Investigation of the effects of Carbonic Anhydrase-IX inhibition on B-RAF protein in melanoma cell line

CA-IX, an important biomarker in melanoma, is an crucial enzyme that is highly expressed in tumor cells and its inhibition has been extensively studied. It has been discovered that this transmembrane enzyme affects some pathways that have roles in carcinogenesis via IC tails. The MAPK pathway, which is important in the transition from extracellular signals to intracellular responses and regulates many cellular functions, is also associated with tumor formation. It is known that the expression of CA-IX is regulated by components of the MAPK pathway. Activation of this pathway by mutated BRAFV600E stimulates melanoma growth, survival, and angiogenesis. The effect of CA-IX inhibition on the MAPK pathway has not been determined until now. The main purpose of this study to investigate the effect of inhibition of CA-IX on B-Raf protein which has a critical role in MAPK pathway. In this study, AZA and VMF inhibitors were treated to A375 melanoma cells which has BRAFV600E mutation. Cell proliferation was determined by WST-1 assay. Furthermore, CA9 and HIF1A expressions in these cells were investigated using qRT-PCR method. Also, the effect of AZA treatment on CA-IX protein levels was demonstrated by ELISA test. The localizations and expressions of HIF-1α, CA-IX, B-Raf and ERK proteins were examined by immunofluorescence in AZA-treated and non-treated melanoma cells. Besides, HIF-1α, B-Raf, p-B-Raf, ERK and p-ERK proteins were examined by Western Blot analysis. The preliminary results indicate that inhibition of CA-IX by AZA causes decreased p-B-Raf and p-ERK protein levels. According to the results of this study; effect of AZA treatment on B-Raf in the MAPK pathway suggests that CA inhibitors can be considered as a potential agent for this pathway.