Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo.

Purpose The unique metabolism of breast cancer cells provides interest in exploiting this phenomenon therapeutically. Metformin, a promising breast cancer therapeutic, targets complex I of the electron transport chain leading to an accumulation of reactive oxygen species (ROS) that eventually lead to cell death. Inhibition of complex I leads to lactate production, a metabolic byproduct already highly produced by reprogrammed cancer cells and associated with a poor prognosis. While metformin remains a promising cancer therapeutic, we sought a complementary agent to increase apoptotic promoting effects of metformin while attenuating lactate production possibly leading to greatly improve efficacy. Dichloroacetate (DCA) is a well-established drug used in the treatment of lactic acidosis which functions through inhibition of pyruvate dehydrogenase kinase (PDK) promoting mitochondrial metabolism. Our purpose was to examine the synergy and mechanisms by which these two drugs kill breast cancer cells. Methods Cell lines were subjected to the indicated treatments and analyzed for cell death and various aspects of metabolism. Cell death and ROS production was analyzed using flow cytometry, Western blot analysis, and cell counting methods. Images of cells were taken with phase contrast microscopy or confocal microscopy. Metabolism of cells was analyzed using the Seahorse XF24 analyzer, lactate assays, and pH analysis. Results We show that when DCA and metformin are used in combination, synergistic induction of apoptosis of breast cancer cells occurs. Metformin-induced oxidative damage is enhanced by DCA through PDK1 inhibition which also diminishes metformin promoted lactate production. Conclusions We demonstrate that DCA and metformin combine to synergistically induce caspase-dependent apoptosis involving oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in expanding breast cancer therapies.

Doxorubicin (DOX) is a potent, commonly used anthracycline antibiotic for a wide range of cancers, but its dose is limited by its strong cardiotoxic effects. The glucose analog 2-deoxy-D-glucose (2-DG) has been shown to enhance the antitumor efficacy of DOX in tumor cell lines and in animals. However, the cardiac effects of 2-DG in these experiments were not determined. In the current study, we examined whether 2-DG could protect from DOX cardiotoxicity in a BALB/c mouse breast cancer model. A total of 32 mice were injected with 10 5 4T1 mouse mammary epithelial tumor cells in the right hind leg. The tumor was allowed to progress for 1 week before dividing the mice into four groups: saline, 2-DG, DOX, and 2-DG with DOX treatment. 2-DG was given 150 mg/kg i.p. Monday through Friday; DOX was given 5 mg/kg i.p. Wednesday. The study was conducted for 3 weeks and echocardiography was performed the day before sacrifice. Two of the eight mice in the DOX group died before echocardiography, while none died in the 2-DG with DOX group. Echocardiography indicated similar cardiac function between saline and 2-DG groups as measured by fractional shortening (36.3±1.31% vs 37.7±0.942%, p>0.05). DOX markedly reduced cardiac function, which was almost completely recovered by 2-DG (25.1±1.06% vs 36.7±1.76%, p<0.01). Serum CK-MB content, a common clinical marker for heart damage, was increased ~6 fold in the DOX group (67.5±19.4 vs 400±145, p<0.023). 2-DG tended to reduce DOX-triggered CK-MB release, but it did not reach significant levels likely due to the small sample size and high variation (279±68.7 vs 400±145, p>0.05). Nonetheless, 2-DG largely eliminated DOX-induced apoptosis in the heart as shown by a DNA laddering assay. 2-DG or DOX alone increased cardiac autophagic flux as measured by the difference in LC3-II protein levels in the absence and presence of the lysosomal inhibitor, bafilomycin A1. Paradoxically, autophagic flux was not notably elevated when mice were treated with 2-DG and DOX simultaneously. These results demonstrate the ability of 2-DG to reduce DOX cardiotoxicity without affecting its antitumor activity, suggesting that 2-DG can improve the therapeutic window for DOX, allowing greater flexibility in designing different regimens for treating cancers.

The unusual metabolism of aerobic glycolysis in cancer cells suggests that this phenomenon can be exploited for improved therapies. Metformin, a drug used to treat type 2 diabetics, has clinical evidence of cancer prevention, and some studies show that cancer patients have a better response when it is used with neoadjuvant therapy. One of the cellular targets of metformin is the inhibition of Complex I in the mitochondrial electron transport chain. This leads to an accumulation superoxide species that can cause oxidative damage to cells and eventually lead to cell death. Based on this, we hypothesized that drugs which promote mitochondrial metabolism will synergize with metformin to increase superoxide production and promote cell death of cancer cells. Cancer cells using aerobic glycolysis display a high level of glucose uptake that is processed through glycolysis to pyruvate, which is then converted to lactate and secreted from the cells. Diverting pyruvate to mitochondrial metabolism rather than its conversion to lactate was expected to enhance metformin cytotoxicity. Dichloroacetate (DCA) is well established drug that has been tested in humans for the treatment of certain metabolic diseases. DCA works through inhibition of pyruvate dehydrogenase kinase (PDK), thus reducing entry of pyruvate into mitochondrial oxidative phosphorylation pathways. Consequently DCA is expected to promote mitochondrial metabolism and reduce lactate secretion by cancer cells. We demonstrate that DCA and metformin synergistically kill cultured breast cancer cells although the drug combination has no effect on non-transformed MCF10A breast epithelial cells. The enhanced cell death of the breast cancer cells displayed is through apoptosis, as shown by caspase-dependent increases in cleaved PARP. DCA treatment was associated with reduced lactate secretion by the cells and increased pH of the growth medium which suggested it promoted oxidative phosphorylation. The combination of two drugs also promoted increased mitochondrial superoxide production compared to metformin alone which was associated with increased oxidative damage. These findings indicate that, as expected, DCA promotes metformin-mediated cell death by enhancing mitochondrial metabolism of pyruvate. Therefore, it is possible that the combination of metformin with DCA, or similar compounds, could enhance cancer therapy. Citation Format: Allison B. Haugrud, Yongxian Zhuang, Wilson K. Miskimins. DCA and metformin synergistically induce apoptosis in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1869. doi:10.1158/1538-7445.AM2013-1869

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