The Cancer Preventive Flavonoid Silibinin Causes Hypophosphorylation of Rb/p107 and Rb2/p130 Via Modulation of Cell Cycle Regulators in Human Prostate Carcinoma DU145 Cells

Abstract

Phosphorylation status of retinoblastoma (Rb) and related proteins is important to drive cell cycle progression. In hyperphosphorylated state, they are growth stimulatory, but their hypophosphorylation is growth inhibitory. Here we assessed whether silibinin causes hypophosphorylation of Rb-related proteins as its growth inhibitory response in human prostate cancer (PCA) DU145 cells. Silibinin treatment of cells resulted in a strong increase (up to 2.3-and 5.4-fold) in the levels of hypophosphorylated Rb/p107 and Rb2/p130, respectively, but a strong decrease (91, 78 and 45%) in protein levels of transcription factors E2F3, E2F4 and E2F5, respectively. In the studies analyzing whether this effect of silibinin is via modulation of cell cycle regulators, silibinin-treated cells showed a strong increase (up to 13- and 6-fold) in Cip1/p21 and Kip1/p27 levels, respectively. Silibinin treatment also resulted in 90 and 70% decrease in CDK4 and CDK2 levels, respectively, but did not alter the protein levels of cyclin D1 and cyclin E. Consistent with its effect on G1 cell cycle regulators, silibinin treated cells exhibited a strong G1 arrest, almost complete growth inhibition, and morphological changes suggestive of differentiation. Together, these results suggest that silibinin caused hypophosphorylation of Rb-related proteins may in part be responsible for its cancer preventive and anti-carcinogenic efficacy in different cancer models including PCA. Key Words:Silibinin, Retinoblastoma, Cell cycle, CDKI, CDK, Cyclin