Curcumin inhibits phorbol ester-induced expression ...

Carcinogenesis Advance Access published July 3, 2003

Accepted for publication in Carcinogenesis

Curcumin inhibits phorbol ester-induced expression of cyclooxygenase-2 in mouse skin through suppression of extracellular signal-regulated kinase activity and NFκB activation

Kyung-Soo Chun1, Young-Sam Keum1, Seong Su Han1, Yong-Sang Song2, Su-Hyeong Kim2, and Young-Joon Surh1,3 1

College of Pharmacy and 2Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 151-741, Seoul, Korea

__________________________ 3

Corresponding author: Prof. Young-Joon Surh College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku Seoul 151-742, South Korea

Phone : + 82 2 880-7845; Fax : + 82 2 874-9775; E-mail : [email protected]

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Copyright (c) 2003 Oxford University Press

Downloaded from http://carcin.oxfordjournals.org/ by guest on June 4, 2013

[Running title : Inhibition of COX-2 expression by curcumin in mouse skin]

NIK, NF-κB-inducing kinase; IKK, IκB kinase; PDTC, pyrrolidine dithiocarbamate; TPCK, N-α-p-tosyl-L-lysine chloromethylketone; MEKK1, MAP kinase/ERK kinase kinase-1; AP-1, activator protin 1

Key words: Curcumin; Cyclooxygenase; carcinogenesis

NF-κB;

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Anti-tumor

promotion;

Mouse

skin


Foot Notes: 1. This manuscript was prepared from a dissertation by K.-S. Chun in partial fulfillment of the requirement for the Ph.D. degree at the Seoul National University. 2. To whom requests for reprints should be addressed, at College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku, Seoul 151-742, South Korea. Phone: +82 2 880 7845; Fax: +82 2 874 9775; E-mail: [email protected] 3. The abbreviations used are: COX, cyclooxygenase; PG, prostaglandin; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; DT, dithiothreitol;, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; iNOS, inducible nitric oxide synthase; MEK, MAP kinase kinase;

Abstract

NF-κB activation in mouse skin, which was associated with its blockade of degradation of the inhibitory protein IκBα and also of subsequent translocation of the p65 subunit to nucleus. TPA treatment resulted in rapid activation via phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein (MAP) kinases, which are upstream of NF-κB. The MEK1/2 inhibitor U0126 strongly inhibited NF-κB activation, while p38 inhibitor SB203580 failed to block TPA-induced NF-κB activation in mouse skin. Furthermore, U0126 blocked the IκBα phosphorylation by TPA, thereby blocking the nuclear translocation of NF-κB. Curcumin inhibited the catalytic activity of ERK1/2 in mouse skin. Taken together, suppression of COX-2 expression by inhibiting ERK activity and NF-κB activation may represent molecular mechanisms underlying previously reported anti-tumor promoting effects of this phytochemical in mouse skin tumorigenesis.

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Recently, there have been considerable efforts to search for naturally occurring substances for the intervention of carcinogenesis. Many components derived from dietary or medicinal plants have been found to possess substantial chemopreventive properties. Curcumin, a yellow colouring ingredient of turmeric (Curcuma longa L., Zingiberaceae), has been shown to inhibit experimental carcinogenesis and mutagenesis, but molecular mechanisms underlying its chemopreventive activities remain unclear. In the present work, we assessed the effects of curcumin on 12-O-tetradecanoylphorbol13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2) in female ICR mouse skin. Topical application of the dorsal skin of female ICR mice with 10 nmol TPA led to maximal induction of COX-2 mRNA and protein expression at about 1 h and 4 h, respectively. When applied topically onto shaven backs of mice 30 min prior to TPA, curcumin inhibited the expression of COX-2 protein in a dose-related manner. Immunohistochemical analysis of TPA-treated mouse skin revealed enhanced expression of COX-2 localized primarily in epidermal layer, which was markedly suppressed by curcumin pretreatment. Curcumin treatment attenuated TPA-stimulated

Introduction

The eukaryotic transcription factor NF-κB plays a central role in general inflammatory as well as immune responses. The 5′-flanking region of the cox-2 promoter contains NF-κB binding sites. In line with this notion, NF-κB has been shown to be a critical regulator of COX-2 expression in many cell lines (12,13). The intracellular signaling cascades controlling NF-κB activation are highly complex and involve the distinct set of kinases. Of the potential protein kinases involved in the activation of NF-κB, mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK)-signaling pathways have been well characterized (14,15). Recently, much attention has been devoted to identifying cancer chemopreventive phytochemicals of dietary and medicinal origin (16). One such compound is curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], the major yellow coloring pigment found in turmeric (Curcuma longa Linn, Zingiberaceae), which has been used for centuries in traditional oriental medicine to treat mainly inflammatory disorders. The anti-inflammatory properties of curcumin have also been verified in experimental studies (16). In addition, the compound has been shown to exert anticarcinogenic or anti-mutagenic effects in many animal models and also in cultured cells (reviewed in 16 and references therein). Thus, curcumin inhibits the development of

