Linsitinib (OSI-906) antagonizes ATP-binding cassette subfamily G member 2 and subfamily C member 10-mediated drug resistance

Abstract

In this study we investigated the effect of linsitinib on the reversal of multidrug resistance (MDR) mediated by the overexpression of the ATP-binding cassette (ABC) subfamily members ABCB1, ABCG2, ABCC1 and ABCC10. Our results indicate for the first time that linsitinib significantly potentiate the effect of anti-neoplastic drugs mitoxantrone (MX) and SN-38 in ABCG2-overexpressing cells; paclitaxel, docetaxel and vinblastine in ABCC10-overexpressing cells. Linsitinib moderately enhanced the cytotoxicity of vincristine in cell lines overexpressing ABCB1, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, linsitinib significantly increased the intracellular accumulation and decreased the efflux of [(3)H]-MX in ABCG2-overexpressing cells and [(3)H]-paclitaxel in ABCC10-overexpressing cells. However, linsitinib, at a concentration that reversed MDR, did not significantly alter the expression levels of either the ABCG2 or ABCC10 transporter proteins. Furthermore, linsitinib did not significantly alter the intracellular localization of ABCG2 or ABCC10. Moreover, linsitinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner. Overall, our study suggests that linsitinib attenuates ABCG2- and ABCC10-mediated MDR by directly inhibiting their function as opposed to altering ABCG2 or ABCC10 protein expression.

Keywords: ABCC10; ABCG2; Linsitinib; Multi-drug resistance; Tyrosine kinase inhibitor.

Fig. 1.

The chemical structure of linsitinib and the effect of linsitinib on the cell lines used in the study. (A) The chemical structure of linsitinib (cis-3-[8-amino-1-(2-phenyl-7-quinolinyl)imidazo[1,5-a]pyrazin-3-yl]-1-methylcyclobutanol). (B) Cytotoxicity of linsitinib in HEK293/pcDNA3.1, ABCG2-482-R2, ABCG2-482-G2 and ABCG2-482-T7 cell lines. (C) Cytotoxicity of linsitinib in H460 and H460/MX20 cell lines. (D) Cytotoxicity of linsitinib in HEK293/pcDNA3.1 and HEK293/ABCC10 cell lines. (E) Cytotoxicity of linsitinib in SW620 and SW620/AD300 cell lines. (F) Cytotoxicity of linsitinib in HEK293/pcDNA3.1 and HEK293/ABCC1 cell lines.

Fig. 2.

The effect of linsitinib on the accumulation of [³H]-MX and [³H]-paclitaxel, respectively. (A) The accumulation of [³H]-MX was significantly increased in ABCG2-482-R2, ABCG2-482-G2 and ABCG2-482-T7 cell lines in the presence of linsitinib. (B) The accumulation of [³H]-paclitaxel was increased in HEK293/ABCC10 cell line in the presence of linsitinib. *indicates p < 0.05 or **indicates p < 0.01 versus the control group. Error bars represent the SD. The experiments were performed at least three independent times.

Fig. 3.

The effect of linsitinib on the efflux of [³H]-MX and [³H]-paclitaxel, respectively. (A) A time course (0, 30, 60, 120 min) versus percentage of intracellular [³H]-MX remaining (%) was plotted using HEK293/pcDNA3.1 and ABCG2-482-R2 cell lines in presence or absence of linsitinib or FTC; (B) A time course (0, 30, 60, 120 min) versus percentage of intracellular [³H]-paclitaxel remaining (%) was plotted using HEK293/pcDNA3.1 and HEK293/ABCC10 cell lines in presence or absence of linsitinib or cepharanthine. *indicates p < 0.05 versus the control group. Error bars represent the SD. The experiments were performed at least three independent times.

Fig. 4.

The effect of linsitinib on the expression levels of ABCG2 and ABCC10 transporters, respectively. (A and B) The expression levels of ABCG2 protein in H460 and H460/MX20 cell lysates were shown. (C and D) The expression levels of ABCC10 protein in HEK293/pcDNA3.1 and HEK293/ABCC10 cell lysates were shown. Image J was used to analyze the grayscale ratios. The grayscale ratios were proportional to the ABCG2 or ABCC10 protein levels. The differences were statistically non-significant (p > 0.05). A representative result was shown and similar results were obtained in two other experiments.

Fig. 5.

The effect of linsitinib treatment on the subcellular localization of ABCG2 and ABCC10, respectively. (A) H460/MX20 cells were treated with 2 μM linsitinib for 72 h. The subcellular localization of ABCG2 was analyzed by immunofluorescence. ABCG2 staining is shown in green. DAPI (blue) counterstains the nuclei. (B) HEK293/ABCC10 cells were treated with 2 μM linsitinib for 72 h. The subcellular localization of ABCC10 was analyzed by immunofluorescence. ABCC10 staining is shown in green. DAPI (blue) counterstains the nuclei. A representative result was shown and similar results were obtained in two other experiments.

Fig. 6.

The effect of linsitinib on ATP hydrolysis by ABCG2. Crude membranes (10 μg protein/reaction) from High-five cells expressing ABCG2 were incubated with increasing concentrations of linsitinib (0–80 μM) in the presence and absence of 0.3 mM vanadate, in ATPase assay buffer as described in Section 2. The mean values are plotted and error bars represent the SD. The experiments were performed at least three independent times.

Fig. 7.

XP Glide predicted binding mode of linsitinib with homology modeled ABCG2. The docked conformation of linsitinib as ball and stick model is shown within the large cavity of ABCG2. Important amino acids are depicted as sticks with the atoms colored as carbon – green, hydrogen – white, nitrogen – blue, oxygen – red and sulfur – yellow, whereas linsitinib is shown with the same color scheme as above except carbon atoms are represented in orange. Dotted black line indicates hydrogen bonding interactions, whereas dotted red line indicates electrostatic interactions. ABCG2 is represented as ribbons based on residue charge (hydrophobic-yellow, basic-blue, acidic-red).