Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis

elifesciences.org Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 1 of 18 Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis William W Wheaton1†, Samuel E Weinberg1†, Robert B Hamanaka1, Saul Soberanes1, Lucas B Sullivan1, Elena Anso1, Andrea Glasauer1, Eric Dufour2, Gokhan M Mutlu1, GR Scott Budigner1, Navdeep S Chandel1* 1Department of Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, United States; 2Institute of Biomedical Technology, University of Tampere, Tampere, Finland Abstract Recent epidemiological and laboratory-based studies suggest that the anti-diabetic drug metformin prevents cancer progression. How metformin diminishes tumor growth is not fully understood. In this study, we report that in human cancer cells, metformin inhibits mitochondrial complex I (NADH dehydrogenase) activity and cellular respiration. Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin. Metformin also reduced hypoxic activation of hypoxia-inducible factor 1 (HIF-1). All of these effects of metformin were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In vivo, the administration of metformin to mice inhibited the growth of control human cancer cells but not those expressing NDI1. Thus, we have demonstrated that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on its ability to inhibit mitochondrial complex I. DOI: 10.7554/eLife.02242.001 Introduction Metformin is widely used to treat patients with type II diabetes mellitus who have high levels of circulating insulin (Nathan et al., 2009). Metformin suppresses liver gluconeogenesis thereby reducing glucose release from the liver (Inzucchi et al., 1998; Viollet et al., 2012). In several recent retrospective studies, investigators have observed an association between metformin use and diminished tumor progression in patients suffering from different types of cancers (Evans et al., 2005; Bowker et al., 2006; Dowling et al., 2012). These data have prompted several prospective clinical trials to determine the efficacy of metformin as an anti-cancer agent. However, the underlying mechanism by which metformin diminishes tumor growth is not fully understood. There are two postulated mechanisms by which metformin reduces tumor growth. Metformin may act at the organismal level, reducing levels of circulating insulin, a known mitogen for cancer cells. Alternatively, metformin may act in a cancer cell autonomous manner. Metformin is known to inhibit mitochondrial complex I in vitro (Ota et al., 2009; El-Mir et al., 2000; Owen et al., 2000) and it is thus possible that this targeting of the electron transport chain could inhibit tumor cell growth (Birsoy et al., 2012). This latter hypothesis has been questioned as cancer cells have the ability to survive on ATP produced exclusively by glycolysis. Furthermore, cancer cells have been shown to conduct glutamine-dependent reductive carboxylation to generate the TCA cycle intermediates required for cell proliferation when the electron transport chain is inhibited (Mullen et al., 2012; Fendt et al., 2013). Thus, it is not clear whether inhibition of complex I by metformin would result in decreasing tumor growth. In the present study, we directly tested whether inhibition of cancer cell mitochondrial complex I by metformin was required to decrease cell proliferation in vitro and tumor progression in vivo. *For correspondence: nav@ northwestern.edu †These authors contributed equally to this work Competing interests: The authors declare that no competing interests exist. Funding: See page 16 Received: 09 January 2014 Accepted: 15 April 2014 Published: 13 May 2014 Reviewing editor: Chi Van Dang, University of Pennsylvania, United States Copyright Wheaton et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. RESEARCH ARTICLE Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 2 of 18 Research article Results Human HCT116 p53−/− colon cancer cells have previously shown to be sensitive to metformin (Buzzai et al., 2007). To determine if metformin treatment inhibited cellular oxygen consumption in these cells, we treated HCT116 p53−/− cells with increasing concentrations of metformin in media containing the metabolic substrates glucose, pyruvate, and