Conceived and designed the experiments: CRAJ XC EHB ZSC. Performed the experiments: TS YHK YL AC YZ AKT EHB JO. Analyzed the data: TS YHK. Contributed reagents/materials/analysis tools: CRAJ YZ XC EHB ZSC. Wrote the paper: TS CRAJ ZSC.
Current address: Department of Pathology, New York University School of Medicine, New York, New York, United States of America
The authors have declared that no competing interests exist.
One of the major mechanisms that could produce resistance to antineoplastic drugs in cancer cells is the ATP binding cassette (ABC) transporters. The ABC transporters can significantly decrease the intracellular concentration of antineoplastic drugs by increasing their efflux, thereby lowering the cytotoxic activity of antineoplastic drugs. One of these transporters, the multiple resistant protein 7 (MRP7, ABCC10), has recently been shown to produce resistance to antineoplastic drugs by increasing the efflux of paclitaxel. In this study, we examined the effects of BCR-Abl tyrosine kinase inhibitors imatinib, nilotinib and dasatinib on the activity and expression of MRP7 in HEK293 cells transfected with MRP7, designated HEK-MRP7-2.
We report for the first time that imatinib and nilotinib reversed MRP7-mediated multidrug resistance. Our MTT assay results indicated that MRP7 expression in HEK-MRP7-2 cells was not significantly altered by incubation with 5 µM of imatinib or nilotinib for up to 72 hours. In addition, imatinib and nilotinib (1-5 µM) produced a significant concentration-dependent reversal of MRP7-mediated multidrug resistance by enhancing the sensitivity of HEK-MRP7-2 cells to paclitaxel and vincristine. Imatinib and nilotinib, at 5 µM, significantly increased the accumulation of [3H]-paclitaxel in HEK-MRP7-2 cells. The incubation of the HEK-MRP7-2 cells with imatinib or nilotinib (5 µM) also significantly inhibited the efflux of paclitaxel.
Imatinib and nilotinib reverse MRP7-mediated paclitaxel resistance, most likely due to their inhibition of the efflux of paclitaxel via MRP7. These findings suggest that imatinib or nilotinib, in combination with other antineoplastic drugs, may be useful in the treatment of certain resistant cancers.
Although the clinical use of surgery, radiation and chemotherapy have decreased the recurrence rates of cancer, cellular resistance to chemotherapeutic drugs is a major obstacle in the treatment of cancer
One of the primary cellular mechanisms that can produce resistance to antineoplastic therapy involves the efflux of drugs from the cancer cells by specific transmembrane transporters or pumps
A number of studies suggest that cancer cells that express the ABC C family transporter MRP7/ABCC10 can develop resistance to various chemotherapeutic drugs. For example, human salivary gland adenocarcinoma (SGA) cells that overexpress MRP7 mRNA and the MRP7 protein display significant resistance to vincristine
The MRP7-overexpressing cells confer resistance to several anticancer drugs including paclitaxel, vincristine and vinblastine
Tyrosine kinase inhibitors (TKIs) can reverse the resistance of cancer cells to antineoplastic drugs through multiple mechanisms. For example, in human SGA cells, MRP7, P-gp, and MRP1 were all detected after prolonged exposure to vincristine
In this study, one of the main goals was to identify TKI compounds that would reverse MRP7-mediated drug resistance. Consequently, it is possible that TKIs, in combination with other antineoplastic drugs, may be useful in the treatment of cancers that express MDR proteins, including the ABC transporters. An important discovery about TKIs was that certain so-called “small molecule” drugs could inhibit TK activity by competing with ATP for binding to the intracellular catalytic domain of receptor TKs, which produced inhibition of various downstream signaling cascades by autophosphorylation
