Human glutathione S-transferase-mediated glutathione conjugation of curcumin and efflux of these conjugates in Caco-2 cells
Usta, M. ; Wortelboer, H.M. ; Vervoort, J.J.M. ; Boersma, M.G. ; Rietjens, I.M.C.M. ; Bladeren, P.J. van; Cnubben, N.H.P. - \ 2007
Chemical Research in Toxicology 20 (2007)12. - ISSN 0893-228X - p. 1895 - 1902.
cancer cells - chemopreventive agent - antioxidant mechanism - drug transporters - dietary curcumin - ethacrynic-acid - rat-liver - apoptosis - enzymes - proliferation
Curcumin, an alpha,beta-unsaturated carbonyl compound, reacts with glutathione, leading to the formation of two monoglutathionyl curcumin conjugates. In the present study, the structures of both glutathione conjugates of curcumin were identified by LC-MS and one- and two-dimensional H-1 NMR analysis, and their formation in incubations with human intestinal and liver cytosol and purified human glutathione S-transferases and also in human Caco-2 cells was characterized. The results obtained demonstrate the site for glutathione conjugation to be the C1 atom, leading to two diastereoisomeric monoglutathionyl curcumin conjugates (CURSG-1 and CURSG-2). The formation of both glutathionyl conjugates appeared to be reversible. The monoglutathionyl curcumin conjugates decompose with a t(1/2) of about 4 h to curcumin and other unidentified degradation products. Both human intestinal and liver cytosol catalyzed curcumin glutathione conjugation. At saturating substrate concentrations, human GSTM1a-1a and GSTA1-1 are shown to be especially active in the formation of CURSG-1, whereas GSTP1-1 and GSTA2-2 have no preference for the formation of CURSG-1 or CURSG-2. GSTT1-1 hardly catalyzes the glutathione conjugation of curcumin. In the Caco-2 human intestinal monolayer transwell model, CURSG-1 and CURSG-2 were formed at a ratio of about 2:1 followed by their excretion, which appeared to be three times higher to the apical (lumen) side than to the basolateral (blood) side. Given that GSTM1a-1a and GSTP1-1 are present in the intestinal epithelial cells, it can be concluded that efficient glutathione conjugation of curcumin may already occur in the enterocytes, followed by an efficient excretion of these glutathione conjugates to the lumen, thereby reducing the bioavailability of (unconjugated) curcumin. In conclusion, the present study identifies the nature of the diastereoisomeric monoglutathionyl curcumin conjugates, CURSG-1 and CURSG-2 formed in biological systems, and reveals that conjugate formation is catalyzed by GSTM1a-1a, GSTA1-1, and/or GSTP1-1 with different stereoselective preference. The formation of glutathione conjugates can already occur during intestinal transport, after which the monoglutathionyl conjugates are efficiently excreted to the intestinal lumen, thereby influencing the bioavailability of curcumin and, as a result, its beneficial biological effects.
Inhibition of multidrug resistance proteins MRP1 and MRP2 by a series of a,ß-unsaturated carbonyl compounds
Wortelboer, H.M. ; Ustafa, M. ; Zanden, J.J. van; Bladeren, P.J. van; Rietjens, I.M.C.M. ; Cnubben, N.H.P. - \ 2005
Biochemical Pharmacology 69 (2005)12. - ISSN 0006-2952 - p. 1879 - 1890.
acid phenethyl ester - organic anion transporter - s-transferase p1-1 - p-glycoprotein - ethacrynic-acid - glutathione-conjugate - cells - curcumin - agents - rat
To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of ¿,ß-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. ¿,ß-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 Sf9-MRP1 or Sf9-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.
Interplay between MRP-inhibition and metabolism of MRP-inhibitors: the case of curcumin
Wortelboer, H.M. ; Usta, M. ; Velde, A.E. van der; Boersma, M.G. ; Spenkelink, A. ; Zanden, J.J. van; Rietjens, I.M.C.M. ; Bladeren, P.J. van; Cnubben, N.H.P. - \ 2003
Chemical Research in Toxicology 16 (2003)12. - ISSN 0893-228X - p. 1642 - 1651.
