The Role Of Endoplasmic Reticulum Stress In Autophagy Of Melanoma

ObjectiveIn this study,we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624.Firstly,we want to investigate whether dabrafenib can cause autophagy in two melanoma cell lines;secondly,whether autophagy induced by dabrafenib is regulated by ER stress;and thirdly,whether decreasing ER stress and autophagy can increase the efficacy of dabrafenib in treating melanoma cells.Methods 1.A375 and MEL624 were regular cultured in vitro.A375 and MEL624 were treated with 0,10,100,250,and 500 nM Da B-Rafenib,After 24 hours,we were analysis and compare The autophagy-related proteins LC3I/II and P62 and the endoplasmic reticulum stress-related proteins p-PERK and CHOP by Western blot.2.Preparation of small interfering RNA against PERK(PERK-siRNA)and non-targeted small interfering RNA(NTC-siRNA)from lipofectamine 2000 transfected melanocytes.Reduction of PERK protein levels by approximately 70 per small interfering RNA against PERK.The PERK-siRNA and NTC-siRNAs of the two cell lines were treated with or without dabrafenib(100 nM).After 24 hours,the cells were collected and detected by Western blot for CHOP,IRE1?,P62,and LC3I/II in both cell lines.3.A375 and MEL624 cell lines were treated with endoplasmic reticulum stress inhibitor 4-phenylbutyrate(4PBA)(10 nM)for 1 hour,followed by treatment with dabrafenib(100 nM)for 24 hours.Collect and cell viability detected by CCK-8.4.Add autophagy inhibitor 3-methyladenosine(3-MA)(2nM)to the cell lines A375 and MEL624,and then treat the two melanoma cells with different concentrations of dabrafenib.After 24 hours,cell viability was measured by CCK-8.Results 1.LC3I/II and P62 expression levels of A375 and MEL624 detected by Western blot, we found that as the dabrafenib concentration increased,the LC3 I/II levels were significantly higher,accompanied by a significant decrease in P62 levels.Using the same method as described above,the expression levels of p-PERK and CHOP in the two cell lines were detected by Western blot,and the expression levels of p-PERK and CHOP were increased with increasing dabrafenib concentration.2.After knocking down the PERK gene with a small interfering RNA,the PERK protein level is reduced by approximately 70%.Western blot analysis showed that the increase in dabrafenib-stimulated endoplasmic reticulum stress signal could be attenuated by knockdown of PERK;at the same time,autophagy signal was also impaired by knockdown of PERK.3.The dabrafenib treatment group had cell survival higher than the 4-phenylbutyrate treatment group.4.Compared with the 3-methyladenosine simultaneous treatment group,the survival rate of the cells in the dabrafenib group was higher.Conclusion 1.B-Raf inhibitors induces both autophagy and ER stress,and in a dose-dependent pattern in melanoma cells.2.The inhibition of ER stress could regulates the autophagy-associated pathways in melanoma cells.3.ER stress response plays a protective role and provides resistance to B-Raf inhibitors mediated cell death in melanoma.4.Autophagy induced by B-Raf inhibitors could protects the melanoma cells from the cytotoxicity of B-Raf inhibitors.