Abstract 5650: Silibinin inhibits epithelial to mesenchymal transition in prostate cancer cells: Role of E-cadherin and beyond

Prostate cancer (PCA) is second leading cause of cancer-related deaths in US men. High morbidity and mortality in PCA patients are due to its metastatic spread to distant organs. Epithelial to mesenchymal transition (EMT) in PCA cells is considered a pre-requisite for acquiring advanced migratory/invasive phenotype, and for subsequent metastasis. Even though multiple signaling mechanisms and cellular changes are involved in EMT, loss of E-cadherin is considered as the single most important biological event in PCA cells for acquiring an invasive mesenchymal phenotype. Accordingly, we hypothesized that promoting E-cadherin expression in PCA cells using non-toxic phytochemicals, such as silibinin, would prevent EMT and subsequent metastasis. Recently, we have established strong efficacy of silibinin against PCA cells and currently it is in phase II clinical trial in PCA patients, but its efficacy and mechanism against EMT in PCA cells are largely unknown. Results of present study showed that silibinin strongly inhibits migratory potential of quiescent PC3 and PC3MM2 PCA cells in wound healing assay (P less than 0.001); and invasive potential of PC3 (by 11-66%), PC3MM2 (by 11-61%) and C4-2B (by 32-71%) cells in transwell matrigel invasion assay. Importantly, cell viability assay confirmed that anti-invasive effect of silibinin was not due to its cytotoxicity. Immunoblot analysis showed that silibinin increases E-cadherin expression in PC3 cells in a dose and time dependent manner, which was at cellular membrane as evidenced by sub-cellular fractional and confocal analyses. Silibinin treatment also decreased the level of transcriptional repressors (Snail1, Slug and ZEB1) and increased E-cadherin mRNA expression. Silibinin treatment also decreased the level of phosphorylated Src and Hakai (an E-3 ubiquitin ligase) suggesting post-translational stabilization of E-cadherin. In other studies, silibinin decreased nuclear and cytoplasmic level of beta-catenin but not in cellular membrane. Morphological examination supported these molecular alterations as silibinin treated PC3 cells were more adherent. Similar E-cadherin increase, beta-catenin decrease and morphological changes with silibinin were also observed in PC3MM2 and C4-2B cells. Next, we generated PC3 cells with stable knock-down of E-cadherin expression (ShEC-PC3) and respective control cells (Sh-PC3). Employing these cells, we found that the anti-migratory/anti-invasive potential of silibinin is only partially dependent on E-cadherin. Since molecules, other than E-cadherin, affected by silibinin treatment (beta-catenin, Snail1, Slug and ZEB1) could also directly influence EMT-related changes, our findings suggest multiple molecular targets of silibinin in its anti-EMT and anti-migratory/anti-invasive potential. Overall, the results of present study suggest silibinin usefulness against PCA metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5650.