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Carcinogenesis Advance Access originally published online on November 23, 2005
Carcinogenesis 2006 27(5):1008-1017; doi:10.1093/carcin/bgi281
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Involvement of p38 MAPK and Nrf2 in phenolic acid-induced P-form phenol sulfotransferase expression in human hepatoma HepG2 cells

Chi-Tai Yeh and Gow-Chin Yen *

Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan

* To whom correspondence should be addressed. Tel: +886 4 2287 9755; Fax: +886 4 2285 4378; Email: gcyen{at}nchu.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Phenolic acids have significant biological and pharmacological properties and some have demonstrated remarkable ability to alter sulfate conjugation. However, the modulation mechanisms of phenolic acids on phenol sulfotransferase expression have not been described. In the present study, we investigated the effects of phenolic acids on the expression of the Phase II P-form of phenol sulfotransferase (PST-P) in human hepatoma HepG2 cells. RT–PCR and western blot data revealed that gallic acid induced increase in PST-P expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in PST-P activity. Actinomycin D and cycloheximide inhibited gallic acid-responsive PST-P mRNA expression, indicating that gallic acid is a requirement for transcription and de novo protein synthesis. Transient transfection of HepG2 cells with a reporter plasmid of the upstream region of the human PST gene caused a significant increase in reporter gene activity after gallic acid exposure. Moreover, gallic acid increased the nuclear levels of Nrf2, a transcription factor governing antioxidant response element (ARE). Electrophoretic mobility shift assay showed increased binding of nuclear proteins to ARE consensus sequence after treatment with gallic acid. While investigating the signaling pathways responsible for PST-P induction, we observed that gallic acid activated the p38 mitogen-activated protein kinase (MAPK) pathway. SB203580, a specific inhibitor of p38 MAPK, abolished gallic acid-induced PST-P protein expression. Similarly, gallic acid also caused an accumulation of Nrf2. Moreover, the protective effects of gallic acid on tert-butyl hydroperoxide-induced toxicity was partially blocked by p38 MAPK and PST-P inhibitors, further demonstrating that gallic acid attenuates oxidative stress through a pathway that involves p38 MAPK and PST-P. These results indicate that gallic acid is a potent inducer of PST-P and that PST-P induction is responsible for the gallic acid-mediated cytoprotection against oxidative damage.

Abbreviations: ARE, antioxidant responsive element; AhR, aryl hydrocarbon receptor; t-BHQ, tert-Butyl hydroquinone; t-BHP, tert-Butyl hydroperoxide; DCNP, 2; 4-Dichloro- 6-nitrophenol; ERK, extracellualr signal-regulated protein kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione S-transfersase; JNK, c-Jun N-terminal kinase; Keap 1, Kelch-like ECH-associated protein-1; MTT, 3-(4,5-Dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide; MAPK, mitogen-activated protein kinase; NQO-1, NAD(P)H: quinine oxidoreductase 1; Nrf2, nuclear factor-E2-related factor-2; ORACROO, oxygen radical absorbance capacity; PST-P, P-form phenosulfotransferases; RT–PCR, reverse transcriptase–polymerase chain rection; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gels; TEAC, trolox equivalent antioxidant capacity; TBST, Tris-buffered saline Tween-20; XRE, xenobiotics responsive element


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
High levels of reactive oxygen species cause damage to cells and are involved in several human pathologies, including chronic disorders and cancer (1). Therefore, the use of compounds with antioxidant properties may help prevent or alleviate disease in which oxidative stress is a primary cause (2). It is known that many antioxidants and dietary components can protect humans and animals against the damage from environmental toxins and carcinogens through the enhancement of cytoprotective machinery. The chemopreventive properties of these agents have been partially ascribed to their ability to induce detoxifying enzymes, which in turn decrease the chemical reactivity of carcinogens and their metabolites through conjugation, reduction and hydrolysis to facilitate their elimination (3). Phenolic acids, especially hydroxycinnamic acid and hydroxybenzoic acid, are secondary plant products and are commonly found in plant-derived foodstuff. In addition, being themselves antioxidant compounds, they activate an antioxidant response that targets cells affecting the expression of Phase II enzymes such as NAD(P)H:quinone oxidoreductase (NQO-1), glutathione S-transferase (GST), {gamma}-glutamylcysteine synthetase and phenol sulfotransferases (PSTs) (4,5).

PSTs are the main Phase II sulfoconjugation enzymes for catecholamines, thyroid hormones and drugs, thereby facilitating biliary or urinary excretion and detoxification (6). Sulfoconjugation plays not only an important role in xenobiotic metabolism, but also a critical role in steroid biosynthesis, the modulation of biological activity and facilitation of the inactivation and elimination of potent endogenous chemicals, including steroids, catecholamines and thyroid hormones. The PSTs are the Phase II enzymes that are transcriptionally regulated by a large variety of stimuli. These include thyroid hormone (7); oxidative stress (8); the cytokines tumor necrosis factor-alpha (TNF-{alpha}), transforming growth factor-beta (TGF-ß) and insulin (9); and phenolic compounds such as gallic acid and ferulic acid (10). Previous studies in our laboratory have shown that p-hydroxybenzoic acid, gentisic acid, ferulic acid, gallic acid and p-coumaric acid increased PST-P activity (5). Moreover, the activity of PST-P could be promoted by the combinations of all these phenolic acids (10). Many reports have shown that harmful substances might accumulate in the body when PST activity is inhibited (11). Moreover, elevation of hepatic sulfotrnasferase activity in mice with resistance to cystic fibrosis (12) and in mice deficient in cerebroside sulfotransferase (13) exhibit a serious impairment of L-selectin metabolism, leading to liver and kidney oxidative damage and inflammation. However, the signaling mechanisms used to activate transcription of Phase II genes such as PST are poorly defined.

Most studies have focused on the activation of the mitogen-activated protein kinases (MAPKs) related to cell growth and stress response. In vertebrates, the three major MAPK pathways are represented by kinase cascades leading to activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPKs (14). Generally, ERK modulates the responses of cell differentiation, whereas JNK and p38 MAPK are activated by stress-associated stimuli, such as heat shock, inflammation, ultraviolet light and irradiation. Many genes encoding detoxification and antioxidant proteins are regulated by Nrf2 (nuclear factor-E2-related factor-2) (15). Recently, Nrf2, a member of the basic-region leucine zipper transcription factor family, was identified as an antioxidant responsive element (ARE)-binding protein (16). Transfection of Nrf2 and ARE constructs has proven that Nrf2 overexpression dramatically increases ARE activity (17). More importantly, Nrf2 knockout mice (Nrf2–/–) possess much lower levels of Phase II detoxifying enzymes and are more susceptible to oxidative stress and carcinogen-induced tumorigenesis than wide-type animals (18). Although the positive effects of phenolic acid on Phase II sulfoconjugation enzyme have been well characterized, the molecular mechanism and signaling cascades leading to these benefits remain largely unknown. In the present study, we investigated the effects of phenolic acids on the expression of the Phase II sulfoconjugation enzyme PST, and examined the roles of Nrf2 and upstream signaling kinases in phenolic acid-induced cytoprotective events.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
p-Hydroxybenzoic acid, gentisic acid (2,5-dihydroxybenzoic acid, 98% purity), ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid, 99% purity), gallic acid (3,4,5-trihydroxybenzoic acid, 98% purity), p-coumaric acid (trans-4-hydroxycinnamic acid), p-nitrophenol, dopamine, sucrose, 2, 4-Dichloro- 6-nitrophenol (DCNP), cycloheximide, antinoymcin D and Na2EDTA were obtained from Sigma Chemical (St Louis, MO). [35S]-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS35) (1.0–1.5 Ci/mmol) was obtained from DuPont New England Nuclear (Boston, MA). Anti-rabbit IgG polyclonal antibody conjugated to peroxidase was purchased from Sigma Chemical. Dubecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and trypsin-EDTA (T/E) were purchased from Gibco BRL (Grand Island, NY). Anti-P-form phenol sulfotransferase (PST-P) antibody was purchased from Calbiochem-Novabiochem (San Diego, CA). Anti-ß-actin antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-protein-disulfide isomerase (anti-PDI) antibody was obtained from StressGen (Victoria, Canada). An antibody against Nrf2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against JNK, phospho-JNK (p–JNK), ERK, phospho-ERK (p-ERK), p38 MAPK and phospho-p38 MAPK (p–p38 MAPK) were obtained from Cell Signaling Technology. The inhibitors of mitogen-activated protein kinase (MAPKs), PD98059, SB203580 and SP600125 were obtained from Biosource (Camarillo, CA). Polyvinyldifluoride (PVDF) membrane for western blotting was obtained from Millipore (Bedford, MA, USA). A TRIzol RNA isolation kit was purchased from Life Technologies (Rockville, MD). Primers for RT–PCR, dNTP, reverse transcriptase and Taq polymerase were obtained from Gibco BRL (Cergy Pontoise, France). All other chemicals were of the highest pure grade available. Molecular mass markers for proteins were obtained from Pharmacia Biotech (Saclay, France).

