Inhibition of Activator Protein-1, NF-κB, and MAPKs and Induction of Phase 2 Detoxifying Enzyme Activity by Chlorogenic Acid

Chlorogenic acid, the ester of caffeic acid with quinic acid, is one of the most abundant polyphenols in the human diet. The antioxidant and anticarcinogenic properties of chlorogenic acid have been established in animal studies. However, little is known about the molecular mechanisms through which chlorogenic acid inhibits carcinogenesis. In this study, we found that chlorogenic acid inhibited the proliferation of A549 human cancer cells in vitro. The results of the soft agar assay indicated that chlorogenic acid suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ cells in a dose-dependent manner. Pretreatment of JB6 cells with chlorogenic acid blocked UVB- or TPA-induced transactivation of AP-1 and NF-κB over the same dose range. At low concentrations, chlorogenic acid decreased the phosphorylation of c-Jun NH2-terminal kinases, p38 kinase, and MAPK kinase 4 induced by UVB/12-O-tetradecanoylphorbol-13-acetate, yet higher doses were required to inhibit extracellular signal-regulated kinases. Chlorogenic acid also increased the enzymatic activities of glutathione S-transferases (GST) and NAD(P)H: quinone oxidoreductase. Further studies indicated that chlorogenic acid could stimulate the nuclear translocation of Nrf2 (NF-E2-related factor) as well as subsequent induction of GSTA1 antioxidant response element (ARE)-mediated GST activity. The phosphatidylinositol 3-kinase pathway might be involved in the activation of Nrf2 translocation. These results provide the first evidence that chlorogenic acid could protect against environmental carcinogen-induced carcinogenesis and suggest that the chemopreventive effects of chlorogenic acid may be through its up-regulation of cellular antioxidant enzymes and suppression of ROS-mediated NF-κB, AP-1, and MAPK activation. Chlorogenic acid, the ester of caffeic acid with quinic acid, is one of the most abundant polyphenols in the human diet. The antioxidant and anticarcinogenic properties of chlorogenic acid have been established in animal studies. However, little is known about the molecular mechanisms through which chlorogenic acid inhibits carcinogenesis. In this study, we found that chlorogenic acid inhibited the proliferation of A549 human cancer cells in vitro. The results of the soft agar assay indicated that chlorogenic acid suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ cells in a dose-dependent manner. Pretreatment of JB6 cells with chlorogenic acid blocked UVB- or TPA-induced transactivation of AP-1 and NF-κB over the same dose range. At low concentrations, chlorogenic acid decreased the phosphorylation of c-Jun NH2-terminal kinases, p38 kinase, and MAPK kinase 4 induced by UVB/12-O-tetradecanoylphorbol-13-acetate, yet higher doses were required to inhibit extracellular signal-regulated kinases. Chlorogenic acid also increased the enzymatic activities of glutathione S-transferases (GST) and NAD(P)H: quinone oxidoreductase. Further studies indicated that chlorogenic acid could stimulate the nuclear translocation of Nrf2 (NF-E2-related factor) as well as subsequent induction of GSTA1 antioxidant response element (ARE)-mediated GST activity. The phosphatidylinositol 3-kinase pathway might be involved in the activation of Nrf2 translocation. These results provide the first evidence that chlorogenic acid could protect against environmental carcinogen-induced carcinogenesis and suggest that the chemopreventive effects of chlorogenic acid may be through its up-regulation of cellular antioxidant enzymes and suppression of ROS-mediated NF-κB, AP-1, and MAPK activation. Epidemiological evidence demonstrates that consumption of healthy foods containing phytochemical compounds might reduce the incidence of cancer and chronic degenerative diseases (1Tamimi R.M. Lagiou P. Adami H.O. Trichopoulos D. J. Intern. Med. 2002; 251: 286-300Crossref PubMed Scopus (75) Google Scholar). Chlorogenic acid, the ester of caffeic acid with quinic acid, is one of the most abundant polyphenols in human diet and has been reported to decrease the incidence of chemical carcinogenesis in several animal models of cancer (2Huang M.T. Smart R.C. Wong C.Q. Conney A.H. Cancer Res. 1988; 48: 5941-5946PubMed Google Scholar, 3Kasai H. Fukada S. Yamaizumi Z. Sugie S. Mori H. Food Chem. Toxicol. 