A Flavonoid Antioxidant, Silymarin, Inhibits Activation of erbBl Signaling and Induces Cyclin-dependent Kinase Inhibitors, Gj Arrest, and Anticarcinogenic Effects in Human Prostate Carcinoma DU145 Cells1

Prostate cancer (PCA) is the most common nonskin malignancy and the second leading cause of cancer deaths in United States males. One prac tical and translational approach to control PCA is to define a mechanismbased anticarcinogenic agent(s). Recently, we showed that silymarin, a flavonoid antioxidant isolated from milk thistle, possesses exceptionally high to complete protective effects against experimentally induced tumorigenesis. Because the epidermal growth factor receptor (erbBl) and other members of the erbB family have been shown to play important roles in human PCA, efforts should be directed to identify inhibitors of this pathway for PCA intervention. In this study, we assessed whether silyma rin inhibits erbBl activation and associated downstream events and mod ulates cell cycle regulatory proteins and progression, leading to growth inhibition of human prostate carcinoma DU145 cells. Treatment of serumstarved cells with silymarin resulted in a significant inhibition of trans forming growth factor «-mediated activation of erbBl but no change in its protein levels. Silymarin treatment of cells also resulted in a significant decrease in tyrosine phosphorylation of an immediate downstream target of erbBl, the adapter protein SHC, together with a decrease in its binding to erbBl. In the studies analyzing cell cycle regulatory molecules, silyma rin treatment of cells also resulted in a significant induction of cyclindependent kinase inhibitors (CDKIs) Cipl/p21 and Kipl/p27, concomi tant with a significant decrease in CDK4 expression, but no change in the levels of CDK2 and CDK6 and their associated cyclins E and Dl, respec tively. Cells treated with silymarin also showed an increased binding of CDKIs with CDKs, together with a marked decrease in the kinase activity of CDKs and associated cyclins. In additional studies, treatment of cells grown in 10% serum with anti-epidermal growth factor receptor mono clonal antibody clone 225 or different doses of silymarin also resulted in significant inhibition of constitutive tyrosine phosphorylation of both erbBl and SHC but no change in their protein levels. Furthermore, whereas silymarin treatment resulted in a significant increase in the protein levels of both Cipl/p21 and Kipl/p27, monoclonal antibody 225 showed an increase only in Kipl/p27. These findings suggest that silyma rin also inhibits constitutive activation of erbBl and that the observed effect of silymarin on an increase in CDKI protein levels is mediated via inhibition of erbBl activation only in the case of Kipl/p27; however, additional pathways independent of inhibition of erbBl activation are possibly responsible for the silymarin-caused increase in Cipl/p21 in DU145 cells. In other studies, silymarin treatment also induced a G, arrest in the cell cycle progression of 1)1 145 cells and resulted in a highly significant to complete inhibition of both anchorage-dependent and an chorage-independent growth of DU145 cells in a doseand time-dependent manner. Taken together, these results suggest that silymarin may exert a strong anticarcinogenic effect against PCA and that this effect is likely to Received 11/11/97; accepted 3/4/98. The costs of publication of this article were defrayed in part hy the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in pan by United States Public Health Service Grant CA 64514 (to R. A.), a Prostate Cancer pilot grant from Case Western Reserve University Cancer Center through the funds provided by United States Public Health Service Grant P30-CA 43703. and an Ohio Cancer Research Associates grant. 2 To whom requests for reprints should be addressed, at the Center for Cancer Causation and Prevention. AMC Cancer Research Center. 1600 Pierce Street. Denver. CO 80214. Phone: (303)239-3580; Fax: (303)239-3560; E-mail: [email protected]. involve impairment of erbBl-SHC-mediated signaling pathway, induction of CDKIs, and a resultant G, arrest.