Cell culture
Human hepatoma cells (HepG2 cells) were obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsin Chu, Taiwan). Cells were grown in DMEM, supplemented with 10% (v/v) FBS (Gibco Brl), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.37% (w/v) NaHCO3, 0.1 mM NEAA, 1 mM sodium pyruvate and 0.03% l-glutamine at 37°C, in a humidified atmosphere of 95% air and 5% CO2. The culture medium was renewed each day. Cells were detached weekly and transfered with 0.1% trypsin and 10 µM EDTA in PBS.

Assay of phenol sulfotransferase activity
Induction of the PST-P by phenolic acid was determined by the phenol sulfotransferase assay (5). The cells were grown in 12-well plates (Costar 3524, Corning, Corning, NY) for 24 h, and then induced for another 24 h by exposure to the fresh medium containing different concentrations of each tested compound. Each test compound was supplemented with the culture medium at concentrations of 10 and 30 µM, respectively. All phenolic acids were solubilized in MeSO4, and the final solvent concentration within all compounds containing cultures and the appropriate vehicle control cultures was adjusted to 0.1%. Treated cells were scraped off, washed and suspended in ice-cold 5 mM potassium phosphate buffer, pH 7.4, before homogenization to produce a cell homogenate. Aliquots of the cell homogenates were collected and immediately tested for PST-P activity. The incubation mixture containing 100 µl of 0.1 M potassium phosphate buffer (pH 7.0), 20 µl of the HepG2 cell homogenates, 20 µl of the substrate and 20 µl of [35S]-labeled PAPS (final concentration 6.7 µM) was added at successive intervals to tubes at 37°C in a water bath, and the reaction was terminated after 20 min by the addition of 0.1 M barium acetate (200 µl). Any unreacted PAPS, free sulfate or protein was precipitated by two additions of 0.1 M barium hydroxide (200 µl) followed by 0.1 M zinc sulphate (200 µl). After centrifugation (11 500x g for 3 min), 500 µl of the supernatant was thoroughly mixed with 4 ml scintillant, and radioactivity was measured by liquid scintillation spectrometry. The protein content of cell homogenates was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA); PST-P acitivity was expressed as pmol/min/mg of protein. All samples were assayed in triplicate in three independent experiments.

RNA extraction and RT–PCR
The expression value of PST-P was quantified by semi-quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) analysis, using glyceraldehydes 3-phosphate dehydrogenase (GAPDH) mRNA as an internal standard. HepG2 cells (1 x 106 in 10 ml medium) were plated in 100 mm tissue culture dishes. After preincubation for 24 h, HepG2 cells were subjected to a time-course by phenolic acids in 0.1% MeSO4. Cellular RNA was extracted by a TRIzol RNA isolation kit (Life Technologies) as described in the manufacturer's manual. RNA concentration and purity were determined based on measurement of the absorbances at 260 and 280 nm. After adding RNase inhibitor (20 U), the total RNA was stored at –70°C. The primer sequences and product sizes were as follows: PST-P forward 5'-ATGGAGCTGATCCAGGACAC-3', reverse 5'-TGACCTACCGT-CCCAGGCCC-3', 987 bp; and GAPDH forward 5'-GACCCCTTCATTGACCTCAAC-3', reverse 5'-CATACCAGGAAATGAGCTTG-3', 300 bp. Briefly, from each sample, 250 ng of RNA was reverse-transcribed using 200 U of Superscript II reverse transcriptase, 20 U of RNase inhibitor, 0.6 mM of dNTP and 0.5 µg/µl of oligo (dT) 12–18. Then, PCR analyses were performed on the aliquots of the cDNA preparations to detect PST-P and GAPDH (the internal standard) gene expression using the FailSafe PCR system (Epicenter Technologies, Madison, WI). The reactions were carried out in a final concentration volume of 50 µl containing 50 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MnCl2, 0.2 mM dNTP, 2 U of Taq DNA polymerase and 50 pmol of 5' and 3' primers. After initial denaturation for 2 min at 95°C, 30 cycles of amplification (at 95°C for 1 min, 60°C for 1 min and 72°C for 1.5 min) were performed, followed by a 7 min extension at 72°C. A 10 µl aliquot from each PCR reaction was electrophoresed in a 1.8% agarose gel containing 0.2 µg/ml ethidium bromide. The gel was then photographed under ultraviolet transillumination. For quantification, the PCR bands on the photograph of the gel were scanned by a densitometer linked to a computer analysis system. We normalized the PST-P signal relative to the corresponding GAPDH signal from the same sample and expressed the data as the PST-P/GAPDH ratio.

Construction of the reporter vector for PST-P promoter
The PST-P promoter was amplified by PCR using sense 5'-GCATGGGTACCGACAAGTGCAAAAGTCATGAACGTA-3' and anti-sense 5'-GCATCGCTCGAGGTTCCTGCGTCAGGGGCCAGA-3' primers. Both primers were designed based on GeneBank accession no. U34804 [GenBank] (additional restriction sites are indicated by underlines). The synthesized 2320 bp fragments were cloned into a TA-vector (Promega Corporation, Madison, WI). The 2320 bp fragments of the PST-P promoter with KpnI and XhoI sites were subcloned into the promoterless firefly luciferase vector pGL3-Basic (Promega Corporation, Madison, WI) resulting in a pGL3/hPST-P plasmid.

Transient transfection and assays for reporter gene activity
HepG2 cells were seeded into 35 mm plates at a concentration of 1 x 105 cells/plate 1 day before transfection. Cells were transiently transfected with 3 µg of plasmid DNA containing 1 µg of the Renilla luciferase construct, pRL-TK (Promega Corporation, Madison, WI), to control transfection efficiency, and 2 µg of the appropriate pGL3/hPST-P promoter firefly luciferase construct. The next day, cells were transfected with the pGL3/hPST-P plasmid and pRL-TK (internal control plasmid) using Lipofectamine (Promega Corporation, Madison, WI), according to the manufacturer's instructions. After transfection (12 h), the medium was replaced with complete medium, and incubation was continued for another 12 h. Transfected cells were then treated with gallic acid for 12 h, and lysed cells were collected. Luciferase activity was recorded by a FLUOstar galaxy luminescence plate reader (BMG LabTechologies, GmbH, Offenburg, Germany) and the dual luciferase kit (Promega Corporation, Madison, WI) according to the manufacture's instructions. Luciferase activity of reporter plasmids was normalized to luciferase activity of the internal control plasmid.