2000; 38: 467-471Crossref PubMed Scopus (190) Google Scholar). However, the molecular mechanisms for its anti-carcinogenic properties are poorly understood. Thus, elucidation of such mechanisms of action is essential before the possibility for chlorogenic acid application in chemoprevention can be considered. AP-1 1The abbreviations used are: AP-1, activator protein-1; MAPK, mitogen-activated protein kinases; MAPKK, MAPK kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MKK4, MAP kinase kinase 4 (alternatively designated SEK1 or MEK4); NQO1, NAD(P)H:quinone oxidoreductase; GST, glutathione S-transferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; ECIS, electric cell-substrate impedance sensing; ROS, reactive oxygen species; ARE, antioxidant response element; FBS, fetal bovine serum; PI, phosphatidylinositol. and/or NF-κB signal transduction pathways are known to be important molecular targets of chemopreventive strategies (4Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar, 5Bode A.M. Dong Z. Lancet Oncol. 2000; 1: 181-188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6Yang S.C. Maliakal P. Meng X. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (846) Google Scholar). Activation of AP-1 or NF-κB induces the expression of target genes that are involved in many disease processes, such as inflammation, neoplastic transformation, tumor progression, metastasis, and angiogenesis (5Bode A.M. Dong Z. Lancet Oncol. 2000; 1: 181-188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6Yang S.C. Maliakal P. Meng X. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (846) Google Scholar, 7Bernstein L.R. Colburn N.H. Science. 1989; 244: 566-569Crossref PubMed Scopus (206) Google Scholar). AP-1 activity has been shown to be involved in the tumor promotion and progression of various types of cancers (4Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar, 5Bode A.M. Dong Z. Lancet Oncol. 2000; 1: 181-188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6Yang S.C. Maliakal P. Meng X. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (846) Google Scholar). Active NF-κB is found in the nucleus of many different cancer cells (8Darnell Jr., J.E. Nat. Rev. Cancer. 2002; 2: 740-749Crossref PubMed Scopus (961) Google Scholar). The potential proapoptotic effect of NF-κB on normal cells and its antiapoptotic and cytoprotective effect on tumor cells have also been reviewed (8Darnell Jr., J.E. Nat. Rev. Cancer. 2002; 2: 740-749Crossref PubMed Scopus (961) Google Scholar, 9Sonis S.T. Crit. Rev. Oral Biol. Med. 2002; 13: 380-389Crossref PubMed Scopus (176) Google Scholar). The components of AP-1 are activated by three distinct but parallel MAPKs: ERKs, JNKs, and p38 kinase. Each consists of a module of three kinases: MAPK, a MAPK kinase (MAPKK) that is responsible for the phosphorylation of MAPK, and a MAPKK kinase that phosphorylates and activates MAPKK (10Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 11Ding M. Shi X. Dong Z. Chen F. Lu Y. Castranova V. Vallyathan V. J. Biol. Chem. 1999; 274: 30611-30616Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). NF-κB can also be activated at the MAPKK kinase level, by MAPK/ERK kinase 1 or 3 (5Bode A.M. Dong Z. Lancet Oncol. 2000; 1: 181-188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 12Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (352) Google Scholar). AP-1, NF-κB, and associated MAPK signal transduction pathways are believed to be crucial in cell transformation and tumor promotion (13Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 14Huang C. Ma W.Y. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (187) Google Scholar, 15Chen F. Ding M. Lu Y. Leonard S.S. Vallyathan V. Castranova V. Shi X. J. Environ. Pathol. Toxicol. Oncol. 