Celecoxib inhibits phorbol ester-induced expression of COX-2 and activation of AP-1 and p38 MAP kinase in mouse skin

Kyung-Soo Chun1, Su-Hyeong Kim2, Yong-Sang Song2 and Young-Joon Surh1,3

1 College of Pharmacy and 2 Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, 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
 
Celecoxib, the first US FDA-approved selective cyclooxygenase-2 (COX-2) inhibitor initially developed for the treatment of adult rheumatoid arthritis and osteoarthritis, was reported to reduce the polyp burden in patients with familial adenomatous polyposis. This specific COX-2 inhibitor also protects against experimentally induced carcinogenesis, but molecular mechanisms underlying its chemopreventive activities remain largely unresolved. In the present work, we found that celecoxib inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of COX-2 in female ICR mouse skin when applied topically 30 min prior to TPA as determined by both immunoblot and immunohistochemical analyses. In another study, celecoxib attenuated the DNA binding activity of activator protein 1 (AP-1) through suppression of c-Jun and c-Fos expression in TPA-treated mouse skin. In addition, celecoxib inhibited both the catalytic activity and phosphorylation of p38 mitogen-activated protein (MAP) kinase. In the same animal model, TPA treatment resulted in rapid activation via phosphorylation of extracellular signal-regulated protein kinase (ERK)1/2 and p38 MAP kinase, which are upstream of AP-1 in mouse skin. In order to clarify the roles of p38 and ERK in TPA-induced AP-1 activation, we utilized the pharmacologic inhibitors of these enzymes. The p38 inhibitor SB203580 blocked TPA-mediated AP-1 activation, while the MEK1/2 inhibitor U0126 was not inhibitory despite suppression of c-Fos expression in mouse skin. Furthermore, SB203580 markedly inhibited COX-2 expression induced by TPA. Taken together, these findings suggest that celecoxib down-regulates COX-2 by blocking activation of p38 MAP kinase and AP-1, which may represent molecular mechanisms underlying antitumor promoting effects of this drug on mouse skin tumorigenesis.

Abbreviations: AP-1, activator protein 1; COX, cyclooxygenase; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated protein kinase; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; NSAIDs, non-steroidal anti-inflammatory drugs; PGs, prostaglandins; SDS, sodium dodecyl sulfate; TPA, 12-O-tetradecanoylphorbol-13-acetate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prostaglandins (PGs) with multiple physiological functions play crucial roles in maintaining the homeostasis of the cell. The key regulatory step in the PG biosynthesis is catalyzed by cyclooxygenase (COX). There are two isoforms of COX, designated as COX-1 and COX-2 (1). COX-1 is a house-keeping enzyme that is constitutively expressed in most tissues. COX-2, on the other hand, is expressed transiently by a wide spectrum of growth factors and pro-inflammatory cytokines under certain pathophysiological conditions (2,3). Besides its role in inflammation, COX-2 has been implicated in the pathogenesis of several types of malignancies (49).

Aspirin and some other non-steroidal anti-inflammatory drugs (NSAIDs) with COX-2 inhibitory activity have received attention because of their protective effects against a variety of human malignancies as well as experimentally induced carcinogenesis (10,11). To be applied to general populations, a chemopreventive agent must have an acceptable safety profile in addition to therapeutic effectiveness. Celecoxib, a selective COX-2 inhibitor, is currently available for the treatment of chronic inflammatory conditions such as adult rheumatoid arthritis and osteoarthritis. Recently, celecoxib has been reported to reduce the burden of polyps in patients with familial adenomatous polyposis (12,13). Celecoxib has also been shown to inhibit experimentally induced tumorigenesis in several animal models, including azoxymethane-induced colon tumorigenesis in F344 rats (14), 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammry carcinogenesis (15,16), ultraviolet (UV)-induced mouse skin tumor formation (1720), and N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in male B6D2F1 mice and female Fischer 344 rats (21). However, the exact molecular mechanisms underlying chemopreventive effects of celecoxib are not fully elucidated yet, and it is still controversial whether or not its antitumorigenic effects are mediated predominantly through suppression of COX-2 and PG synthesis.

