Fig. 4.

Ivermectin inhibited P-gp expression and increased the intracellular drug accumulation. a-c Protein level of P-gp (a), mRNA level of MDR1 (b), or intracellular VCR concentrations (c) in HCT-8 cells treated with 25 nM VCR and/or 3 μM ivermectin (IVM) for 48 h were determined. The protein level was detected by Western blotting analysis and mRNA level was determined by qPCR using GAPDH as the internal control, and intracellular VCR concentrations were determined by HPLC. d and e Cell viability of HCT-8 cells transfected with the plasmid pGenesil-P-gp (P-gp shRNA) (d) or the plasmid pcDNA3.1(+)-P-gp (e) and then treated with 25 nM VCR and/or 3 μM IVM for 48 h. Cell viability was detected by MTT assay. Cells transfected with control shRNA (shCtrl)/empty vector pcDNA3.1(+) (mock) or treated with vehicle serve as control. f The P-gp expression in the HCT-8 xenografts was detected by immunofluorescence (upper panel) and immunohistochemical staining (lower panel). Green: P-gp protein. Scale bars: 200 μm. g VCR accumulation in tumor tissues of the mice was determined by HPLC analysis. Abbreviations: IVM, ivermectin; VCR, vincristine; S, vincristine-sensitive HCT-8 cells/xenografts; R, vincristine-resistant HCT-8 cells/xenografts. Data in a is the representative of three independent experiments. Data in b and c represent the mean ± SD (n = 3). Data in d and e represent the mean ± SD (n = 5). Data in g represent the mean ± SD (n = 6 mice in each group). Statistical significances were determined using one-way ANOVA followed by Dunnett’s test. ^*P < 0.05, ^**P < 0.01, compared with the respective vechicle controls; ^#P < 0.05, ^##P < 0.01, compared with the corresponding columns with the same color in the S group; ^&P < 0.05, ^&&P < 0.01, comparison between the two columns