Investigation of metabolic processes in cultured melanoma cell lines

TASK-3 channels are thought to promote proliferation and/or survival of malignantly transformed cells. Our previous results suggested mitochondrial expression of TASK-3 channels so these channels are legitimate proteins that regulate the electrochemical gradient for ATP synthesis and could play crucial role in maintaining the mitochondrial function in hypoxic or impaired metabolic environments. To investigate the effect of TASK-3 gene silencing on cell proliferation and viability we performed controlled culturing condition changes (reduced glucose concentration and serum deprivation, hypoxic condition). Mitochondrial DNA content, reactive oxygen species (ROS) synthesis and ATP release of the different melanoma cultures were also studied. Reduction of cell viability (MTT assay) and proliferation (CyQant assay) caused by serum and glucose deprivation as well as in hypoxic culturing condition (4% O2, 96% N2) were significantly larger in TASK 3 knockdown melanoma cells compared to their control counterparts. We also measured the ATP release and mitochondrial DNA content of control and TASK-3 knockdown melanoma cultures using luminescent probe and qPCR, respectively. Both the ATP release and the mitochondrial DNA amount was significantly reduced in TASK-3 deficient melanoma cells. The average ROS release (measured by flow cytometry using the fluorescent dye dihydroethidium) showed no significant difference between control (139.8 +/- 3.8 a.u.) and scrambled transfected cells (147.5 +/- 3.7 a.u.), however this value was significantly lower in TASK-3 knockdown cells (107.2 +/- 4.8 a.u; n=9, p was less than 0.05). We conclude that reduced TASK-3 expression results in decreased DNA content (number of mitochondria) and mitochondrial activity which presumably cause reduction of the rate of chemical reactions in the mitochondria. It is also suggested that TASK-3 expression in the mitochondria could be essential in supporting the survival and proliferation of malignantly transformed melanoma cells by increasing their hypoxia and starvation tolerance.