2000; 19: 231-238PubMed Google Scholar, 16Gilmore T.D. Koedood M. Piffat K.A. White D.W. Oncogene. 1996; 13: 1367-1378PubMed Google Scholar). Because of the critical roles of NF-κB, AP-1, and MAPK signaling in carcinogenesis, they have been proposed as targets for chemopreventive agents (5Bode A.M. Dong Z. Lancet Oncol. 2000; 1: 181-188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). It has been reported that the induced AP-1 activity and neoplastic transformation can be blocked by chemopreventive agents, such as pyrrolidine dithiocarbamate (4Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar), tea polyphenols (6Yang S.C. Maliakal P. Meng X. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (846) Google Scholar), resveratrol (17Yu R. Hebbar V. Kim D.W. Mandlekar S. Pezzuto J.M. Kong A.N. Mol. Pharmacol. 2001; 60: 217-224Crossref PubMed Scopus (133) Google Scholar), and blackberry extract (18Feng R. Bowman L.L. Lu Y. Leonard S.S. Shi X. Jiang B.H. Castranova V. Vallyathan V. Ding M. Nutr. Cancer. 2004; 50: 80-89Crossref PubMed Scopus (30) Google Scholar). Many of these inhibitory agents have been shown to be active not only in the JB6 transformation model but also in mouse skin tumor promotion in vivo (5Bode A.M. Dong Z. Lancet Oncol. 2000; 1: 181-188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Thus, the mouse epidermal cell line, JB6, provides a validated model for screening cancer chemopreventive agents and elucidating their mechanisms at the molecular level (4Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar, 5Bode A.M. Dong Z. Lancet Oncol. 2000; 1: 181-188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The transcription factor Nrf2 is a member of the basic leucine zipper NF-E2 family and plays an essential role in the antioxidant response element (ARE)-mediated expression of phase 2 detoxifying enzymes and stress-inducible genes (19Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3226) Google Scholar, 20Motohashi H. Yamamoto M. Trends Mol. Med. 2004; 10: 549-557Abstract Full Text Full Text PDF PubMed Scopus (1397) Google Scholar, 21Katsuoka F. Motohashi H. Engel J.D. Yamamoto M. J. Biol. Chem. 2005; 280: 4483-4490Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Inducers of phase 2 and antioxidative enzymes are known to enhance the detoxication of environmental carcinogens in animals, often leading to protection against neoplasia (22Kensler T.W. Environ. Health Perspect. 1997; 105: 965-970Crossref PubMed Scopus (240) Google Scholar, 23Balogun E. Hoque M. Gong P. Killeen E. Green C.J. Foresti R. Alam J. Motterlini R. Biochem. J. 2003; 371: 887-895Crossref PubMed Scopus (904) Google Scholar, 24Kwak M.K. Itoh K. Yamamoto M. Kensler T.W. Mol. Cell. Biol. 2002; 22: 2883-2892Crossref PubMed Scopus (475) Google Scholar, 25Hayes J.D. McMahon M. Cancer Lett. 2000; 174: 103-113Crossref Scopus (306) Google Scholar). Regulation of both basal and inducible expression of cytoprotective genes is mediated in part by the ARE, a cis-acting sequence found in the 5′-flanking region of genes encoding many phase 2 enzymes, including heme oxygenase-1, glutathione S-transferase (GST) A1, NAD(P)H:quinone oxidoreductase (NQO1), and Nrf2 itself (23Balogun E. Hoque M. Gong P. Killeen E. Green C.J. Foresti R. Alam J. Motterlini R. Biochem. J. 2003; 371: 887-895Crossref PubMed Scopus (904) Google Scholar, 24Kwak M.K. Itoh K. Yamamoto M. Kensler T.W. Mol. Cell. Biol. 2002; 22: 2883-2892Crossref PubMed Scopus (475) Google Scholar, 25Hayes J.D. McMahon M. Cancer Lett. 2000; 174: 103-113Crossref Scopus (306) Google Scholar, 26Thimmulappa R.K. Mai K.H. Srisuma S. Kensler T.W. Yamamoto M. Biswal S. Cancer Res. 2002; 62: 5196-5203PubMed Google Scholar, 27Favreau L. Pickett C.B. J. Biol. Chem. 1995; 270: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Among these defensive systems, GST may block JNK-induced Jun activation and subsequently inhibit mitogenic signaling induced by oncogenic Ras-p21 (28Villafania A. Anwar K. Amar S. Chie L. Way D. Chung