Activation of a novel checkpoint kinase 1-dependent pro-metaphase checkpoint by diallyl trisulfide, a cancer chemopreventive constituent of processed garlic.

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Epidemiological data indicate that increased consumption of Allium vegetables, such as garlic, may reduce the risk for different types of malignancies including cancer of the prostate. Allium vegetable derived organosulfur compounds including diallyl trisulfide (DATS) are highly effective in affording protection against cancer in animal models induced by a variety of chemical carcinogens. More recent studies have revealed that garlic constituents including DATS can suppress proliferation of cultured cancer cells by inhibiting cell cycle progression and causing apoptosis. Even though considerable progress has been made towards our understanding of the signaling pathways responsible for DATS-induced apoptosis, the mechanism by which DATS blocks cell cycle progression remains elusive. We have shown previously that DATS treatment causes G2 phase as well as mitotic arrest in PC-3 and DU145 human prostate cancer cells in association with activation of checkpoint kinase1 (Chk1), which is an intermediary of DNA damage checkpoints. The present study extends these observations and demonstrates that the DATS treated PC-3 cells are arrested mainly in pro-metaphase stage characterized by condensed chromatin, nuclear envelope disruption, and spindle microtubules formation. The DATS-arrested mitotic cells accumulated phosphorylated form of securin and cyclin B1, which are substrates of anaphase promoting complex (APC) suggesting inactivation of APC on treatment with DATS. The DATS treated PC-3 cells also exhibited hyperphosphorylation of APC components Cdc20 and Cdh1. The DATS-induced mitotic arrest, accumulation of cyclin B1 and securin phosphorylation were significantly attenuated by transient transfection with a Chk1 specific siRNA. The U2OS osteosarcoma cells expressing tetracycline-inducible kinase dead ATR, an upstream kinase responsible for activation of Chk1, were also resistant to DATS-induced pro-metaphase arrest as well as accumulation of cyclinB1 and hyperphosphorylation of securin. Even though Chk1 physically interacted with securin as revealed by immunoprecipitation-immunoblotting experiment, the DATS-induced pro-metaphase arrest was maintained in PC-3 cells transfected with securin specific siRNA. In conclusion, the present study reveals existence of a novel Chk1-dependent pro-metaphase checkpoint in human cancer cells that is strikingly different from the recently described ionizing radiation (IR)-induced checkpoint, which is Chk1-dependent but leads to accumulation of telophase cells. However, similar to the IR-induced checkpoint, the DATS-mediated pro-metaphase arrest in our model leads to increased number of multinucleated cells. This study was supported in part by NCI grants CA113363, CA115498 and CA101753.