Study On The Mechanism Of Ferroptosis In Bladder Cancer Induced By Erianin From Dendrobium Chrysotoxum Lindl. Extract

Background: Bladder cancer is one of the most common urinary system tumors in humans.The early clinical symptoms of bladder cancer are not typical,and some patients with bladder cancer are already in the advanced stage when they are found.However,drugs for the treatment of advanced bladder cancer are limited,so the development of new drugs with high efficiency and low toxicity is of great significance for patients with bladder cancer.Erianin is a natural compound extracted from Dendrobium chrysotoxum Lindl.,a traditional Chinese medicine.It has been found that erianin can induce ferroptosis in cancer cells.Our previous study found that erianin can induce ferroptosis in lung cancer cells,but the relationship between erianin,ferroptosis and bladder cancer has not been reported in the literature.Methods: CCK-8 kit was used to detect cell viability,Annexin V-FITC/PI kit was used to detect cell apoptosis,PI kit to detect cell cycle,DCFH-DA staining to detect reactive oxygen species levels,Western Blot to detect cell protein levels,molecular docking to predict drug action targets,sh RNA transfection to knockdown target genes,real-time fluorescence quantitative PCR to detect RNA levels,immunohistochemistry to detect tissue protein level and so on.These methods investigated the effect and pharmacological mechanism of the action of erianin on bladder cancer cellsResults:(1)We found that erianin induced the death of bladder cancer cells KU-19-19 and RT-4 by CCK-8 and flow apoptosis.(2)In order to explore the way erianin induced the death of bladder cancer cells,we used the pancaspase inhibitor Z-VADFMK,Necrostatin-1,autophagy inhibitor CQ,and ferri ion chelating agent DFO in combination with erianin,and found that only DFO could antagonize the efficacy of erianin on bladder cancer cells.There was no statistical significance in the antagonism of other antagonistic effects on erianin.(3)Erianin could activate ROS,accumulate MDA,and consume GSH in bladder cancer cells,and active oxygen inhibitor NAC and GSH supplement can also significantly reverse the efficacy of erianin,and Western Blot analysis showed that erianin can inhibit the expressions of ferroptosis related proteins Nrf2 and GPX4.These results suggested that erianin might induce bladder cancer cell death through ferroptosis pathway.(4)In vivo,we confirmed that erianin induced bladder cancer cell death through ferroptosis.(5)Molecular docking results showed that erianin and Nrf2 had a good combination.In order to further confirm the possibility of molecular docking,TBHQ,the activator of Nrf2,was used in combination with erianin.CCK-8 and Western Blot experiments found that TBHQ could antagonize the effect of erianin on bladder cancer cells.The ferroptosis of bladder cancer cells induced by erianin was reversed.(6)Next,knockdown and overexpression of Nrf2 were performed on bladder cancer cells,and it was found that knockdown of Nrf2 could promote the changes in the levels of ferroptosis related indicators and related proteins of erianin,while overexpression of Nrf2 could reverse the changes in the levels of erianin related proteins.Nrf2 may be an important target of ferroptosis induced by erianin in bladder cancer.(7)In order to further investigate the mechanism of ferroptosis in bladder cancer cells induced by erianin,RT-PCR and Western Blot experiments showed that erianin might play a role in ferroptosis through Nrf2/ HO-1signaling pathway.Conclusions: Erianin induced bladder cancer cell death by ferroptosis.Nrf2 may be the key target of ferroptosis induced by erianin in bladder cancer cells.Erianin may induce ferroptosis in bladder cancer cells by regulating Nrf2/HO-1 signaling pathway.