersson, Janson, Behnam-Motlagh, Henriksson, and Grankvist (2006). 

Using the fluorescent dye PBFI in a 96-well plate approach on cultivated lung cancer cells, we were able to demonstrate how intracellular potassium ion depletion causes apoptosis. In Vitro Toxicol. 20, 986–994. The authors are J. Aramburu, M. B. Yaffe, C. Lopez-Rodriguez, L. Cantley, P. G. Hogan, and A. Rao (1999). Science 285, 2129-2133. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Rice, M.E., Bao, L., and Avshalumov, M.V. (2005). Instead of causing ATP depletion, partial mitochondrial inhibition suppresses the release of dopamine from the striatum and elevates H2O2 to promote medium spiny neuron depolarization. 25, 10029–10040 J. Neurosci A. Barrientos and C. Moraes (1999). Physiological effects of mitochondrial complex I impairment titrated. J Biol Chemistry 274, 16188–16197. Bowker-Kinley, M.M., Popov, K.M., Harris, R.A., Wu, and Davis, W.I. (1998). evidence that the mammalian pyruvate dehydrogenase complex is regulated in certain tissues. Biochemical Journal 329, 191–196. R. Brandes and D.M. Bers (1996).

Decreased mitochondrial NADH fluorescence is brought on by increased work in the cardiac trabeculae and is followed by a gradual recovery. Biophysical Journal, 71, 1024–1035. U. Brandt (2006). the enzyme that converts NADH to quinones for energy (complex I). 75, 69–92, Annu. Rev. Biochem. The authors are Caouette, Dongmo, Berube, Fournier, and Daleau (2003). The Kv1.5 channel expressed in a mammalian cell line is altered by hydrogen peroxide. Pharmacol. Naunyn Schmiedebergs 368, 479–486. L.B. Chen (1988). potential of the mitochondrial membrane in live cells. 4, 155–181 in Annu. Rev. Cell Biol. Sharpe, M., Clayton, R., and J.B. Clark (2005).

It has implications for apoptosis and neurodegenerative illness that cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficiency. 92, 840–849 J. Neurochem In addition to Dietl, Haller, Wirleitner, Volkl, Friedrich, and Striessnig (1995). Purinoceptor stimulation by ATP results in the activation of L-type Ca2+ channels in an alveolar epithelial cell (L2). Physiol. Am. 269, L873-L883. The authors are T. Dohi, E. Beltrami, N. R. Wall, J. Plescia, and D. C. Altieri (2004). Apoptosis is prevented by mitochondrial survivin, which also encourages tumor growth. 114, 1117–1127 J. Clin. Invest. January 2007, Cancer Cell 11, 37-51 The Cancer Cell, Elsevier Inc. A Cancerous Metabolic-Electrical Remodeling Hogg, P.J., and Don, A.S. (2004). Drug targets for cancer: mitochondria. 372–378 in Trends Mol. Med. 10, Buzzai, M., Karnauskas, R., Harris, M.H., Plas, D.R., Zhuang, H., Cinalli, R.M., Alavi, A., Rudin, C.M., and Thompson, C.B. Elstrom, R.L., Bauer, D.E. (2004). 

Cancer cells’ aerobic glycolysis is stimulated by Akt. 64, 3892–3899; Cancer Res. Lynch, R.M., J. Eng, and R.S. Balaban (1989). Fluorescence spectroscopy and imaging of isolated cardiac myocytes using nicotinamide adenine dinucleotide. Biophysical Journal 55, 621–630. R.A. Gatenby and R.J. Gillies (2004). Why is aerobic glycolysis more prevalent in cancers? National Cancer Review 4, 891-899. D.J. Granville and R.A. Gottlieb (2003). The therapeutic target for starting cell death is the voltagedependent anion channel (VDAC) in the mitochondria. Current Medical Chemistry 10, 1527–1533. B.G. Heerdt, M.A. Houston, and L.H. Augenlicht (2005). Colonic carcinoma cells’ intrinsic mitochondrial membrane potential and the likelihood that the tumor would advance are related. 65, 9861–9867; Cancer Res. Dang, C.V., and Kim, J.W. (2005). many functions of glycolytic enzymes. 141–150 in Trends Biochem. Sci. In addition to Brown, D.G., Knoechel, T.R., Tucker, A.D., Robinson, C.M., Phillips, C., Taylor, W., Bungay, P.J., and Kasten, S.A. (2006). 