Skin cancer is an important model system for the study of cancer chemoprevention (22). The ease with which clinical and histological changes may be followed in skin cancer is a great advantage over malignancies of other organ systems. The majority of human malignancies are developed through a sequence of distinct pathophysiologic processes in which multiple genetic alterations or defects are accumulated. One of the most well defined animal models that properly reflect the concept of multi-stage carcinogenesis is skin tumor formation in mice (23). This murine model allows a clear-cut distinction between individual stages of carcinogenesis and aids evaluation of potential chemopreventive activities of a wide array of chemical substances and also investigation of their underlying mechanisms (24). The mechanistic studies then guide us in developing prevention and treatment strategies. Dietary or topical celecoxib has the ability to attenuate inflammation and tumorigenesis induced in UV-irradiated murine cutaneous tissue (1720,25). More recently, topically applied celecoxib has been reported to significantly suppress 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative damage and hyperplasia in mouse skin (26). Our study also reveals the inhibition of TPA-induced mouse skin tumor promotion by topically applied celecoxib (27).

As an initial approach elucidating the molecular mechanisms underlying the anti-inflammatory and antitumor promoting activity of celecoxib in mouse skin, we sought to examine its effects on TPA-induced expression of COX-2 and the activation of upstream mitogen-activated protein (MAP) kinases and transcription factors regulating COX-2 induction. Here, we report that topically applied celecoxib inhibits the tumor promoter-induced COX-2 expression and activation of p38 MAP kinase and activator protein 1 (AP-1) transcription factor in mouse skin in vivo.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Celecoxib (CelebrexTM) was supplied from Pharmacia Korea. TPA was obtained from Alexis Biochemicals (San Diego, CA). U0126 and SB203580 were purchased from Tocris Cookson (Avonmouth, UK). 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/Bio-link 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. Celecoxib and TPA were dissolved in 200 µl of acetone and applied to the dorsal shaven area.

Measurement of PGE2
The female ICR mice were topically treated on their shaven backs with indicated doses of celecoxib 30 min prior to TPA (10 nmol) application and were killed by cervical dislocation 5 h later. The pulverized skin was homogenized with ice-cold ethanol and centrifuged for 10 min at 3000 g. Supernatant was diluted to 15% with respect to ethanol by adding 0.1 M sodium formate, and pelleted protein was dissolved in 8 M urea. The diluted supernatant was applied to a pre-activated AmprepTM C-18 reverse phase cartridge (Amersham Pharmacia Biotech, Buckinghamshire, UK), and eicosanoids were released by ethyl acetate containing 1% methanol. The extract was evaporated to dryness under a gentle stream of nitrogen and resuspended in enzyme immunoassay buffer. The amounts of PGE2 were measured by using the PGE2 enzyme-immunoassay kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer's protocol. The quantity of dissolved protein was determined by the BCA method.

Western blot analysis
For isolation of protein from mouse skin, the dorsal skin was excised, and after the fat was removed on ice, the remaining skin tissues were 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 the following antibodies: COX-2 and COX-1 polyclonal antibodies (Cayman Chemical, Ann Arbor, MI), extracellular signal-regulated protein kinase (ERK) and c-Jun polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), and p38, phospho-p38, phospho-ERK and c-Fos monoclonal antibodies (Santa Cruz Biotechnology). Equal lane loading was assessed using actin (Sigma-Aldrich, St Louis, MO). The blots were rinsed three times with PBST buffer for 5 min each. Washed blots were incubated with 1:5000 dilution of the horseradish peroxide (HRP)-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).

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, 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 minimize non-specific staining, each section was treated with 3% hydrogen peroxide in methanol for 15 min. Slides were incubated with 1:50–100 dilutions of the monoclonal mouse anti-COX-2 antibody (BD Transduction Laboratories, Franklin Lakes, NJ) at room temperature for 60 min in Tris–HCl-buffered saline containing and 0.05% Tween-20 and then developed using the HRP EnVisionTM System (Dako, Glostrup, Denmark). The peroxidase binding sites were detected by staining with 3,3'-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark). Specificity of immunostaining was assessed by quenching the primary antisera by pre-adsorbing with the COX-2 blocking peptide (Cayman Chemical). Finally, counterstaining was performed using Mayer's hematoxylin.

