Ethyl Caffeate Suppresses NF-Jb Activation and Its Downstream Inflammatory Mediators, Inos, COX-2, and PGE2 in Vitro Or in Mouse Skin

British Journal of Pharmacology (2005) 146, 352–363 & 2005 Nature Publishing Group All rights reserved 0007–1188/05 $30.00 www.nature.com/bjp Ethyl caffeate suppresses NF-jB activation and its downstream inflammatory mediators, iNOS, COX-2, and PGE2 in vitro or in mouse skin

1Yi-Ming Chiang, 1Chiu-Ping Lo, 1Yi-Ping Chen, 2Sheng-Yang Wang, 1Ning-Sun Yang, 3Yueh-Hsiung Kuo & *,1Lie-Fen Shyur

1Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC; 2Department of Forestry, National Chung-Hsing University, Taichung, Taiwan, ROC and 3Department of Chemistry, National Taiwan University, Taipei, Taiwan, ROC

1 Ethyl caffeate, a natural phenolic compound, was isolated from Bidens pilosa, a medicinal plant popularlyused for treating certain inflammatorysyndromes.The purpose of this studywas to investigate the structural activity, and the anti-inflammatory functions and mechanism(s) of ethyl caffeate. 2 Ethyl caffeate was found to markedly suppress the lipopolysaccharide (LPS)-induced nitric oxide À1 (NO) production (IC50 ¼ 5.5 mgml ), mRNA and protein expressions of inducible nitric oxide synthase (iNOS), and prostaglandin E2 (PGE2) production in RAW 264.7 macrophages. 3 Transient gene expression assays using human cox-2 promoter construct revealed that ethyl caffeate exerted an inhibitoryeffect on cox-2 transcriptional activityin 12- O-tetradecanoylphorbol-13- acetate (TPA)-treated MCF-7 cells. 4 Immunohistochemical studies of mouse skin demonstrated that TPA-induced COX-2 expression was significantlyinhibited byethylcaffeate with a superior effect to that of celecoxib, a nonsteroidal anti-inflammatorydrug. 5 The phosphorylation and degradation of inhibitor kB(IkB) and the translocation of nuclear transcription factor-kB (NF-kB) into the nucleus, as well as the activation of mitogen-activated protein kinases (MAPKs) induced byLPS in macrophages, were not affected byethylcaffeate. Ethyl caffeate, however, could inhibit NF-kB activation byimpairing the binding of NF- kB to its cis-acting element. These results suggest that ethyl caffeate suppresses iNOS and COX-2 expressions partly through the inhibition of the NF-kB Á DNA complex formation. 6 Structure–activityrelationship analysessuggested that the catechol moietyand a,b-unsaturated ester group in ethyl caffeate are important and essential structural features for preventing NF- kB Á DNA complex formation. This studyprovides an insight into the probable mechanism(s) underlying the anti-inflammatory and therapeutic properties of ethyl caffeate. British Journal of Pharmacology (2005) 146, 352–363. doi:10.1038/sj.bjp.0706343; published online 25 July2005

Keywords: Ethyl caffeate; Bidens pilosa; NF-kB; COX-2; iNOS; PGE2 Abbreviations: COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MAPK, mitogen- activated protein kinase; NF-kB, nuclear factor-kB; NO, nitric oxide; PGE2, prostaglandin E2; TPA, 12-O- tetradecanoylphorbol-13-acetate

Introduction

The nuclear factor-kB (NF-kB) is essential for host defense NF-kB-bound IkB complexes at two conserved serine residues and inflammatoryresponses to microbial and viral infections within the IkB N-terminal regulatorydomain. This targets I kB (Li & Verma, 2002). In response to extracellular stimuli, such for ubiquitin-dependent degradation and allows the liberated as bacterial lipopolysaccharide (LPS), tumor-necrosis factor-a NF-kB dimers to be translocated to the nucleus and bind to (TNF-a), or other inflammatorymediators, the transcription cognate DNA enhancer sequences that lead to the transcrip- factor NF-kB is often activated and subsequentlyfacilitates tion of various genes (Karin et al., 2002). The other major the transcription of a number of genes involved in inflamma- extracellular signal transduction pathwaystimulated by tion, such as cyclooxygenase-2 (COX-2), inducible nitric oxide inflammatorymediators is the mitogen-activated protein synthase (iNOS), and specific cytokines (Bayon et al., 2003). kinase (MAPK) pathway(Guha & Mackman, 2001). MAPKs The inhibitor of kB(IkB) kinase (IKK) complex is a central are a familyof serine/threonine protein kinases composed of element of NF-kB-related signaling. IKK phosphorylates the p44 and p42 isoforms (also known as extracellular signal receptor-activated kinase ERK1 and ERK2), p38, and c-Jun NH -terminal kinase (JNK) (Nishida & Gotoh, 1993; Han *Author for correspondence at: Institute of BioAgricultural Sciences, 2 Academia Sinica, No 128, Sec 2, Academia Road, Nankang, Taipei, et al., 1994). It has been found that LPS, the keymediator Taiwan, ROC; E-mail: [email protected] in the inflammation response, can induce activation of these Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate 353

