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Carcinogenesis Advance Access originally published online on August 31, 2006
Carcinogenesis 2007 28(2):363-371; doi:10.1093/carcin/bgl151
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

cis-9,trans-11-Conjugated linoleic acid down-regulates phorbol ester-induced NF-{kappa}B activation and subsequent COX-2 expression in hairless mouse skin by targeting I{kappa}B kinase and PI3K-Akt

Dal-Mi Hwang1, Joydeb Kumar Kundu1, Jun-Wan Shin1, Jung-Chul Lee1, Hyong Joo Lee2 and Young-Joon Surh1,*

1 National Research Laboratory of Molecular Carcinogenesis and Chemoprevention, College of Pharmacy Seoul 151-742, South Korea
2 Department of Food Science and Technology, College of Agriculture and Life Sciences, Seoul National University Seoul 151-742, South Korea

*To whom correspondence should be addressed at: College of Pharmacy, Seoul National University, Shillim-dong, Kwanak-ku, Seoul 151-742, Korea. Tel: +82 2 880 7845; Fax: +82 2 874 9775; Email: surh{at}plaza.snu.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Conjugated linoleic acid (CLA) has been reported to inhibit mouse skin carcinogenesis, particularly in the promotion stage, but underlying molecular mechanisms remain poorly understood. Since persistent induction of cyclooxygenase-2 (COX-2) is frequently implicated in carcinogenesis, we investigated the effect of cis-9,trans-11-CLA (9Z,11E-CLA) on the tumor promoter-induced COX-2 expression in HR-1 hairless mouse skin in vivo. Topical application of 9Z,11E-CLA caused significant inhibition of COX-2 expression at 6 h induced by 10 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) in HR-1 mouse skin. Since NF-{kappa}B is known to regulate COX-2 gene expression, we determined the effect of 9Z,11E-CLA on TPA-induced activation of this transcription factor. Treatment of mouse skin with 9Z,11E-CLA reduced TPA-induced DNA binding as well as nuclear translocation of NF-{kappa}B by blocking phosphorylation and subsequent degradation of I{kappa}B{alpha}. In addition, 9Z,11E-CLA attenuated TPA-induced phosphorylation of extracellular signal-regulated protein kinase, p38 mitogen-activated protein kinase and Akt. To further elucidate the molecular mechanism underlying the inactivation of NF-{kappa}B by 9Z,11E-CLA, we investigated its effect on TPA-induced activation of I{kappa}B kinase (IKK), an upstream kinase that regulates NF-{kappa}B via phosphorylation and degradation of I{kappa}B{alpha}. 9Z,11E-CLA treatment down-regulated phosphorylation and catalytic activities of IKK{alpha}/ß in TPA-treated mouse skin. Co-treatment of mouse skin with the IKKß-specific inhibitor SC-514 (1 µmol) attenuated TPA-induced activation of Akt and NF-{kappa}B, and also the expression of COX-2 in hairless mouse skin. Taken together, 9Z,11E-CLA inhibits NF-{kappa}B driven-COX-2 expression by blocking the IKK and PI3K-Akt signaling in TPA-treated hairless mouse skin in vivo, which may account for its previously reported anti-tumor promoting effects.

Abbreviations: CLA, Conjugated linoleic acid; COX-2, cyclooxygenase-2; TPA, 12-O-tetradecanoylphorbol-13-acetate; NF-{kappa}B, nuclear factor-kappaB; ERK, extracellular signal-regulated protein kinase; IKK, I{kappa}B kinase


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
A close association between inflammation and cancer has long been suspected (1). There is now growing evidence supporting that chronic inflammation may lead to malignancies of different organs including stomach, colon, breast, skin, prostate, pancreas, etc. (2–4). It has been estimated that ~15% of all cancers are somehow linked to inflammation (3). In response to diverse pro-inflammatory stimuli, such as cytokines, endotoxins, growth factors, and tumor promoters including phorbol ester, prostaglandins are produced in abundance through metabolic conversion of arachidonic acid by the enzyme cyclooxygenase-2 (COX-2), which is inappropriately upregulated in various premalignant and malignant tissues (5–7). Moreover, genetically engineered COX-2 overexpressing transgenic mice (8) are highly susceptible to spontaneous tumor formation, while COX-2 knock out animals (9) are less prone to experimentally induced tumorigenesis. Therefore, targeting COX-2 is a rational approach for cancer chemoprevention (10).

