Abstract
Abstract Multidrug resistance proteins (MRP) confer resistance to drugs from different classes. MRP4 and MRP5 are related MRP proteins and can transport organic anions. Hence their overexpression has been associated with resistance to several purine analogs, such as 6-mercaptopurine and adefovir (PMEA; 9′-(2′-phosphonylmethoxyethyl)adenine)), and antifolates, such as methotrexate (MTX). 6MP itself is not a substrate for MRP4 and 5, but its monophosphate is, in contrast to the di- (DP) and triphosphates (TP). Similarly MTX (a monoglutamate) is a substrate for various MRPs but the higher glutamates are usually not. Resistance to MTX was not observed at a long exposure because of accumulation of polyglutamates, but a 4-hr exposure to MTX conferred resistance. The aim of this study was to investigate whether MRP4 and 5 overexpression would confer resistance to the widely used nucleoside analogs cytarabine (ara-C) and gemcitabine (GEM), and the novel L-nucleoside analog Troxacitabine (TROX). As model systems we used HEK293 cells transfected with either MRP4 (HEK/MRP4; 59-fold increased MRP4 level) or MRP5 (HEK/MRP5i; 991-fold increased MRP5 level) and determined sensitivity after an exposure for 4 hr (followed by 68 hr drug-free medium) and 72 hr. Uptake and accumulation of GEM and TROX were measured with HPLC coupled to either UV or MS-MS, that of ara-C with the radioactive drug. HEK293 cells were moderately sensitive to ara-C (IC50 1.8 µM) and TROX (2.1 µM) and more sensitive to GEM (46 nM) at 72 hr exposure. At 4 hr exposure, the IC50s increased 5-, 5- and 10-fold, respectively. At 4 and 72 hr exposure a 2-3-fold resistance to TROX and ara-C was found in HEK/MRP4 cells, and a 2.5-fold resistance to ara-C in HEK/MRP5 cells. No resistance to GEM was found. PMEA showed a 3-fold resistance in both cell lines. Resistance in the MRP4/5 cells could be reversed by the specific inhibitors probenecid and indomethacine. In order to explain this resistance, accumulation and retention of the active nucleotides were evaluated. GEM-TP even tended to be higher in the MRP4/5 cells. However, accumulation of ara-C nucleotides after 4 hr was 3- and 2-fold lower in MRP4 and MRP5 cells, respectively. After washing with drug-free medium (DFM), the concentration of ara-CTP declined more rapidly in MRP4 compared to the wild-type. Although TROX accumulation was similar in the 3 cell lines, in MRP4 and MRP5 cells it decreased 4- and 2-fold faster, respectively, after the DFM period. TROX-phosphates were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells. In conclusion, MRP4 and MRP5 overexpression confer resistance to TROX and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and TROX-nucleotides in HEK/MRP4-5 cells. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B65.
Published Version
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