c-MET tyrosine kinase inhibitors reverse drug resistance mediated by the ATP-binding cassette transporter B1 (ABCB1) in cancer cells

Abstract

This study investigated the potential of MET kinase inhibitors, cabozantinib, crizotinib, and PHA665752, in reversing multidrug resistance (MDR) mediated by ABCB1 in cancer cells. The accumulation of the fluorescent probe, Rhodamine 123, was assessed using flow cytometry and fluorescence microscopy in MDR MES-SA/DX5 and parental cells. The growth inhibitory activity of MET inhibitors as monotherapies and in combination with chemotherapeutic drugs was evaluated by MTT assay. CalcuSyn software was used to analyze the combination index (CI) as an index of drug-drug interaction in combination treatments. Results showed that at concentrations of 5, and 25 μM, c-MET inhibitors significantly increased Rhodamine 123 accumulation in MDR cells, with ratios up to 17.8 compared to control cells, while exhibiting no effect in parental cells. Additionally, the combination of c-MET inhibitors with the chemotherapeutic agent doxorubicin synergistically enhanced cytotoxicity in MDR cells, as evidenced by combination index (CI) values of 0.54 ± 0.08, 0.69 ± 0.1, and 0.85 ± 0.07 for cabozantinib, crizotinib, and PHA665752, respectively. While all three c-MET inhibitors stimulated ABCB1 ATPase activity in different manners at certain concentrations, PHA-665752 suppressed it at high concentration. In silico analysis also suggested that the transmembrane domains (TMD) of ABCB1 transporters could be considered potential target for these agents. Our results suggest that c-MET inhibitors can serve as promising MDR reversal agents in ABCB1-medicated drug-resistant cancer cells.

Keywords: ABCB1; Combination therapy; Drug resistance; P-glycoprotein; Tyrosine kinase inhibitors.

Fig. 1

Effect of c-MET inhibitors on intracellular accumulation of rhodamine 123 (Rho123) in ABCB1-overexpressing MES-SA/DX5 (A, B) and parental MES-SA cells (C, D). The cells were treated with cabozantinib, crizotinib, and PHA 665752 as c-MET inhibitors and verapamil, a reference inhibitor, at concentrations of 1.5, 5, 25 μM. After treatment, the cells were incubated with 5 μM Rho123, for 20 min. The fluorescence intensity of Rho123 was then measured using flow cytometric analysis to assess the inhibitory effect of c-MET inhibitors on ABCB1. Rho123 accumulation in ABCB1-overexpressing MES-SA/DX5 (A, B) and parental MES-SA cells (C, D) was quantified. The data represented as the ratio of Rho123 accumulation in the presence of the compounds to that in their absence. Error bars represent the standard error of the mean (S.E.M.) and the data is calculated based on 3 independent experiments. There were significant differences between Rho123 accumulation in the absence and presence of test drugs (*p < 0.05, **p < 0.001)

Fig. 2

Effects of c-MET inhibitors on Rho123 accumulation by fluorescence microscopy in MES-SA/DX5 cells. MES-SA/DX5 cells were plated in 6-well plates at a density of 2 × 10^5 cells per mL and allowed to grow for two days at 37 °C. After treatment with cabozantinib, crizotinib, PHA-665752, and verapamil (as a positive control) at a concentration of 25 μM for one hour, Rho 123 was added to each well to reach a final concentration of 50 μM. The cells were incubated with Rho 123 for an additional 20 min, followed by three washes with ice-cold phosphate-buffered saline (PBS). Finally, the cells were resuspended in cold PBS and examined using a Nikon Eclipse Ti-U fluorescent microscope equipped with a blue filter (510–560 nm)

Fig. 3

The anti-proliferative effects of combined treatment of doxorubicin and cabozantinibin MES-SA/DX5 cells. The viability of doxorubicin-resistant MES-SA/DX5 cells was assessed using the MTT assay. To investigate the synergistic interactions between the two drugs, the cells were treated with cabozantinib and doxorubicin at a fixed ratio for 72 h in 96-well plates. Combination analysis was performed using CalcuSyn software. A Displays the decrease in proliferation of the MES-SA/DX5 cancer cell line following the fixed-ratio combination of cabozantinib and doxorubicin. The x-axis represents the concentrations of the drugs administered alone, and in combination at a fixed-ratio of 3:1 (0.5/0.5 µM, 1/1 µM, 2/2 µM, and 4/4 µM). B Shows the CI-FA plot, where “Fa” and “CI” denote fraction affected and combination index values, respectively, showing the synergistic effect of cabozantinib and doxorubicin (values below the horizontal line indicate synergism). The 95% confidence interval is shown as ± 1.96 standard deviations from the mean. C A representative isobologram plot of cabozantinib–doxorubicin combination treatment. Isobologram analysis demonstrates the individual drug concentrations (x-axis for cabozantinib and y-axis for doxorubicin) required to achieve 90% growth inhibition (Fa = 0.9), 75% growth inhibition (Fa = 0.75), and 50% growth inhibition (Fa = 0.5). The combination data points that fall to the left of the additivity curve, indicate a synergistic interaction between the two drugs. Figures 3–4 B-C present representative images from a single experiment.* Combination therapy was significantly different from monotherapy (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