MAPK proteins in macrophage and other cell types (Ham- reagents: aspirin, curcumin, 3-(4,5-dimethylthiazol-2-yl)-2,5- bleton et al., 1996; Chen & Wang, 1999; Guha & Mackman, diphenyltetrazolium bromide (MTT), ethyl cinnamate, ethyl 2001). Activation of p38, but not p44/p42 MAPK, byLPS 3,4-dihydroxyhydrocinnamate, catechol, 12-O-tetradecanoyl- resulted in the stimulation of NF-kB-specific DNA–protein phorbol-13-acetate (TPA), LPS, indomethacin, and DTT were binding and the subsequent expression of iNOS and nitric purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). oxide (NO) release in RAW 264.7 macrophages (Chen & Celecoxib (CELEBREXt) was from PHARMACIA ((Packer) Wang, 1999). NF-kB and MAPKs are therefore known as Pharmacia Ltd., Northumberland, U.K.). Silica gel (230–400 important targets for anti-inflammatorymolecules. mesh) and silica gel 60 F254 TLC and RP-18 F254S TLC plates Improper activation or upregulation of iNOS or COX-2 were purchased from Merck (Germany). RP-18 silica gel has been shown to be associated with the pathophysiologies (75C18-OPN) was purchased from Cosmosil (Kyoto, Japan). of certain types of human cancers as well as inflammatory All other chemicals and solvents used in this studywere of disorders (Beyaert, 2003). Therefore, the identification of HPLC grade. naturallyoccurring phytocompoundsthat can suppress or downregulate the functions of iNOS or COX-2, or the Isolation and elucidation of structure of ethyl caffeate activation of their upstream transcriptional factor NF-kB, from B. pilosa extract maylead to the discoveryof important anti-inflammatory therapeutics. Since inflammation is closelylinked to the B. pilosa Linn. var. radiata (Asteraceae) was collected on the promotion of certain tumors, substances with potent anti- campus of Academia Sinica, Taiwan, in 2001 and a voucher inflammatoryactivities are anticipated to exert chemopreven- specimen (No. 11519) deposited at the herbarium of the tive effects on carcinogenesis (Surh et al., 2001). For instance, Institute of Botany, Academia Sinica, Taiwan. The fresh whole phenolic compounds, particularlythose present in edible and plant of B. pilosa was crushed to give 2.5 kg of raw material, medicinal plants, have been reported to possess substantial which was extracted with 25 l of 70% (v vÀ1) ethanol at room anticancer or cancer chemopreventive properties (Park & temperature. The total crude extract was evaporated in vacuo Pezzuto, 2002). to yield a residue (130 g) that was then suspended in water (1 l) Bidens pilosa (Asteraceae), commonlyknown as ‘hairy and successivelypartitioned with ethylacetate (1 l Â 3 times) beggar-ticks’ or ‘Spanish needles’, is widelydistributed in and n-butanol (1 l Â 3 times), yielding EA, BuOH, and Water tropical and subtropical regions, and has been reported to fractions. Each fraction was evaporated on a rotaryevapora- possess antihyperglycemic (Ubillas et al., 2000), antihyper- tor under reduced pressure to remove organic solvent and then tensive (Dimo et al., 2001; 2002), antiulcerogenic (Tan et al., lyophilized until dry and weighed to determine the yields. The 2000), hepatoprotective (Chin et al., 1996), immunosuppres- yields of the EA, BuOH, and Water fractions were 14.1, 15.2, sive and anti-inflammatory(Pereira et al., 1999), antileukemic and 69.0%, respectively, of the total crude extract in dry (Chang et al., 2001), antimalarial (Branda˜ o et al., 1997), and weight. On the basis of bioactivity-guided fractionation, a antimicrobial (Khan et al., 2001) properties. B. pilosa has portion of the EA fraction (18.0 g) was subjected to column traditionallyor anecdotallybeen used for the management of chromatographyon silica gel, eluted with a CH Cl /MeOH inflammatorydiseases, and stomach and liver disorders. 2 2 gradient solvent system, to give a total of eight fractions However, the cellular and molecular mechanisms underlying (Fractions A–H). The Fraction E (4.3 g eluted from EA the anti-inflammatoryproperties of B. pilosa extract and its fraction with 5% CH Cl /MeOH) was further chromato- derived active compound are currentlynot well defined. In our 2 2 graphed on a silica gel column and eluted in a hexane/ethyl previous study, we identified an ethyl acetate (EA) fraction acetate gradient solvent system to give a total of nine fractions partitioned from the ethanolic extract of fresh whole B. pilosa (Fractions E1–E9). Ethyl caffeate (372.8 mg) was isolated from plants that significantlyinhibited the LPS-induced NO the Fraction E4 (0.7 g) byRP-18 silica gel column elution with production in RAW 264.7 cells (IC ¼ 36 mgmlÀ1) (Chiang 50 50% MeOH/H O. et al., 2004). In the present study, a bioactive phytocompound, 2 Ethyl caffeate: colorless solid; MP 147–1491C; IR n : 3448, ethyl caffeate that exhibits potent inhibitory effects on NO max 1673, 1595 cmÀ1; EI-MS m/z (rel intensity): 208 [M] þ (80), 180 production in macrophages was identified from the bioactive (20), 163 (100); 1H and 13C NMR data (acetone-d )in EA fraction using bioactivity-guided fractionation. Ethyl 6 agreement with published data (Lamidey et al., 2002). caffeate was then investigated for its anti-inflammatory mechanisms in vitro in LPS-stimulated macrophages, and in vivo using TPA-treated mouse skin system. The effects of Cell line and cell culture ethyl caffeate on the activation of NF-kB, MAPKs, as well as on the downstream mediators of inflammation, such as iNOS, RAW 264.7 cells, a murine macrophage cell line, were

COX-2 and prostogladin E2 (PGE2), were investigated. obtained from the American Type Culture Collection (ATCC, MD, U.S.A.) and cultured as recommended byATCC at 37 1C in Dulbecco’s modified essential medium (DMEM) supple- Methods mented with 10% fetal bovine serum (FBS), 100 U mlÀ1 À1 penicillin, and 100 mgml streptomycin in a 5% CO2 Materials incubator. Cells between 5 and 20 passages were used for experiments at a densityof 1 Â 106 cells mlÀ1 except for the NO

IR spectra were recorded on a Perkin-Elmer 983G spectro- production, PGE2 production, and MTT assays. MCF-7 cells photometer. 1H and 13C NMR spectra were obtained from a obtained from ATCC (MD, U.S.A.) were grown in RPMI Varian UnityPlus 400 spectrometer. EIMS were obtained on 1640 media, supplemented with 10% FBS, 100 U mlÀ1 a JEOL JMS-HX 300 mass spectrometer. The chemicals and penicillin, and 100 mgmlÀ1 streptomycin.

British Journal of Pharmacology vol 146 (3) 354 Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate

Measurement of NO and cell viability assays and NF-kB p65) of total proteins were prepared according to Natarajan et al. (1996) with minor modifications. Briefly, RAW 264.7 cells were seeded in 96-well plates at a densityof 1.0 Â 107 cells were washed with cold phosphate-buffered 2 Â 105 cells wellÀ1. The cells were treated with test compounds saline (PBS) and suspended in 250 ml of hypotonic buffer (0.1–20 mgmlÀ1) for 1 h and then incubated for 24 h in fresh (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM DMEM with or without 1 mgmlÀ1 of LPS. Nitrite (a stable EGTA, 1 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate, oxidative end product of NO) accumulation in the medium 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2.0 mgmlÀ1 of RAW 264.7 cells was determined bythe Griess reaction leupeptin, 2.0 mgmlÀ1 aprotinin, and 0.5 mg mlÀ1 benzami- (Schmidt & Kelm, 1996). Briefly, 100 ml cell culture super- dine). The cells were allowed to swell on ice for 10 min, after natants were reacted with 100 ml of Griess reagent (1 : 1 mixture which 20 ml of 2% Nonidet P-40 was added. The cells were of 0.1% N-(1-naphthyl)ethylenediamine in H2O and 1% then vortexed vigorouslyfor 10 s, and the homogenate was sulfanilamide in 5% phosphoric acid) in a 96-well plate, and centrifuged at 12,000 Â g for 5 min at 41C. The supernatants absorbance at 540 nm was recorded using an ELISA reader containing cytosolic proteins were collected. The nuclear (Labsystems Multiskan MS). Results were expressed as total pellets were resuspended in 50 ml ice-cold hypertonic buffer mM nitrite produced in vehicle control (untreated cells) or in (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM cells treated with LPS over the 24 h period in the absence or EGTA, 1 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate, presence of phytocompounds. 1mM PMSF, 2.0 mgmlÀ1 leupeptin, 2.0 mgmlÀ1 aprotinin, and In parallel to the Griess assays, cell viabilities were 0.5 mg mlÀ1 benzamidine), and incubated on ice for 40 min determined using the MTT-based colorimetric assayas with intermittent mixing. Samples were centrifuged at described elsewhere (Scudiero et al., 1988). Briefly, 1 Â 104 12,000 g for 10 min at 41C, and the supernatant (nuclear RAW 264.7 cells treated for 24 h with vehicle or phytocom- extract) was either used immediatelyor stored at À701C. pound were examined for cell viability. Viability of the The protein content was measured bya dye-binding macrophages treated with vehicle (0.5% DMSO) onlywas assay(Bio-Rad Protein Assay).Proteins (20 mg) were separated defined as 100% viable. Survival of macrophage cells after on a 5À20% gradient sodium dodecyl sulfate–polyacrylamide treatment with phytocompounds was calculated using the gel and then electrotransferred to polyvinylidene difluoride following formula: viable cell number (%) ¼ OD570 (treated (PVDF) membrane (Immobilon, Millipore, Bedford, MA, À1 cell culture)/OD570 (vehicle control) Â 100. U.S.A.). The blot was incubated in blocking buffer (3% w v skim milk in TBS buffer) for 30 min, and then incubated, RTÀPCR analysis respectively, with monoclonal antibodies against PARP (Transduction Laboratories, Lexingtons, KY, U.S.A.), RAW 264.7 cells were seeded in six-well plates at a densityof GA3PDH (Biogenesis, England, U.K.), actin, a-tubulin (Oncogene Science, Cambridge, U.K.), phosphorylated 3 Â 106 cells wellÀ1. The cells were treated with test compounds (0.5–5 mgmlÀ1) for 1 h and then incubated for 6 h in fresh form of MAPK family(p42/44, SAPK/JNK, and p38) (New DMEM with or without 1 mgmlÀ1 of LPS. After two washes England Biolabs, Bevery, MA, U.S.A.), iNOS, IkBa,or NF-kB p65 (RelA) (Santa Cruz Biochemicals, Santa Cruz, with ice-cold PBS, the cells were harvested and total cellular s CA, U.S.A.) overnight at 41C. After incubation with the appro- RNA was isolated using TRIZOL Reagent (Invitrogen) according to the manufacturer’s instructions. For each priate anti-rabbit, anti-goat, or anti-mouse IgG conjugated to horseradish peroxidase, the immunoreactive bands RTÀPCR reaction, 2 mg of total RNA was used to synthesise first-strand cDNA using SuperScriptTM II Reverse Transcrip- were visualized with the enhanced chemiluminescence tase (Invitrogen). For amplification of the iNOS gene, a pair of reagents (ECL, Amersham Pharmacia Biotech, Amersham primers were used: Place, U.K.). iNOS, 50-CAGAAGCAGAATGTGACCATC-30 (sense), 50-CTTCTGGTCGATGTCATGAGC-30 (antisense). Transfection of reporter gene constructs and luciferase assay The cDNA sequence of GA3PDH as an internal control was also amplified using the following primers: Chimeric luciferase reporter genes, pPGL3-Basic vector 50-CCATCAATGACCCCTTCATTGACC-30 (sense), (Promega, Madison, WI, U.S.A.) containing the full length 50-GAAGGCCATGCCAGTGAGCTTCC-30 (antisense). or a series of deleted 50 flanking promoter regions of human cox-2 gene were constructed. A specific reverse primer 1 Parameters of PCR reactions were: 94 C for 2 min for one (antisense), À1/À28, cycle, and then 941C for 1 min, 551C for 30 s, and 721C for 1 min for 30 cycles. The amplified PCR products were analyzed 50-TATATAAGCTTCGCAGCGGCGGGCAGGG-30 and with 1% agarose gel electrophoresis, and visualized with four forward primers (sense), ethidium bromide staining. 50-GGCGCGGTACCGGAGAGGAGGGAAAAAT-30; 50-GGCGCGGTACCCTGGGTTTCCGATTTTC-30; 50-ATTCGGGTACCCCCGACGTGACTTCCTC-30; and Western blot analysis 50-ACCATGGTACCTTCCAGCTGTCAAAATC-30 Total cellular proteins (for detecting iNOS, GA3PDH, actin, were used for the amplification of À247/À1, À320/À1, À646/ IkBa, p-SAPK/JNK, p-p38 MAPK, and p-p44/42 MAPK À1, and À1334/À1 (full length) bp regions of human cox-2 proteins) were prepared according to Lo et al. (2002). promoter, respectively, flanked by KpnI and HindIII restric- Cytosolic fraction and nuclear fraction (for PARP, a-tubulin, tion sites from human lymphocyte genomic DNA using PCR

