The CpG Island Methylation Regulated Expression of Endothelial Proangiogenic Genes in Response to β-Carotene and Arachidonic Acid

Abstract

β-carotene (BC) and arachidonic acid (AA) were demonstrated to modulate carcinogenesis by influencing angiogenesis. DNA methylation is an epigenetic mechanism regulating gene expression. The aim of this study was to investigate whether BC and AA change DNA methylation and expression of the proangiogenic genes, which might help explain their impact on carcinogenesis. Human umbilical vein endothelial cells (HUVECs) and endothelial progenitors (EPCs) were incubated with BC or AA for 24 h. Based on microarray results, we selected 18 genes for DNA methylation analysis. CpG island methylation was quantified using the combined bisulphite restriction analysis method and methylation sensitive restriction enzymes. Relative gene expression was quantified using a quantitative real-time PCR (qRT-PCR) method. Incubation with AA significantly decreased methylation of the promoters of both KDR (P = 0.048) and Notch4 (P = 0.027) genes in HUVECs. In EPCs, BC increased methylation of the connexin 43 gene (P = 0.036). qRT-PCR showed that AA (P = 0.059) and BC (P = 0.044) upregulated the KDR gene expression in HUVECs. Connexin 43 gene expression was induced in the presence of 1 μM (P = 0.039) and 3 μM (P = 0.043) BC in EPCs. No significant changes in the Notch4 gene expression were found. The impact of BC and AA on carcinogenesis may be due, at least in part, to changes in expression of angiogenic genes and these transcriptional effects may be mediated by changes in methylation of CpG islands in the gene promoters. However more research is necessary to confirm this hypothesis.