Based on crystal structures of human pyruvate dehydrogenase kinase 2 containing natural and synthetic ligands, regulatory roles of the N-terminal domain have been identified. 402–415 in Biochemistry, vol. S.V. Komarova, A. Pereverzev, J.W. Shum, S.M. Sims, and S.J. Dixon (2005). Convergent calcium/calcineurin/NFAT pathway signaling by acidosis and receptor activator of NF-kB ligand (RANKL) in osteoclasts. USA: 102nd National Academy of Sciences, 2643–2648. Giatromanolaki, A., Sivridis, E., Gatter, K.C., and Harris, A.L. Koukourakis, M.I. (2005). Expression of pyruvate dehydrogenase and pyruvate dehydrogenase kinase in tumor-associated stroma and non-small cell lung cancer. 7, 1-6 Neoplasia Murphy, A.N., Andreyev, and Kushnareva, respectively (2002). Reactive oxygen species production mediated by complex I is modulated by cytochrome c and the oxidation-reduction status of NAD(P)+. Biochemical Journal 368, 545–553. Gealy, R., Takimoto, K., Levitan, E.S., Trimmer, J.S. (1995). In pituitary cells, membrane depolarization prevents Kv1.5 voltage-gated K+ channel gene transcription and protein expression. 270, 6036–6041, J. Biol. Chem. F. Macian (2005). Key controllers of T-cell activity and development are NFAT proteins. Journal of Immunology 5, 472-484. Haromy, A., Hashimoto, K., Wu, X., Dyck, J.R., McMurtry, M.S., and Michelakis, E.D. (2004).

 Dichloroacetate causes the death of pulmonary artery smooth muscle cells, which both prevents and cures pulmonary hypertension. 95 Circ Res, 830–840. M.S. McMurtry, S.L. Archer, D.C. Altieri, S. Bonnet, A. Haromy, G. Harry, S. Bonnet, L. Puttagunta, and E.D. Michelakis (2005). A targeted induction of pulmonary vascular apoptosis by gene therapy that targets survivin results in the reversal of pulmonary arterial hypertension. 115, 1479–1491 J. Clin. Invest. E.D. Michelakis, V. Hampl, A. Nsair, X. Wu, G. Harry, A. Haromy, A. Haromy, R. Gurtu, and S.L. Archer (2002). Variations in vascular oxygen sensing are explained by diversity in mitochondrial activity. Res. Circ. 90, 1307–1315. E.D. Michelakis, B. Thebaud, E.K. Weir, and S.L. Archer (2004). Resistance artery smooth muscle cells’ mitochondrial O2-sensor controls the redox modulation of O2-sensitive K+ channels during hypoxic pulmonary vasoconstriction. 37, 1119–1136 J Mol Cell Cardiol. Namely, D. Namgaladze, I. Shcherbyna, J. Kienhofer, H. W. Hofer, and V. Ullrich (2005). The vascular endothelium’s calcineurin signaling is targeted by superoxide. 334, 1061–1067, Biochem. Biophys. Res. Commun. J.G. Pastorino, J.B. Hoek, and N. Shulga (2005). By phosphorylating voltage-dependent anion channels, glycogen synthase kinase 3b activation inhibits hexokinase II’s ability to attach to mitochondria and increases chemotherapy-induced cytotoxicity. 65, 10545-10554; Cancer Res. S. Pitkanen and B.H. Robinson (1996). Superoxide dismutase is induced and superoxide radical generation is elevated in the presence of a mitochondrial complex I deficit. 98, 345–351 J. Clin. Invest. D.R. Plas and C.B. Thompson (2002). 

