Dauricine Mediates ROS Generation And Inhibits PI3K/Akt/mTOR Pathway To Induce Autophagic Apoptosis Of Bladder Cancer Cells

Purpose:In order to verify whether dauricine can mediate the generation of ROS and inhibit the PI3K/Akt/mTOR pathway to induce autophagy and apoptosis of bladder cancer cells.Methods:MTT method was used to detect the effects of different concentrations of dauricine on cell proliferation after 24 hours;The clone formation experiment was used to detect the inhibitory effects of dauricine on bladder cancer cells;Western blot analysis was used to detect bladder cancer after treatment with different concentrations of dauricine cell-associated apoptosis and autophagy protein expression;Immunofluorescence detection of bladder cancer cell LC3 spots was used to detect after treatment with different concentrations of dauricine.After siRNA transfection to silence autophagy-related genes,clone formation experiment detects the inhibitory effects of dauricine on bladder cancer cells;Western blot analysis verified whether dauricine mediates the PI3K/Akt/mTOR pathway through ROS.Results:The survival rate of the three cell lines were detected by MTT method decreased and were dose-dependent;The clone formation experiment detected the increased inhibitory effect of dauricine on two bladder cancer cells;Western blot experiments confirmed that Bax and Cleaved-The expressions of caspase 3,Cleaved-caspase 9,LC3,ATG5,Beclin1 and SQSTM1 were all increased;Immunofluorescence test confirmed that dauricine treatment of bladder cancer cells induced GFP-LC3 extensive localization;Knockdown Atg5 or Beclin 1 clone formation experiments confirmed that dauricine Alkali significantly inhibits bladder cancer cells;PI3K activation significantly reduces the transformation of LC3-Ⅱ in bladder cancer cells treated with dauricine;After dauricine treatment,the level of reactive oxygen species ROS increased significantly;Western blot analysis showed that the activation of LC3 and PI3K signaling pathways was inhibited after dauricine and ROS scavenger N-acetyl-L-cysteine combined treatment of bladder cancer cells.Conclusion:Dauricine inhibits cell growth by inducing autophagic apoptosis in bladder cancer cells.It is further found that it may induce bladder cancer cell autophagy apoptosis by mediating ROS generation and then inhibiting PI3K/Akt/mTOR signaling pathway.