EFFECT OF 5-AZACYTIDINE ON miRNA EXPRESSION IN HUMAN BREAST CANCER CELLS WITH DIFFERENT SENSITIVITY TO CYTOSTATICS

Abstract

Aim: To analyze expression of miRNA in human breast cancer cells, sensitive and resistant to cisplatin and doxorubicin, and to explore possible modification of drug sensitivity via treatment of cells with 5-azacytidine (5-aza), a demethylating agent. Materials and Methods: The study was performed on wild-type MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP resistant to doxorubicin and cisplatin, respectively. Cells were treated with 5-aza, cisplatin, doxorubicin and their combinations. Relative expression levels of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were examined, using qRT-PCR. The MTT assay was used to monitor cell viability. Results: We compared miRNA expression profiles in MCF-7/S and drug resistant MCF-7/Dox and MCF-7/DDP cells. Changes of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were observed in both resistant cell lines. The most significant differences were found for miRNA-200b (decreased in 50.0 ± 2.6 and 63.0 ± 3.1 times for MCF-7/Dox and MCF7/DDP cells, respectively) and for oncogenic miRNA-221 levels (increase in 62.0 ± 5.7 times for MCF-7/Dox and 83.8 ± 7.2 times for MCF-7/DDP cells). 5-aza treatment caused an increase of miRNA-10b, -122, -200b levels in MCF-7/S cells, miRNA-34a, -10b, -122, -200b and -320a levels in MCF-7/Dox cells and miRNA-34a, -10b, -200b and -320a levels in MCF-7/DDP cells. Pretreatment of all studied lines with 5-aza resulted in the increase of their sensitivity to studied cytostatics. In particular, the IC50 of doxorubicin decreased by 2-, 4- and 3-fold for cell lines MCF-7/S, MCF-7/Dox and MCF-7/DDP cells, respectively, and IC50 of cisplatin in studied cultures decreased by 3-, 2- and 1.5-fold, respectively. Conclusions: It was shown that use of 5-aza can modify sensitivity of breast cancer cells to cytotoxic drugs not only by it’s demetylation effect, but also by changes in expression of miRNAs, involved in cell proliferation, migration and drug resistance development.

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

“…In breast cancer cells, the sensitizing role of AzaC to Dox via controlling the level of miRNA, which is responsible for cell proliferation, has been recently reported (20). AzaC also sensitizes platinum-resistant ovarian cancer cells to carboplatin via inducing caspase-3 expression (21).…”

“…It has also been demonstrated that DNMTIs and HDACIs with doxorubicin and cyclophosphamide can re-activate pro-apoptotic genes P53 and P21 in breast cancer patients (19). Recently, the sensitizing effects of AzaC on the activity of Dox have been reported at the miRNA expression level in human breast cancer cells (20). However, this sensitizing effect is still not clearly understood at the protein expression level in breast cancer MCF7 cells.…”

Epithelial-Mesenchymal Transition (EMT) as a Therapeutic Target

“…In contrast, activation of EMT markers and stemness genes correlated with CpG island hypomethylation [Davalos et al, 2012;Pistore et al, 2017]. Treating cell lines with the hypomethylating agent 5-azacytidine resulted in upregulation of the miRNA-200 family members, which can bind and degrade the mRNA levels of the ZEB EMT-TFs, and was associated with EMT reversal and increased therapeutic sensitivity [Chekhun et al, 2016]. However, treatments with DNA demethylating agents, like 5-azacytidine, on various in vitro and in vivo solid cancer models gave conflicting results of its effects on EMT and metastasis.…”

Modifying Effects of 5-Azacytidine on Metal-Containing Proteins Profile in Guerin Carcinoma With Different Sensitivity to Cytostatics

“…Fold change = 2−ΔΔCt [8], where ΔCt �target -control� is equal to the difference �etween threshold cycles for miRNA �target� and the threshold cycle for RNU4� �control�:…”