274 - Protective effects of novel derivatives of vitamin D3 and lumisterol against UVB-induced damage in human keratinocytes involve activation of Nrf2 and P53 defense mechanisms

Abstract

Objective Ultraviolet B (UVB) plays an important role in the photo-damage of skin through formation of cyclobutane pyrimidine dimers (CPD), the primary mutagenic DNA photoproducts, and regulation of the p53 protective responses against genotoxic damage. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor regulating antioxidant genes, also has a protective role against photo-oxidative damage of the skin. Since the protective role of the active form of vitamin D (1,25(OH)2D3) against UVB induced damage is recognized, we tested whether novel CYP11A1-derived D3-hydroxyderivatives and lumisterol including its hydroxy-derivatives can protect against UVB-induced damage in human keratinocytes. Materials and Methods 1,25(OH)2D3, biochemically generated 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, and 1,20,23(OH)3D3, synthetic lumisterol and its hydroxy-derivatives generated by the action of CYP11A1 including 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3), were used for testing. Cells were treated with compounds for 24 h pre-and post-UVB irradiation at doses of 25, 50, 75, and 200 mJ/cm2. Oxidant formation was determined by the DCFA-DA method. DNA damage was assessed using the comet assay. Nuclear expressions of CPD, phospho-p53 and Nrf2 were assayed by immunofluorescence and western blotting, respectively. Results Trea tment with compounds at concentrations 10-9, 10-8, and 10-7 M showed a dose-dependent reduction of oxidant formation in comparison to non-treated cells. Compounds (10-7 M) were also able to protect against DNA damage and/or induce DNA repair for cells irradiated with UVB (50 mJ/cm2) by reducing CPD levels and the tail moment of comet images. They stimulated p53 phosphorylation at Ser-15 and increased its concentration in the nucleus, and enhanced Nrf2 translocation to the nucleus. Conclusion 1,25(OH)2D3, CYP11A1-derived D3-hydroxyderivatives as well as lumisterol and its hydroxy-derivatives protect keratinocytes against UVB-induced damage via activation of DNA damage protective pathways that include Nrf2 activation and p53-phosphorylation. These compounds represent promising anti-photodamaging agents.