title: Induction of Noxa sensitizes human colorectal cancer cells expressing Mcl-1 to the small-molecule Bcl-2/Bcl-xL inhibitor, ABT-737.
abstract: OBJECTIVE
  The intrinsic drug resistance of colorectal cancers is related in part to overexpression of prosurvival Bcl-2 family proteins. We determined the effects of ABT-737, a small-molecule inhibitor of Bcl-2/Bcl-xL but not Mcl-1, on apoptosis induction alone and in combination with CPT-11 and explored mechanisms underlying their cooperativity.
  
  
  METHODS
  Human colorectal carcinoma cell lines (HCT116 wild-type and Bax(-/-), HT-29, and RKO) were incubated with ABT-737 alone and combined with CPT-11 or bortezomib, and cell viability, caspase cleavage, and Annexin V labeling were measured. In drug-treated cell lines, protein-protein interactions were analyzed by immunoprecipitation. Lentiviral short hairpin RNA was used to knockdown Noxa expression.
  
  
  RESULTS
  ABT-737 induced apoptosis in a dose-dependent manner and its coadministration with the topoisomerase I inhibitor, CPT-11, resulted in a synergistic cytotoxic effect. Apoptosis induction by the drug combination was associated with enhanced caspase-8, caspase-9, and caspase-3 activation and poly(ADP-ribose) polymerase cleavage that were completely abrogated in Bax knockout cells. ABT-737 unsequestered the BH3-only protein Bim from its complex with Bcl-xL or Bcl-2 and disrupted the interaction of Bcl-xL with Bak. CPT-11 treatment up-regulated Noxa expression, as did bortezomib, and enhanced Noxa/Mcl-1 complexes. CPT-11 also disrupted the Mcl-1/Bak interaction. Knockdown of Noxa using short hairpin RNA lentiviral constructs was shown to significantly attenuate the cytotoxic effect of CPT-11 or bortezomib combined with ABT-737 and inhibited caspase-3 cleavage.
  
  
  CONCLUSIONS
  Induction of Noxa by CPT-11 or bortezomib can sensitize colorectal cancer cells expressing Mcl-1 to ABT-737. Up-regulation of Noxa may therefore represent an important strategy to enhance the therapeutic efficacy of ABT-737 against colorectal cancer and other solid tumors.
authors.affiliation: Miles and Shirley Fiterman Center for Digestive Diseases and Division of Oncology, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: Kenji
authors.initials: K
authors.lastname: Okumura
authors.forename: Shengbing
authors.initials: S
authors.lastname: Huang
authors.forename: Frank A
authors.initials: FA
authors.lastname: Sinicrope
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001713:Biphenyl Compounds / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D000077146:Irinotecan; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009596:Nitrophenols / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology*; D051020:bcl-X Protein / Q000037:antagonists & inhibitors*
chemical_list: C501332:ABT-737; C494811:BCL2L1 protein, human; D001713:Biphenyl Compounds; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009596:Nitrophenols; C498702:PMAIP1 protein, human; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D051020:bcl-X Protein; D000077146:Irinotecan; D002166:Camptothecin