Inhibiting the function of ABCB1 and ABCG2 by the EGFR tyrosine kinase inhibitor AG1478

Abstract

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific EGFR tyrosine kinase inhibitor (TKI); its promising pre-clinical results have led to clinical trials. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCC1 and ABCG2 is one of the main causes of multidrug resistance (MDR) and usually results in the failure of cancer chemotherapy. However, the interaction of AG1478 with these ABC transporters is still unclear. In the present study, we have investigated this interaction and found that AG1478 has differential effects on these transporters. In ABCB1-overexpressing cells, non-toxic doses of AG1478 were found to partially inhibit resistance to ABCB1 substrate anticancer drugs as well as increase intracellular accumulation of [3H]-paclitaxel. Similarly, in ABCG2-overexpressing cells, AG1478 significantly reversed resistance to ABCG2 substrate anticancer drugs and increased intracellular accumulation of [3H]-mitoxantrone as well as fluorescent compound BODIPY-prazosin. AG1478 also profoundly inhibited the transport of [3H]-E(2)17betaG and [3H]-methotrexate by ABCG2. We also found that AG1478 slightly stimulated ABCB1 ATPase activity and significantly stimulated ABCG2 ATPase activity. Interestingly, AG1478 did not inhibit the photolabeling of ABCB1 or ABCG2 with [125I]-iodoarylazidoprazosin. Additionally, AG1478 did not alter the sensitivity of parental, ABCB1- or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drug and had no effect on the function of ABCC1. Overall, we conclude that AG1478 is able to inhibit the function of ABCB1 and ABCG2, with a more pronounced effect on ABCG2. Our findings provide valuable contributions to the development of safer and more effective EGFR TKIs for use as anticancer agents in the clinic.

Figure 1. Effect of AG1478 on the survival of the overexpressing ABCB1 or ABCG2 cells

The results of MTT cytotoxicity (A, C) and colony formation assays (B, D) were used to measure cell survival as described in “Materials and Methods”. Data points represent the means ± SD of triplicate determinations. The representative results from three independent experiments are shown.

Figure 2. AG1478 increases the accumulation of [³H]-paclitaxel in ABCB1 overexpressing cells, [³H]-mitoxantrone in the ABCG2 overexpressing cells

The accumulation of [³H]-paclitaxel (A) or [³H]-mitoxantrone (B) was measured after cells were pre-incubated with or without AG1478, verapamil or FTC for 1 h at 37°C and then incubated with 0.1 μM [³H]-paclitaxel or 0.02 μM [³H]-mitoxantrone for another 2 h at 37°C. Data points represent the means ± SD of triplicate determinations. * and ** represent p < 0.05 and p < 0.01, respectively, for values versus those in the control group. Independent experiments were performed at least three times, and a representative experiment is shown.

Figure 3. AG1478 inhibits the ABCG2-mediated efflux of BODIPY-prazosin and transport of E₂17βG and methotrexate

(A) Each cell lines were incubated in 250 nM BODIPY-prazosin alone (heavy solid line) or with 2.5 μM AG1478 (solid line), 10 μM AG1478 (dashed line) and 10 μM FTC (shaded histogram) for 30 min at 37°C, after which the cells were washed, and allowed to efflux for 1 h at 37°C in substrate-free media continuing with or without inhibitors. All samples were analyzed on a flow cytometer. (B) Membrane vesicles were prepared from HEK293/pcDNA3.1 and ABCG2-482-R5 cells. The rates of the uptake of [³H]-E₂17βG and [³H]-methotrexate into membrane vesicles (10 μg protein/reaction) with different concentrations of AG1478 and FTC were measured as described in “Materials and Methods”. Data points represent the means ± SD of triplicate determinations. * and ** represent p < 0.05 and p < 0.01 respectively, for values versus those in the control group. At least three independent experiments were performed, and a representative experiment is shown.

Figure 4. AG1478 does not affect the expression and localization of ABCB1 and ABCG2

(A) KB-C2 (upper panel) and HEK293/ABCG2-482-R5 (lower panel) cells were treated as indicated and Western Blot was performed. Results from a representative experiment are shown. Similar results were obtained in two other trials. (B) The localization of ABCB1 and ABCG2 by immunofluorescence was done on paraformaldehyde fixed cells using the indicated antibodies as outlined in “Materials and Methods”. ABCB1 and ABCG2 specific staining is shown in green and the nuclear DNA stained by propidium iodide is shown in red.

Figure 5. Effect of AG1478 with or without verapamil or FTC on the ATPase activity of ABCB1 and ABCG2

The Vi-sensitive ATPase activity of ABCB1 and ABCG2 with different concentrations of AG1478 (A), and in the presence of 5 μM AG1478, or 50 μM verapamil, or both together (for ABCB1), and 10 μM FTC (for ABCG2) alone or in combination with 5 μM AG1478 (B)were measured as described in ‘Materials and methods’. Mean values are given, and the error bars represent standard error from at least three independent experiments.

Figure 6. AG1478 does not affect the [¹²⁵I]-IAAP photoaffinity labeling of ABCB1 and ABCG2

The photoaffinity labeling of ABCB1 (A) and ABCG2 (B) with [¹²⁵I]-IAAP was performed as mentioned in “Materials and Methods”. The lower panels show the quantify results from at least two independent experiments. Cyclosporine A, verapamil and FTC were used as positive controls. CSA: cyclosporine A; Vera: verapamil. * and ** represent p < 0.05 and p < 0.01, respectively, for values versus those in the control group.