To investigate polypeptide from Chlamy Farreri (PCF)'s protective effect against skin cancer, we used a cellular model of ultraviolet B (UVB)-induced malignant transformation. The human keratinocyte cell line HaCaT was repeatly exposed to UVB (10 mJ/cm(2), 20 times) and malignant transformation was confirmed by Gimesa staining, cell cycle analysis and various assays [anchorage independent growth, matrix metalloproteinase-9 (MMP9) activity, plating efficiency]. The malignant transformation was found to be effectively prevented by PCF pretreatment (2.84mM for 2h prior to each UVB exposure). We investigated the mechanism of PCF-mediated action by determining its effect on DNA methylation status of the tumour suppressor genes [P16 and ras association domain family 1 A (RASSF1A)] in the UVB-transformed cells. Both genes were found to be hypermethylated by chronic UVB exposure. The expression levels of P16, RASSF1A, DNA methyltransferases (DNMTs) and DNA damage inducible protein a (GADD45a) were measured by reverse transcriptase-polymerase chain reaction and western blotting. While chronic UVB exposure was found to suppress the expression of P16 and RASSF1A, it enhanced the expression of DNMT3b. In the early phase of UVB-induced malignant transformation, the GADD45a expression was increased, however, it declined with a continued irradiation of the cells. The UVB-induced DNA hypermethylation of P16 and RASSF1A and subsequent gene silencing was reversed by PCF treatment. The inhibition of DNMTs expression suggested that PCF blocked DNA methylation and thereby the silencing of tumour suppressor genes. Furthermore, the PCF-mediated substantial increase in GADD45a expression indicated that PCF promoted demethylation of tumour suppressor genes via GADD45a induction.
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