Molecular Mechanism Of Ivermectin Induced Autophagy In Bladder Cancer Cells

Objective: To investigate the potential molecular mechanism of ivermectin inhibition of bladder cancer cell proliferation,To explore the effect of ivermectin on bladder cancer cells and its relationship with autophagy in a preliminary way.To gain an in-depth understanding and characterise the functional changes of autophagy proteins during the formation of autophagosomes in bladder cancer cells and to search for new targets for the prevention and treatment of bladder cancerMethods: Two kinds of bladder cancer cells T24,5637 and normal SV-HUC-1 cells were used to screen the drug concentration of ivermectin by CCK-8 assay.After IC50 was analyzed,the experimental groups were divided into 0/5/10/20 μM.The clone formation experiments were observed,the difference between the individual ivermectin concentrations.Tunnel test detects the effect of bladder on changes in the positive rate of apoptotic cells.The trend of anti-apoptotic proteins Bcl-2 and Cleaved-casepase-3 in T24 and 5637 cells treated with ivermectin,as well as the changes of autophagy-related proteins LC3,Atg5,Beclin1,P62 and Hedgehog pathway protein SHH were studied by Western Blot,Gli1,Su Fu changes.The expression of LC3 and P62 proteins was detected by immunofluorescence assay.The formation of autophagosomes in bladder cancer cells in the 10 μM ivermectin-treated group was observed by transmission electron microscopy.Results: Bladder cancer cell lines T24 and 5637 had higher IC50 concentrations compared to SV-HUC-1 cells.The proliferation capacity of bladder cancer cell clusters was inhibited in the clonogenic experimental and control groups compared to the control group.There was a decreasing trend in the 5 μM,10 μM and 20 μM experimental groups;in the Western Blot assay,0 μM,5 μM,10 μM,and 20 Bcl-2 protein showed a decreasing trend in the experimental group at 0 μM,5 μM,10 μM and 20 μM,and in contrast Cleaved-Casepase-3 protein showed an increasing trend,in addition,autophagy-related proteins LC3 II,Beclin1,Atg5 showed a dose-dependent increase,and P62 protein showed the opposite trend.The tunel experiment confirmed an increase in apoptosis protein positivity in the 10 μM ivermectin experimental group compared to the control group.In flow cytometry,there was an increasing trend of apoptosis at 5 μM,10 μM and 20 μM.In immunofluorescence experiments,LC3 protein expression was found to be increased in the experimental group after 10 μM treatment compared to the DMSO control group,and P62 cytoplasmic expression was decreased in the experimental group at 10 μM.Meanwhile,in transmission electron microscopy,it was found that there were significantly more autophagic vesicles in the 10 μM experimental group than in the DMSO control group.Western blot detected the Hedgehog pathway proteins after ivermectin treatment,SHH and Gli1 proteins decreased with increasing drug concentration,and Su Fu increased with increasing drug concentration.Conclusion: Ivermectin can induce apoptosis and autophagy in bladder cancer cells by inducing changes in the Hedgehog pathway under the influence of the Hh pathway.The ability of ivermectin to inhibit the proliferation of bladder cancer cells compared to normal bladder epithelial cells may provide a possible anti-tumour treatment option for ivermectin.