The BTK Inhibitor Ibrutinib (PCI-32765) Overcomes Paclitaxel Resistance in ABCB1- and ABCC10-Overexpressing Cells and Tumors

Abstract

Paclitaxel is one of the most widely used antineoplastic drugs in the clinic. Unfortunately, the occurrence of cellular resistance has limited its efficacy and application. The ATP-binding cassette subfamily B member 1 (ABCB1/P-glycoprotein) and subfamily C member 10 (ABCC10/MRP7) are the major membrane protein transporters responsible for the efflux of paclitaxel, constituting one of the most important mechanisms of paclitaxel resistance. Here, we demonstrated that the Bruton tyrosine kinase inhibitor, ibrutinib, significantly enhanced the antitumor activity of paclitaxel by antagonizing the efflux function of ABCB1 and ABCC10 in cells overexpressing these transporters. Furthermore, we demonstrated that the ABCB1 or ABCC10 protein expression was not altered after treatment with ibrutinib for up to 72 hours using Western blot analysis. However, the ATPase activity of ABCB1 was significantly stimulated by treatment with ibrutinib. Molecular docking analysis suggested the binding conformation of ibrutinib within the large cavity of the transmembrane region of ABCB1. Importantly, ibrutinib could effectively enhance paclitaxel-induced inhibition on the growth of ABCB1- and ABCC10-overexpressing tumors in nude athymic mice. These results demonstrate that the combination of ibrutinib and paclitaxel can effectively antagonize ABCB1- or ABCC10-mediated paclitaxel resistance that could be of great clinical interest. Mol Cancer Ther; 16(6); 1021-30. ©2017 AACR.

Figure 1.

The effect of ibrutinib on the accumulation and efflux of [³H]-paclitaxel in ABCB1- or ABCC10-overexpressing cells. A, Ibrutinib effectively increased the accumulation of [³H]-paclitaxel in KB-C2 cells. Columns are the mean of triplicate determinations; bars, SDs. B, The efficacy of ibrutinib on the efflux of [³H]-paclitaxel from KB-3–1 and KB-C2 cells was measured. A time-dependent versus percentage of intracellular [³H]-paclitaxel was plotted (0, 30, 60, and 120 minutes). Data shown were means ± SDs for independent determinations in triplicate. C, Ibrutinib effectively increased the accumulation of [³H]-paclitaxel in HEK293/ABCC10 cells. D, The effect of ibrutinib on the efflux of [³H]-paclitaxel from HEK293/pcDNA3.1 and HEK293/ABCC10 cells was measured. Three independent experiments were performed.

Figure 2.

The effect of ibrutinib on ABCB1 and ABCC10 expression levels, ABCB1 ATPase activity, and its docking in the homology model of ABCB1. A, The effect of ibrutinib on the protein levels of ABCB1 was tested by Western blot analysis after the KB-C2 cells were treated with 5 μmol/L ibrutinib for 0, 24, 48, and 72 hours. The expression levels of ABCB1 were normalized by β-actin. The differences were statistically not significant (P > 0.05). B, The effect of ibrutinib on the protein levels of ABCC10; the expression levels of ABCC10 were normalized by β-actin. C, The effect of ibrutinib (0–80 μmol/L) on ATP hydrolysis by ABCB1. The mean values are plotted, and error bars represent the SD. The experiments were performed at least three independent times. D, XP-Glide predicted binding mode of ibrutinib with homology-modeled ABCB1. The docked conformation of ibrutinib as ball and stick model is shown within the large hydrophobic cavity of ABCB1. Important amino acids are depicted as sticks with the atoms colored as carbon, green; hydrogen, white; nitrogen, blue; oxygen, red; sulfur, yellow, whereas ibrutinib is shown with the same color scheme as above except carbon atoms are represented in orange. Dotted black line, hydrogen bonding interactions. Molecular surface of homology-modeled ABCB1 was colored by residue charge (hydrophobic, yellow).

Figure 3.

Ibrutinib enhanced the effect of paclitaxel on the growth of an ABCB1-overexpressing KB-C2 cell xenograft model in athymic nude mice. A, Images of excised KB-C2 tumor tissues from different mice are shown on the 18th day after treatment (n = 7). B, Changes in tumor volume with time in ABCB1-overexpressing xenograft model are shown. C, Mean weight (n = 7) of the excised KB-C2 tumors from the mice treated with vehicle, ibrutinib, paclitaxel, or the combination of ibrutinib and paclitaxel. Error bars, SD. *, P < 0.05 versus vehicle group; #, P < 0.05 versus the paclitaxel group. D, The average body weight before and after treatments.

Figure 4.

Ibrutinib enhanced the effect of paclitaxel on the growth of an ABCC10-overexpressing HEK293/ABCC10 cell xenograft model in athymic nude mice. A, Images of excised HEK293/ABCC10 tumors tissues from different mice are shown on the 18th day after treatment (n = 7). B, Changes in tumor volume with time in ABCC10-overexpressing xenograft model are shown. C, Mean weight (n= 7) of the excised HEK293/ABCC10 tumors from the mice treated with vehicle, ibrutinib, paclitaxel, or the combination of ibrutinib and paclitaxel. Error bars, SD. *, P < 0.05 versus vehicle group; #, P < 0.05 versus the paclitaxel group. D, The average body weight before and after treatments.