multidrug-resistance protein - glutathione-s-transferase - chemopreventive agent curcumin - organic anion transporter - p-glycoprotein - drug-resistance - reduced glutathione - rat hepatocytes - ethacrynic-acid - leukotriene c-4
The multidrug resistance proteins MRP1 and MRP2 are efflux transporters with broad substrate specificity, including glutathione, glucuronide, and sulfate conjugates. In the present study, the interaction of the dietary polyphenol curcumin with MRP1 and MRP2 and the interplay between curcumin-dependent MRP inhibition and its glutathione-dependent metabolism were investigated using two transport model systems. In isolated membrane vesicles of MRP1- and MRP2-expressing Sf9 cells, curcumin clearly inhibited both MRP1- and MRP2-mediated transport with IC50 values of 15 and 5 muM, respectively. In intact monolayers of MRP1 overexpressing Madin-Darby canine kidney (MDCKII-MRP1) cells, curcumin also inhibited MRP1-mediated activity, although with a 3-fold higher IC50 value than the one observed in the vesicle model. Interestingly, MRP2-mediated activity was hardly inhibited in intact monolayers of MRP2-overexpressing MDCKII (MDCKII-MRP2) cells upon exposure to curcumin, whereas the IC50 value in the vesicle incubations was 5 muM. The difference in extent of inhibition of the MRPs by curcumin in isolated vesicles as compared to intact cells, observed especially for MRP2, was shown to be due to a swift metabolism of curcumin to two glutathione conjugates in the MDCKII cells. Formation of both glutathione conjugates was about six times higher in the MDCKII-MRP2 cells as compared with the MDCKII-MRP1 cells, a phenomenon that could be ascribed to the significantly lower glutathione levels in the cell line. The efflux of both conjugates, identified in the present study as monoglutathionyl curcumin conjugates, was demonstrated to be mediated by both MRP1 and MRP2. From dose-response curves with Sf9 membrane vesicles, glutathionylcurcumin conjugates appeared to be less potent inhibitors of MRP1 and MRP2 than their parent compound curcumin. In conclusion, curcumin clearly inhibits both MRP1- and MRP2-mediated transport, but the glutathione-dependent metabolism of curcumin plays a crucial role in the ultimate level of inhibition of MRP-mediated transport that can be achieved in a cellular system. This complex interplay between MRP inhibition and metabolism of MRP inhibitors, the latter affecting the ultimate potential of a compound for cellular MRP inhibition, may exist not only for a compound like curcumin but also for many other MRP inhibitors presently or previously developed on the basis of vesicle studies.
Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin
Zanden, J.J. van; Hamman, O. Ben; Iersel, M.L. van; Boeren, J.A. ; Cnubben, N.H.P. ; Bello, M. Lo; Vervoort, J.J.M. ; Bladeren, P.J. van; Rietjens, I.M.C.M. - \ 2003
Chemico-Biological Interactions 145 (2003)2. - ISSN 0009-2797 - p. 139 - 148.
site-directed mutagenesis - human placenta - quinone methide - ethacrynic-acid - active-site - pi - identification - consequences - inactivation - conjugation
In the present study, the inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by the flavonoid quercetin has been investigated. The results show a time- and concentration-dependent inhibition of GSTP1-1 by quercetin. GSTP1-1 activity is completely inhibited upon I h incubation with 100 muM quercetin or 2 h incubation with 25 muM quercetin, whereas 1 and 10 muM quercetin inhibit GSTP1-1 activity to a significant extent reaching a maximum of 25 and 42% inhibition respectively after 2 h. Co-incubation with tyrosinase greatly enhances the rate of inactivation, whereas co-incubation with ascorbic acid or glutathione prevents this inhibition. Addition of glutathione upon complete inactivation of GSTP1-1 partially restores the activity. Inhibition studies with the GSTP1-1 mutants C47S, C101S and the double mutant C47S/C101S showed that cysteine 47 is the key residue in the interaction between quercetin and GSTP1-1. HPLC and LGMS analysis of trypsin digested GSTP1-1 inhibited by quercetin did not show formation of a covalent bond between Cys 47 residue of the peptide fragment 45-54 and quercetin. It was demonstrated that the inability to detect the covalent quercetin-peptide adduct using LGMS is due to the reversible nature of the adduct-formation in combination with rapid and preferential dimerization of the peptide fragment once liberated from the protein. Nevertheless, the results of the present study indicate that quinone-type oxidation products of quercetin likely act as specific active site inhibitors of GSTP1-1 by binding to cysteine 47. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.