title: Smac mimetic reverses resistance to TRAIL and chemotherapy in human urothelial cancer cells.
abstract: OBJECTIVE
  inhibitors of apoptosis proteins (IAPs) have been shown to contribute to resistance of neoplastic cells to chemotherapy and to biologic antineoplastic agents. Consequently, new agents are being developed targeting this family of proteins. In a panel of bladder cancer cell lines, we evaluated a Smac mimetic that antagonizes several IAPs for its suitability for bladder cancer therapy. Experimental design: A panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-A alone, TRAIL alone, chemotherapy alone, compound-A plus TRAIL, and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally, siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting.
  
  
  RESULTS
  single-agent compound-A had little effect, but compound-A augmented TRAIL- and chemotherapy-induced apoptosis. Immunoblotting showed that combination treatment with compound-A and TRAIL resulted in cleavage of procaspase-3 and procaspase-7, activation of which irreversibly commits cells to apoptosis. Immunoprecipitation of XIAP showed displacement of active caspase-3 fragments from XIAP, supporting the proposed mechanism of action. Furthermore, siRNA-mediated silencing of XIAP similarly sensitized these cells to apoptosis.
  
  
  METHODS
  a panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-Alone, TRAIL alone, Chemotherapy alone, compound-A plus TRAIL and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting.
  
  
  CONCLUSIONS
  our results suggest that targeting of XIAP with the Smac mimetic compound-A has the potential to augment the effects of a variety of chemotherapeutic and biologic therapies in bladder cancer.
authors.affiliation: Department of Urology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Adam R
authors.initials: AR
authors.lastname: Metwalli
authors.forename: Sanaz
authors.initials: S
authors.lastname: Khanbolooki
authors.forename: Goodwin
authors.initials: G
authors.lastname: Jinesh
authors.forename: Debasish
authors.initials: D
authors.lastname: Sundi
authors.forename: Jay B
authors.initials: JB
authors.lastname: Shah
authors.forename: Marissa
authors.initials: M
authors.lastname: Shrader
authors.forename: Woonyoung
authors.initials: W
authors.lastname: Choi
authors.forename: Laura M
authors.initials: LM
authors.lastname: Lashinger
authors.forename: Srinivas
authors.initials: S
authors.lastname: Chunduru
authors.forename: David J
authors.initials: DJ
authors.lastname: McConkey
authors.forename: Mark
authors.initials: M
authors.lastname: McKinlay
authors.forename: Ashish M
authors.initials: AM
authors.lastname: Kamat
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D053938:DNA Fragmentation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000627:therapeutic use*; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D019459:Urothelium / Q000473:pathology; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D009842:Oligopeptides; D034741:RNA, Small Interfering; C532637:SMAC peptide; D053221:TNF-Related Apoptosis-Inducing Ligand; D051636:X-Linked Inhibitor of Apoptosis Protein; D053148:Caspase 3; D053179:Caspase 7
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