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Supplement: International Research Conference on Food, Nutrition, and Cancer

Inhibition of Phorbol Ester–Induced COX-2 Expression by Epigallocatechin Gallate in Mouse Skin and Cultured Human Mammary Epithelial Cells^1,2

Joydeb Kumar Kundu^*,3, Hye-Kyung Na^*, Kyung-Soo Chun^*, Young-Kyung Kim, Sang Jun Lee, Sang Sup Lee^*, Ok-Sub Lee, Young-Chul Sim and Young-Joon Surh^*,4

^* College of Pharmacy, Seoul National University, Seoul 151-742, South Korea and Amore-Pacific Corporation R&D Center, Youngin-si 449-729, Gyonggi-do, South Korea

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

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Green tea polyphenols are reported to possess substantial antiinflammatory and chemopreventive properties. However, the molecular mechanism of chemopreventive activity of green tea polyphenols is not fully understood. An abnormally elevated level of cyclooxygenase-2 (COX-2) is implicated in the pathogenesis of carcinogenesis. In the present study, we found that pretreatment of the green tea extract enriched with catechin and epigallocatechin gallate (EGCG) by gavage inhibited COX-2 expression induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin. Similarly, EGCG downregulated COX-2 in TPA-stimulated human mammary epithelial cells (MCF-10A) in culture. To further elucidate the underlying mechanism of COX-2 inhibition by green tea extract and EGCG, we examined their effects on the activation of extracellular signal–regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), which are upstream enzymes known to regulate COX-2 expression in many cell types. Pretreatment with EGCG as well as green tea extract caused a decrease in the activation of ERK. In addition, EGCG inhibited the catalytic activity of ERK and p38 MAPK, suggesting that these signal-transducing enzymes could be potential targets for previously reported antitumor promoting activity of EGCG.

KEY WORDS: • chemoprevention • epigallocatechin gallate (EGCG) • cyclooxygenase-2 (COX-2) • mitogen-activated protein kinase (MAPK) • human mammary epithelial cell (MCF-10A) • mouse skin carcinogenesis

Cyclooxygenase (COX)⁵, the key enzyme that catalyzes the rate-limiting step in prostaglandin (PG) biosynthesis, exists in at least two isoforms, designated as COX-1 and COX-2. Although COX-1 is a housekeeping enzyme, being constitutively expressed in almost all mammalian tissues, COX-2 in contrast is barely detectable under normal physiological conditions. Like other early-response gene products, COX-2 can be induced rapidly and transiently by proinflammatory mediators and mitogenic stimuli including cytokines, endotoxins, growth factors, oncogenes, and phorbol ester. Several lines of compelling evidence from genetic and clinical studies indicate that improper upregulation of COX-2 is implicated in carcinogenesis, particularly in the promotion and progression stages (1). The elevated expression of COX-2 is reported in multiple malignancies, including those of esophagus, stomach, breast, pancreas, lung, colon, skin, urinary bladder, and prostate (2). Mice genetically engineered to overexpress COX-2 are found to be susceptible to tumorigenesis (3) and, conversely, knockout of COX-2 results in altered epidermal differentiation and reduced tumor formation and progression (4). Thus, COX-2 is recognized as a molecular target of many chemopreventive as well as antiinflammatory agents (5,6).

Numerous dietary phytochemicals are shown to downregulate COX-2 expression, whereby they exert chemopreventive activity (6). Of the chemopreventive phytochemicals of dietary origin, the green tea polyphenol epigallocatechin gallate (EGCG; structure shown in Fig. 1) is extensively investigated (7). EGCG is reported to possess antioxidant, antimutagenic, antiinflammatory, and antiangiogenic activities (8–13). Although the chemopreventive potential of EGCG is verified in laboratory as well as population-based studies, the molecular mechanism by which EGCG inhibits the carcinogenic process is not fully understood (14).

View larger version (14K): [in this window] [in a new window]   FIGURE 1  The chemical structure of epigallocatechin gallate (EGCG).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Chemicals

A green tea polyphenol fraction containing 80% catechin (>60% EGCG) was supplied from Amore-Pacific Corporation, R&D Center (Yongin-si, South Korea). EGCG was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan), and TPA was obtained from Alexis Biochemicals (San Diego, CA). Dulbecco's modified Eagle's medium/F-12 nutrient (DMEM/F12), L-glutamine, horse serum, and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY). Enhanced chemiluminescence detection reagent (ECL) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). All other chemicals used were in the purest form available commercially.

