Using LC-MS/MS-based targeted proteomics to monitor the pattern of ABC transporters expression in the development of drug resistance

Abstract

Purpose: The overexpression of ATP-binding cassette transporters (ABC transporters), mainly including permeability glycoproteins (P-gp), multidrug resistance (MDR)-related protein 1 (MRP1), and breast cancer resistance proteins (BCRP), is one of the main reasons for the development of MDR which directly leads to chemotherapy failure. However, most of the currently used detection methods in MDR-related studies are qualitative or semiquantitative, but not quantitative. As a result, the measurement criteria of different experiments are not unified. Moreover, there are many contradictory results of the studies of the induction effect of drugs on ABC transporters. So, it is necessary to establish a quantitative assay for the quantification of P-gp, MRP1, and BCRP to study the mechanism of drug resistance.

Methods: In this paper, a novel and advanced liquid chromatography/mass spectrometry (MS)/MS-based targeted proteomics method for the quantification of P-gp, MRP1, and BCRP was developed and validated. Then, the cell lines MCF-7, HepG-2, and SMMC-7721 were, respectively, induced by different concentrations of doxorubicin (adriamycin [ADM]), mitoxantrone (MX), and methotrexate (MTX), to establish resistance cell lines. The method established was used to quantify the expression of P-gp, MRP1, and BCRP.

Results: The result showed that the induction effects of drugs on protein were relatively stable and selective. ADM, MX, and MTX could induce overexpression of P-gp, MRP1, and BCRP. And, the induction effect of different drugs on proteins was selective. The pattern of overexpression of ABC transporters in the three types of resistance cell lines was different.

Conclusion: During the development of drug resistance, the cell type and patch, but not drug type, were the most important determinant factors of the overexpression level of ABC transporters in resistance cell lines. This study provides a good foundation for understanding the development of drug resistance in cell lines and can be used to explain the contradictory results in other published studies as described above.

Keywords: BCRP; MRP1; P-gp; multidrug resistance; quantification.

Figure 1

Chromatograms for surrogate peptides. Notes: Total ion chromatograms for the developed LC–MS/MS method applied to measure surrogate peptides and their stable isotope-labeled IS peptides in human serum albumin (A) and in membrane protein (B). Abbreviations: P-gp, permeability glycoprotein; MRP1, multidrug resistance-related protein 1; BCRP, breast cancer resistance proteins; cps, counts per second; LC, liquid chromatography; MS, mass spectrometry; IS, internal standards.

Figure 2

Digestion efficiency of different ABC transporters in membrane protein (each 200 μg). Notes: Digestion efficiency of different ABC transporters in membrane protein (each 200 μg) after tryptic digestion (1:40) at 37°C for 2, 4, 16, and 24 hours. All experiments were performed in triplicate (mean ± SD is given). Abbreviations: P-gp, permeability glycoprotein; MRP1, multidrug resistance-related protein 1; BCRP, breast cancer resistance proteins.

Figure 3

Transporter proteins expression in parental cell lines. Abbreviations: P-gp, permeability glycoprotein; MRP1, multidrug resistance-related protein 1; BCRP, breast cancer resistance proteins.

Figure 4

Analysis of transporter proteins expression and corresponding genes expression. Notes: Analysis of transporter proteins expression induced by drugs (P-gp/ADM, A; P-gp/MX, B; P-gp/MTX, C; MRP1/ADM, G; MRP1/MX, H; MRP1/MTX, I; BCRP/ADM, M; BCRP/MX, N; BCRP/MTX, O) and corresponding gene expression (ABCB1/ADM, D; ABCB1/MX, E; ABCB1/MTX, F; ABCC1/ADM, J; ABCC2/MX, K; ABCC2/MTX, L; ABCG2/ADM, P; ABCG2/MX, Q; ABCG2/MTX, R) in resistance cell lines. Data are given in mean ± SEM. *P<0.01, significant differences between parental cells and drug-resistant cell lines; **P<0.01, significant differences between drug-resistant cell lines treated with the same chemotherapeutic drugs. Abbreviations: P-gp, permeability glycoprotein; MRP1, multidrug resistance-related protein 1; BCRP, breast cancer resistance proteins; ADM, adriamycin; MX, mitoxantrone; MTX, methotrexate; SEM, standard error of mean.

Figure 5

The overexpression of ABC transporters in cells and its effect on the development of drug resistance.