D.L. Adler V. Ronai Z. Brandt-Rauf P.W. Yamaizumii Z. Kung H.F. Pincus M.R. Ann. Clin. Lab. Sci. 2000; 30: 57-64PubMed Google Scholar). As the endogenous inhibitor of apoptosis signal-regulating kinase 1, GST may also inhibit apoptosis signal-regulating kinase 1-activated JNK and p38 signaling pathways (29Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Thus, GSTs display broad substrate specificity and are associated with cancer chemopreventive and cytoprotective effects (26Thimmulappa R.K. Mai K.H. Srisuma S. Kensler T.W. Yamamoto M. Biswal S. Cancer Res. 2002; 62: 5196-5203PubMed Google Scholar, 30Yoshitani S.I. Tanaka T. Kohno H. Takashima S. Int. J. Oncol. 2001; 19: 929-939PubMed Google Scholar). Studies with nrf2-disrupted mice indicated that Nrf2 was essential for the induction of GST and NQO1 activities in vivo by different classes of chemopreventive agents, including dithiole-thiones, isothiocyanates, and phenolic antioxidants (31MacMahon M. Itoh K. Yamamoto M. Chanas S.A. Henderson C.J. McLellan L.I. Wolf C.R. Cavin C. Hayes J.D. Cancer Res. 2001; 61: 3299-3307PubMed Google Scholar, 32Kwak M.K. Itoh K. Yamamoto M. Sutter T.R. Kensler T.W. Mol. Med. 2001; 7: 135-145Crossref PubMed Google Scholar). The likely importance of these protective enzymes is highlighted by recent observations that nrf2-null mice were considerably more sensitive to the tumorigenicity of benzo[a]pyrene (33Ramos-Gomez M. Kwak M.K. Dolan P.M. Itoh K. Yamamoto M. Talalay P. Kensler T.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3410-3415Crossref PubMed Scopus (995) Google Scholar) and form higher levels of DNA adducts following exposure to carcinogens (34Ramos-Gomez M. Dolan P.M. Itoh K. Yamamoto M. Talalay P. Kensler T.W. Carcinogenesis. 2003; 24: 461-467Crossref PubMed Scopus (159) Google Scholar). Thus, the induction of phase 2 gene expression is an effective strategy for achieving protection against carcinogenesis (22Kensler T.W. Environ. Health Perspect. 1997; 105: 965-970Crossref PubMed Scopus (240) Google Scholar). Given the animal data suggesting cancer prevention properties of chlorogenic acid, we tested the effects of chlorogenic acid on proliferation of cancer cells and neoplastic transformation induced by tumor promoter in JB6 cells. To elucidate the mechanism of the anti-tumorigenic effect of chlorogenic acid, we also investigated the effects of chlorogenic acid on NF-κB, AP-1, and MAPK activation induced by tumor promotors as well as on the induction of Nrf2 transactivation and phase 2 enzyme activities. Our results demonstrate possible chemoprevention activity of chlorogenic acid and characterize mechanisms of the inhibitory actions on tumor promotion and the inductive effects on Nrf2 transactivation and phase 2 enzyme activity. Materials—Chlorogenic acid, 1-chloro-2,4-dinitrobenzene, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, NADP, FAD, menadione, glucose 6-phosphate, yeast glucose-6-phosphate dehydrogenase, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma. LY294002 was obtained from Calbiochem. Eagle's minimal essential medium and Dulbecco's modified Eagle's medium were obtained from Whittaker Biosciences (Walkersville, MD). Opti-MEM I medium was from Invitrogen. Fetal bovine serum (FBS), gentamicin, and l-glutamine were purchased from Invitrogen. Fugene 6 Transfection reagent was from Roche Applied Science. Antibody against MKK4 or Nrf2 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-SEK1/MKK4 and PhosphoPlus MAPK antibody kits were purchased from Cell Signaling Technology (Beverley, MA). The luciferase assay kit was obtained from Promega (Madison, WI). Plasmids and Cell Culture—AP-1 or NF-κB luciferase reporter plasmid is the construct containing the collagenase promoter bearing AP-1- or NF-κB-binding sites that drives a luciferase reporter gene. A DNA sequence containing the GST A1 ARE (-833 to -533 from the start codon) was prepared by PCR from the mouse GST A1 promoter (-1094 to -10), which was isolated from mouse brain cDNA and inserted into a luciferase reporter vector (ARE-TATA Luc+) (35Kwak M.K. Kensler T.W. Casero Jr., R.A. Biochem. Biophys. Res. Commun. 