A wide array of cellular signaling components comprising proline-directed serine/threonine kinases and a panel of redox-regulated transcription factors are involved in the regulation of COX-2 expression in response to pro-inflammatory stimuli (11). It has been reported that topical application of the prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces COX-2 mRNA transcription and protein expression in mouse skin (12). The 5'-flanking region of cox-2 gene contains several binding motifs for different transcription factors (13) including the eukaryotic transcription factor nuclear factor-kappaB (NF-{kappa}B). NF-{kappa}B, predominantly as a heterodimer of p65 and p50, binds with the kappaB consensus sequence located in the cox-2 gene promoter, thereby regulating COX-2 protein expression (13). In an unstimulated state, NF-{kappa}B is retained in cytosol as an inactive complex with its inhibitory protein I{kappa}B{alpha}, which blocks the nuclear translocation of NF-{kappa}B (14). The phosphorylation of I{kappa}B{alpha} and its subsequent ubiquitination and proteasomal degradation upon inflammatory stimuli, stress or growth factors make NF-{kappa}B free to translocate to the nucleus and bind to {kappa}B regulatory elements (15,16).

The I{kappa}B{alpha} phosphorylation is triggered by the activation of I{kappa}B kinase (IKK) complex (17), which is composed of two catalytic subunits (i.e. IKK{alpha} and IKKß) and a regulatory subunit IKK{gamma}/NEMO (NF-{kappa}B essential modifier) (17). Recent studies provide genetic evidence that IKKß-dependent NF-{kappa}B activation creates an essential link between inflammation and cancer (17,18). Although the role of IKK{alpha} is still unclear, it has recently been reported that IKK{alpha} can directly phosphorylate the NF-{kappa}B subunit p65 at Ser 536, which is required for NF-{kappa}B activation (19). Besides IKKs, several other protein kinases are also reported to regulate NF-{kappa}B activation (20). Of these upstream kinases, the role of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated protein kinase (ERK) and p38 MAPK, in regulating NF-{kappa}B activation has been well-documented (12,20,21). Another serine/threonine kinase, Akt that promotes cell survival by preventing apoptosis (22), has also been reported to regulate COX-2 expression through the NF-{kappa}B/I{kappa}B pathway (23).

Conjugated linoleic acid (CLA) refers to a derivative of naturally occurring essential fatty acid and includes different isomers of linoleic acid (cis9, trans12-octadecadienoic acid). cis9,trans-11-CLA (9Z,11E-CLA) is a predominant isomer, while cis10,trans12-CLA represents only one of the minor isomers of CLA. Much attention has been given to CLA due to its health beneficial activities including anti-carcinogenic, anti-atherosclerotic, anti-diabetic and anti-obese effects (24). In a pioneering study, Ha and colleagues (25) demonstrated the inhibitory effect of CLA on chemically induced mouse skin carcinogenesis. Since then, there has been an increasing body of data on the anti-carcinogenic effects of CLA in various tumor models (26–29). Recently, it has been reported that 9Z,11E-CLA significantly inhibits mouse skin tumorigenesis (30), but underlying molecular mechanisms remain to be elucidated. In this study, we examined the effect of 9Z,11E-CLA on TPA-induced COX-2 expression in HR-1 hairless mouse skin in vivo, and explored possible underlying molecular mechanisms.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
9Z,11E-CLA and rabbit polyclonal COX-2 antibody were purchased from Cayman Chemical Co. (Ann Arbor, MI). TPA was obtained from Alexis Biochemicals (San Diego, CA). SC-514 was purchased from Calbiochem (San Diego, CA). LY294002 was procured from Tocris (Ellisville, MO). Primary antibodies for p65, p50, I{kappa}B{alpha}, p-IKK{alpha}/ß, IKK{alpha}, pERK, ERK, pp38 and p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and antiphospho-I{kappa}B{alpha}, antiphospho-p65-(Ser-536) and anti-IKKß were products of Cell Signaling Technology (Beverly, MA). Anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies were bought from Zymed Laboratories (San Francisco, CA). An oligonucleotide probe containing the NF-{kappa}B consensus sequence of the mouse COX-2 promoter region was purchased from Promega (Madison, WI). The enhanced chemiluminescence (ECL) detection kit and [{gamma}-32P]ATP were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK), and the BCA protein assay reagent was supplied from Pierce Biotechnology (Rockford, IL). All other chemicals used were in the purest form available commercially.

Animal treatment
The male HR-1 hairless mice (6–7 weeks of age) supplied from Sankyo Laboservice Corporation (SLC, Japan) were housed in climate-controlled quarters (24 ± 1°C at 50% humidity) with a 12 h light/12 h dark cycle. 9Z,11E-CLA and TPA were dissolved in 200 µl of acetone and applied topically to the dorsal skin area. All experiments in this study were performed using three mice in each treatment group.

