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Introduction BRCA1 is involved in DNA double strand break repair. BRCA1 function disruption is associated with cancer development mainly in hormone-dependent breast and ovarian tissue. The aim of the current research is the study of the effect of hypoxia, estrogens, phytoestrogens and anti-estrogens on BRCA1 and oestrogen receptors α (ERα) expression in MCF-7 hormone-dependent cancer cell line. Material and methods MCF-7 cells were cultivated in hypoxia conditions (1% O2) with or without tirapazamine (TPZ) (10 µM). MCF-7 cells were cultured in steroid-free media with estrogens or anti-estrogens (17β-estradiol (E2) (10 nM), genistein (0.5 µM), tamoxifen (5 µM), fulvestrant (0.1 µM)). Immunofluorescent method associated with flow cytometry was used for quantitative estimation of BRCA1 and ERα expression using specific anti-BRCA1 (SD118) and anti-ERα (SP-1, ab27614) antibodies, secondary antibodies conjugated with DyLight 650 (ab98729, ab98510), Navios flow cytometer (Beckman Coulter). The level and intensity of marker expression was analysed using the program FlowJo 10.0 with statistical approach of Kolmogorov-Smirnov. Following quantitative parameters of marker expression were determined: level (the ratio of specifically fluorescent cells to the total number of cells), intensity (the ratio of specific cell fluorescence to the cell fluorescence obtained with secondary fluorescent antibodies), index (the product of the level and intensity of the marker expression divided by 100). Results and discussions 1. In hypoxia conditions the decrease in BRCA1 and ERα expression index in 2,0 fold and 3,1 fold, respectively, was observed. 2. Hypoxic cytotoxin TPZ increased BRCA1 expression index 1,6 fold while it decreased ERα expression index 4,1 fold. 3. Phytoestrogen genistein increased BRCA1 expression index 1,6 fold and reduced ERα expression index 2,9 fold. 4. E2 and anti-estrogens reduced ERα expression without any influence on BRCA1 expression. Conclusion 1. Taken together, the results of this study demonstrate that the hypoxia has an inhibiting effect on BRCA1 network and decreases hormonal stimulation of breast cancer cell growth. 2. The data obtained point to the different mechanisms of BRCA1 and steroid hormones expression regulation with phytoestrogens in adenocarcinoma breast cancer cells and open further perspectives for their complex investigation. (RFBR grants №№ 15-04-06991-a, 16-34-01049-mol-a and RSF grant 14-15-00362 (cell culture experiments)). |