glutamine for 24 hr. Subsequently, we measured cellular oxygen consumption. Metformin inhibited cellular oxygen consumption of HCT 116 p53−/− cells at concentrations (0.25–1.0 mM) similar to those reported to affect bioenergetics and gluconeogenesis in primary hepatocytes in vitro (Figure 1A; Foretz et al., 2010; Miller et al., 2013). To determine whether metformin's inhibition of cellular oxygen consumption depended on mitochondrial complex I, we stably overexpressed the Saccharomyces cerevisiae protein NDI1 in HCT 116 p53−/− cells (hereon referred to as NDI1-HCT 116 p53−/− cells). NDI1 is a single-subunit NADH dehydrogenase, which oxidizes NADH in a process similar to the multi-subunit mammalian complex I; however without proton pumping or ROS generation (Seo et al., 1998). By contrast, mammalian complex I contains 45 subunits that pumps protons and generates ROS. NDI1-HCT 116 p53−/− cells demonstrated a slight, non-significant elevation in basal cellular oxygen consumption compared to control cells and were completely resistant to the effects of metformin on cellular oxygen consumption (Figure 1—figure supplement 1, Figure 1B). To ensure that the inhibition of cellular oxygen consumption by metformin was a direct effect of metformin on complex I, we examined mitochondrial respiratory function in saponin-permeabilized cells. Saponin removes cholesterol from plasma membranes, allowing the entry of metabolic substrates directly to mitochondria (Jamur and Oliver, 2010). In the presence of ADP and the complex I substrates pyruvate and malate, metformin fully inhibited oxygen consumption in permeabilized Control-HCT 116 p53−/− cells (Figure 1C). By contrast, metformin had no effect on pyruvate/malate-driven oxygen consumption in NDI1-HCT 116 p53−/− cells (Figure 1D). Metformin also had no effect on oxygen consumption in saponin-permeabilized cells respiring on the complex II substrate succinate in the presence of ADP (Figure 1E). Interestingly, in saponin-permeabilized cells, metformin significantly inhibited complex I-dependent respiration at a much lower concentration than that required to inhibit oxygen consumption of intact cells, suggesting that transport across the plasma membrane is a barrier to metformin's inhibition of complex I. Metformin is known to slowly accumulate in cells in which its eLife digest Metformin is widely used to reduce the high blood sugar levels caused by diabetes. Recently, several studies have suggested that patients taking metformin who also develop cancer have tumors that grow more slowly than average. As clinical trials have already started to investigate if metformin is an effective anti-cancer treatment, it is important to understand how it might restrict tumor growth. Researchers have proposed two ways that metformin could affect tumors. First, insulin is known to prompt cancer cells to divide, so the slower rate of tumor growth could just be a side-effect of the metformin reducing the amount of insulin in the blood. Alternatively, metformin could target cancer cells more directly by cutting the energy supply produced by their mitochondria. Metformin has been shown to disrupt complex I of the electron transport chain that is used by cells to generate energy. However, it is not known if disrupting complex I would actually stop cells dividing because they can generate energy in other ways. Wheaton, Weinberg et al. have now demonstrated that metformin does target complex I in cancer cells, and that its effects depend on the amount of glucose available for cells to convert, without involving mitochondria, into energy. When there is plenty of glucose, metformin slows down the rate at which cancer cells divide, which slows down tumor growth. When the cells are deprived of glucose, metformin kills the cells instead. Metformin also inhibits the pathways that regulate hypoxia inducible factors (HIFs), which are part of a system that helps cells to survive low-oxygen conditions, a prominent feature of many tumors. This means that metformin may combat cancer more effectively if used alongside other treatments that reduce the availability of both oxygen and glucose inside cells. Metformin could also potentially treat conditions that are linked to overactive