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The Induction of Prostaglandin E2 Production, Interleukin-6 Production, Cell Cycle Arrest, and Cytotoxicity in Primary Oral Keratinocytes and KB Cancer Cells by Areca Nut Ingredients Is Differentially Regulated by MEK/ERK Activation

2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês

10.1074/jbc.m404465200

1083-351X

Mei‐Chi Chang, Hui‐Lin Wu, Jang‐Jaer Lee, Po‐Hsuen Lee, Hsiao-Hwa Chang, Liang‐Jiunn Hahn, Bor‐Ru Lin, Yi‐Jane Chen, Jiiang‐Huei Jeng,

Phytochemical Studies and Bioactivities

There are about 200–600 million betel quid (BQ) chewers in the world. BQ chewing is one of the major risk factor of hepatocarcinoma, oropharyngeal, and esophagus cancers in Taiwan, India, and Southeast Asian countries. Thus, the precise molecular mechanisms deserve investigation. We used cultured primary keratinocytes and KB cells, RT-PCR, flow cytometry, Western blotting, and ELISA to evaluate whether alterations in early gene expression is crucial in the carcinogenic processes of BQ. We observed the induction of c-Fos mRNA expression in human gingival keratinocyte (GK) and KB carcinoma cells by areca nut (AN) extract and arecoline. A maximal increment in c-fos gene expression was shown at about 30 min after challenge. AN extract (100–800 μg/ml) and arecoline (0.1–0.8 mm) also stimulated ERK1/ERK2 phosphorylation with a maximal stimulation at 5–10 min of exposure. Pretreatment by U0126 (30 μm), a MEK inhibitor, markedly inhibited the c-Fos, cyclooxygenase-2 (COX-2), and IL-6 mRNA expression of the KB epithelial cells. In addition, U0126 and PD98059 (50 μm) also decreased AN extract- and arecoline-associated PGE2 and IL-6 production in GK and KB cells. However, U0126 by itself arrested the cells in G0/G1 phase, but was not able to prevent AN- and arecoline-induced cell death or apoptosis. In contrast, U0126 enhanced the AN-induced apoptosis of KB cells. AN ingredients thus play a significant role in the pathogenesis of oropharyngeal cancer by activation of MEK1/ERK/c-Fos pathway, which promotes keratinocyte inflammation, cell survival, and affects cell cycle progression. There are about 200–600 million betel quid (BQ) chewers in the world. BQ chewing is one of the major risk factor of hepatocarcinoma, oropharyngeal, and esophagus cancers in Taiwan, India, and Southeast Asian countries. Thus, the precise molecular mechanisms deserve investigation. We used cultured primary keratinocytes and KB cells, RT-PCR, flow cytometry, Western blotting, and ELISA to evaluate whether alterations in early gene expression is crucial in the carcinogenic processes of BQ. We observed the induction of c-Fos mRNA expression in human gingival keratinocyte (GK) and KB carcinoma cells by areca nut (AN) extract and arecoline. A maximal increment in c-fos gene expression was shown at about 30 min after challenge. AN extract (100–800 μg/ml) and arecoline (0.1–0.8 mm) also stimulated ERK1/ERK2 phosphorylation with a maximal stimulation at 5–10 min of exposure. Pretreatment by U0126 (30 μm), a MEK inhibitor, markedly inhibited the c-Fos, cyclooxygenase-2 (COX-2), and IL-6 mRNA expression of the KB epithelial cells. In addition, U0126 and PD98059 (50 μm) also decreased AN extract- and arecoline-associated PGE2 and IL-6 production in GK and KB cells. However, U0126 by itself arrested the cells in G0/G1 phase, but was not able to prevent AN- and arecoline-induced cell death or apoptosis. In contrast, U0126 enhanced the AN-induced apoptosis of KB cells. AN ingredients thus play a significant role in the pathogenesis of oropharyngeal cancer by activation of MEK1/ERK/c-Fos pathway, which promotes keratinocyte inflammation, cell survival, and affects cell cycle progression. Smoking, betel quid (BQ) 1The abbreviations used are: BQ, betel quid; AN, areca nut; COX-2, cyclooxygenase-2; DMEM, Dulbecco's modified Eagle's medium; GK, gingival keratinocyte; IL, interleukin; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PGE2, prostaglandin E2; RT, reverse transcriptase; ERK, extracellular signal-regulated kinase; TNF, tumor necrosis factor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; G3PDH, glyceraldehyde-3-phosphate dehydrogenase. chewing, and the consumption of alcohol, coffee, and tea are the five most popular oral habits in the world. There are about 200 to 600 million betel quid (BQ) chewers distributed across India, Sri Lanka, Pakistan, Taiwan, many other Southeast Asian countries, and South Africa (1RC IA IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. 