Targeting mitochondrial glycerol 3-phosphate dehydrogenase and mitochondrial complex III in PC-3 prostate cancer cells

Abstract

Cancer is the second main cause of death worldwide. Reactive oxygen species (ROS) play a special role in tumor development and growth. The mitochondrial glycerol 3-phosphate dehydrogenase (mG3PDH) and the mitochondrial complex III (CIII) have a high proportion of cellular ROS production. Due to inadequate blood supply tumors very often are characterized by low oxygen pressure. ROS as well as the oxygen supply regulate the stability of the hypoxic inducing factor (HIF) which induces the expression of key glycolytic (iso)enzymes (i.e. lactate dehydrogenase A (LDHA), pyruvate kinase type M2 (M2-PK)) required for energy production, synthesis of building blocks and cancer cell proliferation. ROS have been shown to induce a dimerization as well as a reduction of M2-PK activity thereby diverting glucose flux towards the synthesis of cell building blocks. In tumor cells besides glycolysis, glutaminolysis is an important pathway to provide energy and metabolic precursors for cell building blocks which are both important in cells with high proliferation rate such as tumor cells. The aim of this study was to investigate the impact of targeting mG3PDH and mitochondrial CIII on cell proliferation and metabolism of PC-3 prostate cancer cells which are characterized by high mG3PDH activity. The impact of mG3PDH and CIII inhibition on cell proliferation was investigated in presence of 21% O2 which corresponds to the oxygen concentrations in the air and is used in most of the published cell culture studies as well as in presence of 1.5% O2 which corresponds to the mean oxygen concentration in a variety of solid tumors. In order to simulate therapy over several days long-term incubation periods (96 hours) of PC-3 cells with the corresponding inhibitors were performed. In the first part of the study two substances published as inhibitors of mG3PDH (iGP-1 and RH02211) were investigated. mG3PDH together with cytosolic glycerol 3-P dehydrogenase (cG3PDH) is involved in the transfer of hydrogen from cytosolic NADH + H+ produced within the cytosolic glyceraldehyde 3-P dehydrogenase (GAPDH) reaction into the mitochondrial electron transport chain in order to restore the cytosolic NAD+ for glycolysis. Both iGP-1 and RH02211 induced an inhibition of PC-3 cell proliferation. The RH02211-induced inhibition of cell proliferation was independent upon oxygen supply. The inhibitory effect of both inhibitors on PC-3 cell proliferation was weakened when pyruvate was supplemented into the cultivation medium of the cells suggesting that extracellular pyruvate is an escape mechanism for the inhibition of cell proliferation by both RH02211 and iGP-1. Pyruvate is the substrate via which the LDH oxidizes NADH + H+ to NAD+. A nutrient medium without supplementation of pyruvate is closer to the physiological pyruvate concentrations in the blood. When cultivated in pyruvate supplemented medium PC-3 control cells shifted from production of pyruvate to consumption of extracellular pyruvate and glycolytic as well as glutaminolytic conversion rates decreased which points to a severe impact of extracellular pyruvate on the metabolism of the cells. In contrast to the first published description which showed a decrease of H2O2 production in pyruvate supplemented PC-3 cells treated for 15 minutes with 1-30 µM RH02211 in our experiments 16 µM RH02211 induced an increase of H2O2 production as well as an increase of oxygen consumption independent upon the pyruvate concentration in the medium. Although the H2O2 production rates increased together with the glycolytic conversion rates in pyruvate starved RH02211 treated PC-3 cells the tetramer : dimer ration of M2-PK, the LDH isoenzyme equipment as well as the composition of the glycolytic enzymes were not impaired. In pyruvate supplemented cultivation medium RH02211 did not impair the glycolytic conversion rates. iGP-1 induced an increase of glycolysis and glutaminolysis in pyruvate starved PC-3 cells. In the second part of the study three different commercially available CIII inhibitors (Antimycin A, Myxothiazol and S3QEL-2) that target CIII by different mode of actions were investigated. All three inhibitors induced a dose dependent inhibition of PC-3 cells proliferation in pyruvate starved medium. The inhibition of cell proliferation by CIII inhibition was weakened by hypoxia when Qi site was inhibited (AA) but not when CIII Qo was inhibited (Myx and S3QEL-2). Measurements of metabolic nutrients and products in the cell cultivation supernatants of the cells point to an activation of glycolysis by AA, Myx and S3QEL-2 independently upon oxygen supply. Glutaminolysis was downregulated in AA and Myx treated cells at both 21% and 1.5% O2 as well as in S3QEL-2 treated cells at 1.5% O2 but not at 21% O2. These results indicate that glucose became the main energetic source for PC-3 cell proliferation when CIII was inhibited. The increase in glycolysis in AA, Myx and S3QEL-2 treated cells was not linked with changes in the activity of the glycolytic enzymes as well as with changes in H2O2 production. Both AA and Myx strongly decreased PC-3 cell respiration. In contrast to the first published description which showed no impact of S3QEL-2 on respiration of HEK 293 cells treated for 3 hours with 34 µM S3QEL-2 in our experiments a slight but significant decrease in oxygen consumption was found in presence of 12 µM S3QEL-2. The weaker effect of S3QEL-2 on mitochondrial respiration compared to Myx may be an explanation for the significantly higher IC50 values of S3QEL-2 in comparison to Myx for the inhibition of PC-3 cell proliferation.