Preparation of nuclear extracts
HepG2 cells (1 x 107) in dishes were washed three times with cold phosphate-buffered saline (PBS) and harvested by centrifugation at 1100 r.p.m. for 10 min. The cell pellet was carefully resuspended in three pellet volumes of cold buffer containing 20 mM HEPES (pH 7.0), 0.15 mM EDTA, 0.015 mM EGTA, 10 mM KCl, 1% Nonidet-40, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate and 1 mM Na3VO4. The homogenate was then centrifuged at 500x g for 20 min, and the nuclear pellet was resuspended in five pellet volumes of cold buffer containing 10 mM HEPES (pH 8.0), 25% glycerol, 0.1 M NaCl, 0.1 mM EDTA, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate and 1 mM Na3VO4. After centrifugation at 500x g for 20 min, nuclei were resuspended in two pellet volumes of hypertonic cold buffer containing 10 mM HEPES (pH 8.0), 25% glycerol, 0.4 M NaCl, 0.1 mM EDTA, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate and 1 mM Na3VO4, and were incubated for 30 min at 4°C on a rotating wheel. Nuclear debris was removed by centrifugation at 900x g for 20 min at 4°C; one part of the supernatant was resolved by sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) and submitted to immunoblot analysis using anti-Nrf2 and anti-PDI antibodies. The rest of the supernatant was used for electrophoretic mobility shift assay (EMSA). Protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).

Electrophoretic mobility shift assay (EMSA)
EMSA was performed using a DNA-protein binding detection kit (Gibco BRL) according to the manufacture's protocol. Briefly, oligonucleotides for ARE (5'-CTACGATTTCTGCTTAGTCATTGTCTTCC-3') and Specific protein-1 (5'-ATTCGATCGGGGCGGGGCGAGC-3') were labeled with [{gamma}-32P]ATP by T4 polynucleotides kinase and purified on a Nick column (Amersham Pharmacia Biotech, NJ). The reaction mixture (20 µl) contained 4 µl of 5x binding buffer [containing 20% glycerol, 5 mM MgCl2, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM DTT, 0.25 mg/ml poly dI–dC and 50 mM Tris–HCl (pH 7.5)], 15 µg of nuclear extract and sterile water. The reaction mixture was preincubated without the probe at room temperature for 10 min. The probe (1 µl, containing 106 c.p.m.) was then added, and DNA-binding reactions were carried out for 50 min at room temperature. In some analyses, specificity of the binding was determined by competition experiments, which were carried out by adding a 20-fold molar excess of an unlabeled ARE to the reaction mixture before the labeled probe was added. Specific protein-1 (SP-1) oligonucleotide was used as a negative control for competition experiments. After 50 min incubation at room temperature, 2 ml of 0.1% bromophenol blue was added, and samples were electrophoresed through a 6% non-denaturating polyacrylamide gel at 150 V for 2 h. Finally, the gel was dried and exposed on an X-ray film.

Western blotting
The gallic acid-treated and -untreated cells were rinsed twice with PBS (pH 7.0) and the total proteins were extracted by adding 200 µl of cold lysis buffer (50 mM Tris–HCl (pH 7.4), 1 mM NaF, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethanesulfonyl fluoride, 1% NP-40 and 10 µg/ml leupeptin) to the cell pellets, on ice, for 30 min; this was followed by centrifugation at 10 000 g for 30 min at 4°C. Western blotting was performed according to the method used by Maiti and Chen (19). The proteins in the cytosolic fraction (supernatant) were measured by Bradford assay with bovine serum albumin (BSA) as the standard. The samples (50 µg of protein) were mixed with 5x sample buffer containing 0.3 M Tris-HCl (pH 6.8), 25% 2-mercaptoethanol, 12% SDS, 25 mM EDTA, 20% glycerol and 0.1% bromophenol blue. The mixtures were boiled at 95°C for 5 min and then subjected to 12% SDS-polyacrylamide minigels at a constant current of 20 mA. Electrophoresis was carried out on SDS–PAGE. Following electrophoresis, proteins on the gel were electrotransferred onto an immobile membrane (PVDF; Millipore, Bedford, MA) with a transfer buffer composed of 25 mM Tris–HCl (pH 8.9), 192 mM glycine and 20% methanol. The membrane was then washed with Tris-buffered saline (10 mM Tris, 150 mM NaCl) containing 0.05% Tween-20 (TBST) and blocked in TBST containing 5% non-fat dried milk. Each membrane was further incubated overnight at 4°C with its respective specific antibodies [PST-P (1:2000), Nrf2 (1:2000), p-ERK (1:2000), ERK (1:2000), p-JNK (1:1000), JNK (1:2000), p-p38 MAPK (1:1000), p38 MAPK (1:2000), PDI (1:2000) and ß-actin (1:5000)]. After hybridization with primary antibodies, the membrane was washed with TBST three times, incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 45 min at room temperature and washed with TBST three times. The final detection was performed with ECLTM (enhanced chemiluminescence) western blotting reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Detection of cell viability using MTT reduction assay
Cell proliferation was determined by the 3-(4,5-Dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) assay. HepG2 cells were seeded onto 96-well plates at a concentration of 1 x 104 cells/well in DMEM plus 10% FBS. After incubating for 24 h, some of the cells were treated with 30 µM gallic acid for 16 h. After changing the medium, t-BHP (tert-butyl hydroperoxide; 0, 20, 40, 60, 80 and 100 µM) was added to the media for 8 h. The controls were treated with 0.1% MeSO4 only. Dye solution (10 µl), specific for the MTT assay, was added to each well for an additional 4 h incubation at 37°C. After the addition of MeSO4 (100 µl/well), the absorbance at 570 nm (formation of formazan) and 630 nm (reference) were recorded by a Fluostar Galaxy plate reader (BMG Lab Technology, GmbH, Offenburg, Germany). The percent viability of the treated cells was calculated as follows: (A570nm–A630nm)sample/(A570nm–A630nm)control x 100.

Statistical analysis
Each experiment was performed in triplicate and repeated three times. The results were expressed as means ± SD. Statistical comparisons were made by one-way analysis of variance (ANOVA), followed by a Duncan multiple-comparison test. Differences were considered significant when the P-values were <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Induction of PST-P activity by phenolic acids
To determine the influence of phenolic acids on Phase II PST-P activity, we used human hepatoma HepG2 cells, which have been widely used in drug metabolism and chemoprevention studies (20). HepG2 cells possess a robust metabolic system responsive to various environmental stimuli and stresses, which may be attributed to their well-developed microsomal system and hepatic origin. The effects of various phenolic acids on PST-P activity were measured after treatment with 10 and 30 µM of phenolic acids. We found that treatments below 30 µM of phenolic acids had no significant effect on the cell growth of HepG2 cells (data not shown). The result showed that PST-P activity of the control (without phenolic acids) was 22 ± 3 pmol/min/mg protein. Moreover, the PST-P activity was influenced by the addition of phenolic acids. The induction of PST-P activity, after 24 h of exposure to different concentrations (10 and 30 µM, respectively) of each phenolic acid, is shown in Figure 1A. Gentisic acid, p-hydroxybenzoic acid, p-coumaric acid and gallic acid were found to increase PST-P activity. Among the phenolic acids tested, the greatest proportionate induction of PST-P activity was observed in the treatment with either gallic acid or gentisic acid; both induced PST-P activity by 2.5- and 3.3-fold, respectively, at a concentration of 30 µM. In concentrations of 30 µM, both p-coumaric acid and p-hydroxybenzoic acid moderately increased PST-P activity, by 1.4- and 1.8-fold. However, no significant effect was found in the treatment with ferulic acid in this in vitro model. Gallic acid, a trihydroxybenzoic acid, has been found to cause a significant increase in the activity of PST-P in a dose-dependent manner (Figure 1B); thus, we proceeded using gallic acid, the strongest inducer, to elucidate its molecular mechanism on the induction of PST-P. Because the concentration of gallic acid at 30 µM was capable of inducing maximal activity of PST-P in HepG2 cells, all subsequent experiments involving gallic acid were performed using a concentration of 30 µM.


