Calcitriol (1,25-dihydroxycholecalciferol), the most active form of
vitamin D, has selective anti-proliferative effects on
tumor-derived endothelial cells (TDEC) compared with
Matrigel-derived endothelial cells (MDEC). Although both cell types have an intact
vitamin D receptor-signaling axis, this study demonstrates that upon treatment with
calcitriol, 24-hydroxylase (CYP24)
mRNA,
protein and enzymatic activity were markedly induced in MDEC in a time-dependent manner but not in TDEC. Furthermore, treatment of MDEC with a CYP24
small interfering RNA restored sensitivity to
calcitriol. To investigate the lack of CYP24 induction in TDEC, we examined methylation patterns in the promoter regions of the CYP24 gene in these two cell types. We identified two putative CpG island regions located at the 5' end. Using methylation-specific PCR and
bisulfite sequencing, we determined that these CpG islands were hypermethylated in TDEC but not in MDEC. These data may explain the recruitment of
vitamin D receptor to the promoter region in MDEC but not TDEC, as revealed by
chromatin immunoprecipitation analyses. Treatment of TDEC with the
DNA methyltransferase inhibitor
5-aza-2'-deoxycytidine restored
calcitriol-mediated induction of CYP24, which led to loss of sensitivity to
calcitriol growth inhibitory effects. CYP24 promoter hypermethylation was also observed in endothelial cells isolated from other
tumors but not in endothelial cells isolated from normal mouse tissues. These observations indicate that the methylation status of the CYP24 promoter differs in endothelial cells isolated from different microenvironments (tumor versus normal) and that methylation silencing of CYP24 contributes to selective
calcitriol-mediated growth inhibition in endothelial cells.