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There has been accumulating evidence for the association between inflammatory tissue damage and the process of cancer development (1). Cyclooxygenase (COX), an important enzyme involved in mediating the inflammatory process, catalyzes the ratelimiting step in the synthesis of prostaglandins (PGs) from arachidonic acid. There are two isoforms of COX, designated COX-1 and COX-2 (2). COX-1 is expressed constitutively in most tissues and appears to be responsible for maintaining normal physiological functions. In contrast, COX-2 is detectable in only certain types of tissues and is induced transiently by growth factors, proinflammatory cytokines, tumor promoters, and bacterial toxins (1,3). Expression of COX-2 has been reported to increase in human colorectal adenocarcinoma (4) and other malignancies such as breast, cervical, prostate, and lung tumors (5). Genetic knock-out or pharmacological inhibition of COX-2 has been shown to protect against intestinal polyposis in mice (6). Administration of the selective COX-2 inhibitor celecoxib at 400 mg twice a day for 6 months reduced the number of polyps significantly in patients with familial adenomatous polyposis (7). In addition, celecoxib has been found to inhibit experimentally induced colon, breast, bladder, and skin carcinogenesis (8-11).

chemically induced tumors of oral cavity, skin, forestomach, duodenum and colon in rodents (17-20). Curcumin has a variety of biochemical activities that are related to its chemopreventive action. These include antioxidation (21,22), inactivation of AP-1 (23) and NF-κB (24), inhibition of PG biosynthesis (25), and inhibition of activity and expression of ornithine decarboxylase (20,26). The anti-inflammatory properties of curcumin are considered to contribute to its anti-tumor promoting activity. In the present study, we examined the effect of curcumin on COX-2 induction by the tumor promoter 12-O-tetradecanoylphorbol-13acetate (TPA) in mouse skin in vivo. To further elucidate the molecular mechanisms by which curcumin regulates cox-2 gene expression, we also investigated its effect on activation of upstream signaling enzymes, such as ERK or p38 MAP kinase, and the

Materials and methods Chemicals Curcumin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). TPA was obtained from Alexis Biochemicals (San Diego, CA, USA). All other chemicals used were in the purest form available commercially. Animal treatment Female ICR mice (6 to 7 week of age) were supplied from the Dae-Han Korea Experimental Animal Center (Daejeon, Korea). The animals were housed in climatecontrolled quarters (24+1℃ at 50% humidity) with a 12-h light/12-h dark cycle. The dorsal side of skin was shaved using an electric clipper, and only those animals in the resting phase of the hair cycle were used in all experiments. Curcumin and TPA were dissolved in 200 µl of acetone and applied to the dorsal shaven area. Western blot analysis The mice were topically treated on their shaven backs with indicated doses of curcumin 30 min before 10 nmol TPA treatment and were killed by cervical dislocation at the indicated times. For isolation of protein from mouse skin, the dorsal skin was excised, and the fat was removed on ice, immediately placed in liquid nitrogen and pulverized in mortar. The pulverized skin was homogenized on ice for 20 s with a Polytron tissue homogenizer and lysed in 2 ml ice-cold lysis buffer [150 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), 20 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM Na3VO4,

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transcription factor NF-κB in mouse skin in vivo.

protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Mannheim, Germany)] for 10 min. Lysates were centrifuged at 12,000 g for 20 min, and supernatant containing 30 µg protein was boiled in sodium dodecyl sulfate (SDS) sample loading buffer for 10 min before electrophoresis on 12% SDS-polyacrylamide gel. After electrophoresis for 2 h, proteins in SDS-polyacrylamide gel were transferred to PVDF membrane (Gelman Laboratory, Ann Arber, MI), and the blots were blocked with 5% non-fat dry milk-PBST buffer [phosphate-buffered saline (PBS) containing 0.1% Tween-20] for 60 min at room temperature. The membranes were incubated for 2 h at room temperature with 1:1000 dilution of COX-2, p65, IκBα, and ERK polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA),