Preparation of nuclear extracts
Nuclear extract was prepared from mouse skin as described previously (28). 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, 125 µl of 10% Nonidet P-40 (NP-40) solution was added, followed by centrifugation for 2 min at 14 800 g. The resulting pellets 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 the protein concentration.

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 AP-1 oligonucleotide probe (5'-CGC TTG ATG AGT CAG CCG GAA C-3') was labeled with [{gamma}-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 volume of 25 µl containing 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% glycerol, 0.1 mg/ml sonicated salmon sperm DNA, 10 µg of nuclear extracts and 100 000 c.p.m. of the labeled probe. A 100-fold excess of unlabeled oligonucleotide (competitor) was added where necessary. To verify the composition of AP-1, supershift assays were carried out by pre-incubation with rabbit c-Jun or c-Fos polyclonal antibody prior to the addition of the labeled AP-1 oligonucleotide. 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 for 2 h. Finally, the gel was dried and exposed to X-ray film.

MAP kinase assay (non-radioactive)
The catalytic activities of p38 MAP kinase and ERK were carried out by using a non-radioactive MAP kinase assay kit (Cell Signaling Technology, Beverly, MA). The protein fraction from dorsal skin of mice was prepared as described above for western blot analysis. Monoclonal phospho-specific antibodies to p38 and ERK were used to selectively immunoprecipitate an active form of each MAP kinase in mouse skin. The resulting immunoprecipitate was then incubated with ATF-2 and Elk-1 fusion protein as substrates for p38 and ERK, respectively, in the presence of ATP and kinase buffer, allowing immunoprecipitated active MAP kinases to phosphorylate their substrates. Phosphorylation of ATF-2 and Elk-1 was measured by immunoblot analysis with phospho-ATF-2 and phospho-Elk-1 antibodies, respectively. Briefly, collected tissues were lysed in 300 ml of lysis buffer [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 µg/ml leupeptin]. The lysates were centrifuged, and the supernatant was incubated with specific immobilized phospho-p38 kinase and phospho-ERK monoclonal antibodies with gentle rocking for overnight at 4°C. The beads were washed twice each with 500 µl 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 the aforementioned MAP kinase substrates was selectively measured by immunoblotting with specific antibodies detecting phosphorylation of ATF-2 and Elk-1 at Thr71 and Ser383, respectively.

MAP kinase inhibitor studies
In order to determine the possible involvement of MAP kinases in signaling pathways leading to COX-2 induction in TPA-treated mouse skin, we utilized the U0126 and SB203580, which are pharmacological inhibitors of MEK1/2 (upstream of ERK) and p38, respectively. Their effects on catalytic activities of MAP kinase, AP-1 activation and COX-2 expression were investigated by the kinase assay, EMSA and western blot analysis, respectively, following the procedures described above.

Statistical analysis
Data are expressed as means ± SD. Statistical significance of changes was determined by the Student's t-test. Differences resulting in P values <0.05 were considered to be statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of MAP kinase inhibitors on TPA-induced AP-1 DNA binding activity and COX-2 expression
The intracellular signaling cascades controlling AP-1 activation are highly complex and may involve different kinases. Of the potential upstream kinases involved in activation of AP-1, the MAP kinases have been well characterized (29). MAP kinase family proteins have been shown to play an important role in regulating AP-1 activation in various types of cultured cells via multiple mechanisms (30). AP-1 heterodimers or homodimers are constitutively localized within the nucleus, and transactivation of AP-1 is achieved through phosphorylation of its activation domain by a specific MAP kinase (31). The MAP kinase family includes ERK, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38.

We have shown previously that TPA treatment resulted in rapid activation via phosphorylation of ERK and p38 MAP kinases in mouse skin (32). However, the JNK activity remained unchanged even after TPA treatment. The phosphorylation of Elk-1 by ERK enhances its ability to form a complex with serum response element and results in serum response element-dependent activation of the c-fos promoter (31). The phosphorylation of ATF-2 by p38 MAP kinase augments the TPA response element (TRE)-dependent transcriptional activity of c-jun (31). However, it remained unclarified whether MAP kinases could participate in activation of AP-1 in mouse skin in vivo. To properly assess the possible involvement of p38 MAP kinase and ERK in TPA-stimulated AP-1 signaling, we have utilized pharmacological inhibitors of these kinases. SB203580 is known to selectively inhibit p38 MAP kinase whilst U0126 is an ultrapotent inhibitor of MEK1/2 responsible for activation of ERK. As shown in Figure 1, SB203580 and U0126 at 4 µmol each suppressed the activity of p38 MAP kinase and phosphorylative activation of ERK1/2, respectively.



