Curcumin inhibits phorbol ester-induced expression of cyclooxygenase-2 in mouse skin through suppression of extracellular signal-regulated kinase activity and NF-{kappa}B 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, College of Medicine, Seoul National University, Seoul 151-741, South Korea
2 Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul 151-741, South Korea

3 To whom correspondence should be addressed Email: surh{at}plaza.snu.ac.kr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 coloring 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- tetradecanoylphorbol-13-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 ~1 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 pre-treatment. Curcumin treatment attenuated TPA- stimulated NF-{kappa}B activation in mouse skin, which was associated with its blockade of degradation of the inhibitory protein I{kappa}B{alpha} 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-{kappa}B. The MEK1/2 inhibitor U0126 strongly inhibited NF-{kappa}B activation, while p38 inhibitor SB203580 failed to block TPA-induced NF-{kappa}B activation in mouse skin. Furthermore, U0126 blocked the I{kappa}B{alpha} phosphorylation by TPA, thereby blocking the nuclear translocation of NF-{kappa}B. Curcumin inhibited the catalytic activity of ERK1/2 in mouse skin. Taken together, suppression of COX-2 expression by inhibiting ERK activity and NF-{kappa}B activation may represent molecular mechanisms underlying previously reported antitumor promoting effects of this phytochemical in mouse skin tumorigenesis.

Abbreviations: AP-1, activator protin 1; COX, cyclooxygenase; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; ERK, extra-cellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; PG, prostaglandin; SDS, sodium dodecyl sulfate; TPA, 12-O-tetradecanoylphorbol-13-acetate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 rate-limiting 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, pro-inflammatory 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 knockout 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 (811).

The eukarytoic transcription factor NF-{kappa}B plays a central role in general inflammatory as well as immune responses. The 5'-flanking region of the cox-2 promoter contains NF-{kappa}B binding sites. In line with this notion, NF-{kappa}B has been shown to be a critical regulator of COX-2 expression in many cell lines (12,13). The intracellular signaling cascades controlling NF-{kappa}B activation are highly complex and involve the distinct set of kinases. Of the potential protein kinases involved in the activation of NF-{kappa}B, mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase/stress-activated protein kinase-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 antimutagenic effects in many animal models and also in cultured cells (reviewed in 16 and references therein). Thus, curcumin inhibits the development of chemically induced tumors of oral cavity, skin, forestomach, duodenum and colon in rodents (1720). Curcumin has a variety of biochemical activities that are related to its chemopreventive action. These include antioxidation (21,22), inactivation of activator protin (AP)-1 (23) and NF-{kappa}B (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 antitumor promoting activity. In the present study, we examined the effect of curcumin on COX-2 induction by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (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 transcription factor NF-{kappa}B in mouse skin in vivo.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 (6–7 weeks of age) were supplied from the Dae-Han/Biolink Experimental Animal Center (Daejeon, Korea). The animals were housed in climate-controlled quarters (24 ± 1°C 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, 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 Arbor, 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, I{kappa}B{alpha} and ERK polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-p65 and phospho-I{kappa}B{alpha} polyclonal antibodies (Cell Signaling Technology, Beverly, MA) and p38, phospho-p38 and phospho-ERK monoclonal antibodies (Santa Cruz Biotechnology). Equal lane loading was assessed using actin (Sigma Chemical Co.) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (Trevigen, Gaithersburg, MD). 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 conjugated-secondary antibody (Zymed Laboratories, San Francisco, CA) and then washed again three times with PBST buffer. The transferred proteins were visualized with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).

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). 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). After being fixed by UV irradiation, membranes were pre-hybridized for 30 min at 68°C in ExpressHyb hybridization solution (Clontech Laboratories, Palo Alto, CA). Hybridization was carried out for 1 h at 68°C with a cox-2 cDNA probe (Cayman Chemical, Ann Arbor, MI) labeled with [{alpha}-32P]dCTP (NEN Life Science Products, Boston, MA) using a Rediprime II labeling system (Amersham Pharmacia Biotech). After hybridization, the membranes were washed twice for 30 min at room temperature in low-stringency buffer (2x SSC and 0.05% SDS) and twice for 40 min at 50°C in high-stringency buffer (0.1x SSC and 0.1% SDS). Washed membranes were then autoradiographed on the X-ray film using an intensifying screen at 70°C. Each band was quantified using a BAS2000 image analyzer (Fuji Photo Film Co., Tokyo, Japan).

