Abstract 3664: Silibinin induces cell cycle arrest and apoptotic death via targeting hedgehog signaling in basal cell carcinoma cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Skin cancer is the most common malignancy in the United States with the number of incidences persistently rising due to the depletion of the ozone layer and lack of adequate solar protection. Non-melanoma skin cancer (NMSC), composed of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), encompasses the vast majority of skin cancers with over 2 million new cases each year. Silibinin, an active bioflavanone was previously shown to have major protective effect against ultraviolet B radiation (UVB)-induced SCC through promoting DNA damage repair as well as inhibiting inflammation and angiogenesis. To date, however, the efficacy of silibinin has not been tested against BCC, which alone accounts for about 80% of NMSC incidences. BCC mostly causes disfigurement, pain and morbidity, and warrants greater attention for its prevention and treatment. The hallmark of BCC has been the constitutive activation of hedgehog (Hh) signaling pathway, mostly through the loss of function mutations in the patched (PTCH) gene. In the present study, we utilized ASZ001 cell line, derived from a mouse BCC tumor induced by UV-radiation in a PTCH1 heterozygous mouse, to evaluate the anti-cancer efficacy of silibinin against BCC. ASZ001 cells lack both copies of the PTCH1 gene and closely resembles the PTCH1 status in human BCC. We treated ASZ001 cells with either DMSO (control) or various doses of silibinin (25-100 μM), and performed trypan blue exclusion, clonogenic, apoptosis and cell cycle distribution assays. Results showed that silibinin treatment (25-100 μM) decreases the total cell number by 38-51% (p<0.001), 67-84% (p<0.001) and 40-95% (p<0.05-p<0.001) after 24, 48 and 72 h of treatment. Clonogenic assay further confirmed the decrease in cell viability by silibinin, and we observed 18% (p<0.05), 92% (p<0.001) and 100% (p<0.001) decrease in colony formation with 25, 50 and 100 μM doses of silibinin, respectively, in ASZ001 cells. Trypan blue exclusion assay showed that silibinin treatment (25-100 μM) also increased the cell death by 2 folds (p<0.05), 3 folds (p<0.001), and 4 folds (p<0.001) after 24, 48 and 72 h of treatment while annexin/PI staining confirmed that silibinin-induced cell death in ASZ001 cells is mainly apoptotic in nature. FACS analyses revealed that silibinin treatment significantly modulated the cell cycle distribution in ASZ001 cells, and we observed an S-phase arrest at 50 μM silibinin dose and a G2M arrest at 100 μM silibinin dose after 24 and 48 h of treatment. Molecular studies showed that silibinin treatment strongly decreases the expression of hedgehog signaling transcriptional factor Gli1, which is typically overexpressed in BCC and is known to regulate the expression of several oncogenes. Together, these results provide the first evidence of silibinin's efficacy against BCC cells, and suggest that silibinin could be useful in the prevention and/or treatment of both types of NMSCs. Citation Format: Cynthia Tilley, Gagan Deep, Chapla Agarwal, Rajesh Agarwal. Silibinin induces cell cycle arrest and apoptotic death via targeting hedgehog signaling in basal cell carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3664. doi:10.1158/1538-7445.AM2013-3664