P21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells
P21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells
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DOI：
10.1158/1535-7163.mct-07-0104
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发表时间：
2007-10-01
影响因子：
5.7
通讯作者：
Agarwal, Rajesh
中科院分区：
文献类型：
--
作者：
Roy, Srirupa;Kaur, Manjinder;Agarwal, Rajesh
最近的研究表明，水飞蓟宾诱导p21/Cip 1和p27/Kip 1和G，逮捕无关的前列腺癌细胞，而不管p53的状态，但是，这种诱导的生物学意义和机制还没有研究。在这里，使用两种不同的前列腺癌细胞系DU 145和22130，代表雄激素非依赖性和雄激素依赖性阶段的恶性肿瘤，首先，我们研究了p21和p27诱导在水飞蓟宾介导的G1阻滞的重要性。通过RNA干扰单独沉默p21和p27显示了G，阻滞的显著逆转;然而，它们的同时消融显示了22 Rv 1而不是DU 145细胞中G，阻滞的额外逆转。这些结果表明，而这些分子的相对重要性可能是细胞系特异性的，它们的诱导水飞蓟宾是必不可少的，其G1逮捕效果。接下来，进行研究以检查它们的诱导机制，其中环己酰亚胺追踪实验表明水飞蓟宾增加p21和p27蛋白半衰期。这种效应伴随着Skp 2水平的强烈降低及其与p21和p27的结合，以及磷酸化Thr（187）p27的强烈降低，而蛋白酶体活性没有显著变化，表明了翻译后机制。使用Skp 2-小干扰RNA转染的细胞进一步阐明了Skp 2的作用，其中用水飞蓟宾处理观察到降低的G、阻滞和减弱的Cip/Kip诱导。此外，水飞蓟宾引起p21和p27 mRNA水平的显着增加，以及它们的启动子活性的增加，也表明了转录机制。总之，我们的研究结果首次确定了p21和p27诱导及其调控机制在水飞蓟宾介导的细胞周期阻滞中的核心作用。
Recent studies have shown that silibinin induces p21/Cip 1 and p27/Kip1 and G, arrest indifferent 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 DU 145 and 22130, representing androgen-independent and androgen-dependent stages of malignancy, first we investigated the importance of p21 and p27 induction in silibininmediated G, arrest. Silencing p21 and p27 individually by RNA interference showed marked reversal in G, arrest; however, their simultaneous ablation showed additional reversal of G, 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, 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, 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.