Immunohistochemical staining of COX-2
The dissected skin was prepared for immunohistochemical analysis of COX-2 localization. Four-micrometer 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 1:50–100 dilutions of the monoclonal mouse anti-COX-2 antibody (Cayman Chemical) at room temperature for 60 min in Tris-buffered saline 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). Finally, counterstaining was performed using Mayer's hematoxylin.

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°C 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) according to the manufacturer's protocol. Briefly, the NF-{kappa}B oligonucleotide probe (5'-AGT TGA GGG GAC TTT CCC AGG C-3') was labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase and purified on a Nick column (Amersham Pharmacia Biotech). The binding reaction was carried out in a total volume 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 100000 c.p.m. 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% non-denaturating polyacrylamide gel at 150 V in a cold room for 2 h. Finally, the gel was dried and exposed to X-ray film.

MAP kinase assay (non-radioactive)
Kinase assays for determining the catalytic activities of p38 and ERK were carried out by using a non-radioative MAP kinase assay kit (Cell Signaling Technology) as described in the protocol provided by the manufacturer. 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 µM ATP and 2 µg 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibitory effects of curcumin on TPA-induced COX-2 expression in mouse skin
It has been shown previously 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 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 pre-treatment (Figure 4). Curcumin-mediated suppression of COX-2 expression was verified by western blot analysis (Figure 5).



View larger version (34K):
[in this window]
[in a new window]
 
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 [{alpha}-32P]dCTP. Quantification of cox-2 mRNA signal intensities was normalized to those of GAPDH mRNA.

 


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 2. Dose-related expression of COX-2 protein (A) and its mRNA (B) in TPA-treated mouse skin. Dorsal skins of female ICR mice were treated topically with acetone alone or with various doses 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.

 


View larger version (82K):
[in this window]
[in a new window]
 
Fig. 3. COX-2 immunostaining in mouse skin. Paraffin-embedded tissues from TPA-treated 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.

 


View larger version (79K):
[in this window]
[in a new window]
 
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-colored product (arrow). (A) Skin treated with acetone alone. (B) Skin treated with TPA. (C) Skin treated with curcumin 30 min prior to TPA.

 


View larger version (42K):
[in this window]
[in a new window]
 
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 volume 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 representative of two independent experiments. Quantification of COX-2 expression was normalized to actin using a densitometer.

 
Inhibition of TPA-induced NF-{kappa}B DNA binding activity by curcumin
Because NF-{kappa}B 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 previous studies demonstrated an apparent increase in epidermal NF-{kappa}B DNA binding as early as 10 min after 10 nmol TPA application, which was abolished by the excess unlabeled probe (27). Effects of curcumin on NF-{kappa}B activation were examined with varying doses of the compound topically applied 30 min before, simultaneously with or 30 min after the TPA treatment. Pre-treatment of curcumin strongly inhibited TPA-induced NF-{kappa}B activation, whereas co- and post-treatment exhibited weaker inhibitory effects on NF-{kappa}B DNA binding in TPA-stimulated mouse skin (Figure 6A).



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6. Effect of curcumin on NF-{kappa}B 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 prepared and incubated with the radiolabeled oligonucleotides containing the NF-{kappa}B consensus sequence for analysis by the EMSA. Lane 1, free probe alone (no nuclear extracts). (B and C) Nuclear and cytoplasmic extracts from mouse skin treated 10 nmol TPA for 1 h, with and without curcumin (1, 5 or 25 µmol) pre-treatment, were assayed for p65, I{kappa}B{alpha} (B) and phospho-I{kappa}B{alpha} (C), respectively, by western blot analysis. NE, nuclear extract; CE, cytoplasmic extracts. The western blot is representative of two separate experiments.