Figure 1
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  		Fig. 1. Induction of PST-P activity by phenolic acids. HepG2 cells were treated with 10 and 30 µM phenolic acids for 24 h, and PST-P activity was measured as described in Materials and methods. The results are presented as the ratios of PST-P activities in the treated cells to vehicle control. Each value of the ratio of PST-P activity represents the mean ± SD of three independent experiments. *P < 0.05 versus vehicle control. The mean uninduced activity in the vehicle control cells was 22 ± 3 pmol/min/mg protein.

 
Gallic acid induces PST-P expression
The effect of gallic acid treatment on PST-P mRNA expression was further examined in HepG2 cells by RT–PCR analyses. As shown in Figure 2A, exposure to gallic acid (30 µM) increases PST-P mRNA levels in HepG2 cells in a time-dependent manner. A marked induction of PST-P mRNA expression was observed after a 6 h exposure of gallic acid; maximum induction of PST-P mRNA expression was observed at 24 h. Western bolt analyses using polyclonal anti-PST-P antibodies confirmed that the increase in PST-P mRNA levels by gallic acid was accompanied by an increase in PST-P protein expression (Figure 2B). To know the induction time of PST-P activity by gallic acid, a time-course study was also undertaken. Significant induction of PST-P activity was observed at 24 h after the addition of 30 µM gallic acid (Figure 2C). The enhanced PST-P mRNA and protein expression in HepG2 cells corresponded to the induction of PST-P activity after treatment with gallic acid, suggesting that the observed induction of PST-P activity by gallic acid is due to the increase in PST-P protein synthesis through activation of gene transcription.


Figure 2
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  		Fig. 2. Induction of PST-P mRNA and protein by gallic acid in HepG2 cells. HepG2 cells were incubated in the absence or in the presence of gallic acid (30 µM) for the indicated times. (A) Cellular RNA was extracted by invitrogen RNA isolation kit and RT–PCR was performed as described in Materials and methods. Expression of PST-P mRNA was analyzed by RT–PCR. GAPDH, the housekeeping gene, was used as an internal control. (B) Whole cell lysates were subjected to western blot analysis using a polyclonal anti-PST-P antibody. (C) The activity of PST-P was measured as described in Materials and methods. The results are presented as the ratios of PST-P activities in the treated cells to vehicle control. Each value of the ratio of PST-P activity represents the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 versus vehicle control. The mean uninduced activity in the vehicle control cells was 22 ± 3 pmol/min/mg protein.

 
Transcriptional and translational regulation of PST-P induction by gallic acid
To examine the mechanism that causes PST-P expression to increase in response to gallic acid, we first examined whether PST-P mRNA expression was dependent on the absence or presence of RNA synthesis inhibitors actinomycin D and cycloheximide followed by RT–PCR analyses. As shown in Figure 3, gallic acid-mediated induction of PST mRNA was completely abolished in the presence of actinomycin D (5 µg/ml). These data suggest that gallic acid increased PST-P gene expression by enhancing gene transcription. To further determine regulation of PST-P expression by gallic acid, HepG2 cells were pretreated with the protein synthesis inhibitor cycloheximide (10 µg/ml) prior to treatment with gallic acid and then analyzed for PST-P mRNA expression. Cycloheximide significantly attenuated the upregulation of PST-P mRNA steady-state levels in response to gallic acid treatment, suggesting that synthesis of new proteins was also required for gallic acid-induced PST-P mRNA expression (Figure 3).


Figure 3
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  		Fig. 3. Effects of actinomycin D and cycloheximide on PST-P mRNA in HepG2 cells after gallic acid treatment. HepG2 cells were pretreated with either actinomycin D (5 µg/ml) or cycloheximide (10 µg/ml) for 4 h and then treated with 30 µM gallic acid for 24 h. Total RNA was extracted and analyzed for PST-P mRNA expression by RT–PCR. GAPDH, the housekeeping gene, was used as an internal control. The intensity of indicated PST-P mRNA was detected by densitometric analysis and expressed as folds of control. Data shown are representative of three independent experiments.

 
Induction of PST-P promoter-driven luciferase expression by gallic acid
To determine whether increased transcription of the PST-P gene was mediated by its 5'-untranslated region, the luciferase reporter plasmid, flanked by ~2300 bp upstream of 5'-untranslated region of the human PST-P gene, was constructed as described in Material and methods. This construct was transiently transfected into HepG2 cells for 24 h followed by increasing concentrations of gallic acid from 1 to 30 µM. After incubation for another 12 h, cells were harvested and analyzed for luciferase activity. As shown in Figure 4, luciferase activity increased in response to the increased concentrations of gallic acid from 1 to 30 µM. Furthermore, the activity of the PST-P promoter increased 3.2-fold in cells exposed to 30 µM of gallic acid. This result was in agreement with the findings given in Figure 1B.


Figure 4
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  		Fig. 4. PST-P promoter activity in relation to the concentration of gallic acid. HepG2 cells were transiently transfected with pGL3/hPST-P and pRL-TK for 24 h, followed by treatment with increasing concentrations of gallic acid, as described in Materials and methods. Ferulic acid (FA, 30 µM) was used as a control. Luciferase activities of the reported plasmid were normalized to those of the internal control plasmid and are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 versus vehicle control.

 
Gallic acid stimulates Nrf2 protein expression
Nrf2 is essential for ARE-mediated induction of Phase II detoxifying enzymes. To assess whether gallic acid increased the binding activity of Nrf2 to ARE, a nuclear extract isolated form HepG2 cells treated with gallic acid was probed with a radiolabeled ARE sequence. The EMSA revealed that gallic acid at a concentration of 30 µM increased ARE binding activity in a time-dependent manner. ARE binding activity began to increase 6 h after exposure of cells to gallic acid and extended to 12 h (Figure 5A). Competition experiments using excess amounts of either unlabeled ARE or SP-1 oligonucleotide confirmed the specificity of protein binding to the ARE. However, the addition of a 20-fold excess of an unlabeled ARE to the activated nuclear extract completely abolished ARE binding, while excess unlabeled SP-1 oligonucleotide failed to inhibit the DNA binding (Figure 5B). To further investigate the nuclear translocation of Nrf2 induced by gallic acid, HepG2 cells were treated with 30 µM gallic acid for 0, 1, 3, 6 and 12 h. Nrf2 proteins in the nuclear compartments of the cells were detected by western blot (Figure 5C). After gallic acid treatment, Nrf2 protein in the nuclear fraction increased compared with the control, whereas tert-Butyl hydroquinone (t-BHQ) strongly induced nuclear-induced nuclear translocation of Nrf2 (Figure 5C). These data suggested that induction of PST-P by gallic acid might be mediated by the activation of the transcription factor Nrf2.


Figure 5
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  		Fig. 5. Effect of gallic acid on the activation of nuclear factor Nrf2 binding to the ARE. (A) EMSA analysis of the ARE transcription complex. Nuclear extracts were prepared from HepG2 cells treated with gallic acid (30 µM) for 1–12 h. Nuclear extract (10 µg) was then incubated for 30 min with 5 ng of radiolabeled oligonucleotide containing the ARE sequence and separated on a 4% PAGE. Arrowhead indicates the ARE binding complex. (B) Competition assays with 20-fold molar excess of unlabeled ARE oligonucleotide or SP-1 oligonucleotide added to the reaction mixture that included radiolabeled ARE-containing oligonucleotide (5 ng) and nuclear extracts prepared form HepG2 cells treated with gallic acid (30 µM) for 6 h. (C) Immunoblot analysis of the level of Nrf2 in the nuclear fraction of HepG2 cells treated with gallic acid (30 µM) for 1–12 h or t-BHQ (30 µM) for 6 h. Immunoreactive protein bound with rabbit anti-Nrf2 antibody was visualized by an ECLTM kit after incubation with HRP-conjugated secondary antibody. Anti-protein-disulfide isomerase (PDI) antibody was used for normalization. Results were confirmed by three separate experiments, and a representative immunoblot is shown.