Animal treatment

Female ICR mice (6–7 wk of age, average body weight 25 g) were supplied from the Dae-Han Experimental Animal Center (Daejeon, South 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 animals in the resting phase of the hair cycle were used. EGCG was dissolved in 0.5% sodium carboxymethylcellulose to obtain a concentration of 5 mg/mL for gastric intubation. TPA (10 nmol) was dissolved in 200 µL of acetone for topical application. After food was withheld for 8 h, mice were given EGCG by gavage at a dose of either 20 or 50 mg/kg body weight 1 h before topical application of TPA to the shaved back.

MCF-10A cell culture

Human mammary epithelial cells (MCF-10A) were cultured in DMEM/F-12 medium supplemented with 5% heat-inactivated horse serum, 10 µg/mL insulin, 100 ng/mL cholera toxin, 0.5 µg/mL hydrocortisone, 20 ng/mL recombinant EGF, 2 mmol/L L-glutamine and 100 units/mL penicillin/streptomycin at 37°C in a 5% CO₂ /95% air atmosphere. EGCG and TPA were dissolved in dimethyl sulfoxide and were diluted to the desired concentrations with culture medium.

Western blot analysis

Animals were killed by cervical dislocation at the indicated times. For isolation of protein from mouse skin, the dorsal skin was excised, the fat was removed on ice, and the skin was immediately placed in liquid nitrogen and pulverized with a 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 mmol/L NaCl, 0.5% Triton X-100, 50 mmol/L Tris-HCl (pH 7.4), 20 mmol/L EGTA, 1 mmol/L dithiothreitol (DTT), 1 mmol/L Na₃VO₄, protease inhibitor cocktail tablet (Cell Signaling Technology, Inc., Beverly, MA)] for 10 min. Lysates were centrifuged at 14,800 x g for 15 min, and supernatant containing 30 µg protein was boiled in sodium dodecylsulfate (SDS) sample loading buffer for 10 min before electrophoresis on 12% SDS-polyacrylamide gel. After electrophoresis for 2 h, proteins in SDS-polyacrylamide gel were transferred to PVDF membrane (Gelman Laboratory, Ann Arber, MI), and the blots were blocked with 5% nonfat 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 with 1:500 dilution of COX-2 (Cayman product for mouse skin and Santa Cruz product for MCF-10A cells), and ERK polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), and 1:1000 dilution of phospho-ERK monoclonal antibodies (Santa Cruz Biotechnology). Equal lane loading was assessed using actin (Sigma Chemical Co., 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 peroxidase conjugated-secondary antibody (Zymed Laboratories, Inc., San Francisco, CA) and then washed again three times with PBST buffer. The transferred proteins were visualized with an ECL detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Preparation of nuclear extracts

The nuclear extract from mouse skin was prepared as described previously (16). Briefly, scraped dorsal skin was homogenized in 1 mL of ice-cold hypotonic buffer A [10 mmol/L HEPES, pH 7.8; 10 mmol/L KCl; 2 mmol/L MgCl₂; 1 mmol/L DTT; 0.1 mmol/L EDTA; 0.1 mmol/L phenylmethylsulfonylfluoride (PMSF)]. After 25 min incubation on ice, the nucleoprotein complexes were lysed with 75 µL of 10% Nonidet P-40 (NP-40) solution, followed by centrifugation for 2 min at 14,800 x g. The nuclei were washed once with 400 µL of buffer A plus 25 µL of 10% NP-40, centrifuged, resuspended in 250 µL of buffer C (50 mmol/L HEPES, pH 7.8; 50 mmol/L KCl; 300 mmol/L NaCl; 0.1 mmol/L EDTA; 1 mmol/L DTT; 0.1 mmol/L PMSF; and 10% glycerol), and centrifuged for 5 min at 14,800 x g. The supernatant containing nuclear protein was collected and stored at -70°C after determination of protein concentrations.

Electrophoretic mobility shift assay (EMSA)

EMSA was performed using a DNA-protein binding detection kit (Gibco BRL, Grand Island, NY) according to the manufacturer's protocol. Briefly, the oligonucleotides for AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA C-3') and CCAAT/enhancer binding protein-ß (C/EBPß) (5'-CAT GGG CTC TGA TTG GCT GCT TTG) were labeled with [-³²P]ATP by T4 polynucleotide kinase and purified on a Nick column (Amersham Pharmacia Biotech). The binding reaction was carried out in a total volume of 25 µL containing 10 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 mg/ml sonicated salmon sperm DNA, 10 µg of nuclear extracts, and 100,000 cpm of the labeled probe. After 50-min incubation at room temperature, 2 µL of 0.1% bromophenol blue was added, and samples were electrophoresed through a 6% nondenaturating polyacrylamide gel at 150 V for 2 h. Finally, the gel was dried and exposed to an X-ray film.

