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Research Articles: Therapeutics, Targets, and Development

Activation of a novel ataxia-telangiectasia mutated and Rad3 related/checkpoint kinase 1–dependent prometaphase checkpoint in cancer cells by diallyl trisulfide, a promising cancer chemopreventive constituent of processed garlic

Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Requests for reprints: Shivendra V. Singh, Department of Pharmacology, University of Pittsburgh Cancer Institute, Hillman Cancer Center Research Pavilion, 2.32A, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3263; Fax: 412-623-7828. E-mail: singhs{at}upmc.edu

Abstract

Diallyl trisulfide (DATS), a cancer chemopreventive constituent of garlic, inhibits growth of cancer cells by interfering with cell cycle progression, but the mechanism is not fully understood. Here, we show the existence of a novel ataxia-telangiectasia mutated and Rad3 related (ATR)/checkpoint kinase 1 (Chk1)–dependent checkpoint partially responsible for DATS-mediated prometaphase arrest in cancer cells, which is different from the recently described irradiation–induced mitotic exit checkpoint. The PC-3 human prostate cancer cells synchronized in prometaphase by nocodazole treatment and released to DATS-containing medium remained arrested in prometaphase, whereas the cells released to normal medium exited mitosis and resumed cell cycle. The mitotic arrest was maintained even after 4 h of culture of DATS-treated cells (4-h treatment) in drug-free medium. The DATS-arrested mitotic cells exhibited accumulation of anaphase-promoting complex/cyclosome (APC/C) substrates cyclin A and cyclin B1 and hyperphosphorylation of securin, which was accompanied by increased phosphorylation of the APC/C regulatory subunits Cdc20 and Cdh1. The DATS-mediated accumulation of cyclin B1 and hyperphosphorylation of securin, Cdc20, and Cdh1 were partially but markedly attenuated by knockdown of Chk1 or ATR protein. The U2OS osteosarcoma cells expressing doxycycline-inducible kinase dead ATR were significantly more resistant not only to DATS-mediated prometaphase arrest but also to the accumulation of cyclin B1 and hyperphosphorylation of securin, Cdc20, and Cdh1 compared with cells expressing wild-type ATR. However, securin protein knockdown failed to rescue cells from DATS-induced prometaphase arrest. In conclusion, the present study describes a novel signaling pathway involving ATR/Chk1 in the regulation of DATS-induced prometaphase arrest. [Mol Cancer Ther 2007;6(4):1249–61]

Grant support: U.S. Public Health Service grants CA113363 and CA115498, awarded by the National Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Current address for A. Herman-Antosiewicz: Department of Molecular Biology, University of Gdask, Kladki 24, 80-822 Gdansk, Poland.

¹ A. Herman-Antosiewicz, and S.V. Singh, unpublished observations.

Received 8/ 9/06; revised 1/24/07; accepted 2/26/07.