Metformin Induces Autophagy In Bladder Cancer Cells And Its Mechanism

Objective:Bladder cancer is an important global public health issue.In the latest global cancer statistics in 2018,the new incidence of bladder cancer ranks fourth among malignant tumors,and the mortality rate ranks eighth.In my country,bladder cancer is the most popular malignant tumor of the urinary system,and it is gradually expanding its impact on the health of our people.In recent years,more and more evidence shows that metformin not only has a significant hypoglycemic effect,but also plays an important anti-tumor effect in many types of cancer.This mechanism is mainly related to the activation of AMPK and the reduction of m TOR signaling pathway and protein expression.As a potential new target for cancer treatment,autophagy,its autophagy-related m TOR signaling pathway,plays an important role in tumor cell growth,proliferation and survival.Therefore,the research on this subject can provide a theoretical basis for the prevention and treatment of metformin in clinical urinary system tumors.Methods:The bladder cancer cell line BIU-87 was used for the experiment,and the MTT colorimetric experiment was used to detect the viability of BIU-87 cells at different concentrations of metformin at different times;the transmission electron microscope(TEM)was used to observe the different concentrations and different times of the metformin treatment group.The morphology and quantity of phagosomes;the autophagy staining detection kit(MDC method)was used to detect the morphology and quantity of bladder cancer cell autophagosomes using a fluorescent microscope;the Western blot method was used to detect the effects of different concentrations of metformin for 6 hours,12 hours,and 24 hours.Phage marker proteins LC3-I,LC3-II expression.The Western blot method was used to detect the effects of metformin at different concentrations for 24 hours and 48 hours,respectively,to detect the expression of phospho-p70s6k,phospho-4EBP1 mediated by the autophagy signaling pathway mTORC1;the expression of phospho-AKT and Ac tin mediated by mTORC2;analysis of the m TOR signaling pathway and its The changes and expression of downstream proteins illustrate some of the signaling pathway mechanisms related to autophagyResults:Metformin has a significant inhibitory effect on the proliferation of bladder cancer cells,and the inhibitory effect is concentration-dependent and time-dependent(p<0.05).Transmission electron microscopy results show that:compared with the control group,the formation of autophagosomes can be observed in each treatment group of metformin,and consistent with the results of MDC staining,the number of autophagosomes formed by metformin acting on bladder cancer cells increased significantly,and It was concentration-dependent(p<0.05).Western-blot detection showed that the expression of LC3B(LC3-Ⅰ,LC3-Ⅱ)protein in the metformin treatment group was significantly higher than that in the control group(p<0.05)and the autophagy mechanism may be through the inhibition of mTORC1 and mTORC1 in the mTOR pathway in bladder cancer cells.mTORC2 pathway downstream protein expression,such as phospho-p70S6K1,phospho-4EBP1,phospho-AKT,Ac tin.Conclusions:Metformin can significantly inhibit cell viability after acting on human bladder cancer cell BIU-87,and the inhibitory effect is significantly concentration-dependent and time-dependent.After metformin acted on human bladder cancer cell BIU-87,the expression of autophagy marker protein LC3B(ie LC3-Ⅰ/Ⅱ<1)was detected,and the ratio of LC3-II/Actin was significantly increased,indicating that metformin can induce autophagy,resulting in Phagocytosis.Metformin produces autophagy after acting on human bladder cancer cell BIU-87,forming autophagosomes.With the increase of metformin concentration,the number of autophagosomes formed increased significantly,and the number of autophagosomes formed was significantly concentration-dependent.After metformin acted on human bladder cancer cell BIU-87,the expression of mTORC1-mediated phospho-p70s6k and phospho-4EBP1 protein gradually decreased,and the protein expression was concentration-dependent and time-dependent.After metformin acted on human bladder cancer cell BIU-87,the expression of mTORC2-mediated phospho-AKT protein gradually decreased,and the expression of this protein was concentration-dependent and time-dependent.This study will enable us to effectively intervene in metformin induced autophagy,and provide effective guidance in the application of clinical urinary system antitumor;metformin drug therapy may become a highly innovative,developmental,and safe and effective new method for the treatment of bladder tumors.