INVESTIGATION OF THE EFFECTS OF EGFR INHIBITION ON CLAUDINS IN LUNG CANCER CELL LINES
Project no. 171 · Status: Kapanmış · 2018
Summary
Tight junctions are membranous structures present in the epithelial, endothelial and mesothelial cells. These are multiprotein complexes that links between adjacent cells of both epithelial and endothelial cell layers. It was reported that YM201636, which is a PIKfyve inhibitor, inhibits the endocytic recycling of tight junction proteins CLDN1 and CLDN2 and disrupt the sturucters of tight junctions. Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein present in the cell membrane and it has critical importance in the regulation of cell proliferation. To inhibit EGFR function in the cancer cells, molecules that inhibit intracellular tyrosine kinase signaling or neutralizing antibodies against EGFR and its ligands are used. Canertinib (CI-1033) is a tyrosine kinase inhibitor that inhibits EGFR, HER2 and HER4 in vitro conditions. In this study, it is aimed to elucidate the relationship between EGFR and claudins, which is a structural component of tight junctions. For this purpose, EGFR mutant and wild-type lung cancer cell lines HCC827 and H1299 were used, and the cytotoxic and apoptotic effects of CI-1033 and YM201636 agents were investigated with their effects on EGFR and claudin mRNA and protein expressions. XTT was used in the cytotoxicity analysis, Annexin / PI staining in the apoptosis assays, quantitative RT-PCR in determining mRNA expressions, immunofluorescence staining and western blot methods were used in determining protein expressions. In order to evaluate the malignancy potential of the cells, cell migration and anchorage independent growth were quantitated. The IC50 for CI-1033 was determined as 2.17 and 16.42 µM for the HCC827 and H1299 cell lines respectively. YM201636 showed cytotoxic effect at 11.07 µM in the EGFR mutant HCC827 cell line whereas EGFR did not affect cell viability at 75 µM concentration in wild-type H1299 cells. The agents YM201636 caused a significant increase in EGFR, CLDN1, CLDN3 and CLDN5 mRNA expression levels in HCC827 cells and there was no significant change in claudin mRNA levels after YM201636 in H1299 cells. CI-1033 and YM201636 inhibitors exerted different effects on CLDN expression in EGFR mutant and wild-type cell lines. Key Words: lung cancer; tight junctions; claudins; EGFR