2003; 305: 662-670Crossref PubMed Scopus (73) Google Scholar). The JB6 promotion-sensitive (P+) mouse epidermal cell line and JB6 cells, stably transfected with either AP-1-luciferase reporter plasmid (4Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar) or a NF-κB-luciferase reporter plasmid (a gift from Dr. Chuanshu Huang, New York University School of Medicine), were cultured in Eagle's minimal essential medium containing 5% FBS, 2 mm l-glutamine, and 50 μg/ml gentamicin. The human lung cancer epithelial cell line, A549, was obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium containing 10% FBS. The cells were grown at 37 °C in a 5% CO2 atmosphere. In all experiments, exponentially growing cells were used. Assay of the Antioxidant Capacity of Chlorogenic Acid—The antioxidant capacity of chlorogenic acid was determined using a 2,2′-azio-diethylbenzthiazoline sulfonate test set (Randox Laboratories Ltd.). The principle of the assay depends on production of the radical cation 2,2′-azio-diethylbenzthiazoline sulfonate+ in incubation medium containing the substrates (H2O2 and peroxidase), which is a blue-green color and can be detected at 600 nm. Antioxidants in the sample cause the suppression of this color production to a degree that is proportional to their concentrations. The assays were calibrated against standards and expressed as μmol/liter. Assay of AP-1 or NF-κB Activity—Confluent monolayers of JB6 stable transfectants were trypsinized, and 2 × 104 viable cells suspended in 0.5 ml of Eagle's minimal essential medium supplemented with 5% FBS were seeded to each well of a 48-well plate. Plates were incubated overnight at 37 °C in a humidified atmosphere of 5% CO2. The medium was then switched to 0.1% FBS Eagle's minimal essential medium and culture for 24 h to minimize basal activity of AP-1 or NF-κB. The cells were pretreated with or without chlorogenic acid for 1 h and then exposed to TPA (20 ng/ml) or UVB (4 kJ/m2) irradiation. The cells were extracted with 100 μl of 1× lysis buffer provided in the luciferase assay kit by the manufacturer. Luciferase activity was measured using a Monolight luminometer, model 3010. The results are presented as relative AP-1 or NF-κB activity compared with untreated controls (36Ding M. Lu Y. Bowman L. Huang C. Leonard S. Wang L. Vallyathan V. Castranova V. Shi X. J. Biol. Chem. 2004; 279: 10670-10676Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Cell Proliferation Assay by Electric Cell-Substrate Impedance Sensing (ECIS)—The ECIS model 1600R (Applied BioPhysics, Troy, NY) was used to measure the influence of chlorogenic acid on the proliferation of human cancer A549 cells or JB6 cells. The ECIS assay has been used for continuous measurement of cell micromotion, attachment, spreading, and growth (37Lo C.M. Keese C.R. Giaever I. Biophys. J. 1995; 69: 2800-2807Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 38Giaever I. Keese C.R. Nature. 1993; 366: 591-592Crossref PubMed Scopus (667) Google Scholar). The cells (1 × 104) were suspended in 400 μl of Dulbecco's modified Eagle's medium with or without chlorogenic acid and seeded on electrodes. The cells were equilibrated in the incubator for 15 min. The rate of cell proliferation on the microelectrode was monitored for 72 h as real-time changes in resistance. Anchorage-independent Transformation Assay—The effect of chlorogenic acid on TPA-induced cell transformation was investigated in JB6 P+ cells using the soft agar assay as described previously (4Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar, 39Dong Z. Birrer M.J. Watts R.G. Matrisian L.M. Colburn N.