Western blot analysis
The male HR-1 mice were topically exposed to TPA (10 nmol) on their backs with or without 9Z,11E-CLA (0.25 or 1.0 mg equivalent to 0.89 or 3.57 µmol, respectively) and killed by cervical dislocation at the indicated times. In other experiments, SC-514 (0.2 or 1 µmol) or LY294002 (0.5 or 5 µmol) was co-treated with TPA. Control animals were treated with acetone only. For isolation of protein from mouse skin, the dorsal skin was excised and the fat was removed on ice. The fat-free skin tissues were then 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 1 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 DTT, 1 mM Na3VO4, protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Mannheim, Germany)] for 10 min. Lysates were centrifuged at 14 800x g for 15 min. Supernatant was collected and total protein concentration was quantified by using the BSA protein assay kit. Cell lysate (30 µg) was boiled in SDS sample loading buffer for 5 min before electrophoresis on 12% SDS–polyacrylamide gel. After electrophoresis for 1.5 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 1 h at room temperature. The membranes were incubated for 4 h at room temperature with 1:1000 dilution of primary antibodies for COX-2, ERK, pERK and p38 and for 12 h at 4°C with 1:500 dilution of primary antibodies of pIKK{alpha}/ß, IKK{alpha}, IKKß, phospho-p65-(Ser-536) and phospho-p38. Equal lane loading was assessed using actin (Sigma–Aldrich, St Louis, MO). The blots were washed three times with PBST buffer at 5 min intervals followed by incubation with 1:5000 dilution of the HRP-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA) for 1 h and then washed again three times with PBST buffer. The transferred proteins were visualized with an ECL detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer's instructions.

Preparation of cytosolic and nuclear extracts from mouse skin
The nuclear extract from mouse skin was prepared as described previously (12). In brief, scraped dorsal skin 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, 70 µl of 10% Nonidet P-40 (NP-40) solution was added, and the mixture was then centrifuged for 2 min at 14 800x g. The supernatant was collected as cytosolic fraction. The precipitated 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 800x g. The supernatant containing nuclear proteins was collected and stored at –70°C after determination of the protein concentrations.

Electrophoretic mobility shift assay (EMSA)
EMSA for NF-{kappa}B DNA binding 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'-GAGGGGATTCCCTTA-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 reaction mixture containing 5 µg of incubation buffer [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 [{gamma}-32P]ATP-end labeled oligonucleotide. An 100-fold excess of unlabeled oligonucleotide (competitor) was added where necessary. After 50 min incubation at room temperature, 2 µl of 0.1% bromophenol blue was added, and samples were electrophoresed through 6% non-denaturating polyacrylamide gel at 150 V for 2 h. Finally, the gel was dried and exposed to an X-ray film.

In vitro kinase assay (radioactive)
The induction of I{kappa}B kinase{alpha}/ß (IKK{alpha}/ß) activity by topically applied phorbol ester in mouse skin and its modulation by 9Z,11E-CLA were examined by the IKK{alpha} and ß kinase assays. Whole cell extracts prepared from mouse skin were used to assay the IKK activity according to the protocol described by Bharti et al. (31). Briefly, animals were treated with TPA, with or without 9Z,11E-CLA, on dorsal skin and sacrificed at specified times. Cytosolic extracts (200 µg) were subjected to immunoprecipitation using an IKK{alpha} or IKKß antibody. The kinase reaction was carried out by the addition of the substrate protein (GST-I{kappa}B{alpha}, Santa Cruz Biotechnology) to the immunoprecipitate in the presence of 0.5 µCi [{gamma}-32P]ATP followed by incubation at 30°C for 30 min. The reaction was terminated by adding 2.5x SDS sample buffer, boiled at 99°C for 5 min, vortexed and centrifuged at 14 000x g for 2 min. Supernatant was subjected to SDS–PAGE, and the gel was stained with Coomassie brilliant blue and destained with destaining solution (glacial acetic acid:methanol:distilled water, 1:4:5, v/v). The destained gel was dried and exposed to X-ray film to determine the level of GST–pI{kappa}B{alpha} by autoradiography.

Statistical evaluation
Values were expressed as the mean ± SEM. of at least three independent experiments. Statistical significance was determined by Student's t-test, and a P-value of less than 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
9Z,11E-CLA inhibited TPA-induced COX-2 expression in hairless mouse skin
It has been reported that the prototype tumor promoter TPA is a potent inducer of COX-2 expression in various cell lines (32,33) and mouse skin in vivo (12). As shown in Figure 1a, topical application of TPA (10 nmol) onto dorsal skins of HR-1 hairless mice induced COX-2 expression maximally at 6 h. Topical application of 9Z,11E-CLA (0.25 or 1 mg) to mouse skin immediately after TPA treatment for 6 h diminished COX-2 expression in a dose-dependent manner (Figure 1b).