HEK293 cells and the MRP7 cDNA were generously provided by Dr. Gary Kruh (University of Illinois at Chicago, Chicago, IL). The transfected HEK-MRP7-2 cells and empty vector transfected HEK293-pcDNA3.1 cells were established from HEK293 cells through electroporation
DMEM, bovine serum and penicillin/streptomycin were purchased from Hyclone (Logan, UT). Nilotinib (Tasigna®) (
Confluent monolayer cells in T-25 flask were harvested and rinsed twice with cold PBS. The cell extracts were prepared using the Radioimmunoprecipitation assay buffer [1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µM p-APMSF, 10 µM leupeptin, and 10 µM aprotinin] for 30 min on ice with occasional rocking followed by centrifugation at 12,000 rpm at 4°C for 15 min. The protein concentrations of cell lysates were determined by the Bradford protein assay
Equal amount of total cell lysates (40 µg) were resolved by 4–12% sodium dodecyl sulfate polycrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto nitrocellulose membranes
Drug sensitivity was analyzed using a slightly modified MTT colorimetric assay
After drug incubation of 72 h, 20 µl MTT (4 mg/ml) was added to each well and the plate was further incubated for 4 h, allowing viable cells to develop from the yellow-colored MTT into dark-blue formazan crystals. Subsequently, the medium was gently removed without agitating the adhesive monolayer of cells, and 100 µl of DMSO was added into each well to dissolve the formazan crystals. The plates were well shaken for 5 min, and an OPSYS microplate reader read the absorbance at 570 nm from DYNEX Technologies Inc. (Chantilly, VA). The degree of resistance was calculated by dividing the IC50 for the MDR cells by that of the parental cells, whereas the degree of MDR reversal was calculated by dividing the IC50 of the cells with the anticancer drug in the absence of inhibitor by that obtained in the presence of the inhibitor. The concentrations required to inhibit growth by 50% of the control cells were calculated from survival curves using the Bliss method
The antineoplastic drugs used included paclitaxel, vincristine and doxorubicin at varying concentrations up to a final concentration of 10, 1, and 1 µM, respectively. The BCR-Abl TKIs, such as nilotinib and imatinib, were subsequently used at nontoxic concentrations of 1, 2.5 and 5 µM to screen against vincristine and doxorubicin. In this study, we first selected a single nontoxic dose (5 µM) to examine the effects of TKIs on MRP7-mediated resistance to paclitaxel. Once we determined which TKIs had the most significant reversal effect, such as imatinib and nilotinib, we subsequently selected three concentrations (1, 2.5 or 5 µM) for each TKI to determine whether their reversal effects were concentration-dependent to paclitaxel, vincristine and doxorubicin.
The HEK293-pcDNA3.1 parental cells and HEK-MRP7-2 transfected cells were seeded in two T75 flasks and incubated with DMEM supplemented with 10% bovine serum at 37°C. After the cells were 60 to 95% confluent, each inhibitor was added to separate flasks and the cells were incubated for 1 h. The cells were then trypsinized and two aliquots (48×106 cells) from each cell line were suspended in the medium. Subsequently, cells were suspended in the medium containing [3H]-paclitaxel at a concentration of 0.1 µM with or without the TKIs nilotinib and imatinib (5 µM) for 1 h at 37°C. One hour later, the incubation medium was replaced by the medium containing the TKIs without paclitaxel. Aliquots (1×106 cells) were collected at various time points (0, 20, 60, and 120 min). The cells were then washed with ice-cold PBS and each sample was placed in scintillation fluid to measure the radioactivity in a Packard TRI-CARB 1900CA liquid scintillation counter from Packard Instrument Inc. (Downers Grove, IL).