 
Involvement of the p38 pathway in the induction of PST-P expression by gallic acid in HepG2 cells
Recently, studies on Phase II detoxifying enzyme induction by oxidative stress stimuli have shown that pathways involving MAPKs are responsible for the transduction of signals initiating gene activation (21). To determine whether a similar signal mechanism is responsible for the upregulation of PST-P expression by gallic acid in HepG2 cells, we examined the activation states of three MAPK subfamilies, JNK, ERK and p38, in HepG2 cells. Cells were exposed to gallic acid and then immunoblots were performed using anti-phospho-JNK, ERK and p38. As shown in Figure 6A, gallic acid increased the levels of phosphorylated JNK and p38. The same blots were probed with the antibody to total JNK, ERK and p38 as protein loading controls. The gallic acid-mediated increase in PST-P protein expression was completely blocked by SB203580 (a specific inhibitor of p38) and moderately blocked by SP600125 (a specific inhibitor of JNK), whereas a similar concentration of PD98059 (a specific inhibitor of ERK) had no significant effect (Figure 6B). These results indicated that a kinase in the p38 pathway might be involved in the regulation of PST-P expression by gallic acid.


Figure 6
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  		Fig. 6. Effect of gallic acid on phosphrylations of the MAPKs in HepG2 cells. (A) The cells incubated in the absence or presence of gallic acid (30 µM) for the indicated times were subjected to western blot analysis using phosphor-specific antibodies to JNK, ERK or p38. As controls, the same cell lysates were subjected to western blot analysis using corresponding non-phospho-specific antibodies to detect total JNK, ERK or p38. (B) Cells were pretreated with PD98059 (10 µM), SP600125 (10 µM) or SB203580 (20 µM) for 2 h and then exposed to 30 µM gallic acid for 24 h in the presence of inhibitor. Western blot analysis was performed using specific antibodies for PST-P and ß-actin. The intensity of indicated PST-P proteins was detected by densitometric analysis and expressed as folds of control. Data shown are representative of three independent experiments.

 
Stabilization of Nrf2 is dependent on phosphorylation by MAPK pathway
It has been previously reported that the MAPK signaling pathway may be involved in the regulation of the ARE response (15). We sought to determine whether phosphorylation mediated by this pathway may play a role in promoting Nrf2 stability. In this experiment, HepG2 cells were pretreated with either SP600125 or SB203580 for 2 h followed by gallic acid treatment for 6 h. The lysates were analyzed by immunoblotting with both anti-Nrf2 and anti-PDI antibodies. The results of this experiment showed that both of these compounds attenuated the inducing effects of gallic acid on the Nrf2 protein level, by 60 and 70%, respectively (Figure 7). These data suggest a link between phosphorylation and Nrf2 stability and a positive effect from the p38/JNK MAPK pathway.


Figure 7
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  		Fig. 7. Phosporylation by the p38/JNK MAPK pathway increases Nrf2 stability. (A) HepG2 cells were treated with control (lane 1), or 30 µM gallic acid (lane 2) for 6 h, 10 µM SP600125 (lane 3), 20 µM SB203580 (lane 4) for 2 h, followed by gallic acid for 6 h. The total cell lysates were analyzed by immunoblotting with rabbit anti-Nrf2 and anti-PDI antibodies. (B) The result in A was quantitated using a densitometer, and the Nrf2 value was plotted after normalization with those of anti-PDI. The blots shown are representative of three independent experiments with similar results.

 
Effect of gallic acid on t-BHP-induced oxidative injury in HepG2 cells
To examine whether gallic acid-elevated cellular PST-P mRNA expression could lead to cytoprotection against oxidative injury, HepG2 cells were pretreated with and without 30 µM gallic acid for 16 h. After changing the medium, cells were exposed to different concentrations of t-BHP (0, 20, 40, 60, 80 and 100 µM) for 8 h. Cell viability was determined by an MTT assay. As shown in Figure 8A, cell viability of HepG2 cells pretreated with gallic acid was significantly higher at all concentrations of t-BHP compared with untreated cells. Next, we analyzed the long-term effect of gallic acid after a 12 h incubation, a time consistent with the induction of PST-P expression through the MAPKs pathway described previously in this study. The cytoprotective effect of gallic acid in the presence of PST-P inhibitor DCNP and the p38 MAPK inhibitor SB203580 was compared. HepG2 cells were maintained in low serum medium for 12 h, then pretreated with vehicle (50 µM DCNP or 20 µM SB203580) for 15 min, exposed to gallic acid for 12 h as indicated in Figure 8B, incubated with 200 µM t-BHP and then immediately analyzed by MTT assay. As shown in Figure 8B, DCNP significantly blocked the cytoprotective effect of gallic acid after incubation for 12 h. Similarly, inhibition of p38 MAPK by 20 µM SB203580 partially reduced the cytoprotective effect of gallic acid after incubation for 12 h. These results indicate that induction of PST-P by gallic acid through the p38 MAPK pathway is essential to elicit the cytoprotective effect of gallic acid against oxidative stress.


Figure 8
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  		Fig. 8. Effect of gallic acid on t-BHP-induced oxidative injury in HepG2 cells. (A) HepG2 cells were either untreated or treated with 30 µM gallic acid for 16 h. After changing the medium, cells were exposed to different concentrations of t-BHP (0, 20, 40, 60, 80 and 100 µM) for 8 h. Cell viability was determined by an MTT assay. *P < 0.05 and **P < 0.01 versus corresponding group without gallic acid pretreatment. (B) HepG2 cells were maintained in low serum medium for 12 h pretreated with either 50 µM DCNP or 20 µM SB203580 for 15 min and then exposed to gallic acid for 12 h as indicated. During the last 30 min of gallic acid incubation, cells were challenged with 200 µM t-BHP and then analyzed by MTT assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Chemoprevention is one of the most promising areas in cancer research. Potential chemopreventive agents may function by a variety of mechanisms directed at all major stages of carcinogenesis. One proposed mechanism for cellular protection against the chemical and neoplastic effects of carcinogens involves the induction of Phase II detoxification enzymes (22). A large body of evidence, based on preclinical and clinical research, also indicates that modulation of the body's Phase II detoxification enzymes could provide an effective approach for cancer prevention (23). Understanding the molecular mechanism of Phase II detoxification enzyme modulation is critically important in designing rational cancer preventive agents. Phenolic acids have been shown to be protective agents against chemically induced cardiotoxicity, mutagenesis and carcinogenesis (24). Although the mechanism of phenolic acids has not been fully elucidated, recent studies have shown the induction of Phase I and Phase II enzymes to be one major mechanism (25). More recently, Yeh et al. (5,10) have demonstrated that Phase II PSTs induction by phenolic acids may also play an important role in chemoprevention.

