Vitamin C protects against UV irradiation-induced apoptosis through reactivating silenced tumor suppressor genes p21 and p16 in a Tet-dependent DNA demethylation manner in human skin cancer cells.

AIM

DNA methylation plays important roles in various kinds of carcinogenesis. Vitamin C could induce Tet-dependent DNA demethylation in embryonic stem cells. Therefore, the antagonizing activity of vitamin C on ultraviolet (UV)-induced apoptosis was investigated in this study.

METHODS

Apoptosis of human epidermoid carcinoma A431 cells and p16-knockout (KO) or p21-KO fibroblasts was assessed by a fluorescence-activated cell sorter. Real-time PCR and western blot were used to determine the relative expression levels of p12, p21, and Tet1/2/3 genes. The global DNA methylation levels were determined using MethylFlash Methylated DNA Quantification Kit in A431 cells with or without vitamin C treatment. To examine the DNA demethylation activity of vitamin C, DNA immunoprecipitation (DIP)-qPCR was performed to determine the relative levels of 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) in p16 and p21 promoter regions containing cytosine-phosphorothiolated guanine (CpG) islands.

RESULTS

The increasing apoptosis of A431 cells under prolonged UV irradiation was remarkably decreased by the combination of vitamin C treatment, suggesting that vitamin C protects against UV-induced apoptosis. Concurrently, vitamin C induced a significant reduction of global DNA methylation in a time- and dose-dependent manner in A431 cells. Vitamin C also reactivated the expression of p16 and p21 at mRNA and protein levels. Mechanistically, about 27% 5hmC-positive cells were observed in vitamin C-treated A431 cells, and the 5hmC enrichment at p16 and p21 promoter regions was also largely increased by vitamin C. Moreover, the expression of p16 and p21 was decreased in Tet1/2 double-knockdown cells, in which the inhibitory effect of vitamin C on UV-induced apoptosis was dismissed. Furthermore, the inhibition of UV-induced apoptosis on vitamin C treatment nearly disappeared in p16- or p21-knockout primary cultured fibroblasts.

CONCLUSION

These results demonstrate that vitamin C effectively antagonizes UV-induced apoptosis through regulation of Tet activity, DNA demethylation, and subsequent tumor suppressor gene activation in skin cancer cells.