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© 2006 American Society for Nutrition J. Nutr. 136:1150-1155, May 2006

Biochemical, Molecular, and Genetic Mechanisms

Cocoa Polyphenols Inhibit Phorbol Ester-Induced Superoxide Anion Formation in Cultured HL-60 Cells and Expression of Cyclooxygenase-2 and Activation of NF-{kappa}B and MAPKs in Mouse Skin In Vivo1,2

Ki Won Lee*,3, Joydeb Kumar Kundu{dagger}, Sue Ok Kim{dagger}, Kyung-Soo Chun{dagger}, Hyong Joo Lee*,4 and Young-Joon Surh{dagger},4

* Department of Food Science and Technology, School of Agricultural Biotechnology, and {dagger} College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea

4 To whom correspondence should be addressed. E-mail: surh{at}plaza.snu.ac.kr; leehyjo{at}snu.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We investigated the antioxidant and antiinflammatory activities of a flavonoid-rich polyphenolic fraction of cocoa. Cocoa polyphenol (CP) was fractionated from commercial cocoa powder and contained 468 mg/g of gallic acid–equivalent phenolics and 413 mg/g epicatechin-equivalent flavonoids. CP exhibited a dose-dependent free radical–scavenging activity as determined by both 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2'-diphenyl-1-picrylhydrazyl radical scavenging assays. CP also dose-dependently inhibited xanthine oxidase activity and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide-anion generation in cultured human promyeolcytic leukemia HL-60 cells. Oral administering of CP (4, 20, 40, and 200 mg/kg body weight) to ICR mice 1 h prior to TPA (10 nmol) inhibited ear edema at 5 h in a dose-dependent manner. The levels of COX-2 expression induced in mouse skin after 4-h treatment with topical TPA (10 nmol) was also diminished significantly by pretreating CP (40 or 200 mg/kg) for 30 min. CP at the same doses inhibited TPA-induced nuclear translocation of p65 and subsequent DNA binding of NF-{kappa}B at 1 h by blocking the degradation of I{kappa}B{alpha} in mouse skin. Moreover, phosphorylation of p38 mitogen-activated protein kinase in ICR mouse skin, measured 4 h after TPA treatment, was suppressed by oral pretreatment of CP (40 or 200 mg/kg). Although extracellular signal–regulated protein kinase 1/2 phosphorylation was unaffected, CP inhibited the catalytic activity of extracellular signal–regulated protein kinase 1/2 in TPA-stimulated mouse skin. Since cellular proinflammatory and prooxidant states are closely linked to tumor promotion, the antioxidant and antiinflammatory properties of CP may constitute the basis of possible antitumor promoting effects of this phytochemical.

KEY WORDS: • cocoa polyphenols • antiinflammation • cyclooxygenase-2 • nuclear factor-{kappa}B • mouse skin

In recent years, there has been substantial progress in identifying a variety of chemopreventive phytochemicals from our daily diet (1,2). However, little attention has been given to the chemopreventive potential of cocoa, which is the main ingredient of widely consumed chocolates and cocoa beverages (3). High concentrations of flavonoids, predominantly as flavonol oligomers of monomeric base units known as procyanidins, are present in cocoa (4). Flavonols and procyanidins of cocoa have been shown to inhibit the growth and biosynthesis of polyamines in human colon cancer (Caco-2) cells (5). Our recent study has revealed that cocoa contains more phenolic phytochemicals and a higher antioxidant capacity than tea and red wine (6). Consuming chocolate has been reported to increase the total antioxidant capacity of human blood plasma in vivo (7). Previous studies indicate that cocoa powder extract and polyphenols prolong the lag time of LDL oxidation (8,9). Cocoa flavonoids inhibit both the dioxygenase and 5,6-leukotriene A4 synthase activities of human 5-lipoxygenase (10). Moreover, orally administered cocoa powder or cocoa liquor inhibits chemically induced carcinogenesis in experimental animals (11,12).