 
Effects of curcumin on TPA-induced phosphorylation and degradation of I{kappa}B{alpha} and nuclear translocation of p65
One of the most critical steps in NF-{kappa}B activation is the dissociation of I{kappa}B, which is mediated through phosphorylation and subsequent proteolytic degradation of this inhibitory subunit. To determine whether the inhibitory effect of curcumin on NF-{kappa}B DNA binding was due to its suppression of I{kappa}B{alpha} degradation via phosphorylation, the cytoplasmic levels of I{kappa}B{alpha} and phospho-I{kappa}B{alpha} were determined by western blot analysis. Topical application of TPA led to phosphorylation and degradation of I{kappa}B{alpha}, which were significantly repressed by curcumin pre-treatment (Figure 6B and C). This finding is consistent with NF-{kappa}B DNA binding affected by the same treatment (Figure 6A). We also measured the level of p65, the functionally active subunit of NF-{kappa}B, 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 I{kappa}B{alpha} phosphorylation.

Effects of curcumin on TPA-induced activation of MAP kinases
MAP kinases are known to regulate NF-{kappa}B activation by multiple mechanisms. Accumulating evidence indicates that NF-{kappa}B 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 inactivating NF-{kappa}B 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.



View larger version (38K):
[in this window]
[in a new window]
 
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-{kappa}B 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 killed 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 ATF-2 fusion protein (substrate for activated p38) in the presence of 100 mM ATP. ATF-2 phosphorylation was measured by western blotting of non-radioactive 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.

 


View larger version (57K):
[in this window]
[in a new window]
 
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, 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.

 
Inhibition of TPA-induced NF-{kappa}B DNA binding activity and COX-2 expression by MAP kinase inhibitors
To determine which of the MAP kinases is involved in activation of NF-{kappa}B in mouse skin, we investigated the inhibitory effect of the pharmacological inhibitors of MAP kinases on NF-{kappa}B 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-{kappa}B DNA binding activity, whereas the p38 MAP kinase inhibitor SB203580 failed to (Figure 9A). This result suggests that the activation of NF-{kappa}B 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 I{kappa}B protein, which leads to increased nuclear translocation of NF-{kappa}B and subsequent DNA binding in various cell systems (3033). To investigate the possible role of ERK in I{kappa}B phosphorylation and degradation, we measured the levels of phosphorylated I{kappa}B{alpha} with and without U0126 pre-treatment. As illustrated in Figure 9B, U0126 blocked the phosphorylation of I{kappa}B{alpha}, 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 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 TPA-treated mouse skin. These data suggest that ERK plays a central role in intracellular signaling cascades mediating TPA-induced COX-2 expression in mouse skin.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 9. Effect of MEK1/2 inhibitor U0126 on TPA-induced NF-{kappa}B 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, 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-I{kappa}B{alpha}, as described in the text. (C) U0126 was applied topically 30 min prior to TPA. Mice 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.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years considerable efforts have been made to develop chemopreventive agents that could inhibit, retard or reverse the multi-stage 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 anticarcinogenesis (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 antitumor 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 (18,19,3739). 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 non-steroidal 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 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 products. 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 (4244). We have found that topically applied curcumin abrogated iNOS expression in TPA-stimulated mouse skin (K.-S.Chun et al., unpublished observation). Besides aforementioned pro-inflammatory enzymes, curcumin may also target other molecules such as NAD(P)H:oxidoreductase (45) and ornithine decarboxylase (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 NF-{kappa}B (12,13). Curcumin blocked tumor promoter-mediated NF-{kappa}B transactivation by inhibiting the NF-{kappa}B-inducing kinase (NIK)/I{kappa}B kinase (IKK) signaling complex, probably at the level of IKK{alpha}/ß (46). When the effect of curcumin on NF-{kappa}B activation by TPA was examined in mouse skin, pre-treatment of curcumin was found to be most effective in terms of inhibiting NF-{kappa}B DNA binding, whereas co- or post-treatment caused a weaker effect. The inhibitory effect of curcumin on NF-{kappa}B activation by TPA was due to inhibition of I{kappa}B degradation and p65 translocation to the nucleus. Our previous study revealed that curcumin could also suppress NF-{kappa}B activation through direct interruption of NF-{kappa}B DNA binding in TPA-pre-treated nuclear extract (47). Production of PGE2 and 6-ketoPGF1{alpha} in LPS-stimulated J774 macrophages was reported to be reduced by the antioxidant pyrrolidine dithiocarbamate (PDTC) and the serine protease inhibitor, N-{alpha}-p-tosyl-L-lysine chloromethylketone (TPCK), both of which are inhibitors of NF-{kappa}B (12). Suppression of the prostanoid production by PDTC or TPCK was not mediated through direct inhibition of COX-2 activities since these NF-{kappa}B inhibitors did not influence the catalytic activity of the enzyme when added to the new media after bacterial lipopolysaccharide challenge (12). According to our previous study, topical application of PDTC resulted in dose-related suppression of TPA-induced activation of NF-{kappa}B 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-{kappa}B activation.

