Dichloroacetic acid upregulates apoptosis of ovarian cancer cells by regulating mitochondrial function

Abstract

Background: Metabolic reprogramming is a characteristic of tumor cells and is considered a potential therapeutic target. Even under aerobic conditions, tumor cells use glycolysis to produce energy, a phenomenon called the "Warburg effect". Pyruvate dehydrogenase kinase 1 (PDK1) is a key factor linking glycolysis and the tricarboxylic acid cycle. Dichloroacetic acid (DCA) reverses the Warburg effect by inhibition of PDK1 to switch cytoplasmic glucose metabolism to mitochondrial oxidative phosphorylation (OXPHOS).

Methods: Cell viability was examined using a standard MTT assay. Glucose consumption and l-lactate production were measured using commercial colorimetric kits, and intracellular lactate dehydrogenase (LDH) activity was evaluated using cell lysates and an LDH Quantification Kit. Real-time PCR was used to detect the expression of related genes. The production of total ROS was evaluated by staining with dichlorofluorescin diacetate.

Results: Comparison of various aspects of glucose metabolism, such as expression of key enzymes in glycolysis, lactate production, glucose consumption, mitochondrial oxygen consumption rate, and citric acid production, revealed that A2780/DDP cells were primarily dependent on glycolysis whereas A2780 cells were primarily dependent on mitochondrial OXPHOS. Mitochondrial uncoupling protein 2 (UCP2) protects against mitochondrial ROS while allowing energy metabolism to switch to glycolysis. Treatment of A2780 cells with various concentrations of DCA resulted in decreased expression of UCP2, a metabolic switch from glycolysis to mitochondrial OXPHOS, and an increase in oxidative stress induced by ROS. These effects were not observed in A2780/DDP cells with higher UCP2 expression suggesting that UCP2 might induce changes in mitochondrial functions that result in different sensitivities to DCA.

Conclusion: Our results show that a drug targeting tumor metabolic changes affects almost the entire process of glucose metabolism. Thus, it is necessary to comprehensively determine tumor metabolic functions to facilitate individualized antitumor therapy.

Keywords: DCA; glucose; glycolysis; metabolism; mitochondrial function.

Figure 1

DCA decreases A2780 cell survival and induces apoptosis compared with A2780/DDP cells. Notes: (A) Cisplatin-sensitive human ovarian cancer cells A2780 and their cisplatin-resistant clones A2780/DDP were treated with DCA at 0, 20, 40, 80, and 160 mM for 24 hours and then analyzed by MTT assay. (B) A2780 and A2780/DDP cells were treated with DCA at the indicated doses for 24 hours followed by annexin V/PI staining. (C) Total cell death was quantified by flow cytometric analysis. (D) A2780 and A2780/DDP cells were treated with the indicated doses of DCA for 24 hours. Equal amounts of protein were subjected to Western blotting to determine the levels of the indicated proteins. Data are presented as mean ± SE, n=3. *P<0.05 compared with respective controls. Abbreviations: DCA, dichloroacetic acid; PI, propidium iodide.

Figure 2

Analysis of glycolysis and mitochondrial functions in A2780 and A2780/DDP cells. Notes: (A) Consumption of glucose, (B) production of lactic acid, and (C) LDH activity were measured after incubation under basal conditions. (D) Equal amounts of protein were subjected to Western blotting to determine the levels of the indicated proteins in A2780 and A2780/DDP cells. (E, F) Total RNA was isolated from A2780 and A2780/DDP cells to quantify mRNA expression of LDHA and HK2. (G) OCR, (H) mitochondrial citrate, (I) mitochondrial ROS, and (J) mitochondrial potential were measured in A2780 and A2780/DDP cells after incubation under basal conditions (data were normalized to protein concentrations). Data are presented as mean ± SE, n=3. *P<0.05, **P<0.01 compared with A2780 cells. Abbreviations: LDH, lactate dehydrogenase; OCR, oxygen consumption rate; PDH, pyruvate dehydrogenase.

Figure 3

DCA decreases glycolysis in A2780 cells compared with A2780/DDP cells. Notes: (A, B) Cells were treated with DCA at the indicated doses for 24 hours, and the culture supernatants were subjected to analysis of glucose consumption and lactate production. (C) LDH activity was measured after treatment with DCA for 24 hours. (D) Equal amounts of protein were subjected to Western blotting to determine the levels of the indicated proteins. Data are presented as mean ± SE, n=3. *P<0.05 compared with the respective controls. Abbreviations: DCA, dichloroacetic acid; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase.

Figure 4

DCA increases mitochondrial OXPHOS and ROS production in A2780 cells compared with A2780/DDP cells. Notes: Mitochondrial citrate (A) and the OCR (B) were measured in the presence of DCA at the indicated doses for 24 hours. Mitochondrial ROS (C) and overall ROS (E) were detected in A2780 and A2780/DDP cells treated with DCA at the indicated doses for 24 hours by measuring fluorescence intensities using fluorescence microscopy (×200) or flow cytometry, respectively. (D) Expression levels of UCP2 protein in A2780 and A2780/DDP cells after DCA treatment. Data are presented as mean ± SE, n=3. *P<0.05, **P<0.01 compared with the respective controls. Abbreviations: DCA, dichloroacetic acid; OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation.

Figure 4

DCA increases mitochondrial OXPHOS and ROS production in A2780 cells compared with A2780/DDP cells. Notes: Mitochondrial citrate (A) and the OCR (B) were measured in the presence of DCA at the indicated doses for 24 hours. Mitochondrial ROS (C) and overall ROS (E) were detected in A2780 and A2780/DDP cells treated with DCA at the indicated doses for 24 hours by measuring fluorescence intensities using fluorescence microscopy (×200) or flow cytometry, respectively. (D) Expression levels of UCP2 protein in A2780 and A2780/DDP cells after DCA treatment. Data are presented as mean ± SE, n=3. *P<0.05, **P<0.01 compared with the respective controls. Abbreviations: DCA, dichloroacetic acid; OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation.

Figure 5

Cell viability and expression of UCP2 in A2780 and A2780/DDP cells after gene silencing of PDK1 combined with DCA. Notes: (A) shPDK1 combined with DCA further decreased the survival rate of A2780 cells, whereas there was no obvious effect in A2780/DDP cells. (B) The expression of UCP2 protein in shPDK1 A2780 cells and A2780/DDP cells. Data were expressed relative to the control group. *P<0.05, **P<0.01 compared with the respective controls. #P<0.05, A2780/DDP vs A2780. Abbreviation: DCA, dichloroacetic acid.