Based On SLC7A11/RIP3/Nrf2 To Study The Molecular Mechanism Of Shikonin-Induced Necroptosis Of Human Bladder Cancer And Its Anti-tumor Metastasis Effect

Objective: Bladder cancer is one of the top ten malignant tumors over the global,accounting for the first place in the incidence of urogenital tumors in China,ranking fourth among men.The treatment of human bladder cancer usually involves cystectomy or chemoradiation,but many patients can’t have an operation.Platinum-containing drugs are often used in clinical treatment,which have large toxic side effects and easy drug resistance.Patients who are PD-L1 positive can receive 5 kinds of programmed death receptor ligands for immunotherapy.Erastin,a type I ferroptosis agonist,targets to inhibit SLC7A11-induced ferroptosis in a variety of tumor cells,but its role in human bladder cancer is not clear.Studies have reported that shikonin can kill many tumor cells,and has been repeatedly reported as an inducer of necroptosis of cancer cells,but its strong non-selective cytotoxicity will limit its clinical application.The link between necroptosis and ferroptosis is unclear.In this project,we explored the molecular mechanism of human bladder cancer cell death induced by low concentrations of Erastin and Shikonin targeting on SLC7A11/RIP3/Nrf2 and and its anti-tumor metastasis effect.Methods and results:(1)MTT assay detected the effect of type I ferroptosis agonist Erastin on cell viability of different human bladder cancer cell lines(EJ,T24,BIU,J82).We find that Erastin has no significant inhibition on the proliferation of human bladder cancer cells,and human bladder cancer cells are not sensitive to Erastin;(2)In order to study whether targeting SLC7A11 can promote the effect of lowconcentration shikonin on the proliferation of human bladder cancer cells,Erastin and shikonin were combined to treat 4 human bladder cancer cells.MTT assay showed that Erastin promotes shikonin to reduce cell viability in a concentration-dependent and time-dependent manner,and human bladder cancer cells EJ and T24 are more sensitive;(3)In order to study whether targeting SLC7A11 can promoted low-concentration shikonin-induced death of human bladder cancer cells as necroptosis,the changes in the expression of necroptosis-related proteins were detected by Western blotting.The results demonstrated that RIP1,RIP3,and p-MLKL protein expression increased.Erastin has a synergistic effect on shikonin-induced necroptosis in human bladder cancer.RIP1 specific inhibitor Nec-1 can partially reverse the targeted SLC7A11 promotion of shikonin-induced cell death by reducing the expression of necroptosis proteins;(4)In order to study the relationship between Erastin’s promotion of low-concentration shikonin-induced necroptosis and ROS,flow cytometry was used to detect the ROS production of EJ and T24 cells.The results showed that Erastin promoted shikonininduced human bladder cancer cells to produce excessive ROS and reduced the mitochondrial membrane potential.The ROS inhibitor NAC almost completely reversed cell death.At this time,detection of protein changes revealed that the expression of necroptosis-related proteins was reduced.It shows that Erastin promotes low concentration of shikonin to induce excessive ROS production in human bladder cancer cells to induce necroptosis;(5)The above experiment found that targeting SLC7A11 can promoted low concentration of shikonin to induce cells to produce excessive ROS,which caused oxidative damage.Western blotting was used to detect its influence on the Nrf2 signaling pathway.The results showed that the protein expression of p-Nrf2 increased in a time-dependent manner during the first 12 h,then it decreased from 12 h to 24 h.The downstream NQO1 and HO-1 protein expression changes are consistent with pNrf2.Immunofluorescence detection found that the protein expression of Nrf2 decreased and transferred from the nucleus to the cytoplasm.The above results indicate that targeting SLC7A11 promotes the activation of low concentrations of shikonin and then inhibits the Nrf2 pathway;(6)In the study of targeting SLC7A11 to promote the inhibition of shikonin on the Nrf2 pathway,it was found that P62 protein was degraded.In order to verify whether shikonin targeting SLC7A11 induces autophagy,immunofluorescence detected an increase in LC3 fluorescent spots and an increase in fluorescence intensity.It demonstrated that the expression of p-P70S6 K decreased,m TOR was inhibited,P62 was degraded,Beclin1 expression increased,LC3 accumulated,and autophagy occurred.The autophagy inhibitor LY294002 promotes cell death induced by the two compounds by inhibiting cytoprotective autophagy;(7)The relationship between targeting SLC7A11 to promote shikonin-induced cell death and ferroptosis was further studied.It reduced the expression of SLC7A11 and GPX4,but the ferroptosis inhibitor Fer-1 instead promoted cell death.Western blotting results show that Fer-1 promotes the death of human bladder cancer cells by inhibiting autophagy and further inhibiting the Nrf2 signaling pathway;(8)In order to study the effect of low concentration shikonin targeting SLC7A11 on the invasion and metastasis ability of human bladder cancer cells.Transwell results showed that it inhibited the invasion and metastasis ability of human bladder cancer cells.Western blotting showed that the low concentration of shikonin targeting SLC7A11 increased the protein expression of E-Cadherin and decreased the expression of Vimentin.Conclusion: Targeting SLC7A11 promotes low concentration of shikonin to induce necroptosis of human bladder cancer cells by producing excessive ROS.The activation of the Nrf2 signaling pathway is inhibited,which further promotes cell death.Targeting SLC7A11 promotes protective autophagy in human bladder cancer cells.The expression of ferroptosis proteins SLC7A11 and GPX4 decreased,and the ferroptosis inhibitor Fer-1 promoted cell death by inhibiting autophagy and further inhibiting Nrf2.Targeting SLC7A11 promotes the inhibition of low-concentration shikonin on the invasion and metastasis of human bladder cancer cells.