title: Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells.
abstract: BACKGROUND
  Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer.
  
  
  METHODS
  The cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment.
  
  
  RESULTS
  Advanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells.
  
  
  CONCLUSIONS
  Our results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.
authors.affiliation: Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
authors.forename: Ji-Fan
authors.initials: JF
authors.lastname: Lin
authors.affiliation: Division of Urology, Department of Surgery, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Division of Urology, School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
authors.forename: Yi-Chia
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
authors.forename: Shan-Che
authors.initials: SC
authors.lastname: Yang
authors.affiliation: Division of Urology, Department of Surgery, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Division of Urology, School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
authors.forename: Te-Fu
authors.initials: TF
authors.lastname: Tsai
authors.affiliation: Division of Urology, Department of Surgery, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
authors.forename: Hung-En
authors.initials: HE
authors.lastname: Chen
authors.affiliation: Division of Urology, Department of Surgery, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Division of Urology, School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
authors.forename: Kuang-Yu
authors.initials: KY
authors.lastname: Chou
authors.affiliation: Division of Urology, Department of Surgery, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Division of Urology, School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan; Department of Urology, Taipei Medical University, Taipei, Taiwan.
authors.forename: Thomas I-Sheng
authors.initials: TI
authors.lastname: Hwang
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D000068338:Everolimus / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D013329:Structure-Activity Relationship; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000473:pathology*
chemical_list: D000970:Antineoplastic Agents; D000068338:Everolimus; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases
keywords: RAD001; apoptosis; autophagy; bladder cancer; chloroquine