Hypoxia-induced Autophagy Promotes Gemcitabine Resistance In Human Bladder Cancer Cells Through Hypoxia-inducible Factor 1α Activation

Objective:Overcoming the chemoresistance of bladder cancer is a pivotal obstacle in clinical treatments.Hypoxia widely exists in solid tumors and has been demonstrated to contribute to chemoresistance through hypoxia-inducible factor 1α(HIF-1α)-mediated autophagy in several types of cancer.However,it is unclear whether HIF-la-mediated autophagy and chemoresistance occur in bladder cancer.Therefore,we investigated whether hypoxia-induced cytoprotective autophagy counteracted gemcitabine-induced apoptosis through increasing HIF-1αexpression.Method:1.Detecting the expression of the hypoxia-specific indicator HIF-la in 20 paired bladder cancer and adjacent non-cancerous tissues using immunohistochemistry;2.CCK8 and Western blot were used to detect the proliferation inhibition rate of bladder cancer cells and the difference of cleaved caspase3 and PARP cleavage in normoxic and hypoxic conditions.The IC50 values were calculated with spss 17.0;3.Treated bladder cancer cells with hypoxia alone or in combination with gemcitabine,western blot was used to detect the autophagic marker proteins LC3 and P62.Autophagy was also observed by monitoring the formation of GFP spots using confocal microscopy,autophagosomes were observed by transmission electron microscopy;4.Autophagy inhibitor 3MA was used to inhibit autophagy,CCK8,Western blot and Annexin V-PI flow cytometry were used to detect the change of proliferative rate,apoptotic protein and apoptotic rate of bladder cancer cells treated with gemcitabine under hypoxia;5.Western blot was used to detect the expression of HIF-1α,BNIP3,Beclinl and LC3 in bladder cancer cells under hypoxia.The expression of HIF-la was knocked down by RNAi technology.The changes of HIF-la,BNIP3,Beclinl and LC3 protein expression were detected by Western blot.CCK8,Western blot,Annexin V-PI flow cytometry was used to detect the change of the inhibitory rate,apoptotic protein and apoptotic rate of bladder cancer cells after knockdown of HIF-1α under hypoxiaResults:The expression of HIF-la was significantly higher in bladder cancer than in adjacent tissues(P<0.05).Gemcitabine-induced apoptosis during hypoxia was significantly reduced compared with that observed under normoxia.In addition,hypoxia activated autophagy and enhanced gemcitabine-induced autophagy in bladder cancer lines T24 and BIU-87.Combined treatment using gemcitabine and an autophagy inhibitor 3MA under hypoxia significantly increased gemcitabine cytotoxicity.Furthermore,it was demonstrated that hypoxia-activated autophagy depended on the HIF-la/BNIP3/Beclinl signaling pathway.Suppressing HIF-la inhibited autophagy,BNIP3 and Beclinl,as well as enhanced gemcitabine-induced apoptosis in bladder cancer cells under hypoxiaCONCLUSIONS:The results of the present study demonstrated that that hypoxia-induced cytoprotective autophagy resists gemcitabine-induced apoptosis by increasing HIF-la expression.Therefore,targeting HIF-la-associated pathways or autophagy in bladder cancer may be a successful strategy to enhance the sensitivity of bladder cancer chemotherapy.