Another transcription factor that plays an important role in controlling cox-2 gene is AP-1. A role of AP-1 in COX-2 induction has been demonstrated in various cell lines (4951). In our previous data, topical application of curcumin suppressed TPA-stimulated AP-1 activation by directly blocking the binding of the pre-activated 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-{kappa}B 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-{kappa}B 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 regulate NF-{kappa}B activation via multiple mechanisms. A substantial body of data indicates that NF-{kappa}B activation is modulated by MAP kinase/ERK kinase kinase-1 (MEKK1), a kinase upstream of MAP kinases (5254). 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.

To determine which MAP kinase play an important role in activation of NF-{kappa}B in mouse skin, we investigated the inhibitory effects of the ultrapotent MEK1/2 inhibitor U0126 and the p38 inhibitor SB203580 on NF-{kappa}B activation. Interestingly, only U0126 abolished the NF-{kappa}B 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-{kappa}B activation in mouse skin.

Dhawan and Richmond (30) showed that in Hs294T cells, ERK regulation of NF-{kappa}B activation involves increased I{kappa}B phosphorylation with concomitant elevation in the NF-{kappa}B DNA binding activity. An increase in phosphorylation of I{kappa}B is responsible for enhanced degradation of this inhibitory subunit, which leads to increased nuclear localization of NF-{kappa}B and subsequent DNA binding. In our present study, U0126 inhibited the TPA-induced phosphorylation of I{kappa}B{alpha} in mouse skin. Most inhibitors of NF-{kappa}B activation, such as silymarin (60) and oleandrin (61), exert their anti-inflammatory effects through suppression of phosphorylation and degradation of I{kappa}B{alpha}. Thus, we examined whether curcumin could also block NF-{kappa}B activation by inhibiting I{kappa}B{alpha} phosphorylation. Topical application of curcumin suppressed TPA-induced I{kappa}B{alpha} phosphorylation in a dose-dependent manner. Based on these findings, we suggest that the ERK signaling pathway regulates NF-{kappa}B activation through inhibition of I{kappa}B phosphorylation. These results may explain the molecular mechanism responsible for the inhibitory effect of curcumin on NF-{kappa}B activation in TPA-treated mouse skin (Figure 10).



View larger version (28K):
[in this window]
[in a new window]
 
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 I{kappa}B (present study). Curcumin can also directly interfere with NF-{kappa}B DNA binding (47).

 
In conclusion, the present study demonstrates that curcumin inhibits induction of COX-2 in TPA-treated mouse skin in vivo. Since improper and abnormal over-expression 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 and NF-{kappa}B 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.


