Sensitization of metformin-cytotoxicity by dichloroacetate via reprogramming glucose metabolism in cancer cells

Abstract

To investigate sensitization of metformin-cytotoxicity, cancer cells were treated with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK). Metformin-cytotoxicity was mainly dependent on glucose availability and reducing power generated by pentose phosphate pathway, whereas DCA cotreatment enhanced metformin-cytotoxicity via reprogramming glucose metabolism by inhibiting PDK and increasing mitochondrial respiration. DCA cotreatment elicited cell death rather than cell survival despite high glucose and high GSH condition. In conclusion, DCA sensitized metformin-cytotoxicity by reprogramming glucose metabolism in part from aerobic glycolysis to mitochondrial oxidation, evidenced by measurements of glucose consumption, lactate release, and the ratio of oxygen consumption rate/extracellular acidification rate.

Introduction

Growing evidences indicate that metabolic perturbations of cancer cells such as aerobic glycolysis or glutamine addiction are inevitable for carcinogenesis beyond the epiphenomenon [13], [31], and enormous amount of efforts have been exhausted to find out drug-able targets and candidate chemicals on cancer metabolism [5], [30]. Based on the epidemiological, preclinical and clinical data, metformin, a biguanide used for treatment of diabetes mellitus, became one of the most attractive and promising drugs targeting for cancer metabolism. Although the mechanism of metformin is not fully elucidated, intracellular function of metformin has been known to inhibit respiratory chain complex I [8], [21]. At present, many evidences indicate that AMPK activation is a master node of anti-tumor effects of metformin [3], [16], [23], therefore, LKB1^−/− cancer cells are more resistant to metformin-induced cytotoxicity in the in vitro culture system [22], [34]. In contrast, AMPK activation has been shown to protect cancer cells from energy stress via regulation of NADPH homeostasis [12], therefore, LKB1^−/− cancer cells are more sensitive to phenformin-induced metabolic stress in mouse lung cancer model [24]. In the confusing contexts, there are reports that anti-tumor effects metformin are dependent on glucose concentration in culture medium [11], [17], [25]; Glucose-deprivation significantly enhances metformin-cytotoxicity [17], whereas metformin-induced AMPK activation is inefficient under high glucose (25 mM) condition [25]. Therefore, in-depth understanding of biochemical mechanism of metformin-cytotoxicity needs to be clarified before using metformin as an anticancer agent focusing on glucose metabolism.

Warburg effect frequently observed in cancer cells is important not only for energy generation but also for maintaining reduced status under hostile tumor microenvironment. Glucose is the main source of reducing power, NADPH, via pentose phosphate pathway [1], [9], [22]. Indeed, 2-deoxyglucose (2DG)-mediated cancer cell death is dependent mainly on the intolerable oxidative stress in addition to energy crisis [15]. Based on the concept that increased glycolysis protects cancer cells from oxidative stress, metformin-increased glycolysis has been thought to be capable of protecting cells from mitochondrial oxidative stress resulting from the inhibition of respiratory chain complex I [35]. Therefore, we investigated whether metformin-induced mitochondrial stress can be augmented by oxidative stress due to GSH depletion under glucose deprivation or H₂O₂ treatment under glucose-sufficient condition. Based on the reports that inhibition of glycolysis by 2-DG enhances cytotoxic effects of metformin [2], [6], [14] and that dichloroacetate (DCA) reduces glycolysis by activating pryruvate dehydrogenase (PDH) [32] along with oxidative metabolism and antitumor activity [20].

We explored in the present study the effects of DCA on the metformin-cytotoxicity and the regulation of glucose metabolism. As opposed to the effects of metformin alone, the combined treatment of cancer cells with metformin and DCA significantly enhanced PDH activity, but not glycolysis, thus recovered mitochondrial respiration in cancer cells. The reprogramming of glucose metabolism was tightly associated with severe oxidative stress. In summary, DCA-mediated reprogramming of aerobic glycolysis to mitochondrial oxidation augmented metformin-induced mitochondrial and cellular redox stress that was sufficient to induce massive cell death despite high glucose level.