Nilotinib potentiates anticancer drug sensitivity in murine ABCB1-, ABCG2-, and ABCC10-multidrug resistance xenograft models

Abstract

A panel of clinically used tyrosine kinase inhibitors were compared and nilotinib was found to most potently sensitize specific anticancer agents by blocking the functions of ABCB1/P-glycoprotein, ABCG2/BCRP and ABCC10/MRP7 transporters involved in multi-drug resistance. Nilotinib appreciably enhanced the antitumor response of (1) paclitaxel in the ABCB1- and novel ABCC10-xenograft models, and (2) doxorubicin in a novel ABCG2-xenograft model. With no apparent toxicity observed in the above models, nilotinib attenuated tumor growth synergistically and increased paclitaxel concentrations in ABCB1-overexpressing tumors. The beneficial actions of nilotinib warrant consideration as viable combinations in the clinic with agents that suffer from MDR-mediated insensitivity.

Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Figures

Figure 1. Resistance reversal of ABCB1, ABCG2 and ABCC10 anticancer substrates with or without TKI's

The results of the MTT cytotoxicity assays were used to measure cell survival and the bar graph represents the change in resistance-fold (RF) in the presence or absence of TKI's in resistant cells. Resistance-fold was calculated by dividing the IC50 value for paclitaxel or mitoxantrone with or without the TKIs in the resistant cells shown in A-E with IC50 value for paclitaxel or mitoxantrone without inhibitors in the respective parental cells (not shown) as described in Materials and Methods. The MDR reversal effect of TKIs at 1 μmol/L in combination with paclitaxel is shown in ABCB1 overexpressing A. HEK293/ABCB1 B. KB-C2 cells; in combination with mitoxantrone in C. HEK293/R2 D. H460/MX20 cells; and in combination with paclitaxel in E. HEK293/MRP7 (ABCC10) cells is shown. The data points represent the mean ± SD of at least three independent experiments performed in triplicate. Statistically, *, P < 0.05; **,P < 0.01, versus the control group (Student t-test).

Figure 2. The effect of nilotinib on ABCB1 xenograft models

Potentiation of antitumor effects of paclitaxel by nilotinib in ABCB1 overexpressing (KB-C2) oral epidermoid carcinoma xenograft model is shown. Atleast two independent experiments were carried out using athymic NCR nude mice implanted s.c. with KB-C2 cells. A. A representative picture of the excised KB-C2 tumor sizes from different mice is shown on the 18th day after implantation. B. The bar graph represents the mean tumor weight (n=6-10) of the excised KB-C2 tumor from different mice. The treatments were as follows: (a) vehicle (q3d × 6), (b) paclitaxel (18 mg/kg, i.p., q3d × 6) (c) nilotinib (75 mg/kg, p.o., q3d × 6) (d) paclitaxel (18 mg/kg, i.p., q3d × 6) + nilotinib (75 mg/kg, p.o., q3d × 6, given 1 h before giving paclitaxel). Each column represent the mean determinations and the bars represent SD. *, P < 0.05; **, P < 0.001 versus the control group. C. Changes in mean body weight before and after treatment for ABCB1-xenograft model are shown in the bar graph. D. Changes in tumor volume with time in ABCB1-xenograft model are shown. Points represent mean tumor volume for each group (n=6) after implantation. Each point on line graph represent the mean tumor volume (mm3) at a particular day after implantation and the bars represent SD. *, P < 0.05 versus the vehicle group; *,#, P < 0.05 versus paclitaxel alone group.