British Journal of Pharmacology vol 146 (3) Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate 355 and cloning into pPGL3-Basic vector. The PCR products were antibody(CaymanChemical, Ann Arbor, MI, U.S.A.) at verified byDNA sequencing. The promoterless pPGL3-Basic room temperature for 1–2 h. The slides were developed using vector was used as a negative control plasmid for luciferase the HPR EnVisionTM system (Dako, Glostrup, Denmark), assays. and the peroxidase-binding sites were detected bystaining with MCF-7 cells were seeded in 24-well plates at a densityof 3,30-diaminobenzidine tetrahydrochloride (Dako). Finally, 1.2 Â 105 cells wellÀ1 and cotransfected with pCOX-2-Luc and counterstaining was performed using Mayer’s hematoxyline. pRL-TK-Luc (Promega, Madison, WI, U.S.A.) plasmids using LipofectAMINE reagent (Invitrogen) as suggested in the Electrophoretic mobility shift assay (EMSA) manufacturer’s protocol. Following transfection (after 4 h), the medium was replaced with fresh RPMI medium and cells EMSAs were performed (with a commerciallyavailable kit, were allowed to recover for 16 h. The transfected cells were LightShiftt Chemiluminescent EMSA Kit (Pierce Biotechnol- then treated with vehicle (0.1% DMSO), 50 ng mlÀ1 TPA ogy, Rockford, IL, U.S.A.)) according to the manufacturer’s alone, or TPA mixed with test compound at indicated instructions. The binding reaction was optimized in binding concentrations for 6 h. Cell lysates were prepared using Passive buffer containing 10% glycerol, 100 mM KCl, 1.5 mM MgCl2, Lysis Buffer (Promega) and then employed for luciferase and 0.3 mM EDTA. Nuclear extracts (4 mg) were incubated activityassays.The promoter activityin arbitraryunit (AU) with biotin end-labeled, 22-mer double-stranded NF-kB was obtained from the ratio of fireflyluciferase activity oligonucleotide, 50-ATGTGAGGGGACTTTCCCAGGC-30 (pCOX-2-Luc) to that of Renilla luciferase (pRL-TK-Luc). (underlining indicates NF-kB-binding site) for 20 min at room temperature. The DNA–protein complexes were separated Measurement of prostaglandin E2 (PGE2) production from nonbound oligonucleotides on a native 5À10% gradient in macrophages polyacrylamide gel. The specificity of NF-kB DNA binding was examined bysupershifting of the DNA–protein complex

PGE2 production was determined according to a modified band byanti-p65 antibody,and bycompetition with the procedure of Hwang et al. (2002). Briefly, RAW 264.7 cells unlabeled NF-kB binding oligonucleotide. An antibody were seeded in 96-well plates at a densityof 1 Â 104 cells wellÀ1 against Ref-1 (Santa Cruz Biochemicals, Santa Cruz, CA, and incubated for 18 h. The cells were pretreated with 500 mM U.S.A.) was employed as a negative control. of aspirin for 3 h to inactivate endogenous COX-1, washed For in vitro binding assays, nuclear extracts from LPS- twice with PBS and then incubated with the test compounds stimulated RAW 264.7 cells (1 mgmlÀ1, 30 min) were treated (0.1–20 mgmlÀ1) for 1 h. The cells were then incubated for 16 h with different concentrations of test compounds at 371C for in fresh DMEM with or without 1 mgmlÀ1 of LPS. After 30 min and analyzed for NF-kB binding byEMSA as incubation, culture media were recovered for PGE2 measure- described above. ment. The amount of PGE2 was determined using the ACEt Competitive Enzyme Immunoassay according to the supplier’s instructions (EIA, Cayman Chemical, Ann Arbor, MI, Results U.S.A.). Ethyl caffeate significantly inhibits NO production, Immunohistochemical study of COX-2 expression and iNOS gene and protein expressions in mouse skin NO is a critical signaling molecule produced at inflammatory The immunohistochemical studywas performed according to sites byiNOS, which is often expressed in response to LPS, Chun et al. (2004) with minor modifications. Female ICR mice interferon-g, and a varietyof proinflammatorycytokines were supplied from the LaboratoryAnimal Center (College of (MacMicking et al., 1997). In this study, the effects of ethyl Medicine, National Taiwan University, Taipei, Taiwan). Mice caffeate on NO production in RAW 264.7 cells stimulated were topicallytreated on their shaven backs with vehicle byLPS were investigated. Curcumin, the major yellow pigment (acetone, 200 ml siteÀ1) or TPA (10 nmol 200 mlÀ1 siteÀ1) for 4 h. of turmeric (Curcuma longa L., Zingiberaceae) known for its For the compound treatments, mice were treated with anti-inflammatoryand anticancer activities (Lin, 2004), was compound at the indicated doses first for 30 min, then further used in parallel as a positive control in this study. As treated with TPA for 4 h, and finallykilled bycervical shown in Figure 1, the nitrite level (equivalent to the NO dislocation. Sections (4 mm) of formalin-fixed, paraffin- level) in culture supernatants was markedlyelevated from embedded tissue were cut onto silanized glass slides and 1.570.3 mM to 33.074.6 mM after a 24 h treatment with LPS. deparaffinized three times with xylene for 5 min each prior to Ethyl caffeate significantly inhibited LPS-induced NO produc- rehydration through a graded alcohol bath. For antigen tion in a dose-dependent manner. At 2 mgmlÀ1, NO production retrieval, the deparaffinized sections were heated and boiled was 35% inhibited, relative to LPS only(graybar) in ethyl in 10 mM citrate buffer (pH 6.0) for 10 min and then rinsed caffeate-treated cells, whereas no detectable effect was with PBS containing 0.05% Tween-20 (PBST) buffer for observed in the curcumin-treated cells (open bar) (Figure 1).

