p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells

• Published in: Molecular cancer therapeutics 2007-10, Vol.6 (10), p.2696-2707
• Main Authors: Roy, Srirupa, Kaur, Manjinder, Agarwal, Chapla, Tecklenburg, Marianne, Sclafani, Robert A, Agarwal, Rajesh
• Format: Article
• Language: English
• Subjects: Antioxidants - pharmacology; Blotting, Western; Bromodeoxyuridine; cell cycle; Cell Differentiation - drug effects; Cell Proliferation - drug effects; chemoprevention; Cyclin-Dependent Kinase Inhibitor p21 - biosynthesis; Cyclin-Dependent Kinase Inhibitor p27 - biosynthesis; G1 Phase - drug effects; Humans; Immunoprecipitation; Luciferases - metabolism; Male; Phosphorylation - drug effects; Promoter Regions, Genetic; prostate cancer; Prostatic Neoplasms - drug therapy; Prostatic Neoplasms - metabolism; Prostatic Neoplasms - pathology; Proteasome Endopeptidase Complex - drug effects; Proteasome Endopeptidase Complex - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Small Interfering - pharmacology; S-Phase Kinase-Associated Proteins - antagonists & inhibitors; S-Phase Kinase-Associated Proteins - genetics; S-Phase Kinase-Associated Proteins - metabolism; silibinin; Silybin; Silybum marianum; Silymarin - pharmacology
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.MCT-07-0104

Recent studies have shown that silibinin induces p21/Cip1 and p27/Kip1 and G 1 arrest in different prostate cancer cells irrespective of p53 status; however, biological significance and mechanism of such induction have not been studied. Here, using two different prostate cancer cell lines DU145 and 22Rv1, representing androgen-independent and androgen-dependent stages of malignancy, first we investigated the importance of p21 and p27 induction in silibinin-mediated G 1 arrest. Silencing p21 and p27 individually by RNA interference showed marked reversal in G 1 arrest; however, their simultaneous ablation showed additional reversal of G 1 arrest in 22Rv1 but not DU145 cells. These results suggest that whereas relative importance of these molecules might be cell line specific, their induction by silibinin is essential for its G 1 arrest effect. Next, studies were done to examine mechanisms of their induction where cycloheximide-chase experiments showed that silibinin increases p21 and p27 protein half-life. This effect was accompanied by strong reduction in Skp2 level and its binding with p21 and p27 together with strong decrease in phosphorylated Thr 187 p27 without considerable change in proteasomal activity, suggesting a posttranslational mechanism. Skp2 role was further elucidated using Skp2-small interfering RNA–transfected cells, where decreased G 1 arrest and attenuated Cip/Kip induction were observed with silibinin treatment. Further, silibinin caused a marked increase in p21 and p27 mRNA levels together with an increase in their promoter activity, also indicating a transcriptional mechanism. Together, our results for the first time identify a central role of p21 and p27 induction and their regulatory mechanism in silibinin-mediated cell cycle arrest. [Mol Cancer Ther 2007;6(10):2696–707]