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).

Figure 1 with 1 supplement with 1 supplement see all Download asset Open asset 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. https://doi.org/10.7554/eLife.02242.003

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 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 Control-HCT 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).

Figure 2 with 2 supplements with 2 supplements see all Download asset Open asset 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. https://doi.org/10.7554/eLife.02242.005

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.

Figure 3 with 2 supplements with 2 supplements see all Download asset Open asset 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. https://doi.org/10.7554/eLife.02242.008

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 wild-type 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 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).

Figure 4 Download asset Open asset 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. https://doi.org/10.7554/eLife.02242.011

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.

Figure 5 Download asset Open asset 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. https://doi.org/10.7554/eLife.02242.012

An emerging function of mitochondria distinct from their ability to perform biosynthetic and bioenergetic reactions is the generation of H 2 O 2 , 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 H 2 O2 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 H 2 O 2 by SOD1 (Han et al., 2003; Muller et al., 2004). We measured production and subsequent release of H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 release (Figure 6B). Previous reports have shown that metformin does not substantially increase H 2 O 2 production in isolated liver mitochondria and that metformin diminishes mitochondrial H 2 O 2 production in response to paraquat, which induces mitochondrial ROS production (Batandier et al., 2006; Algire et al., 2012).

Figure 6 Download asset Open asset Metformin reduces HIF-1 activation through inhibition of mitochondrial complex I. (A and B) H 2 O 2 levels emitted by mitochondria isolated from Control-HCT 116 p53−/− and NDI1-HCT 116 p53−/− cells respiring on 2 mM malate and 10 mM pyruvate. Mitochondria were treated with 1 mM Metformin, 500 nM rotenone, 500 nM Antimycin, or left untreated. H 2 O 2 levels were measured using Amplex Red. (C) Levels of HIF1α protein in Control-HCT 116 p53−/− and NDI1-HCT 116 p53−/− cells treated with 0 or 1 mM metformin for 24 hr, then placed in normoxia (21% O 2 ), hypoxia (1.5% O 2 ) or treated with Deferoxamine (DFO) for 8 hr. (D) Quantification of HIF1α protein levels from panel C. (E) Hypoxic-induced expression of HIF target genes in Control-HCT 116 p53−/− and NDI1-HCT 116 p53−/− treated with 0, 0.5 mM or 1 mM metformin for 24 hr following treatment with normoxia or hypoxia for 16 hr. Error bars are SEM (n = 3 for Amplex Red; Blot is representative of four independent blots quantified in D, n = 4 for gene expression). * indicates significance p<0.05. https://doi.org/10.7554/eLife.02242.013

One biological consequence of mitochondrial-generated H 2 O 2 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 Control-HCT 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 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 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).

Figure 7 with 3 supplements with 3 supplements see all Download asset Open asset Metformin inhibits mitochondrial complex I to diminish tumor growth. (A) Average tumor volume in mice injected with 3 × 106 Control-HCT 116 p53−/− or NDI1-HCT 116 p53−/− cells injected into the left flank of J:Nu mice. Mice were given ad libitum, water free of metformin (squares) or were treated with 250 mg/kg of metformin in the drinking water starting 4 days post tumor injection (triangles). (B) Average tumor mass from mice injected with 3 × 106 Control-HCT 116 p53−/− or NDI1-HCT 116 p53−/− cells injected into the left flank of J:Nu mice after 32 days. (C) Average daily water consumption of mice treated with metformin (1.25 mg/ml). (D) HIF target genes expression measured in Control-HCT 116 p53−/− or NDI1-HCT 116 p53−/− tumors treated with metformin. Error bars are SEM (n = 8 per group for tumor study, n = 8 for H 2 O consumption, error bars represent standard deviation of two cages with four mice house in each cage, n = 3 for gene expression). * indicates significance p<0.05. https://doi.org/10.7554/eLife.02242.014

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.