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Induction of Noxa Sensitizes Human Colorectal Cancer Cells Expressing Mcl-1 to the Small-Molecule Bcl-2/Bcl-x_L Inhibitor, ABT-737

Authors' Affiliations: Miles and Shirley Fiterman Center for Digestive Diseases and Division of Oncology, Mayo Clinic, Rochester, Minnesota

Requests for reprints: Frank A. Sinicrope, Miles and Shirley Fiterman Center for Digestive Diseases and Division of Oncology, Mayo Clinic, 200 1st Street Southwest, Rochester, MN 55905. E-mail: sinicrope.frank{at}mayo.edu.

Purpose: 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-x_L but not Mcl-1, on apoptosis induction alone and in combination with CPT-11 and explored mechanisms underlying their cooperativity.

Experimental Design: 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-x_L or Bcl-2 and disrupted the interaction of Bcl-x_L 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.