Molecular Mechanism Of Autophagy And Necroptosis Of Bladder Cancer Cells Induced By Shikonin

Objective:Bladder cancer accounts for the first place in the incidence rate of urogenital tumors in China,and has been increasing year by year.Most bladder cancer can be diagnosed in the early stage,but the recurrence and progression rate is very high.The 5-year survival rate of stage ? bladder cancer is only 15%,which seriously endangers the health and quality of life of patients.In clinical treatment of bladder cancer,conventional drugs are not effective because of a series of adverse reactions.However,some natural medicines have the advantages of mild efficacy and small adverse reactions in anti-tumor.A large number of literatures have proved that shikonin can inhibit the proliferation of a variety of cancer cells,but the mechanism of action on bladder cancer cells is still unclear.In this paper,the EJ cells of human bladder cancer were studied as the main research object,and the mechanism of shikonin inducing the death of bladder cancer cells was preliminarily discussed,which provided the theoretical basis and experimental basis for the research and development of shikonin,and also provided new ideas for the treatment of bladder cancer.Methods:After stimulating bladder cancer cells at different concentrations and time with the shikonin,then we can define how it would affect the cell proliferation with the MTT assay and cell cloning experiments.If we treat the EJ cells stimulated by the shikonin using different methods,flow cytometry was used to detect the changes of intracellular reactive oxygen species and mitochondrial membrane potential.Western blot was used to detect the expression levels of autophagy,necroptosis,and Nrf2 pathway-related proteins.Immunofluorescence was used to detect the effects of shikonin on the expression of LC3 and Nrf2 protein.Immunoprecipitation was used to detect the formation of complex between RIP 1 and RIP3,Nrf2 and Keap 1,respectively.And nuclear plasm separation experiment was used to detect the change of Nrf2 protein expression level.In addition,reactive oxygen scavenger NAC,autophagy inhibitor LY294002 and RIP1 inhibitor Nec-1 were respectively used to pretreat EJ cells,then combined with shikonin to detect the changes of autophagy,necroptosis and Nrf2 pathway-related protein expression by Western blot.The effect of NAC on the expression level of RIP1-RIP3 complex was detected by immunoprecipitation.MTT was used to detect the effect of shikonin combined with NAC,LY294002,Nec-1,ML385,z-VAD on the survival rate of EJ cells,respectively.Results:1.Shikonin decreased the survival rate of bladder cancer cells(EJ,J82,BIU87,T24,UMUC3)and inhibited cell proliferation in a concentration-and time-dependent manner;while cell cloning experiments further showed that shikonin could significantly inhibit the proliferation of EJ cells.2.Shikonin significantly increased the expression level of autophagy-related proteins LC3,Beclinl,ATG5,ATG7,ATG12 in the EJ and J82 cells of human bladder cancer,and showed an obvious relationship with both the concentration and the time.Immunofluorescence assay showed that the expression level of LC3 protein increased significantly with the prolonged time of shikonin acting on EJ cells.3.Shikonin's stimulating the EJ and J82 cells of human bladder cancer,which can effectively promote the expression level of the RIP1?RIP3?p-MLKL,also showing the dependence of concentrations and time.Immunoprecipitation results showed that RIP1 and RIP3 complex were increased in shikonin-treated EJ cells.MTT assay results showed that the inhibitor z-VAD had no effect on the survival rate of EJ cells,while the inhibitor Nec-1 significantly reversed the survival rate of EJ cells.4.Shikonin stimulated EJ cells to reduce the mitochondrial membrane potential,and significantly increase intracellular reactive oxygen species levels in a concentration-dependent manner.5.The expression level of Nrf2 pathway related proteins in EJ and J82 cells was up-regulated by shikonin in a time-dependent manner.Immunoprecipitation assay showed that the complex of Nrf2 and Keap1 was decreased in shikonin-treated EJ cells.Nucleocytoplasmic separation and immunofluorescence assay showed that Nrf2 entered the nucleus with the most expression level at 12h.MTT assay showed that the inhibitor ML385 could enhance the toxicity of shikonin to EJ cells.6.The expression level of key executive proteins in autophagy,necroptosis and Nrf2 pathway was significantly down-regulated after the combination of reactive oxygen species scavenger NAC and shikonin in EJ and J82 cells.Immunoprecipitation results showed that shikonin-induced RIP 1 and RIP3 complex was significantly reduced by NAC pretreatment.MTT assay showed that NAC almost completely reversed the cell survival rate induced by shikonin.7.The inhibitor LY294002 further upregulated shikonin-induced RIP1 and RIP3 protein expression levels in EJ and J82 cells,while Nec-1 could increase the protein expression levels of LC3 and Beclinl induced by shikonin.MTT assay showed that shikonin-induced cell viability was decreased in EJ cells pretreated with inhibitors 3-MA and LY294002,respectively,but the combination of Nec-1 and shikonin significantly increased EJ cell viability.Conclusion:By overproducing ROS and causing oxidative stress response,shikonin activates the Nrf2 pathway and induces autophagy and necroptosis,thus inhibiting the proliferation of bladder cancer cells.In addition,inhibition of autophagy can significantly enhance shikonin-induced necroptosis.Therefore,shikonin can be used as a potential drug for the treatment of bladder cancer,and reasonable inhibition of autophagy can effectively increase the sensitivity of bladder cancer cells to shikonin,which may become a promising treatment.