Thiopurine Metabolism and Identification of the Thiopurine Metabolites Transported by MRP4 and MRP5 Overexpressed in Human Embryonic Kidney Cells

Abstract

Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methylthio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.

Highly Potent Activation of Nrf2 by Topical Tricyclic Bis(Cyano Enone): Implications for Protection against UV Radiation during Thiopurine Therapy

“…Of note, in our previous experience (unpublished), such systemic effects on any of these systems were not observed when sulforaphane, another potent Nrf2 activator, was topically administered under similar experimental conditions. MRP4 participates in the export of nucleoside monophosphate analogs (41)(42)(43), and its inducible gene expression is known to be regulated by Nrf2 (40,44,45). Furthermore, silencing of mrp4 increases the 6-TG incorporation in DNA of Hepa1c1c7 cells treated with this thiopurine (36).…”

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes

“…Spectral karyotyping of these cells matched those described by others (Kytola et al, 2000). Parental HEK293 cells (293 human embryonic kidney cells) and the MRP4-overexpressing HEK293/ 4.63 cells were gifts of P Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands) (Wielinga et al, 2002). HEK293/4.63 cells were reported to express significantly more MRP4 (Reid et al, 2003) without overexpression of other ABC drug transporters and were cultured as previously described (Wu et al, 2005).…”

Evidence for dual mode of action of a thiosemicarbazone, NSC73306: a potent substrate of the multidrug resistance–linked ABCG2 transporter

“…For instance, the presence of NSC73306 was unable to overcome MRP1-mediated etoposide resistance, MRP4-mediated NSC251820 resistance, or MRP5-mediated ZD1694 resistance. Etoposide, NSC251820, and ZD1694 are established substrates of MRP1, MRP4, and MRP5, with relative RFs of 140, 7, and 7, respectively (27,38,39). MK-571 (25 Amol/L) and/or quercetin (10 Amol/L) were used as positive controls to reverse drug resistance conferred by MRPs (39).…”

“…Doxorubicin (0.2 Ag/mL) was added to the COR-L23R cell culture medium (26). Parental expressing human embryonic kidney 293 (HEK293) cells, MRP4-expressing HEK293/4.63 cells, and MRP5-expressing HEK293/5I (27,28) were maintained in DMEM (Life Technologies, Invitrogen), supplemented with 10% FCS and 100 units of penicillin/streptomycin/mL at 37jC in 5% CO 2 humidified air. G418 (80 Ag/mL) was added to the MRP1-HEK293 cell culture medium (29).…”