In the present study, we examined the PST-P-inducing potency of phenolic acids and its molecular mechanism in human HepG2 cells. The results of our study showed that gallic acid is capable of strongly inducing the expression of PST-P in a concentration-dependent manner (Figure 1). These results are in agreement with our previous findings (5), in which gallic acid, gentisic acid, p-hydroxybenzoic acid and p-coumaric acid were found to enhance the activity of PST-P. These phenolic compounds, especially gallic acid, gentisic acid, p-hydroxybenzoic acid and p-coumaric acid exhibited strong antioxidant activity in the oxygen radical absorbance capacity (ORACFormula) assay and the trolox equivalent antioxidant capacity (TEAC) assay. Thus, a significant correlation was found between the promotion effect in PST-P activity and the antioxidant capacity of phenolic compounds (5). The observed effects of phenolic acids in the present study indicate that PST-P can detoxify carcinogens. Indeed, numerous animal and cell culture studies have shown that naturally occurring polyphenols can exert anticarcnogenic and cytoprotective effects against diverse chemical carcinogens and mutagens via induction of Phase II detoxification enzymes (26). On the basis of the induction potency of PST-P activity by five phenolic acids in cultured human HepG2 cells, gallic acid, showing the strongest potent induction, was used to investigate the molecular mechanisms of phenolic acids on PST-P activity in HepG2 cells.

Flavonoids and other dietary phenolics have been shown to strongly inhibit neoplastic transformation in mammary organ cultures and epithelial cells, inhibit benzo[a]pyrene DNA adduct formation and induce the Phase II metabolizing enzymes GST, NQO and UGT (27). Phase II enzyme induction may explain the chemopreventive effect of polyphenolic compounds in inhibiting heterocyclic amine-induced colonic aberrant crypt foci formation in rats (28). The synthetic flavonoid 4'-bromoflavone was the most potent in vivo inducer of NQO and reduced glutathione (GSH) synthesis enzymes and prevented mammary carcinogenesis in rats induced by polycyclic aromatic hydrocarbons (29). Dietary flavonoid/phenolic-mediated induction of UGT may be important for the glucuronidation and detoxification of colon and other carcinogens, as well as for the metabolism of therapeutic drugs (30). Studies of the mechanisms, by which polyphenolic compounds are involved in chemoprevention, constitute an increasingly active area of research. The effects of polyphenolic compounds on Phase I enzymes, such as cytochromes P450, or on Phase II enzymes, such as GST and NQO-1, appear to involve multiple mechanisms (31). Gallic acid, and its catechin derivatives, has demonstrated excellent chemopreventive effects in many target organs challenged with various carcinogens. A number of studies indicate that gallic acid is a potent inducer of Phase II drug metabolism enzymes; this molecular mechanism is thought to involve transcriptional upregulation of Phase II genes (4). To our knowledge, this is the first reported analysis of human PST-P expression in response to in vitro exposure to gallic acid. Gallic acid stimulated a time-dependent PST-P mRNA and PST-P protein induction without significant toxicity in our experimental conditions. We further confirmed that the gallic acid-induced increases in PST-P mRNA and protein were accompanied by corresponding increases in PST-P enzyme activity (Figure 2). Thus, our data provide clear evidence that gallic acid is a potent inducer of PST-P in human hepatoma HepG2 cells. Furthermore, the stimulation PST-P expression by most inducers has been shown to occur primarily as a consequence of transcriptional regulation of the PST-P gene. In fact, cis-acting DNA sequences involved in induction by various agents have been identified in the PST-P gene from several species (32). In this study, we have demonstrated that activation of the transcription of PST-P promoter/luciferase reporter activity was induced in relation to increasing concentrations of gallic acid (Figure 4). In addition to the finding that transcription of PST-P is induced by gallic acid, we also found that both actinomycin D (a transcriptional inhibitor) and cycloheximide (a translation inhibitor) eliminated gallic acid-mediated PST-P mRNA expression (Figure 3). Based on our findings, we suggest that PST-P is also subject to post-transcriptional control in HepG2 cells exposed to gallic acid. From our results, we suggest that PST-P gene induction by gallic acid is primarily regulated at the transcription level.

The transcriptional activation of the Phase II enzymes has been traced to either the cis-acting transcriptional enhancer called ARE or, alternatively, to the electrophile response element. It has been shown that the transcription factor Nrf2 positively regulates the ARE-mediated expression of Phase II detoxification enzyme genes and stress-induced genes (15). The activity of Nrf2 is normally suppressed in the cytosol by specific binding to the chanperone Keap 1. Nrf2 and Keap 1 have been reported to be the primary sensors in the cellular response to oxidative stress (33). The cytoplasmic protein Keap 1 interacts with Nrf2 and inhibits its nuclear translocation. In addition, the disulfide bond in Keap 1 is thought to be a sensor for oxidative conditions. Signals associated with oxidative stress disrupt the binding between Nrf2 and Keap 1, resulting in the translocation of Nrf2 into the nucleus. In the nucleus, Nrf2 forms a heterodimer with small-Maf protein and activates the transcription of oxidative stress-related proteins via an ARE (34). However, upon stimulation by electrophilic agents or compounds that possess the ability to modify thiol groups, Keap 1 repression of Nrf2 activity is lost, allowing Nrf2 protein to translocate into the nucleus and potentate the ARE sequence (35). This mechanism of gene activation leads to the synthesis of highly specialized proteins that efficiently protect mammalian cells from various forms of stress and, consequently, reduce the propensity of tissues and organisms to develop disease or malignancy (36). Hence, activation of Nrf2, which controls constitutive and inducible expression of Phase II detoxifying genes, may be one of the protective mechanisms against xenobiotics (37). The most significant finding in this study is the demonstration of the involvement of the Nrf2 pathway in gallic acid-mediated PST-P gene induction. The electrophoretic mobility shift assay revealed that the nuclear ARE binding activity was significantly increased by gallic acid treatment in HepG2 cells. Additionally, gallic acid also increased the Nrf2 nuclear translocation (Figure 5), suggesting that increased expression of the Nrf2 protein may play a key role in gallic acid-induced PST-P gene activation.

Various polyphenol or flavone compounds, natural and synthetic, produce effects similar to gallic acid in the increase of Phase II activity. Such agents have been classified as monofunctional (Phase II) or bifunctional inducers with the capacity to increase both Phase I and Phase II enzymes (38). Recent studies have demonstrated that gallic acid has mixed aryl hydrocarbon receptor (AhR) agonist/antagonist activities, allowing it to bind to the AhR and induce cytochrome P450 1A1 transcription as well as inhibit 3-methylcholanthrene-induction of cytochrome P450 1A1 expression (39). These studies suggest that gallic acid could be a natural dietary ligand of the AhR. The ability to induce both cytochrome P450 1A1 and PST-P enzymes suggests the likelihood that gallic acid can operate as a bifucntional inducer. It has long been recognized that nearly all bifunctional inducers can activate xenobiotics responsive element (XRE) and ARE pathways concomitantly. The most common explanation is an indirect link. Briefly, bifunctional compounds first activate the AhR–XRE pathway, inducing Phase I enzymes, including cytochrome P450 1A1, which in turn metabolize these compounds. The resulting electrophilic intermediary metabolites further activate the Nrf2–ARE pathway.

MAPKs signaling cascades are stimulated by many extracellular stimuli, such as growth factors, cytokines and various environment stresses, and serve as a common signal transduction pathway shared by signals involved in proliferation, differentiation, functional activation and stress response (40). Activation of MAPKs may therefore mediate many or even opposite cellular process and the specific outcome of these events may depend on the specific stimuli and cellular context. The p38 MAPK has been reported to participate in the activation of the ARE-mediated gene by inducing xenobiotics (41). Previous studies have shown that overexpression or activation of MAPKs differentially affects Nrf2 activity and Phase II detoxifying enzymes (42). In addition, it has been shown that induction of GST by green tea polyphenol extract (GTP) treatments is mediated by c-Jun N-terminal kinase pathways (43). To identify other upstream regulatory mechanisms involved in gallic acid-induced signaling events, MAPK pathways were also examined in the present study. We found that the p38 MAPK pathway was involved in the induction of PST-P expression by gallic acid in HepG2 cells. Compared with the untreated HepG2 cells, gallic acid-treated cells had higher levels of p-p38 while the levels of p-ERK were not changed. Inhibition of the p38 MAPK pathway by SB203580 inhibitor almost completely blocked gallic acid-induced PST-P protein expression, suggesting that gallic acid induces PST-P expression via the p38 MAPK pathway (Figure 6). Furthermore, using inhibitors of the p38 MAPK pathway, it appears that this pathway may lead to phosphorylation of Nrf2 and its increased stability (Figure 7). In a previous study, Shen et al. (44) reported that activation of MAPK pathways induced ARE-mediated gene expression via the Nrf2-dependent mechanism.