Oxidative stress and inflammation are implicated in multistage carcinogenesis (13–15). Reactive oxygen species (ROS)5, produced as typical by-products of eicosanoid metabolism during the inflammatory tissue damage, can alter the course of normal biochemical processes, leading to preneoplastic transformation of cells (16,17). The generation of the superoxide anion is elevated during oxidative burst or inflammation. Xanthine oxidase activity is elevated during the promotional phase of tumorigenesis (18). Tumor promoters, in particular, phorbol ester, generate superoxide anion radicals in epithelial cells and leukocytes. One of the key enzymes that mediates inflammatory response is cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis. Evidence suggests that inappropriate induction of COX-2 plays a pivotal role in tumor promotion and progression (19–21). Therefore, antioxidant and antiinflammatory phytochemicals that target COX-2 are potential antitumor promoting agents.

The redox-sensitive eukaryotic transcription factor, nuclear factor-{kappa}B (NF-{kappa}B), has been known to regulate COX-2 expression and is a critical target for chemoprevention with antiinflammatory substances (22,23). In response to oxidative and proinflammatory stimuli, NF-{kappa}B is activated, at least in part, by a series of upstream kinases, including those belonging to the mitogen-activated protein kinase (MAPK) family. The activated form of MAPK-family proteins, such as extracellular signal–regulated protein kinase (ERK), c-Jun NH2-terminal kinase, and p38 MAPK, can phosphorylate and activate transcription factors, thereby altering the expression of COX-2 (15,24). A typical tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has been shown to be a potent stimulator of COX-2 expression in various cell lines as well as in mouse skin in vivo (25,26). Previous studies from this laboratory revealed that topical application of TPA on mouse skin resulted in the activation of MAPK and aforementioned transcription factors that regulate COX-2 expression (22,26). In the present work, we attempted to evaluate the antioxidant and antiinflammatory activities of a flavonoid-rich polyphenolic fraction, prepared from commercially available cocoa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Chemicals. Gallic acid, epicatechin, vitamin C, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as diammonium salt, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Folin and Ciocalteu's phenol reagent were obtained from Sigma Chemical. Trolox was purchased from Aldrich Chemical. 2,2'-Azobis(2-amidinopropane)dihydrochloride was obtained from Wako Chemicals. 12-O-Tetradecanoylphorbol-13-acetate was obtained from Alexis Biochemicals. All other chemicals used were in the purest form available commercially.

    Sample preparation. Commercial cocoa powder (50 g) was extracted with 500 mL of 50% (v:v) aqueous ethanol under reflux for 6 h. After extracting, the solution was filtered off to collect the extract, with this process repeated twice. The collected cocoa extract was loaded onto a styrene-based adsorption resin column (60 mm x 450 mm, HP-20, Mitsubishi), washed with 20% (v:v) aqueous ethanol, and then eluted with 60% (v:v) aqueous ethanol. The eluted cocoa polyphenol fraction was concentrated at 40 or 50°C under reduced pressure and used to determine antioxidant and antiinflammatory activity and the polyphenol content.

    Measurement of the total phenolic content. The total phenolic content of cocoa polyphenol (CP) was measured by following Folin-Ciocalteu's method. The assay was replaced 5 times. The total phenolic content in CP was determined as mg/g of gallic acid equivalent.

    Determination of the total flavonoid content. The total flavonoid content was measured by a colorimetric assay method developed by Zhishen et al. (27). The assay was repeated 5 times. The total flavonoid content is expressed as mg/g of epicatechin equivalent.

    Assessment of ABTS radical-scavenging activity. The method developed by van den Berg et al. (28,29) was used with slight modification for assessing the ABTS radical-scavenging activity of CP as described previously.

    Assessment of DPPH radical-scavenging activity. The DPPH radical-scavenging activity of CP was measured by the method described by Brand-Williams et al. (28,30) with minor modifications as described previously.

    Measurement of hypoxanthine/xanthine oxidase activity. The method developed by Gotoh and Niki (31) was used with a slight modification. Briefly, samples were added at different concentrations to the reaction solution containing 100 µL of 30 mmol/L EDTA (pH 7.4), 10 µL of 30 mmol/L hypoxanthine in 50 mmol/L NaOH, and 200 µL of 1.42 mmol/L nitroblue tetrazolium. After a 3-min reaction, 100 µL of 0.5 kU/L xanthine oxidase was added to the mixture and the volume was adjusted to 3 mL with 50 mmol/L phosphate buffer (pH 7.4) and incubated at room temperature for 20 min. Absorbance was then measured at 560 nm relative to that of a prepared blank.

