Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53

Seung Joon Baek, Leigh C. Wilson and Thomas E. Eling,1

Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA

Abstract

Dietary phenolic substances including resveratrol, a stillbene compound, are found in several fruits and vegetables, and these compounds have been reported to have anti-oxidant, anti-inflammatory and antitumorigenic activities. However, the molecular mechanisms underlying the antitumorigenic or chemopreventive activities of these compounds remain largely unknown. The expression of NAG-1 [non-steroidal anti-inflammatory (NSAID) drug-activated gene-1], a member of the transforming growth factor-beta (TGF-ß) superfamily, has been shown to be associated with pro-apoptotic and antitumorigenic activities. Here, we have demonstrated that resveratrol induces NAG-1 expression and apoptosis in a concentration-dependent manner. Resveratrol increases the expression of p53, tumor suppressor protein, prior to NAG-1 induction, indicating that NAG-1 expression by resveratrol is mediated by p53 expression. We also show that the p53 binding sites within the promoter region of NAG-1 play a pivotal role to control NAG-1 expression by resveratrol. Derivatives of resveratrol were examined for NAG-1 induction, and the data suggest that resveratrol-induced NAG-1 and p53 induction is not dependent on its anti-oxidant activity. The data may provide linkage between p53, NAG-1 and resveratrol, and in part, a new clue to the molecular mechanism of the antitumorigenic activity of natural polyphenolic compounds.

Abbreviations: COX, cyclooxygenase;; FACS, fluorescence-activated cell sorter;; MTS/PMS, Owen's reagent-phenazine methosulphate;; NAG-1, NSAID-activated gene-1;; NSAIDs, non-steroidal anti-inflammatory drugs;; PCR, polymerase chain reaction;; RIPA, radioimmunoprecipitation;; TGF-ß, transforming growth factor-beta;; TPA, 12-O-tetradecanoylphorbol acetate.

Introduction

Several non-nutritive components in fruits, vegetables, herbs and spices have been found to inhibit tumor formation in experimental animals (1). Epidemiological studies have also suggested that nutrition plays an important role in carcinogenesis. Approximately 30% of cancer morbidity and mortality can potentially be prevented with the proper adjustment of diets. Many of the investigations studying the relationship between diet and cancer development have focused on resveratrol as this phenolic chemical has potent antitumorigenic and anti-inflammatory properties (2). Resveratrol is found in many plants, particularly in grape skin, and a significant amount of resveratrol is present in red wine. Resveratrol inhibits the development of pre-neoplastic lesions in mammary glands of carcinogen-treated mice and reduces tumor formation as measured by the two-stage model of skin cancer (2,3). In addition to antitumorigenic activity, resveratrol is thought to be responsible for the reduced risk of cardiovascular disease associated with moderate consumption of red wine (4). Resveratol has anti-inflammatory activity as it suppresses carragenen-induced pedal edema, an effect attributed to suppression of prostaglandin synthesis via inhibition of cyclooxygenase (COX) activity (5). Furthermore, resveratrol inhibits TPA-induced COX-2 transcription (6). The mechanism by which resveratrol exerts anti-inflammatory and antitumorigenic activities may be related to the inhibition of either COX transcription or inhibition of COX activity, but further evidence is required to support this hypothesis.

The molecular mechanism of the tumor inhibition by resveratrol is not clear, but it appears to alter several biological processes of potential importance to tumor development. For example, resveratrol inhibits ribonucleotide reductase (7), DNA polymerase (8) and COX-2 transcription (6) and acts as an agonist for the estrogen receptor (9). Therefore, the molecular mechanism of resveratrol in antitumorigenesis is probably due to multiple actions. Recently, resveratrol was reported to trigger apoptosis (10,11), and many reports suggest that resveratrol induces apoptosis in cell culture (11–14). We have investigated the stimulation of apoptosis in cultured cells by some inhibitors of prostaglandin H synthase (COX). These COX inhibitors induced apoptosis and the expression of a protein that we called NAG-1 [non-steroidal anti-inflammatory (NSAID)-activated gene-1; also known as PTGF-ß], which is a transforming growth factor-beta (TGF-ß) superfamily protein. NAG-1 has antitumorigenic activity and stimulates apoptosis in colorectal and other cancer cell lines (15). NAG-1 basal expression is up regulated by Sp1, Sp3 and COUP-TF1 transcriptional factors (16), and by activators of the p53 tumor suppressor gene (17,18). Therefore, we decided to investigate the relationship between NAG-1, p53 and resveratrol.

In this study, the effect of resveratrol on the induction of NAG-1 expression and apoptosis in colorectal and other cell lines was investigated. We report that resveratrol enhances NAG-1 expression, induces apoptosis and suppresses cell growth. Furthermore, resveratrol induces p53 protein, which regulates NAG-1 expression in the promoter region. We propose a novel mechanism that NAG-1 may mediate the antitumorigenic activity of resveratrol.

Materials and methods

Cell lines and reagents
Cell lines were purchased from ATCC (Rockville, MD). Human colorectal carcinoma cells, HCT-116, HCT-15 and human osteosarcoma cells, U2OS, were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS) and gentamicin (10 µg/ml). A549 lung epithelial carcinoma cells were grown in RPMI 1640 medium supplemented with 10% FBS and gentamicin. T-RexTM-U2OS cells were purchased from Invitrogen and maintained in DMEM with antibiotics. Resveratrol was purchased from Sigma (St Louis, MO) and dissolved in dimethylsulfoxide (DMSO). All resveratrol derivatives were generously provided by Dr Sang Kook Lee (Ewha Womans University, Seoul, Korea).

