Antineoplastic action of 5-aza-2′-deoxycytidine and histone deacetylase inhibitor and their effect on the expression of retinoic acid receptor β and estrogen receptor α genes in breast carcinoma cells

Abstract.

Purpose: During tumorigenesis several cancer-related genes can be silenced by aberrant methylation. In many cases these silenced genes can be reactivated by exposure to the DNA methylation inhibitor, 5-aza-2′-deoxycytidine (5-AZA-CdR). Histone acetylation also plays a role in the control of expression of some genes. The aim of this study was to determine the antineoplastic activities of 5-AZA-CdR and trichostatin A (TSA), either administered alone or in combination, in MDA-MB-231 breast carcinoma cells. The effects of these drugs (alone and in combination) on the expression of the tumor suppressor gene, retinoic acid receptor (RARβ) and of the estrogen receptor α gene (ERα), whose expression is lost in the cell line used in the study, were also investigated. Methods: MDA-MB-231 cells were treated with 5-AZA-CdR and TSA and the antitumor activity of these drugs was determined by clonogenic assay. Total RNA was extracted from the treated cells and RT-PCR was used to determine the effect of the treatment on the expression of RARβ and ERα. Methylation-sensitive PCR analysis was used to confirm that lack of expression of both genes was due to hypermethylation of their promoter regions. A single nucleotide primer extension assay was also used to quantify the reduction in DNA methylation following drug treatment. Results: Both 5-AZA-CdR and TSA alone showed significant antineoplastic activity. The combination of the two drugs was synergistic with respect to MDA-MB-231 cell kill. 5-AZA-CdR alone weakly activated the expression of both RARβ and ERα. TSA alone only activated RARβ, but not ERα. The combination of these agents appeared to produce a greater activation of both genes. Conclusions: The interesting interaction between 5-AZA-CdR and TSA in both cell kill and cancer-related gene reactivation provides a rationale for the use of inhibitors of DNA methylation and histone deacetylation in combination for the chemotherapy of breast cancer.