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Research Articles: Therapeutics, Targets, and Development

Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line

¹ Department of Molecular Biology, University of Gdask, Gdask, Poland and ² Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

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

Abstract

Previous studies have indicated that D,L-sulforaphane (SFN), a synthetic cancer chemopreventive analogue of cruciferous vegetable-derived isomer (–)-1-isothiocyanato-(4R)-(methylsulfinyl)-butane, activates a checkpoint kinase 2 (Chk2)–dependent G₂-M phase cell cycle arrest in p53-deficient human prostate cancer cells. Because p53 is a downstream target of Chk2 kinase and known to regulate G₂-M transition by transcriptional regulation of cyclin-dependent kinase (Cdk) inhibitor p21^Cip1/Waf1 (p21), the present study was undertaken to determine the role of p21 in SFN-induced cell cycle arrest using wild-type p53–expressing cell line LNCaP. The SFN treatment caused a modest increase in S phase fraction and a marked increase in G₂-M fraction in LNCaP cells in a concentration- and time-dependent manner. The SFN-induced S phase arrest correlated with a reduction in protein levels of cyclin D1, cyclin E, Cdk4, and Cdk6, whereas activation of the G₂-M checkpoint was accompanied by induction of cyclin B1 and down-regulation of Cdk1 and Cdc25C protein levels. The SFN-treated LNCaP cells were also arrested in mitosis as revealed by immunofluorescence microscopy and increased Ser¹⁰ phosphorylation of histone H3, a sensitive marker for mitotic cells. The SFN treatment increased activating phosphorylation of Chk2 (Thr⁶⁸) that was accompanied by induction of p53 and p21. The SFN-induced mitotic arrest was statistically significantly increased by small interfering RNA–based knockdown of p21. However, p21 protein knockdown did not have any appreciable effect on SFN-induced cytoplasmic histone-associated DNA fragmentation (apoptosis). In conclusion, the present study indicates that induction of p21 protects against SFN-induced mitotic arrest in LNCaP cells. [Mol Cancer Ther 2007;6(5):1673–81]

Grant support: National Cancer Institute/USPHS grants CA115498 and CA101753.

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: A. Herman-Antosiewicz and H. Xiao contributed equally to this work.

Received 12/28/06; revised 3/26/07; accepted 3/30/07.

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