Benzyl isothiocyanate (BITC), a highly promising cancer chemopreventive constituent of many edible cruciferous vegetables, causes apoptotic cell death in MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) human breast cancer cell lines but the mechanism of cell death is not fully understood. We now demonstrate that the BITC-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) due to inhibition of complex III of the mitochondrial respiratory chain (MRC). The BITC-induced ROS production and apoptosis were significantly inhibited by overexpression of catalase and Cu,Zn-superoxide dismutase as well as pharmacological inhibition of MRC using rotenone and diphenyleneiodonium chloride. Mitochondrial DNA deficient Rho-0 variant of MDA-MB-231 cells, generated by culture in the presence of 1 mM sodium pyruvate, 1 mM uridine, and 2.5 micromolar ethidium bromide over a period of seven weeks, were nearly completely resistant to BITC-mediated ROS generation and apoptosis induction. The Rho-0 MDA-MB-231 cells also resisted BITC-mediated mitochondrial translocation (activation) of multidomain proapoptotic protein Bax. Biochemical assays revealed inhibition of complex III-linked ubiquinol cytochrome c reductase activity in BITC-treated MDA-MB-231 cells as early as 1 h of treatment. The BITC treatment caused activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which function upstream of Bax activation in apoptotic response to various stimuli. Pharmacological inhibition of both JNK and p38 MAPK conferred partial yet significant protection against BITC-induced apoptosis. Activation of JNK and p38 MAPK resulting from BITC exposure was abolished by overexpression of catalase. The BITC-mediated conformational change of Bax was markedly suppressed by ectopic expression of catalytically inactive mutant of JNK kinase 2 [JNKK2(AA)], an upstream kinase in JNK signal transduction pathway. In conclusion, the present study offers a mechanistic model for BITC-induced apoptosis involving ROS, which are mitochondria-derived and serve to function upstream of JNK/p38 MAPK activation and Bax translocation to the mitochondria. This investigation was supported by US PHS grant CA129347, awarded by the National Cancer Institute.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 15.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO