Recent studies have shown that
silibinin induces p21/Cip1 and p27/Kip1 and G1 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 G1 arrest. Silencing p21 and p27 individually by RNA interference showed marked reversal in G1 arrest; however, their simultaneous ablation showed additional reversal of G1 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 G1 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 G1 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.