Published OnlineFirst August 11, 2009; DOI: 10.1158/1535-7163.MCT-09-0290 2348 Curcumin blocks prostaglandin E2 biosynthesis through direct inhibition of the microsomal prostaglandin E2 synthase-1 Andreas Koeberle,1 Hinnak Northoff,2 anti-inflammatory activities. [Mol Cancer Ther 2009;8 and Oliver Werz1 (8):2348–55] 1Department of Pharmaceutical Analytics, Pharmaceutical Institute, University of Tuebingen, and 2Institute for Clinical and Introduction Experimental Transfusion Medicine, University Medical Center Curcumin (diferuloylmethane; Fig. 1), an antioxidant poly- Tuebingen, Tuebingen, Germany phenol from Curcuma longa (tumeric), is a major ingredient of the curry spice tumeric and has been used for the therapy Abstract of inflammatory and infectious diseases in ayurvedic med- icine. Results from preclinical and clinical studies indicate Prostaglandin E (PGE ) plays a crucial role in the apparent 2 2 chemopreventive, antiproliferative, proapoptotic, antimeta- link between tumor growth and chronic inflammation. Cy- static, antiangiogenic, and anti-inflammatory effects of cur- clooxygenase (COX)-2 and microsomal PGE synthase-1, 2 cumin (for review, see refs. 1, 2). The pleiotropic activities of which are overexpressed in many cancers, are functional- curcumin are supposed to be linked to its interference with ly coupled and thus produce massive PGE in various tu- 2 the expression or activation of multiple key signaling mole- mors. Curcumin, a polyphenolic β-diketone from tumeric cules, including peroxisome proliferator–activated receptor with anti-carcinogenic and anti-inflammatory activities, γ, p53, nuclear factor-E2–related factor, nuclear factor κB was shown to suppress PGE formation and to block the 2 (nuclear factor κB), activator protein-1, protein kinase C, expression of COX-2 and of microsomal PGE synthase-1. 2 protein kinase A, focal adhesion kinase, protein kinase B, Here, we identified microsomal PGE synthase-1 as a mo- 2 tumor necrosis factor-α,interleukin1β, chemokines, p300 lecular target of curcumin and we show that inhibition of histone acetyl transferase, cyclooxygenase (COX)-2, 5-lipox- microsomal PGE synthase-1 activity is the predominant 2 ygenase, and matrix metalloproteinase-9 (1, 2). Numerous mechanism of curcumin to suppress PGE biosynthesis. 2 molecular targets of curcumin have been identified thus Curcumin reversibly inhibited the conversion of PGH to 2 far, including COX-1 (IC =25–50 μmol/L;refs.3,4),5- PGE by microsomal PGE synthase-1 in microsomes of 50 2 2 lipoxygenase (IC =0.7μmol/L; ref. 3), glycogen synthase interleukin-1β–stimulated A549 lung carcinoma cells with 50 kinase-3β (IC = 0.07 μmol/L; ref. 5), DNA topoisomerase an IC of 0.2 to 0.3 μmol/L. Closely related polyphenols 50 50 II (at 50 μmol/L; ref. 6), inhibitor of NFκBkinase(IC = (e.g., resveratrol, coniferyl alcohol, eugenol, rosmarinic 50 20 μmol/L; ref. 7), protein kinase C (IC =15μmol/L; acid) failed in this respect, and isolated ovine COX-1 and 50 ref. 8), and xanthine oxidase (IC = 200–400 μmol/L). How- human recombinant COX-2 were not inhibited by curcu- 50 ever, many of these interactions are characterized by low minupto30μmol/L. In lipopolysaccharide-stimulated affinities as reflected by the respective high IC values in human whole blood, curcumin inhibited COX-2–derived 50 functional assays, and the pharmacologic relevance of most PGE formation from endogenous or from exogenous 2 of these target interactions is uncertain. arachidonic acid, whereas the concomitant formation of Prostaglandin E (PGE ) is a potent lipid mediator that is COX-2–mediated 6-keto PGF α and COX-1–derived 12(S)- 2 2 1 closely linked to inflammation and cancer. The biosynthesis hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was sup- of PGE requires transformation of arachidonic acid by pressed only at significant higher concentrations. Based 2 COX-1 or COX-2 [enzyme commission (EC) 1.14.99.1] to on the key function of PGE in inflammation and carcinogen- 2 PGH , which is subsequently converted by PGE synthases esis, inhibition of microsomal PGE synthase-1 by curcumin 2 2 2 (EC 5.3.99.3) to PGE (9). Whereas the cytosolic PGE provides a molecular basis for its anticarcinogenic and 2 2 synthase is constitutively expressed and preferentially couples to COX-1, the microsomal PGE2 synthase-1 is functionally linked to COX-2. COX-2 and microsomal PGE2 synthase-1 Received 3/27/09; revised 5/19/09; accepted 5/20/09; published are induced by proinflammatory stimuli, and both enzymes OnlineFirst 8/11/09. are overexpressed in various cancers (9, 10). Curcumin was Grant support: Deutsche Forschungsgemeinschaft (WE 2260/8-1). – shown to lower PGE2 formation in cellular models (3, 11 14), The costs of publication of this