Azacytidine-induced Chemosensitivity to Doxorubicin in Human Breast Cancer MCF7 Cells

Abstract

Background/Aim: It is necessary to select an appropriate strategy that makes cancer cells more sensitive to chemotherapeutic drug(s). The aim of this study was to further explore the mechanism(s) behind drug sensitization in the breast cancer MCF7 cell line using 5-azacytidine (AzaC) pretreatment in doxorubicin (Dox) chemotherapy. Materials and Methods: Cells were treated with either AzaC or Dox alone or in combination (AzaC+Dox) for 24 and 48 h. Cells were also pretreated with AzaC for a week before replacement with Dox at varying concentrations in another group of experiments (AzaC/Dox) for 24 and 48 h. The cells in the groups were subjected to cytochemical analyses, including 3( 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT), intracellular reactive oxygen species (ROS) and cell death by dual staining with Hoechst33342 (Hoechst) and propidium iodide (PI). Cells were also analyzed for determining specific alterations of possible target proteins by western blot. Results: Compared to a single (AzaC and Dox) or simultaneous drug exposure (AzaC+Dox), cellular activity was significantly reduced in AzaC/Dox group of cells in a dose-and time-dependent manner (p<0.05). The clonogenic potential of cells was also significantly inhibited in AzaC/Dox compared to Dox and AzaC group after 7 days (p<0.05). The rates of ROS-generated and PI-stained cells were significantly higher in the AzaC+Dox and AzaC/Dox groups compared to those of individual drug groups at 48 h (p<0.05). These observations were accompanied by activation of ERK1/2 and p38MAPK, as well as up-regulation of BAX and P53 in the AzaC/Dox group. However, the levels of BCL-2were down-regulated in Dox and AzaC+Dox among a different group of cells. Elevated levels of activated caspase-3 were observed only in AzaC/Dox group of cells. The levels of phosphonuclear factor-kappa B (pNF-kappa B)(Thr) decreased with increasing dosage of Dox in different groups, while down-regulation of heat shock protein 70 (HSP70) was observed only in the AzaC+Dox group of cells. Conclusion: AzaC significantly increases the sensitivity of MCF7 cells to Dox via activation of ERK1/2, P53, BAX and caspase-3. Moreover, the inhibition of pNF-kappa B(Thr) and HSP70 might also contribute to an increase in the sensitivity of MCF7 cells to Dox.