The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites

• Published in: Molecular cancer therapeutics Vol. 4; no. 5; pp. 855 - 863
• Main Authors: Pratt, Susan, Shepard, Robert L., Kandasamy, Ramani A., Johnston, Paul A., Perry, William, Dantzig, Anne H.
• Format: Journal Article
• Language: English
• Published: United States American Association for Cancer Research 01.05.2005
• Subjects: 5-fluorouracil; Adenosine Triphosphate - pharmacology; Antimetabolites, Antineoplastic - pharmacokinetics; Antimetabolites, Antineoplastic - pharmacology; Apoptosis - drug effects; Biological Transport - drug effects; Cells, Cultured; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Floxuridine - metabolism; Fluorouracil - pharmacokinetics; Fluorouracil - pharmacology; Humans; Kidney - cytology; Kidney - drug effects; Kidney - metabolism; Metabolites; MRP-5 (ABCC5); Multidrug resistance protein; Multidrug Resistance-Associated Proteins - metabolism; Phosphorylation - drug effects; Thioguanine - pharmacology; Thymidylate Synthase - metabolism
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.MCT-04-0291

5′-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5′-fluoro-2′-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5′-fluoro-2′-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were ∼9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 μmol/L [ 3 H]5-FdUMP, [ 3 H]5-FUMP, [ 3 H]dUMP, and not [ 3 H]5-FUdR, or [ 3 H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a K m of 1.1 mmol/L and V max of 439 pmol/min/mg protein. Uptake of 250 μmol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3′,5′-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 μmol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.