All experiments were repeated at least three times and the differences were determined by two-tailed Student's t-test. The a priori statistical significance was set at
In this study, the two cell lines utilized were HEK293 cells transfected with either the MRP7 expression vector or the empty vector control (pcDNA3.1). Immunoblot analysis was performed to detect the expression level of MRP7 protein in the aforementioned lines. MRP7 protein (MW 171 kD) was expressed in HEK-MRP7-2 cells, but not in HEK293-pcDNA3.1 cells (
(A) Expression of MRP7 in HEK293-pcDNA3.1 (lane 1) and MRP7-transfected cells (lane 2). (B) Expression of P-gp in HEK293-pcDNA3.1 (lane 1), HEK-MRP7-2 (lane 2), KB-3-1 (lane 3) and KB-C2 cells (lane 4). (C) Effect of 5 µM of imatinib on the expression level of MRP7 (HEK-MRP7-2) for 36 and 72 h, respectively. (D) Effect of 5 µM of nilotinib on the expression level of MRP7 (HEK-MRP7-2) for 36 and 72 h, respectively. Equal amounts (40 µg protein) of total cell lysate were used for each sample. The nitrocellulose membranes were immunoblotted with primary antibody against MRP7 or actin at 1∶500 dilution, or P-gp at 1∶500 dilution at 4°C overnight, and then incubated with HRP-conjugated secondary antibody at 1∶1000 dilutions at room temperature for 3 h.
To evaluate the effect of imatinib/nilotinib on the expression of MRP7, the HEK-MRP7-2 cells were incubated with 5 µM of imatinib or nilotinib for 36 and 72 h, respectively. The incubation of HEK-MRP7-2 cells with imatinib or nilotinib did not significantly alter the expression of the protein levels of MRP7 at different time points (
To determine the drug resistance profile of MRP7, the sensitivity of HEK-MRP7-2 transfected cells to specific antineoplastic drugs was compared to that of the vector-only control cells, HEK293-pcDNA3.1. The HEK-MRP7-2 cells exhibited a significant higher level of resistance to paclitaxel and vincristine (9.5- and 6.7-fold resistance compare to the control cells, respectively) (
HEK293-pcDNA3.1 | HEK-MRP7-2 | |||||||||
Compound | IC50 | ± | SD |
RF |
DMF |
IC50 | ± | SD |
RF |
DMF |
21.85 | ± | 1.89 | (1.00) | 207.03 | ± | 19.69 | (9.47) | |||
+ Nilotinib 1 µM | 10.15 | ± | 0.94 | (0.46) | 2.15 | 26.34 | ± | 1.34 | (1.21) | 7.86 * |
+ Nilotinib 2.5 µM | 8.61 | ± | 0.32 | (0.39) | 2.54 | 15.87 | ± | 0.94 | (0.73) | 13.05 ** |
+ Nilotinib 5 µM | 7.63 | ± | 0.15 | (0.35) | 2.86 | 9.90 | ± | 0.42 | (0.45) | 20.91 ** |
+ Imatinib 1 µM | 16.17 | ± | 0.77 | (0.74) | 1.35 | 44.43 | ± | 2.07 | (2.03) | 4.66 * |
+ Imatinib 2.5 µM | 13.70 | ± | 0.82 | (0.63) | 1.59 | 22.75 | ± | 2.36 | (1.04) | 9.10 * |
+ Imatinib 5 µM | 9.76 | ± | 0.82 | (0.45) | 2.24 | 16.63 | ± | 2.36 | (0.76) | 12.45 ** |
+ Dasatinib 2.5 µM | 19.87 | ± | 0.94 | (0.91) | 1.10 | 204.11 | ± | 13.45 | (9.34) | 1.01 |
9.86 | ± | 1.89 | (1.00) | 66.38 | ± | 9.69 | (6.73) | |||
+ Nilotinib 1 µM | 8.40 | ± | 0.10 | (0.85) | 1.17 | 31.73 | ± | 0.91 | (3.22) | 2.09 |
+ Nilotinib 2.5 µM | 7.60 | ± | 0.32 | (0.77) | 1.30 | 9.71 | ± | 1.44 | (0.98) | 6.84 * |
+ Nilotinib 5 µM | 6.64 | ± | 2.46 | (0.67) | 1.48 | 7.39 | ± | 0.77 | (0.75) | 8.98 * |
+ Imatinib 1 µM | 7.33 | ± | 0.75 | (0.74) | 1.35 | 27.16 | ± | 2.02 | (2.75) | 2.44 |
+ Imatinib 2.5 µM | 7.08 | ± | 0.82 | (0.72) | 1.39 | 9.62 | ± | 0.36 | (0.98) | 6.90 * |
+ Imatinib 5 µM | 7.10 | ± | 0.56 | (0.72) | 1.39 | 8.05 | ± | 0.58 | (0.82) | 8.25 * |