Figure 1
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  		Fig. 1 Inhibitory effects of 9Z,11E-CLA on TPA-induced COX-2 expression in mouse skin. (a) Dorsal skins of male HR-1 mice were treated topically with acetone alone or TPA (10 nmol in 0.2 ml acetone) for indicated time periods. Data are representative of two different sets of animals showing similar trend of COX-2 expression. (b) Mice were treated topically with TPA in the presence or absence of 9Z,11E-CLA (0.25 or 1 mg) dissolved in 0.2 ml acetone. Control animals were treated with acetone in lieu of TPA. Mice were sacrificed after 6 h of TPA treatment. Total cell lysates were analyzed for COX-2 expression by immunoblotting. Quantification of COX-2 immunoblot was normalized to that of actin followed by statistical analysis of relative image density. n = 3 per treatment group; *P < 0.001 (control versus TPA alone; TPA alone versus 9Z,11E-CLA 1·0 mg plus TPA); **P < 0.05 (9Z,11E-CLA 0.25 mg plus TPA versus TPA alone).

 
TPA-induced NF-{kappa}B activation was negated by 9Z,11E-CLA in mouse skin
The promoter region for cox-2 gene contains binding sites for various transcription factors including NF-{kappa}B (34,35), which regulates COX-2 expression in response to diverse stimuli. Treatment of dorsal skins of hairless mice with TPA (10 nmol) increased DNA binding of NF-{kappa}B maximally at 3 h, which was nullified by the addition of excessive amounts of unlabeled probe (Figure 2a). To determine the effects of 9Z,11E-CLA on TPA-induced NF-{kappa}B activation, nuclear extracts from TPA-treated mouse skin with or without 9Z,11E-CLA treatment, were analyzed by EMSA using the oligonucleotide harbouring the NF-{kappa}B consensus motif. As illustrated in Figure 2b, co-treatment of mouse skin with 9Z,11E-CLA suppressed TPA-induced NF-{kappa}B DNA binding in HR-1 hairless mouse skin. 9Z,11E-CLA also abrogated TPA-induced phosphorylation and subsequent degradation of I{kappa}B{alpha} (Figure 2c). Since the predominant form of NF-{kappa}B is a heterodimer of p65/RelA-p50 (14,36), we studied the effects of 9Z,11E-CLA on the nuclear translocation of p65 and p50. Our study revealed that the TPA-induced nuclear translocation of p65/RelA and p50 was inhibited by topical application of 9Z,11E-CLA (Figure 2d). The transcriptional activation of NF-{kappa}B is regulated by the phosphorylation of its functionally active subunit p65/RelA at serine 536 residue in its transcriptional activation domain (TAD) (37). Topically applied 9Z,11E-CLA inhibited phosphorylation of p65/RelA at serine 536 induced by TPA (Figure 2e).


Figure 2
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  		Fig. 2 The inhibitory effect of 9Z,11E-CLA on TPA-induced NF-{kappa}B activation in hairless mouse skin in vivo. (a) Male HR-1 mice were treated topically with 10 nmol TPA for different time periods. Epidermal nuclear extract (10 µg) was probed with radiolabeled NF-{kappa}B oligonucleotide. Lane 1, free probe alone (no nuclear extracts); lane 2, acetone control; lanes 3–7, TPA alone for 1, 3, 6, 12 and 24 h, respectively; lane 8, TPA-treated sample plus excess unlabeled oligonucleotide. Data are representative of three different sets of animals showing similar trend of NF-{kappa}B DNA binding. (b–e) Inhibitory effects of 9Z,11E-CLA on TPA-induced NF-{kappa}B activation. Dorsal skin of male HR-1 mice was co-treated with either 9Z,11E-CLA (0.25 or 1 mg) or acetone only. After 3 h, animals were sacrificed, and epidermal cytosolic and nuclear extracts were prepared. (b) The nuclear extract (10 µg) was subjected to evaluate the effect of 9Z,11E-CLA on TPA-induced NF-{kappa}B DNA binding. (c) The cytoplasmic fraction from mice treated with acetone, TPA alone, and 9Z,11E-CLA (0.25 or 1 mg) plus TPA were subjected to western blot analysis to examine the expression of pI{kappa}B{alpha} (n = 3 per treatment group; aP < 0.005, control versus TPA; bP < 0.001, 9Z,11E-CLA plus TPA versus TPA only) and I{kappa}B{alpha} (n = 3 per treatment group; cP < 0.005, control versus TPA; dP < 0.05, 9Z,11E-CLA plus TPA versus TPA only) using specific antibodies. (d) Nuclear protein (50 µg) was separated by SDS–PAGE, and immunoblot was performed by using primary antibodies specific to detect p65 and p50. Quantification of p65 immunoblot was normalized to that of actin followed by statistical analysis of relative image density. n = 3 per treatment group; *P < 0.001 (control versus TPA alone), **P < 0.05 (9Z,11E-CLA plus TPA versus TPA alone). (e) The effect of 9Z,11E-CLA on TPA-induced phosphorylation of p65. Whole epidermal tissue lysates prepared from mouse skin treated with TPA in the presence or absence of 9Z,11E-CLA were subjected to western blot analysis and immunobloted by using a specific antibody to detect p65 phosphorylated at serine 536 residue. Data are representative of three different set of animals giving similar trend.

