Promoter methylation of tumour suppressor genes is involved in silencing of their transcription and can be a target for epigenetic therapy. The aim of the present studies was to examine the effects of combined action of natural compounds [i.e. all-trans retinoic acid (ATRA), vitamin D₃ (Vit.D₃), resveratrol (RES)] and anticancer agents [i.e. nucleoside analogues: 2-chloro-2’-deoxyadenosine (2CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A), 5-aza-2’-deoxycytidine (5-aza-dCyd)] on promoter methylation and expression of tumour suppressor genes (i.e. BRCA1, RARbeta2, PTEN, APC) as well as on expression of DNA methyltransferase (DNMT1) and p21 genes in non-invasive MCF-7 and invasive MDA-MB-231 breast cancer cells.

Methylation of promoters of the tested genes and expression of the genes on the mRNA level were estimated using methylation-sensitive restriction analysis (MSRA) and real-time PCR, respectively.

In MCF-7 cells all tested compounds (at IC₅₀ concentrations) reduced RARbeta2, PTEN and APC promoter methylation, whereas the effects of the tested compounds on the gene expression were more differentiated. PTEN mRNA level increased by 30% in the cases of all compounds, expression of RARbeta2 increased over 2-fold after treatment with nucleoside analogues and APC expression increased by 20-50% after treatment with nucleoside analogues and Vit.D₃.

In invasive MDA-MB-231 cells, only 5-aza-dCyd (at IC₅₀ concentration) reduced PTEN methylation (by 25%). The reduction of PTEN methylation by other tested compounds required several-fold higher concentrations than IC₅₀. Moreover, the changes of PTEN promoter methylation did not result in alteration of PTEN expression, with the exception of Vit.D₃ which presence led to increase of PTEN expression by 35% and slight decrease of DNMT1 expression.

Combination of ATRA, Vit.D₃ and RES with nucleoside analogues gave varied results among that the most striking were: (i) in MCF-7 cells Vit.D₃ significantly enhanced 2CdA effect on PTEN methylation (decrease by 63%) and expression (1.5-fold increase), which was accompanied by 2-fold decrease of DNMT1 and 1.7-fold increase of p21 expressions; (ii) in both cell lines ATRA improved stimulation of RARbeta2 expression by 2CdA and 5-aza-dCyd; (iii) in MCF-7 cells RES enhanced inhibitory effect of F-ara-A on RARbeta2 and APC methylation as well as on DNMT1 expression (2-fold); simultaneously we observed an increase of p21 and APC mRNA levels (4.5- and 2-fold, respectively).

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