RT Journal Article SR Electronic T1 Inhibition of Retinoblastoma Protein (Rb) Phosphorylation at Serine Sites and an Increase in Rb-E2F Complex Formation by Silibinin in Androgen-dependent Human Prostate Carcinoma LNCaP Cells: Role in Prostate Cancer Prevention 1 This work was supported in part by United States Army Medical Research and Materiel Command Prostate Cancer Program DAMD17-98-1-8588. In part, this work was done during the tenure of the authors at AMC Cancer Research Center, Denver, CO 80262.1 JF Molecular Cancer Therapeutics JO Mol Cancer Ther FD American Association for Cancer Research SP 525 OP 532 VO 1 IS 7 A1 Tyagi, Alpana A1 Agarwal, Chapla A1 Agarwal, Rajesh YR 2002 UL http://mct.aacrjournals.org/content/1/7/525.abstract AB Several studies have identified silibinin as an anticarcinogenic agent. Recently, we showed that silibinin inhibits cell growth via G1 arrest, leading to differentiation of androgen-dependent human prostate carcinoma LNCaP cells (X. Zi and R. Agarwal, Proc. Natl. Acad. Sci. USA, 96: 7490–7495, 1999). Here, we extend this study to assess the effect of silibinin on total retinoblastoma protein (Rb) levels and its phosphorylation status, levels of E2F family members, and Rb-E2F binding in LNCaP cells. Compared with controls, silibinin resulted in an increase in total Rb levels that was largely attributable to an increase in unphosphorylated Rb (up to 4.1-fold). This effect of silibinin was mainly attributable to a large decrease (70–97%) in the amount of Rb phosphorylated at specific serine sites. In other studies, silibinin showed a moderate effect on E2F1 but up to 98 and 90% decreases in E2F2 and E2F3 protein levels, respectively. Silibinin treatments also resulted in an increase in the amount of Rb binding to E2F1 (3.8-fold), E2F2 (2.2-fold), and E2F3 (2.2-fold). Cyclin-dependent kinases (CDKs), together with their catalytic subunit cyclins, phosphorylate Rb, which makes transcription factor E2Fs free from Rb-E2F complexes, resulting in cell growth and proliferation. Conversely, CDK inhibitors inhibit this phosphorylation, maintaining E2Fs bound to Rb, which causes growth inhibition. On the basis of our data showing that silibinin induces both unphosphorylated Rb levels and Rb-E2F binding, we also assessed its effect on upstream cell cycle regulators. Silibinin-treated cells showed up to 2.4- and 3.6-fold increases in Cip1/p21 and Kip1/p27 levels, respectively, and a decrease in CDK2 (80%), CDK4 (98%), and cyclin D1 (60%). Consistent with these results, silibinin showed both G1 arrest and growth inhibition. Together, these findings identify modulation of Rb levels and its phosphorylation status as a molecular mechanism of silibinin-induced neuroendocrine differentiation of human prostate carcinoma LNCaP cells and suggest that this could be a novel approach for prostate cancer prevention by silibinin.