    Acknowledgments
 
This work was supported by the grant (00PJ1-PG3-21400-0052) from the Ministry and Welfare, Republic of Korea.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Prescott,S.M. and Fitzpatrick,F.A. (2000) Cyclooxygenase-2 and carcinogenesis. Biochim. Biophys. Acta, 1470, M69–78.[CrossRef][ISI][Medline]
  2. Funk,C.D., Funk,L.B., Kennedy,M.E., Pong,A.S. and Fitzgerald,G.A. (1991) Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment. FASEB J., 5, 2304–2312.[Abstract/Free Full Text]
  3. Gately,S. (2000) The contributions of cyclooxygenase-2 to tumor angiogenesis. Cancer Metastasis Rev., 19, 19–27.[ISI][Medline]
  4. Sano,H., Kawahito,Y., Wilder,R.L., Hashiramoto,A., Mukai,S., Asai,K., Kimura,S., Kato,H., Kondo,M. and Hla,T. (1995) Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res., 55, 3785–3789.[Abstract]
  5. Howe,L.R., Subbaramaiah,K., Brown,A.M. and Dannenberg,A.J. (2001) Cyclooxygenase-2: a target for the prevention and treatment of breast cancer. Endocr. Relat. Cancer, 8, 97–114.[Abstract/Free Full Text]
  6. Oshima,M., Dinchuk,J.E., Kargman,S.L., Oshima,H., Hancock,B., Kwong,E., Trzaskos,J.M., Evans,J.F. and Taketo,M.M. (1996) Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase-2 (COX-2). Cell, 87, 803–809.[ISI][Medline]
  7. Tive,L. (2000) Celecoxib clinical profile. Rheumatology, 39 (suppl. 2), 21–28.[Abstract]
  8. Kawamori,T., Rao,C.V., Seibert,K. and Reddy,B.S. (1998) Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res., 58, 409–412.[Abstract]
  9. Harris,R.E., Alshafie,G.A., Abou-Issa,H. and Seibert,K. (2000) Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase-2 inhibitor. Cancer Res., 60, 2101–2103.[Abstract/Free Full Text]
  10. Fischer,S.M., Lo,H.H., Gordon,G.B., Seibert,K., Kelloff,G., Lubet,R.A. and Conti,C.J. (1999) Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis. Mol. Carcinogen., 25, 231–240.[CrossRef][ISI][Medline]
  11. Grubbs,C.J., Lubet,R.A., Koki,A.T., Leahy,K.M., Masferrer,J.L., Steele,V.E., Kelloff,G.J., Hill,D.L. and Seibert,K. (2000) Celecoxib inhibits N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder cancers in male B6D2F1 mice and female Fischer-344 rats. Cancer Res., 60, 5599–5602.[Abstract/Free Full Text]
  12. D'Acquisto,F., Iuvone,T., Rombola,L., Sautebin,L., Di Rosa,M. and Carnuccio,R. (1997) Involvement of NF-{kappa}B in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Lett., 418, 175–178.[CrossRef][ISI][Medline]
  13. Kojima,M., Morisaki,T., Izuhara,K., Uchiyama,A., Matsunari,Y., Katano,M. and Tanaka,M. (2000) Lipopolysaccharide increases cyclooxygenase-2 expression in a colon carcinoma cell line through nuclear factor-{kappa}B activation. Oncogene, 19, 1225–1231.[CrossRef][ISI][Medline]
  14. Janssen-Heininger,Y.M., Poynter,M.E. and Baeuerle,P.A. (2001) Recent advances towards understanding redox mechanisms in the activation of nuclear factor kappaB. Free Radic. Biol. Med., 28, 1317–1327.[CrossRef][ISI]
  15. Schulze-Osthoff,K., Ferrari,D., Riehemann,K. and Wesselborg,S. (1997) Regulation of NF-{kappa}B activation by MAP kinase cascades. Immunobiology, 198, 35–49.[ISI][Medline]
  16. Surh,Y.-J. (1999) Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances. Mutat. Res., 428, 305–327.[ISI][Medline]
  17. Conney,A.H., Lou,Y.R., Xie,J.G., Osawa,T., Newmark,H.L., Liu,Y., Chang,R.L. and Huang,M.-T. (1997) Some perspectives on dietary inhibition of carcinogenesis: studies with curcumin and tea. Proc. Soc. Exp. Biol. Med., 216, 234–245.[Abstract]