5 min. Each section was treated with 3% hydrogen peroxide The IC50 values for inhibiting NO production in ethyl caffeate- in methanol for 15 min to diminish nonspecific staining. The and curcumin-treated cells were 5.5 mgmlÀ1 and 6.5 mgmlÀ1, sections were then washed with blocking solution (PBS–1% respectively(Figure 1). BSA, PBA) for 30 min and then PBST twice for 5 min each. To evaluate further whether inhibition of NO production in The slides were incubated with 2% normal goat serum in PBA RAW 264.7 cells was affected by the cytotoxic effects of ethyl for 30 min to minimize the immunological binding of antibody, caffeate and curcumin, viabilities of test cells were determined and then incubated with a 1 : 500 dilution of polyclonal COX-2 using MTT assays. We found that ethyl caffeate had little or

British Journal of Pharmacology vol 146 (3) 356 Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate

50 approximately56% reduction was observed when compared ethyl caffeate curcumin to the macrophages treated with LPS only, as determined 40 LPS only À1 vehicle using densitometryanalysis.At a dose of 2 mgml , the level of iNOS mRNA in ethyl caffeate-treated cells was similar to

M) 30 * µ that in vehicle control (non-LPS or phytocompound treated)

* cells. Western blot analysis further demonstrated that a 20 **

Nitrite ( more significant inhibition at the protein level of iNOS was observed in ethyl caffeate-treated (0.5 mgmlÀ1) macrophages, 10 ** with onlyapproximately30% of the iNOS protein level seen ** in the cells treated with LPS alone (Figure 3b). These results 0 indicate that the inhibition of NO production in LPS- Vehicle LPS 0.1 0.5 1.0 2.0 5.0 10.0 control stimulated macrophages byethylcaffeate is mediated through −1 Concentration (µg ml ) the transcriptional as well as translational downregulation Figure 1 Effects of ethyl caffeate and curcumin on LPS-induced of iNOS. NO production in activated RAW 264.7 cells. Cells were treated with or without the indicated concentrations of test compounds for 1 h, then followed bythe addition of LPS (1 mgmlÀ1) for 24 h. Vehicle controls were obtained from cells treated with 0.5% DMSO Effect of ethyl caffeate on COX-2 expression and PGE2 only. NO in the culture medium was measured as described in the production in MCF-7 cells or macrophages Methods. The data are representative of three experiments and expressed as mean7s.d. Statistical analyses between LPS and ethyl Tumor promoter TPA was reported to act as a potent inducer caffeate or curcumin treatment were performed using Student’s of COX-2 expression in various cell types, for example, t-test. Significant inhibition is indicated by* and **, with a P-value o0.05 and o0.01, respectively. macrophages and monocytic cells (Mestre et al., 2001). In order to verifywhether the anti-inflammatoryproperties of ethyl caffeate contribute to its antitumor promoting activity, 120 we examined the effect of ethyl caffeate on COX-2 gene (cox-2) ethyl caffeate activityin TPA-treated MCF-7 cells, a human mammary 100 curcumin adenocarcinoma cell line. Transient transfection experiments * * * * using a cox-2 promoter-luciferase reporter assaywere 80 performed. One full length (À1334/À1), as well as a series of deleted variants of cox-2 promoter-luciferase constructs 60 * * (À646/À1, À320/À1 and À247/À1) were constructed for this 40 * study(Figure 4a). As shown in Figure 4b, TPA significantly induced the transcriptional activityof the À1334/À1 (full Relative cell viability (%) viability cell Relative 20 length) cox-2 promoter in MCF-7 cells (a 2.7-fold increase, * black bar), as compared to that of the control cells (without 0 TPA treatment, white bar). The TPA-induced cox-2 promoter 0.1 0.5 1 2 5 10 20 activityin MCF-7 cells was downregulated byethylcaffeate −1 Concentration (µg ml ) in a dose-responsive manner. The addition of 10, 50, and Figure 2 Effect of ethyl caffeate and curcumin on cell viability of 100 mgmlÀ1 ethyl caffeate to TPA-treated MCF-7 cells resulted RAW 264.7 cells. Cells were treated with or without the indicated in the decrease of full-length cox-2 promoter (À1334/À1) À1 concentrations (0.1À20 mgml ) of test compounds for 24 h. activities, by72, 57, and 48% (graybars), respectively, Percentage of viable cells was determined using MTT assay. Data representative of three experiments were expressed as mean7s.d. as compared to that of the cells treated with TPA only The statistical analyses between vehicle control and ethyl caffeate or (black bar). No cytotoxic effects on MCF-7 cells treated curcumin treatment were performed using Student’s t-test. Po0.01 with ethyl caffeate were observed at the same dosages (10À (*) is considered statisticallysignificant. 100 mgmlÀ1) over 6 h, as determined using MTT assay(data not shown). Aspirin (45 mgmlÀ1 ¼ B250 mM) and indomethacin (3.5 mgmlÀ1 ¼ B10 mM), two well-known nonsteroidal anti- no cytotoxicity to RAW 264.7 cells at concentrations of inflammatorydrugs (NSAID), used as reference controls in 10 mgmlÀ1 or below, whereas curcumin had a significant this experiment, exhibited comparable inhibitoryeffects to cytotoxic effect at 5 mgmlÀ1, as less than 50% of tested cells that of ethyl caffeate (at concentrations between 50 and were detected as viable after treatment with 5 mgmlÀ1 curcumin 100 mgmlÀ1 ¼ B240À480 mM) on the À1334/À1 cox-2 promo- for 24 h (Figure 2). These results indicate that the inhibition of ter activityin MCF-7 cells. NO production induced byLPS in macrophages byethyl An IL-10 promoter-driven luciferase reporter gene assay caffeate (Figure 1) was not due to the toxicityof the compound was also employed in parallel in this study to examine whether to the tested cells. the regulation of cox-2 promoter activitybyethylcaffeate The effects of ethyl caffeate on iNOS gene and protein is highlyspecific. The same doses of ethylcaffeate and expression, which mediate the synthesis of NO in LPS- experimental conditions used in the cox-2 promoter assay stimulated RAW 264.7 cells, were also investigated. RTÀPCR were employed in this IL-10 reporter assay. We found that analysis for specific mRNA in total RNA samples extracted ethyl caffeate did not affect TPA-induced IL-10 promoter from RAW 264.7 cells was performed. The result (Figure 3a) activityeven in the presence of 100 mgmlÀ1 ethyl caffeate shows that ethyl caffeate (1 mgmlÀ1) significantlyinhibited (data not shown). This result suggests that ethyl caffeate the iNOS mRNA expression in LPS-treated macrophages; does not regulate transcriptional activityof IL-10, and that

