Dysregulation And Effect Of Bim In Melanoma Cells In The State Of ER Stress

Background & Objective: At present there is fateful obstacle that solid tumor cells including melanoma cells are largely resistance to treatment in the clinical treatment.This is largely due to the resistance to the apoptosis induced by clinical drugs in tumor cells.Endoplasmic reticulum(ER) stress-induced apoptosis increasingly plays an important role following the traditional apoptosis pathway,death receptor pathway and the mitochondrial pathway.Many chemotherapeutic drugs trigger ER stress and subsequent activation of the unfolded protein response (UPR) that either protects the cell by alleviating the ER stress condition,or eliminates the cell by induction of apoptosis in too strong or too long stess.Recent research has revealed that melanoma cells are not sensitive to ER-tress induced apoptosis and the mechanism is not quite clear now.It is known that induction of apoptosis by ER stress involves BH3-only protein family that plays important roles in mitochondrial pathway.The BH3-only protein Bim has been shown to play an important role in ER stress-induced apoptosis in diverse cell types.The aim of this study is to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under ER stress. These results will give an effective molecular target for clinically treatment of melanoma.Methods: The melanoma cell lines Mel-RM and MM200 and control group HEK293 cells were treated with tunicamycin,a naturally occurring antibiotic that induces ER stress by inhibition of glycosylation,at varying doses for different periods.Apoptotic cells were quantitated using the Annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm apoptotic cell death.Western blotting was used to measure activation of caspase-3 and -9, and the expression of Bim,GRP78,CHOP,and FOXO1 at the protein level.The expression of Bim, CHOP and FOXO1 at the mRNA level was quantitated by qPCR.The siRNA technique was used to inhibit the expression of Bim.The expression levels of Bim after siRNA Bim were measured by Western blot. The apoptosis of Bim in TM-induced melanoma cell lines was measured by flowcytometry.Results: Treatment of melanoma cells with the classic ER stress inducer tunicamycin did not induce significant apoptosis and activation of the caspase cascade,whereas it caused marked activation of caspase-3 and -9,and apoptosis in HEK293 cells that were used as a control.Significantly,Induction of ER stress resulted in up-regulation of Bim in HEK293 cells,but the expression of Bim was not increased by ER stress in melanoma cells at either the protein or the mRNA level.siRNA knockdown of Bim protected HEK293 cells against ER stress-induced apoptosis, suggesting that the lack of Bim up-regulation may be a protective mechanism in melanoma cells under ER stress. While the transcription factor CHOP that is known to induce Bim transcription was increased,another transcription factor FOXO1 that can also transcriptionally activate Bim was decreased, by ER stress in melanoma cells.Conclusion The lack of Bim up-regulation contributes to resistance of melanoma cells to ER stress-induced apoptosis and may be a mechanism by which melanoma cells adapt to ER stress conditions.We propose that down-regulation of FOXO1 may be responsible for dysregulation of Bim in melanoma cells upon ER stress.