| Objective:In this study,sulfasalazine (SAS) was used as oxidative stress inducer to replicate oxidative stress model.The cisplatin-sensitive cells (SKOV3,A2780)and drug-resistant cells (SKOV3/DDP,OVCAR-3) of human ovarian epithelial cancer were used as the research objects to study the effect of programmed necrosis pathway in drug-resistant cells (SKOV3/DDP,OVCAR-3) and the possible regulatory mechanism of multifunctional protein p62/SQSTM1 (Abbreviated as p62 protein) in this pathway.To reveal the effect of necroptosis pathway in cisplatin-resistant human ovarian epithelial cancer cells and its regulatory mechanism,and to provide new ideas for targeting programmed necrosis pathway and overcoming drug resistance of human ovarian epithelial cancer.Method:(1)The effects of SAS on the survival rates of human ovarian epithelial cancer SKOV3,SKOV3/DDP,A2780 and OVCAR-3 cells were detected by MTT assay.(2)The effects of SAS combined with Necrostatin-1 (Nec-1),a programmed necrosis inhibitor,on the survival rates of human ovarian epithelial cancer SKOV3,SKOV3/DDP,A2780 and OVCAR-3 cells were detected by MTT assay.(3)The effect of Nec-1 combined with SAS on the survival rate of human ovarian epithelial cancer SKOV3 and SKOV3/DDP cells was detected by RTCA.(4)Western blotting was used to detect the expression of RIP1,RIP3,MLKL and phosphorylated protein in human ovarian epithelial cancer SKOV3,SKOV3/DDP,A2780 and OVCAR-3 cells.(5)The expression of RIP1 and RIP3 complex was detected by immunoprecipitation.(6)Western blotting was used to detect the effect of SAS on the expression of p62 protein in human ovarian epithelial cancer SKOV3,SKOV3/DDP,A2780 and OVCAR-3 cells.(7)Western blotting was used to detect the effect of overexpression of zz domain mutation p62 protein (zz-p62) on programmed necrosis-related protein in human epithelial ovarian cancer SKOV3 cells and human epithelial ovarian cancer A2780 cells.Results:(1)MTT results showed that the cell survival rates of human ovarian epithelial cancer SKOV3,SKOV3/DDP,A2780 and OVCAR-3 cells treated with SAS at different concentrations decreased with the increase of SAS concentration,and showed a dose-dependent manner.SAS 0.5 mM acted on human ovarian epithelial cancer SKOV3 cells,SAS 2 mM acted on human ovarian epithelial cancer SKOV3/DDP cells.The cell survival rates were (81.9 ±1.4)% and (80.3±1.5)%.SAS 0.5 mM acted on human ovarian epithelial cancer A2780 cells,SAS 2 mM acted on human ovarian epithelial cancer OVCAR-3 cells,and the cell survival rates were (78.0±1.9)% and (77 ±5.6)%.Therefore,SAS 0.5 m was selected.M acted on human epithelial ovarian cancer SKOV3 cells,SAS 2 mM acted on human epithelial ovarian cancer SKOV3/DDP cells,SAS 0.5 mM acted on human epithelial ovarian cancer A2780 cells,SAS 2 mM acted on human epithelial ovarian cancer OVCAR-3 cells for subsequent experiments.Compared with human epithelial ovarian cancer SKOV3 and A2780 cells,SAS at the same concentration could not significantly reduce the survival rate of human epithelial ovarian cancer SKOV3/DDP and OVCAR-3 cells.(2)The survival curve of RTCA represents the number of cells,and the slope of the curve represents the growth rate of cells.Firstly,the survival curve of RTCA was observed.Compared with SAS group,the survival curve of human epithelial ovarian cancer SKOV3 cells treated with Nec-110 mu M combined with SAS group was lower,while the survival curve of human epithelial ovarian cancer SKOV3/DDP cells was higher.Continue to observe the slope of RTCA real-time curve.The results showed that the sl ope of curve in SAS group was lower than that in control group.Compared with SAS group,there was no difference in the slope of curve growth between Nec-110 mu M a nd SAS group.In human epithelial ovarian cancer SKOV3/DDP cells,the slope of cu rve in SAS group was lower and increased negatively compared with that in control gr oup.Compared with the control group,the curve slope of Nec-110 um combined with SAS increased significantly.(3)Western blotting results showed that the expression of programmed necrosis-related protein phosphorylated RIP1,RIP3 and MLKL in human ovarian epithelial cancer SKOV3/DDP cells treated with SAS increased at 12 hours after SAS treatment,while the expression of programmed necrosis-related protein pho sphorylated RIP1 and RIP3 increased at 12 hours after SAS treatment,while the expre ssion of programmed necrosis-related protein phosphorylated MLKL did not change.The phosphorylated RIP1,RIP3 and MLKL of human ovarian epithelial cancer SKO V3 and A2780 cells treated with SAS were not expressed.(4)The results of immunoprecipitation showed that RIP1 and RIP3 complex were expressed in human ovarian epithelial cancer SKOV3/DDP cells treated with SAS.(5)Western blotting results showed that the expression of p62 protein in human ovarian epithelial cancer SKOV3/DDP cells increased 12 hours after SAS treatment,and p62 protein in human ovarian epithelial cancer OVCAR-3 cells increased 6 hours after SAS treatment,while p62 protein was not expressed in human ovarian epithelial cancer SKOV3 and A2780 cells after SAS treatment.(6)Western blotting results showed that p62 protein deleted in zz domain was overexpressed in human ovarian epithelial cancer SKOV3 cells and A2780 cells,and the ratio of phosphorylation of RIP1,RIP3 and MLKL by programmed necrosis-related proteins was decreased.Conclusion:(1)Compared with cisplatin-sensitive cells of human epithelial ovarian cancer,cisplatin-resistant cells SKOV3/DDP and OVCAR-3 of human epithelial ovarian cancer are insensitive to SAS.When SKOV3/DDP and OVCAR-3 cells are injured by SAS,programmed necrosis pathway exists.Necrostatin-1 (Nec-1) inhibits programmed necrosis,which indicates that the survival rate of the cells increases,suggesting that it is resistant to SAS.The pathway of programmed necrosis in drug cells may be a new way of anti-cancer drug treatment.(2)Compared with cisplatin-sensitive cells of human ovarian epithelial cancer,oxidative stress induced damage to cisplatin-resistant cells SKOV3/DDP and OVCAR-3,increased the expression of p62 protein,overexpressed the mutation of zz domain in cisplatin-sensitive cells SKOV3 and A2780,decreased the expression of programmed necrosis-related protein phosphorylated RIP1,RIP3 and MLKL,suggesting that p62 protein may regulate the process.Sexual necrosis pathway provides a theoretical basis for finding therapeutic targets to overcome drug resistance. |
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