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Fig. 1. Effect of MAP kinase inhibitors on TPA-induced activation of p38 and ERK in mouse skin. (A) Female ICR mice were treated topically with acetone or with 2 or 4 µmol SB203580 30 min prior to 10 nmol TPA. Animals were killed by cervical dislocation 1 h later. The activity for p38 kinase was determined by the immune complex assay as described in ‘Materials and methods’. Tissue lysates containing 200 µg protein were treated with a specific immobilized phospho-p38 MAP kinase monoclonal antibody. The resulting immunoprecipitates were then incubated with ATF-2 fusion protein in the presence of 100 µM ATP. Phosphorylation of ATF-2 was measured by western blotting of non-radioactive labeled samples using the phospho-ATF-2 antibody. (B) Mice were treated topically with either the solvent alone or U0126 (1 or 4 µmol) 30 min prior to TPA application. Mice were killed 1 h after the TPA treatment, and expression of ERK was measured by western blot analysis.

 
To determine which of the MAP kinases is involved directly in activation of AP-1 in mouse skin, we have compared the effects of the aforementioned MAP kinase inhibitors on AP-1 activation by TPA. SB203580, but not U0126, abrogated the induction of the AP-1 DNA binding activity (Figure 2A). This result indicates that activation of AP-1 can be mediated, at least in part, via the p38 MAP kinase-dependent pathway in TPA-treated mouse skin. The specificity and composition of AP-1 were verified by competition with an excess of the cold probe and supershift with antibodies against c-Jun and c-Fos that are typical components of AP-1. The AP-1 DNA binding induced by TPA could be suppressed competitively by excess amounts of a cold (non-radiolabeled) oligonucleotide bearing specific AP-1 binding motif (Figure 2B). Antibodies directed against c-Jun and c-Fos proteins did not supershift the induced AP-1 band, but instead suppressed its binding to labeled DNA. Such competition without concomitant retardation of AP-1 DNA complex may probably be due to the spatial location of antibody binding sites at the close proximity of the consensus sequence of AP-1 in mouse skin. In this case, antibody binding to c-Jun or c-Fos would interfere with oligonucleotide binding of AP-1.



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Fig. 2. Effects of MAP kinase inhibitors on TPA-induced activation of AP-1, expression of c-Jun and c-Fos and COX-2 expression. (A) Nuclear extracts from mouse skin treated with acetone or 10 nmol TPA for 1 h were assayed 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. Each MAP kinase inhibitor was applied topically 30 min prior to TPA treatment. (B) Nuclear protein from skin of mice treated with TPA for 1 h was incubated with 0-, 10- or 100-fold excess of unlabeled AP-1 oligonucleotide or with an antibody against c-Jun and c-Fos, and subjected to EMSA. The first lane in (A) and (B) represents probe only (no nuclear extract added to the incubation mixture). (C) Dorsal skin of female ICR mice was treated topically with acetone or a MAP kinase inhibitor (4 µmol) 30 min prior to topical application of 10 nmol TPA. After 1 h, mice were killed, and epidermal nuclear extracts were prepared for western blot analysis. (D) SB203580 (4 µmol) was applied topically 30 min prior to 10 nmol TPA. Mice were killed 4 h after the TPA treatment. COX-2 protein was determined by immunoblot analysis, as described under ‘Materials and methods’. All experiments were repeated at least twice.

 
To examine whether these MAP kinase signaling pathways are involved in the expression of major AP-1 components in TPA-treated mice, we have examined the effects of SB203580 and U0126 on TPA-induced expression of c-Jun and c-Fos. The p38 MAP kinase inhibitor SB203580 blocked c-Jun expression without affecting c-Fos induction, whereas the ERK inhibitor U0126 preferentially suppressed c-Fos expression at the pharmacologically effective dose (i.e. 4 µmol) capable of inhibiting the corresponding MAP kinases (Figure 2C).