Figure 3. The effect of nilotinib on ABCG2 xenograft model

Potentiation of antitumor effects of doxorubicin by nilotinib in ABCG2 overexpressing NSCLC H460/MX-20-xenograft model is shown on the 18th day after implantation. At least two independent experiments were carried out using athymic NCR nude mice implanted s.c. with H460/MX-20 cells. A. A representative picture of the excised H460/MX-20 tumor sizes from different mice is shown on the 18th day after implantation. B. The bar graph represents the mean tumor weight (n=6-10) of the excised H460/MX-20 tumor from different mice. The treatment were as follows: (a) vehicle (q3d × 6), (b) doxorubicin (1.8 mg/kg, i.p., q3d × 6) (c) nilotinib (75 mg/kg, p.o., q3d × 6) (d) doxorubicin (1.8 mg/kg, i.p., q3d × 6) + nilotinib (75 mg/kg, p.o., q3d × 6, given 1 h before giving doxorubicin). Each column represent the mean determinations and the bars represent SD. *, P < 0.05; **, P < 0.001 versus the control group. C. Changes in mean body weight before and after treatment for ABCG2-xenograft model are shown in the bar graph. D. Changes in tumor volume with time in ABCG2-xenograft model are shown. Points represent mean tumor volume for each group (n=6) after implantation. Each point on line graph represent the mean tumor volume (mm3) at a particular day after implantation and the bars represent SD. *, P < 0.05 versus the vehicle group; *,#, P < 0.05 versus doxorubicin alone group.

Figure 4. The effect of nilotinib on ABCC10/MRP7 xenograft model

Potentiation of antitumor effects of paclitaxel by nilotinib in human embryonic kidney (HEK293) cells transfected with ABCC10/MRP7-xenograft model is shown on the 18th day after implantation. Atleast two independent experiments were carried out using athymic NCR nude mice implanted s.c. with HEK293/MRP7. A. A representative picture of the excised HEK293/MRP7 tumor sizes from different mice is shown on the 18th day after implantation. B. The bar graph represents the mean tumor weight (n=6-10) of the excised HEK293/MRP7 tumor from different mice. The treatments were as follows: (a) vehicle (q3d × 6), (b) paclitaxel (18 mg/kg, i.p., q3d × 6) (c) nilotinib (75 mg/kg, p.o., q3d × 6) (d) paclitaxel (18 mg/kg, i.p., q3d × 6) + nilotinib (75 mg/kg, p.o., q3d × 6, given 1 h before giving paclitaxel). Each column represent the mean determinations and the bars represent SD. *, P < 0.05; **, P < 0.001 versus the control group. C. Changes in mean body weight before and after treatment for ABCC10-xenograft model are shown in the bar graph. D. Changes in tumor volume with time in ABCC10-xenograft model are shown. Points represent mean tumor volume for each group (n=6) after implantation. Each point on line graph represent the mean tumor volume (mm3) at a particular day after implantation and the bars represent SD. *, P < 0.05 versus the vehicle group; *,#, P < 0.05 versus paclitaxel alone group.

Figure 5. The effect of nilotinib on plasma and tumor concentration of paclitaxel in ABCB1-xenograft model

Paclitaxel concentration in KB-C2 tumor (box – paclitaxel tumor AUC) A. plasma (box – paclitaxel plasma AUC) B. KB-C2 tumor/plasma AUC ratio C. with or without nilotinib treatment is shown. In the combination group, 75 mg/kg nilotinib was given orally, 1 h before giving 18 mg/kg paclitaxel via tail vein injection. Mice (n=5-6) were euthanized via cardiac puncture at different intervals and tumor and plasma was harvested and stored in -80°. Paclitaxel was quantified as described in materials and methods. Columns, means (n = 3-6); bars, SD. * P < 0.05, compared with only paclitaxel receiving group (unpaired student t-test).

Figure 6. XP-Glide predicted binding mode of nilotinib with homology modeled ABCB1 and ABCG2

The docked conformations of nilotinib as ball and stick model are shown within the large hydrophobic cavity of A. ABCB1 and B. ABCG2. Important amino acids are depicted as sticks with the atoms colored as carbon – green, hydrogen – white, nitrogen – blue, oxygen – red, sulfur – yellow, whereas nilotinib is shown with the same color scheme as above except carbon atoms are represented in orange and fluorine in light green. Dotted black line indicates hydrogen-bonding interactions, whereas dotted red line indicates electrostatic interactions. ABCB1 and ABCG2 are represented as macromodel surfaces based on residue charge (hydrophobic-yellow).