  18. Huang,M.-T., Smart,R.-C., Wong,C.-Q. and Conney,A.H. (1988) Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res., 48, 5941–5946.[Abstract]
  19. Huang,M.-T., Lou,Y.-R., Ma,W., Newmark,H.L., Reuhl,K.R. and Conney,A.H. (1994) Inhibitory effects of dietary curcumin on forestomach, duodenal and colon carcinogenesis in mice. Cancer Res., 54, 5841–5847.[Abstract]
  20. Huang,M.-T., Ma,W., Lu,Y.-P., Chang,R.L., Fischer,C., Manchard,P.S., Newmark,H.L. and Conney,A.H. (1995) Effects of curcumin, demethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion. Carcino-genesis, 16, 2493–2497.[Abstract]
  21. Joe,B. and Lokesh,B.R. (1994) Role of capsaicin, curcumin and dietary n-3 fatty acids in lowering the generation of reactive oxygen species in rat peritoneal macrophages. Biochim. Biophys. Acta, 1224, 255–263.[ISI][Medline]
  22. Reddy,A.C. and Lokesh,B.R. (1994) Studies on the inhibitory effects of curcumin and eugenol on the formation of reactive oxygen species and the oxidation of ferrous iron. Mol. Cell Biochem., 37, 1–8.
  23. Huang,T.S., Lee,S.C. and Lin,J.K. (1991) Suppression of c-Jun/AP-1 activation by an inhibitor of tumor promotion in mouse fibroblast cells. Proc. Natl Acad. Sci. USA, 88, 5292–5296.[Abstract]
  24. Singh,S. and Aggarwal,B.B. (1995) Activation of transcription factor NF-{kappa}B is suppressed by curcumin (diferuloylmethane). J. Biol. Chem., 270, 24995–25000.[Abstract/Free Full Text]
  25. Huang,T.S., Lysz,T. and Conney,A.H. (1992) Inhibitory effects of curcumin on tumor promotion and arachidonic acid metabolism in mouse epidermis. In Wattenberg,L.W. (ed.), Cancer Chemoprevention. CRC Press Inc., Boca Raton, pp. 375–391.
  26. Lu,Y.P., Chang,R.L., Huang,M.-T. and Conney,A.H. (1993) Inhibitory effects of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced increase in ornithine decarboxylase mRNA in mouse epidermis. Carcinogenesis, 14, 293–297.[Abstract]
  27. Han,S.S., Keum,Y.S., Seo,H.J., Chun,K.-S., Lee,S.S. and Surh,Y.-J. (2001) Capsaicin suppresses phorbol ester-induced activation of NF-{kappa}B/Rel and AP-1 transcription factors in mouse epidermis. Cancer Lett., 164, 119–126.[CrossRef][ISI][Medline]
  28. Liu.,X.H. and Rose,D.P. (1996) Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res., 56, 1525–1527.
  29. Subbaramaiah,K., Chung,W.J., Michaluart,P., Telang,N., Tanabe,T., Inoue,H., Jang,M., Pezzuto,J.M. and Dannenberg,A.J. (1998) Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J. Biol. Chem., 273, 21875–21882.[Abstract/Free Full Text]
  30. Dhawan,P. and Richmond,A. (2002) A Novel NF-{kappa}B-inducing kinase-MAPK signaling pathway up-regulates NF-{kappa}B activity in melanoma cells. J. Biol. Chem., 277, 7920–7928.[Abstract/Free Full Text]
  31. Foehr,E.D., Bohuslav,J., Chen,L.F., DeNoronha,C., Geleziunas,R., Lin,X., O'Mahony,A. and Greene,W.C. (2000) The NF-{kappa}B-inducing kinase induces PC12 cell differentiation and prevents apoptosis. J. Biol. Chem., 275, 34021–34024.[Abstract/Free Full Text]
  32. Sonoda,Y., Kasahara,T., Yamaguchi,Y., Kuno,K., Matsushima,K. and Mukaida,N. (1997) Stimulation of interleukin-8 production by okadaic acid and vanadate in a human promyelocyte cell line, an HL-60 subline. J. Biol. Chem., 272, 15366–15372.[Abstract/Free Full Text]
  33. Briant,L., Robert-Hebmann,V., Sivan,V., Brunet,A., Pouyssegur,J. and Devaux,C. (1998) Involvement of extracellular signal-regulated kinase module in HIV-mediated CD4 signals controlling activation of nuclear factor-kappa B and AP-1 transcription factors. J. Immunol., 160, 1875–1885.[Abstract/Free Full Text]