Northern blot analysis The pulverized skin was homogenized on ice for 20 s with a Polytron for isolation of total RNA by using TRIzol® reagent (Roche Molecular Biochemicals, Mannheim, Germany). For Northern blot analysis, 20 µg of total RNA was subjected to electrophoresis on 1.2% agarose-formaldehyde gel and transferred to a Hybond-Nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). After fixed by UV irradiation, membranes were prehybridized for 30 min at 68℃ in ExpressHyb hybridization solution (Clontech Laboratories, Palo Alto, CA, USA). Hybridization was carried out for 1 h at 68℃ with a cox-2 cDNA probe (Cayman Chemical, Ann Arbor, MI, USA) labeled with [α-32P]dCTP (NEN Life Science Products, Boston, MA, USA) using a Rediprime Ⅱ labeling system (Amersham Pharmacia Biotech, Buckinghamshire, UK). After hybridization, the membranes were washed twice for 30 min at room temperature in low-stringency buffer (2×SSC and 0.05% SDS) and twice for 40 min at 50℃ in high-stringency buffer (0.1×SSC and 0.1% SDS). Washed

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phospho-p65, and phospho-IκBα polyclonal antibodies (Cell Signaling Technology, Inc., Beverly, MA, USA), and p38, phospho-p38, phospho-ERK monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Equal lane loading was assessed using actin (Sigma Chemical Co., St. Louis, MO, USA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (Trevigen, Gaithersburg, MD, USA). The blots were rinsed three times with PBST buffer for 5 min each. Washed blots were incubated with 1:5000 dilution of the horseradish peroxidase conjugatedsecondary antibody (Zymed Laboratories, Inc., San Francisco, CA, USA) and then washed again three times with PBST buffer. The transferred proteins were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).

membranes were then autoradiographed on the x-ray film (Agfa-Gevaert, NV, USA) using an intensifying screen at 70℃. Each band was quantified using a BAS2000 image analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan). Immunohistochemical staining of COX-2 The dissected skin was prepared for immunohistochemical analysis of COX-2

Preparation of nuclear extracts The nuclear extract from mouse skin was prepared as described previously (27). Briefly, scraped dorsal skin of mice was homogenized in 1 ml of ice-cold hypotonic buffer A [10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF)]. After 15 min incubation on ice, the nucleoprotein complexes were lysed with 125 µl of 10% Nonidet P-40 (NP-40) solution, followed by centrifugation for 2 min at 14,800 g. The nuclei were washed once with 400 µl of buffer A plus 25 µl of 10% NP-40, centrifuged, resuspended in 150 µl of buffer C [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 10% glycerol], and centrifuged for 5 min at 14,800 g. The supernatant containing nuclear proteins was collected and stored at -70℃ after determination of protein concentrations. Electrophoretic mobility shift assay (EMSA) EMSA was performed using a DNA-protein binding detection kit (GIBCO BRL, Grand Island, NY, USA) according to the manufacturer's protocol. Briefly, the NF-κB oligonucleotide probe (5'-AGT TGA GGG GAC TTT CCC AGG C-3') was labeled with

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localization. Four-µm sections of formalin-fixed, paraffin-embedded tissue were cut onto silanized glass slides and deparaffinized three times with xylene for 10 min each and rehydrated through graded alcohol bath. The deparaffinized sections were heated and boiled twice for 6 min in 10 mM citrate buffer, pH 6.0 for antigen retrieval. To diminish non-specific staining, each section was treated with 3% hydrogen peroxide in methanol for 15 min. For the detection of COX-2, slides were incubated with 1:50100 dilutions of the monoclonal mouse anti-COX-2 antibody (Cayman Chemical, Ann Arbor, MI, USA) at room temperature for 60 min in TBST [Tris-buffered saline (TBS) containing and 0.05% Tween-20] and then developed using the HPR EnVisionTM System (Dako, Glostrup, Denmark). The peroxidase binding sites were detected by staining with 3,3'-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark). Finally, counterstaining was performed using Mayer's hematoxylin.

[γ-32P]ATP by T4 polynucleotide kinase and purified on a Nick column (Amersham Pharmacia Biotech, Buckinghamshire, UK). The binding reaction was carried out in a total vol. of 25 µl containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% (v/v) glycerol, 0.1 mg/ml sonicated salmon sperm DNA, 10 µg of nuclear extracts, and 100,000 cpm of the labeled probe. After 50 min incubation at room temperature, 2 µl of 0.1% bromophenol blue was added, and samples were electrophoresed through a 6% nondenaturating polyacrylamide gel at 150 V in a cold room for 2 h. Finally, the gel was dried and exposed to x-ray film.