HIFs, such as pulmonary hypertension. DOI: 10.7554/eLife.02242.002 Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 3 of 18 Research article Figure 1. Metformin inhibits mitochondrial complex I function. (A) Relative mitochondrial oxygen consumption rate (OCR) of intact Control-HCT 116 p53−/− and (B) NDI1-HCT 116 p53−/− cells treated with metformin in complete media for 24 hr. (C) Relative complex I (2 mM malate, 10 mM pyruvate, 10 mM ADP)-driven oxygen consumption rate of saponin permeabilized Control-HCT 116 p53−/− cells and (D) NDI1-HCT 116 p53−/− cells treated with metformin for 20 min in mitochondrial assay buffer. (E) Relative complex II-driven oxygen consumption rate of saponin permeabilized Control-HCT 116 p53−/− cells treated with 10 mM succinate and 10 mM ADP in the presence of 1 mM metformin or the complex II inhibitor 3-Nitropropionic acid (3-NPA). (F) Representative western blot and quantification of levels of OCT1 protein in Control BFP-HCT 116 p53−/− and NDI1-HCT 116 p53−/− cells. Error bars are SEM (OCR: n = 4; OCT1: n = 4). * indicate significance p<0.05. DOI: 10.7554/eLife.02242.003 The following figure supplements are available for figure 1: Figure supplement 1. NDI1 expression slightly increases oxygen consumption. DOI: 10.7554/eLife.02242.004 Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 4 of 18 Research article uptake is mediated by organic cation transporters (OCTs) (Emami Riedmaier et al., 2013). To ensure that NDI1-HCT 116 p53−/− cells are not refractory to metformin because of a change in metformin uptake, we analyzed the expression of OCT 1 in both control and NDI1-HCT 116 p53−/− cells. Expression of OCT1 protein did not change with the presence of NDI1 (Figure 1F). We next sought to determine if metformin-dependent inhibition of complex I resulted in changes in proliferation and survival of HCT116 p53−/− cells. Metformin did not induce cell death in ControlHCT 116 p53−/− or NDI1-HCT 116 p53−/− cells in the presence of glucose (Figure 2A,B), however, in the absence of glucose, metformin induced cell death in Control-HCT 116 p53−/− but not in NDI1-HCT 116 p53−/− cells (Figure 2C,D). Metformin diminished cell proliferation in Control-HCT 116 p53−/− cells but not in NDI1-HCT 116 p53−/− cells in media containing glucose (Figure 2E,F). These results indicate that the metformin-dependent inhibition of complex I decreases cell proliferation in the presence of glucose and increases cell death under glucose deprivation. These inhibitory effects of metformin were not specific to HCT116 p53−/− cells as metformin inhibited oxygen consumption and cellular proliferation of Control-HCT 116 p53+/+ cells and Control-A549 human lung cancer cells but not NDI1-HCT 116 p53+/+ or NDI1-A549 cells (Figure 2—figure supplement 1 and 2). Taken together, these results indicate that the anti-proliferative and cell death promoting effects of metformin require mitochondrial complex I inhibition. We also examined whether phenformin, a more lipophilic biguanide, also exert its anti-proliferative effects on cancer cells through inhibition of complex I. Phenformin inhibited oxygen consumption in Control-HCT 116 p53−/− cells and saponin-permeabilized Control HCT 116 p53−/− cells at 100-fold lower concentration compared to metformin (Figure 3A,C). Expression of NDI1 rescued the phenformin-mediated decrease in oxygen consumption (Figure 3B,D). Phenformin diminished cell proliferation in the control but not NDI1 expressing HCT116 p53−/− cells (Figure 3E,F), and did not induce cell death in media containing glucose, similar to metformin (Figure 3G,H). Collectively, these results indicate that phenformin also exerts its biological effects through inhibition of mitochondrial complex I. To determine whether metformin and phenformin diminish proliferation and survival of cells lacking endogenous complex I activity, we utilized a variant of CCL16 hamster fibroblasts that harbors a mutation in complex I (B2-CCL16) (Seo et al., 1998). Metformin inhibited proliferation of wild-type Control-CCL16 hamster fibroblasts but not that of B2-CCL16 cells or of B2-CCL16 cells reconstituted with NDI1 (NDI1-CCL16). Metformin and phenformin inhibited cellular oxygen consumption in wildtype CCL16 but