Figure 4

CMC stem cells are resistant to doxorubicin: reversal by verapamil pretreatment. a) Cumulative dose–response curves of the effects of doxorubicin (DOX) on cell viability, measured by MTT assays, in CSC (CMC CSC) and differentiated (CMC DIFF) canine mammary carcinoma (CMC) cells. A statistically significant reduction in cell viability of CMC DIFF was observed (*p < 0.05 for 0.1 μM DOX, ***p < 0.001 for higher concentrations vs. control), while CMC CSC were not affected. Data represent the mean ± s.e.m. b) DOX intracellular distribution in CMC CSC untreated (left) or treated (right) with the calcium channel blocker verapamil, to inhibit ABC transporter activity. CSCs were labelled with the lipid green dye DiO to highlight cell shape. Subcellular DOX fluorescence (red) localization is mainly confined to cytoplasm (co-localization with DiO, yellow) of resistant cells, while the fluorescent accumulation of DOX in the nuclei is markedly increased by verapamil. Original magnification 20X. c) Dose–response analysis of verapamil on the cytotoxic activity of DOX: reversal of resistance was significantly achieved starting from 0.1 μM (*p < 0.05, **p < 0.01; ***p < 0.001 vs. respective value of DOX alone). Data represent the mean ± s.e.m. d) Mean IC₅₀ values calculated using nonlinear regression curve fit analysis in CMC cells exposed to DOX alone or in combination with verapamil.