MAPK assay (nonradioactive)

Kinase assays for determining the catalytic activities of p38 and ERK were carried out by using a nonradioative MAPK assay kit (Cell Signaling Technology, Inc., Beverly, MA) as described by the protocol provided by the manufacturer. Collected tissues were lysed in 300 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; 1 g/mL leupeptin). The lysates were centrifuged, and the supernatant was incubated with specific immobilized phospho-p38 MAPK 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 mmol/mL Tris-HCl, pH 7.5; 5 mmol/L glycerolphosphate; 2 mmol/L DTT; 0.1 mmol/L Na₃VO₄; 10 mmol/L MgCl₂). The kinase reactions were carried out in the presence of 100 µmol/L ATP and 2 µg of activating transcription factor-2 (ATF-2; p38 substrate) or Elk-1 (ERK substrate) at 30°C for 30 min. Phosphorylation of ATF-2 and Elk-1 was selectively measured by immunoblotting with specific antibodies detecting phosphorylation of ATF-2 and Elk-1 at Thr-71 and Ser-383, respectively.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Inhibitory effects of the green tea extract and EGCG on TPA-induced COX-2 expression in mouse skin

TPA was shown to be a potent stimulator of COX-2 expression in various cell lines (17–19). Recently, Chun et al. (19) reported that topical application of TPA (10 nmol) can enhance both the level of COX-2 protein and its mRNA transcripts in mouse skin (19). Intragastric administration of the green tea extract at 1 h before topical application of TPA on the shaved back of female ICR mice resulted in a dose-related decrease in the levels of COX-2 protein 4 h after TPA administration (Fig. 2). Under the same experimental conditions, the expression of the housekeeping enzyme COX-1 was barely changed.

View larger version (56K): [in this window] [in a new window]   FIGURE 2  Inhibitory effects of green tea extract on phorbol ester–induced cyclooxygenase (COX)-2 expression and extracellular signal–regulated protein kinase (ERK)1/2 phosphorylation. Female ICR mice were given GTE at a dose of either 20 or 50 mg/kg body weight by gastric intubation. After 1 h, mice were treated topically with 0.2 mL acetone or 10 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA in 0.2 mL acetone), and killed 4 h later. Control animals were treated with acetone in lieu of TPA. Proteins were analyzed for COX-2, COX-1, ERK1/2, and pERK1/2 by immunoblotting. The immunoblot is representative of three independent experiments. GTE, green tea extract.

View larger version (48K): [in this window] [in a new window]   FIGURE 3  Effects of EGCG on TPA-induced COX-2 expression (A) and activation of MAPK (B). Mice were given indicated amount of EGCG by gavage. Control animal was given 0.5% sodium carboxymethylcellulose alone. After 1 h, animals were treated topically with 0.2 mL acetone or 10 nmol TPA. (A) Animals were killed 4 h after the TPA treatment. Protein was analyzed for COX-2 and COX-1 by immunoblotting. (B) Animals were killed 1 h after the TPA treatment. Total protein was isolated from the dorsal skin and quantified. Protein was analyzed for ERK1/2 and pERK1/2 by immunoblotting. For measurement of ERK1/2 and p38 kinase activity, cell lysates containing 200 µg protein for each kinase assay were treated with a specific immobilized phospho-ERK1/2 or phospho-p38 kinase monoclonal antibody, respectively. The resulting immunoprecipitate was then incubated with Elk-1 fusion protein for the ERK kinase assay and ATF-2 fusion protein for the p38 kinase assay in the presence of 100 mmol/L ATP. Elk-1 phosphorylation as a measure of the ERK activity and ATF-2 phosphorylation as a measure of the p38 MAPK activity were determined by a nonradioactive method using phospho-Elk-1 and phospho-ATF-2 antibody, respectively. Data represent two independent experiments that gave rise to a similar trend.