    Measurement of superoxide-anion radical-scavenging activity. Inhibitory tests of TPA-induced generation of superoxide anion radical in dimethylsulfoxide (DMSO)-differentiated HL-60 human promyelocytic leukemia (HL-60) cells were done as previously reported (32). HL-60 human promyelocytic leukemia cells were obtained from the Korea Cell Line Bank.

    Animals. Female ICR mice (6–7 wk of age; mean body weight, 25 g), supplied by the Dae-Han Experimental Animal Center, were housed in a climate-controlled environment (24 ± 1°C at 50% relative humidity) with a 12-h light/12-h dark cycle. The experimental protocols were approved by the Animal Care and Use Committee of the Seoul National University.

    Measurement of mouse ear edema. Mouse ear edema was performed as described previously (33). Briefly, groups of 6 female ICR mice were treated with CP dissolved in 0.5% sodium carboxymethylcellulose by gavage at doses of 4, 20, 40, and 200 mg/kg body weight 1 h prior to topical application of TPA (10 nmol). The mice were killed 5 h later, and ear punches were prepared. The increase in the weight of ear punch from TPA-treated mice compared to that of control is indicative of inflammation.

    Western blot analysis. The female ICR mice (6–8 wk of age) received CP (40 or 200 mg/kg body weight) by gastric intubation 30 min before topical application 10 nmol TPA. Animals were killed by cervical dislocation 1 h (I{kappa}B{alpha} and p65) or 4 h (COX-2 and MAPKs) later. The protein extracts from the dorsal skin were prepared as described previously (34). After quantification of protein concentration, an aliquot of whole cell lysate containing 30 µg of protein was boiled in a 5x sodium dodecylsulfate (SDS) sample loading buffer for 5 min before electrophoresis on a 12% SDS-polyacrylamide gel. Proteins in the SDS-polyacrylamide gel were transferred to a PVDF membrane (Gelman Laboratory) and the blots were blocked with 5% nonfat dry milk PBST buffer (phosphate buffered saline containing 0.1% Tween-20) for 1 h at room temperature. The membranes were incubated for 2 h with 1:4000 dilutions of actin (Sigma Chemical) and ERK; 12 h with a 1:500 dilution of p65 (Santa Cruz Biotechnology); and 12 h with 1:1000 dilutions of COX-2 (Cayman Chemical), phospho-ERK, and phospho-p38 antibodies (Santa Cruz Biotechnology). Equal lane loading was assessed using actin (Sigma Chemical). The blots were washed 3 times with PBST buffer for 5 min each. Washed blots were incubated with a 1:5000 dilution of the horseradish peroxidase–conjugated secondary anti-rabbit or anti-mouse antibodies (Zymed Laboratories) and then washed again 3 times with PBST buffer. Levels of expression for a specific protein were visualized by treating the blots with an ECL detection kit (Amersham Pharmacia Biotech) and exposing them to X-ray film.

    Electrophoretic mobility shift assay. The cytosolic and nuclear extracts from mouse skin were prepared as described previously (34). Electrophoretic mobility shift assay (EMSA) was performed using a DNA protein–binding detection kit (Gibco BRL) according to the manufacturer's instructions as described previously (33). 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 performed in 25 µL of a solution containing 10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 1 mmol/L DTT, 1 mmol/L EDTA, 4% (v:v) glycerol, 0.1 g/L sonicated salmon-sperm DNA, 10 µg of nuclear extracts, and the labeled probe at 1.665 GBq. After 50 min of incubation at room temperature, 2 µL of 0.1% bromophenol blue was added, and the samples were electrophoresed through a 6% nondenaturing polyacrylamide gel at 150 V in a cold room for 2 h. Finally, the gel was dried and exposed to X-ray film.