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 609-613Crossref PubMed Scopus (370) Google Scholar). The cells (1 × 104) were exposed to TPA (20 ng/ml) in the presence or absence of different concentrations of chlorogenic acid, in 1 ml of 0.33% basal medium Eagle agar containing 15% FBS over 3.5 ml of 0.5% agar containing 15% FBS Eagle's minimal essential medium. The cultures were maintained in a 37 °C, 5% CO2 incubator for 2 weeks, and the anchorage-independent colonies were counted. Protein Kinase Phosphorylation Assay—The cells were extracted with 1× SDS sample buffer. Immunoblots for phosphorylation of ERKs, JNKs, and p38 kinase were carried out as described in the protocol of the manufacturer, using phosphospecific antibodies against phosphorylated sites of ERKs, JNK, and p38 kinase. Nonphosphospecific antibodies against ERKs and p38 kinase proteins provided in the assay kits were used to normalize the phosphorylation assay, using the same transferred membrane blot (36Ding M. Lu Y. Bowman L. Huang C. Leonard S. Wang L. Vallyathan V. Castranova V. Shi X. J. Biol. Chem. 2004; 279: 10670-10676Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Enzyme Activity Assay—The activities of the typical phase 2 enzymes, NQO1 and GST, were measured spectrophotometrically as described previously (35Kwak M.K. Kensler T.W. Casero Jr., R.A. Biochem. Biophys. Res. Commun. 2003; 305: 662-670Crossref PubMed Scopus (73) Google Scholar, 40Habig W.H. Pabst M.J. Jakoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar). For the NQO1 assay, the cells were grown in 96-well plates, treated with chlorogenic acid, and lysed by 0.8% digitonin. The reaction solution (25 mm Tris-HCl (pH 7.4), 0.06% bovine serum albumin, 5 μm FAD, 1 mm glucose 6-phosphate, 30 μm NADP, 300 units of glucose-6-phosphate dehydrogenase, 725 μm 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and 50 μm menadione) was added into the wells, and the reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was measured at 610 nm. NQO1 induction by chlorogenic acid was expressed as ratios of treated over vehicle control (35Kwak M.K. Kensler T.W. Casero Jr., R.A. Biochem. Biophys. Res. Commun. 2003; 305: 662-670Crossref PubMed Scopus (73) Google Scholar). For total GST assay, JB6 cells were incubated with chlorogenic acid and then lysed with 200 μl of lysis buffer (0.25 m sucrose, 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and 1% Triton X-100) for 30 min. Cytosolic fractions were prepared by centrifugation and stored at -70 °C until use. Assays were conducted in a thermostated compartment at 25 °C, using 1-chloro-2,4-dinitrobenzene as the substrate. Cytosolic protein (45 μg) was added to 800 μl of reaction mixture containing 100 mm KH2PO4 (pH 6.5) and 1 mm glutathione. The reaction was initiated by adding 1 mm 1-chloro-2,4-dinitrobenzene, and the formation of thioether at 5 min was measured at 340 nm. Total enzymatic activity of GST was expressed as nmol/min/mg protein. Immunocytochemistry of Nrf2—After treatment with chlorogenic acid for 3 h, JB6 cells grown on Lab-TEK chamber slides were fixed in 100% methanol for 30 min at room temperature. The cells were then washed with phosphate-buffered saline followed by blocking with 5% bovine serum albumin in phosphate-buffered saline for 1 h. The cells were incubated with polyclonal rabbit anti-Nrf2 antibody (1:100) in phosphate-buffered saline containing 0.5% bovine serum albumin overnight at 4 °C. After they were washed four times with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology) for an additional 1 h in the dark. Counterstaining with 4′,6-diamidino-2-phenylindole verified the location and integrity of nuclei. Stained cells were washed and examined using a laser-scanning confocal microscope, Zeiss LSM 510 (Thornwood, NY). Preparation of Nuclear Extracts and Nrf2 Nuclear Translocation Assay—Nuclear extracts from JB6 cells were prepared as described previously (15Chen F. Ding M. Lu Y. Leonard S.S. Vallyathan V. Castranova V. Shi X. J. Environ. Pathol. Toxicol. Oncol. 