 
9Z, 11E-CLA suppressed phosphorylation and catalytic activity of IKK in TPA-treated mouse skin
Since the activation of IKK depends on the phosphorylation of its subunit IKKß at a specific serine residue in its activation loop (38,39), we examined the effect of 9Z,11E-CLA on TPA-induced IKK phosphorylation. Topical application of TPA increased the phosphorylation of IKK{alpha}/ß at Ser 181, which was abolished by 9Z,11E-CLA treatment (Figure 3a). We also performed an in vitro radioactive kinase assay to measure the catalytic activities of IKK{alpha} and IKKß. As shown in Figure 3b, TPA application led to a significant induction of IKK{alpha} activity in mouse skin in vivo, which was suppressed by 9Z,11E-CLA. Likewise, increased activation of IKKß by TPA was reduced by 9Z,11E-CLA treatment (Figure 3c).


Figure 3
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  		Fig. 3 Effect of topically applied 9Z,11E-CLA on TPA-induced activation of IKK in hairless mouse skin. Dorsal skins of mice were treated with TPA (10 nmol) with or without 9Z,11E-CLA (0.25 or 1 mg). Control animals were treated with acetone only. Animals were killed 3 h after TPA treatment. Whole epidermal tissue lysates were subjected to western blot analysis and in vitro kinase assay. (a) Inhibitory effect of 9Z,11E-CLA on TPA-induced phosphorylation of IKK{alpha}/ß (serine 181). Quantification of pIKK{alpha}/ß immunoblot was normalized to that of actin. n = 3 per treatment group; *P < 0.032 (control versus TPA alone), **P < 0.05 (TPA alone versus 9Z,11E-CLA plus TPA). (b and c) Effect of 9Z,11E-CLA on TPA-induced IKK{alpha}/ß activity in mouse skin. Whole tissue extracts prepared from mouse skin treated with TPA for 3 h with or without 9Z,11E-CLA were immunoprecipitated with primary antibodies specific to IKK{alpha} (b) or IKKß (c), and immunoprecipitate was reacted with GST-I{kappa}B{alpha} substrate protein in the presence of [{gamma}-32P]ATP as described in Materials and methods. Autoradiogram is a representative of triplicate set experiments.

 
IKKß regulates TPA-induced COX-2 expression and NF-{kappa}B DNA binding in hairless mouse skin in vivo
As mentioned above, release of NF-{kappa}B from its inhibitory protein I{kappa}B{alpha} is essential for the activation of NF-{kappa}B. Phosphorylation of I{kappa}B{alpha} is a precedent step for its degradation, which is mainly dependent on the activation of the upstream kinase IKKß (17). To investigate the role of IKKß in TPA-induced COX-2 expression in mouse skin, we utilized the IKKß selective inhibitor SC-514 (0.2 or 1 µmol). Co-treatment of mouse skin with SC-514 and TPA abolished the NF-{kappa}B DNA binding (Figure 4a) as well as COX-2 expression (Figure 4b).


Figure 4
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  		Fig. 4 Effects of SC-514, a specific inhibitor of IKKß, on TPA-induced DNA binding of NF-{kappa}B and expression of COX-2. Dorsal skins of male HR-1 mice were treated topically with SC-514 (0, 0.2 or 1 µmol) dissolved in 0.2 ml acetone before TPA treatment. Control animals were treated only with acetone. (a) Mice were sacrificed 3 h after TPA treatment and 10 µg of protein from nuclear extract was incubated with the radiolabeled oligonucleotides containing the NF-{kappa}B consensus sequence for analysis by EMSA. (b) Mice were killed 6 h after TPA application, and whole lysate (30 µg) was analyzed for COX-2 expression by immunobloting. Quantification of COX-2 immunoblot was normalized to that of actin. n = 3 per treatment group; *P < 0.012 (SC-514 plus TPA versus TPA alone). ND, not detectable.

