Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line Anna Herman-Antosiewicz, 1 Hui Xiao, 2 Karen L. Lew, 2 and Shivendra V. Singh 2 1 Department of Molecular Biology, University of Gdan ´sk,Gdan ´sk, Poland and 2 Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Abstract Previous studies have indicated that D,L-sulforaphane (SFN), a synthetic cancer chemopreventive analogue of cruciferous vegetable-derived isomer (À)-1-isothiocya- nato-(4R )-(methylsulfinyl)-butane, activates a checkpoint kinase 2 (Chk2)–dependent G 2 -M phase cell cycle arrest inp53-deficienthumanprostatecancercells.Becausep53 is a downstream target of Chk2 kinase and known to regulate G 2 -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 p21inSFN-inducedcellcyclearrestusingwild-typep53– expressing cell line LNCaP. The SFN treatment caused a modestincreaseinSphasefractionandamarkedincrease in G 2 -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 G 2 -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 Ser 10 phosphorylation of histone H3, a sensitive marker for mitotic cells. The SFN treatment increased activating phosphorylation of Chk2 (Thr 68 ) 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] Introduction Epidemiologic studies continue to support the premise that dietary intake of cruciferous vegetables may lower the risk of different types of malignancies including prostate cancer (1 – 4). Anticarcinogenic effect of cruciferous vege- tables is attributed to organic isothiocyanates, which are generated due to myrosinase-mediated hydrolysis of corresponding glucosinolates (5, 6). Broccoli is a rather rich source of the isothiocyanate compound (À)-1-isothio- cyanato-(4R )-(methylsulfinyl)-butane (L-SFN). The L-SFN and its synthetic analogue D,L-sulforaphane (SFN) have generated a great deal of research interest due to their remarkable anticancer effects (7 – 14). For instance, L-SFN or SFN has been shown to afford significant protec- tion against chemically induced cancer in animal models (8, 11 – 13). Cancer chemoprevention by L-SFN or SFN has been observed against 9,10-dimethyl-1,2-benzanthracene – induced mammary cancer in rats, azoxymethane-induced colonic aberrant crypt foci in rats, benzo[a ]pyrene-induced forestomach cancer in mice, and 7,12-dimethylbenz(a )an- thracene/12-O -tetradecanoylphorbol 13-acetate – induced skin tumorigenesis in mice (8, 11 – 13). More recently, SFN administration was shown to significantly inhibit lung metastasis induced by B16F-10 melanoma cells in C57BL/6 mice (14). Evidence is accumulating to indicate that SFN can suppress proliferation of cultured cancer cells by causing cell cycle arrest and apoptosis induction (15 – 24). Interest- ingly, normal epithelial cells, including a normal human prostate epithelial cell line PrEC and an immortalized normal human bronchial epithelial cell line BEAS-2B, are significantly more resistant to apoptosis induction by SFN compared with cancer cells (21). Consistent with these cellular results, oral administration of SFN significantly retards growth of PC-3 human prostate cancer xenografts in nude mice without causing weight loss or any other side effects (18). Inhibition of histone deacetylase activity by SFN has also been observed in human benign prostate hyperplasia epithelial cell line BPH-1 and PC-3 and LNCaP human prostate cancer cells (23). An understanding of the mechanism(s) by which SFN causes cell cycle arrest and apoptosis induction is critical for its further clinical development because this knowledge Received 12/28/06; revised 3/26/07; accepted 3/30/07. 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. 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@upmc.edu Copyright C 2007 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-06-0807 1673 Mol Cancer Ther 2007;6(5). May 2007 Research. on October 7, 2021. © 2007 American Association for Cancer mct.aacrjournals.org Downloaded from