+ Dasatinib 2.5 µM | 9.19 | ± | 0.88 | (0.93) | 1.07 | 62.74 | ± | 7.11 | (6.36) | 1.06 |
30.10 | ± | 0.75 | (1.00) | 38.45 | ± | 0.36 | (1.28) | |||
+ Nilotinib 1 µM | 25.73 | ± | 0.10 | (0.85) | 1.17 | 31.73 | ± | 0.91 | (1.05) | 1.21 |
+ Nilotinib 2.5 µM | 33.77 | ± | 0.32 | (1.12) | 0.89 | 36.52 | ± | 1.44 | (1.21) | 1.05 |
+ Nilotinib 5 µM | 31.36 | ± | 2.46 | (1.04) | 0.96 | 33.25 | ± | 1.77 | (1.10) | 1.16 |
+ Imatinib 1 µM | 31.52 | ± | 0.75 | (1.05) | 0.95 | 42.18 | ± | 2.02 | (1.40) | 0.91 |
+ Imatinib 2.5 µM | 34.14 | ± | 0.82 | (1.13) | 0.88 | 42.96 | ± | 0.36 | (1.43) | 0.90 |
+ Imatinib 5 µM | 32.77 | ± | 0.56 | (1.09) | 0.92 | 38.75 | ± | 0.58 | (1.29) | 0.99 |
+ Dasatinib 2.5 µM | 27.33 | ± | 1.57 | (0.91) | 1.10 | 32.47 | ± | 1.33 | (1.08) | 1.18 |
Values represent the mean±SD of at least three independent experiments performed in triplicate.
Fold resistance was the IC50 values for paclitaxel, vincristine, and doxorubicin of HEK293-pcDNA3.1 in the presence of either nilotinib, imatinib or dasatinib, or the transfected cells HEK-MRP7-2 with or without the reversing agents, divided by the IC50 values for paclitaxel, vincristine, and doxorubicin of HEK293-pcDNA3.1 cells without the reversing agents. Cell survival was determined by the MTT assay as described in “Section 2”.
Dose-modifying factor was the ratio of IC50 values without reversal agent to the IC50 values with reversal agents. * Significantly different from the control transfected as assayed by the Student's t-test (
We tested several BCR-Abl TKIs to determine if they could reverse the resistance of HEK293 cells overexpressing MRP7 to the antineoplastic drug paclitaxel and vincristine. The magnitude of reversal produced by the TKIs to paclitaxel was variable (
Two cell lines, HEK293-pcDNA3.1 and HEK-MRP7-2, are represented as HEK293 and MRP7, respectively. After seeding and culturing cells for 24 h, equal amounts of PBS or the reversal agents were added into HEK293-pcDNA3.1 cells (shown as
In addition to paclitaxel, we also examined the effect of the selected TKIs to sensitize cells to another anticancer drug, vincristine. Similar to the findings with paclitaxel, nilotinib and imatinib (1, 2.5 and 5 µM) significantly reversed MRP7-mediated vincristine resistance (2.1-, 6.8- and 9.0-fold, respectively, for nilotinib; 2.4-, 6.9- and 8.2-fold, respectively, for imatinib) in a concentration-dependent manner (
Overall, imatinib and nilotinib significantly reversed MRP7-mediated resistance to paclitaxel and vincristine, but not doxorubicin. Furthermore, this reversal was concentration-dependent (
In order to determine the mechanism by which imatinib and nilotinib surmount or reverse MRP7-mediated paclitaxel resistance, their effect on the accumulation of [3H]-paclitaxel in MRP7-transfected cells was examined. The intracellular concentration of [3H]-paclitaxel in HEK-MRP7-2 cells was 30% of that accumulated by the HEK293-pcDNA3.1 cells (
The intracellular paclitaxel accumulations in HEK293-pcDNA3.1 and HEK-MRP7-2 cells were measured after the incubation with 0.1 µM paclitaxel. Intracellular accumulation of paclitaxel in HEK293-pcDNA3.1 cells in the absence of imatinib and nilotinib were shown in the left bars (▪). Intracellular accumulation of paclitaxel in the presence of 5 µM of imatinib in HEK293-pcDNA3.1 cells was shown on the right (□). Intracellular accumulation of paclitaxel in HEK-MRP7-2 cells in the presence of 5 µM of nilotinib was shown on the right (
