Inhibition of multidrug resistance proteins MRP1 and MRP2 by a series of a,b-unsaturated carbonyl compounds Heleen M. Wortelboer a, * , Mustafa Usta a , Jelmer J. van Zanden b , Peter J. van Bladeren c,d , Ivonne M.C.M. Rietjens b,c , Nicole H.P. Cnubben a a Physiological Sciences, TNO Quality of Life, 3700 AJ Zeist, The Netherlands b Division of Toxicology, Wageningen University, PO Box 8000, 6700 EAWageningen, The Netherlands c Wageningen University/TNO Centre for Food Toxicology, PO Box 8000, 6700 EAWageningen, The Netherlands d Nestle ´ Research Centre, PO Box 44, CH-1000 Lausanne 26, Switzerland Received 7 February 2005; accepted 7 April 2005 Abstract To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of a,b-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. a,b-Unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf 9-MRP1 or Sf 9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1- mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition. # 2005 Elsevier Inc. All rights reserved. Keywords: MRP1; MRP2; a,b-Unsaturated carbonyl compounds; Inhibition 1. Introduction One of the mechanisms underlying multidrug resistance (MDR) in mammalian tumor cells has been assigned to enhanced removal of drugs due to overexpression of efflux transporter proteins, such as P-glycoprotein (Pgp/ABCB1), the multidrug resistance proteins MRP1/ABCC1, and to a lesser extent MRP2/ABCC2 [1,2]. These membrane- embedded proteins all belong to the ATP-binding cassette (ABC) transporter protein family differing and MRP2 have many substrates in common, among them glutathione-, glucuronide- and significantly. Also unconjugated drugs, such as daunomycin, vinca alkaloids, methotrexate, fluor- ouracil and chlorambucil are transported, in which process the tripeptide glutathione plays an essential role [3]. For vincristine and daunorubicin, it has been suggested that their transport out of the cell is accompanied by co-trans- port of reduced glutathione (GSH) whereas for other www.elsevier.com/locate/biochempharm Biochemical Pharmacology 69 (2005) 1879–1890 Abbreviations: ACRO, acrolein; BDCUR, bisdemethoxycurcumin; Calcein-AM, calcein acetoxymethylester; CAPE, caffeic acid phenethyl ester; CINN, cinnamaldehyde; CITR, citral; CROT, crotonaldehyde; CsA, cyclosporin A; CUR, curcumin; DCUR, demethoxycurcumin; EA, etha- crynic acid; EASG, glutathionylconjugate of ethacrynic acid; GST, glu- tathione S-transferase; HBSS, Hanks’ balanced salt solution; HEX, trans-2- hexenal; MDCK cells, Madin-Darby canine kidney cells; MDR, multidrug resistance protein; Pgp, P-glycoprotein; MRP, multidrug resistance protein; Sf 9, Spodoptera frugiperda insect cells * Corresponding author. Tel.: +31 30 6944484; fax: +31 30 6960264. E-mail address: wortelboer@voeding.tno.nl (H.M. Wortelboer). 0006-2952/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2005.04.001