not in CCL16-NDI1 cells (Figure 3—figure supplement 1 and 2). When these cells were cultured in galactose-substituted media, both metformin and phenformin induced cell death only in wild-type CCL16 cells. Survival of NDI1-CCL16 cells was not affected by metformin or phenformin (Figure 3—figure supplements 1 and 2). The B2-CCL16 cells die in galactose in the absence of metformin or phenformin since they harbor a mutation in complex I. Taken together, these results confirm that the anti-proliferative effects of metformin and phenformin require mitochondrial complex I inhibition. The positive charge of metformin has been proposed to account for it is accumulation within the matrix of mitochondria that exhibit a robust inner mitochondria membrane potential (Owen et al., 2000). Alternatively, the non-polar hydrocarbon-side chain of the drug could promote binding to complexes within mitochondrial membranes. We tested whether the mitochondrial membrane potential is necessary for metformin-dependent inhibition of complex I. Saponin-permeabilized Control-HCT 116 p53−/− cells were induced to respire on pyruvate/malate in the presence of either ADP or CCCP. Although both ADP and CCCP induce mitochondrial respiration, only CCCP depolarizes mitochondrial inner membrane potential. Metformin inhibited ADP but not CCCP stimulated oxygen consumption indicating that the metformin-mediated inhibition of mitochondrial complex I required polarized mitochondria (Figure 4A,B). Rotenone, an irreversible inhibitor of complex I, does not require polarized mitochondria to inhibit mitochondrial oxygen consumption (Figure 4C,D). Our results suggest that metformin would not be effective in suppressing complex I activity of intact cells if mitochondrial inner membrane potential was disrupted. Metformin-mediated inhibition of the electron transport chain diminishes proton pumping, which might depolarize the mitochondrial membrane, thus limiting accumulation of the drug. However, we did not observe a reduction in the mitochondrial inner membrane potential measured using TMRE fluorescent dye in Control and NDI1 HCT116 p53−/− cells after metformin treatment (Figure 4E,F). When electron transport function is inhibited, the ATP synthase can function in reverse such that it uses ATP generated by glycolysis to pump protons across the inner mitochondrial membrane, maintaining membrane potential (Appleby et al., 1999). The ATP synthase Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 5 of 18 Research article Figure 2. Metformin decreases cell proliferation by inhibiting mitochondrial complex I. (A) Percentage of live Control-HCT 116 p53−/− or (B) NDI1-HCT 116 p53−/− treated with metformin for 72 hr in media containing 10 mM glucose. (C) Percentage of live Control-HCT116 p53−/− or (D) NDI1-HCT 116 p53−/− treated with metformin for 24 hr followed by glucose withdrawal for 16 hr. (E) Cell number of Control-HCT 116 p53−/− cells and (F) NDI1-HCT 116 p53−/− cells 24, 48, and 72 hr post treatment with 0.5 mM or 1 mM metformin in complete media. Error bars are SEM (n = 4). * indicates significance p<0.05. DOI: 10.7554/eLife.02242.005 The following figure supplements are available for figure 2: Figure supplement 1. Metformin decreases cellular proliferation through inhibition of mitochondrial complex I function in HCT 116 p53+/+ cells. DOI: 10.7554/eLife.02242.006 Figure supplement 2. Metformin decreases cellular proliferation through inhibition of mitochondrial complex I function in A549 cells. DOI: 10.7554/eLife.02242.007 Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 6 of 18 Research article Figure 3. Phenformin decreases cell proliferation by inhibiting mitochondrial complex I. (A) Relative mitochondrial oxygen consumption rate (OCR) of intact Control-HCT 116 p53−/− and (B) NDI1-HCT 116 p53−/− cells treated with phenformin in complete media for 24 hr. (C) Relative complex I (2 mM malate, 10 mM pyruvate, 10 mM ADP)-driven oxygen consumption rate of saponin permeabilized Control-HCT 116 p53−/− cells and (D) NDI1-HCT 116 p53−/− cells treated with phenformin for 20 min in mitochondrial assay buffer. (E) Cell number of Control-HCT 116 p53−/− cells and (F) NDI1-HCT 116 p53−/− cells 24, 48, and 72 hr post treatment with 0 or 5 