Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemopreventive agent.
Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.
Choi
Sunga
S
Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Singh
Shivendra V
SV
D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D018890:Chemoprevention; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D017879:Isothiocyanates; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000187:drug effects; D024101:Mitochondrial Proteins / Q000378:metabolism; D011467:Prostate / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology; D011487:Protein Conformation / Q000187:drug effects; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
D016588:Anticarcinogenic Agents; C506593:Apaf1 protein, mouse; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; D002352:Carrier Proteins; D015938:Complement Membrane Attack Complex; C499605:Diablo protein, mouse; D006023:Glycoproteins; D017879:Isothiocyanates; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C047610:SC5b-9 protein complex; D013454:Sulfoxides; D013861:Thiocyanates; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D003165:Complement System Proteins; D045304:Cytochromes c; C505409:Casp3 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C016766:sulforaphane