Tyrphostin RG14620 selectively reverses ABCG2-mediated multidrug resistance in cancer cell lines

Abstract

The multidrug resistance (MDR) phenotype associated with the overexpression of ATP-binding cassette (ABC) drug transporters ABCB1, ABCC1 and ABCG2 is a major obstacle in cancer chemotherapy. Numerous epidermal growth factor receptor (EGFR) inhibitors have previously been shown capable of reversing MDR in ABCG2-overexpressing cancer cells. However, most of them are not transporter-specific due to the substantial overlapping substrate specificity among the transporters. In this study, we investigated the interaction between ABCG2 and tyrphostin RG14620, an EGFR inhibitor of the tyrphostin family, in multidrug-resistant cancer cell lines. We found that at nontoxic concentrations, tyrphostin RG14620 enhances drug-induced apoptosis and restores chemosensitivity to ABCG2-overexpressing multidrug-resistant cancer cells. More importantly, tyrphostin RG14620 is selective to ABCG2 relative to ABCB1 and ABCC1. Our findings were further supported by biochemical assays demonstrating that tyrphostin RG14620 stimulates ATP hydrolysis and inhibits photoaffinity labeling of ABCG2 with IAAP, and by a docking analysis of tyrphostin RG14620 in the drug-binding pocket of this transporter. Taken together, our findings indicate that tyrphostin RG14620 is a potent and selective modulator of ABCG2 that may be useful to overcome chemoresistance in patients with drug-resistant tumors.

Keywords: ABC transporter; ABCG2; EGFR inhibitor; Multidrug resistance; Tyrphostin RG14620.

Fig. 1

Tyrphostin RG14620 inhibits ABCG2-mediated fluorescent drug efflux. The accumulation of fluorescent calcein (0.5 μM calcein-AM) in HEK293 cells (A and B, left panels), ABCB1-transfected MDR19-HEK293 cells (A, right panel) and ABCC1-transfected MRP1-HEK293 cells (B, right panel), or 1 μM pheophorbide A (PhA) in HEK293 cells (C, left panel) and in ABCG2-transfected R482-HEK293 cells (C, right panel), or in human colon S1 cells (D, left panel) and their ABCG2-overexpressing variant S1-M1-80 cancer cells (D, right panel), or in human lung H460 cells (E, left panel) and their ABCG2-overexpressing variant H460-MX20 cancer cells (E, right panel), was measured in the absence (solid lines) or presence of 20 μM tyrphostin RG14620 (shaded, solid lines) or a reference inhibitor (dotted lines) for ABCB1 (3 μM tariquidar), ABCC1 (25 μM MK-571) or ABCG2 (3 μM Ko143), and analyzed immediately by flow cytometry as described previously [68]. Representative histograms of three independent experiments are shown.

Fig. 2

Effect of tyrphostin RG14620 on drug resistance mediated by ABCB1, ABCC1 or ABCG2. Drug-sensitive parental HEK293 cells (A-C, left panels) and HEK293 cells transfected with human ABCB1 (A, right panel), ABCC1 (B, right panel) or ABCG2 (C, right panel), as well as (D) drug-sensitive S1 colon cells (left panel) and their ABCG2-overexpressing variant S1-M1-80 cells (right panel) and (E) drug-sensitive H460 cells (left panel) and their ABCG2-overexpressing variant H460-MX20 (right panel) lung cancer cells were treated with doxorubicin, etoposide or mitoxantrone in the absence (open circles) or presence of tyrphostin RG14620 at 0.1 μM (filled circles), 0.2 μM (open squares), 0.5 μM (filled squares) 1.0 μM (open triangles) or 2.0 μM (filled triangles), respectively, as described in Materials and methods. Points, mean values from at least three independent experiments; bars; SEM.

Fig. 3

Effect of tyrphostin RG14620 on ABCG2 protein expression in human cancer cell lines. Immunoblot detection of human ABCG2 protein expression in (A) ABCG2-overexpressing H460-MX20 lung cancer cells and (B) quantification of ABCG2 in total cell lysate protein (10 μg/lane) from cells treated with DMSO (vehicle control) or increasing concentrations of tyrphostin RG14620 (RG) for 72 h as described previously [68]. α-tubulin was used as an internal control for equal loading. Values are presented as mean ± SEM calculated from three independent experiments.

Fig. 4

Tyrphostin RG14620 enhances topotecan-induced apoptosis in ABCG2-overexpressing S1-M1-80 cancer cells. (A) Drug-sensitive human colon S1 cancer cells (top panels) and ABCG2-overexpressing drug-resistant variant S1-M1-80 cancer cells (lower panels) were treated with either DMSO (control), 5 μM tyrphostin RG14620 (+ RG14620), 5 μM topotecan (+ topotecan) or a combination of 5 μM topotecan and 5 μM of tyrphostin RG14620 (topotecan + RG14620) for 48 h. Cells were isolated and analyzed by flow cytometry as described previously [25]. Representative dot plots and the mean values of three independent experiments are shown. (B) Quantification of topotecan-induced apoptosis in human colon S1 and S1-M1-80 cells. Values are presented as mean ± S.D. calculated from three independent experiments. ^**p < 0.01; ^***p < 0.001, versus the same treatment without tyrphostin RG14620.

Fig. 5

Tyrphostin RG14620 stimulates vanadate-sensitive ABCG2 ATPase activity. The effect of 0 – 5 μM of tyrphostin RG14620 on vanadate-sensitive ABCG2 ATP hydrolysis was determined by endpoint P_i assay, as described previously [3]. Points indicate the mean of at least three independent experiments; bars indicate the SEM. The inset shows the ATPase activity in the presence of lower concentrations from 0 to 0.5 μM of RG compound.

Fig. 6

Binding of tyrphostin RG14620 to ABCG2 protein. (A) Tyrphostin RG14620 (RG14620) inhibits photoaffinity labeling of ABCG2 with [¹²⁵I]IAAP. Membrane protein (50–75 μg protein) from MCF-7-FLV1000 cells was incubated in the absence or presence of 10 μM of RG14620 before 3–6 nM [¹²⁵I]IAAP (2,200 Ci/mmol) was added to the samples, as detailed in Materials and methods. Photo-labeled ABCG2 was processed and visualized as described previously [46, 68]. Representative autoradiogram and values represent mean ± S.D. calculated from three independent experiments. (B) Binding mode of RG14620 with human ABCG2 structure (PDB: 5NJG) as predicted by Acclerys Discovery Studio 4.0 software as described in Materials and methods. RG14620 is shown as a molecular model with the atoms colored as carbon- gray, hydrogen-light gray, chlorine-green, sulfur-yellow, nitrogen-blue and oxygen-red. The same color scheme is used for interacting amino acid residues.

Fig. 7

Comparison of the levels of anticancer drug accumulation in ABCG2-overexpressing MDR cancer cells in the absence or presence of tyrphostin RG14620. In the absence of tyrphostin RG14620 (left panel), anticancer drugs (red circles) are transported out of cancer cells by ABCG2, thereby reducing their intracellular concentration and rendering chemotherapy ineffective. In the presence of tyrphostin RG14620 (green triangle, right panel), anticancer drugs are no longer pumped out by ABCG2, resulting in effective killing of cancer cells.