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.
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 muM 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 Km and Vmax values of 1.3 +/- 0.2 mM and 44 +/- 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 < or = 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.
Breedveld
Pauline
P
Department of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
Pluim
Dick
D
Cipriani
Greta
G
Dahlhaus
Femke
F
van Eijndhoven
Maria A J
MA
de Wolf
Cornelia J F
CJ
Kuil
Annemieke
A
Beijnen
Jos H
JH
Scheffer
George L
GL
Jansen
Gerrit
G
Borst
Piet
P
Schellens
Jan H M
JH
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D001692:Biological Transport; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D004285:Dogs; D005260:Female; D005492:Folic Acid / Q000493:pharmacokinetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D008942:Mitoxantrone / Q000493:pharmacokinetics*; D009363:Neoplasm Proteins / Q000378:metabolism*; D011817:Rabbits; D000077185:Resveratrol; D013267:Stilbenes / Q000493:pharmacokinetics*; D019772:Topotecan / Q000493:pharmacokinetics*
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D013267:Stilbenes; D019772:Topotecan; D005492:Folic Acid; D008942:Mitoxantrone; D000077185:Resveratrol; D008727:Methotrexate