µM phenformin in complete media. (G) Percentage of live Control-HCT 116 p53−/− or (H) NDI1-HCT 116 p53−/− treated with metformin for 72 hr followed in complete media. Error bars are SEM (Relative OCR n = 5; Cell number n = 4). * indicates significance p<0.05. DOI: 10.7554/eLife.02242.008 Figure 3. Continued on next page Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 7 of 18 Research article inhibitor, Oligomycin A, diminished TMRE fluorescence in Control-HCT 116 p53−/− cells treated with metformin suggesting that in the presence of metformin, intact cells maintain their mitochondrial membrane potential by reversal of the ATP synthase (Figure 4E). Rotenone irreversibly inhibits complex I, which contributes to its high toxicity in vivo. Because metformin is well tolerated, we sought to determine if metformin might reversibly bind to complex I. Saponin-permeabilized Control-HCT 116 p53−/− cells were treated with pyruvate and malate to maintain the mitochondrial inner membrane potential. Metformin was then added, followed by injection of either ADP or CCCP. Metformin inhibited ADP, but not CCCP-stimulated oxygen consumption (Figure 5A,B). As metformin accumulation requires mitochondrial membrane polarization (Figure 4A,B), these results indicate that metformin reversibly inhibits mitochondrial complex I. If the metformin that accumulated in the mitochondrial matrix irreversibly inhibited complex I, then oxygen consumption would have remained attenuated in CCCP-treated cells after metformin treatment. An emerging function of mitochondria distinct from their ability to perform biosynthetic and bioenergetic reactions is the generation of H2O2, which promotes signaling in normal and cancer cells (Hamanaka and Chandel, 2010; Sena and Chandel, 2012). Mitochondrial complexes I and III produce superoxide into the mitochondrial matrix, where it is converted quickly to H2O2 by SOD2 (Brand, 2010). Mitochondrial complex III also generates superoxide into the mitochondrial intermembrane space where it escapes through VDACs to cytosol and is converted into H2O2 by SOD1 (Han et al., 2003; Muller et al., 2004). We measured production and subsequent release of H2O2 from isolated mitochondria in the presence of metformin, rotenone, or antimycin A (complex III inhibitor) using pyruvate and malate as substrates. Consistent with previous reports, rotenone and antimycin increased the release of H2O2 from mitochondria isolated from Control-HCT 116 p53−/− cells (Figure 6A,B; St-Pierre et al., 2002; Muller et al., 2004). In contrast, metformin did not substantially increase H2O2 release, suggesting that metformin and rotenone act on different sites of complex I (Figure 6A). When mitochondria were isolated from NDI1–HCT 116 p53−/− cells, only antimycin lead to a significant increase in H2O2 release (Figure 6B). Previous reports have shown that metformin does not substantially increase H2O2 production in isolated liver mitochondria and that metformin diminishes mitochondrial H2O2 production in response to paraquat, which induces mitochondrial ROS production (Batandier et al., 2006; Algire et al., 2012). One biological consequence of mitochondrial-generated H2O2 is hypoxic stabilization of the hypoxia-inducible factors (HIFs) (Chandel et al., 2000). HIFs are involved in metabolic adaptation of tumor cells to hypoxia (Semenza, 2012). Metformin reduced hypoxic stabilization of HIF-1α in ControlHCT 116 p53−/− but not in NDI1-HCT 116 p53−/− (Figure 6C,D). Metformin did not decrease deferoxamine (DFO) stabilization of HIF-1α protein. DFO is an iron chelator known to directly stabilize HIF-1α protein independent of upstream signaling events. Metformin also significantly diminished hypoxic activation of HIF-dependent target genes, vascular endothelial growth factor (VEGF), and carbonic anhydrase 9 (CA9) in Control-HCT 116 p53−/− but not in NDI1-HCT 116 p53−/− (Figure 6E). Thus, metformin is an effective agent to reduce hypoxic activation of HIF-1. Finally, we directly tested whether tumor cell autonomous inhibition of mitochondrial complex I by metformin was required to decrease tumor progression in vivo. As our NDI1-HCT 116 p53−/− cells are refractory to multiple effects of metformin in vitro, we reasoned that if