t-BHP is a potent oxidizer capable of reacting with a wide range of biological molecules. Among the t-BHP-mediated deleterious alterations are oxidation of thiol-containing biomolecules, oxidation of proteins, lipid peroxidation as well as base modification and DNA single-strand breaks (45). These adverse effects are largely responsible for the cytoxicity mediated by t-BHP in target tissue/cells (46). However, whether upregulation of PST-P also affords cytoprotection against t-BHP-mediated toxicity in HepG2 cells has not been investigated. The result presented in this study clearly showed that incubation of HepG2 cells with gallic acid resulted in high resistance to t-BHP-induced cell death (Figure 8A). The involvement of PST-P in the cytoprotective action of gallic acid was examined using the PST-P and p38 MAPKs inhibitor of DCNP and SB203580, respectively. DCNP and SB203580 abrogated the protective effect of gallic acid on t-BHP-induced cell death (Figure 8B). Plant polyphenols are well known antioxidants and have been shown to protect cultured cells from oxygen stress. Furthermore, phenolic antioxidants exhibit anti-inflammatory, anti-atherosclerotic and anticarcinogenic activities (47). The anticarcinogenic activity is due to induction of the Phase II detoxifying gene through an ARE (48). Gallic acid, and its catechin derivatives, has been demonstrated to have excellent chemopreventive effects in many target organs challenged with various carcinogens. We found previously that gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenolic acid present in fruits and vegetables, was the best inducer in PST-P (5). The above results indicated that the gallic acid-mediated cytoprotection against t-BHP toxicity was significantly reduced by the induction of PST-P. These observations strongly suggested that intracellular PST-P appeared to be an important factor in gallic acid-mediated cytoprotection against t-BHP toxicity in HepG2 cells.

Phenolic acids are widely distributed in plants and are present in considerable amounts in the human diet. The intake of hydroxybenzoic and hydroxycinnamic acid has been estimated to be ~11 and 211 mg/day, respectively (49). Moreover, a recent study demonstrated that plasma concentrations of gallic acid in humans given a single dose of gallic acid (two acidum gallicum tablets) could reach 52.3 µM (50). The effect of increased gallic acid levels in plasma on PST-P remains to be proven. In the present study, the ability of this phenolic acid to modulate PST-P was found at concentrations that may well be achievable in human plasma. Our result presents an induction response of PST-P to various phenolic acids in human hepatoma cells. Therefore, the significant induction of PST-P by the phenolic acid used in this study appears to be important and suggests that phenolic acids could be used as chemopreventive agents in sulfate conjugation.

In conclusion, the results of the present study support the hypothesis that dietary polyphenols increase expression of Phase II enzymes, which, as a group, may effectively inhibit various forms of carcinogeneisis and chemical-induced cellular damage. The observations indicated that PST-P gene expression in HepG2 cells was enhanced via p38, MAPK and Nrf2. Thus, the inducibility of PST-P expression by gallic acid in HepG2 cells appears to be determined by multiple signaling molecules, indicating that increased PST-P activity is an important element in gallic acid-mediated cytoprotection against oxidative stress. PST is a key enzyme in drug metabolism, bile acid detoxification and the regulation of intratissue active hormone levels; therefore, increased expression of PST will promote the efficiency of detoxification. These findings provide some understanding of the antioxidant properties of phenolic acids.


