ABC multidrug transporters such as P-glycoprotein (P-gp) and BCRP can make tumor cells resistant to many anticancer drugs. In addition, they can limit the oral availability and penetration of these drugs in certain tissues, thus possibly interfering with the clinical efficacy of cancer chemotherapy at several levels.1 Oral administration of anticancer drugs is rarely feasible because of low and variable oral availability, and this is at least partly caused by ABC transporters. Oral administration has many advantages over i.v. administration as it is less invasive, easier to use for the patient in a chronic regimen and more cost-effective because of decreased hospitalization. We previously demonstrated that the oral availability of the taxane paclitaxel is severely limited by the paclitaxel-transporting P-gp, due to its presence and activity in the apical membranes of hepatocytes and intestinal epithelial cells.2 P-gp also limits the brain and fetal penetration of taxanes.3, 4 In view of their clinical importance, we wanted to know whether taxanes are also transported by other pharmacokinetically important ATP binding cassette (ABC) transporters. While BCRP/Bcrp1 has similar pharmacokinetic functions as P-gp, paclitaxel is not substantially transported by BCRP or murine Bcrp1.5
Recent publications suggest that another ABC transporter, multidrug resistance protein 2 (MRP2, ABCC2), might have broader pharmacokinetic and clinical relevance than previously thought. Like P-gp, MRP2 occurs in the bile canalicular membrane and in the apical membranes of pharmacologically important epithelia such as small intestine, brain endothelial cells and placental trophoblasts.1, 4, 6, 7 In rats it has been shown to limit the oral availability of one of its substrates and to contribute to hepatobiliary and intestinal excretion.1 Moreover, substantial levels of MRP2 are detected in a variety of human tumors.8 Based on these observations, the in vivo pharmacologic effects of MRP2 might be similar to those of P-gp. MRP2 transports or confers resistance to the anticancer drugs vinblastine,9 methotrexate,10 doxorubicin11 and cisplatin,12 whereas mixed results have been reported for etoposide.11, 13
MRP2 also transports HIV protease inhibitors (HPIs), and this transport is stimulated in intact cells in the presence of probenecid or sulfinpyrazone.14 In vesicular transport studies, we found that an extensive range of drugs, including one of the transported HPIs, saquinavir, could also stimulate MRP2-mediated transport.15 Stimulation appears to be a consequence of direct activation of MRP2 (rather than upregulation of the amount of protein) as increased transport is observed instantaneously in in vitro vesicle uptake experiments.15 Probenecid is frequently used as an adjunct to antimicrobial therapy, and other MRP2-stimulating drugs (many of which possibly not yet recognized as such) might be coadministered during cancer chemotherapy. Thus, MRP2 in intestinal and other epithelia and in tumors might limit the oral, tissue and tumor uptake of its transported drug substrates; and these effects could be exacerbated by unintentional MRP2 stimulation.
Here, we show that MRP2 transports paclitaxel and docetaxel, that this transport (and that of vinblastine and etoposide) is stimulated by probenecid, that MRP2 confers substantial paclitaxel resistance and that this resistance is boosted by probenecid.
Material and methods
[3H]Vinblastine, [3H]etoposide, [3H]inulin and [14C]inulin were from Amersham (Little Chalfont, UK). [3H]Docetaxel and [3H]paclitaxel were from Moravek Biochemicals (Brea, CA). Paclitaxel was from Sankyo (Tokyo, Japan). GlaxoSmithKline (Uxbridge, UK) kindly provided GF120918 (Elacridar). Deionized water was obtained using the Milli-Q Plus system (Millipore, Bedford, MA). All other chemicals were from Sigma-Aldrich (Steinheim, Germany).
Generation and culturing of polarized MDCKII cell lines stably expressing human MRP2 has been described.9 In short, the MDCKII cell line16 was transduced with either neomycin (control transduction) or human MRP2 expression vectors. From the MDCKII-Neo lines, clone 6 (very low expression of endogenous Mrp2) was used and from the MDCKII-MRP2 lines, clone 17 (very high expression of MRP2 in the apical membrane). The MDCKII lines and derived clones were cultured in DMEM with Glutamax (Life Technologies, Breda, the Netherlands) supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin and 10 % (v/v) FCS (Life Technologies, complete medium). Before the start of experiments, the MDCKII-Neo and -MRP2 lines were cultured in medium containing G418 (200 μg/ml) for 1 week, but G418 was never present during the experiment.