British Journal of Pharmacology vol 146 (3) Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate 357

a b − Ethyl caffeate --0.5 1 2 5 --0.5 1 2 5 (µg ml 1)

LPS - + + + + + - + + + + + (1 µg ml−1)

iNOS

GA3PDH

1.2 1.2

1 1

0.8 0.8

0.6 0.6

0.4 0.4

iNOS/GA3PDH

Relative ratio of 0.2 0.2

0 0 Vehicle LPS 0.5 1 2 5 Vehicle LPS 0.5 1 2 5 − − Ethyl caffeate (µg ml 1) Ethyl caffeate (µg ml 1) Figure 3 Effects of ethyl caffeate on LPS-induced iNOS expression in RAW 264.7 cells. (a) Cells were treated with the indicated concentrations of ethyl caffeate for 1 h followed by the addition of LPS (1 mgmlÀ1) for 6 h. Total cellular RNA (2 mg) was subjected to RT–PCR and the final PCR product resolved using 1% agarose gel electrophoresis. (b) Cells were treated with the indicated concentrations of ethyl caffeate for 1 h followed by the addition of LPS (1 mgmlÀ1) for 18 h. Total cellular proteins (20 mg) were resolved bySDS–PAGE, then transferred to PVDF membrane and detected with specific antibodies as described in the Methods. Quantification of iNOS expression was normalized to GA3PDH using a densitometer. inhibition of cox-2 promoter activitybyethylcaffeate is highly in RAW 264.7 cells was effectivelyinhibited bythe ethyl specific. caffeate treatment (45%) at a dose of 5 mgmlÀ1, as quantita- A comparative studyof the transfection experiments with tivelydetermined using densitometry.We next determined the full-length cox-2 promoter (À1334/À1) or deletion constructs, amount of PGE2, a critical inflammatorymediator, which is containing NFkB-, NF-IL6-, and CRE-binding sites (À646/ one of the major products produced bythe enzymaticreaction À1), NF-IL6, and CRE-binding sites (À320/À1), and a single of COX-2. Figure 5b shows that ethyl caffeate at 1 mgmlÀ1

CRE site (À247/À1), respectively, (Figure 4c) was performed markedlysuppressed the PGE 2 production in LPS-stimulated in MCF-7 cells with or without the TPA treatment. Similar RAW 264.7 cells. Ethyl caffeate (2–5 mgmlÀ1) treatment caused luciferase activities (a 2.5–2.6-fold higher activitythan that of total inhibition of PGE2, whereas only40 À78% was inhibited the basal COX-2 reporter activityin control cells without TPA bycurcumin at the same doses. Indomethacin used as a treatment, white bars) were observed for the À1334/À1 and positive control in this studyexhibited total inhibition of PGE 2 À646/À1 promoter constructs in TPA-treated MCF-7 cells production at a concentration between 0.5 and 1 mgmlÀ1. (black bars) (Figure 4c). These results indicate that the À646/ À1 promoter region of cox-2 promoter, containing NFkB-, NF-IL6-, and CRE-binding sites, was able to act as the Effects of ethyl caffeate on TPA-induced COX-2 minimal responsive element for TPA to induce luciferase expression in mouse skin activityin MCF-7 cells. The À320/À1 and À247/À1 deletion promoter constructs, harboring NF-IL6- and CRE-binding In order to examine whether ethyl caffeate can inhibit COX-2 sites, and a single CRE site, respectively, were observed to be expression in vivo, we topicallyapplied ethylcaffeate or slightlyor not responsive to the TPA treatment in MCF-7 cells celecoxib, a well-known NSAID with potent COX-2 inhibitory (black bars), as theyexhibited similar luciferase activities to the activity, on TPA-treated mouse skin and conducted immuno- controls (white bars). histochemical analyses. Upon treatment with acetone (vehicle We further examined the effect of ethyl caffeate on the control) for 4 h, specific COX-2 immunostaining was detect- transcriptional activityof cox-2 in TPA-treated MCF-7 cells. able in the dermal sebaceous gland (Figure 6). In contrast, In the presence of 20 mgmlÀ1 of ethyl caffeate (gray bars), 67 COX-2 expression increased dramaticallyin the epidermal and 80% of luciferase activities were found in TPA-treated layer upon TPA treatment (10 nmol) for 4 h. TPA-induced cells transfected with À1334/À1 and À646/À1 promoter COX-2 expression was significantlyabolished byethylcaffeate constructs, respectively, compared to those of the test cells in a dose-dependent manner. An inhibition of COX-2 protein harboring the same constructs treated with TPA only(100%, expression by1 mg 200 mlÀ1 siteÀ1 ethyl caffeate (24 mM) was black bars). Little or no effects were observed for the À320/À1 comparable to the inhibitoryeffect of 1 mg 200 mlÀ1 siteÀ1 and À247/À1 promoter activities when MCF-7 cells were celecoxib (13 mM), and, strikingly, 48 mM ethyl caffeate (2 mg cotreated with TPA and ethyl caffeate (20 mgmlÀ1). 200 mlÀ1 siteÀ1) was observed to be more effective than 10 mg The COX-2 protein expression in LPS-stimulated macro- 200 mlÀ1 siteÀ1 celecoxib treatment (131 mM) (Figure 6). Similar phages was further examined using Western blot analysis. results were obtained in three independent repeated experi- Figure 5a shows that LPS-mediated COX-2 expression (100%) ments.

British Journal of Pharmacology vol 146 (3) 358 Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate a CRE a NF-IL6 − (-707/-700) (-266/-258) Ethyl caffeate--- 0.5 1 2 5 (µg ml 1) GATA-1 ATG (-823/-818) NF-κB NF-κB CRE −1 NF-IL6 (-584/-567) (-363/-343) (-193/-187) LPS - + + + + + (1 µg ml ) (-846/-838) 5' 3' Luc COX-2 -1334 -646 -320 -247 -1

-1334/-1 GA3PDH -646/-1 1.2 -320/-1 -247/-1 1

DH b 12 0.8 0.6 10 /GA3P * 0.4

Relative ratio

8 COX-2 0.2 * * 6 ** ** 0 DMSO LPS 0.5 1 2 5 4 − Ethyl caffeate (µg ml 1)

Luciferase activity 2 b 1000 0 curcumin 800 Control TPA 2.5 10 50 100 Aspirin Indom. ) ethyl caffeate

