Abstract
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 G(2)/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 14-3-3beta. 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 G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/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 G(2)/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.
Highlights
Epidemiological studies have revealed an inverse correlation between the dietary intake of cruciferous vegetables and the risk for certain types of cancers, including prostate cancer [1,2,3,4,5]
Previous studies have shown that SFN-treated HT29 colon cancer cells and Jurkat T-leukemia cells are arrested in the G2/M phase [24, 27], which may be of importance in the anticarcinogenic effect of SFN, but no attempts were made to define the mechanism of the cell cycle arrest
We demonstrate that SFN treatment causes an irreversible G2/M phase cell cycle arrest in PC-3 cells which is associated with a marked decrease in the expression of key G2/M-regulating proteins, including cyclin B1, cell division cycle 25B (Cdc25B) and Cdc25C
Summary
Epidemiological studies have revealed an inverse correlation between the dietary intake of cruciferous vegetables and the risk for certain types of cancers, including prostate cancer [1,2,3,4,5]. We provide evidence to indicate that cell cycle arrest in SFN-treated PC-3 cells is caused by the generation of reactive oxygen species (ROS) and ataxia telangiectasia-mutated (ATM)/checkpoint kinase 2 (Chk2)-mediated phosphorylation of Cdc25C at Ser-216.
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