HG-829 is a potent noncompetitive inhibitor of the ATP-binding cassette multidrug resistance transporter ABCB1

Abstract

Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrug-resistant malignancies.

Figure 1

HG-829 does not alter cellular expression of Pgp. A, the original compound HG-829 (PGE2799041) was used after conversion to its hydrochloride salt. B, K562-R cells overexpress Pgp (shaded dark histogram) compared with the parental cell line (shaded light histogram). Treatment with HG-829, 1 μmol/L, for 48 hours did not affect Pgp expression in the parental (dotted line) or resistant cell line (heavy solid line). C, Pgp protein levels were not affected by HG-829 as shown by Western blotting in K562-R cells treated for 24 hours with 0.5, 1, 2.5 μmol/L, or drug vehicle (dimethyl sulfoxide). K562-S did not express Pgp. Densitometric analysis is representative of 2 independent experiments (mean ± SEM).

Figure 2

HG-829 inhibits Pgp-mediated rhodamine efflux in Pgp-overexpressing cells. K562-R (A) and MDR-1–transfected HEK-293 cells (MDR-19; B) were incubated with 0.5 μg/mL rhodamine 123; cells were then washed and incubated in rhodamine-free medium with or without modulators for 1 hour. Both cell lines export rhodamine (shaded histogram) after rhodamine intake (dotted histogram). Rhodamine efflux is inhibited in the presence of HG-829, 2.5 μmol/L in the media (heavy solid line; K562-R, P = 0.03; MDR-19, P < 0.01) and to a lesser degree in the presence of 2.5 μmol/L cyclosporin A (CsA; dashed lines). Representative results from 1 of at least 3 experiments are shown. C, HG-829 significantly inhibited rhodamine efflux at concentrations as low as 0.07 μmol/L. Values represent mean ± SEM of 4 independent experiments. Efflux comparison versus media alone: HG-829 2.5 μmol/L, P = 0.002; 1.25 μmol/L, P = 0.003; 0.62 μmol/L, P = 0.002; 0.31 μmol/L, P = 0.002; 0.15 μmol/L, P = 0.01; 0.07 μmol/L, P = 0.02; cyclosporin A 2.5 μmol/L, P = 0.023. The zero values represent rhodamine intake (dotted) and efflux (white) without any modulator (D) HG-829 shows prolonged inhibition of Pgp-mediated rhodamine efflux. Cells were incubated with 2.5 μmol/L HG-829 or cyclosporin A for 1 hour, followed by 0.5 μg/mL rhodamine 123. Control samples were incubated with rhodamine alone. Cells were then washed and incubated in rhodamine-free medium. HG-829 promoted rhodamine retention for at least 8 hours (1 hour, P < 0.0001; 2 hours, P < 0.0001; 4 hours, P = 0.0374; 8 hours, P = 0.0145). Cyclosporin A only showed statistically significant retention at 1 hour (P = 0.0002). Values represent media ± SEM of 3 independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 3

Kinetics of calcein AM uptake by K562-R cells. A, persistent exclusion of calcein AM is observed in the resistant cells (circles). The presence of HG-829 significantly increased cellular fluorescence, HG-829 0.5 μmol/L (inverted triangles), 1 μmol/L (squares), 1.5 μmol/L (rhomboids), 2 μmol/L (stars). Data represent the mean of triplicates. B, HG-829 significantly increased accumulation of calcein AM in cells (0.5 μmol/L, P = 0.0015 and 1 μmol/L, P = 0.0019) compared with control. Cyclosporin A (CsA) significantly increased calcein AM accumulation at 1 μmol/L (P = 0.05). Bars represent the mean (expressed as percentage of control) ± SD of 2 independent experiments with 5 replicates at 19 minutes. C, HG-829 displays noncompetitive efflux kinetics. Double reciprocal plot of rate versus calcein AM concentration at several HG-829 concentrations. No inhibitor (circles), HG-829 0.5 μmol/L (inverted triangles), 1 μmol/L (squares), 1.5 μmol/L (rhomboids), and 2 μmol/L (stars). The data illustrated are representative results from 1 of 3 experiments. DMSO, dimethyl sulfoxide.

Figure 4

HG-829 enhances antineoplastic cytotoxicity in cells that overexpress Pgp. K562-R (A); MDR-1–transfected HEK-293 cells (MDR-19; B); H460/VBL (C). Cells were treated with HG-829 or cyclosporin A as control and the respective drug for 72 hours, then incubated with MTT dye, and quantified for the level of MTT uptake. Parental cells (open circles); resistant cells (filled circles); HG-829 0.5 μmol/L (triangles); and HG-829 1 μmol/L (hatched squares). The mean of 4 wells was calculated for each concentration of the drug and reported as the percentage of control. Data represent media ±SEM of 2 independent experiments. Each experiment was repeated 4 to 6 times.

Figure 5

HG-829 enhancement of antineoplastic cytotoxicity extends to a wide range of Pgp substrates, including paclitaxel (A), vincristine (B), and etoposide (C) and cells overexpressing MRP1 (ABCG1; D) or breast cancer resistance protein (ABCG2; E). Cells were treated with HG-829 and corresponding antineoplastic for 72 hours, then incubated with MTT dye, and quantified for level of MTT uptake. Parental cells (open circles); resistant cells (filled circles); HG-829 0.5 μmol/L (triangles); HG-829 1 μmol/L (hatched squares); HG-829 2 μmol/L (inverted triangles); HG-829 3 μmol/L (open rhomboids); and HG-829 4 μmol/L (asterisks). The mean of 4 wells was calculated for each concentration of the drug and reported as the percentage of control. Data represent media ±SEM of 2 independent experiments. Each experiment was repeated 4 to 6 times.

Figure 6

HG-829 inhibits Pgp activity in vivo. SCID mice were implanted s.c. with K562-R (A) and K562 parental cells (B). There was a significant decrease in tumor size of K562-R xenografts treated with HG-829 þ daunorubicin versus vehicle (P < 0.01) and versus daunorubicin or HG-829 alone. The animals implanted with K562 parental cells responded to daunorubicin treatment with no statistical difference in tumor size between daunorubicin + HG-829 or daunorubicin alone. Daunorubicin + HG-829 versus HG-829 vehicle, P < 0.01; daunorubicin versus HG-829 vehicle, P < 0.01; daunorubicin + HG-829 versus HG-829 alone, P < 0.01; daunorubicin versus HG-829 alone, P < 0.05. Daunorubicin 2 mg/kg i.p. every other day (open circles); daunorubicin + HG-829 (filled asterisks); HG-829 25 mg/kg i.p. per day (triangle); and HG-829 vehicle (inverted triangle). Values represent the media ± SEM of 7 animals per group. Similar results were obtained in 2 independent experiments.