metformin acted directly on mitochondrial complex I within the tumor cells to reduce tumorigenesis then NDI1-HCT 116 p53−/− xenograft tumors would not be inhibited in their growth. However, if metformin acts at the organismal level to diminish tumorigenesis then NDI1-HCT 116 p53−/− xenograft tumor growth would be suppressed similar to control tumors. Control-HCT 116 p53−/− cells subcutaneously injected into the left flank of nude mice rapidly grew in vivo, while tumors from mice fed metformin through drinking water ad libitum starting 4 days post-implantation exhibited a marked reduction in growth (Figure 7A,B). NDI1-HCT 116 p53−/− xenograft growth was resistant to metformin therapy (Figure 7A,B), suggesting The following figure supplements are available for figure 3: Figure supplement 1. Metformin inhibits mitochondrial complex I of CCL16 cells. DOI: 10.7554/eLife.02242.009 Figure supplement 2. Phenformin inhibits mitochondrial complex I of CCL16 cells. DOI: 10.7554/eLife.02242.010 Figure 3. Continued Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 8 of 18 Research article that the metformin carries out its tumor inhibitory effects in a cancer cell autonomous manner through inhibition of mitochondrial complex I. Importantly, the consumption of water containing metformin was similar between control and NDI1 tumor barring mice (Figure 7C). Transcripts for the HIF target Figure 4. Metformin inhibition of complex I requires an intact mitochondrial inner membrane potential. (A) Complex I (2 mM malate, 10 mM pyruvate)-driven oxygen consumption rate of saponin permeabilized Control-HCT 116 p53−/− cells over time. At t = 5 min permeabilized cells were treated with either 10 mM ADP to induce respiration with an intact mitochondrial membrane potential or (B) 10 µM CCCP to induce respiration in absence of mitochondrial membrane potential. At t = 12 min 1 mM metformin was added to cells. At t = 48 min antimycin A was added. (C) Complex I (2 mM malate, 10 mM pyruvate)-driven oxygen consumption rate of saponin-permeabilized Control-HCT 116 p53−/− cells. At t = 5 min permeabilized cells were treated with either 10 mM ADP to induce respiration with an intact mitochondrial membrane potential or (D) 10 µM CCCP to induce respiration in absence of mitochondrial membrane potential. At t = 15 min, 1 μM rotenone was added to cells. At t = 25 min antimycin A was added. (E) Mitochondrial membrane potential measured by TMRE staining of Control-HCT116 p53−/− cells or (F) NDI1-HCT 116 p53−/− in the presence of 1 mM Metformin, 10 µM CCCP or 2.5 µM Oligomycin A. Error bars are SEM (n = 4). * indicates significance p<0.05. DOI: 10.7554/eLife.02242.011 Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 9 of 18 Research article genes CA9 and VEGF were diminished in control tumors treated with metformin but not in NDI1 expressing tumors (Figure 7D,E). The blood glucose, plasma lactate, insulin, and IGF-1 level displayed no differences between the metformin-treated animals and control animals at the end of the study (Figure 7—figure supplement 1), consistent with previous reports (Tomimoto et al., 2008). To further bolster our conclusions, we examined tumor growth of A549 cells expressing NDI1 and shRNA targeting mammalian NDUFS3, a subunit of the human mitochondrial complex I (Vogel et al., 2007). A549 cells are null for the tumor suppressor LKB1 and are known to be responsive to metformin therapy (Rocha et al., 2011). Furthermore, LKB1-deficient tumors are more susceptible to the related biguanide phenformin (Shackelford et al., 2013). We replaced the endogenous mitochondrial complex I by expressing the NDI1 protein in A549 cells stably expressing shRNA against NDUFS3 (Figure 7—figure supplement 2A), referred to as NDI1-shNDUFS3-A549 cells. The Control-A549 cells contain the empty vectors with selection markers BFP and puromycin for NDI1 and shRNA, respectively. NDI1-NDUFS3-A549 cells were resistant to the metformin-mediated reduction in cellular and mitochondrial oxygen consumption and cell proliferation (Figure 7—figure supplement 2B–G). Metformin also decreased HIF-1a protein levels in control but not in NDI1-NDUFS3-A549 cells (Figure 7—figure supplement 3A). Control-A549 cells subcutaneously injected into