Cells were seeded on microporous polycarbonate membrane filters (Transwell 3414; Costar, Corning, NY) at a density of 1.0 × 106 cells/well in 2 ml of complete medium. Cells were grown for 3 days, with medium replacement every day. Two hours before the start of the experiment, complete medium in the apical and basolateral compartments was replaced with Optimem medium containing 5 μM GF120918 and, if applicable, the appropriate concentration of transport modulators. We demonstrated that paclitaxel transport was completely abolished in an MDR1 transduced cell line with high levels of MDR1 expression in the presence of the MDR1/BCRP inhibitor GF120918 (5 μM, data not shown). At t = 0 hr, the experiment was started by replacing the medium from both compartments with medium that was but contained 5 μM of the radiolabeled drug (approx. 3 kBq/well) and either [3H]- or [14C]inulin (approx. 3 kBq/well) in the appropriate compartment. The latter compounds were added to check for tightness of the cell layers. Cells were incubated at 37°C in 5% CO2, and 50 μl aliquots were taken each hour, up to 4 hr. The radioactivity in these aliquots was measured by the addition of 4 ml of scintillation fluid (Ultima-gold; Packard, Meriden, CT) and subsequent liquid scintillation counting. Inulin leakage was tolerated up to 1% per hour in each well but never exceeded 3% over 4 hr, unless stated otherwise. The percentage of radioactivity appearing in the opposite compartment, of the total amount initially applied, was measured and plotted. In a control transport experiment, levels of paclitaxel were also determined by direct injection of transport medium obtained from the acceptor compartments into the HPLC system described by Sparreboom et al.17 and confirmed that this radioactivity consisted almost entirely of native paclitaxel (data not shown). The amount of radiolabeled drug associated with the cell layer at the end of the experiment was determined by liquid scintillation counting of the excised filter after washing with ice-cold PBS.
Drug sensitivity assays
Growth inhibition (50% inhibitory concentration, IC50) assays were performed by seeding 350 MDCKII-Neo, 400 MDCKII-Parent or 550 MDCKII-MRP2 cells per well in 96-well plates in complete medium containing 15 nM of the histone deacetylase inhibitor trichostatin A, to enhance expression of the transduced MRP2 gene.11 After cell attachment (24 hr), 200 nM of the P-gp/BCRP inhibitor GF120918 was applied with paclitaxel in a dilution series of 2-fold concentration steps, with each concentration in sextuplicate wells. The concentration of GF120918 was lower than that in the transport experiments, to circumvent toxicity, but still sufficient to inhibit P-gp-mediated transport. After 4 days, when control wells were still subconfluent, cells were lysed in situ and nucleic acids were stained with a proprietary dye (Cyquant/Sybr Green 1; Molecular Probes, Eugene, OR) and quantified by fluorescence (485 nm excitation, 530 nm emission).
IC50 values ± SDs were determined across 3 independent experiments, unless stated otherwise. In general, statistical analysis was performed using Student's t-test (2-tailed, unpaired). Differences between 2 sets of data were considered statistically significant at p < 0.05.
Taxanes are MRP2 substrates and their transport can be stimulated by probenecid
MDCKII cell lines transduced with human MRP2 cDNA or a neomycin-selectable marker were cultured on porous membranes in Transwell plates and tested for their capacity to mediate transepithelial transport of 5 μM [3H]paclitaxel and [3H]docetaxel (Fig. 1). As taxanes are also P-gp substrates, the P-gp inhibitor GF120918 (5 μM) was added to suppress endogenous P-gp function at a concentration that does not inhibit MRP2 function.18 The MDCK-Neo line was used as it contains only a very low amount of endogenous canine Mrp2.9, 14
Human MRP2 transported both paclitaxel and docetaxel efficiently, as can be seen by increased transport in the apical direction and decreased transport in the basolateral direction in the MDCKII-MRP2 line compared to the MDCKII-Neo line (Fig. 1). Active transport is represented clearly by the relative ratio of paclitaxel transport (i.e., the percentage of apically directed transport divided by basolaterally directed translocation after 4 hr, denoted r), which was more than 6-fold increased (from 1.4 ± 0.1 to 9.0 ± 0.6) in the MRP2 line compared to the Neo line (Fig. 1a,b). In line with increased efflux transport, the amount of [3H]paclitaxel associated with the cell layers (denoted Ap and Bl) was about 3-fold decreased in MRP2-overexpressing cells (Fig. 1a,b). In a control transport experiment, it was determined by HPLC17 that the [3H]paclitaxel appearing in the opposite compartment consisted virtually entirely of native paclitaxel (data not shown).
