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

Abstract

Previous studies have indicated that D,L-sulforaphane (SFN), a synthetic cancer chemopreventive analogue of cruciferous vegetable-derived isomer (À)-1-isothiocyanato-(4R)-(methylsulfinyl)-butane, activates a checkpoint kinase 2 (Chk2) -dependent G 2 -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 G 2 -M transition by transcriptional regulation of cyclin-dependent kinase (Cdk) inhibitor p21Cip1/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 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 SFNinduced 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 histoneassociated 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]

Sulforaphane Suppresses Polycomb Group Protein Level via a Proteasome-Dependent Mechanism in Skin Cancer Cells

“…One difference, however, was an increase in cyclin B1 in A431 and HaCaT but not in SCC-13 cells. SFN-dependent induction of cyclin B1 has been reported in the context of G 2 /M cell cycle arrest (Gamet-Payrastre et al, 2000;Herman-Antosiewicz et al, 2007). Although an increase in cyclin B1 is a pro-proliferation response, it did not seem to counter the ability of SFN to suppress proliferation of the skin cell lines.…”

“…Cip1 and altered expression of cyclin B1, cdc25b, and cdc25c in different cancer cells (Gamet-Payrastre et al, 2000;Singh et al, 2004b;Herman-Antosiewicz et al, 2007). Figure 2A shows that SFN treatment of SCC-13 cells increases the number of G 2 /M events from 11 to 34% and is associated with a concomitant decrease in G 1 events from 48 to 25%.…”

Sulforaphane Induction of p21Cip1 Cyclin-dependent Kinase Inhibitor Expression Requires p53 and Sp1 Transcription Factors and Is p53-dependent

“…In cancer cells, SFN regulation of p21 Cip1 expression is frequently p53-independent (5,18,33). This includes HT-29 colon cancer cells, which express mutant p53 (33), LnCaP prostate cancer cells, which express wild-type p53 (18), and PC-3 prostate cancer cells, which lack p53 (5).…”

Honokiol causes G0-G1 phase cell cycle arrest in human prostate cancer cells in association with suppression of retinoblastoma protein level/phosphorylation and inhibition of E2F1 transcriptional activity

“…A control nonspecific siRNA (UUCUCCGAACGUGUCACGUdTdT) was from Qiagen. The p21-and p53-targeted siRNA previously validated in our laboratory (26,27) were purchased from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. The sequences of the p21-and p53-targeted siRNA are not revealed by the manufacturer.…”