  34. Weinstein,I.B. (1991) Cancer prevention: recent progress and future opportunities. Cancer Res., 51 (18 suppl.), 5080s–5085s.[Abstract]
  35. DiGiovanni, J. (1992) Multistage carcinogenesis in mouse skin. Pharmacol. Ther., 54, 63–128.[CrossRef][ISI][Medline]
  36. Bhimani,R.S., Troll,W., Grunberger,D. and Frenkel,K. (1993) Inhibition of oxidative stress in HeLa cells by chemopreventive agents. Cancer Res., 53, 4528–4533.[Abstract]
  37. Limtrakul,P., Lipigorngoson,S., Namwong,O., Apisariyakul,A. and Dunn,F.W. (1997) Inhibitory effect of dietary curcumin on skin carcinogenesis in mice. Cancer Lett., 116, 197–203.[CrossRef][ISI][Medline]
  38. Singh,S.V., Hu,X., Srivastava,S.K., Singh,M., Xia,H., Orchard,J.L. and Zaren,H.A. (1998) Mechanism of inhibition of benzo[a]pyrene-induced forestomach cancer in mice by dietary curcumin. Carcinogenesis, 19, 1357–1360.[Abstract]
  39. Tao,L., Li,K. and Pereira,M.A. (1997) Chemopreventive agents-induced regression of azoxymethane-induced aberrant crypt foci with the recovery of hexosaminidase activity. Carcinogenesis, 18, 1415–1418.[Free Full Text]
  40. Zhang,F., Altorki,N.K., Mestre,J.R., Subbaramaiah,K. and Dannenberg,A.J. (1999) Curcumin inhibits cyclooxygenase-2 transcription in bile acid- and phorbol ester-treated human gastrointestinal epithelial cells. Carcinogenesis, 20, 445–451.[Abstract/Free Full Text]
  41. Bennett,A. (1986) The production of prostanoids in human cancers, and their implications for tumor progression. Prog. Lipid Res., 25, 539–542.[CrossRef][ISI][Medline]
  42. Wilson,K.T., Fu,S., Ramanujam,K.S. and Meltzer,S.J. (1998) Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in Barrett's esophagus and associated adenocarcinoma. Cancer Res., 58, 2929–2934.[Abstract]
  43. Takahashi,M., Fukuda,K., Ohata,T. and Wakabayashi,K. (1997) Increased expression of inducible and endothelial nitric oxide synthases in rat colon tumors induced by azoxymethane. Cancer Res., 57, 1233–1237.[Abstract]
  44. Ambs,S., Merriam,W.G., Bennett,W.P., Felley-Bosco,E., Ogunfusika,M.O., Oser,S.M., Klein,S., Shields,P.G., Billiar,T.R. and Harris,C.C. (1998) Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression, Cancer Res., 58, 334–341.[Abstract]
  45. Nakamura,Y., Murakami,A., Ohto,Y., Torikai,K., Tanaka,T. and Ohigashi,H. (1998) Suppression of tumor promoter-induced oxidative stress and inflammatory responses in mouse skin by a superoxide generation inhibitor 1'-acetoxychavicol acetate. Cancer Res., 58, 4832–4839.[Abstract]
  46. Plummer,S.M., Holloway,K.A., Manson,M.M., Munks,R.J., Kaptein,A., Farrow,S. and Howells,L. (1999) Inhibition of cyclooxygenase-2 expression in colon cells by the chemopreventive agent curcumin involves inhibition of NF-{kappa}B activation via the NIK/IKK signalling complex. Oncogene, 18, 6013–6020.[CrossRef][ISI][Medline]
  47. Surh,Y.-J., Han,S.S., Keum,Y.-S., Seo,H.-J. and Lee,S.S. (2000) Inhibitory effects of curcumin and capsaicin on phorbol ester-induced activation of eukaryotic transcription factors, NF-{kappa}B and AP-1. Biofactors, 12, 107–112.[ISI][Medline]
  48. Surh,Y.-J., Chun,K.-S., Cha,H.-H., Han,S.S., Keum,Y.-S., Park, K.-K. and Lee,S.S. (2001) Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-{kappa}B activation. Mutat. Res., 480–481, 243–268.[ISI]
  49. Guo,Y.S., Hellmich,M.R., Wen,X.D. and Townsend,C.M.,Jr (2001) Activator protein-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression in intestinal epithelial cells. J. Biol. Chem., 276, 22941–22947.[Abstract/Free Full Text]