Collected tissues were lysed in 200 µl of lysis buffer per sample [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerolphosphate, 1 mM Na3VO4, 1 g/ml leupeptin]. The lysates were centrifuged, and the supernatant was incubated with specific immobilized phospho-p38 and phospho-ERK monoclonal antibodies with gentle rocking for overnight at 4°C. The beads were washed twice each with 500 ml of lysis buffer and the same volume of kinase buffer [25 mM Tris-HCl (pH 7.5), 5 mM glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2 ]. The kinase reactions were carried out in the presence of 100 mM ATP and 2 mg of ATF-2 or Elk-1 at 30°C for 30 min. Phosphorylation of ATF-2 and Elk-1 was selectively measured by immunoblotting with specific antibodies detecting phosphorylation of ATF-2 and Elk-1 at Thr71 and Ser383, respectively. Results Inhibitory effects of curcumin on TPA-induced COX-2 expression in mouse skin It has previously been shown that TPA is a potent stimulator of COX-2 expression in various cell lines (28,29). We determined whether this was also the case in mouse skin. When 10 nmol of TPA was applied topically to the shaven backs of female ICR mice, the COX-2 protein level increased in a time related manner with maximal expression observed at 4 h (Figure 1A). Under the same experimental conditions, cox-2 mRNA expression peaked 1 h after the TPA application (Figure 1B). TPA application caused dose-dependent increases in both COX-2 protein and mRNA expression (Figure 2). To

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MAP kinase assay (Nonradioactive) Kinase assays for determining the catalytic activities of p38 and ERK were carried out by using a nonradioative MAP kinase assay kit (Cell Signaling Technology, Inc., Beverly, MA, USA) as described in the protocol provided by the manufacturer.

determine localization of COX-2 in mouse skin, we conducted immunohistochemical analysis. In acetone-treated control skin, specific COX-2 immunostaining was barely detectable in dorsal layer. Upon treatment with TPA for 4 h, the expression of COX-2 increased mainly in the epidermal layer (Figure 3), which was suppressed by curcumin pretreatment (Figure 4). Curcumin-mediated suppression of COX-2 expression was verified by Western blot analysis (Figure 5). Inhibition of TPA-induced NF-κB DNA binding activity by curcumin Because NF-κB is known to play a critical role in regulating the induction of COX-2, we have determined whether curcumin could suppress activation of this transcription factor in nuclear extracts obtained from the mouse skin stimulated with TPA. Our

unlabeled probe (27). Effects of curcumin on NF-κB activation were examined with varying doses of the compound topically applied 30 min before, simultaneously with or 30 min after the TPA treatment. Pretreatment of curcumin strongly inhibited TPAinduced NF-κB activation, whereas co- and post-treatment exhibited weaker inhibitory effects on NF-κB DNA binding in TPA-stimulated mouse skin (Figure 6A). Effects of curcumin on TPA-induced phosphorylation and degradation of IκBα and nuclear translocation of p65 One of the most critical steps in NF-κB activation is the dissociation of IκB, which is mediated through phosphorylation and subsequent proteolytic degradation of this inhibitory subunit. To determine whether the inhibitory effect of curcumin on NF-κB DNA binding was due to its suppression of IκBα degradation via phosphorylation, the cytoplasmic levels of IκBα and phospho-IκBα were determined by Western blot analysis. Topical application of TPA led to phosphorylation and degradation of IκBα, which were significantly repressed by curcumin pretreatment (Figure 6B, 6C), which was consistent with NF-κB DNA binding affected by the same treatment (Figure 6A). We also measured the level of p65, the functionally active subunit of NF-κB, in nucleus. Upon TPA treatment, the nuclear translocation of p65 increased, which was blocked by curcumin (Figure 6B). These results indicate that curcumin inhibits TPA-induced translocation of p65 to the nucleus through blockade of IκBα phosphorylation. Effects of curcumin on TPA-induced activation of MAP kinases MAP kinases are known to regulate NF-κB activation by multiple mechanisms.

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previous studies demonstrated an apparent increase in epidermal NF-κB DNA binding early as 10 min after 10 nmol TPA application which was abolished by the excess

Accumulating evidence indicates that NF-κB activation is modulated by ERK as well as p38 MAP kinase. As shown in Figure 7, ERK and p38 in mouse skin were phosphorylated in response to TPA treatment. Phosphorylation of each MAP kinase was evident at 30 min following TPA application and was sustained up to 4 h. To confirm that phosphorylation of ERK and p38 reflected their enhanced catalytic activity, kinase assays were performed. In parallel with elevated phosphorylation, the activities of ERK and p38 increased rapidly after TPA application (Figure 7). However, the JNK activity remained unchanged even after TPA treatment (data not shown). After verifying the ERK and p38 activation in TPA-stimulated mouse skin, we examined whether curcumin could down-regulate the aforementioned MAP kinases, thereby