article were defrayed in part by the – payment of page charges. This article must therefore be hereby marked in whole blood (15), and in vivo (16 19). Besides direct inhibi- – μ advertisement in accordance with 18 U.S.C. Section 1734 solely to tion of COX-1 and -2 (IC50 =25 50 mol/L for COX-1 and indicate this fact. >50μmol/L for COX-2 (3, 4)), impaired activation of activa- Requests for reprints: Oliver Werz, Department of Pharmaceutical Analytics, tor protein-1 and the NFκB signaling pathway, resulting in Pharmaceutical Institute, University of Tuebingen, Auf der Morgenstelle 8, D-72076 Tuebingen, Germany. Phone: 49-7071-2978793; reduced expression of COX-2 (13, 20) and microsomal Fax: 49-7071-294565. E-mail: [email protected] PGE2 synthase-1 (11), might be responsible. However, the Copyright © 2009 American Association for Cancer Research. effects of curcumin on prostanoid biosynthesis are diverse, doi:10.1158/1535-7163.MCT-09-0290 depending on the distinct assays used. For example, the Mol Cancer Ther 2009;8(8). August 2009 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst August 11, 2009; DOI: 10.1158/1535-7163.MCT-09-0290 Molecular Cancer Therapeutics 2349 Figure 1. Structure of curcumin and related polyphenols. α μ conversion of arachidonic acid to PGD2 and PGF2 was penicillin (100 U/mL), and streptomycin (100 g/mL) at blocked in epidermal microsomes (21), but curcumin in- 37°C in a 5% CO2 incubator. After 3 d, confluent cells α creased the formation of PGF2 and the stable PGI2 degra- were detached using 1× trypsin/EDTA solution and reseeded α β– 6 2 dation product 6-keto PGF1 in interleukin-1 stimulated at 2 × 10 cells in 20 mL medium in 175-cm flasks. A549 cells (11). Here, we identified microsomal PGE2 For isolation of human platelets, venous blood was taken synthase-1 as functional and highly susceptible molecular from healthy adult donors (Blood Center of the University target of curcumin. Our data show that suppression of Hospital Tuebingen) who did not take any medication for at PGE2 biosynthesis in cell-based assays is primarily due to least 7 d, and leukocyte concentrates were prepared by cen- interference with microsomal PGE2 synthase-1 rather than trifugation (4,000 × g; 20 min; 20°C). Cells were immediately with COX enzymes, and this interaction occurs at low con- isolated by dextran sedimentation and centrifugation on centrations that may be achieved in vivo. Nycoprep cushions (PAA). Platelet-rich plasma was ob- tained from the supernatants, mixed with PBS (pH 5.9; 3:2 v/v), and centrifuged (2,100 × g;15min;roomtempera- Materials and Methods ture), and the pelleted platelets were resuspended in PBS Reagents (pH 5.9)/0.9% NaCl (1:1 v/v). Platelets were finally resus- Curcumin, purchased from Sigma-Aldrich was dissolved pended in PBS (pH 7.4) and 1 mmol/L CaCl2. in DMSO and kept in the dark at −20°C, and freezing/thaw- Determination of PGE2 and 6-keto PGF1α Formation in ing cycles were kept to a minimum. The thromboxane Lipopolysaccharide-Stimulated Human Whole Blood synthase inhibitor CV4151 (22) and the microsomal PGE2 Peripheral blood from healthy adult volunteers (see synthase-1 inhibitor 2-(2-chlorophenyl)-1H-phenanthro above) was obtained by venipuncture and collected in sy- [9,10-d]-imidazole were generous gifts by Dr. S. Laufer ringes containing heparin (20 U/mL). For determination α (University of Tuebingen) and Dr. M. Schubert-Zsilavecz of PGE2 and 6-keto PGF1 , aliquots of whole blood (0.8 mL) (University of Frankfurt), respectively. Materials used are were mixed with the thromboxane synthase inhibitor DMEM/high glucose (4.5 g/L) medium, penicillin, strepto- CV4151 (1 μmol/L) and with aspirin (50 μmol/L). A total vol- mycin, and trypsin/EDTA solution (PAA); PGH2 (Larodan); ume of 1 mL was adjusted with sample buffer [10 mmol/L β and 11 -PGE2, PGB1, 3-(3-(tert-butylthio)-1-(4-chlorobenzyl)- potassium phosphate buffer (pH 7.4), 3 mmol/L KCl, 5-isopropyl-1H-indol-2-yl)-2,2-dimethylpropanoic acid (MK- 140 mmol/L NaCl, and 6 mmol/L D-glucose]. After preincu- 886), human recombinant COX-2, and ovine COX-1 (Cayman bation with the indicated compounds for 5 min at room Chemical). All other chemicals were obtained from Sigma- temperature, the samples were stimulated with lipopolysac- Aldrich, unless stated otherwise. charide (10 μg/mL) for 5 h at 37°C. Prostanoid formation Cells was stopped on ice, the samples were centrifuged (2,300 × g; α A549 cells were cultured in DMEM/high glucose (4.5 g/L) 10 min; 4°C), and 6-keto PGF1 was quantified in the superna- α medium supplemented with heat-inactivated FCS (10%, v/v), tant using a 6-keto PGF1 High Sensitivity EIA Kit (Assay Mol Cancer Ther 2009;8(8). August 2009 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research.