The MRP2-mediated transport of [3H]paclitaxel was clearly stimulated by the addition of 500 μM probenecid, as indicated by a further increase in transport in the apical direction (p = 0.02), a further decrease in translocation in the basolateral direction (p = 0.002) and a further reduction of cell-bound radioactivity (p < 0.002) in the presence of probenecid (Fig. 1b,d). The relative transport ratio increased from 9.0 ± 0.6 to 23 ± 6. The limited increase in apically directed transport of paclitaxel by MRP2 upon probenecid addition probably reflects that under these conditions paclitaxel uptake across the basolateral membrane was rate-limiting for total transepithelial paclitaxel transport. In view of the short time frame in which these experiments were performed, it is unlikely that increased expression of MRP2 is responsible for the stimulation observed.
[3H]Docetaxel was also efficiently transported by MRP2, and transport was further stimulated by probenecid, possibly even better than with paclitaxel, with a >5-fold improved transport ratio and a nearly 3-fold reduction in cellular accumulation upon probenecid treatment of MDCKII-MRP2 cells (Fig. 1e–h). Modest stimulation of apically directed paclitaxel and docetaxel transport by probenecid was also observed in the Neo line (Fig. 1c,g). This stimulation might be due to endogenous canine Mrp2.
MRP2-mediated transport of etoposide and vinblastine is enhanced by probenecid
In view of the clear stimulation of MRP2-mediated transport of taxanes by probenecid, we tested 2 other anticancer drugs, etoposide and vinblastine (Fig. 2). Some controversy exists as to whether etoposide is transported by MRP2.11, 13, 19 Based on both increased apically directed transport and relative transport ratios, our data indicate significant transport of etoposide by human MRP2 and further stimulation by probenecid (Fig. 2a–d), albeit not to the same extent as for the taxanes. The very low amount of etoposide associated with the cell layers (presumably mainly background binding) precluded useful interpretation of these data. Vinblastine was efficiently transported by MRP2 (Fig. 2e,f), as demonstrated earlier;20 and this transport can also be modestly stimulated by probenecid, as evidenced primarily by decreased concentrations in the cell layer and increased relative ratios of transport (Fig. 2f,h).
Concentration-dependent stimulation of MRP2-mediated paclitaxel and docetaxel transport
Previously, we found that probenecid could enhance MRP2-mediated transport of saquinavir over a wide concentration range.14 We found similar results for transepithelial transport of 5 μM paclitaxel and 5 μM docetaxel (Fig. 3a,b). Without probenecid, the relative transport ratio of paclitaxel was 1.5 in the Neo line and 12 in the MRP2 line. The maximum relative ratios of paclitaxel transport obtained with 350 μM probenecid in the Neo and MRP2 lines were, respectively, 2.3 and 38 (Fig. 3a). These data correspond with the relative levels of endogenous canine Mrp2 in the Neo line (very low) and exogenous human MRP2 in the MRP2 line (high). For docetaxel, the relative transport ratios without probenecid were 1.3 and 6.6 in the Neo and MRP2 lines, respectively. These relative ratios increased maximally to 1.9 and 32 in the presence of 500 μM probenecid.
Apparent inhibition of MRP2-mediated paclitaxel and docetaxel transport was observed at probenecid concentrations exceeding 5 mM, possibly due to a compromised condition of the cells, as [14C]inulin leakage was increased considerably in these cells under these conditions.
MRP2-mediated paclitaxel resistance and effect of probenecid
Short-term transport experiments were performed at comparatively high drug concentrations (5 μM), whereas upon prolonged exposure, paclitaxel is cytotoxic to most cell lines in the low nanomolar range. To test therefore whether MRP2 overexpression can also lead to taxane drug resistance, we determined the resistance to paclitaxel in the MDCKII-Neo and MDCKII-MRP2 lines in a growth inhibition assay (Fig. 4). The parental MDCKII cells, which contain somewhat more endogenous canine Mrp2 than the Neo line, were also included. GF120918 (200 nM) was present to suppress endogenous P-gp activity. Figure 4 shows that the MRP2 line was 15-fold resistant compared to the Neo line and 9-fold compared to the parental cells, demonstrating that MRP2 overexpression can substantially increase the resistance to paclitaxel (p < 0.001).
