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

摘要

We tested whether novel CYP11A1-derived vitamin Dsub3/sub- and lumisterol-hydroxyderivatives, including 1,25(OH)sub2/subDsub3/sub, 20(OH)Dsub3/sub, 1,20(OH)sub2/subDsub3/sub, 20,23(OH)sub2/subDsub3/sub, 1,20,23(OH)sub3/subDsub3/sub, lumisterol, 20(OH)Lsub3/sub, 22(OH)Lsub3/sub, 20,22(OH)sub2/subLsub3/sub, and 24(OH)Lsub3/sub, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24?h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200?mJ/cmsup2/sup, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100?nM showed a dose-dependent reduction in oxidant formation. At 100?nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50–200?mJ/cmsup2/sup of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR , HO-1 , CAT , SOD1, and SOD2 , with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)sub2/subDsub3/sub or CYP11A1-derived vitamin Dsub3/sub- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin Dsub3/sub and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.