Because TPA-induced COX-2 expression is reported to be regulated, at least in part, by a series of upstream kinases collectively known as MAPKs (19), we determined the effect of the green tea extract and EGCG on TPA-induced activation of ERK, a representative MAPK involved in a wide array of cellular signaling cascades. ERK1/2 activation via phosphorylation was reported to peak at 1 h and was sustained up to 4 h after topical application of TPA in mouse skin (19). Pretreatment of green tea extract at 50 mg/kg body weight (per oral) suppressed TPA-induced ERK1/2 activation (Fig. 2). Likewise, EGCG pretreatment was found to inhibit ERK1/2 activation in TPA-treated mouse skin (Fig. 3B). Topical application of TPA in mouse skin was reported to stimulate the catalytic activities of ERK and p38 MAPK in parallel with their elevated phosphorylation (19). EGCG, at a dose that inhibited COX-2 induction, suppressed the TPA-induced enhancement of the catalytic activities of ERK and p38 MAPK as determined by the kinase assay (Fig. 3B).

Effects of EGCG on TPA-induced DNA binding of AP-1 and C/EBPß in mouse skin

Certain transcription factors including nuclear factor-B (NF-B), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein-ß (C/EBPß), independently or in combination, play a role in the induction of COX-2 (20–23). To investigate the possible inhibitory effect of EGCG on one or more of these transcription factors, the nuclear fractions from TPA-treated mouse skin with or without EGCG pretreatment were analyzed by electromobility gel shift assay. Because AP-1, a heterodimer of c-Jun and c-Fos, is a downstream target of c-Jun-NH₂-terminal kinase (JNK) and p38 MAPK, the inhibition of the catalytic activity of p38 MAPK by EGCG led us to investigate whether EGCG could suppress activation of this transcription factor in mouse skin stimulated with TPA. Contrary to our expectation, TPA-induced DNA binding of AP-1 was not affected by EGCG at a dose that decreased the catalytic activity of p38 (Fig. 4A). Similarly, EGCG failed to block the DNA binding of another transcription factor C/EBPß in TPA-treated mouse skin (Fig. 4B).

View larger version (84K): [in this window] [in a new window]   FIGURE 4  Effect of EGCG on the activation of activator protein-1 (AP-1) (A), and C/EBPß (B) in mouse skin treated with TPA. Female ICR mice were given EGCG at a dose of 50 mg/kg body weight by gastric intubation. Animals in the control and TPA-alone group were given 0.5% sodium carboxymethylcellulose. After 1 h, mice were treated topically with 0.2 mL acetone or 10 nmol TPA, and killed 1 h later. The epidermal nuclear extracts were prepared and incubated with the radiolabeled oligonucleotides containing either AP-1 or C/EBPß consensus sequence for analysis by electrophoretic mobility shift assay. Lane 1, free probe alone (no nuclear extract); Lane 2, acetone control; Lane 3, TPA alone; Lane 4, EGCG (50 mg/kg) plus TPA.

The induction of COX-2 by TPA was also verified in cultured human mammary epithelial cells. Thus, treatment of MCF-10A cells with 10 nmol/L TPA led to a dramatic increase in COX-2 expression in MCF-10A cells (Fig. 5). When MCF-10A cells were exposed to EGCG concurrently with TPA for 4 h, expression of COX-2 protein was inhibited in a concentration-dependent manner.

View larger version (34K): [in this window] [in a new window]   FIGURE 5  Inhibitory effects of EGCG on TPA-induced COX-2 expression in MCF-10A cells. (A) Time course of TPA-induced COX-2 expression in MCF-10A cells. MCF-10A cells were treated with dimethyl sulfoxide or 10 nmol/L TPA for 4, 6, and 8 h. Protein was analyzed for COX-2 expression by immunoblotting. (B) MCF-10A cells were treated with indicated concentrations of EGCG in the presence or absence of TPA (10 nmol/L) for 4 h. The Western blot represents three independent experiments.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The molecular signaling mechanisms involved in the induction of COX-2 in response to various external stimuli have not been fully clarified. One of the most extensively investigated intracellular signaling cascades involved in cellular proinflammatory responses involves the MAPK pathway. Accumulating evidence indicates that enzymes of the MAPK family play a role in regulating COX-2 expression. The Parke-Davis MEK inhibitor PD98059 partially blocked lipopolysaccharide-induced COX-2 expression in RAW 264.7 cells and also in lysophosphatidic acid–stimulated rat mesangial cells, which supports the association of ERK activation with PG production (37). Lipopolysaccharide-induced expression of COX-2 was blunted by SB203580, the p38 MAPK inhibitor, which resulted in decreased PGE₂ production in RAW 264.7 cells (38). Similar effects were observed in lipopolysaccharide-stimulated monocytes (39).