the left flank of nude mice rapidly grew in vivo, while tumors from mice fed metformin through drinking water ad libitum starting 2 weeks prior to tumor induction exhibited a marked reduction in growth over 45 days (Figure 7—figure supplement 3B). By contrast, NDI1-NDUFS3-A549 xenografts were completely resistant to metformin therapy, suggesting that metformin carries out its tumor inhibitory effects in a cell autonomous manner through inhibition of mitochondrial complex I in these cells (Figure 7—figure supplement 3C). NDI1-NDUFS3-A549 xenografts grew slower than control xenografts in untreated mice. An alternative explanation for the resistance of NDI1-NDUFS3-A549 cells to metformin could be that effects of metformin are blunted in slower-growing cells. However, based on our results from HCT116 cells in vivo and extensive analysis of A549 cells in vitro, we find that it is likely that NDI1 expressing A549 cells are also resistant to metformin in vivo due to rescue of complex I activity by the NDI1 protein. Discussion The mechanisms by which metformin inhibits cancer growth is not fully understood. Metformin has been previously shown to inhibit mitochondrial complex I, yet it is not known whether metformin exhibits its anti-tumor effects through inhibition of complex I. It is important to note that many Figure 5. Metformin reversibly inhibits mitochondrial complex I. (A) Complex I (2 mM malate, 10 mM pyruvate)-driven oxygen consumption rate of saponin permeabilized Control-HCT 116 p53−/− cells over time. At t = 5 min permeabilized cells were exposed to 1 mM metformin. At t = 25 min respiration was stimulated with either 10 mM ADP to induce respiration with an intact mitochondrial membrane potential or (B) 10 µM CCCP to induce respiration lacking membrane potential with 10 mM ADP. At t = 42 min antimycin A was added. For mitochondrial membrane potential error bars are SEM (n = 4). For oxygen consumption rates, error bars are standard deviation (n = 6). * indicates significance p<0.05. DOI: 10.7554/eLife.02242.012 Human biology and medicine Wheaton et al. eLife 2014;3:e02242. DOI: 10.7554/eLife.02242 16 of 18 Research article between 0.5 µg/ml and 2 µg/ml (Graham et al., 2011). This treatment created four experimental groups: Control-HCT 116 p53−/− with H2O (n = 8), Control-HCT 116 p53−/− with Metformin (n = 8), NDI1-HCT 116 p53−/− with H2O (n = 8), NDI1-HCT 116 p53−/− with Metformin (n = 8). Experiments are from two independent cohorts of four mice each. Tumors were measured three times per week using calipers and tumor volume was determined using the equation (3.14/6 × L × W2). At the completion of the study, mice were euthanized and the tumors were extracted and weighed. For A549s, 3 × 106 Control-A549 or NDI1-NDUFS3-A549 cells were injected into the left flank of female nude mice (Nu:Nu) administered metformin (250 mg/kg) in their drinking water for 2 weeks prior to cell injection and continuously administered throughout the experiment. All mouse work was done in accordance with Northwestern University Institutional Animal Care and Use Committee. Statistical analysis Data are presented as the mean ± SEM. Statistical significance was determined using 1-way ANOVA with a Bonferroni posttest correction, 2-way ANOVA when two variables were present, or the students t test comparing control to experimental conditions for p<0.05. For all differences uncovered, a student t test was performed to verify differences between control and experimental groups. Acknowledgements We are grateful to the following people for providing reagents: E Scheffler (CCL16-B2 cells), Dr Takao Yagi (CCL16-NDI1 cells). This work is supported by grants from the NIH (R01CA123067, 5P01HL071643) to NSC, NIH (ES015024) to GMM and Veterans Administration Merit Award (GRSB). The work was also supported by NIH training grant T32 GM08061 to LBS and 5T32HL076139-10 to SW. Additional information Funding Funder Grant reference number Author National Institutes of Health RO1 CA123067 Navdeep S Chandel National Institutes of Health T32GM08061 Lucas B Sullivan National Institutes of Health T32HL076139 Samuel E Weinberg The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. 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