37. International Agency for Research on Cancer, Lyon1985Google Scholar, 2Jeng J.H. Chang M.C. Hahn L.J. Oral Oncol. 2001; 37: 477-492Crossref PubMed Scopus (356) Google Scholar). In Taiwan, about one-tenth of the total population (about 2–2.8 million people) have a BQ chewing habit. Some even chew BQ all day with a consumption of more than 25 quids/day (3Ko Y.C. Chiang T.A. Chang S.J. Hsieh S.F. J. Oral Pathol. Med. 1992; 21: 261-264Crossref PubMed Scopus (312) Google Scholar). Chewing BQ has long been considered to be a major risk factor for oral leukoplakia, oral submucous fibrosis, and oral cancer (1RC IA IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. 37. International Agency for Research on Cancer, Lyon1985Google Scholar). Moreover, BQ chewing has been shown to increase the incidence of cancer in oral cavity (1RC IA IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. 37. International Agency for Research on Cancer, Lyon1985Google Scholar, 2Jeng J.H. Chang M.C. Hahn L.J. Oral Oncol. 2001; 37: 477-492Crossref PubMed Scopus (356) Google Scholar), oropharynx, pharynx (4Tominaga S. Cancer Lett. 1999; 143: S19-S23Crossref PubMed Scopus (38) Google Scholar, 5Ho P.S. Ko Y.C. Yang Y.H. Shieh T.Y. Tsai C.C. J. Oral Pathol. Med. 2002; 31: 213-219Crossref PubMed Scopus (74) Google Scholar), esophagus (6Sankaranarayanan R. Duffy S.W. Padmakumary G. Nair S.M. Day N.E. Padmanabhan T.K. J. Cancer. 1991; 49: 485-489Crossref PubMed Scopus (70) Google Scholar, 7Phukan R.K. Ali M.S. Chetia C.K. Mahata J. Br. J. Cancer. 2001; 85: 661-667Crossref PubMed Scopus (117) Google Scholar, 8Wu M.T. Wu D.C. Hsu H.K. Kao E.L. Lee J.M. Br. J. Cancer. 2003; 89: 1202-1204Crossref PubMed Scopus (21) Google Scholar), and liver (9Parkin D.M. Srivatanakul P. Khlat M. Chenvidhya D. Chotiwan P. Insiripong S. L'Abbe K.A. Wild C.P. Int. J. 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Epidemiological studies have indicated a multifactor etiology for these malignant diseases (14Spitz M.R. Semin. Oncol. 1994; 21: 281-288PubMed Google Scholar). Histological observation of upper end esophageal mucosa of BQ chewers by endoscopy showed marked submucous fibrosis (66%), atrophy of the squamous epithelium (52%), hyperkeratosis (52%), parakeratosis (30%), dyskeratosis (14%), acanthosis (14%), and mild dysplasia (2%) (15Misra S.P. Misra V. Dwivedi M. Gupta S.C. Postgrad. Med. J. 1998; 74: 733-736Crossref PubMed Scopus (29) Google Scholar). Thus, the molecular pathogenesis of how BQ components contribute to carcinogenesis deserves further delineation. BQ usually comprises a piece of areca nut (AN), influorescence Piper betle, and lime with or without Piper betle leaves. AN contains many polyphenols and several alkaloids such as arecoline, guvacoline, arecaidine, and guvacine. Many experiments have demonstrated that AN extract or its alkaloids possess cytotoxic or genotoxic effects on several kinds of cells in vitro (1RC IA IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. 37. International Agency for Research on Cancer, Lyon1985Google Scholar, 2Jeng J.H. Chang M.C. Hahn L.J. Oral Oncol. 2001; 37: 477-492Crossref PubMed Scopus (356) Google Scholar). Arecoline, a major AN alkaloid, is mutagenic to mammalian cells (16Shirname L.P. Menon M.M. Bhide S.V. Carcinogenesis. 