| Summary: | multidrug resistance (MDR) remains a major problem in the fight against tumors. MDR – multidimensional phenomenon, which occurs as a consequence of activation of several systems, associated both with the release of xenobiotics from the cell by ABC-transporters, and with stimulation of pathways that enhance cell survival. One of the possible regulators of MDR is multifunctional protein YB-1. YB-1 acting as a transcription factor in cell’s nucleus, can upregulate expression of MDR1, as well as some other ABC-transporters genes. Furthermore, YB-1 is secreted by cells and binds to the protein Notch3, and that stimulates proliferation and migration of cells. Also, resistance of cells may increase upon stimulation of VEGF-VEGER systems. Our aim was to study the mechanisms of MDR at various levels in the pairs of parental and resistant breast cancer cells: changes in the expression and localization of YB-1, evaluation expression of ABC-transporters, activation of protein Notch, as well as changes in the VEGF system.
Materials and methods: Long-term cultivation of cells MCF-7 (breast adenocarcinoma) and HBL-100 (transformed cells) in the presence of doxorubicin to obtain resistant sublines, MTT assay,real-time PCR, immunocytochemistry, flow cytofluorometry.
Results: we obtained the sublines of cells MCF-7 and HBL-100 resistant to doxorubicin: MCF-7/D7 and HBL-100/D85, respectively. The level of resistance to doxorubicin in resistant MCF-7/D6 was 7 times higher than in parent MCF-7 (IC50 was 0.7 ± 0.01 mcM vs 0.1 ± 0.002 mcM, respectively), and the level of resistance HBL-100/D85 was 44.7 times higher than the HBL-100 (IC50 8.5 ± 0.17 mcM vs 0.19 ± 0.03 mcM). Also, MCF-7/D7 acquired cross-resistance to cisplatin (6.7 ± 0.1 mcM vs 48 ± 0.2 mcM) and paclitaxel (2 ± 0.03 nM vs 50 ± 1 nM), but not to vinblastine and 5-fluorouracil, and HBL-100/D85 – about 100 times to paclitaxel (13 ± 0.2 nM vs 1100 ± 19 nM) and vinblastine (2.4 ± 0.3 nM vs 250 ± 5 nM), but not to the cisplatin and 5-fluorouracil. The expression profile of ABC-transporter genes showed significant activation of the MDR1 expression in subline HBL-100/D85 (more than 1000 times), but not in MCF-7/D6.
Expression of other genes of the family ABC-transporters - MRP1, BCRP, MVP - increased slightly. Expression of mRNA YB-1 was almost unchanged. YB-1 protein detected in cytoplasm of parental lines, but it detected in nuclei in resistant sublines. Also, protein Notch3 translocated from membrane into nucleus. Evaluation of VEGF expression profile revealed that in a couple of VEGFA-VEGFR2 didn’t occur any changes. However, we observed a significant upregulation of expression mRNA VEGFC and down-regulation of mRNA VEGFR3 in resistant sublines MCF-7/D6 and HBL-100/D85.
Conclusion: the results of our study show that the appearance of MDR associated with various changes in cells. Common changes in the resistant sublines were translocation of protein YB-1 and Notch3 in cells nuclei, as well as the dynamics of expression changes in pair VEGFCVEGFR3.The last observation is an interesting finding and requires more detailed study.
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