To test whether probenecid might also boost MRP2-mediated paclitaxel resistance, growth inhibition assays were performed in the presence of 250 μM probenecid, a concentration that was by itself not significantly cytotoxic to the cells (data not shown). Figure 4 shows that probenecid had comparatively little effect on paclitaxel resistance in the Neo line and parental cells, whereas it increased resistance in the MRP2 line by another 2.7-fold (p < 0.01).
We have identified the taxane anticancer drugs paclitaxel and docetaxel as efficiently transported substrates of MRP2 in vitro. We further found that MRP2-mediated transport of these cytostatic drugs and of the known MRP2 substrates vinblastine and etoposide can be stimulated significantly by probenecid and, for paclitaxel, over a wide concentration range of probenecid. Finally, we demonstrated that MRP2 overexpression can lead to substantial paclitaxel resistance and that probenecid can further enhance this drug resistance phenotype. Similar results were reported earlier for P-gp-mediated drug resistance.21 Our findings may be relevant both for the pharmacokinetics and administration of taxanes and for the occurrence of taxane resistance in tumors.
Oral administration of taxanes is hampered by low oral availability; therefore, they must be administered i.v. It is thus important that mechanisms limiting the oral availability of these drugs are investigated, to possibly improve future therapy regimens for taxanes. Previously, we demonstrated in mice that the oral availability of paclitaxel is substantially limited due to P-gp function.2 Since the localization and distribution of MRP2 in liver and intestine is similar to that of P-gp, our present findings suggest that MRP2 function might likewise limit oral availability and contribute to hepatobiliary and intestinal drug excretion of paclitaxel and docetaxel, as previously shown for other substrates.1
The stimulation of transport of taxanes by MRP2 with probenecid or other coadministered drugs could give rise to clinically relevant drug–drug interactions. Zelcer et al.15 demonstrated that a large variety of clinically used compounds can stimulate MRP2. Certain MRP2 modulators can inhibit MRP2-mediated transport of some substrates (e.g., probenecid inhibits methotrexate transport by MRP210) and stimulate transport of others [e.g., probenecid stimulates transport of taxanes, etoposide, vinblastine and HPIs (this study and Huisman et al.14). Probenecid is frequently orally administered at high dosages, e.g., with penicillin and/or cephalosporins to boost plasma concentrations of these antibiotics. With the present knowledge, it may be advisable to circumvent the use of probenecid and other MRP2-stimulating drugs during chemotherapy of cancer patients with MRP2 substrate drugs, also as probenecid can stimulate MRP2 over a wide concentration range (Fig. 3) and can enhance MRP2-mediated drug resistance (Fig. 4). The oral availability of the anticancer drugs might be (further) decreased as a combined result of decreased uptake from the gut and increased hepatobiliary excretion, and elimination of parenterally administered drugs might be increased, with potentially adverse consequences for therapeutic efficacy.
For some malignancies, P-gp-mediated MDR can worsen the clinical perspective and P-gp inhibition can improve this situation.22 Next to the previously identified anticancer drug substrates of MRP2,9, 10, 11, 13 this study adds the clinically important taxanes. Sandusky et al.8 demonstrated considerable levels of MRP2 in tumors originating from lung, breast, ovarian, renal and colon carcinomas obtained even from treatment-naive patients. MRP2 might therefore play a role in intrinsic and acquired drug resistance in these tumors. We note, e.g., that paclitaxel (next to several other MRP2 substrate drugs) is indicated for use against non-small cell lung, breast and ovarian carcinomas. However, low upregulation of MRP2 might not be sufficient to mediate significant resistance to MRP2 substrate drugs, as indicated by the study of Henness et al.23
Taken together, our data suggest that MRP2 function might be clinically relevant for a wider range of important anticancer drugs than was formerly assumed and that other drugs, such as probenecid, can substantially stimulate the MRP2-mediated transport of these drugs and possibly exacerbate the MDR phenotype. Given the high MRP2 expression in several human tumors and in important pharmacologic barriers, it may be of interest to develop effective, unequivocal MRP2 inhibitors for further optimization of chemotherapy regimens.
We thank Dr. J.W. Jonker and especially Dr. J.D. Allen for useful comments during the study. We thank Mr. J. Lagas for performing control experiments, Prof. Dr. P. Borst for providing the MDCKII cell line and transduced clones and Dr. R.A.F.M. Chamuleau, Dr. R. Hoekstra, Dr. J.W. Jonker, Dr. G. Merino, Dr. E.M. Huisman-Rocchi and Mr. A.E. van Herwaarden for critical reading of the manuscript.