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.

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 G 2 -M fraction. The DATS-induced cell cycle arrest in PC-3 cells was associated with increased Tyr 15 phosphorylation of cyclin-dependent kinase 1 (Cdk1) and inhibition of Cdk1/cyclinB1 kinase activity. The DATStreated PC-3 and DU145 cells also exhibited a decrease in the protein level of Cdc25C and an increase in its Ser 216 phosphorylation. The DATS-mediated decrease in protein level and Ser 216 phosphorylation of Cdc25C as well as G 2 -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 G 2 -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 G 2 -M phase cell cycle arrest in human prostate cancer cells is caused by ROS-mediated destruction and hyperphosphorylation of Cdc25C.

We have shown previously that generation of reactive oxygen species (ROS) is a critical event in G 2 -M phase cell cycle arrest caused by diallyl trisulfide (DATS), which is a highly promising anticancer constituent of processed garlic. Using DU145 and PC-3 human prostate cancer cells as a model, we now report a novel mechanism involving c-Jun NH 2 -terminal kinase (JNK) signaling axis, which is known for its role in regulation of cell survival and apoptosis, in DATS-induced ROS production. The DATS-induced ROS generation, G 2 -M phase cell cycle arrest and degradation, and hyperphosphorylation of Cdc25C were significantly attenuated in the presence of EUK134, a combined mimetic of superoxide dismutase and catalase. Interestingly, the DATS-induced ROS generation and G 2 -M phase cell cycle arrest were also inhibited significantly in the presence of desferrioxamine, an iron chelator, but this protection was not observed with iron-saturated desferrioxamine. DATS treatment caused a marked increase in the level of labile iron that was accompanied by degradation of light chain of iron storage protein ferritin. Interestingly, DATSmediated degradation of ferritin, increase in labile iron pool, ROS generation, and/or cell cycle arrest were significantly attenuated by ectopic expression of a catalytically inactive mutant of JNK kinase 2 and RNA interference of stressactivated protein kinase/extracellular signal-regulated kinase 1 (SEK1), upstream kinases in JNK signal transduction pathway. In conclusion, the present study provides experimental evidence to indicate existence of a novel pathway involving JNK signaling axis in regulation of DATS-induced ROS generation.

Professional tennis players experienced an intensified inflammatory response after the completed tournament season, which may lead to overreaching. Applying whole-body cryostimulation in conjunction with moderate-intensity training was more effective for the recovery process than the training itself. The 5-day exposure to cryostimulation twice a day ameliorated the cytokine profile, resulting in a decrease in tumor necrosis factor α and an increase in interleukin 6.

Purpose-Present study was undertaken to elucidate the mechanism of cellular responses to D,Lsulforaphane (SFN), a highly promising cancer chemopreventive agent.Methods-Mitochondrial DNA deficient Rho-0 variants of LNCaP and PC-3 cells were generated by culture in the presence of ethidium bromide. Apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation and activation of caspase-3. Immunoblotting was performed to determine the expression of apoptosis-and cell cycle-regulating proteins. Generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and cell cycle distribution were measured by flow cytometry.Results-The Rho-0 variants of LNCaP and PC-3 cells were significantly more resistant to SFNinduced ROS generation, apoptotic DNA fragmentation, disruption of MMP, cytosolic release of cytochrome c, and G2/M phase cell cycle arrest compared with corresponding wild-type cells. SFNinduced autophagy, which serves to protect against apoptotic cell death in PC-3 and LNCaP cells, was also partially but markedly suppressed in Rho-0 variants compared with wild-type cells. SFN statistically significant inhibited activities of mitochondrial respiratory chain enzymes in LNCaP and PC-3 cells.Conclusion-These results indicate, for the first time, that mitochondria-derived ROS serve to initiate diverse cellular responses to SFN exposure in human prostate cancer cells.