The Effect of Low pH on Breast Cancer Resistance Protein (ABCG2)-Mediated Transport of Methotrexate, 7-Hydroxymethotrexate, Methotrexate Diglutamate, Folic Acid, Mitoxantrone, Topotecan, and Resveratrol in In Vitro Drug Transport Models

Abstract

Some cellular uptake systems for (anti)folates function optimally at acidic pH. We have tested whether this also applies to efflux from cells by breast cancer resistance protein (BCRP; ABCG2), which has been reported to transport folic acid, methotrexate, and methotrexate di-and triglutamate at physiological pH. Using Spodoptera frugiperda-BCRP membrane vesicles, we showed that the ATP-dependent vesicular transport of 1 M methotrexate by BCRP is 5-fold higher at pH 5.5 than at physiological pH. The transport of methotrexate was saturable at pH 5.5, with apparent K m and V max values of 1.3 Ϯ 0.2 mM and 44 Ϯ 2.5 nmol/mg of protein/min, respectively, but was linear with drug concentration at pH 7.3 up to 6 mM methotrexate. In contrast to recent reports, we did not detect transport of methotrexate diglutamate at physiological pH, but we did find transport at pH 5.5. We also found that 7-hydroxy-methotrexate, the major metabolite of methotrexate, is transported by BCRP both at physiological pH and (more efficiently) at low pH. The pH effect was also observed in intact BCRP-overexpressing cells: we found a 3-fold higher level of resistance to both methotrexate and the prototypical BCRP substrate mitoxantrone at pH 6.5 as at physiological pH. Furthermore, with MDCKII-BCRP monolayers, we found that resveratrol, which is a neutral compound at pH Յ 7.4, is efficiently transported by BCRP at pH 6.0, whereas we did not detect active transport at pH 7.4. We conclude that BCRP transports substrate drugs more efficiently at low pH, independent of the dissociation status of the substrate.Uptake of weak acid and weak base chemotherapeutic drugs by tumors is greatly influenced by the dissociation properties of the drug itself and by the cellular pH gradient (i.e., the difference of extracellular pH in the tumor and the intracellular pH maintained by the cells) (Tannock and Rotin, 1989;Boyer and Tannock, 1992;Kozin et al., 2001;Mahoney et al., 2003). Whereas the median pH value in normal tissues is 7.5, in many tumor tissues, the extracellular pH is more acidic and may be as low as 5.8 (Tannock and Rotin, 1989). This is a consequence of a high rate of lactic acid production in tumors even under aerobic conditions (Tannock and Rotin, 1989;Boyer and Tannock, 1992;Gatenby and This work has been presented previously in abstract form: Breedveld P, Pluim D, Cipriani G, Dahlhaus F, van Eijndhoven MAJ, Borst P, and Schellens JHM (2005) The effect of low pH on BCRP(ABCG2)-mediated transport of methotrexate, 7-hydroxymetotrexate, methotrexate diglutamate, folic acid and resveratrol in in vitro drug transport models. N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; SN-38, 7-ethyl-10-hydroxycamptothecin; solution A, formic acid and acetonitrile 5%; solution B, formic acid and acetonitril 23%; Ko143,2,3,4,6,7,12,2Ј:1,6]pyrido [3,4-b]indol-3-yl)-propionic acid tert-butyl ester.

Transport of Diclofenac by Breast Cancer Resistance Protein (ABCG2) and Stimulation of Multidrug Resistance Protein 2 (ABCC2)-Mediated Drug Transport by Diclofenac and Benzbromarone

“…We also tested whether diclofenac could inhibit the transport of MTX by BCRP in vesicular uptake experiments performed at pH 7.4 and pH 5.5. Figure 2 shows that the net transport of MTX by BCRP was higher at pH 5.5 compared with pH 7.4, which is consistent with previous findings (Breedveld et al, 2007). The ATP-dependent transport of MTX by BCRP was inhibited by diclofenac in a concentrationdependent manner under both pH conditions, as shown in Fig.…”

“…Transport of [ 3 H]MTX was studied at pH 7.4 and pH 5.5. The latter condition was included because the net transport of MTX by BCRP was reported to be higher at pH 5.5 (Breedveld et al, 2007). Vesicular uptake buffers for incubations with Sf9 vesicles consisted of 50 mM Tris and 250 mM sucrose, and the pH was adjusted with HCl to either 7.4 or 5.5.…”

Organic Anion-Transporting Polypeptide 1B1 Mediates Transport of Gimatecan and BNP1350 and Can Be Inhibited by Several Classic ATP-Binding Cassette (ABC) B1 and/or ABCG2 Inhibitors

“…Radiolabeled drugs were analyzed by liquid scintillation counting (Tri-Carb 2100 CS Liquid Scintillation Analyzer; Canberra Industries, Meriden, CT) and the nonradiolabeled drugs by fluorescence measurements at specific excitation/ emission wavelengths of 370/480 (CPT-11), 380/535 (SN-38), 380/420 (lurtotecan), or 370/420 (BNP1350) using a microplate reader (Infinite M200; Tecan Trading AG, Männedorf, Switzerland). The tested concentrations of the nonradiolabeled CPTs were chosen within the linear range of the calibration curve of the fluorescence measurements, and the concentrations of the radiolabeled drugs were based on our experience with these compounds (Breedveld et al, 2007;Marchetti et al, 2007).…”

Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides

“…We therefore tested cladribine transport in an indirect fashion by determining its ability to competitively inhibit uptake of [ 3 H]methotrexate e into Sf9 vesicles. The experiments were done at pH 7.4 rather than at pH 5.5, where transport rates are higher (45), to make the results obtained comparable with those of the Transwell experiments done at pH 7.4. The affinity of Abcg2 for methotrexate, not determined previously, proved to be similar to that of ABCG2 at about 2 mmol/L, in line with published data (45 -47).…”