   Acknowledgments
 
This research work was partially supported by the National Science Council, the Republic of China, under the grant NSC92-2313-B005-067.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Cooke,M.S., Evans,M.D., Dizdaroglu,M., and Lunec,J. (2003) Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J., 17,1195–214.[Abstract/Free Full Text]
  2. Collins,A.R. (2005) Assays for oxidative stress and antioxidant status: applications to research into the biological effectiveness of polyphenols. Am. J. Clin. Nutr., 81, 261–267.
  3. Kensler,T.W. (1997) Chemoprevention by inducers of carcinogen detoxification enzymes. Environ. Health Perspect., 105, 965–970.
  4. Ow,Y.Y., and Stupans,I. (2003) Gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes. Curr. Drug Metab., 4, 241–248.[CrossRef][ISI][Medline]
  5. Yeh,C.T. and Yen,G.C. (2003) Effects of phenolic acids on human phenol sulfotransferase in relation to their antioxidant activity. J. Agric. Food Chem., 51, 1474–1479.[Medline]
  6. Runge-Morris,M.A. (1997) Regulation of expression of the rodent cytosolic sulfotransferases. FASEB J., 11, 109–117.[Abstract]
  7. Richard,K., Hume,R., Kaptein,E., Stanley,E.L., Visser,T.J. and Coughtrie,M.W. (2001) Sulfation of thyroid hormone and dopamine during human development: ontogeny of phenol sulfotransferases and arylsulfatase in liver, lung, and brain. J. Clin. Endocrinol. Metab., 86, 2734–2742.[Abstract/Free Full Text]
  8. Maiti,S., Grant,S., Baker,S.M., Karanth,S., Pope,C.N. and Chen,G. (2004) Stress regulation of sulfotransferases in male rat liver. Biochem. Biophys. Res. Commun., 323, 235–241.[CrossRef][ISI][Medline]
  9. Davies,E., Tsang,C.W., Ghazali,A.R., Harris,R.M. and Waring,R.H. (2004) Effects of culture with TNF-alpha, TGF-beta and insulin on sulphotransferase (SULT 1A1 and 1A3) activity in human colon and neuronal cell lines. Toxicol. In Vitro, 18, 749–754.[CrossRef][ISI][Medline]
  10. Yeh,C.T.; Shih,P.S. and Yen,G.C. (2004) Synergistic effect of antioxidant phenolic acids on human phenol sulfotransferase activity. J. Agric. Food Chem., 52, 4139–4143.[Medline]
  11. Falany,C.N. (1991) Molecular enzymology of human liver cytosolic sulfotransferase. Trends Pharmacol. Sci., 44, 255–259.
  12. Falany,J.L., Greer,H., Kovacs,T., Sorscher,E.J. and Falany,C.N. (2002) Elevation of hepatic sulphotransferase activities in mice with resistance to cystic fibrosis. Biochem. J., 364, 115–120.[ISI][Medline]
  13. Ogawa,D., Shikata,K., Honke,K. et al. (2004) Cerebroside sulfotransferase deficiency ameliorates L-selectin- dependent monocyte infiltration in the kidney after ureteral obstruction. J. Biol. Chem., 279, 2085–2090.[Abstract/Free Full Text]
  14. Obata,T., Brown,G.E. and Yaffe,M.B. (2000) MAP kinase pathways activated by stress: the p38 MAPK pathway. Crit. Care Med., 28, 67–77.[CrossRef][ISI][Medline]
  15. Kobayashi,M., and Yamamoto,M. (2005) Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. Antioxid. Redox Signal, 7, 385–394.[CrossRef][ISI][Medline]
  16. Katsuoka,F., Motohashi,H., Engel,J.D. and Yamamoto,M. (2005) Nrf2 transcriptionally activates the mafG gene through an antioxidant response element. J. Biol. Chem., 280, 4483–4490.[Abstract/Free Full Text]
  17. Venugopal,R. and Jaiswal,A.K. (1996) Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase 1 gene. Proc. Natl. Acad. Sci. USA, 93, 14960–14965.[Abstract/Free Full Text]
  18. Enomoto,A., Itoh,K., Nagayoshi,E., Haruta,J., Kimura,T., O'Connor,T., Harada,T. and Yamamoto,M. (2001) High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol. Sci., 59, 169–177.[Abstract/Free Full Text]
  19. Maiti,S. and Chen,G. (2003) Tamoxifen induction of aryl sulfotransferase and hydroxysteroid sulfotransferase in male and female rat liver and intestine. Drug Metab. Dispos., 31, 637–644.[Abstract/Free Full Text]
  20. Xie,T., Belinsky,M., Xu,Y. and Jaiswal,A.K. (1995) ARE- and TRE-mediated regulation of gene expression: response to xenobiotics and antioxidants. J. Biol. Chem., 270, 6894–6900.[Abstract/Free Full Text]
  21. Yu,R., Lei,W., Mandlekar,S., Weber,M.J., Der,C.J., Wu,J. and Kong,A.T. (1999) Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals. J. Biol. Chem., 274, 27545–27552.[Abstract/Free Full Text]
  22. Shureiqi,I., Reddy,P. and Brenner,D.E. (2000) Chemoprevention: general perspective. Crit. Rev. Oncol. Hematol., 33, 157–167.[ISI][Medline]
  23. Kensler,T.W., Qian,G.S., Chen,J.G. and Groopman,J.D. (2003) Translational strategies for cancer prevention in liver. Nat. Rev. Cancer., 3, 321–329.[CrossRef][ISI][Medline]
  24. Abdel-Wahab,M.H., El-Mahdy,M.A., Abd-Ellah,M.F., Helal,G.K. Khalifa,F. and Hamada,F.M. (2003) Influence of p-coumaric acid on doxorubicin-induced oxidative stress in rat's heart. Pharmacol. Res., 48, 461–465.[CrossRef][ISI][Medline]
  25. Krajka-Kuzniak,V., Szaefer,H. and Baer-Dubowska,W. (2004) Modulation of 3-methylcholanthrene-induced rat hepatic and renal cytochrome P450 and phase II enzymes by plant phenols: protocatechuic and tannic acids. Toxicol. Lett., 152, 117–126.[CrossRef][ISI][Medline]
  26. Wilkinson,J. and Clapper,M.L. (1997) Detoxification enzymes and chemoprevention. Proc. Soc. Exp. Biol. Med., 216, 192–200.[Abstract]
  27. Steele,V.E., Kelloff,G.J., Balentine,D., Boone,C.W., Mehta,R., Baheri,D., Sigman,C.C., Zhu,S. and Sharma,S. (2000) Comparative chemopreventive mechanisms of green tea, black tea and selected polyphenol extracts measured by in vitro bioassays. Carcinogenesis, 21, 63–67.[Abstract/Free Full Text]
  28. Dashwood,R.H., Xu,M., Hernaez,J.F., Hasaniya,N., Youn,K. and Razzuk,A. (1999) Cancer chemopreventive mechanisms of tea against heterocyclic amine mutagens from cooked meat. Proc. Soc. Exp. Biol. Med., 220, 239–243.[Abstract]
  29. Song,L.L., Kosmeder,J.W., Lee,S.K., Gerhauser,C. And Pezzuto,J.M. (1999) Cancer chemopreventive activity mediated by 4'-bromoflavone, a potent inducer of phase II detoxification enzymes. Cancer Res., 59,578–585.[Abstract/Free Full Text]
  30. Tukey,R.H., Strassburg,C.P. and Mackenzie,P.I. (2002) Pharmacogenomics of human UDP-glucuronosyltransferases and irinotecan toxicity. Mol. Pharmacol., 62, 446–450.[Free Full Text]
  31. Ahmad,N. and Mukhtar,H. (1999) Green tea polyphenols and cancer: biological mechanisms and practical implications. Nutr. Rev., 57, 78–83.[ISI][Medline]
  32. Duanmu,Z., Kocarek,T.A. and Runge-Morris,M. (2001) Transcriptional regulation of rat hepatic aryl sulfotransferase (SULT1A1) gene expression by glucocorticoids. Drug Metab. Dispos., 29, 1130–1135.[Abstract/Free Full Text]
  33. Nguyen,T., Sherratt,P.J., Nioi,P., Yang,C.S. and Pickett,C.B. (2005) Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1. J. Biol. Chem., 280, 32485–32492.[Abstract/Free Full Text]
  34. Hong,F., Sekhar,K.R., Freeman,M.L. and Liebler,D.C. (2005) Specific patterns of electrophile adduction trigger Keap1 ubiquitination and Nrf2 activation. J. Biol. Chem., 280, 31768–31775.[Abstract/Free Full Text]
  35. Motohashi,H. and Yamamoto,M. (2004) Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol. Med., 10, 549–557.[CrossRef][ISI][Medline]
  36. Iida,K., Itoh,K., Kumagai,Y., Oyasu,R., Hattori,K., Kawai,K., Shimazui,T., Akaza,H. and Yamamoto,M. (2004) Nrf2 is essential for the chemopreventive efficacy of oltipraz against urinary bladder carcinogenesis. Cancer Res., 64, 6424–6431.[Abstract/Free Full Text]
  37. Chan,K., Han,X.D. and Kan,Y.W. (2001) An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen. Proc. Natl. Acad. Sci. USA, 98, 4611–4616.[Abstract/Free Full Text]
  38. Prochaska,H.J., Santamaria,A.B. and Talalay,P. (1992) Rapid detection of inducers of enzymes that protect against carcinogens. Proc. Natl. Acad. Sci. USA, 89, 2394–2398.[Abstract/Free Full Text]
  39. Krajka-Kuzniak,V., Szaefer,H. and Baer-Dubowska,W. (2004) Modulation of 3-methylcholanthrene-induced rat hepatic and renal cytochrome P450 and phase II enzymes by plant phenols: protocatechuic and tannic acids. Toxicol. Lett., 152, 117–126.[CrossRef][ISI][Medline]
  40. Seger,R. and Krebs,E.G. (1995) The MAPK signaling cascade. FASEB J., 9, 726–735.[Abstract]
  41. Kong,A.N., Owuor,E., Yu,R., Hebbar,V., Chen,C., Hu,R. and Mandlekar,S. (2001) Induction of xenobiotic enzymes by the MAP kinase pathway and the antioxidant or electrophile response element (ARE/EpRE). Drug Metab. Rev., 33, 255–271.[CrossRef][ISI][Medline]
  42. Yu,R., Chen,C., Mo,Y.Y., Hebbar,V., Owuor,E.D., Tan,T.H. and Kong,A.N. (2000) Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism. J. Biol. Chem., 275, 39907–39913.[Abstract/Free Full Text]
  43. Yu,R., Jiao,J.J., Duh,J.L., Gudehithlu,K., Tan,T.H. and Kong,A.N. (1997) Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated phase II enzyme gene expression. Carcinogenesis, 18, 451–456.[Abstract/Free Full Text]
  44. Shen,G., Hebbar,V., Nair,S., Xu,C., Li,W., Lin,W., Keum,Y.S., Han,J., Gallo,M.A. and Kong,A.N. (2004) Regulation of Nrf2 transactivation domain activity.The differential effects of mitogen-activated protein kinase cascades and synergistic stimulatory effect of Raf and CREB-binding protein. J. Biol. Chem., 279, 23052–23060.[Abstract/Free Full Text]
  45. Di Simplicio,P. and Rossi,R. (1994) The time-course of mixed disulfide formation between GSH and proteins in rat blood after oxidative stress with tert-butyl hydroperoxide. Biochim. Biophys. Acta., 1199,245–252.[Medline]
  46. Yen,G.C., Yeh,C.T. and Chen,Y.J. (2004) Protective effect of Mesona procumbens against tert-butyl hydroperoxide-induced acute hepatic damage in rats. J. Agric. Food Chem., 52, 4121–4127.[Medline]