Silibinin induced growth arrest and apoptosis in oral cancer cells

Oral cancer is the fifth leading cause of cancer-related deaths in male population in Taiwan. Despite recent advances in radiotherapy and chemotherapy, the survival of patients with oral cancer has not improved significantly. Continued investigation of new chemotherapeutic agents is in urgent need.Our recent studies have shown that survivin is overexpressed in oral cancer, but not in normal tissues. It have shown that silibinin, a flavonoid isolated from Silybum marianum, inhibits survivin expression in various human cancer cell lines. We also found silibinin inhibited survivin expression in human oral cancer cell lines SAS and Ca9-22, supporting the idea that silibinin might develop as a specific strategy for cancer treatment. We further investigated the effects and mechanisms of silibinin on SAS and Ca9-22 cells. Silibinin significantly inhibited the proliferation of SAS and Ca9-22 cells in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that silibinin treatment induced apoptosis following G1 arrest. Western blotting showed silibinin treatment induced DR5, FADD, Bax, caspase-8, -9 activation and cytochrome C released . Silibinin-induced apoptosis was also inhibited by caspase 8 inhibitor (Z-LEHD-FMK) or caspase 9 inhibitor (Z-IETD-FMK) in SAS and Ca9-22 cells. The result showed that silibinin-induced apoptosis by activating intrinsic- and extrinsic-apoptosis pathway. Pretreatment of cells with N-acetyl cysteine ( NAC ) inhibibited the increase of DR5, Bax expression and PARP cleavage. ROS played an important role for silibinin-induced apoptosis.We further evaluated the potential synergistic effect of silibinin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in OSCC cell lines. Compared with either TRAIL (20 ng/ml ) or silibinin (150 μM)treated alone, co-administration of both drugs synergistrically induces apoptosis in both SAS and Ca9-22 cell lines. Our results indicated that silibinin sensitizes OSCC cells to TRAIL-mediated apoptosis. This may be regarded as a novel strategy for treatment of OSCC.