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Ultraviolet B (UVB) radiation is a complete carcinogen and considered the major etiologic factor for non-melanoma skin cancer (NMSC) induction and growth. Previous studies have shown that silibinin, a naturally occurring flavonoid, affords strong protection against UVB-induced DNA damage and apoptosis in the skin epidermis of animal model. In the present study, using mouse epidermal JB6 cells we investigated the mechanistic aspects of silibinin’s protective response against UVB-induced cellular damage. We observed that silibinin pretreatment inhibits apoptosis induced by moderate doses of UVB (30-50 mJ/cm2). This protective response was accompanied by a gradual reduction in UVB-induced cyclobutane pyrimidine dimer (CPD) levels, in a time-dependent manner, with upto 54% reduction in 48 hrs. Since delay in cell cycle progression provides extended time to the damaged cells for repair, we next investigated whether silibinin’s protective effect is mediated via modulation of cell cycle events in UVB-damaged cells. We found that silibinin reverses UVB-induced S phase arrest by retaining greater percentage of cells in G1 phase of the cell cycle. In order to further elucidate the mechanistic aspect of silibinin’s protective response, we performed cell synchronization assays, where cells were synchronized at G0/G1 by serum starvation and released in serum-containing media, in the presence or absence of 100 uM silibinin, and irradiated when maximal cells were at either G1 or S phase, followed by cell cycle analysis as a function of time. Whereas UVB-alone irradiated cells showed only a transient delay in cell cycle progression, silibinin pretreated cells showed a prolonged and prominent delay in progression from both G1 to S and S to G2/M phases of the cell cycle, possibly providing more time for DNA repair. In immunoblot studies, compared to UVB irradiated cells, silibinin pretreated cells showed a further upregulation of both phospho (Ser 15) and p53 as well as GADD45α, a downstream target of p53 involved in DNA repair and cell cycle arrest. Next we used p53-siRNA to identify the precise role of p53 in the cell cycle events, and observed a reduction in UVB-induced S phase arrest in p53-knock down cells and that silibinin pretreatment failed to reverse S phase arrest by UVB. In addition both UVB and silibinin failed to induce GADD45α levels in p53 knockdown cells indicating that GADD45α is regulated in a p53-dependent manner in JB6 cells. These studies suggest that the protective response of silibinin against UVB-induced damage is mainly through cell cycle delay, where p53 and GADD45α play important roles.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA