Background. The tumor consists of different malignant cells like ordinary cancer cells and cancer stem cells. It is necessary to use therapeutic agents targeting all heterogeneous tumor cells population for successful treatment. The research aimed to study the mono- and combination therapeutic effects of anticancer drugs such as methotrexate (0.1 and 1 μg/ml) and salinomycin (1-40 μM). Two anticancer drugs with different cytotoxic mechanisms were tested in 2D and 3D culture.
Methods. The cells MCF-7 were cultivated in standard conditions in 2D and 3D culture. MCF-7 spheroids were generated with 2% carboxymethyl cellulose on an orbital shaker (PSU-10i, Biosan, Latvia) at 80 rpm for 3-5 hours at non-adhesive conditions. Monolayer and spheroid cultures were incubated with salinomycin (1-40 μM) and methotrexate (0.1 and 1 μg/ml). MTT-test was used to detect the cytotoxicity effect of antitumor drugs. Expression markers of cancer stem cells (bmi-1, CD44, CD133) and cytokeratin (clone AE1/AE3) were performed by immunochemistry. Morphometric study on 3D culture was carried out using the AxioVision 2000 (Zeiss) program.
Results. The results demonstrated that the IC50 for Sal was 19 μg/ml, IC50 for Met 67,4 μg/ml, IC50 for Sal+Met 0,1μg/ml – 42.7 μg/ml, IC50 for Sal+Met1μg/ml – 34,5 μg/ml. The IC50 for Sal 1-40 μM + Met 1 μg/ml had twice smaller than the IC50 for Sal and Met separately. The results of immunochemistry showed that after incubation with Met 10 μg/ml expression level of cancer stem cells markers were higher 2-3 times than Sal 4 μM + Met 0,1 μg/ml. The expression of cytokeratin decreased by 25% and 0% compared to control after combination Sal 40 μM + Met 0.1 and Sal 40 μM + Met 1 μg/ml respectively. The next stage of the work was the study of drugs in a 3D culture. The results of culturing spheroids with (Sal 40μM +Met 1μg/ml; Sal 40μM + Met 0,1 μg/ml) was decreased spheroid size by 82% and 73% in accordance. Cytotoxicity of antitumor drugs on tumor spheroids with Sal 4 μM + Met 1 μg/ml and Sal 4 μM + Met 0,1 μg/ml were reflected in decreasing spheroid volume by 40% and 36%. The decreasing of spheroids size was a dose-dependent tendency from 4μM to 40 μM of Sal.
Conclusion. Our result demonstrated that 2D and 3D cultures were sensitive to mono- and combination therapy. The decrease of size spheroid culture and decrease in live-cell viability after incubation with Sal 4-40 μM + Met 0,1-1 μM is a good marker for synergistic activity. The data showed that Sal + Met hasn't cytotoxic influence on the cancer stem cell population.