Table 1.
Number of ABCB1 transcripts per µg of total RNA in each of the cell lines.
Figure 1.
Effects of CDKi and the model ABCB1 inhibitor LY on ABCB1-mediated efflux of HOE in MDCKII-ABCB1 cells.
0% and 100% respectively indicate the fluorescence of unaffected control cells and the maximal fluorescence observed in assays with a particular CDKi. Presented data are means ± SD obtained from three independent experiments performed in triplicate.
Figure 2.
Time-dependent accumulation of HOE in MDCKII cells.
MDCKII-ABCB1 (A) and MDCKII parent (B) cells were incubated in the absence (control □) or presence of purvalanol A (▪), roscovitine (▴), olomoucine II (▾) flavopiridol (⧫) or SNS-032 (○) at their respective IC50 concentrations. A potent ABCB1 inhibitor, LY (•), was used as a positive control for ABCB1 inhibition. Presented data are means ± SD obtained from three independent experiments performed in triplicate. The statistical significance (*P<0.05; **P<0.01; ***P<0.001) of differences in HOE levels detected in treated and control cells was determined using unpaired t tests.
Figure 3.
Effect of the tested CDKi on intracellular accumulation of DNR in MDCKII-ABCB1 cells.
(A) Inhibition, expressed as the ratio between the median fluorescence with and without the indicated inhibitors during the efflux period, normalized to the effect in the parental cell line MDCKII (see Materials and Methods for details). LY (1 µM) was used as a model inhibitor. Presented data are means ± SD obtained from three independent experiments performed in duplicate. P values of differences between observed inhibition ratios and the null hypothetical value of 1 (no ABCB1 inhibition) were determined by unpaired two-tailed t tests: *P<0.05; **P<0.01; ***P<0.001. (B) Representative histograms of data obtained in assays with each compound at 20 µM: control with no inhibitor (light grey), tested compound (black), 1 µM LY (dark grey).
Figure 4.
Effects of CDKi on the ATPase activity of ABCB1-Sf9 membrane preparations.
Vanadate-sensitive ATPase activity in the presence of purvalanol A, olomoucine II, roscovitine, flavopiridol, or SNS-032 in activation (•) and inhibition (□) experiments. Presented data are means ± SD representative of at least two experiments performed in duplicate. The significance of differences linked to the absence and presence of CDKi in the basal activity of the transporter in activation assays (†P<0.05; ††P<0.01; †††P<0.001) and the activity of the activated transporter in inhibition assays (*P<0.05; **P<0.01; ***P<0.001) were determined using unpaired t tests.
Figure 5.
Cytotoxicity and combination experiments in MDCKII-ABCB1 and parental cell lines.
Cytotoxic effect of (A, E) purvalanol A (▪), (B, F) olomoucine II (▾), (C, G) roscovitine (▴) or daunorubicin (•) and their combination (⧫) on MDCKII-ABCB1 or MDCKII parent cells. For combinations, concentrations corresponding to particular points in the graph are sums of concentrations of the individual drugs administered in fixed concentration ratios (Table 2), based on the ratio of their respective EC50 values. Presented data are means ± SD obtained from at least three independent experiments performed in triplicate. (D, H) The cytotoxic effect (combination index, CI, plot) of CDKi and daunorubicin combinations on MDCKII-ABCB1 or MDCKII parent cells, obtained using CompuSyn software. Fractional effects (Fa) were calculated from the cell viability values of individual compounds; Fa = 0 means no antiproliferative effect, Fa = 1 means 100% antiproliferative effect. CI = 0.9–1.1 indicates additive effect, CI <0.9 synergism and CI > 1.1 antagonism.
Table 2.
Dose reduction index (DRI) values for drug combinations scheduled after 72 h of simultaneous treatment.
Figure 6.
Cytotoxicity and combination experiments in HCT-8 and HepG2 cell lines.
Cytotoxic effect of (A, E) purvalanol A (▪), (B, F) olomoucine II (▾), (C, G) roscovitine (▴) or daunorubicin (•) and their combination (⧫) on HCT-8 cells or HepG2. For combinations, concentrations corresponding to particular points in the graph are sums of concentrations of the individual drugs administered in fixed concentration ratios (Table 2), based on the ratio of their respective EC50 values. Presented data are means ± SD obtained from at least three independent experiments performed in triplicate. (D, H) The cytotoxic effect (combination index, CI, plot) of CDKi and daunorubicin combinations on MDCKII-ABCB1 or MDCKII parent cells, obtained using CompuSyn software. Fractional effects (Fa) were calculated from the cell viability values of individual compounds; Fa = 0 means no antiproliferative effect, Fa = 1 means 100% antiproliferative effect. CI = 0.9–1.1 indicates additive effect, CI <0.9 synergism and CI >1.1 antagonism.