Based on the above results, it is possible that the increase in intracellular paclitaxel produced by imatinib and nilotinib could be due to: (1) a decrease in the efflux of paclitaxel and/or, (2) an increase in the uptake of paclitaxel. Therefore, the next experiment was conducted to determine if the increase in paclitaxel accumulation produced by imatinib and nilotinib was due to an inhibition of paclitaxel efflux. HEK-MRP7-2 cells and HEK293-pcDNA3.1 cells were incubated with paclitaxel and a time course for intracellular drug accumulation was determined (
The percentage of the paclitaxel released was plotted as a function of time. After 1 h of incubation of the TKIs, [3H]-paclitaxel was co-incubated in HEK293-pcDNA3.1 with TKI (
This study was the first to identify that the BCR-Abl TKIs imatinib and nilotinib, but not dasatinib, could reverse MRP7-mediated MDR in a concentration-dependent manner.
HEK293 cells transfected with the MRP7 recombinant gene HEK-MRP7-2, and HEK293-pcDNA3.1 cells transfected with empty vector, were used for the tests of MRP7 efflux function. These transfected cell lines have previously been used in studies designed to determine the effects of cepharanthine on the reversal of MRP7-mediated resistance to paclitaxel
Previously, it has been reported that cepharanthine and nilotinib significantly reversed P-gp-mediated MDR in HEK-MRP7-2 cells
In this study, we found that specific TKIs: 1) significantly decreased resistance to paclitaxel in MRP7-overexpressing cells (
In order to extend the findings obtained with paclitaxel, we examined the effect of specific TKIs on the response of MRP7-expressing cells to another antineoplastic drug, vincristine, which is a substrate for MRP7
It is well established that MRP7, P-gp and MRP1 are all drug efflux pumps responsible for the extracellular transport of a variety of antineoplastic drugs. Consequently, when these pumps are present in the tumor cells concurrently, each of the pumps contributes to the efflux and decrease of intracellular drug concentrations. This latter action ultimately leads to drug levels that are no longer cytotoxic, leading to failure of therapy. For instance, nilotinib has been identified as an inhibitor of P-gp and the BCRP efflux pumps
Future experiments, based on the current findings, include the: 1) determination of the effects of newly synthesized TKIs to reverse MRP7-mediated drug resistance, and 2) analysis of the effects of TKIs on the efflux activity of other ABC transporters.
In summary, the results of this study are the first to indicate that the BCR-Abl TKIs imatinib and nilotinib, but not dasatinib, at concentrations that did not produce cytotoxicity, significantly reversed MRP7-mediated MDR to paclitaxel and vincristine. Furthermore, these TKIs might be inhibitors for multiple MDR efflux pumps, such as P-gp and BCRP for nilotinib
We thank Dr. Gary Kruh (University of Illinois at Chicago, Chicago, IL) for providing HEK293 cells, MRP7 cDNA and his encouragement. We thank Novartis Pharmaceuticals Corporation for providing us nilotinib and the clinical data of nilotinib in reference #26 (Basel, Switzerland). We thank Dr. Michael M. Gottesman (NCI, Bethesda, MD) for providing KB-3-1 cells and Dr. Shin-ichi Akiyama (Kagoshima University, Japan) for providing KB-C2 cells and polyclonal antibody against human MRP1.