225 GENE MUTATION AND ALTERED EXPRESSION OF NBS1 IN HBV ASSOCIATED HEPATOCELLULAR CARCINOMAS

Abstract

Background and Aims: The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D33) inhibits cell growth and induces cell differentiation and apoptosis in numerous tumors, such as colon, breast and prostate cancers. The role of 1,25-D3 in the development of the hepatocellular carcinoma has not yet been established. The aim of the present study was to examine the gene expression of the vitamin D inactivating CYP24A1 as well as the activating 1-alpha-hydroxylase (CYP27B1) enzyme after 1,25-D3 treatment in hepatocellular carcinoma cell lines (1,25-D). Methods: HepG2 and HUHneo (Dept. of Molecular Virology, Univ. of Heidelberg) cells were treated with 4nM concentrations of 1,25-D3 in Optimem medium and were incubated in 24-well plates in two parallels for treatment duration of 30, 60, 120 minutes, 5, 8, 10, 12, 14, 24, 26, 28 hours. Total RNA was isolated from each sample with Roche High Pure Total RNA Isolation kit. Five hundred ng of total RNA was reverse-transcribed to cDNA. The expression differences of selected genes were analyzed by Taqman probe-based quantitative real-time PCR. Relative quantification was carried out from collected data (threshold cycle numbers) by Applied Biosystems 7500 System SDS software 1.3. Results: CYP24A1 mRNA expression significantly (p < 0.001) increased in response to 1,25-D3 administration in a timedependent manner in both cell lines. In HepG2 cell line the CYP24A1 mRNA expression showed 5300-fold elevation reaching the maximum value at 8 hours. In the HUHneo cells the increase was 152-fold of the baseline level, and the curve reached the maximum at 14 hours. There was no change in CYP27B1 gene activity of the cells treated with 1,25-D3. The activating effect of vitamin D3 on CYP24 mRNA expression was at 28 hours still detectable. Conclusions: Our novel data raise the possibility that the significant increase in the CYP24A1 gene expression of hepatocellular carcinoma cell lines following vitamin D administration may stimulate the inactivation of 1,25-D3, thus “protecting” the tumor cells against the anti-cancer effects of 1,25-D3. This is the first report on the role of vitamin D3 on the CYP24A1 mRNA transcription in hepatocellular carcinoma cells in vitro.