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

Purpose: During tumorigenesis several cancer-related genes can be silenced by aberrant methylation. In many cases these silenced genes can be reactiva ted by exposure to the DNA methylation inhibitor, 5-aza-2 ' -deoxycytidine (5-AZA-CdR). Historic acetylation also plays a role in the control of expre ssion of some genes. The aim of this study was to determine the antineoplas tic activities of 5-AZA-CdR and trichostatin A (TSA), either administered a lone or in combination, in MDA-MB-231 breast carcinoma cells. The effects o f these drugs (alone and in combination) on the expression of the tumor sup pressor gene, retinoic acid receptor (RAR beta) and of the estrogen recepto r alpha gene (ER alpha), whose expression is lost in the cell line used in the study, were also investigated. Methods: MDA-MB-231 cells were treated w ith 5-AZA-CdR and TSA and the antitumor activity of these drugs was determi ned 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 RAR beta and ER alpha. Methylation-sensitive PCR analysis was used to c onfirm that lack of expression of both genes was due to hypermethylation of their promoter regions. A single nucleotide primer extension assay was als o used to quantify the reduction in DNA methylation following drug treatmen t. 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 bo th RAR beta and ER alpha. TSA alone only activated RAR beta, but not ER alp ha. The combination of these agents appeared to produce a greater activatio n 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 historic deacety lation in combination for the chemotherapy of breast cancer.