Effect of hypoxia factors gene silencing on ROS production and metabolic status of A375 malignant melanoma cells.

The innate response of melanocytes to exogenous or endogenous stress stimuli like extreme pH and temperature, metabolite and oxygen deficiency or a high UV dose initiates a cellular stress response. This process activates adaptive processes to minimize the negative impact of the stressor on the pigment cell. Under physiological conditions, a non-cancer cell is directed to apoptosis if the stressor persists. However, malignant melanoma cells will survive persistent stress thanks to distinct "cancerous" signaling pathways (e.g. MEK) and transcription factors that regulate the expression of so-called "survival genes" (e.g. HIF, MITF). In this survival response of cancer cells, MEK pathway directs melanoma cells to deregulate mitochondrial metabolism, to accumulate reduced species (NADH), and to centralize metabolism in the cytosol. The aim of this work was to study the effect of gene silencing in malignant melanoma A375 cells on metabolic processes in cytosol and mitochondria. Gene silencing of HIF-1α, and miR-210 in normoxia and pseudohypoxia, and analysis of its effect on MITF-M, and PDHA1 expression. Detection of cytosolic NADH by Peredox-mCherry Assay. Detection of OCR, and ECAR using Seahorse XF96. Measurement of produced O with MitoTracker Red CMXRos. H NMR analysis of metabolites present in cell suspension, and medium. By gene silencing of HIF-1α and miR-210 the expression of PDHA1 was upregulated while that of MITF-M was downregulated, yielding acceleration of mitochondrial respiratory activity and thus elimination of ROS. Hence, we detected a significantly reduced A375 cell viability, an increase in alanine, inositol, nucleotides, and other metabolites that together define apoptosis. Based on the results of measurements of mitochondrial resipiratory activity, ROS production, and changes in the metabolites obtained in cells under the observed conditions, we concluded that silencing of HIF-1α and miR-210 yields apoptosis and, ultimately, apoptotic cell death in A375 melanoma cells.