Study On The Molecular Mechanism Of Reverse Resistance Of Type Ⅱ Ferroptosis Inducer To Human Bladder Cancer Cells Based On GPX4/Nrf2

Background: Bladder cancer is the ninth most common cancer in the world and the fourth most common cancer in men.Glutathione peroxidase 4(GPX4)plays a key role in the regulation of ferroptosis.When GPX4 is inhibited,it can induce ferroptosis,thereby effectively avoiding the problem of tumor resistance.The expression of GPX4 in a variety of tumor tissues is significantly higher than that in normal tissues,and is negatively correlated with the prognosis of patients with multiple types of cancer.In addition,inhibiting the activity of Nrf2 can significantly enhance the killing effect of ferroptosis inducers on cancer cells.Objective: This project uses Nrf2 and GPX4 as potential targets for inducing ferroptosis in bladder cancer,and explores the effect of targeting Nrf2/GPX4 on bladder cancer cells and its molecular mechanism,providing new ideas for targeted therapy of bladder cancer.Methods: Firstly,the Hoglund database and Western blotting were used to analyze the relationship between GPX4 and bladder cancer;secondly,bladder cancer cells EJ and T24 were selected as the research objects,MTT and clone formation experiments were used to detect the effect of targeting GPX4 alone and targeting Nrf2/GPX4 effect on cell proliferation;thirdly,with a variety of inhibitors intervention,flow cytometry,immunofluorescence,MDA detection,and Western blotting to determine the death type of bladder cancer cells induced by targeting Nrf2/GPX4;finally,Wound-healing and Transwell were used to detected the effect of targeting Nrf2/GPX4 on the metastasis.Results:(1)The survival data of 224 bladder cancer patients in the Hoglund database showed that the overall survival rate of patients with low GPX4 expression was significantly better than that of patients with high GPX4 expression;Western blotting showed that the expression of GPX4 in malignant bladder cancer cells EJ and T24 was significantly higher than that in benign bladder cancer cell BIU.These results indicated that the expression of GPX4 was positively correlated with the malignancy of bladder cancers.(2)MTT and clone formation results showed that targeting GPX4 alone had no significant effect on the survival rate of bladder cancer cells,but targeting Nrf2/GPX4 could significantly reduce the survival rate of bladder cancer cells.(3)Western blotting results showed that targeting Nrf2/GPX4 could down-regulate ferroptosis-related proteins GPX4,x CT and Nrf2 pathway-related proteins,the expression of autophagy-related protein p62 was also reduced,while the expression of LC3 was increased;MDA results showed that targeting Nrf2 /GPX4 significantly increased the level of lipid peroxidation in bladder cancer cells;JC-1fluorescent probe results showed that targeting Nrf2/GPX4 significantly reduced the mitochondrial membrane potential of bladder cancer cells;immunofluorescence results showed that targeting Nrf2/GPX4 significantly accumulated LC3 in bladder cancer cells.These results indicated that targeting Nrf2/GPX4 might induce ferroptosis and autophagy in bladder cancer cells,and inhibite the Nrf2 signaling pathway.(4)The addition of ferroptosis inhibitors(Fer-1,DFO,Trolox)almost completely reversed the death of bladder cancer cells induced by targeting Nrf2/GPX4;when adding autophagy inhibitors(Baf-A1)or RIPK1 inhibitors(Nec-1),cell death was partially inhibited;if pan-Caspase inhibitor(Z-VAD-FMK),RIPK3inhibitor(GSK872,GSK840)or ROS scavenger(NAC)were added,cell death was not inhibited.These results suggested that the death of bladder cancer cells induced by targeting Nrf2/GPX4 was mainly ferroptosis.(5)Western blotting showed that ferroptosis inhibitors(Fer-1,DFO,Trolox)restored the expression of autophagy-related proteins LC3,p62 and Nrf2pathway-related proteins;similarly,autophagy inhibitor(Baf-A1)also restored ferroptosis-related protein GPX4 and Nrf2 pathway-related proteins.These results suggested that targeting Nrf2/GPX4 induced ferroptosis and autophagy in bladder cancer cells to promote each other,and both inhibited the Nrf2 signaling pathway.(6)Western blotting results showed that RIPK1 inhibitor(Nec-1)significantly down-regulated autophagy-related protein LC3,while p62 was significantlyup-regulated,and the expression levels of ferroptosis-related protein and Nrf2pathway-related proteins were also partially up-regulated.These results suggested that RIPK1 inhibitor(Nec-1)might affect the ferroptosis and Nrf2 signaling pathways by inhibiting autophagy,and increased the survival rate of cells.(7)Wound-healing and Transwell results showed that targeting Nrf2/GPX4 inhibited the metastasis of bladder cancer cells.Conclusion: The Nrf2/GPX4 signaling pathway was activated in bladder cancer cells.Targeting Nrf2/GPX4 could induce bladder cancer cells to regulate cell death through ferroptosis and autophagy,but had no significant correlation with apoptosis,necroptosis and ROS.In addition,targeting Nrf2/GPX4 could also significantly inhibit the metastasis of bladder cancer cells.