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 the tumor promotion in mouse skin in vivo. Topical application of TPA on the ears of CD1 mice and skin of SENCAR mice induced a rapid and sustained activation of ERK but not of p38 MAPK (40,41). According to a recent study from this laboratory, treatment of dorsal skin of female ICR mice with TPA significantly enhanced both catalytic activities and phosphorylation of p38 MAPK and ERK1/2 (19). Chun et al. (19) suggested that ERK may play a regulatory role in the signaling pathway mediating TPA-induced COX-2 expression in mouse skin. In the present study, intragastric administration of EGCG as well as green tea extract inhibited TPA-induced activation of ERK1/2 and the catalytic activity of both ERK1/2 and p38 MAPK suggesting that these kinases may be the molecular targets for the antitumor promoting activity of EGCG in mouse skin. Vayalil et al. (42) reported a similar inhibitory effect of EGCG on MAPK activation in SKH-1 hairless mouse skin treated with a single or multiple exposure to UVB in which topically applied EGCG prevented depletion of antioxidant enzymes and inhibited phosphorylation of ERK1/2, JNK, and p38 MAPK (42). Although EGCG inhibits activation of MAPKs induced by mitogens, UV, bacterial toxins, or cytokines, EGCG alone was also found to induce some of the aforementioned signaling molecules during the apoptotic process (43,44).

Regulatory elements on the murine cox-2 promoter harbors one site for cyclic AMP response element binding, two sites for C/EBPß, and one NF-B binding site (20–23). Another eukaryotic transcription factor, AP-1, is also involved in COX-2 induction in various cell lines (45–47). Because NF-B, AP-1, and C/EBPß are representative transcription factors known to play a role in regulating the expression of COX-2 and several members of the MAPK family activate these transcription factors, we investigated the effect of EGCG on TPA-induced activation of these transcription factors in mouse skin. We initially examined the effect of EGCG on AP-1 activation in TPA-treated mouse skin. Although EGCG has been reported to inhibit TPA-induced DNA binding activity of AP-1 in cultured keratinocytes and arsenite-induced activation of AP-1 in mouse JB6 cells (48,49), we found no inhibitory effect of EGCG on the DNA binding of AP-1 in TPA treated mouse skin in vivo. Therefore, it seems likely that EGCG exerts differential effects on activation of transcription factors and upstream signaling kinases depending on the cell or tissue type. EGCG also failed to block TPA-induced DNA binding of C/EBPß. Nomura et al. (50) reported that EGCG prevented TPA-induced DNA binding activity of NF-B by blocking phosphorylation of the inhibitory counterpart of nuclear factor-B (IB) in mouse JB6 cells (50). The effect of EGCG on NF-B activation by TPA in mouse skin is under investigation.

	FOOTNOTES

 
¹ Presented as part of a symposium, "International Research Conference on Food, Nutrition, and Cancer," given by the American Institute for Cancer Research and the World Cancer Research Fund International in Washington, D.C., July 17–18, 2003. This conference was supported by Balchem Corporation; BASF Aktiengesellschaft; California Dried Plum Board; The Campbell Soup Company; Danisco USA, Inc.; Hill's Pet Nutrition, Inc.; IP-6 International, Inc.; Mead Johnson Nutritionals; Roche Vitamins, Inc.; Ross Products Division; Abbot Laboratories; and The Solae Company. Guest editors for this symposium were Helen A. Norman and Ritva R. Butrum.

² This work was supported by the Biogreen Project, Republic of Korea.

³ Joydeb Kumar Kundu is supported by a doctoral scholarship for foreign scholars from Seoul National University.

⁵ Abbreviations used: AP-1, activator protein-1; COX, cyclooxygenase; DTT, dithiothreitol; ECL, enhanced chemiluminescence; EGCG, epigallocatechin gallate; ERK, extracellular signal–regulated protein kinase; EMSA, electrophoretic mobility shift assay; JNK, c-Jun-NH₂-terminal kinase; NF-B, nuclear factor-B; MAPK, mitogen-activated protein kinase; PBST, phosphate-buffered saline Tween; PG, prostaglandin; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecylsulfate, TPA, 12-O-tetradecanoylphorbol-13-acetate.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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