1985; 5: 501-503Crossref Scopus (81) Google Scholar) and causes chromosomal aberration in Chinese hamster ovary cells in vitro or mouse bone marrow cells in vivo (17Stich H.F. Stich W. Lam P.P. Mutat. Res. 1981; 90: 355-363Crossref PubMed Scopus (69) Google Scholar, 18Panigrahi G.B. Rao A.R. Mutat. Res. 1982; 103: 197-204Crossref PubMed Scopus (73) Google Scholar). Sundqvist et al. (19Sundqvist K. Liu Y. Nair J. Bartsch H. Arvidson K. Graftstrom R.C. Cancer Res. 1989; 49: 5294-5298PubMed Google Scholar) have shown that AN extract and nitroso-derivatives of AN alkaloids are cytotoxic and genotoxic to buccal epithelial cells. AN extract induces the differentiation, DNA strand breaks and DNA protein cross-links in cultured buccal mucosal epithelial cells (19Sundqvist K. Liu Y. Nair J. Bartsch H. Arvidson K. Graftstrom R.C. Cancer Res. 1989; 49: 5294-5298PubMed Google Scholar, 20Sundqvist K. Graftstrom R.C. Int. J. Cancer. 1992; 52: 305-310Crossref PubMed Scopus (46) Google Scholar). We have demonstrated that AN extract can induce DNA strand breaks and is cytotoxic to oral mucosal fibroblasts (21Jeng J.H. Kuo M.L. Hahn L.J. Kuo M.Y.P. J. Dent. Res. 1994; 73: 1043-1049Crossref PubMed Scopus (136) Google Scholar). AN extract also induces unscheduled DNA synthesis and morphological alterations in cultured gingival keratinocytes (GK) (22Jeng J.H. Hahn L.J. Lin B.R. Chan C.P. Hsieh C.C. Chang M.C. J. Oral Pathol. Med. 1999; 28: 64-71Crossref PubMed Scopus (92) Google Scholar). These results indicate that AN ingredients may be important to the pathogenesis of BQ chewing-related oral cancer. In addition to the genotoxic stress, tissue inflammation, and release of inflammatory mediators such as prostanoids, interleukin-1α (IL-1α), IL-6, and TNF-α have been suggested to be a key factor for carcinogenesis of gastrointestinal systems including colorectum, stomach, esophagus, liver, and pancreas (23Roberts R.A. Kimber I. Carcinogenesis. 1999; 20: 1397-1401Crossref PubMed Scopus (47) Google Scholar, 24Orlando R.C. Rev. Gastroenterol. Disord. 2002; 2: S2-S8PubMed Google Scholar, 25Ohshima H. Tatemichi M. Sawa T. Biochem. Biophys. Acta. 2003; 417: 3-11Google Scholar, 26Tsuji S. Kawai N. Tsujii M. Kawano S. Hori M. Aliment. Pharmacol. Ther. 2003; 18: 82-89Crossref PubMed Scopus (47) Google Scholar, 27Gasparini G. Longo R. Sarmiento R. Morabito A. Lancet Oncology. 2003; 4: 605-615Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Elevation of cyclooxygenase-2 expression in the squamous cell carcinoma of the hypopharynx, esophagus, and oral cavity has also been reported (28Peng J. Su C. Chang H. Chai C. Hung W. Human Pathol. 2002; 33: 101-104Crossref Scopus (49) Google Scholar, 29Zimmermann K.C. Sarbia M. Weber A. Borchard F. Gabbert H.E. Schor K. Cancer Res. 1999; 59: 198-204PubMed Google Scholar). Recently, BQ components have been shown to induce keratinocyte inflammation by stimulation the prostanoids, interleukin-6 and TNF-α production of gingival keratinoytes (GK), and KB cancer cells (28Peng J. Su C. Chang H. Chai C. Hung W. Human Pathol. 2002; 33: 101-104Crossref Scopus (49) Google Scholar, 30Jeng J.H. Ho Y.S. Chan C.P. Wang Y.J. Hahn L.J. Lei D. Hsu C.C. Chang M.C. Carcinogenesis. 2000; 21: 1365-1370Crossref PubMed Google Scholar, 31Jeng J.H. Wang Y.J. Chiang B.L. Lee P.H. Chan C.P. Ho Y.S. Wang T.M. Lee J.J. Hahn L.J. Chang M.C. Carcinogenesis. 2003; 24: 1301-1315Crossref PubMed Scopus (157) Google Scholar). However, the precise mechanisms responsible for induction of prostanoids and IL-6 production are not fully clear. Induction of COX-2 and IL-6 gene expression has been associated with the MEK1/ERK-1/2/c-Fos/AP-1 activation in colon carcinoma cells, intestinal epithelial cells, and other kind of cells (32Glinghammar B. Rafter J. Gastroenterology. 2001; 120: 401-410Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 33Guo Y.S. Cheng J.Z. Jin G.F. Gutkind J.S. Hellmich M.R. Townsend C.M. J. Biol. Chem. 2002; 277: 48755-48763Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 34Huang Y. Zhang Q. Toxicol. Appl. Pharmacol. 