After treatment for the indicated time period, cells were harvested in the medium to collect live and dead cells. 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 malignancy 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 circulation 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 including oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in AT7519 trifluoroacetate expanding breast AT7519 trifluoroacetate malignancy therapies. studies have concluded that metformin inhibits growth of many types of malignancy cells including those from breast cancer, colon cancer, AT7519 trifluoroacetate prostate malignancy, ovarian malignancy, and gliomas [9C12]. Metformin is known to activate AMP-activated protein kinase (AMPK) which leads to inhibition of protein synthesis and cell growth . However, activation of AMPK alone is not enough to lead to apoptotic cell death . Studies have shown that metformin accumulates in the mitochondria and mildly inhibits complex I of the electron transport chain, an event that takes place upstream of AMPK activation [15C18]. As complex I is usually inhibited, impeded electron passage prospects to superoxide production within the mitochondrial matrix, damaging mitochondrial proteins, lipids, and nucleic acids. In studies in which metformin has been shown to promote cell death, apoptosis is the main pathway [10, 12, 19]. We have previously shown that metformin induces both caspase-dependent and poly(ADP-ribose) polymerase (PARP) dependent cell death in most breast malignancy cell lines while being non-cytotoxic to non-transformed breast epithelial cells . PARP-dependent cell death was associated with major alterations in mitochondrial shape and function, leading to the conclusion that mitochondrial damage in malignancy cells is a key mediator of metformin-induced cell death. Based on these observations, we hypothesized that compounds that promote mitochondrial oxidative metabolism would enhance metformin-induced mitochondrial damage and synergize with metformin in killing cancer cells. As metformin treatment also promotes production of lactate , such a compound would ideally also combat this effect. DCA is also an orally available drug with well-studied pharmacokinetics and has been tested for the treatment of lactic acidosis (a potential side effect of metformin) and mitochondrial deficiencies . DCA is an inhibitor of pyruvate dehydrogenase kinase (PDK) which phosphorylates pyruvate dehydrogenase (PDH), rendering it inactive . PDH is the enzyme responsible for catalyzing the transformation of pyruvate to acetyl-CoA for access into the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation. In malignancy cells, PDK activity is usually often elevated, acting as a gatekeeper to reduce the flux of pyruvate from your cytoplasm into mitochondria metabolism. This is thought to be an important component of metabolic reprogramming in malignancy cells, leading to reduced glucose oxidation and the production of lactate [24C26]. By inhibiting PDK, DCA enhances PDH activity, allowing pyruvate to enter the TCA cycle rather than being converted to lactate and secreted . In this study, we examined the antitumor activity and interplay of two metabolism targeting drugs, metformin and DCA. We show that DCA enhances the cytotoxicity of metformin to breast malignancy cells through a mechanism involving oxidative damage while simultaneously lowering lactate production by metformin, potentially providing a dual therapeutic advantage. Methods Chemicals & Reagents The following chemicals, reagents, and packages were purchased through Sigma-Aldrich unless normally noted: metformin (1, 1-dimethylbiguanide), sodium dichloroacetate, 0.4% trypan blue answer, Vectashield mounting medium for fluorescence containing 4,6 diamidino-2-phenylindole (DAPI) YWHAS (Vector Laboratories), Lactate Assay Kit (Eton Biosciences), caspase inhibitor OPH-109 (MP Biomedicals), Coomassie Brilliant Blue R250 (Bio-Rad Laboratories), paraformaldehyde, SYTOX? Green (Life Technologies), Triton AT7519 trifluoroacetate X-100 (Eastman), and PARP inhibitor II INH2BP (Epigentek). Cell Culture MCF-7 and T47D human breast malignancy cell lines and MCF10A human mammary epithelial cells were purchased from ATCC. The 66CL4 mouse mammary carcinoma cell collection was provided by Dr. Fred.