    ERK activity assay (nonradioactive). An in vitro kinase assay for determining the catalytic activity of ERK was performed, using a nonradioative p44/p42 kinase assay kit (Cell Signaling Technology) according to the protocol provided by the manufacturer. Collected tissues were lysed in 500 mL of lysis buffer per sample (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L glycerolphosphate, 1 mmol/L Na3VO4, and 1 g/mL leupeptin). Tissue lysates were centrifuged (14,800 x g; 15 min), and the supernatant was incubated with specific immobilized phospho-ERK monoclonal antibodies with gentle rocking overnight at 4°C. The beads were washed twice each with 500 µL of lysis buffer and the same volume of kinase buffer (25 mmol/L Tris-HCl, pH 7.5, 5 mmol/L glycerolphosphate, 2 mmol/L DTT, 0.1 mmol/L Na3VO4, and 10 mmol/L MgCl2). The kinase reaction was performed in the presence of 100 mmol/L ATP and 2 µg of Elk-1 (an ERK substrate) fusion protein at 30°C for 30 min. The phosphorylation of Elk-1 was measured by immunoblotting with a specific antibody detecting phosphorylation of Elk-1 at serine 383.

    Statistical analysis. Values are means ± SEM and data was analyzed using 1-way ANOVA followed by Duncan's multiple range test. Differences were considered significant at P < 0.01.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Antioxidant activities of CP. CP extracted from commercial cocoa powder contained 468 mg/g gallic acid–equivalent phenolics and 413 mg/g epicatechin-equivalent flavonoids. Similar to our previous observations with pure phenolic phytochemicals (28), CP exhibited a dose-dependent free radical-scavenging activity as determined by both ABTS and DPPH radical-scavenging assays (Fig. 1A). The elevation of xanthine oxidase activity and generation of the superoxide anion during the promotional phase of tumorigenesis have been reported previously (18). CP dose-dependently inhibited xanthine oxidase activity (Fig. 1B). Treatment with CP also suppressed the TPA-induced generation of superoxide anion in cultured HL-60 cells (Fig. 1B) without affecting cell viability (data not shown).


Figure 1
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  		FIGURE 1  CP scavenges free radicals and inhibits xanthine oxidase activity and TPA-induced superoxide anion formation. The ABTS and DPPH radical scavenging activities of CP (A). Inhibitory activity of CP on xanthine oxidase activity and TPA-induced superoxide anion radical generation (B). Values are means ± SEM, n = 6. Means for a variable without a common letter differ, P < 0.01.

 
    CP inhibits TPA-induced ear edema and COX-2 expression in mouse skin. Intragastric administering of CP significantly decreased TPA-induced ear edema by 58 and 87% at doses of 40 and 200 mg/kg body weight, respectively (P < 0.01, Table 1). Oral administering of CP at 0.5 h prior to topical application of TPA on the shaved back of female ICR mice resulted in a dose-dependent reduction in TPA-induced COX-2 expression (Fig. 2).


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  		TABLE 1 Inhibitory effects of CP on TPA-induced mouse ear edema

 

Figure 2
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  		FIGURE 2  Inhibitory effect of CP on TPA-induced expression of COX-2 in mouse skin. Relative band intensities of COX-2 were determined in 3 independent experiments using an image analyzer and plotted as a percentage of the total forms of actin. Values are means ± SEM, (n = 3). Means for a variable without a common letter differ, P < 0.01.

 
    Inhibitory effects of CP on TPA-induced DNA binding activity of NF-{kappa}B, nuclear translocation of p65, and degradation of I{kappa}B{alpha}. Because NF-{kappa}B plays a major role in regulating the COX-2 gene expression, we examined the effects of CP on the activation of this transcription factor in mouse skin stimulated with TPA. Pretreatment with CP exhibited an inhibitory effect on NF-{kappa}B DNA binding (Fig. 3A) in TPA-treated mouse skin. To elucidate a possible mechanism underlying the inhibition of TPA-induced DNA binding of NF-{kappa}B by CP in mouse skin, we examined the effect of CP on the expression of I{kappa}B{alpha} and p65. Consistent with the inhibition of NF-{kappa}B DNA binding, pretreatment with CP inhibited TPA-induced nuclear localization of p65 (Fig. 3B) as well as degradation of I{kappa}B{alpha} (Fig. 3C).