−1 −1 -1 (-TPA) 45 µg ml 3.5 µg ml l − indomethacin

+ Ethyl caffeate (µg ml 1) 600

(pg m (pg c 20 2 400 control ###

### PGE 16 TPA only 200 TPA + ethyl caffeate * 12 * 0 012345 8 Concentration (µg ml-1) Figure 5 Effects of ethyl caffeate on LPS-induced COX-2 expres- Luciferase activity 4 sion and PGE2 production in RAW 264.7 cells. (a) Cells were treated 0 with the indicated concentration of ethyl caffeate for 1 h followed by -247/-1 -320/-1-646/-1 -1334/-1 the addition of LPS (1 mgmlÀ1) for 18 h. Total cellular proteins (20 mg) were resolved bySDS–PAGE, then transferred to PVDF Figure 4 Characterization the effect of ethyl caffeate on transcrip- membrane and detected with specific antibodies as described in the tional activities of cox-2 promoter. (a) Map of full-length cox-2 Methods. Quantification of COX-2 expression was normalized to promoter. Arrows indicate the full-length and three serial deletion GA3PDH using a densitometer. (b) Aspirin-pretreated RAW 264.7 cox-2 promoter constructs. (b) MCF-7 cells transfected with full- cells were treated with the indicated concentrations of ethyl caffeate length of pCOX-2-Luc constructs ( 1334/ 1) and internal control À À for 1 h and then stimulated with LPS for 16 h. PGE2 in the culture plasmid pRL-TK-Luc were left unstimulated (Control) or stimu- medium was measured as described in the Methods. Indomethacin lated with TPA in the absence (black bar) or presence (graybars) of was used as a positive control. The data are representative of three the indicated concentrations of ethyl caffeate, aspirin, and indo- experiments and expressed as mean7s.d. methacin for 6 h. Total lysate (10 mg) was subjected to dual luciferase reporter assay. The promoter activity in arbitrary unit (AU) was obtained from the ratio of fireflyluciferase activity(pCOX-2- Luc) to that of Renilla luciferase (pRL-TK-Luc). Significant 30 min of LPS stimulation, the phospho-MAPK proteins were inhibition is indicated by* and **, with a P-value o0.05 and 0 significantlyinduced; however, no significant inhibition on the o0.01, respectively. (c) Sequential 5 -deletions of the full-length cox- 2 promoter was cloned as described in the Methods. Specific protein levels of phospho-MAPK proteins were observed upon promoter activities of À1334/À1, À646/À1, À320/À1, and À247/À1 ethyl caffeate treatment of murine macrophages at concentra- constructs were obtained as described in (b). The data are tions between 1 and 10 mgmlÀ1 (ca., 4.8À48 mM) (Figure 7). 7 representative of three experiments and expressed as mean s.d. Recently, it has been shown that octyl caffeate, a semisynthe- Statistical analyses between control and TPA only (#) or control and TPA þ ethyl caffeate (*) were performed using Student’s t-test. sized compound from caffeic acid, at 50 mM, can suppress LPS/ Significant inhibition is indicated by* and ###, with a P-value INF-g-induced expressions of SAPK/JNK, p38, and p42/44 o0.05 and o0.01, respectively. proteins in rat aortic smooth muscle cells (Hsiao et al., 2003). It is not clear whether the differences in cell type (murine macrophages vs rat aortic smooth muscle cells) with respect to Ethyl caffeate does not affect LPS-induced activation specific inducing conditions (i.e., LPS induction vs LPS and of MAPK in macrophages INF-g coinduction) between the previous (Hsiao et al., 2003) and this studycould have resulted in the different effects on the The activation of SAPK/JNK, p38, and p42/44 (ERK 1/2) activation of MAPK proteins bythe two structurallysimilar MAPK proteins known to be involved in the regulation of compounds, that is, octyl caffeate and ethyl caffeate. iNOS or COX-2 expression were further investigated to determine if there was anylink between these signaling molecules and the anti-inflammatorymechanism(s) of ethyl Ethyl caffeate does not affect NF-kB translocation caffeate. Since phosphorylation is required for the activation of MAPKs, immunoblotting analyses using phospho-specific The translocation of NF-kB to the nucleus is preceded by antibodies against MAPK proteins were performed. After phosphorylation, ubiquitination, and proteolytic degradation

British Journal of Pharmacology vol 146 (3) Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate 359

as internal controls to ensure that there was no cross contamination during protein extractions of the cellular and nuclear fractions.

Ethyl caffeate inhibits LPS-induced NF-kB activation through prevention of NF-kB binding to DNA

LPS has been reported to act as a potent inducer of NF-kB activation in macrophages (Bayon et al., 2003). We used an in vitro binding assayto examine the effect of ethylcaffeate to perturb the abilityof active NF- kB to bind to a biotin- labeled oligonucleotide containing kB DNA elements using EMSA. Nuclear extracts from LPS-stimulated cells were incubated with increasing concentrations of ethyl caffeate for Figure 6 Immunohistochemical studyon the inhibitoryeffect of 30 min at 371C. Inhibition of NF-kB binding to DNA byethyl ethyl caffeate on COX-2 expression in TPA-treated murine skin. caffeate was observed in a dose-dependent manner in that the Dorsal skins of female ICR mice were treated topicallywith acetone compound significantlyinhibited the NF- kB binding to DNA (vehicle control) or TPA only(10 nmol) for 4 h, or treated with the at 10 mgmlÀ1 with virtuallya complete inhibition at 20 mgmlÀ1 indicated concentrations of ethyl caffeate or celecoxib for 30 min and then treated with TPA for 4 h. Paraffin-embedded tissues from (Figure 9a). It has been shown that reducing agents (e.g., DTT) vehicle, TPA only, and compound/TPA-treated mice were immu- can reverse the inhibitoryeffect of a number of compounds, nostained for specific COX-2 protein and counter-stained with for example, caffeic acid phenethyl ester, L-1-tosylamido-2- hematoxylin, as described in the Methods. Immunohistograms were phenylethyl chloromethyl ketone (a serine protease inhibitor), taken with an Olympus DP-70 camera on a Nikon ECLIPSE E800 and herbimycin A, on NF-kB activation (Natarajan et al., microscope (mangnification: Â 200). 1996). We, therefore, further examined the abilityof DTT to reverse the effect of ethyl caffeate in preventing the formation Ethyl caffeate (µg ml−1) --12 510 of NF-kB DNA complex. As shown in Figure 9a, DTT at 50 mM had no significant effect on LPS-dependent activation of − LPS (1 µg ml 1) - + + + + + NF-kB, but it completelyreversed the inhibition induced by ethyl caffeate. This result is in agreement with the previous p-SAPK/JNK reports (Natarajan et al., 1996), suggesting the important role of sulfhydryl groups in the TNF- or LPS-dependent activation p-p38 of NF-kB. p-p42 We next investigated the effect of ethyl caffeate on inhibition p-p44 of NF-kB activation in macrophages byEMSA. As shown in Figure 9b, the induction of specific NF-kB DNA binding Actin activitybyLPS in RAW 264.7 cells was moderatelyinhibited 1 Figure 7 Effects of ethyl caffeate on MAPKs phosphoration in by10 mgmlÀ ethyl caffeate. The relative level of NF-kB DNA RAW 264.7 cells. Cells were treated with various concentrations of binding complex in ethyl caffeate-treated macrophages acti- À1 ethyl caffeate for 1 h followed by the addition of LPS (1 mgml ) for vated byLPS at 60, 120, and 180 min were 1.0, 0.7, and 0.6, 30 min. Total cellular proteins were prepared for Western blot analysis of phospho-MAPKs proteins by specific anti-phospho- respectively, compared to those (1.0) in the cells activated with MAPKs antibodies. LPS only(without compound treatment). The NF- kB DNA complex in the LPS-treated cells was supershifted (band labeled with asterisk) when coincubated with specific antibody of IkB (Karin & Lin, 2002). Western blot analysis was against p65 (Rel A protein of NF-kB) and was unaffected by performed to determine whether ethyl caffeate can abolish the the addition of a nonspecific antibodyagainst Ref-1 (redox NF-kB translocation in macrophages. In this regard, we factor 1, a cellular redox/DNA repair protein). Moreover, the determined the protein distribution of NF-kB (Rel A) and specific binding of NF-kB DNA to nuclear protein could be phospho-IkBa in cytosolic and nuclear proteins as well as the completelyprevented with the addition of excess, unlabeled protein level of IkBa in cytosolic proteins (correlated to the consensus NF-kB oligonucleotide. Taken together, these degradation of IkBa protein) of LPS-stimulated RAW 264.7 results demonstrate that NF-kB DNA binding can be very cells treated with ethyl caffeate or vehicle control. First, we specificallyinhibited byethylcaffeate. observed that the phosphorylation as well as the degradation of IkB induced byLPS in macrophages was not affected by Structure–activity relationship studies of ethyl caffeate treatment with ethyl caffeate (Figure 8a and b). We further inhibition of NF-kB DNA complex formation examined the appearance of the p65 subunit (Rel A) of NF-kB in the cytosolic and nuclear extracts of control and ethyl To delineate the role of ethyl caffeate in the inhibition of NF- caffeate-treated cells. Results shown in Figure 8c revealed that kB Á DNA complex formation, ethyl caffeate and its structural ethyl caffeate treatment for 1 h, at concentrations between 1 analogs, ethyl 3,4-dihydroxyhydrocinnamate, ethyl cinnamate, and 20 mgmlÀ1, did not reduce nuclear localization of the Rel and catechol, were used for in vitro NF-kB DNA-binding A protein, since equal levels of RelA proteins were observed assays. Figure 10 shows that ethyl caffeate and ethyl 3,4- in the nuclear proteins. Poly(ADP-ribose) polymerase (PARP), dihydroxyhydrocinnamate (containing no a,b-unsaturated a nuclear protein, and a-tubulin, a cytosolic protein, were used ester group) completelyinhibited NF- kB DNA binding at 50

