Role of cellular interaction and cell phenotype in azacytidine to doxorubicin sensitivity in breast cancer cell-derived clones

Background: Breast malignancies are the most diverse tumors, having the highest level of cellular heterogeneity at various stages of development. This heterogeneous nature of cells is even observed in a controlled growth environment. Cellular phenotype, expression of cell surface markers, epithelial to mesenchymal transition, and cellular niche play a pivotal role in the heterogeneity of breast cancer cells. Heterogeneity in breast cancer cells is one of the leading causes of drug resistance where only a portion of cells respond to the therapeutic drugs while the other cells escape from therapies. Furthermore, cellular heterogeneity is one of the major barriers to designing therapeutic strategies. Therefore, it is important to design multidimensional strategies that consider various aspects, including cell type, cellular microenvironment, and the type of anticancer drugs to treat breast cancer. Aim: This study aimed to investigate the responses of cells from various single-cell-derived breast cancer clones to 5-azacytidine (AzaC) and doxorubicin (Dox). Furthermore, the role of AzaC in the sensitization of breast cancer cells to Dox was studied in breast cancer clones. Materials and methods: Single cell-derived clones were established from four distinct breast cancer cell lines including MCF7, MDA-MB-231, MCF7-GAPDH-RFP and MDA-GAPDH-RFP. Following one week of clonal development, the clones were subjected to separate or combined treatments of 5 mu M AzaC and 500 nM Dox for 48 h. In the sequential group (AzaC/Dox), clones were exposed to 5 mu M AzaC for one week, followed by 500 nM Dox for 48 h. Morphological and immunocytochemical analyses were performed by using crystal violet staining and immunolabeling, respectively. Furthermore, the expression levels of specific protein markers, including alpha smooth muscle actin (alpha-SMA), vimentin (VIM), cytokeratin-8 (CK-8), cytokeratin-19 (CK-19) and platelet endothelial cell adhesion molecule-1 (PECAM-1), were evaluated in response to the drugs. In addition, the expressed target protein markers were quantified in five independent sets of clones from each treatment group (n = 5). Results: After 48 h of treatment, the sizes of the colonies in all treatment groups were markedly decreased as compared to the control group. In the AzaC group, colonies exhibited irregular shapes and scattered cell patterns. In Dox- and AzaC+Dox-treated clones, the cell number in each colony evidently decreased, while in AzaC/Doxtreated clones, a fraction of clonal cells showed a similar phenotype to mesenchymal-like cells. Immunocytochemistry showed a significant decrease in the number of alpha-SMA- and VIM-expressing cells in the drug-treated groups. Notably, in MCF7- GAPDH-RFP and MDA-GAPDH-RFP clones, VIM expression remained high despite the low cell number. Overall, CK-8 was down-regulated while CK-19 was strongly expressed in AzaC-treated MDAMB-231 clones. Despite the small cell number, both markers exhibited up-regulation in the drug-treated MCF7and MDA-GAPDH-overexpressed clones. Likewise, there was a notable increase in the expression of PECAM-1 in drug-treated MCF7- and MDA-RFP clones. Conclusion: This study demonstrates that AzaC-sensitizes breast cancer cells to Dox in single cell-derived clones. Furthermore, the high expression pattern of the target protein markers in drug-treated clones reflects their role in cell survival and drug resistance. Further studies are needed to uncover the detailed molecular mechanism involved in cell survival and drug resistance against AzaC and Dox in breast cancer cell-derived clones.