Figure 3
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  		FIGURE 3  Inhibitory effect of CP on the activation of NF-{kappa}B in mouse skin treated with TPA. CP pretreatment inhibited the TPA-induced DNA binding activity of NF-{kappa}B in mouse skin (A). The EMSA was performed in duplicate with both experiments producing similar results. Lane 1, free probe alone (no nuclear extract); lane 2, acetone control; lane 3, TPA alone; lane 4, CP (40 mg/kg) plus TPA; and lane 4, CP (200 mg/kg) plus TPA. CP blocked TPA-induced nuclear translocation of p65 in mouse skin (B). Nuclear extracts were analyzed with Western blot to examine the p65 protein expression. Data represent 2 independent experiments exhibiting a similar trend. CP inhibited TPA-induced degradation of I{kappa}B{alpha} (C). Cytoplasmic extracts were assayed for I{kappa}B{alpha} expression by Western blot analysis. Data represent 2 independent experiments exhibiting a similar trend.

 
    Effects of CP on TPA-induced activation of ERK and p38 MAPK. ERK and p38 MAPK are involved in upregulation of COX-2 expression via NF-{kappa}B signaling in TPA-stimulated mouse skin (26,34), and we investigated whether CP could suppress the TPA-induced activation of these MAPKs through phosphorylation. Pretreatment with CP suppressed the TPA-stimulated phosphorylation of p38 MAPK (Fig. 4A). Although pretreatment with CP did not affect TPA-induced phosphorylation of ERK1/2 (Fig. 4B), CP attenuated the catalytic activity of ERK1/2 as revealed by a reduced expression of phosphorylated Elk-1, which is a substrate of ERK1/2 (Fig. 4C).


Figure 4
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  		FIGURE 4  Effects of CP on TPA-induced activation of MAPKs. CP inhibits TPA-induced phosphorylation of p38 (A) but not ERK1/2 (B) in mouse skin. Total protein was isolated from the dorsal skin and analyzed for ERK1/2, phospho-ERK1/2 (p-ERK1/2), p38, and phospho-p38 (p-p38) by immunoblotting. Relative band intensities were determined in 3 independent experiments, using an image analyzer. Values are means ± SEM (n = 3). Means for a variable without a common letter differ, P < 0.01. CP inhibits TPA-induced ERK1/2 kinase activity in mouse skin (C). Elk-1 phosphorylation as a measure of the ERK activity was determined by Western blot analysis, using phospho-Elk-1 antibody (pElk). Data represent 3 independent experiments exhibiting a similar trend.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Regular consumption of fruits and vegetables, particularly those rich in flavonoids, can reduce the risk of cancer (1,2). Cocoa extracted from the fruits of Theobroma cacao is one of the most widely consumed beverages. Flavonoids in cocoa are mostly procyanidin oligomers that exist as complex forms of flavan-3-ol monomer. Procyanidin oligomers derived from chocolate have been reported to be more effective than monomeric epicatechin (on a molar basis) in preventing oxidation and nitration reactions caused by peroxynitrite (4). Cocoa contains more phenolic phytochemicals and exhibits a higher antioxidant capacity than teas and red wine (6). Because cocoa extract inhibited chemically induced tumorigenesis in the mammary gland, pancreas (11), and lungs (12) in experimental animals, we investigated the antioxidant and antiinflammatory effects of CP in an initial attempt to assess the antitumor-promoting potential of this phytochemical. Our study showed that CP exhibited strong free radical–scavenging activity as determined by radical-scavenging assays using ABTS and DPPH.

It has been shown that ROS, originating from the superoxide anion through xanthine oxidase, can cause certain forms of cancer (18). Although the processes that produce skin inflammation by topical application of TPA during tumor promotion are not yet completely understood, it is known that TPA stimulates phospholipase A2 and increases the release of arachidonic acid and prostaglandins (35). Moreover, the ROS generated by oxidative stimuli, such as TPA, plays an important role in causing inflammation (15). It is possible that the activation of the arachidonate cascade and generation of free radicals after TPA exposure are interrelated. Several chemopreventive agents exhibited antioxidant activity through their ability to scavenge oxygen radicals and suppress generation of the superoxide-anion radical in cells (36,37). The inhibitory effects of CP on the generation of the superoxide anion in cultured HL-60 cells treated with TPA and those on xanthine oxidase activity suggest a potential role for CP in preventing tumor promotion.