 
9Z, 11E-CLA inhibited activation of ERK and p38 MAPK in TPA-stimulated mouse skin
MAPKs have been reported as important regulators of NF-{kappa}B activation and subsequent COX-2 expression in mouse skin treated with TPA (12,21). We observed that topical application of TPA induced activation of ERK and p38 MAPK via phosphorylation which peaked at 3 h in HR-1 hairless mouse skin (data not shown). Western blot analysis revealed that 9Z,11E-CLA repressed TPA-induced phosphorylation of ERK and p38 MAPK in a dose-dependent manner (Figure 5).


Figure 5
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  		Fig. 5 Effects of 9Z,11E-CLA on TPA-induced phosphorylation of ERK and p38 MAPK in mouse skin. Male HR-1 hairless mice were treated with TPA in the presence or absence of 9Z,11E-CLA (0.25 or 1 mg) for 3 h. Total tissue lysates were separated by 12% SDS–polyacrylamide gel, transferred to PVDF membrane, and immunoblotted by using primary antibodies specific to detect ERK, p38, phospho-ERK and phospho-p38. The immunoblot is representative of three independent experiments eliciting a similar pattern, and the intensity of phospho-p38 was normalized to p38 followed by statistical analysis in comparison to control. n = 3 per treatment group; *P < 0.05 (TPA alone in comparison to control, and TPA alone versus 9Z,11E-CLA plus TPA).

 
Akt appears to be a potential target of 9Z,11E-CLA in suppressing TPA-induced activation of COX-2 and NF-{kappa}B in mouse skin
Phosphoinositide 3-kinase (PI3K) and its downstream target Akt/protein kinase B (PKB) are known to regulate NF-{kappa}B activation and COX-2 expression (40,41). This prompted us to examine the role of Akt/PKB in mediating the inhibitory effects of 9Z,11E-CLA on TPA-induced NF-{kappa}B activation and COX-2 expression in mouse skin. Topical application of 9Z,11E-CLA inhibited TPA-induced phosphorylation of Akt (Figure 6a). The TPA-induced Akt phosphorylation was nullified by topical application of a specific PI3K inhibitor, LY294002 in HR-1 hairless mouse skin (Figure 6b). LY294002, at pharmacologically effective doses, repressed TPA-induced NF-{kappa}B DNA binding (Figure 6c) and COX-2 expression (Figure 6d). These findings suggest that the inhibitory effect of 9Z,11E-CLA on COX-2 expression in TPA-treated mouse skin may be mediated via blockage of the PI3K-Akt signaling.


Figure 6
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  		Fig. 6 Inhibitory effects of 9Z,11E-CLA on TPA-induced phosphorylation of Akt and that of LY294002 on TPA-induced COX-2 expression and NF-{kappa}B activation in hairless mouse skin in vivo. (a) Dorsal skins of hairless mice were treated topically with either acetone or 9Z,11E-CLA (0.25 or 1 mg) followed by TPA treatment. Control animals were treated with acetone in lieu of TPA. The expression of phospho-Akt was measured by western blot analysis of whole tissue lysates, and immunoblots were quantified and adjusted to Akt by statistical analysis of densitometric data. n = 3 per treatment group; *P < 0.05 (control versus TPA alone), **P < 0.01 (TPA alone versus 9Z,11E-CLA plus TPA). (b) Dorsal skins of hairless mice were treated with LY294002 (0.5 or 5 µmol) immediately before TPA (10 nmol) treatment. Animals were killed at 3 h after TPA treatment and total protein was analyzed for pAkt expression by immunoblotting. (c) Animals were treated with LY294002 as indicated in Figure 6b. Nuclear extracts (10 µg) were incubated with radiolabeled oligonucleotides containing the NF-{kappa}B consensus sequence for analysis by the EMSA. Blots are representative of three independent experiments. (d) Animals were treated with LY294002 as indicated in Figure 6b, and sacrificed after 6 h. Whole tissue lysates were subjected to western blot analysis for measuring the expression of COX-2.

 
IKKß regulates TPA-induced Akt activation in hairless mouse skin
According to an earlier study, Akt can activate IKK{alpha} through phosphorylation (36), but a direct relationship between IKKß and Akt has yet to be established. Since 9Z,11E-CLA inhibited TPA-induced COX-2 expression by blocking activation of IKKß and Akt, we attempted to link these two upstream kinases. As illustrated in Figure 7, co-treatment of mouse skin with the IKKß inhibitor SC-514 attenuated TPA-induced phosphorylation of Akt (Figure 7), suggesting that IKKß regulates the activation of Akt in TPA-stimulated hairless mouse skin in vivo.


