| Abstract [eng] |
The aim of the study was to evaluate the anticancer activity of sunitinib analogues in human breast cancer cell cultures. Work tasks: 1. To study the effect of sunitinib analogues on the viability of human breast cancer cells; 2. To analyse the effect of sunitinib analogues on the human breast cancer cell clonogenicity; 3. To determine the method of death of human breast cancer cells caused by sunitinib analogues; 4. To study the activity of sunitinib analogues in three-dimensional cultures (spheroids) of human breast cancer cells. The object of the study is seventeen new analogues of sunitinib synthesized at Cagliari University (Italy) and sunitinib used in the clinical practice. Methods. The cytotoxic activity of the compounds was investigated in three breast cancer cell lines: MDA-MB-231, MCF-7, BT-474 and non-cancerous cells – fibroblasts. Initially, the compounds that most inhibited cell viability were selected for each cell line under investigation. The effect of sunitinib analogues on cell viability was determined by the MTT assay. Subsequently, the effect of the most active compounds on cell clonogenicity was investigated, assessing how cells are able to form colonies. The method of cell death caused by the compounds was determined by fluorescence microscopy using double staining with Propidium Iodide and Hoechst 33342. Cell spheroids (three-dimensional culture) were produced by 3D Bioprinting method. Photos were analysed by the ImageJ program. The data were processed by Microsoft Office Excel 2016 calculating the mean and standard deviations of the results obtained in three independent experiments. The averages were compared using Student's t criterion, and differences were considered statistically significant when the significance level p <0.05. Results. Sunitinib analogues possessed higher anticancer activity than the drug sunitinib that was used in clinics. Compound 1 showed the highest anticancer activity against all three breast cancer cell lines (EC50 value for MDA-MB-231, MCF-7 and BT-474 cell line was 69.3 ± 7.6; 73.3 ± 9.9; 94. 3 ± 5.1 nM respectively; p <0.05). The viability of MDA-MB-231 cells was also strongly inhibited by compound 6 (258.3 ± 23.2 nM; p <0.05). The compounds possessed lower activity against fibroblasts compared to cancer cells, except compound 7. Compound 8 also had a greater effect on fibroblast viability than MCF-7 cell line. Sunitinib analogues inhibited the formation of MCF-7 and BT-474 cell colonies. Compound 17 had the greatest effect on MCF-7 stem cell proliferation (the number of colonies decreased to 32.2 % and the area decreased by 22.4 %), the formation of BT-474 colonies was mostly reduced by compound 1 (up to 25.5 %). For only a small number of cells of the MDA-MB-231 cell line, analogues of sunitinib induced cell apoptosis and necrosis. No significant effect was observed on the necrosis of the MCF-7 cell line and apoptosis was induced by compounds 7 and 8. The apoptosis of BT-474 cells was caused only by the maximum used concentration of compound 1 (90 % of the EC50 value) and necrosis by the maximum concentration of compound 1 and compound 9. The three-dimensional cultures of MDA-MB-231 and BT-474 cells were less sensitive to SNT analogues than two-dimensional culture. The growth of MDA-MB-231 spheroids was inhibited by compounds 7 and 17, and BT-474 by compound 9. Conclusion. Six of seventeen studied sunitinib analogues have the antitumor activity against MDA-MB-231, MCF-7 and BT-474 breast cancer cell lines and many of them are more active than sunitinib. The compounds inhibit the cell viability, colony formation and cause apoptosis and necrosis, also inhibit the spheroid growth, that is why they are perspective for a further research. |