  50. Subbaramaiah,K., Lin,D.T., Hart,J.C. and Dannenberg,A.J. (2001) Peroxisome proliferator-activated receptor gamma ligands suppress the transcriptional activation of cyclooxygenase-2. Evidence for involvement of activator protein-1 and CREB-binding protein/p300. J. Biol. Chem., 276, 12440–12448.[Abstract/Free Full Text]
  51. Subbaramaiah,K., Norton,L., Gerald,W. and Dannenberg,A.J. (2002) Cyclooxygenase-2 is overexpressed in HER-2/neu-positive breast cancer: evidence for involvement of AP-1 and PEA3. J. Biol. Chem., 277, 18649–18657.[Abstract/Free Full Text]
  52. Bonvin,C., Guillon,A., van Bemmelen,M.X., Gerwins,P., Johnson,G.L. and Widmann,C. (2002) Role of the amino-terminal domains of MEKKs in the activation of NF kappa B and MAPK pathways and in the regulation of cell proliferation and apoptosis. Cell Signal., 14, 123–131.[CrossRef][ISI][Medline]
  53. Reiser,C.O.A., Lanz,T., Hoffmann,F., Hofer,G., Rupprecht,H.D. and Goppelt-Strube,M. (1998) Lysophosphatidic acid-mediated signal-transduction pathways involved in the induction of the early-response genes prostaglandin G/H synthase-2 and Egr-1: a critical role for the mitogen-activated protein kinase p38 and for Rho proteins. Biochem. J., 330, 1107–1114.[ISI][Medline]
  54. Wang,D. and Richmond,A. (2001) Nuclear factor-kappa B activation by the CXC chemokine melanoma growth-stimulatory activity/growth-regulated protein involves the MEKK1/p38 mitogen-activated protein kinase pathway. J. Biol. Chem., 276, 3650–3659.[Abstract/Free Full Text]
  55. Hwang,D., Jang,B.C., Yu,G. and Boudreau,M. (1997) Expression of mitogen-inducible cyclooxygenase induced by lipopolysaccharide mediation through both mitogen-activated protein kinase and NF-{kappa}B signaling pathways in macrophages. Biochem. Pharmacol., 54, 87–96.[CrossRef][ISI][Medline]
  56. Pouliot,M., Baillargeon,J., Lee,J.C., Cleland,L.G. and James,M.J. (1997) Inhibition of prostaglandin endoperoxide synthase-2 expression in stimulated human monocytes by inhibitors of p38 mitogen-activated protein kinase. J. Immunol., 158, 4930–4937.[Abstract]
  57. Dean,J.L., Brook,M., Clark,A.R. and Saklatvala,J. (1999) p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J. Biol. Chem., 274, 264–269.[Abstract/Free Full Text]
  58. Jaffee,B.D., Manos,E.J., Collins,R.J., Czerniak,P.M., Favata,M.F., Magolda,R.L., Scherle,P.A. and Trzaskos,J.M. (2000) Inhibition of MAP kinase kinase (MEK) results in an anti-inflammatory response in vivo. Biochem. Biophys. Res. Commun., 268, 647–651.[CrossRef][ISI][Medline]
  59. Liu,Y., Duysen,E., Yaktine,L.A., Au,A., Wang,W. and Birt,D. (2001) Dietary energy restriction inhibits ERK but not JNK or p38 activity in the epidermis of SENCAR mice. Carcinogenesis, 22, 607–612.[Abstract/Free Full Text]
  60. Manna,S.K., Mukhopadhyay,A., Van,N.T. and Aggarwal,B.B. (1999) Silymarin suppresses TNF-induced activation of NF-{kappa}B, c-Jun N-terminal kinase, and apoptosis. J. Immunol., 163, 6800–6809.[Abstract/Free Full Text]
  61. Manna,S.K., Sah,N.K., Newman,R.A., Cisneros,A. and Aggarwal,B.B. (2000) Oleandrin suppresses activation of nuclear transcription factor-kappaB, activator protein-1, and c-Jun NH2-terminal kinase. Cancer Res., 60, 3838–3847.[Abstract/Free Full Text]
  62. Frank,N., Knauft,J., Amelung,F., Nair,J., Wesch,H. and Bartsch,H. (2003) No prevention of liver and kidney tumors in Long-Evans Cinnamon rats by dietar curcumin, but inhibition at other sites and of metastases. Mutat. Res., 523–524, 127–135.[ISI]
Received April 29, 2003; revised June 15, 2003; accepted June 17, 2003.