The Positive Role And Potential Mechanisms Of Androgen Blockade In Regulating Apoptosis Of Bladder Cancer Cells

Objective: To investigate the effects and mechanisms of androgen blockade on apoptosis of bladder cancer cells.Methods: 1.AR-positive UM-UC-3 cells and AR-negative 5637 cells were cultured and maintained in our study.The AR signaling were inhibited by 50 μM AR competitor Bic(bicalutamide)or androgen ablation.The apoptotic cells were quantified by hoechst staining and flow cytometry.In addition,the double or multiple membranes of autophagic vesicles(autophagosomes and autolysosomes),which are considered hallmarks of autophagy,were detected by TEM(transmission electron microscopy).The number of autophagosomes(yellow spots)and autolysosomes(red spots)were quantified using a confocal fluorescence microscope.Furthermore,LC3II/I and apoptosis related protein Bcl-2 and cleaved caspase-3 were detected by western blotting.2.AR specific si RNA was used to downregulate the expression of AR in UM-UC-3 cells,and RNA transcriptome sequencing from control cells or cells treated with si RNAs against AR was performed to investigate the changes of the downstream ATGs of AR.Some ATGs with relatively obvious changes were then quantified using q RTPCR.According to the results of q RT-PCR,a certain ATG with significant changes was selected as our target gene.The specific si RNA was used to silence the target gene.TEM and western blotting were used to investigate the effects of the target gene on autophagy of UM-UC-3 cells.Then,flow cytometry and western blotting were used to investigate the effects of the target gene on apoptosis of UM-UC-3 cells.3.UM-UC-3 cells and(or)5637 cells were treated with 50 μM Bic and(or)100 n M RAPA(rapamycin)or 100 μM CQ for 48 h,and then autophagy and apoptosis were detected with the above methods.Results: Androgen deprivation and androgen receptor competitor bicalutamide induced apoptosis and autophagy in AR-positive UM-UC-3 cells,but there were no similar effects were detected in AR-negative 5637 cells.Androgen deprivation and bicalutamide upregulated the expression of ULK2.ULK2 knockdown inhibited autophagy and apoptosis in bladder cancer cells.RAPA could induced autophagy and apoptosis in 5637 cells.Furthermore,the pro-apoptosis effect of Bic on UM-UM-3 cells could be rescued by the application of CQ and be enlarged by the application of RAPA.Conclusion: Androgen deprivation and AR competitor bicalutamide promoted apoptosis via autophagy induction by upregulating the expression of ULK2 in bladder cancer cells.Targeting the AR with bicalutamide and its downstream ULK2 may be a potential approach for inhibiting bladder cancer progression.