Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters

Abstract

Apatinib, a small-molecule multitargeted tyrosine kinase inhibitor, is in phase III clinical trial for the treatment of patients with non-small-cell lung cancer and gastric cancer in China. In this study, we determined the effect of apatinib on the interaction of specific antineoplastic compounds with P-glycoprotein (ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). Our results showed that apatinib significantly enhanced the cytotoxicity of ABCB1 or ABCG2 substrate drugs in KBv200, MCF-7/adr, and HEK293/ABCB1 cells overexpressing ABCB1 and in S1-M1-80, MCF-7/FLV1000, and HEK293/ABCG2-R2 cells overexpressing ABCG2 (wild-type). In contrast, apatinib did not alter the cytotoxicity of specific substrates in the parental cells and cells overexpressing ABCC1. Apatinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin in the multidrug resistance (MDR) cells. Furthermore, apatinib significantly inhibited the photoaffinity labeling of both ABCB1 and ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. The ATPase activity of both ABCB1 and ABCG2 was significantly increased by apatinib. However, apatinib, at a concentration that produced a reversal of MDR, did not significantly alter the ABCB1 or ABCG2 protein or mRNA expression levels or the phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Importantly, apatinib significantly enhanced the effect of paclitaxel against the ABCB1-resistant KBv200 cancer cell xenografts in nude mice. In conclusion, apatinib reverses ABCB1- and ABCG2-mediated MDR by inhibiting their transport function, but not by blocking the AKT or ERK1/2 pathway or downregulating ABCB1 or ABCG2 expression. Apatinib may be useful in circumventing MDR to other conventional antineoplastic drugs.

Figure 1. Effect of apatinib on the intracellular accumulation of DOX and Rho 123

The accumulation of DOX (A) and Rho 123 (B) was measured by flow cytometric analysis after cells were pre-incubated with or without apatinib, verapamil (VRP) or fumitremorgin C (FTC) for 3 h at 37°C and then incubated with 10 µM DOX or 5 µM Rho 123 for another 3 h or 0.5 h at 37°C, respectively, as described in “Materials and Methods”. The results are presented as fold change in fluorescence intensity relative to untreated control MDR cells. Columns, means of triplicate determinations; bars, SD. **, P < 0.01, versus the MDR control group. Independent experiments were performed at least three times, and a representative experiment is shown.

Figure 2. Effect of apatinib on the ATPase activity ABCB1 and ABCG2, the photoaffinity labeling of ABCB1 and ABCG2 with [¹²⁵I]-Iodoarylazidoprazosin ([¹²⁵I]-IAAP) and the transport kinetics of DOX mediated by the ABCB1 and ABCG2 transporter

(A) Vi-sensitive ATPase activity of ABCB1. (B) beryllium fluoride-sensitive ATPase activity of ABCG2. (B, inset) Effect of lower concentrations of apatinib on ABCG2 ATPase activity. The amount of inorganic phosphate released was quantitated using a colorimetric method. The photoaffinity labeling of ABCB1 (C) and ABCG2 (D) with [¹²⁵I]IAAP was performed using indicated concentrations of apatinib as described in Materials and Methods. The amount of inorganic phosphate released was quantitated using a colorimetric method. Crude membranes from High Five insect cells expressing ABCB1 (C) and from MCF7/FLV1000 cells expressing ABCG2 (D) were incubated with various concentrations of apatinib for 10 min at room temperature, and 3 to 6 nM [¹²⁵I]-IAAP (2,200 Ci/mmol) were then added before illuminated with a UV lamp (365 nm) as described in Materials and Methods. In all three panels, lane 1 is control without apatinib. Effect of apatinib on the transport kinetics of intracellular DOX efflux mediated by the ABCB1 (E) and ABCG2 (F) transporter. The quantity of efflux DOX in MDR cells was measured for 5 min at 37°C at various DOX concentrations (2.5–20 µM) in the presence or absence of apatinib by flow cytometry. Data represent mean ± SD in at least three different experiments. The bars represent the SD value. The K_i values were determined from the double reciprocal Lineweaver-Burk plots in the absence (◆) or presence of 3 µM apatinib (▲).

Figure 3. Effect of apatinib on the expression of ABCB1 and ABCG2 in MDR cells at the protein (A) and mRNA (B) levels

KBv200, MCF-7/adr and S1-M1-80 cells were treated with apatinib at the indicated concentrations for 48 h. A representative result from at least three independent experiments is shown.

Figure 4. Effect of apatinib on the phosphorylation of AKT and ERK1/2

Equal amount of protein was loaded for Western blot analysis as described in “Materials and Methods”. Independent experiments were performed at least three times and result from a representative experiment is shown. 1, untreated control; 2, 0.75 µM apatinib; 3, 1.5 µM apatinib; 4, 3.0 µM apatinib; 5, 10 µM apatinib; and 6, 15 µM apatinib.

Figure 5. Potentiation of antitumor effects of paclitaxel by apatinib in a KBv200 xenograft model in athymic nude mice

(A) Changes in tumor volume with time after tumor cell inoculation. Data points represent mean tumor volume for each group of 12 mice after implantation; bars, SD. (B) Tumor size. The picture was taken on the 17th day after implantation. The various treatments were as follows: control (vehicle alone), apatinib (70 mg/kg,p.o., q3d × 4); paclitaxel (18 mg/kg, i.p., q3d × 4) and paclitaxel (18 mg/kg, i.p., q3d × 4) plus apatinib (70 mg/kg, p.o., q3d × 4, given 1 h before paclitaxel administration).