Induction of Noxa sensitizes human colorectal cancer cells expressing Mcl-1 to the small-molecule Bcl-2/Bcl-x L inhibitor, ABT-737

Kenji Okumura, Shengbing Huang, Frank A Sinicrope

Research output: Contribution to journalArticle

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Abstract

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 cas-pase-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.

Original languageEnglish (US)
Pages (from-to)8132-8142
Number of pages11
JournalClinical Cancer Research
Volume14
Issue number24
DOIs
StatePublished - Dec 15 2008

Fingerprint

irinotecan
Noxae
Colorectal Neoplasms
Apoptosis
Small Interfering RNA
Proteins
Topoisomerase I Inhibitors
Cell Line
Poly(ADP-ribose) Polymerases
Caspase 9
Caspase 8
Annexin A5
Drug Combinations
Caspases
ABT-737
Immunoprecipitation
Drug Resistance
Caspase 3

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Induction of Noxa sensitizes human colorectal cancer cells expressing Mcl-1 to the small-molecule Bcl-2/Bcl-x L inhibitor, ABT-737. / Okumura, Kenji; Huang, Shengbing; Sinicrope, Frank A.

In: Clinical Cancer Research, Vol. 14, No. 24, 15.12.2008, p. 8132-8142.

Research output: Contribution to journalArticle

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title = "Induction of Noxa sensitizes human colorectal cancer cells expressing Mcl-1 to the small-molecule Bcl-2/Bcl-x L inhibitor, ABT-737",
abstract = "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 cas-pase-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.",
author = "Kenji Okumura and Shengbing Huang and Sinicrope, {Frank A}",
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T1 - Induction of Noxa sensitizes human colorectal cancer cells expressing Mcl-1 to the small-molecule Bcl-2/Bcl-x L inhibitor, ABT-737

AU - Okumura, Kenji

AU - Huang, Shengbing

AU - Sinicrope, Frank A

PY - 2008/12/15

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N2 - 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 cas-pase-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.

AB - 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 cas-pase-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.

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