Many in vitro cell culture studies have described a close association between MAP kinase activity and COX-2 expression (33,34). To verify the involvement of p38 MAP kinase in the signaling pathway regulating the TPA-induced COX-2 expression as well as AP-1 activation in mouse skin, we have examined the effect of the p38 MAP kinase inhibitor on TPA-induced COX-2 expression. SB203580 at a pharmacologic dose blocked COX-2 induction, indicative of the possible role of p38 MAP kinase in up-regulating the TPA-induced COX-2 expression through AP-1 activation in mouse skin (Figure 2D).

Inhibitory effects of celecoxib on TPA-induced PGE2 production and COX-2 expression in mouse skin
We have shown previously that TPA, a prototypic tumor promoter and a mitogen, stimulates COX-2 expression and PGE2 production in mouse skin (35,36). As illustrated in Figure 3A, topical application of dorsal skin of female ICR mice with 1 or 10 µmol celecoxib significantly reduced the TPA-induced PGE2 synthesis. In addition, celecoxib pre-treatment resulted in marked inhibition of COX-2 expression in TPA-treated mouse skin without altering the COX-1 level (Figure 3B). The suppression of PGE2 production was more prominent than that of COX-2 expression, suggesting that celecoxib inhibits the COX-2 activity as well as expression in TPA-treated mouse skin. To assess the localization of COX-2 in mouse skin, we conducted an immunohistochemical analysis. In acetone-treated control skin, specific COX-2 immunostaining was barely detectable in the dorsal layer (Figure 4A and B). In contrast, expression of COX-2 increased dramatically in the epidermal basal layer upon treatment with 10 nmol TPA for 4 h (Figure 4C and D). Topical application of celecoxib at 10 µmol abolished epidermal COX-2 expression (Figure 4E and F). Lack of staining following the application of a blocking peptide for the COX-2 antibody demonstrated immunospecificity (Figure 4G and H).



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Fig. 3. Inhibitory effects of celecoxib on phorbol ester-induced PGE2 production and COX-2 expression. (A) Female ICR mice were treated topically with acetone or with 1.0 or 10 µmol of celecoxib 30 min prior to 10 nmol TPA, and animals were killed 5 h after the TPA treatment for PGE2 analysis. Data are expressed as the means ± SD obtained from five mice per group. Significantly different from the group treated with TPA alone (*P < 0.01; **P < 0.005). (B) Mice were treated topically with either the solvent alone or celecoxib (1.0 or 10 µmol) 30 min prior to TPA treatment. Animals were killed by cervical dislocation 4 h later. Protein extracts (30 µg) were loaded onto a 12% SDS–polyacrylamide gel, electrophoresed and subsequently transferred onto PVDF membrane. Immunoblots were probed with a rabbit polyclonal COX-2 antibody. The western blot is representative of three independent experiments.

 


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Fig. 4. Inhibitory effects of celecoxib on phorbol ester-induced COX-2 expression by immunohistochemical staining. Paraffin-embedded tissues from TPA-treated mice were immunostained for COX-2 and counterstained with hematoxylin, as described in ‘Materials and methods’. Positive COX-2 staining yielded a brown-colored product. (A and B) Skin treated with acetone (10x and 40x). (C and D) Skin treated with 10 nmol TPA for 4 h (10x and 40x). (E and F) Skin treated with 10 µmol celecoxib 30 min prior to 10 nmol TPA (10x and 40x). (G and H) Blocking peptide used to show specificity of antibody binding (10x and 40x). The part of stained specimens (A, C, E and G) inside rectangular boxes represent sites of magnification.

 
Inhibition of TPA-induced AP-1 DNA binding activity by celecoxib
Because AP-1 is likely to play a role in regulating the induction of COX-2 in mouse skin, we have determined whether celecoxib could suppress activation of this transcription factor using nuclear extracts obtained from mouse skin stimulated with TPA. When celecoxib was applied topically onto the shaven backs of female ICR mice, it strongly inhibited TPA-induced AP-1 DNA binding (Figure 5A). The same treatment failed to block the TPA-induced DNA binding of NF-{kappa}B and nuclear translocation of its functional subunit p65 (data not shown). To elucidate the molecular mechanism by which celecoxib inhibits AP-1 activation, we examined its effect on expression of c-Jun and c-Fos proteins following TPA treatment. Celecoxib treatment suppressed the expression of both c-Jun and c-Fos in mouse skin (Figure 5B).



