Olmutinib (HM61713) reversed multidrug resistance by inhibiting the activity of ATP-binding cassette subfamily G member 2 in vitro and in vivo

Abstract

Overexpressing of ATP-binding cassette (ABC) transporters is the essential cause of multidrug resistance (MDR), which is a significant hurdle to the success of chemotherapy in many cancers. Therefore, inhibiting the activity of ABC transporters may be a logical approach to circumvent MDR. Olmutinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), which has been approved in South Korea for advanced EGFR T790M-positive non-small cell lung cancer (NSCLC). Here, we found that olmutinib significantly increased the sensitivity of chemotherapy drug in ABCG2-overexpressing cells. Furthermore, olmutinib could also increase the retention of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABC transporter subfamily G member 2 (ABCG2)-overexpressing cells. In addition, olmutinib was found to stimulate ATPase activity and inhibit photolabeling of ABCG2 with [¹²⁵I]-iodoarylazidoprazosin (IAAP). However, olmutinib neither altered ABCG2 expression at protein and mRNA levels nor blocked EGFR, Her-2 downstream signaling of AKT and ERK. Importantly, olmutinib enhanced the efficacy of topotecan on the inhibition of S1-MI-80 cell xenograft growth. All the results suggest that olmutinib reverses ABCG2-mediated MDR by binding to ATP bind site of ABCG2 and increasing intracellular chemotherapeutic drug accumulation. Our findings encouraged to further clinical investigation on combination therapy of olmutinib with conventional chemotherapeutic drugs in ABCG2-overexpressing cancer patients.

Keywords: ABC, adenosine triphosphate (ATP)-binding cassette; ABCG2; ABCG2, ABC transporter subfamily G member 2; ATPase; Chemotherapy; DDP, cisplatin; DMEM, Dulbecco׳s modified Eagle׳s medium; DMSO, dimethyl sulfoxide; DOX, doxorubicin; FTC, fumitremorgin C; IAAP, iodoarylazidoprazosin; MDR, multidrug resistance; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide; MX, methotrexate; Multidrug resistance; Olmutinib; PCR, polymerase chain reaction; Rho 123, rhodamine 123; TKI, tyrosine kinase inhibitor; Tyrosine kinase inhibitor; VRP, verapamil.

Graphical abstract

Figure 1

The structure of olmutinib and cytotoxicity of olmutinib. (A) The structure of olmutinib. MTT cytotoxicity assay was assessed in ABCG2 and ABCB1-overexpressing cells and their parental sensitive cells: (B) ABCB1-negative KB and ABCB1-overexpressing KBv200 cells; (C) ABCG2-negative H460 and ABCG2-overexpressing H460/MX20 cells; (D) ABCG2-negative S1 and ABCG2-overexpressing S1-MI-80 cells; (E) ABCB1-negative HEK293/pcDNA3.1 and ABCG2-overexpressing Wild-type ABCG2–482-T7 and; (F) ABCG2-negative HEK293/pcDNA3.1 and ABCG2-overexpressing mutant ABCG2–482-R2 cells. Cells were treated with varying concentrations of olmutinib for 72 h. Results from three independent experiments are expressed as the mean ± SD.

Figure 2

Olmutinib enhanced the anticancer effect of topotecan in the S1-MI-80 cell xenograft model in nude mice. (A) The changes in tumor volume over time after the S1-MI-80 cell implantation (n = 6). Data shown are mean ± SD of tumor volumes for each group. (B) The image of tumors size in four groups excised from the mice on the 75th day after implantation. (C) Average percentage change in bodyweight after treatments. (D) Mean tumor weight after excising from the mice on the 75th day after implantation (n = 6). The fourtreatment groups were: (1) control (vehicle of olmutinib, p.o., every 5 day and saline i.p. every 5 day); (2) olmutinib (30 mg/kg, p.o., every 5 day); (3) topotecan (2 mg/kg, i.p., every 5 day) and (4) topotecan (2 mg/kg, i.p., every 5 day)+olmutinib (30 mg/kg, p.o., every 5 day given 1 h before giving topotecan). Results are presented as the mean±SD. ^**P<0.01 significantly different from control group.

Figure 3

Effect of olmutinib on the intracellular accumulation of DOX, Rho 123 in MDR cells and their parental cells. The accumulation of DOX (A), Rho 123 (B) in S1 and S1-MI-80 cells was measured by flow cytometric analysis as described in Section Material and methods. The results were presented as fold change in fluorescence intensity relative to control MDR cells. Data are expressed as mean ± SD from three independent experiments; ^**P<0.01 significantly different from control group.

Figure 4

Effect of olmutinib on the efflux of Rho 123, the ATPase activity and the [¹²⁵I]-IAAP photoaffinity labeling of ABCG2. (A) Time course (0, 30, 60, 90, 120 min) of Rho 123 efflux was measured in S1 and S1-MI-80 cells, with or without 1 µmol/L olmutinib. Data are expressed as mean±standard. (B) Olmutinib competed for photolabeling of ABCG2 by [¹²⁵I]-IAAP crude membranes from ABCG2-overexpressing MCF7/FLV1000 cells were incubated with [¹²⁵I]-IAAP and a range of different concentration (0–5 µmol/L) of olmutinib. The samples were then cross-linked by UV illumination, and subjected to SDS-PAGE. A representative autoradiogram from three independent experiments was shown. The relative amount of [¹²⁵I]-IAAP incorporated is plotted against the concentration of olmutinib used in the competition. 100% incorporation refers to the absence of olmutinib. (C) Effect of olmutinib on ABCG2 ATPase activity. The vanadate-sensitive ABCG2 ATPase activity in the presence of the indicated concentrations of olmutinib was evaluated. Data are expressed as mean±SD from three independent experiments.

Figure 5

Effect of olmutinib on the expression of ABCG2 in MDR cells. (A) The protein levels of ABCG2 in MDR cells after different concentrations olmutinib stimulation for 48 h were measured by Western blot analysis (GAPDH as loading control). (B) Olmutinib did not alter the mRNA and protein levels in MDR cells in concentration dependent manners. Real time-PCR was further applied to confirm the unaltered mRNA levels in MDR cells. (C) The cell surface expression of ABCG2 were measured by fow cytometry before and after olmutinib stimulation on MDR cells and their parental cells. All these experiments were repeated at least three times, and representative images and densitometry results are shown in each panel.

Figure 6

Effect of olmutinib on AKT, ERK, and their phosphorylations in MDR and the parental cells. S1-MI-80 and S1 cells were treated with different concentrations of olmutinib for 48 h. (A) The total and the phosphorylation protein level of AKT and ERK were detected by Western blot (GAPDH as loading control). All these experiments were repeated at least three times. (B) Quantitative analysis of AKT, ERK and their phosphorylation.