Benzyl Isothiocyanate Targets Mitochondrial Respiratory Chain to Trigger Reactive Oxygen Species-dependent Apoptosis in Human Breast Cancer Cells

Shivendra V. Singh,Anna A. Powolny,Dong Xiao

doi:10.1074/jbc.m802529200

Abstract

Benzyl isothiocyanate (BITC), a dietary cancer chemopreventive agent, causes apoptosis in MDA-MB-231 and MCF-7 human breast cancer cells, 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. The BITC-induced ROS production and apoptosis were significantly inhibited by overexpression of catalase and Cu,Zn-superoxide dismutase and pharmacological inhibition of the mitochondrial respiratory chain. The mitochondrial DNA-deficient Rho-0 variant of MDA-MB-231 cells was nearly completely resistant to BITC-mediated ROS generation and apoptosis. The Rho-0 MDA-MB-231 cells also resisted BITC-mediated mitochondrial translocation (activation) of Bax. Biochemical assays revealed inhibition of complex III activity in BITC-treated MDA-MB-231 cells as early as at 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)). Interestingly, a normal human mammary epithelial cell line was resistant to BITC-mediated ROS generation, JNK/p38 MAPK activation, and apoptosis. In conclusion, the present study indicates that the BITC-induced apoptosis in human breast cancer cells is initiated by mitochondria-derived ROS.

Highlights

Fraumeni syndrome, atypical hyperplasia of the breast, late age at first full-term pregnancy, early menarche, and late menopause [2,3,4,5,6]
The human mammary epithelial cell line (HMEC) were resistant toward BITC-induced apoptotic DNA fragmentation (Fig. 12B), caspase-3 activation (Fig. 12C), and Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) activation (Fig. 12D). These results indicated that BITC-mediated reactive oxygen species (ROS) generation, JNK activation, and apoptosis was selective toward breast cancer cells
The results of the present study demonstrate that ROS act as key signaling intermediates in BITC-mediated apoptosis in human breast cancer cells, which is not a cell line-specific response because the positive correlation between ROS production and cell death is observed in both MDA-MB-231 and MCF-7 cells (Figs. 1–3)

Summary

EXPERIMENTAL PROCEDURES

Reagents—BITC (purity ϳ98%) was purchased from Sigma. Reagents for cell culture, including RPMI 1640 medium, penicillin, and streptomycin antibiotic mixture, and fetal bovine serum were purchased from Invitrogen. 2 ϫ 105 cells were plated in 60-mm culture dishes, allowed to attach by overnight incubation, and exposed to Me2SO (control) or the desired concentration of BITC for the specified time intervals. Measurement of MRC Enzyme Activities—Cells were plated at a density of 1 ϫ 106 in 100-mm culture dishes, allowed to attach overnight, and treated with Me2SO or the desired concentrations of BITC for specified time periods. The reaction was initiated by the addition of ubiquinone Q2 to the reaction mixture containing 100 ␮g of lysate protein, 50 mM potassium phosphate buffer (pH 7.4), 20 mM sodium succinate, 0.1 mM EDTA, 74 ␮M dichlorophenolindophenol, 1 mM KCN, and 10 ␮M rotenone and followed for 5 min at 30 °C. Cells (2 ϫ 105) were plated in T25 flasks, allowed to attach by overnight incubation, exposed to the desired concentrations of BITC for the specified time periods, and collected by trypsinization. Difference was con- significantly lower in catalase-overexpressing MDA-MB-231 sidered significant at p Ͻ 0.05

RESULTS

DISCUSSION