Hypomethylation and induction of retinoic acid receptor beta 2 by concurrent action of adenosine analogues and natural compounds in breast cancer cells

DNA methylation is considered as a potential cause of aberrations in regulation of gene expression during carcinogenesis. Therefore, changes in DNA methylation patterns may be targets for chemoprevention. In the present study, we investigated effects of all-trans retinoic acid (ATRA), vitamin D3, and resveratrol alone and in combination with adenosine analogues: 2-chloro-2′-deoxyadenosine (2CdA) and 9-beta-d-arabinosyl-2-fluoroadenine (F-ara-A), on methylation and expression of retinoic acid receptor beta 2 (RARbeta2) in MCF-7 and MDA-MB-231 breast cancer cell lines. Alterations in methylation and expression levels after treatment of cells with the tested compounds were evaluated by methylation-sensitive restriction analysis (MSRA) and real-time PCR, respectively. RARbeta2 promoter in the tested fragment was partially methylated in MCF-7 cells and non-methylated in MDA-MB-231 cells. In MCF-7 cells, all compounds, except for resveratrol, inhibited promoter methylation and increased expression of RARbeta2. All natural compounds improved the action of 2CdA and F-ara-A on RARbeta2 methylation and/or expression. Combination of ATRA or vitamin D3 with 2CdA was the most effective. In MDA-MB-231 cells, only 2CdA, F-ara-A, and ATRA induced RARbeta2 expression without any notable effects in combined treatment. Our results demonstrate that both natural compounds and adenosine analogues are able to reduce promoter methylation and/or induce expression of RARbeta2 in non-invasive MCF-7 cells. Furthermore, the natural compounds improve effects of adenosine analogues, however only at early non-invasive stages of carcinogenesis.