British Journal of Pharmacology vol 146 (3) 360 Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate

a 0 5 15 30 45 60 0 5 15 30 45 60 (min) LPS --+ + + + + + - + + + + + (1 µg ml−1) Ethyl caffeate ------+ + + + + + (5 µg ml−1) p-IκBα

Actin b Ethyl caffeate --1202 5 10 (µg ml−1)

LPS -+- + + + + + + (1 µg ml−1)

IκBα Actin

c Cytoplasm Nucleus

Ethyl caffeate -----11 2 51015 20 - 2510205 (µg ml−1) LPS -+- + + + + + + - + + + + + + (1 µg ml−1)

NFκB PARP α-tubulin

Figure 8 Effects of ethyl caffeate on IkBa phosphorylation and degradation, and NF-kB translocation in RAW 264.7 cells. (a) Cells were treated with 1 mgmlÀ1 of LPS in the absence or presence of 5 mgmlÀ1 ethyl caffeate for different time courses as indicated. Total cellular proteins were prepared for Western blot analysis of phospho-IkBa protein byspecific anti-phospho-I kBa antibody. (b) Cells were treated with various concentrations of ethyl caffeate for 1 h followed by the addition of LPS (1 mgmlÀ1) for 11 min. Total cellular proteins were prepared for Western blot analysis of IkBa protein byspecific anti-I kBa monoclonal antibody. (c) Cells were treated with various concentrations of ethyl caffeate for 1 h followed by the addition of LPS (1 mgmlÀ1) for 30 min. Cytosolic and nuclear extracts (20 mg) were prepared for Western blot analysis of p65 of NF-kB protein byspecific anti-p65 NF- kB monoclonal antibody. PARP, a nuclear protein, and a-tubulin, a cytosolic protein, were used as controls to confirm that there was no contamination during extraction of each fraction.

and 100 mM, respectively. Catechol totally blocked the binding to be involved in the iNOS expression mediated byLPS in at 400 mM, whereas ethyl cinnamate, which contains an a,b- mouse macrophages, were examined to elucidate the possible unsaturated ester group but lacks the catechol moiety, did not molecular actions of ethyl caffeate on inhibiting the produc- inhibit NF-kB-binding activityeven at a concentration of tion of NO and iNOS. Although phosphorylation plays an 400 mM. These results therefore suggest that the catechol important role in activating MAPK in LPS signaling, we moietyand the a,b-unsaturated ester group together may found that the activated phosphorylated forms of MAPKs playimportant roles in the inhibition of binding of NF- kB including SAPK/JNK, p38, and p42/44 induced byLPS were to DNA. not affected byethylcaffeate treatment. In addition, the translocation of NF-kB to the nucleus, mediated bythe phosphorylation, ubiquitination, and proteolytic degradation Discussion of IkB, was also not responsive to ethyl caffeate. However, we did observe that the activityof NF- kB binding to the Manylines of evidence have indicated that nitric oxide (NO) is consensus sequence of kB was inhibited byethylcaffeate in a potent proinflammatorymediator and mayhave a multi- activated macrophage cells and in cell-free binding systems. faceted role in mutagenesis and carcinogenesis (Surh et al., We thus propose here that the significant inhibition of NO 2001). iNOS catalyzes the oxidative deamination of L-arginine production observed was likelydue to the downregulation of to produce NO. Therefore, aberrant or excessive expression of transcriptional and translational activities of iNOS caused by iNOS is often implicated in the oncogenesis and pathogenesis the interference of NF-kB activitybyethylcaffeate. of cancer. Compounds that can selectivelyinhibit the Expression of COX-2 has been related to various human undesirable expression of iNOS mayserve as potential cancer cancers, including breast and colon cancers (Bing et al., 2001). chemoprevention candidates. Here, we demonstrate that a In this study, therefore, we used a human mammary natural phenolic compound, ethyl caffeate, isolated from a carcinoma cell line, MCF-7, to evaluate the effect of ethyl medicinal plant, B. pilosa, exhibits potent inhibitoryeffects on caffeate on transcriptional activities of COX-2. Promoter NO production as well as iNOS expression in LPS-activated elements in cox-2 gene for NF-kB, NF-IL6, and CRE have murine macrophages RAW 264.7 cells. Two signaling path- been found to be important in regulating cox-2 gene ways, the MAPK and NF-kB activation mechanisms, known transcription (Mestre et al., 2001). Mestre et al. reported that

British Journal of Pharmacology vol 146 (3) Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate 361

−1 present study, we analyzed the cox-2 transcriptional activity a Ethyl caffeate (µg ml ) - - 2 5 10 20 20 - − mediated byTPA in MCF-7 cells using transfection experi- LPS (1 µg ml 1) - + + + + + + + ments with cox-2 promoter-luciferase full-length and deletion µΜ DTT (50 ) ------+ + constructs. Our results showed that the À646/À1 promoter NF-kB•DNA complex construct, containing mainlythe NF- kB, NF-IL6, and CRE promoter sites, exhibited significant luciferase activities, reach- ing a similar level as that of the À1334/À1 (full-length) b construct, in TPA-treated MCF-7 cells (Figure 4c). Whereas the À320/À1 and À247/À1 deletion promoter constructs, B DNA

κ -1 - - which harbors NF-IL6 and CRE promoter sites and a single Time (min) 60 120 180 NF- CRE site, respectively, were observed to exhibit little or no µ −1 Anti-Ref-1 Anti-Ref-1 An -1 Anti-p65 Anti-p65 Ethyl caffeate ( g ml ) 10 10 10 Anti- 65 ------Cold NF- response to TPA treatment. These results indicated that the µ −1 LPS (1 g ml ) - + + + + + + - + + + + promoter element for NF-kB or the coexistence of the NF-kB, * NF- κB•DNAB DNA NF-IL6, and CRE promoter elements playan important role complex in mediating cox-2 gene transcription in response to TPA