Inhibition of TPA-induced NF-κB DNA binding activity and COX-2 expression by MAP kinase inhibitors To determine which of MAP kinases is involved in activation of NF-κB in mouse skin, we investigated the inhibitory effect of the pharmacological inhibitors of MAP kinases on NF-κB activation by TPA. SB203580 is known to selectively inhibit p38 MAP kinase and U0126 is an ultrapotent inhibitor of MAP kinase kinase (MEK)1/2 responsible for activation of ERK. We have confirmed that SB203580 and U0126 at 4 µmol each suppressed the activity of p38 MAP kinase and phosphorylative activation of ERK1/2, respectively (data not shown). U0126 blocked the NF-κB DNA binding activity, whereas the p38 MAP kinase inhibitor SB203580 failed to (Figure 9A). This result suggests that the activation of NF-κB occurs via the ERK-dependent pathway in mouse skin. Many studies have revealed a close association between the ERK activity and phosphorylation and degradation of IκB protein, which leads to increased nuclear translocation of NF-κB and subsequent DNA binding in various cell systems (30-33). To investigate the possible role of ERK in IκB phosphorylation and degradation, we measured the levels of phosphorylated IκBα with and without U0126 pretreatment. As illustrated in Figure 9B, U0126 blocked the phosphorylation of IκBα, thereby suppressing degradation of this inhibitory protein. In order to further investigate the possible involvement of ERK in the signaling pathway mediating COX-2 induction, we

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inactivating NF-κB and further suppressing the COX-2 induction. Curcumin inhibited catalytic activities of both p38 MAP kinase and ERK1/2. In addition, curcumin inhibited activation of p38 MAP kinase through phosphorylation while it did not much influence the phosphorylation of ERK1/2 in mouse skin (Figure 8). Under the same experimental conditions, the level of the total form of each kinase remained almost constant.

examined the effect of U0126 on the TPA-induced COX-2 expression.

As shown in

Figure 9C, U0126 at 4 µmol almost completely abrogated COX-2 induction in TPAtreated mouse skin. These data suggest that ERK plays a central role in intracellular signaling cascades mediating TPA-induced COX-2 expression in mouse skin. Discussion

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In recent years considerable efforts have been made to develop chemopreventive agents that could inhibit, retard or reverse the multistage carcinogenesis (34). Chemoprevention has become an emerging area of cancer research that, in addition to providing a practical approach to identifying potentially useful inhibitors of malignant transformation, affords opportunities to study the mechanisms of anti-carcinogenesis (34). Chemopreventive agents can act in any stages of carcinogenesis, i.e., initiation, promotion, or progression. The intervention of cancer in the promotion stage, however, seems to be most appropriate and practical since tumor promotion is a reversible event which requires repeated and prolonged exposure to promoting agents (35). Tumor promotion is closely linked to inflammation and oxidative stress (35,36), and it is hence likely that compounds with strong anti-inflammatory and anti-oxidative activities act as anti-tumor promoters as well. Curcumin, a naturally occurring anti-inflammatory and antioxidant compound, has been shown to inhibit experimentally induced tumorigenesis in several animal models, including 7,12-dimethylbenz[a]anthracene-initiated and TPA-promoted skin tumors, benzo[a]pyrene-induced forestomach tumors, and azoxymethane-induced intestinal tumors in mice (18,19, 37-39). There is a growing body of compelling evidence that targeted inhibition of COX-2 expression or activity is valuable for not only alleviating inflammation, but preventing cancer. Therefore, agents that interfere with the intracellular signaling mechanisms governing the transcription of COX-2 are considered to be potential chemopreventives. Treatment of several human gastrointestinal cell lines with curcumin suppressed expression of COX-2 protein and mRNA as well as PGE2 production induced by TPA (40). Since chronic inflammation predisposes to malignancy (41), the inhibition of COX-2 by curcumin is likely to contribute to both anti-inflammatory and chemopreventive effects this phytochemical exerts. While the majority of nonsteroidal anti-inflammatory drugs inhibit the catalytic activity of COX-2, our results indicate that curcumin can inhibit expression of the COX-2 induced by the typical tumor promoter TPA in mouse skin. These data suggest that previously reported chemopreventive properties of curcumin against mouse skin carcinognesis are

