Figure 5.

P-gp inhibitors restore calcein-AM or daunorubicin accumulation in MDR ovarian cancer cells. Panel A: Calcein-AM accumulation. Calcein AM accumulation upon P-gp inhibition was analyzed as described in methods using A2780ADR cells. All experiments had identical components except for the additions indicated. Additions were: DMSO, carrier vehicle at 0.5% final volume; compounds 19, 29, 34 or 45 at 10 µM; VER, 10 μM verapamil; NOV, 60 μM novobiocin; PRO, 250 μM probenecid. Scale bars are 200 µm. Panel B: Time course of calcein accumulation. Fluorescence measurements were made on the entire wells during the imaging experiments described in panel A. The increase in relative fluorescence resulting from accumulated calcein is plotted versus the time of incubation. The indicated additions were as described in panel A. Panel C: Daunorubicin accumulation. Intracellular daunorubicin accumulation in A2780ADR cells was observed similarly to the accumulation of calcein (see panel A). After a 2 hour incubation with 10 µM daunorubicin, fluorescence images of the cells were obtained using a Texas Red filter. Additions were as indicated in panel A. Panel D: Quantification of intracellular daunorubicin accumulation. A2780ADR cells were incubated as described for panel C. After the 2 hour incubation, cells were washed twice with cold RPMI to remove extracellular daunorubicin, and then lysed. The fluorescence of each well was measured at excitation 488/20 nm and emission 575/20 nm. Percent accumulation of daunorubicin in A2780ADR cells was normalized to the parental A2780 cells. Additions were as indicated in panel A. Each histogram represents the average ± S.D. from two independent experiments (n = 6; ****P < 0.0001).