2000; 19: 231-238PubMed Google Scholar). Briefly, cells were treated with or without chlorogenic acid and harvested. The cells were suspended in hypotonic buffer A (10 mm HEPES (pH 7.6), 10 mm KCl, 0.1 mm EDTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride) for 10 min on ice and then vortexed for 10 s. Nuclei were pelleted by centrifugation at 12,000 × g for 20 s and were resuspended in buffer C (20 mm HEPES (pH 7.6), 25% glycerol, 0.4 m NaCl, 1 mm EDTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride) for 30 min on ice. The supernatants containing nuclear proteins were collected after centrifugation at 12,000 × g for 2 min and stored at -70 °C. Proteins that were extracted from either whole cell lysate (30 μg) or nuclei (30 μg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and detected with an Nrf2 antibody. Transfection of Plasmids and Measurement of Luciferase Activity—A DNA sequence containing mouse GST A1 ARE (-833 to -533 from the start codon) was inserted into a luciferase reporter pGL3-Basic vector (ARE-TATA Luc+) as described previously (35Kwak M.K. Kensler T.W. Casero Jr., R.A. Biochem. Biophys. Res. Commun. 2003; 305: 662-670Crossref PubMed Scopus (73) Google Scholar). JB6 cells were plated on 24-well plates at density of 50–60% confluence. The transfection complex containing 0.5 μg of plasmid DNA in Opti-MEM I medium was added to each well. The cells were treated with chlorogenic acid for 18 h following transient transfection of ARE-TATA Luc+. Luciferase activity was measured using the luciferase assay kit as described above. Statistical Analysis—Data are presented as means ± S.E. of n experiments/samples as noted in the figure legends. Significant differences were determined using Student's t test. Significance was set at p ≤ 0.05. The Total Antioxidant Capacity of Chlorogenic Acid—Reactive oxygen species (ROS) have been known to be mutagenic and associated with many diseases. In light of the important role of ROS in tumor promoter-induced AP-1 activation, transformation, and tumor promotion, we measured the antioxidant capacity of chlorogenic acid using the Randox reagent set. As shown in Fig. 1, chlorogenic acid displayed stronger antioxidant activity than that of ascorbic acid over the same concentration range, indicating that chlorogenic acid can effectively scavenge reactive oxygen radicals. Chlorogenic Acid Inhibits the Proliferation of Cancer Cells— The effect of chlorogenic acid on proliferation of a human lung cancer cell line, A549, was determined using the ECIS assay. Cells were grown in the electrode array wells and treated with chlorogenic acid for 72 h. The ECIS resistance, indicating the number of cells in the wells, was monitored for the duration of the experiment. At a concentration of 80 μm, proliferation of A549 cells was significantly suppressed (Fig. 2A). Interestingly, chlorogenic acid had little effect on the proliferation of JB6 cells at the same dose (Fig. 2B). This result suggests that chlorogenic acid may preferentially inhibit tumor cell growth. Effects of Chlorogenic Acid on TPA- or UVB-induced AP-1 and NF-κB Activation—Previous studies have shown that either AP-1 or NF-κB activation is required for neoplastic transformation in JB6 cells (4Li J.J. Westergaard C. Ghosh P. Colburn N.H. Cancer Res. 1997; 57: 3569-3576PubMed Google Scholar, 5Bode A.M. Dong Z. Lancet Oncol. 2000; 1: 181-188Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 41Hsu T.C. Young M.R. Cmarik J. Colburn N.H. Free Radic. Biol. Med. 2000; 28: 1338-1348Crossref PubMed Scopus (251) Google Scholar) and that these two transcription factors play an important role in carcinogenesis. We thus tested the effects of chlorogenic acid on TPA- or UVB-induced AP-1 and NF-κB activity, using a reporter gene assay. Pretreatment of cells with chlorogenic acid markedly inhibited TPA-induced AP-1 and NF-κB activity over a similar concentration range (Fig. 3, A and B). At a concentration of 40 μm chlorogenic acid, TPA-induced AP-1 or NF-κB activation was suppressed by 30 or 42%, respectively. Chlorogenic acid alone had no effect on AP-1 or NF-κB activity. These inhibitory effects were not due to the cytotoxicity of chlorogenic acid on JB6 cells, since the ECIS assay indicated that the proliferation of JB6 cells was not affected by chlorogenic acid even at a concentration of 80 μm (Fig. 2B). It is well known that UVB irradiation acts both a