MP45-19 BRUCE BRIDGES AUTOPHAGY AND APOPTOSIS IN SILIBININ-INDUCED CELL DEATH IN BLADDER CANCER

Abstract

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2016MP45-19 BRUCE BRIDGES AUTOPHAGY AND APOPTOSIS IN SILIBININ-INDUCED CELL DEATH IN BLADDER CANCER Jin Zeng, Wei Liu, Feng Li, Yi Sun, Lei Li, Xinyang Wang, and Dalin He Jin ZengJin Zeng More articles by this author , Wei LiuWei Liu More articles by this author , Feng LiFeng Li More articles by this author , Yi SunYi Sun More articles by this author , Lei LiLei Li More articles by this author , Xinyang WangXinyang Wang More articles by this author , and Dalin HeDalin He More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.296AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Autophagy is a tightly-regulated catabolic process that involves the degradation of intracellular components via lysosomes. Although the pivotal role of autophagy in cell growth, development, and homeostasis has been well understood, its function in bladder cancer (BCa) prevention and intervention remains to be delineated. We previously identified intravesical silibinin, a natural flavonoid, as a novel and effective chemopreventive therapy against BCa, which were associated with its proapoptotic effects. However, underlying mechanisms are poorly understood. The aim of this study was to investigate the function and mechanism of autophagy induced by silibinin in BCa. METHODS Human BCa cell line 5637, RT4 and T24 were used as the model system in vitro and in vivo. The autophagy status in cells was determined by the detection of microtubule-associated light chain 3 (LC3-I/II) using immunofluorescent staining and western blot and the formation of autophagolysosome vacuoles using transmission electron microscopy. The expression of autophagy and apoptosis related molecules after silibinin treatment were examined by western blot. Detection of apoptosis level was measured by caspase 3/7/9 activation, caspase 3/7/9 cleavage and flow cytometry assays. Both nude mice xenografts and orthotopic rat BCa tissues were analyzed for apoptosis and autophagy molecular alterations after oral or intravesical silibinin treatment in vivo. RESULTS Silibinin treatment increased the expression of LC3-II, promoted autophagosome accumulation and autophagolysosome vacuoles formation and enhanced autophagic flux by clearance of p62 in BCa cells. Mechanically, the adenosine 5’-monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway was involved in the regulation of silibinin-induced autophagy. Blocking autophagy with choloroquine or bafilomycin A1 or inhibition of autophagosome formation using Atg5 or Atg7 SiRNA restored p62 levels, inhibited caspase 3 processing and PARP cleavage and thus reversed silibinin-induced cell death and apoptosis. Furthermore, we firstly found that BIR repeat containing ubiquitin-conjugating enzyme (BRUCE), a member of the inhibitor of apoptosis protein family, was degraded by autophagy in BCa cells. Silibinin treatment resulted in a strong decrease in BRUCE expression and consequent caspase 9/3/7 activation. Blocking autophagy with choloroquine or bafilomycin A1 can inhibit the degradation of BRUCE by silibinin. Overexpression of exogenous BRUCE inhibited caspases processing and PARP cleavage and finally led to the inhibition of apoptotic cell death. In vivo data showed that, either oral silibinin or intravesical instillation of silibinin inhibited the growth of xenografts in nude mice or orthotopic bladder tumors in rats respectively, which were associated with autophagy induction, BRUCE downregulation and caspase 3 activation. CONCLUSIONS Our study provides new insights regarding the biological and anti-proliferative activities of silibinin against BCa, focusing on BRUCE-mediated autophagy and apoptosis interaction, which thereby further advocates its clinical effectiveness against BCa. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e615-e616 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Jin Zeng More articles by this author Wei Liu More articles by this author Feng Li More articles by this author Yi Sun More articles by this author Lei Li More articles by this author Xinyang Wang More articles by this author Dalin He More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...