Ratio 1 1 1 0.7 1 0.6 treatment in MCF-7 cells. The inhibitoryeffects of ethyl caffeate on cox-2 promoter were found to act upon the À1334/ À1 (full length) and À646/À1 promoter constructs, inhibiting the full-length promoter the most, suggesting that the very 0 Figure 9 Inhibition of LPS-induced NF-kB activation byethyl upstream 5 flanking region of cox-2, containing the cis-acting caffeate in vitro and in vivo. (a) Nuclear extracts from LPS- element(s), for example, SP-1, GATA-1, or GRE, other than stimulated RAW 264.7 cells were treated with different concentra- the three NF-kB, NF-IL6, and CRE promoter sites, mayplay tions of ethyl caffeate for 30 min at 371C in vitro and analyzed for a role in, or be responsive to, the treatment of ethyl caffeate. NF-kB binding byelectrophoretic mobilityshift assays(EMSA) as described in the Methods. The arrow indicates the gel location of Ethyl caffeate was observed to significantly inhibit NF-kB NF-kB bound to DNA. (b) Cells were treated with various activation not byblocking the degradation of I kBa, but by concentrations of ethyl caffeate for 1 h followed by treatment with preventing the binding of NF-kB and DNA. These observa- LPS (1 mgmlÀ1) for 60, 120, and 180 min. Nuclear extracts were tions are similar to that obtained from caffeic acid phenethyl prepared and assayed for activation of NF-kB byEMSA. Specificity ester (CAPE), an active component of propolis from honeybee of NF-kB band was analyzed by incubation with anti-p65 antibody, anti-Ref-1 antibody, and unlabeled NF-kB oligonucleotide. The hives. CAPE is known to exhibit various bioactivities including arrow indicates the gel location of NF-kB bound to DNA and the anti-inflammatoryand anticarcinogenic properties (Natarajan asterisk indicates the supershift band. et al., 1996). Like CAPE, we found that ethyl caffeate- dependent inhibition of NF-kB activation was reversed bythe reducing agent DTT, suggesting that ethyl caffeate may Ethyl 3,4-dihydroxy- modify a critical sulfhydryl group in NF-kB protein. Struc- Ethyl caffeate hydrocinnamate ture–activityrelationship studies on CAPE, as demonstrated byNatarajan et al. (1996), showed that the number and (µM)

LPS 20 50 100 400 20 50 100 400 placement of hydroxyl groups in CAPE is a critical determi- nant of the extent of inhibition of NF-kB DNA binding. In this study, we further demonstrated that the presence of hydroxyl groups in the catechol moiety of caffeic acid derivatives are essential for their abilityto inhibit NF- kB DNA complex formation, as there was no detectable inhibition Ethyl cinnamate Catechol found for ethyl cinnamate, which contains an a,b-unsaturated ester group but lacks the catechol moiety(Figure 10). In (µM) contrast to ethyl cinnamate, ethyl caffeate containing both LPS 20 50 100 400 20 50 100 400 catechol and an a,b-unsaturated ester group exhibited the most significant inhibitoryeffect (Figure 10). Phytocompounds containing a,b-unsaturated ester groups, for example, Avicin G, a triterpenoid saponin from Acacia Figure 10 In vitro inhibition of NF-kB binding to DNA by victoriae (Haridas et al., 2001), or containing a,b-unsaturated different structural analogs of ethyl caffeate. Nuclear extracts from carbonyl moiety, for example, sesquiterpene lactones helanalin LPS-stimulated RAW 264.7 cells were treated with different (Lyss et al., 1998), have been suggested to prevent the binding 1 concentrations (mM) of test compounds for 30 min at 37 C and of NF-kB to DNA bydirectlytargeting cysteineresidues of analyzed for NF-kB binding byEMSA. p65 subunit of NF-kB. X-raycrystallographystudieshave demonstrated that cysteine residues in p65 play an important the maximal transcriptional activityof cox-2 induced byLPS role in optimal DNA–protein interactions (Kumar et al., 1992; in murine macrophages was observed as a sophisticated Chen et al., 1998). Mutational and other biochemical studies cooperation among NF-kB, NF-IL6, and CRE promoter further showed that these two types of structural groups can sites. In other words, the isolated NF-kB, NF-IL6, and CRE react with nucleophiles, especially the cysteine sulfhydryl promoter sites were less effective than the intact cox-2 groups of p65 bya Michael-type addition (Schmidt, 1997). Our promoter, containing the three promoter sites, in mediating structure–activityrelationship studyshowed that the effective- the cox-2 transcription in LPS-treated macrophages. In the ness of ethyl caffeate and its structural analogs at preventing

British Journal of Pharmacology vol 146 (3) 362 Y.-M. Chiang et al Anti-inflammatory mechanism of ethyl caffeate a O O generate five possible reaction centers and ethyl 3,4-dihydrox- HO [O ] O yhydrocinnamate may only generate four after oxidation O O [R] (Figure 11a and b, as indicated byarrows), therefore, ethyl HO O caffeate maybe more effective in covalentlyinteracting with Ethyl caffeate the p65 protein than ethyl 3,4-dihydroxyhydrocinnamate, b O O possibly via Michael-type addition (Schmidt, 1997). Ethyl HO [O ] O cinnamate, however, which lacks the catechol moiety(Figure O O [R] 11c), cannot be oxidized and thus it cannot block the Cys HO O residues of NF-kB through the proposed ‘oxidation–inhibi- Ethyl 3,4-dihydroxyhydrocinnamate tion’ mechanism. c O In summary, this is to our knowledge the first report to

O demonstrate that a naturally occurring phytocompound, ethyl caffeate, possesses potent anti-inflammatoryactivities. Our

Ethyl cinnamate results reveal that ethyl caffeate exerts its anti-inflammatory effects byattenuating NF- kB activation and suppression of d HO O NF-kB-regulated proteins. This NF-kB regulation in turn [O ] affects iNOS and COX-2 expression at the gene transcriptional [R] HO O or translational levels. The production of PGE2, a growth- Catechol promoting factor in certain carcinoma cell lines and a mediator Figure 11 An ‘oxidation–inhibition’ mechanism proposed for the of inflammation (Tjandrawinata et al., 1997), was also ethyl caffeate and its derivatives to prevent NF-kB binding to DNA. significantly inhibited by ethyl caffeate in this study. The The arrowheads indicate the possible reaction centers of (a) ethyl anti-inflammatoryproperties of ethylcaffeate seem to also caffeate, (b) ethyl 3,4-dihydroxyhydrocinnamate, (c) ethyl cinna- contribute to its antitumor promoting activityas demonstrated mate and (d) catechol. using the TPA-stimulated mouse skin evaluation system. The TPA induced overexpression of COX-2 protein in mouse skin NF-kB DNA binding were as follows: ethyl caffeate4ethyl was found to be drasticallyinhibited byethylcaffeate 3,4-dihydroxyhydrocinnamate4catechol4ethyl cinnamate treatment. Since inadequate or abnormal overexpressions of (Figure 10). On the basis of this result, previous reports COX-2 and iNOS are implicated in the pathogenesis of various described above, and the chemical environment of the target types of human cancers, results obtained from this study phytocompounds, we propose here an ‘oxidation–inhibition’ indicate that ethyl caffeate may have therapeutic applications mechanism for the observed effects of ethyl caffeate and its for inflammatorydisorders or chemopreventive activities structural analogs on the inhibition of NF-kB DNA binding against cancers. (Figure 11). Oxidant(s) present in the cellular environment, for example, nuclear extract, might thus have the abilityto oxidize We are indebted to Dr Pei-Wen Hsiao for valuable suggestions and we the catechol moiety, thereby generating o-benzoquinone that thank other colleagues at the Institute of BioAgricultural Sciences, mayin turn more effectivelyinteract with the Cysresidues of Academia Sinica, for their excellent technical assistance. We thank Dr NF-kB leading to the inhibition of NF-kB binding to the DNA Vanisree Staniforth (Dr Ning-Sun Yang laboratory) for providing us (Figure 11d). Ethyl 3,4-dihydroxyhydrocinnamate containing with the plasmid pIL10-Luc carrying IL-10 promoter and luciferase a catechol, but no a,b-unsaturated ester moiety, inhibits NF- reporter gene. We thank Wei-Yu Chen, M.D. (Department of Pathology, Taipei Medical University, Taiwan) for his excellent kB DNA binding; however, the inhibition is less effective assistance on preparation of paraffin-embedded mouse skin tissue (approximatelytwo-fold lower) than that of ethylcaffeate sections. This work was supported bygrant (AS91IBAS1PP) from the (Figure 10). One possible reason is that ethyl caffeate may Genomic Research Center (GRC), Academia Sinica, Taiwan, R.O.C.

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