Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C.
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.
Singh
Shivendra V
SV
Department of Pharmacology and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA. singhs@msx.upmc.edu
Herman-Antosiewicz
Anna
A
Singh
Ajita V
AV
Lew
Karen L
KL
Srivastava
Sanjay K
SK
Kamath
Ravindra
R
Brown
Kevin D
KD
Zhang
Lin
L
Baskaran
Rajasekaran
R
D021581:Active Transport, Cell Nucleus; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D017879:Isothiocyanates; D008938:Mitosis / Q000187:drug effects*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D020687:cdc25 Phosphatases / Q000378:metabolism*
D016588:Anticarcinogenic Agents; D018797:Cell Cycle Proteins; D017879:Isothiocyanates; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013454:Sulfoxides; D013861:Thiocyanates; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases; C016766:sulforaphane