NF-κB (12,13). Curcumin blocked tumor promoter-mediated NF-κB transactivation by inhibiting the NF-κB-inducing kinase (NIK)/IκB kinase (IKK) signaling complex, probably at the level of IKKα/β (46). When the effect of curcumin on NF-κB activation by TPA was examined in mouse skin, pre-treatment of curcumin was found to be most effective in terms of inhibiting NF-κB DNA binding, whereas co- or posttreatment caused a weaker effect. The inhibitory effect of curcumin on NF-κB activation by TPA was due to inhibition of IκB degradation and p65 translocation to the nucleus. Our previous study revealed that curcumin could also suppress NF-κB activation through direct interruption of NF-κB DNA binding in TPA-pretreated nuclear extract (47). Production of PGE2 and 6-ketoPGF1α in LPS-stimulated J774 macrophages was reported to be reduced by the antioxidant pyrrolidine dithiocarbamate (PDTC) and the serine protease inhibitor, N-α-p-tosyl-L-lysine chloromethylketone (TPCK), both of which are inhibitors of NF-κB (12). Suppression of the prostanoid production by PDTC or TPCK was not mediated through direct inhibition of COX-2 activities since these NF-κB inhibitors did not influence the catalytic activity of the enzyme when added to the new media after baterial lipopolysaccharide challenge (12). According to our previous study, topical application of PDTC resulted in dose-related suppression of TPA-induced activation of NF-κB and also caused reduced COX-2 protein expression in mouse skin (48). These data also support the notion that the naturally occurring anti-inflammatory agent curcumin inhibits TPA-induced COX-2 expression in mouse skin, possibly by blocking NF-κB activation. Another transcription factor that plays an important role in controlling cox-2 gene is activator protein-1 (AP-1). A role of AP-1 in COX-2 induction has been demonstrated in various cell lines (49-51). In our previous data, topical application of

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attributable in part to its inhibition of expression of COX-2 as well as its catalytic activity. TPA treatment can induce or activate a wide array of genes and their protein ptoducts. Like COX-2, elevated expression of inducible nitric oxide synthase (iNOS) and/or its catalytic activity has been observed in several human malignancies also in chemically-induced tumors in experimental animals (42-44). We have found that topically applied curcmin abrogated iNOS expression in TPA-stimulated mouse skin (K.-S. Chun et al., unpublished observation). Besides aforementioned proinflammatory enzymes, curcumin may also target other molecules such as NAD(P)H:oxidoreductase (45) and ornithine decarboxylse (20,26) in exerting its chemopreventive activity. Thus, it is unlikely that COX-2 is the only target for chemoprevention by curcumin in mouse skin. Control of cox-2 induction involves a complex array of regulatory factors including

curcumin suppressed TPA-stimulated AP-1 activation by directly blocking the binding of the preactivated nuclear extract to the AP-1 consensus sequence (47). Therefore, it is plausible that curcumin inhibits COX-2 expression through not only inactivation of NF-κB but also other transcription factors including AP-1 in mouse skin. The molecular signaling mechanisms involved in the induction of COX-2 as well as activation of NF-κB in response to various external stimuli have not been fully clarified. One of the most extensively investigated intracellular signaling cascades involved in pro-inflammatory responses is the MAP kinase pathway. MAP kinases

To determine which MAP kinase play an important role in activation of NF-κB in mouse skin, we investigated the inhibitory effects of the ultrapotent MEK1/2 inhibitor U0126 and the p38 inhibitor SB203580 on NF-κB activation. Interestingly, only U0126 abolished the NF-κB binding activity. In another experiment, we examined an inhibitory effect of U0126 on TPA-induced COX-2 expression. U0126 almost completely abrogated TPA-induced COX-2 protein expression, which supports the idea that ERK may play an important role in the signaling pathway of TPA-induced COX-2 expression and NF-κB activation in mouse skin.

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regulate NF-κB activation via multiple mechanisms. A substantial body of data indicates that NF-κB activation is modulated by MAP kinase/ERK kinase kinase-1 (MEKK1), a kinase upstream of MAP kinases (52-54). There is accumulating evidence indicating that enzymes of the MAP kinase family play a role in cox-2 gene expression. The Parke-Davis MEK inhibitor PD98059 partially blocked LPS-induced COX-2 expression in RAW 264.7 cells and also in lysophosphatidic acid-stimulated rat mesangial cells (55), which further supports the association of ERK activation with COX-2 mediated PG production. LPS-induced expression of COX-2 was blunted by SB203580, the p38 MAP kinase inhibitor, which resulted in decreased PGE2 production in RAW 264.7 cells (56). Similar effects were observed in LPS-stimulated monocytes (57). Although the MAP kinase signaling pathways have been extensively investigated in cultured cell lines, much less is known about the specificity of MAP kinases and extent to which they are activated during the tumor promotion in mouse skin in vivo. Topical application of TPA on the ears of CD1 mice (58) and skin of SENCAR mice (59) induced a rapid and sustained activation of ERK but not of p38 MAP kinase. Most importantly, we have found that treatment of dorsal skins of female ICR mice with TPA significantly enhanced both catalytic activities and phosphorylation of p38 MAP kinase and ERK1/2. Topically applied curcumin inhibited activities of both p38 and ERK1/2 MAP kinases in mouse skin.