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Fig. 5. (A) Effect of celecoxib on AP-1 activation and expression of major AP-1 components in mouse skin treated with TPA. Dorsal skins of mice were treated topically with acetone alone, 10 nmol TPA alone or 1.0 and 10 µmol of celecoxib 30 min before TPA treatment. Mice were killed 1 h after the TPA treatment, and epidermal nuclear extracts were incubated with the radiolabeled oligonucleotide containing the AP-1 consensus sequence for analysis by EMSA. The first lane represents free probe alone (no nuclear extracts). EMSA was performed in duplicate and a representative result is shown. (B) Nuclear extracts from mouse skin treated with 10 nmol TPA for 1 h were assayed for c-Jun and c-Fos by western blot analysis. The western blot is representative of two separate experiments.

 
Inhibitory effects of celecoxib on TPA-induced activation of MAP kinases
Since p38 MAP kinase was found to be implicated in AP-1 activation in TPA-treated mouse skin (Figure 2B), we examined whether celecoxib could down-regulate this particular MAP kinase, thereby inhibiting AP-1 activation and subsequently the COX-2 induction in TPA-treated mouse skin. Since phosphorylation of the MAP kinases leads to their activation, this event was directly assessed using phospho-specific antibodies against ERK and p38 MAP kinase. To confirm that phosphorylation of these kinases confers enhanced catalytic activity, an immune complex kinase assay was also performed. Phosphorylation and the catalytic activity of p38 MAP kinase as well as ERK were evident in 30 min following TPA application, and sustained up to 4 h (32). Celecoxib inhibited both catalytic activity and phosphorylation of p38 MAP kinase (Figure 6A), while it barely suppressed those of ERK1/2 in mouse skin (Figure 6B). Under the same experimental conditions, the levels of total or unphosphorylated forms of both kinases remained almost constant.



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Fig. 6. Effects of celecoxib on TPA-induced activation of p38 MAP kinase (A) and ERK (B). Female ICR mice were treated topically with acetone or with 1.0 or 10 µmol of celecoxib 30 min prior to TPA (10 nmol). After 1 h, mice were killed, and total protein was isolated from the dorsal skin and quantified. The kinase activities were determined by the immune complex assay as described in the legend to Figure 1. Tissue lysates containing 200 µg protein were treated with a specific immobilized phospho-p38 kinase or a phospho-ERK monoclonal antibody. The resulting immunoprecipitates were then incubated with ATF-2 or Elk-1 fusion protein in the presence of 100 µM ATP. Phosphorylation of ATF-2 or Elk-1 was measured by western blotting of non-radioactive labeled samples using the phospho-ATF-2 or phospho-Elk-1 antibody. The phosphorylated form of p38 or ERK was detected by immunoblotting using a corresponding phospho-specific antibody. Data are representative of two independent experiments, which gave rise to a similar trend.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several epidemiological studies have revealed an inverse association between the frequent consumption of NSAIDs and the risk of colorectal cancer (37,38). Laboratory studies have also demonstrated that administration of aspirin, piroxicam, sulindac or ibuprofen inhibits chemically induced colon carcinogenesis (3942). However, prolonged administration of traditional NSAIDs also causes undesired side effects, such as gastrointestinal bleeding, ulceration and renal toxicity as a result of disruption of COX-1-dependent homeostatic functions (43). Because the majority of conventional NSAIDs inhibit COX-1 as well as COX-2, it is logical to search for specific inhibitors of COX-2 as potential candidates for use as chemopreventive agents (43).

Celecoxib is the first US FDA-approved drug with selective COX-2 inhibitory activity for the treatment of adult rheumatoid arthritis and osteoarthritis (44). In a clinical trial with patients suffering from familial adenomatous polyposis, celecoxib efficiently reduced the burden of colonic polyps (12,45). Likewise, celecoxib has been shown to decrease the polyp formation in the Apc mutant Min mouse model of familial adenomatous polyposis (46). Furthermore, celecoxib effectively inhibited the experimentally induced mammary (16), skin (1720) and urinary bladder (21) carcinogenesis.

