We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G₂/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3β. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G₂/M arrest. HCT116 human colon cancer-derived Chk2^-/- cells were significantly more resistant to G₂/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G₂/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.

The present study reports a novel response to sulforaphane, a highly promising anticancer constituent of several edible cruciferous vegetables, in PC-3 and LNCaP human prostate cancer cells involving induction of autophagy. Exposure of PC-3 and LNCaP cells to sulforaphane resulted in several specific features characteristic of autophagy, including appearance of membranous vacuoles in the cytoplasm as revealed by transmission electron microscopy and formation of acidic vesicular organelles as revealed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. The sulforaphane-induced autophagy was associated with up-regulation, processing, and recruitment to autophagosomes of microtubule-associated protein 1 light chain 3 (LC3), which is a mammalian homologue of the yeast autophagy regulating protein Apg8/Aut7p. Treatment of cells with a specific inhibitor of autophagy (3-methyladenine) attenuated localization of LC3 to autophagosomes but exacerbated cytosolic release of cytochrome c as well as apoptotic cell death as revealed by analysis of subdiploid fraction and cytoplasmic histone–associated DNA fragmentation. In conclusion, the present study indicates that induction of autophagy represents a defense mechanism against sulforaphane-induced apoptosis in human prostate cancer cells. To the best of our knowledge, the present study is the first published report to convincingly document induction of autophagy by an isothiocyanate class of dietary chemopreventive agent. (Cancer Res 2006; 66(11): 5828-35)

Molecular mechanism of cell cycle arrest caused by diallyl trisulfide (DATS), a garlic-derived cancer chemopreventive agent, has been investigated using PC-3 and DU145 human prostate cancer cells as a model. Treatment of PC-3 and DU145 cells, but not a normal prostate epithelial cell line (PrEC), with growth suppressive concentrations of DATS caused enrichment of the G₂–M fraction. The DATS-induced cell cycle arrest in PC-3 cells was associated with increased Tyr¹⁵ phosphorylation of cyclin-dependent kinase 1 (Cdk1) and inhibition of Cdk1/cyclinB1 kinase activity. The DATS-treated PC-3 and DU145 cells also exhibited a decrease in the protein level of Cdc25C and an increase in its Ser²¹⁶ phosphorylation. The DATS-mediated decrease in protein level and Ser²¹⁶ phosphorylation of Cdc25C as well as G₂–M phase cell cycle arrest were significantly attenuated in the presence of N-acetylcysteine implicating reactive oxygen species (ROS) in cell cycle arrest caused by DATS. ROS generation was observed in DATS-treated PC-3 and DU145 cells. DATS treatment also caused an increase in the protein level of Cdk inhibitor p21, but DATS-induced G₂–M phase arrest was not affected by antisense-mediated suppression of p21 protein level. In conclusion, the results of the present study indicate that DATS-induced G₂–M phase cell cycle arrest in human prostate cancer cells is caused by ROS-mediated destruction and hyperphosphorylation of Cdc25C.