The anti-proliferative effects of combined treatment of doxorubicin and crizotinib in MES-SA/DX5 cells. The viability of doxorubicin -resistant MES-SA/DX5 cells was assessed using the MTT assay. To investigate the synergistic interactions between the two drugs, the cells were treated with crizotinib and doxorubicin at a fixed ratio for 72 h in 96-well plates. Combination analysis was performed using CalcuSyn software. A Displays the decrease in proliferation of the MES-SA/DX5 cancer cell line following the combination of crizotinib and doxorubicin. The x-axis represents the concentrations of the drugs administered alone, and in combination at a fixed-ratio of 1:1 (1/1 µM, 2/2 µM, 4/4 µM, and 8/8 µM). B Shows the CI-FA plot, where “Fa” and “CI” denote fraction affected and combination index values, respectively, showing the synergistic effect of crizotinib and doxorubicin (values below the horizontal line indicate synergism). The 95% confidence interval is shown as ± 1.96 standard deviations from the mean. C A representative isobologram plot of crizotinib–doxorubicin combination treatment. Isobologram analysis demonstrates the individual drug concentrations (y-axis for crizotinib and x-axis for doxorubicin) required to achieve 90% growth inhibition (Fa = 0.9), 75% growth inhibition (Fa = 0.75), and 50% growth inhibition (Fa = 0.5). The combination data points that fall to the left of the additivity curve, indicate a synergistic interaction between the two drugs. Figures 3–4 B-C present representative images from a single experiment. * Combination therapy was significantly different from monotherapy (*p < 0.05, **p < 0.001)

Fig. 5

The anti-proliferative effects of combined treatment of doxorubicin and PHA-665752 in MES-SA/DX5 cells. The viability of doxorubicin-resistant MES-SA/DX5 cells was assessed using the MTT assay. To investigate the synergistic interactions between the two drugs, the cells were treated with PHA-665752 and doxorubicin at a fixed ratio for 72 h in 96-well plates. Combination analysis was performed using CalcuSyn software. A Displays the decrease in proliferation of the MES-SA/DX5 cancer cell line following the combination of PHA-665752 and doxorubicin. The x-axis represents the concentrations of the drugs administered alone, and in combination at the fixed-ratio of 1:1 (1/1 µM, 2/2 µM, 4/4 µM, and 8/8 µM8). B Shows the CI-FA plot, where “Fa” and “CI” denote fraction affected and combination index values, respectively, showing the synergistic effect of PHA-665752 and doxorubicin (values below the horizontal line indicate synergism). The 95% confidence interval is shown as ± 1.96 standard deviations from the mean. C A representative isobologram plot of PHA-665752-doxorubicin combination treatment. Isobologram analysis demonstrates the individual drug concentrations (x-axis for PHA-665752 and y-axis for doxorubicin) required to achieve 90% growth inhibition (Fa = 0.9), 75% growth inhibition (Fa = 0.75), and 50% growth inhibition (Fa = 0.5). The combination data points that fall to the left of the additivity curve, indicate a synergistic interaction between the two drugs. Figures 3–4 B-C present representative images from a single experiment. * Combination therapy was significantly different from monotherapy (*p < 0.05, **p < 0.01)

Fig. 6

Effects of c-MET inhibitors on ABCB1 ATPase activity. The ABCB1 ATPase activity was assessed using the ADP-Glo™ assay system (Promega, USA). In this assay, 5 μg of recombinant human ABCB1 membranes were incubated with 1X-ABCB1 Assay Buffer as an untreated control. Additionally, 200 μM verapamil (an ATPase stimulator), 100 μM sodium orthovanadate (an ATPase inhibitor), and varying concentrations of c-MET TKIs (1.5, 5, and 25 μM) were added to 384-well white plates. The reaction was started by adding 5 mM ATP and incubating the plates at 37 °C for 40 min. To halt the reaction, 10 μL of ATPase Detection Reagent was added, followed by a 60-min incubation at room temperature. Luminescence was then measured with a Tecan Infinite® 200 PRO Microplate Reader. Data are presented as changes in luminescence (RLU = relative light unit). *p < 0.01, **p < 0.005 compared with control basal activity group. Data are presented as mean ± SEM of three or more experiments

Fig. 7

Docking poses of c-MET inhibitors inside the drug binding domain of ABCB1 transporter. The position of cabozantinib (A), crizotinib (B), and PHA-665752 (C) in the drug binding domain of ABCB1. The yellow, blue, and green dashed lines show the H-bond, Pi-Pi stacking, and Pi-cation interactions. ABCB1 has formed two and one hydrogen bonds with cabozantinib and crizotinib, respectively. PHA-665752 has only constructed one Pi-cation interaction with the target

Fig. 8

The RMSD plots of the ABC transporters. The protein RMSD values of three MD simulations for cabozantinib, crizotinib, and PHA-665752, which are converged at about 4 Å are shown. The convergence of the RMSD values indicates the stability of the system and protein structure during the MD simulation. PHA-665752 has experienced more stability during the simulation compared to cabozantinib (A), crizotinib

Fig. 9

The interactions constructed between the c-MET inhibitors and ABCB1 residues during the MD simulation. The ABCB1 residues with the highest interaction with the ligands during the MD simulation are shown for cabozantinib (A), crizotinib (B) and PHA-665755 (C). The residues Trp232 and Val991 are the main and common target residues for cabozantinib, and crizotinib, while PHA-665752 has a considerable interaction fraction with Phe994