Sulforaphane [SFN; 1-isothiocyanato-4-(methylsulfinyl)-butane] is a cruciferous-vegetable derived chemopreventive agent that has generated a great deal of research interest due to its interesting biological effects including induction of phase 2 carcinogen detoxification enzymes and prevention of chemically-induced cancers in animal models. More recent studies including those from our laboratory have revealed that SFN can inhibit proliferation of cultured cancer cells by causing G2-M phase cell cycle arrest and apoptosis induction, but the mechanism of these effects are not fully understood. In the present study, we have investigated the mechanism of apoptosis induction by SFN using PC-3 and LNCaP human prostate cancer cells as a model. The LNCaP cell line containing wild type p53 was relatively more sensitive to SFN-induced apoptosis compared with p53 deficient PC-3 cells. The SFN treatment caused stabilization of p53 protein in LNCaP cells, and transient transfection with p53 specific siRNA significantly protected against the cell death caused by higher concentrations of SFN. The SFN treatment caused conformational change (activation) of Bax in both cell lines that was more pronounced in the LNCaP cell line than in PC-3 cells. Treatment of PC-3 cells with SFN resulted in a marked decrease in the levels of inhibitor of apoptosis (IAP) family proteins including cIAP1, cIAP2 and XIAP, which was accompanied by cytoplasmic sequestration of p65-nuclear factor κB (NFκB). The effect of SFN on levels of cIAP1, cIAP2 and XIAP proteins was biphasic in LNCaP cells with initial induction followed by a down-regulation in the expression. Luciferase reporter gene assay revealed a biphasic response for the effect of SFN on transcriptional activation of NFκB in LNCaP cells. The HCT116 derived XIAP null cells exhibited increased sensitivity to SFN-induced apoptosis compared with wild type HCT116 cells. The SFN treatment also caused an increase in the protein level of Apaf-1, which was accompanied by transcriptional activation of E2F1. Transcriptional activity of E2F1 is regulated by checkpoint kinase 2 (Chk2). However, the SFN-mediated induction of Apaf-1 in PC-3 cells was not affected by Chk2 protein knockdown. Moreover, SFN-mediated induction of Apaf-1 was maintained in HCT116 derived Chk2 null cells. In conclusion, the present study indicates that the SFN-induced apoptosis in human prostate cancer cells is regulated by p53 stabilization, Bax activation, down-regulation of IAP family proteins and Chk2-independent induction of Apaf-1. This study was supported in part by NCI grants CA101753 and CA115498.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]