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

Abstract

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 O2•− with MitoTracker Red CMXRos. 1H 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.

Influencing Mechanism of Nod-Like Receptor Protein 3 Inflammasome Activation in A375 Cell Activity in Human Cutaneous Malignant Melanoma

“…In some literature, it was documented that NLRP3 inflammasome is very important in the influencing mechanism on activity of human cutaneous MM A375 cells [26]. The activated NLRP3 inflammasome could induce the activation of caspase-1 to promote the maturity and secretion of IL-1β and IL-18 precursors [27], which was similar to the results of the research. The experiment indicated that the levels of IL-18 and IL-1β in group C were lower greatly than the values in groups A and B (P < 0:05).…”

Metabolic Profiling of Thymic Epithelial Tumors Hints to a Strong Warburg Effect, Glutaminolysis and Precarious Redox Homeostasis as Potential Therapeutic Targets

“…For example, low levels of alanine have been observed in various cancer types [45], to which we can now add TCs. While the mechanisms leading to low alanine levels are poorly understood [45], they were associated with apoptosis resistance in melanoma [46] and high aggressiveness in various non-thymic carcinomas [45], which fits well with the particularly high apoptosis S4 were log2 transformed and then the value of each metabolite was standardized (subtracted) with the respective mean value of the log2 transformed values of the normal thymi. Note that aggressive thymomas (B2, B3 thymomas) and TCs form distinct clusters.…”

“…For example, low levels of alanine have been observed in various cancer types [45], to which we can now add TCs. While the mechanisms leading to low alanine levels are poorly understood [45], they were associated with apoptosis resistance in melanoma [46] and high aggressiveness in various non-thymic carcinomas [45], which fits well with the particularly high apoptosis resistance and clinical aggressiveness of TCs [1,3]. Interestingly, there was no metabolite with a consistently higher level in TCs than in B2 and B3 thymomas; even lactic acid levels that were higher in aggressive (B2, B3 and TC) than indolent TETs were lower in TCs than in B2 and B3 thymomas (Figure 1).…”

Machine Learning Algorithms for a Novel Cuproptosis-related Gene Signature of Diagnostic and Immune Infiltration in Endometriosis

“…Spakova et al demonstrated that silencing HIF-1α/miR210 expression increases PDHA1 and decreases MITF-M expression. This promotes mitochondrial respiratory activity, which aids in eliminating reactive oxygen species in melanoma cells [21]. Our results indicate a signi cant role of GLS, NFE2L2, and PDHA1 in EMT pathogenesis.…”