Fischer et al. (17) reported that dietary celecoxib had a significant chemopreventive activity against UV-induced skin carcinogenesis in SKH-HR-1 hairless mice. In a subsequent study, celecoxib in combination with difluoromethylornithine, caused regression of UV-induced skin tumors to a much greater extent than did each compound alone (18). Pentland and colleagues (19) also showed that orally administered celecoxib blocked additional tumor formation after the onset of photocarcinogenesis in hairless mice. Celecoxib at the FDA-approved human equivalent doses of 200 and 400 mg twice daily delayed the skin tumor latency period and reduced the tumor multiplicity (25). Although the above studies employed a UV-induced skin carcinogenesis model with orally administered celecoxib, we found that this selective COX-2 inhibitor suppressed the DMBA-induced mouse skin tumor formation when given topically prior to each TPA treatment during the promotion stage (27). The present study also reveals the ability of topically applied celecoxib to inhibit TPA-induced PGE2 production by COX-2, which may account for the antitumor promoting activity of this compound. Although celecoxib is considered to exert its therapeutic effects through inhibition of the catalytic activity of COX-2, our data clearly indicate that the drug can also block COX-2 expression in TPA-stimulated mouse skin. While our manuscript is under revision, Wilgus and colleagues (20) have reported that topical celecoxib can ameliorate the UVB-induced oxidative damage, inflammation, and papilloma formation in mouse skin.

AP-1 is one of the major eukaryotic transcription factors involved in regulating COX-2 expression (4749). AP-1 is minimally activated under normal physiologic conditions, but is dramatically activated by various pathophysiological stimuli, including phorbol ester, endotoxin, cytokines, reactive oxygen species, etc. (29). c-Jun/c-Jun homodimers and c-Jun/c-Fos heterodimers preferentially bind to the AP-1 consensus sequence TGAC/GTCA (50). The activity of AP-1 is regulated at the level of transcription of c-jun and c-fos genes by protein–protein interactions and also through post-translational modifications of Jun and Fos proteins (5153).

Topical application of celecoxib suppressed expression of c-Jun and c-Fos protein induced by TPA, suggesting that a decrease in the AP-1 DNA binding activity in celecoxib-treated mice occurs as a consequence of down-regulation of its functional subunits. Considering the important role of AP-1 in tumor promotion (5456), the inhibition of activation of this transcription factor and expression of its components is likely to contribute to the antitumor promoting effect of celecoxib on mouse skin carcinogenesis. AP-1 regulates the transcription of a vast variety of genes, some of which are involved in neoplastic transformation and tumor promotion (50,54).

AP-1 activation occurs via three MAP kinase pathways. ERK1/2 is known to phosphorylate Elk-1, and JNK phosphorylates ATF-2 and c-Jun, while p38 MAP kinase catalyzes phosphorylation of both Elk-1 and ATF-2. To determine which MAP kinase(s) play(s) an important role in activation of AP-1 in mouse skin, we investigated the effects of the MEK1/2 inhibitor U0126 and the p38 inhibitor SB203580 on AP-1 activation. While SB203580 effectively inhibited the induction of AP-1 binding activity as well as c-Jun expression, U0126 had no effect on AP-1 activation despite its ability to inhibit c-Fos expression. These results imply that c-Fos may not be the key protein responsible for mediating the activation of AP-1 in TPA-treated mouse skin. The 5'-flanking region of c-jun promoter contains AP-1 binding sites, and c-jun expression is positively auto-regulated through binding of AP-1 to TRE sites on c-jun (31). Przybyszewski et al. (57) have reported that the decrease in TPA-induced AP-1 DNA binding activity in mice under dietary energy restriction is a consequence of lower levels of c-Jun protein. Transgenic mice expressing epidermal-targeted v-fos were found to be sensitized to TPA-induced skin tumor promotion, developing carcinomas as well as papillomas with Ha-ras mutation (58). This finding suggests that mutational activation rather than enhanced expression of c-fos expression is important in benign-to-malignant progression of mouse skin carcinogenesis. In c-fos null mice harboring v-Ha-ras, papillomas were still induced by phorbol ester treatment, but they were not converted to carcinomas, suggesting that c-fos is necessary for the progression of tumors from benign to malignant forms, but may not play a crucial role in the early stage of promotion during papillomagenesis (59). The susceptibility of c-jun knockout mice to spontaneous or chemically induced carcinogenesis could not be assessed because c-jun null embryos were not viable (60). However, Young and colleagues (55) reported that dominant negative c-jun-expressing transgenic mice are not prone to tumor promoter-induced AP-1 transactivation and are resistant to skin tumor promotion without eliciting skin anomalies. These findings suggest that AP-1 signaling mediated by c-Jun is important in mouse skin tumor promotion.