Dhawan and Richmond (30) showed that in Hs294T cells, ERK regulation of NFκB activation involves increased IκB phosphorylation with concomitant elevation in the NF-κB DNA binding activity. An increase in phosphorylation of IκB is responsible for enhanced degradation of this inhibitory subunit, which leads to increased nuclear localization of NF-κB and subsequent DNA binding. In our present study, U0126 inhibited the TPA-induced phosphorylation of IκBα in mouse skin. Most inhibitors of NF-κB activation, such as silymarin (60) and oleandrin (61), exert their antiinflammatory effects through suppression of phosphorylation and degradation of

and NF-κB signaling cascades may provide molecular basis for suppression of tumor promotion as well as inflammation exerted by this chemopreventive phytochemical in mouse skin. Although chemopreventive and chemoprotective properties of curcumin have been extensively investigated and well documented, the compound, under certain pathophysiologic conditions, may exert detrimental effects. According to a recent study by Frank and colleagues, 0.5% curcumin in the diet failed to protect Long-Evans Cinnamon rats against hepatic and renal carcinogenesis, but rather shortened the median survival time, probably due to enhanced oxidative stress induced by this phenolic in the presence of excess copper (62). Therefore, a caution should be made for intake of curcumin by patients suffering from metal storage disorders, such as Wilson’s disease or hepatitis C viral infection. Acknowledgements This work was supported by the grant (00PJ1-PG3-21400-0052) from the Ministry and Welfare, Republic of Korea.

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IκBα. Thus, we examined that whether curcumin could also block NF-κB activation by inhibiting IκBα phosphorylation. Topical application of curcumin suppressed TPAinduced IκBα phosphorylation in a dose-dependent manner. Based on these findings, we suggest that the ERK signaling pathway regulates NF-κB activation through inhibition of IκB phosphorylation. These results may explain the molecular mechanism responsible for the inhibitory effect of curcumin on NF-κB activation in TPA-treated mouse skin (Figure 10). In conclusion, the present study demonstrates that curcumin inhibits induction of COX-2 in TPA-treated mouse skin in vivo. Since improper and abnormal overexpression of COX-2 is implicated in the pathogenesis of various types of human cancers, assessment of the effects of curcumin on cox-2 gene expression may represent a useful surrogate biomarker for the evaluation of its chemopreventive potential. Our findings that curcumin inhibits TPA-induced COX-2 expression by blocking the ERK

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Legends to Figures Fig. 1. TPA induced COX-2 protein (A) and its mRNA (B) expression in mouse skin. A, Dorsal skins of female ICR mice were treated topically with acetone alone or with 10 nmol TPA in acetone for indicated time periods. Protein extracts (30 µg) were loaded onto a 12% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto PVDF membrane. Immunoblots were a probed with a goat polyclonal COX-2 antibody.

B, cox-2 mRNA expression was determined by Northern blot analysis using the cox-2 cDNA probe labeled with [α-32P]dCTP. Quantification of cox-2 mRNA signal intensities was normalized to those of GAPDH mRNA.

Fig. 3. COX-2 immunostaining in mouse skin. Paraffin-embedded tissues from TPAtreated mouse were immunostained for COX-2 and counterstained with hematoxylin, as described in Materials and methods. COX-2 staining gives a brown reaction product. A, COX-2 staining in acetone-treated control skin. B, COX-2 staining in 10 nmol TPA-treated mouse. C, COX-2 staining in 100 nmol TPA-treated mouse. Fig. 4. Inhibitory effects of curcumin on phorbol ester-induced COX-2 expression by immunohistochemistry staining. Female ICR mice were treated topically with curcumin (25 µmol) or acetone alone 30 min before TPA (10 nmol) application. Control mice were treated with acetone in lieu of TPA. Mice were killed 4 h after the TPA treatment, and skin samples were subjected to immunohistochemical analysis with COX-2 antibody as described in Materials and methods. Positive COX-2 staining yielded a brown-coloured product (arrow). A, Skin treated with acetone alone. B, Skin treated with TPA. C, Skin treated with curcumin 30 min prior to TPA. Fig. 5. Inhibitory effects of curcumin on phorbol ester-induced COX-2 expression. Female ICR mice were treated topically with 0.2 ml acetone or curcumin in the same vol. of acetone 30 min prior to 10 nmol TPA, and animals were killed 4 h after the TPA treatment. Protein was analyzed for COX-2 by immunoblotting. The Western blot is

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Fig. 2. Dose-related expression of COX-2 protein (A) and its mRNA (B) in TPAtreated mouse skin. Dorsal skins of female ICR mice were treated topically with acetone alone or with various dose of TPA in acetone. Mice were killed 4 or 1 h later for immunoblot analysis of COX-2 protein (A) or Northern blot analysis of COX-2 mRNA (B), respectively.

representative of 2 independent experiments. normalized to actin using a densitometer.

