GSK1904529A, a Potent IGF-IR Inhibitor, Reverses MRP1-Mediated Multidrug Resistance

Abstract

Overexpression of multidrug-resistant efflux transporters is one of the major causes of chemotherapy failure. MRP1, a 190 kDa efflux transporter, confers resistance to a wide of range of chemotherapeutic drugs. Here we study the cellular effects of GSK1904529A in reversing MRP1-mediated drug resistance. Cytotoxicity of GSK1904529A was determined by MTT assay. Reversal effects of GSK1904529A in combination with MRP1 substrates were determined. The intracellular accumulation and efflux of MRP1 substrate was measured by scintillation counter and protein expression was determined by Western blotting analysis. Cell cycle effects of GSK1904529A in combination with MRP1 substrates were determined by flow cytometric analysis. GSK1904529A, at non-toxic concentrations, enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 cells. Furthermore, GSK1904529A increased the intracellular accumulation of [³ H]-vinblastine by inhibiting the efflux function of MRP1. GSK1904529A did not alter the expression level of MRP1, induced a G0/G1 phase cell cycle arrest. Our results indicated that GSK1904529A significantly increased the sensitivity of MRP1 overexpressing cells to chemotherapeutic agents. Furthermore, GSK1904529A enhanced the efficacy of chemotherapeutic drugs that are substrates of MRP1. J. Cell. Biochem. 118: 3260-3267, 2017. © 2017 Wiley Periodicals, Inc.

Keywords: ABC TRANSPORTERS; GSK1904529A; MRP1; MULTIDRUG-RESISTANCE.

Figure 1. Cytotoxicity of GSK1904529A

(A) The chemical structure of GSK1904529A (N-(2,6-difluorophenyl)-5-[3-[2-[5-ethyl-2-methoxy-4-[4-(4-methylsulfonylpiperazin-1-yl)piperidin-1-yl]anilino]pyrimidin-4-yl]imidazo[1,2-a]pyridin-2-yl]-2-methoxybenzamide). (B) Cytotoxicity of GSK1904529A was determined by the MTT assay in HEK293/pcDNA3.1 and HEK/MRP1 cells. Error bars indicate SD.

Figure 2. GSK1904529A increases the sensitivity of MRP1 overexpressing cells to the substrates of MRP1

HEK293/pcDNA3.1 and HEK/MRP1 cells were treated with vincristine (A), vinblastine (B), and cisplatin (C) alone or in combination with GSK1904529A at 0.01 and 0.1 µM. MTT assay was carried out to determine the change in resistance fold. MK571 at 25 µM was used as a positive control inhibitor of MRP1 and cisplatin was used as negative control substrate drug. Points with error bars represent the mean ± SD. Each of the above figures is a representative of three independent experiments, each done in triplicate.

Figure 3. GSK1904529A increased the intracellular accumulation of [³H]-vinblastine

The accumulation of [³H]-vinblastine was measured after HEK293/pcDNA3.1 (A) and HEK293/MRP1 (B) cells were preincubated with or without GSK1904529A or MK571 for 2 h at 37°C and then incubated with 0.01 µM [³H]-vinblastine for another 2 h at 37°C. Error bars indicate SD. **: p < 0.01 versus the control group.

Figure 4. GSK1904529A decreased the efflux of [³H]-vinblastine

The effect of GSK1904529A on the efflux of [³H]-vinblastine from HEK293/pcDNA3.1 (A) and HEK293/MRP1 (B) cells was measured. A time-dependent decrease of efflux of [³H]-vinblastine was found (0, 30, 60 and 120 min). Data shown are means ± SDs from three independent determinations, in triplicate. * P < 0.05, significantly different from control.

Figure 5. GSK1904529A did not alter the expression levels of MRP1

(A) The effect of GSK1904529A on the protein levels of MRP1 was tested after HEK293/MRP1 cells were treated with 0.1 µM GSK1904529A for 0, 24, 48 and 72 h. (B) The effect of GSK1904529A on the protein levels of MRP1 was tested after HEK293/MRP1 cells were treated with 0.01, 0.05, 0.1 µM GSK1904529A for 72 h. (C and D) ImageJ was used to analyze the grayscale ratios of MRP1/β-actin. The greyscale ratios were proportional to the MRP1 protein levels.

Figure 6. Effect of GSK1904529A on the cell cycle of HEK293/pcDNA3.1 and HEK293/MRP1cells

HEK293/pcDNA3.1 (A) and HEK293/MRP1 (B) cells were treated with vincristine alone or in combination with GSK1904529A. Cells were harvested and stained with propidium iodide (PI) and analyzed by flow cytometer for cell cycle. Quantification of cell population in G0/G1, S, and G2/M phase of the cell cycle was done.