2003; 191: 40-47Crossref PubMed Scopus (18) Google Scholar). Alterations in the expression of MAPK, several proto-oncogenes or tumor suppressor genes have been identified in cancer tissues from different sites and are considered to be important in the sequential stages of chemical carcinogenesis (35Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-I S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (618) Google Scholar, 36Mishima K. Inoue K. Hayashi Y. Oral Oncol. 2002; 38: 468-474Crossref PubMed Scopus (61) Google Scholar, 37Souza R.F. Shewmake K. Terada L.S. Spechler S.J. Gastroenterology. 2002; 122: 299-307Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). In this study, we used cultured primary GK and KB cancer cells to test whether BQ components stimulate cell cycle dysregulation, prostanoids, and IL-6 production in epithelial cells via MEK1/ERK-1/2/c-Fos signal transduction pathways. Materials—Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, penicillin/streptomycin, keratinocyte growth medium (KGM-SFM), pituitary gland extract, and epidermal growth factors etc. were from Invitrogen Life Technologies, Inc. ELISA kits for IL-6 measurement were from BIOSOURCE (BIOSOURCE International, Inc.). Arecoline, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), calfskin type I collagen and bovine plasma fibronectin were obtained from Sigma. PGE2 ELISA kits were purchased from Cayman Chemical Company (Ann Arbor, MI). Ethidium bromide, agarose, and kits for reverse transcription (RT) and PCR were purchased from HT Inc. Total RNA isolation kits were from Qiagen Inc. (Santa Clarita, CA). AN extract was prepared and weighed as previously described (31Jeng J.H. Wang Y.J. Chiang B.L. Lee P.H. Chan C.P. Ho Y.S. Wang T.M. Lee J.J. Hahn L.J. Chang M.C. Carcinogenesis. 2003; 24: 1301-1315Crossref PubMed Scopus (157) Google Scholar, 38Jeng J.H. Chen S.Y. Liao C.H. Tung Y.Y. Lin B.R. Hahn L.J. Chang M.C. Free Rad. Biol. Med. 2002; 32: 860-871Crossref PubMed Scopus (52) Google Scholar). Specific PCR primer sets for COX-2, β-actin, and IL-6 were synthesized by Genemed Biotechnologies, Inc. (San Francisco, CA). Mouse anti-human COX-2 monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). Mouse antihuman-G3PDH and -phospho-ERK-1/2 (p-ERK) antibodies were from Santa Cruz. Protein assay kits were obtained from Bio-Rad. Flow cytometric reagents were obtained from BD Biosciences. KB cancer cells were obtained from American Type Culture Collection. Culture of GK and KB Carcinoma Cells—GK were cultured as described previously with slight modifications (22Jeng J.H. Hahn L.J. Lin B.R. Chan C.P. Hsieh C.C. Chang M.C. J. Oral Pathol. Med. 1999; 28: 64-71Crossref PubMed Scopus (92) Google Scholar, 30Jeng J.H. Ho Y.S. Chan C.P. Wang Y.J. Hahn L.J. Lei D. Hsu C.C. Chang M.C. Carcinogenesis. 2000; 21: 1365-1370Crossref PubMed Google Scholar, 31Jeng J.H. Wang Y.J. Chiang B.L. Lee P.H. Chan C.P. Ho Y.S. Wang T.M. Lee J.J. Hahn L.J. Chang M.C. Carcinogenesis. 2003; 24: 1301-1315Crossref PubMed Scopus (157) Google Scholar). Briefly, healthy human gingival tissues (with a gingivitis index 590 nm. In total, 20,000 cells each were analyzed for the control and the experimental samples. The percentage of cells in sub-G0/G1, G0/G1 phase, S phase and G2/M phase were determined using standard ModiFit software and the CellQuest programs. Effects of MEK1 Inhibitors on AN Extract and Arecoline-induced COX-2 Protein Production of GK and KB Cells—For elucidation of whether MEK1/ERK-1/2 activation mediates the AN and arecoline associated PGE2 production via COX-2 protein expression, KB cells were serum-starved for 24 h, pretreated with Me2SO (control) or PD98059 (12.5, 25, and 50 μm) for 15 min and then exposed to AN extract for 24 h. Cell lysates was prepared as described above and subjected to 12% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membrane was blocked for 30 min at room temperature in a blocking reagent (20 mm Tris, pH 7.4, 125 mm NaCl, 0.2% Tween 20, 5% nonfat dry milk, and 0.1% sodium azide), incubated