Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line.
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 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 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 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.
Herman-Antosiewicz
Anna
A
Department of Molecular Biology, University of Gdask, Gdask, Poland.
Xiao
Hui
H
Lew
Karen L
KL
Singh
Shivendra V
SV
D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D016195:G2 Phase; D015972:Gene Expression Regulation, Neoplastic; D006657:Histones / Q000378:metabolism; D006801:Humans; D017879:Isothiocyanates; D008297:Male; D008856:Microscopy, Fluorescence; D008938:Mitosis; D011471:Prostatic Neoplasms / Q000188:drug therapy*; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D016588:Anticarcinogenic Agents; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D006657:Histones; D017879:Isothiocyanates; D013454:Sulfoxides; C495901:TP53 protein, human; D013861:Thiocyanates; D016159:Tumor Suppressor Protein p53; C016766:sulforaphane