Telatinib reverses chemotherapeutic multidrug resistance mediated by ABCG2 efflux transporter in vitro and in vivo

Abstract

Multidrug resistance (MDR) is a phenomenon where cancer cells become simultaneously resistant to anticancer drugs with different structures and mechanisms of action. MDR has been shown to be associated with overexpression of ATP-binding cassette (ABC) transporters. Here, we report that telatinib, a small molecule tyrosine kinase inhibitor, enhances the anticancer activity of ABCG2 substrate anticancer drugs by inhibiting ABCG2 efflux transporter activity. Co-incubation of ABCG2-overexpressing drug resistant cell lines with telatinib and ABCG2 substrate anticancer drugs significantly reduced cellular viability, whereas telatinib alone did not significantly affect drug sensitive and drug resistant cell lines. Telatinib at 1 μM did not significantly alter the expression of ABCG2 in ABCG2-overexpressing cell lines. Telatinib at 1 μM significantly enhanced the intracellular accumulation of [(3)H]-mitoxantrone (MX) in ABCG2-overexpressing cell lines. In addition, telatinib at 1 μM significantly reduced the rate of [(3)H]-MX efflux from ABCG2-overexpressing cells. Furthermore, telatinib significantly inhibited ABCG2-mediated transport of [(3)H]-E₂17βG in ABCG2 overexpressing membrane vesicles. Telatinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner, indicating that telatinib might be a substrate of ABCG2. Binding interactions of telatinib were found to be in transmembrane region of homology modeled human ABCG2. In addition, telatinib (15 mg/kg) with doxorubicin (1.8 mg/kg) significantly decreased the growth rate and tumor size of ABCG2 overexpressing tumors in a xenograft nude mouse model. These results, provided that they can be translated to humans, suggesting that telatinib, in combination with specific ABCG2 substrate drugs may be useful in treating tumors that overexpress ABCG2.

Keywords: ABC transporter; ABCG2; Multidrug resistance; Telatinib; Tyrosine kinase inhibitor.

Figure 1. The effect of telatinib on intracellular levels and efflux of [³H]-mitoxantrone

(A) The accumulation [³H]-MX in empty vector transfected HEK293/pcDNA3.1, ABCG2-482-R2, ABCG2-482-G2 and ABCG2- 482-T7 cells with or without telatinib treatment. Columns are the mean of triplicate determinations. *, P < 0.05 versus the control group. (B) The effect of telatinib at 1 μM on retention of [³H]-MX over a period of time in HEK293/pcDNA3.1 and ABCG2-482-R2. Data points represent the means ± SD. *, P < 0.05 versus the respective time point of control group. The figures are a representative of three independent experiments each done in triplicates.

Figure 2. The effect of telatinib on ABCG2 protein expression and localization

ABCG2-482-R2 cells were treated with (A) telatinib upto 1 μM for 72 h (upper panel) (B) and telatinib 1 μM over a period of 72 h (upper panel). Quantification of the obtained blots from three independent experiments is shown (lower panel). (C) The localization of ABCG2 by Immunofluorescence in H460 and H460/MX20 after treatment with telatinib 1μM for 72 h. Immunofluorescence staining of cells with primary antibody against ABCG2 and Alexa Fluor 488-conjugated secondary antibody was observed by confocal microscope. ABCG2 specific staining is shown in green and the nuclear DNA stained by DAPI is shown in blue. Representative results are shown and similar results were obtained in two other trials.

Figure 3. The effect of telatinib on ATPase activity of ABCG2

The Vi-sensitive ATPase activity of ABCG2 in membrane vesicles was determined Mean values are given, and the error bars represent standard error from three independent experiments.

Figure 4. Effect of telatinib on the ATP-dependent transport of [³H]-E₂17βG

Membrane vesicles (10 μg) were prepared from HEK293/pcDNA3.1 and ABCG2- 482-R2 cells. The rate of uptake of [³H]-E₂17βG into membrane vesicles was measured for 10 min at 37 °C in uptake medium containing 4 mM of ATP or AMP. For inhibition experiments, membrane vesicles from HEK293/pcDNA3.1 and ABCG2-482-R2 cells were incubated with telatinib or FTC for 1 h on ice, and then transport reactions were carried out for 10 min at 37 °C in an uptake medium containing 4 mM ATP. Each column represent the mean of triplicate determinations and the bars represent SD. *P < 0.05, versus the control group. Experiments were repeated at least three times and a representative experiment is shown.

Figure 5. Glide predicted binding mode of telatinib with homology modeled ABCG2

(A) The docked conformation of telatinib as ball and stick model is shown within the transmembrane region of ABCG2. Important amino acids are depicted as sticks with the atoms colored as carbon – green, hydrogen – white, nitrogen – blue and oxygen – red), whereas the telatinib is shown with the same color scheme as above except carbon atoms are represented in orange and chlorine-dark green. (B) Schematic diagram of the important interactions observed in the complex of telatinib with the binding site residues of human ABCG2.

Figure 6. The effect of telatinib on H460 and H460/MX20 tumor xenograft growth rate

(A) Changes in tumor volume with time in H460 xenograft are shown. (B) Changes in tumor volume with time in H460/MX20 xenograft are shown. Points represent mean tumor volume for each group after implantation. Each point on line graph represent the mean tumor volume (mm³) at a particular day after implantation and the bars represent SD. (C) A representative picture of the excised H460 tumor sizes from different mice is shown on the 18^th day after implantation. (D) A representative picture of the excised H460/MX20 tumor sizes from different mice is shown on the 18^th day after implantation. The treatment were as follows: (a) vehicle (q3d X 6), (b) DOX (1.8 mg/kg, i.p., q3d X 6), (c) telatinib (15 mg/kg, p.o., every 2^nd and 3^rd day) and (d) DOX (1.8 mg/kg, i.p., q3d X 6) + telatinib (15 mg/kg, p.o., every 2^nd and 3^rd day, given 1 h before giving DOX). Each column represents the mean determinations and the bars represent SD. Data are means ± SD for 8 animals. At least two independent experiments were carried out using athymic NCR nude mice. The statistical analysis was carried out on day 18. *, P < 0.05 versus vehicle group; #, P < 0.05 versus DOX and telatinib group in Fig (A) and (B), respectively.

Figure 7. The effect of telatinib on H460 and H460/MX20 tumor xenograft mice

(A) The bar graph represents the mean tumor weight (n=8) of the excised H460 tumor from different mice. (B) The bar graph represents the mean tumor weight (n=8) of the excised H460/MX20 tumor from different mice. (C) Changes in mean body weight before and after treatment for xenograft model are shown in the bar graph. (D) ABCG2 expression analysis by immunohistochemistry in tumor tissues collected from different groups at the end of the xenograft study. There was no ABCG2 expression in the H460 tumors (upper row), while ABCG2 expressed specifically in the cell membrane of H460/MX 20 tumors (lower low, arrows). *, P < 0.05 versus vehicle group; #, P < 0.05 versus DOX and telatinib group in Fig (A) and (B), respectively.