for 2 h with mouse anti-human COX-2 (1:500) and G3PDH antibodies. After final membrane washing, the immunoreactive bands were visualized on Fuji x-ray film after developed by ECL reagents. Statistical Analysis—Four or more separate experiments were performed. Results were analyzed by a paired Student's t test. A p value < 0.05 was considered to show a significant difference between groups. Up-regulation of c-Fos mRNA Expression in GK and KB Cells by AN Extract and Arecoline—An increase of the c-Fos mRNA expression in KB cells was evident following exposure to AN extract (400 μg/ml) for 30 min. Elevated c-Fos expression was still noted after 1 h of exposure, but the level of c-Fos then decreased gradually to the basal level after 2 h of treatment (Fig. 1a). Arecoline (0.4 mm), the major alkaloid in AN extract, also stimulated the expression of c-Fos. The kinetics of its stimulatory effect on c-Fos was very similar to that of AN extract. This stimulatory effect was a transient phenomenon and became less evident after 1 h of exposure (Fig. 1b). We then studied the effect of different doses of AN extract (100–800 μg/ml) or arecoline (0.1–0.8 mm) on c-Fos expression in KB cells. The induction of c-Fos mRNA expression in KB cells was noted after exposure to different concentrations of AN extract (100–800 μg/ml) for 30 min. However, at concentrations higher than 800 μg/ml, the stimulatory effect of AN on c-Fos mRNA expression became less apparent (Fig. 2a). Elevated expression of c-Fos mRNA was also noted after exposure to over 0.1 mm of arecoline for 30 min. However, the stimulatory effect became less evident after exposure to over 0.8 mm arecoline (Fig. 2b). A similar effect of AN extract and arecoline on c-Fos expression in GK was also noted (data not shown). Activation of ERK-1/2 Phosphorylation of KB Cells by AN Extract and Arecoline—AN extract (400 μg/ml) rapidly induced ERK-1/2 phosphorylation of KB cells after 2.5 min of exposure and was sustained for 10 min. The phosphorylation of ERK-1/2 rapidly declined to below the basal level after 30 min of exposure (Fig. 3a). Similarly, arecoline (0.4 mm) also stimulated ERK-1/2 phosphorylation of KB cells after 5 min of exposure with a peak induction at 10 min of exposure (Fig. 3b). AN extract stimulated the ERK phosphorylation in a dose-dependent manner with maximal effect at a concentration of 800 μg/ml (Fig. 3c). Arecoline also induced the phosphorylation of ERK-1/2 at concentrations over 0.1 mm (Fig. 3d). Inhibition of c-Fos mRNA Expression of KB Cells by U0126 — The stimulatory effect of AN extract and arecoline on c-Fos mRNA expression of KB cells was inhibited by U0126 (30 μm), a MEK1 inhibitor. As shown in Fig. 4a, AN extract (400 μg/ml) and arecoline (0.4 and 0.8 mm) stimulated c-Fos mRNA expression of KB cells after 30 min of exposure (lanes 4, 6, and 8). Pretreatment with U0126 for 15 min markedly suppressed AN- and arecoline-induced c-fos gene expression (Fig. 4a, lanes 5, 7, and 9). Quantitatively, AN extract induced the c-Fos expression by 2.74-fold compared with the control, whereas arecoline (0.4 mm) elevate the c-Fos expression by 2.58-fold compared with the control, as revealed by densitometry analysis of multiple blots. Pretreatment with U0126 resulted in a marked attenuation of the AN extract and arecoline-induced c-Fos mRNA expression (Fig. 4b). Modulation of AN- and Arecoline-associated PGE2 and IL-6 Production in GK and KB Cells by U0126 and PD98059 —As shown in Table I, AN extract stimulated PGE2 and IL-6 production by KB cells. In the absence of AN extract, U0126 (30 μm) inhibited the basal level production of PGE2 but not IL-6 production in KB cells. However, U0126 almost completely prevented the AN-extract-induced production of both PGE2 and IL-6 in KB cells (Table I). Similarly, PD98059 (50 μm), another MEK1 inhibitor, also inhibited AN extract-induced PGE2 and IL-6 production by KB cells (data not shown). Exposure of KB cells to AN extract (400–800 μg/ml) sho