Figure 7
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  		Fig. 7 Effect of SC-514 on TPA-induced phosphorylation of Akt in hairless mouse skin. Dorsal skins of hairless mice were treated topically with either acetone or SC-514 (0.2 or 1 µmol) immediately followed by TPA treatment for 3 h. Control animals were treated with acetone in lieu of TPA. The expression of actin and phospho-Akt was measured by western blot analysis of whole tissue lysates, and the immunoblot was quantified and adjusted with actin by statistical analysis in comparison to control using a densitometer (n = 3 per treatment group; *P < 0.05).

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Chemoprevention is a promising strategy to reduce the ever-increasing incidence of cancer. The intervention of multistage carcinogenesis by modulating intracellular signaling pathways may provide molecular basis of chemoprevention with a wide variety of chemopreventive agents (42,43). CLA, a dietary polyunsaturated fatty acid composed of several positional or geometrical isomers of linoleic acid, exhibited anti-carcinogenic effects in experimentally induced tumor models including those of mammary, forestomach, colon, prostate and skin cancer (26–29). The most abundant and biologically active isomer of CLA is 9Z,11E-CLA, which has been shown to inhibit 7,12-dimethylbenz[a]anthracene-initiated and TPA-promoted mouse skin carcinogenesis (30). The present study was aimed at unraveling the molecular basis of the anti-tumor promoting effect of CLA in TPA-stimulated mouse skin.

Recent advances in our understanding of cellular and molecular events in the carcinogenic process have suggested that components of the intracellular signaling network would be potential targets for chemoprevention. Because of casual relationship between inflammation and cancer (1,44), COX-2, a key enzyme involved in inflammatory signaling, has been considered as one of the potential targets for chemoprevention (43). In response to prooxidative/pro-inflammatory stimuli, aberrant activation of intracellular signaling arrays that are mediated through diverse classes of kinases and transcription factors results in an inappropriate expression of COX-2 (11). Although many of the synthetic COX-2 inhibitors have been reported as potential chemopreventive agents, the USA Food and Drug Administration has recently warned the use of selective COX-2 inhibitors because of their cardiovascular risk (http://www.fda.gov/cder/drug/infopage/COX2/default.htm). However, the preclinical and clinical evidence suggesting the efficacy of COX-2 inhibitors in preventing cancer led the scientists to continue cancer prevention research with COX-2 inhibitors (45), especially with relatively safe anti-inflammatory phytochemicals from dietary sources. The inhibition of TPA-induced COX-2 expression by 9Z,11E-CLA in mouse skin may hence represent a rational strategy for skin cancer chemoprevention with this substance. Similar to our study, 9Z,11E-CLA was found to inhibit COX-2 protein expression in human prostate cancer (PC-3) cells (46). Several other studies also reported the inhibitory effect of 9Z,11E-CLA on COX-2 mRNA expression in TNF{alpha}-stimulated vascular smooth muscle cells (47), bacterial lipopolysaccharide (LPS)-treated Raw 264.7 murine macrophages, and TPA-stimulated human breast cancer (MCF-7) cells (48).

The induction of COX-2 in mouse skin is regulated by a series of transcription factors including NF-{kappa}B, which acts as a lynchpin in inflammation-associated carcinogenesis (2). Treatment of dorsal skins of hairless mice with 9Z,11E-CLA suppressed TPA-induced NF-{kappa}B activation by blocking phosphorylation of I{kappa}B{alpha} and subsequent nuclear translocation of p65/RelA and p50 as well as the phosphorylation of p65 at serine-536 residue. Consistent with our findings, 9Z,11E-CLA has been reported to suppress LPS-induced NF-{kappa}B activation in dendritic cells in vitro (49). Besides NF-{kappa}B, TPA-induced cox-2 gene transcription is also regulated by other transcription factors including activator protein-1 (AP-1). 9Z,11E-CLA suppressed AP-1 activation in TPA-stimulated MCF-7 human mammary cancer (48) and in HT29 colon cancer cells (50). The role of other transcription factors such as CREB and CCAAT/enhancer binding protein (CEBP) in COX-2 induction has also been reported (13). Although, TPA has been shown to activate CREB and CEBP in mouse skin (21,51), the effect of 9Z,11E-CLA on the activation of these transcription factors is yet to be established.