Quantification of COX-2 expression was

Fig. 6. Effect of curcumin on NF-κB activation in mouse skin treated with TPA. A, Dorsal skins of mice were treated topically with acetone alone (lane 2), 10 nmol TPA alone (lane 3) or 1 µmol (A), 5 µmol (B), 10 µmol (C) or 25 µmol (D) of curcumin 30 min before, simultaneously with, or 30 min after TPA treatment. Mice were killed 1 h after the TPA treatment (acetone for the control), and epidermal nuclear extracts were

and without curcumin (1, 5, or 25 µmol) pretreatment, were assayed for p65, IκBα, (B) and phospho-IκBα (C) respectively by Western blot analysis. NE, nuclear extract; CE, cytoplasmic extracts. The Western blot is representative of 2 separate experiments. Fig. 7.

Time course of activation of p38 MAP kinase and ERK in TPA-treated mouse

skin. TPA-induced activation of p38 MAP kinse and ERK that are upstream of NF-κB was assessed based on elevated levels of phosphorylated enzymes as well as enhancement of their catalytic activity. A, TPA (10 nmol) was topically applied onto shaven backs of female ICR mice. Mice were sacrificed at indicated time intervals. Total protein was isolated from the dorsal skin and quantified. Cell lysates containing 200 µg protein were treated with a specific immobilized phospho-p38 kinase monoclonal antibody. The resulting immunoprecipitate was then incubated with ATF2 fusion protein (substrate for activated p38) in the presence of 100 mM ATP. ATF-2 phosphorylation was measured by Western blotting of nonradioactive labeled samples using the phospho-ATF-2 antibody. The phosphorylated form of p38 was detected by immunoblotting using a corresponding phospho-specific antibody. B, The protein extracts were prepared at indicated time points from TPA-stimulated skin, and ERK phosphorylation was determined by Western blot analysis. The kinase activity of ERK was determined by the immune complex assay in a manner similar to that described for p38 except that Elk-1 was included as a substrate. Fig. 8. Effects of curcumin on TPA-induced activation of p38 MAP kinase (A) and ERK (B). Female ICR mice were treated topically with acetone or with 1, 5, or 25 µmol of curcumin 30 min prior to TPA.

Mice were killed 1 h after the TPA treatment,

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prepared and incubated with the radiolabelled oligonucleotides containing the NF-κB consensus sequence for analysis by the EMSA. Lane 1, free probe alone (no nuclear extracts). EMSA was performed in duplicated and showed representative. B, C, Nuclear and cytoplasmic extracts from mouse skin treated 10 nmol TPA for 1 h, with

and expression of both kinases was measured by Western blot analysis. The kinase activities were determined by the immune complex assay as described in the legend to Figure 7. Data are representative of two independent experiments which showed a similar trend. Fig. 9. Effect of MEK1/2 inhibitor U0126 on TPA-induced NF-κB DNA binding activity and COX-2 expression. A, Dorsal skin of female ICR mice was treated topically with acetone, U0126, or SB203580 30 min prior to topical application of 10 nmol TPA. After 1 h, mice were killed and epidermal nuclear extracts were prepared for EMSA. Lane 1, free probe alone; lane 2, acetone control; lane 3, TPA alone; lane 4,

were killed 4 h after the TPA treatment. Lane 1, acetone control; lane 2, TPA alone; lane 3, U0126 (4 µmol) + TPA.

All experiments were repeated at least twice.

Fig. 10. Hypothetical mechanism underlying suppression of TPA-induced COX-2 expression by curcumin in mouse skin. Note that curcumin can suppress the activation of ERK1/2 or degradation of IκB (present study). with NF-κB DNA binding (47).

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Curcumin can also directly interfere


U0126 (4 µmol) + TPA; lane 5, SB203580 (4 µmol) + TPA. B, Dorsal skin of female ICR mice was treated with acetone (lane 1), 10 nmol TPA (lane 2) alone, or 4 µmol of U0126 (lane 3). Cytosolic extracts were assayed by Western blot for phospho-IκBα, as described in the text. C, U0126 was applied topically 30 min prior to TPA. Mice

4 2 1 0.5 0

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curcumin U0126