Recently, enzymes of the MAP kinase family have been reported to play a key role in regulating cox-2 gene expression. The Parke-Davis MEK inhibitor PD98059 blocked TPA-induced COX-2 expression in primary mouse keratinocytes (61). Lipopolysaccharide (LPS)-induced COX-2 expression in monocytes was also attenuated by U0126, lending further support to the association between ERK activation and PG production (62). In another study, however, LPS-induced expression of COX-2 was abrogated by the p38 MAP kinase inhibitor SB203580, which resulted in decreased PGE2 production in J774 macrophages (63). Similar effects of SB203580 were observed in LPS-stimulated monocytes (64). However, other investigators reported that topical application of TPA on the ears of CD1 mice (65) and skin of SENCAR mice (66) induced a rapid and sustained activation of ERK but not that of p38 MAP kinase. Most importantly, we have found that treatment of dorsal skin of female ICR mice with TPA significantly enhanced both catalytic activity and phosphorylation of p38 MAP kinase and ERK1/2 (32). However, down-stream effectors of MAP kinase activation responsible for mediating COX-2 induction are poorly defined in mouse skin. In our present study, SB203580 at a pharmacologically effective dose not only blocked TPA-induced AP-1 activation, but almost completely abrogated COX-2 protein expression, suggesting that p38 MAP kinase may play a central role in the signaling pathway mediating TPA-induced COX-2 expression and AP-1 activation in mouse skin. The use of genetically tractable animal models, in which the gene encoding p38 MAP kinase is mutated or deleted, will better clarify the upstream signal events that celecoxib targets in suppressing COX-2 induction in mouse skin.

Current evidence implicating COX-2 in tumor development stems from the observation that NSAIDs can block malignant transformation in several genetic and chemical models of tumor induction. However, this observation is somewhat confounded by data indicating that NSAIDs can exert their anti-neoplastic effects via both COX-2-dependent and -independent mechanisms (67,68). In particular, the molecular events responsible for COX-2 effects on tumor development are still unresolved. Nonetheless, improper and abnormal overexpression of COX-2 is implicated in the pathogenesis of various types of human cancers, and assessment of the effects of celecoxib on cox-2 gene expression may represent a useful surrogate biomarker for the evaluation of its chemopreventive potential. Our present study demonstrates the ability of celecoxib, administered directly to the skin, to inhibit inflammatory cascades leading to COX-2 induction by the tumor promoter, providing an opportunity of exploring the clinical efficacy of the topically applied COX-2 inhibitors for the management of human skin cancer as well as inflammatory disorders. To the best of our knowledge, this is the first report that demonstrates the regulation of p38 MAP kinase and AP-1 signaling pathways by celecoxib in mouse skin in vivo. It would be worthwhile examining the effects of celecoxib on activation of the above signaling molecules as well as induction of COX-2 expression in the same animal model used for tumorigenicity bioassay.

In conclusion, our present study may provide mechanistic basis for the antitumor promoting effect of celecoxib in mouse skin carcinogenesis by addressing the role of AP-1 and p38 MAP kinase in TPA-induced COX-2 expression as schematically represented in Figure 7.



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Fig. 7. Hypothetical mechanism underlying suppression of TPA-induced COX-2 expression by celecoxib in mouse skin. SRE, serum-response element; TRE, TPA-response element; SRF, serum-response factor; FRK, c-Fos-regulating kinase.

 

    Notes
 
A preliminary account of part of this work was presented at the minisymposia during the 93rd and 94th Annual Meetings of the American Association for Cancer Research held in San Francisco (April 6–10, 2002) and Washington DC (July 11–14, 2003), respectively.


    Acknowledgments
 
This study was supported by Grant (02-PJ2-PG3-20802-0003) from the Ministry of Health and Welfare, Republic of Korea.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 23, 2003; revised December 23, 2003; accepted December 30, 2003.