IKK has been reported to be responsible for the phosphorylation of I{kappa}B{alpha} and p65 (52,53). IKK consists of two catalytic subunits, IKK{alpha} and IKKß, and a regulatory subunit IKK{gamma}/NEMO. It is suggested that IKK{alpha} exhibits a catalytic activity of p65 phosphorylation, whereas IKKß is largely responsible for phosphorylation of both I{kappa}B{alpha} and p65 (54). In our study, topical application of the selective IKKß inhibitor, SC-514 blunted TPA-induced COX-2 expression and NF-{kappa}B activation in mouse skin. In addition, the activation of IKK depends on the phosphorylation of its IKKß subunit (38,39). DiDonato et al. (55) also demonstrated that cytokine-stimulated IKK activation was inhibited upon dephosphorylation with PP2A, lending further support to phosphorylation-dependent activation of IKK. Moreover, a study using TNF{alpha}-stimulated HeLa cells expressing site-specific modified ikk suggested that the activation of IKK complex occurred as a consequence of IKKß phosphorylation (38). Therefore, we attempted to examine the effect of 9Z,11E-CLA on TPA-induced IKK activation. Topical application of TPA resulted in increased phosphorylation of IKK (Ser181), which was decreased by 9Z,11E-CLA treatment. Moreover, co-treatment of 9Z,11E-CLA suppressed TPA-induced catalytic activity of IKKß.

Several lines of evidence suggest that ERK and p38 MAPK may regulate transcriptional activation of NF-{kappa}B (56,57). Topical application of 9Z,11E-CLA inhibited phorbol ester-induced activation of ERK and p38 MAPK, suggesting that the compound attenuated TPA-induced NF-{kappa}B activation possibly through inhibition of these upstream kinases, leading to eventual COX-2 suppression.

The serine/threonine kinase Akt is a mitogen-activated survival factor (22,58). It was reported that Akt regulated COX-2 expression in endometrial cancer cells, and inhibition of PI3K with wortmannin and LY294002 suppressed I{kappa}B{alpha} phosphorylation (23). In another study, apigenin inhibited TPA-induced COX-2 expression through Akt inhibition in keratinocytes (41). It has been suggested that the PI3K-Akt pathway contributes to activation of NF-{kappa}B through phosphorylation of I{kappa}B{alpha} (59). In addition, it has also been suggested that PI3K-Akt can phosphorylate IKK{alpha} and activate IKK complex, leading to NF-{kappa}B activation (60). Ozes and colleagues (36) have suggested that Akt constitutively interacts with and phosphorylates IKK{alpha}, and this association is not affected by TNF-{alpha} or wortmannin. Our current study revealed that Akt was activated through phosphorylation in TPA-stimulated HR-1 hairless mouse skin, and co-treatment of LY294002 diminished TPA-induced COX-2 expression and NF-{kappa}B DNA binding, suggesting a regulatory role of Akt in TPA-stimulated COX-2 expression in mouse skin. Therefore, it is plausible that 9Z,11E-CLA inhibits TPA-induced COX-2 expression, at least in part, by attenuating the activation of Akt. While a possible linkage between IKK{alpha} and Akt was reported (36), Tanaka et al. (39) have demonstrated that PDK1, an upstream kinase of Akt, mediates IKKß phosphorylation in HT1080 cells. In contrast, our present study reveals that co-treatment with the IKKß selective inhibitor SC-514 abolishes TPA-induced phosphorylation of Akt, indicating that IKKß is perhaps one of the upstream regulators of Akt activation in TPA-treated mouse skin in vivo (Figure 8).


Figure 8
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  		Fig. 8 A schematic representation of suppression of TPA-induced NF-{kappa}B activation and COX-2 expression in hairless mouse skin by 9Z,11E-CLA. Topical application of TPA activates IKKß, which, in turn, phosphorylates I{kappa}B{alpha} and Akt, thereby facilitates p65/p50 release and contributes to the activation of NF-{kappa}B and subsequent induction of COX-2. Each of these events can be blocked by 9Z, 11E-CLA. The inhibition of TPA-induced ERK and p38 MAPK phosphorylation by 9Z,11E-CLA may also contribute to inactivation of NF-{kappa}B and suppression of COX-2 expression.

 
In conclusion, 9Z,11E-CLA inhibited TPA-induced NF-{kappa}B activation and subsequent COX-2 expression by blocking the IKK and Akt signaling in hairless mouse skin in vivo, which provides a mechanistic basis of the previously reported anti-tumor promoting activity of 9Z,11E-CLA.


   Acknowledgments
 
This work was supported by the National Research Laboratory (NRL) Grant from the Ministry of Science and Technology, Republic of Korea.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 21, 2006; revised August 2, 2006; accepted August 16, 2006.


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