The Study Of CIAP1and Survivin Nuclear Expression In Bladder Cancer Tissues And Cells

Bladder cancer is the most common malignancy of the urinary tract system andrepresents an important cause of morbidity and mortality. Like other tumors, the maincharacter of bladder cancer is apoptotic deficiency with the imbalance of proliferationand the resistance of chemotherapeutics. Thus, attention has increasingly focused onovercoming the unlimited proliferation and the apoptotic deficiency of cancer cells atthe cellular level to improve therapeutic strategies.Inhibitor of apoptosis protein（sIAPs）, a group of apoptosis regulators, can potentlyblock both the intrinsic and the extrinsic apoptotic pathway by inhibiting caspaseactivity and regulating nuclear factor kappa-enhancer binding protein（NF-κB）or tumornecrosis factor-α（TNF-α）pathway. The expression and subcellular localization of IAPsare usually correlated with cancer progression and resistance to chemotherapy, and areassociated with the prognosis. One research revealed that the Survivin nuclear labelingindex（Survivin-N）was a superior biological and prognostic marker for Ta/T1urothelialcarcinomas of the urinary bladder. One of our previous studies demonstrated thatnuclear cIAP1expression（cIAP1-N）may be strongly correlated with bladder cancerstage, grade, tumor recurrence and tumor-related mortality. Therefore, cIAP1andSurvivin nuclear expression may play an important role in the development andprogression of bladder cancer.Based on these researches, our study further explored the correlation betweennuclear cIAP1and Survivin expression in tissues and cells of bladder cancer. Weanalyzed the influence of cIAP1and Survivin subcellular distribution on the functionsof bladder cancer cells. We also examined the influence of cIAP1and Survivinsubcellular distribution on the other IAPs and the drug sensitivity. Our results aimed tooffer theoretical and experimental basis in the research of bladder cancer targetedtherapy using IAPs as target. Objective1. Detect the expression and distribution of cIAP1and Survivin in tissues ofbladder cancer. Analyze the correlation between nuclear cIAP1and Survivin expressionin bladder cancer tissues. Analyze the influence of nuclear cIAP1and Survivinexpression on the prognosis.2. Detect the expression and distribution of cIAP1and Survivin in bladder cancercells. Detect the interaction of cIAP1and Survivin in nucleus. Observe the influence ofcIAP1and Survivin subcellular distribution on apoptosis, cell cycle, immigration andinvasion of bladder cancer cells.3. Detect the influence of cIAP1and Survivin subcellular distribution on theexpression of other IAPs and PTEN. Detect the influence of cIAP1and Survivinsubcellular distribution on the doxorubicin sensitivity.Methods1. The expression and distribution of IAPs in bladder cancer tissues were detectedby Western blot and immunohistochemisty. Correlation between nuclear cIAP1andSurvivin expression was assessed using Spearman’s correlation and linear regressionanalysis. Survival functions and differences were calculated by the Kaplan-Meiermethod and assessed using the log rank statistic. Multivariable survival analyses wereperformed using the Cox proportional hazards regression model. P＜0.05wasconsidered to be statistically significant.2. The subcellular distribution of IAPs in bladder cancer cells was detected byconfocal laser scanning microscope. The interaction of cIAP1and Survivin in nucleuswas detected by Co-Immunoprecipitation. The apoptosis, cell cycle, immigration andinvasion of bladder cancer cells were detected using Hoechst33258,Rhodamine-Phalloidin, flow cytometry, cell Scarification and ECMatrix gel assays.3. The influence of cIAP1and Survivin subcellular distribution on the expressionof other IAPs and PTEN was detected by western blot. The influence of cIAP1andSurvivin subcellular distribution on the doxorubicin sensitivity was detected by MTT,Hoechst33258and Rhodamine-Phalloidin assays.Results1. The study of correlation between nuclear cIAP1and Survivin expressionand their influence on prognosis in bladder cancercIAP1and Survivin appeared cytoplasmic and nuclear distribution in differentgrade bladder cancer tissues. The nuclear expression of cIAP1and Survivin was increased in high grade bladder cancer tissues. Spearman’s correlation and linearregression analysis revealed that there was a significant positive correlation betweencIAP1-N expression and Survivin-N expression. The mean recurrence-free survivaltimes were significantly decreased in the high cIAP1-N and Survivin-N expressiongroups compared with the low expression groups. The expression of cIAP1-N andSurvivin-N was a significant independent prognostic marker for recurrence-free survival,in addition to the presence of muscle-invasive disease and high tumor grade.2. The study of the influence of nuclear cIAP1and Survivin expression on thefunctions of bladder cancer cells.cIAP1and Survivin were restrained in the nucleus after the stimulation of LMB.The expression of nuclear cIAP1and Survivin was increased. Survivin restrained in thenucleus faster than cIAP1. The stimulation of LMB has no influence on the other IAPs.There was an interaction of cIAP1and Survivin in nucleus. Low dose LMB couldchange the subcellular distribution of cIAP1and Survivin, but could not induceapoptosis in bladder cancer cells. The change of cIAP1and Survivin subcellulardistribution could result in G2/M cell cycle arrest and could increase the immigrationand invasion of bladder cancer cells.3. The study of the influence of cIAP1and Survivin subcellular distribution onother apoptosis regulating proteins.The expression of cytoplasmic XIAP and Livin was increased after the stimulationof LMB. The expression of cIAP2had no change. The nuclear PTEN expression did notchange, but the cytoplasmic PTEN expression was decreased after the stimulation ofLMB. The combination of LMB and doxorubicin could increase apoptosis in bladdercancer cells.Conclusions1. There was significant correlation between nuclear cIAP1and Survivinexpression in bladder cancer tissues. cIAP1-N and Survivin-N expression may be amarker in bladder cancer prognosis.2. There was an interaction of cIAP1and Survivin in bladder cancer nucleus. Thechange of cIAP1and Survivin subcellular distribution could result in G2/M cell cyclearrest and could increase the immigration and invasion of bladder cancer cells.3. The change of cIAP1and Survivin subcellular distribution could increasecytoplasmic XIAP and Livin expression and decrease cytoplasmic PTEN expression. The change of cIAP1and Survivin subcellular distribution could increase thedoxorubicin sensitivity in bladder cancer cells.