The regulation of programmed cell death by cell metabolism. 13, 75–78, Trends Endocrinol Metab. Wang, S., Hashimoto, K., Moudgil, R., Harry, G., et al. Pozeg, Z.I., Michelakis, E.D., McMurtry, M.S., Thebaud, B., Wu, X.C., Dyck, J.R. (2003). The O2-sensitive potassium channel Kv1.5 can be transferred into living cells to lower pulmonary hypertension and repair hypoxic pulmonary vasoconstriction in chronically hypoxic rats. 107th printing, 2037-2044. Wolfl, S., Kraft, R., Kalff, R., and Patt, S. Preussat, C. Beetz, M. Schrey (2003). Human gliomas express the voltage-gated potassium channels Kv1.3 and Kv1.5. Neuroscience Letters 346:33–36. Izumo, S., Ma, Q., and Pu, W.T. (2003). Important survival proteins called NFAT transcription factors prevent cardiomyocyte apoptosis in vitro following phenylephrine stimulation. 92 Circ Res, 725-731 Yuan, J.X., and Remillard, C.V. (2004). K+ channel activation is a crucial mechanism in programmed cell death. L49–L67 in Am. J. Physiol., Lung Cell. Mol. Physiol. Minami, E., Chase, E.G., Murry, C.E., and Santana, L.F. are the authors of the study (2004). voltage-gated K+ current decreases brought on by NFATc3 following myocardial infarction. Res. Circ. 94, 1340–1350. Witters, L.A., Gamble, J., Lopaschuk, G.D., and Saddik, M. (1993). 

Regulating fatty acid oxidation in the heart is acetyl-CoA carboxylase. J. Biol. Chem., 268:2583–25845 E.A. Schon and G. Manfredi (2003). mitochondrial malfunction and neuronal aging. 111, 303–312 J. Clin. Invest. S. Shimizu, M. Narita, and Y. Tsujimoto (1999). The mitochondrial channel VDAC’s release of apoptogenic cytochrome c is controlled by Bcl-2 family proteins. 399 Nature, 483–487 P.W. Stacpoole (1989). Dichloroacetate’s medicinal properties. 38, 1124–1144. Metabolism Harman, E.M., Thomas, R.G., Lorenz, A.C., and Stacpoole, P.W. (1988). Lactic acidosis treatment using dichloroacetate. 108, 58–63 Ann Intern Med Nagaraja, N.V., Stacpoole, P.W., and Hutson, A.D. (2003). effectiveness of the lactate-lowering medication dichloroacetate. 683-691. J. Clin. Pharmacol. Barnes, C., Bunch, S.T., Carney, P.R., Fennell, E.M., Felitsyn, N.M., Gilmore, R.L., Greer, M., Henderson, G.N., and others; Stacpoole, P.W. (2006). Dichloroacetate has undergone a controlled clinical trial for the treatment of congenital lactic acidosis in children. Children’s Health 117, 1519–1531. Thompson, C.B., Schumacker, P.T., Chandel, N.S., and M.G. Vander Heiden (1999). Bcl-xL promotes mitochondrial ATP/ADP exchange to stop cell death after growth factor removal. 159–167. Mol. Cell 3. Wang, Z. (2004). K+ channel functions in controlling the growth and death of tumor cells. 448 Pflugers Arch, 274-286 Wang, J., Yang, B., Nattel, S., Cao, L., Han, H., Wang, Y., and Z. (2002). HERG K+ channel, which controls the proliferation and death of tumor cells. Cancer Research, 62(24), 4843–4848. O. Warburg (1930). Regarding the chemistry of tumors (London: Constable). Mediation of neuronal apoptosis by augmentation of outward potassium current. Yu, S.P., Yeh, C.H., Sensi, S.L., Gwag, B.J., Canzoniero, L.M., Farhangrazi, Z.S., Ying, H.S., Tian, M., Dugan, L.L., and Choi, D.W. 1997. 114–117. Science 278. N. Zamzami and G. Kroemer (2001). How the Pandora’s box is opened by the mitochondrion in apoptosis. 67–71. Nat. Rev. Mol. Cell Biol. Cancer Cell 11, 37–51, January 2007, published by Elsevier Inc. Metabolic-electrical Remodeling in Cancer Cells