Because inflammation is causally linked to tumor promotion (38), our study, showing the antiinflammatory effects of CP against TPA-induced mouse ear edema, suggests that CP may possess antitumor-promoting effects. Improper cellular signal transduction that leads to inappropriate upregulation of COX-2, the enzyme that catalyzes the production of inflammatory mediators such as prostaglandins, has been attributed to tumor promotion (2,15,24). In addition, overexpression of COX-2 in genetically engineered mice leads to enhanced tumorigenesis (19), whereas knocking out COX-2 results in reduced tumor formation and progression (20). Thus, COX-2 is recognized as a molecular target of many chemopreventive agents. COX-2, which is barely detected in resting cells, is elevated upon treating cells or tissues with diverse stimuli, including phorbol ester (24,39). Because most polyphenols are probably too hydrophilic to cross the gut wall by passive diffusion and the bioavailability of polyphenols differ greatly depending on factors such as chemical structure, conjugation pattern, enterohepatic recirculation etc. (40–42), CP was administered in the present study at a maximum dose of 200 mg/kg body weight by gavage. This study demonstrated that CP exhibited strong inhibitory activity in TPA-induced edema and COX-2 expression in mouse skin.

The 5'-flanking region of the cox-2 promoter contains consensus sequences for binding with several transcription factors, including NF-{kappa}B (23), which has been reported to regulate TPA-induced COX-2 expression in mouse skin (26). Enhanced DNA binding of NF-{kappa}B depends on the release of this transcription factor from its cytosolic repressor inhibitory protein I{kappa}B{alpha}, which undergoes extensive degradation upon exposure of cells or tissues to oxidative stimuli/tumor promoters. Our study demonstrates that CP strongly suppresses the activation of NF-{kappa}B in TPA-treated mouse skin. Moreover, CP suppresses TPA-induced degradation of I{kappa}B{alpha}, which may represent the molecular mechanism responsible for the inhibitory effect of CP on the activation of NF-{kappa}B.

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. The MAPK pathway is one of the most extensively investigated intracellular signaling cascades involved in proinflammatory responses. MAPKs regulate NF-{kappa}B activation via multiple mechanisms. Accumulating evidence suggests that enzymes of the MAPK family play a role in cox-2 gene expression (24). Although the MAPK-signaling pathways have been extensively investigated in cultured cell lines, much less is known about the specificity of MAPKs and the extent to which they are activated during tumor promotion in mouse skin in vivo. Our previous studies (22,34) demonstrate that treating the dorsal skin of female ICR mice with TPA significantly enhances both the catalytic activities and phosphorylation of p38 MAPK and ERK1/2. In the present study, we found that CP inhibited TPA-induced phosphorylation of p38 MAPK. Our results indicate that CP has no inhibitory effect on the phosphorylation of ERK1/2, but it elicits strong inhibitory effects on the catalytic activity of ERK1/2 when stimulated by TPA. A similar effect has also been observed with curcumin, a well-known chemopreventive phytochemical, which inhibits the kinase activity of ERK without affecting its phosphorylation state in TPA-stimulated mouse skin (26). However, the exact molecular mechanism underlying this phenomenon is not clear.

In conclusion, our study demonstrates that CP prepared from cocoa possesses free radical–scavenging, antioxidant, and antiinflammatory properties. As a mechanistic basis of its antiinflammatory effects, CP inhibits the induction of COX-2 expression, the activation of MAPKs, and NF-{kappa}B signaling in TPA-treated mouse skin, which indicates the role of CP as a potential cancer chemopreventive agent.


   FOOTNOTES
 
1 This work was supported by a research grant from the Korea Science and Engineering Foundation (KOSEF) for Biofoods Research Program, Ministry of Science and Technology, Ministry of Science and Technology, Republic of Korea. Back

2 The first two authors contributed equally to this work. Back

3 Present address: College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea. Back

5 Abbreviations used: ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); COX-2, cyclooxygenase-2; CP, cocoa polyphenol; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ERK, extracellular signal–regulated protein kinase; MAPK, mitogen-activated protein kinase; NF-{kappa}B, nuclear factor-kappa B; ROS, reactive oxygen species; SDS, sodium dodecylsulfate; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back

Manuscript received 12 September 2005. Initial review completed 15 October 2005. Revision accepted 17 January 2006.


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