4051

We have shown previously that sulforaphane (SFN), a constituent of cruciferous vegetables such as broccoli, suppresses proliferation of PC-3 human prostate cancer cells in culture by inducing caspase-mediated apoptosis, and that growth of PC-3 xenograft in nude mice is inhibited significantly upon oral administration of SFN. In the present study, we have extended these observations and elucidated the mechanism for cell cycle arrest in SFN treated cells. A 24 h exposure of PC-3 cells to growth suppressive concentrations of SFN (20 μM) resulted in accumulation of G2/M-phase cells with a concomitant decrease in G0/G1-phase cells. SFN-induced G2/M arrest correlated with a marked decline in the expression of proteins that regulate G2/M progression, including cyclinB1 (53-92% reduction), cell division cycle 25B (Cdc25B; 48-60% reduction) and Cdc25C (42-84% reduction). Consistent with the known function of Cdc25B and Cdc25C in dephosphorylation (activation) of cyclic dependent kinase 1 (Cdk1), Tyr15 phosphorylation of Cdk1 was increased by about 2-fold in cells treated with 20 μM SFN for 16 and 24 h. Interestingly, treatment of cells with SFN also resulted in rapid and sustained phosphorylation of Cdc25C at Ser216. Phosphorylation of Cdc25C at Ser216 has been shown to create a binding site for 14-3-3- protein, which retains this dual specificity phosphatase in the cytosol. Immunoprecipitation using 14-3-3 antibodies followed by Western blotting using anti-Cdc25C antibodies revealed an increase in binding of 14-3-3 with Cdc25C in SFN treated cells when compared with control. Translocation of Cdc25C from nucleus to the cytosol upon treatment with SFN was confirmed by immunohistochemical analysis. SFN-induced phosphorylation of Cdc25C was associated with a rapid and sustained phosphorylation of checkpoint kinase 2 (Chk2) at Thr68. In conclusion, the results of the present study suggest that the mechanism for SFN-induced G2/M arrest in PC-3 cells may involve Chk2 mediated phosphorylation of Cdc25C leading to inactivation of Cdk1/cyclinB kinase complex. This investigation was supported by USPHS grant CA101753, awarded by the National Cancer Institute.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]