RNA isolation and northern blot analysis
When reaching 60–80% confluence in 10 cm plates, the cells were treated at indicated concentrations and times with different compounds or DMSO in the absence of serum. Total RNAs were isolated using TRIzol reagents (Life Technologies, Rockville, MD) according to the manufacturer's protocol. For northern blot analysis, 10 µg of total RNA was denatured at 55°C for 15 min and separated in a 1.2% agarose gel containing 2.2 M formaldehyde and transferred to Hybond-N membrane (Amersham, Piscataway, NJ). After fixing the membrane by UV, the blots were pre-hybridized in hybridization solution (Rapid-hyb buffer, Amersham) for 1 h at 65°C followed by hybridization with cDNA labeled with [{alpha}-32P]dCTP by random primer extension (DECAprimeII kit, Ambion, Austin, TX). The probe used was full-length NAG-1 fragment (15). After 4 h incubation at 65°C, the blots were washed once with 2x standard saline citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) at room temperature and twice with 0.1x SSC/0.1% SDS at 65°C. Messenger RNA abundance was estimated by intensities of the hybridization bands of autoradiographs using Scion Image (Scion Image, Frederick, MD). Equivalent loading of RNA samples was confirmed by hybridizing the same blot with a 32P-labeled ß-actin probe, which recognizes RNA of ~2 kb.

Western blot analysis
The level of NAG-1 expression was evaluated using western blot analysis with anti-human-NAG-1 antibody (15). Cells were grown to 60–80% confluency in 10 cm plates followed by 48 h treatment in the presence of indicated compounds. The media was harvested and concentrated ~15-fold using Centriprep 10 concentrators (Amicon, Bedford, MA). Alternatively, total cell lysates were isolated using RIPA buffer [1x phosphate-buffered saline (PBS), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS]. Proteins (30 µg) were separated by 15% SDS–PAGE and transferred for 1 h onto nitrocellulose membrane (Schleicher and Schuell, Keene, NH). The blots were blocked for 1h with 5% skimmed milk in Tris-buffered saline/Tween 0.05% (TBS-T) and probed with anti-NAG-1 antibody (1:5000 dilution in 1% skimmed milk in TBS-T) at room temperature for 4 h. After washing with TBS-T, the blots were treated with horseradish peroxidase-conjugated secondary antibody for 1 h and washed several times. Proteins were detected by the enhanced chemiluminescence system (Amersham).

Cell proliferation assay
Cell proliferation assay was performed by CellTiter 96 Aqueous Non-Radioactive cell proliferation assay kit (Promega, Madison, WI). The assay was carried out as described in the manufacturer's protocol. In 96 well plates, cells were split into 500 cells/well in 100 µl. After 16 h, cells were treated with various compounds in a 96 well plate and incubated at different time points. MTS/PMS solution (20 ml/well) was added and incubated for 1h. Absorbance at 490 nm was measured using an ELISA plate reader (Molecular Dynamics, Monlo Park, CA).

Cloning of NAG-1 promoter
The luciferase constructs containing the NAG-1 promoter were generated by polymerase chain reaction (PCR) methods using human genomic DNA (Promega). The following primers were used to generate each construct: for pNAG966/+70 clone, sense primer, 5'-TCTAGAACTCTTGACGTCAGATGATC-3', antisense, 5'-TGAGAGCCATTCACCGTCCTGAGTTC-3'; pNAG966/+41 clone, sense, 5'-TCTAGAACTCTTGACGTCAGATGATC-3', antisense, 5'-TGTGCAGGTTGCGGCTCTGAGCTGGG-3'; pNAG133/+70, sense, 5'-CACCCCCAGACCCCGCCCAGCTGTGGTCATTG-3', antisense, 5'-TGAGAGCCATTCACCGTCCTGAGTTC-3'; pNAG133/+41, sense primer, 5'-CACCCCCAGACCCCGCCCAGCTGTGGTCATTG-3', antisense, 5'-TGTGCAGGTTGCGGCTCTGAGCTGGG-3'. After PCR, each fragment was cloned into TA vector (Invitrogen), sequenced and further cloned into pGLBasic3 vector digested with XhoI/HindIII restriction enzymes.

Transfection using the luciferase reporter system
U2OS and HCT-15 cells were plated in 6 well plates at 2x105 cells/well in McCoy's 5A media supplemented with 10% fetal bovine serum. After growth for 16 h, plasmid mixtures containing 1 µg of NAG-1 promoter linked to luciferase and 0.1 µg of pRL-null (Promega) were transfected by lipofectamine (Life Technologies) according to the manufacturer's protocol. After 24 h, the media was changed to serum-free media and resveratrol was added. Cells were harvested in 1x luciferase lysis buffer after 48 h treatment with resveratrol, and luciferase activity was determined and normalized to the pRL-null luciferase activity using the Dual Luciferase Assay Kit (Promega).

Inducible expression of NAG-1 system in U2OS cells
For the generation of stable cell lines with controlled expression of NAG-1, the T-Rex system was utilized. T-RexTM-U2OS cells were purchased from Invitrogen, containing pCDNA6/TR that expresses the Tet repressor protein. The PCR fragment was amplified from pCDNA3.1/NAG-1 (15) using the following primers: 5'-GGAATTCCAACCTGCACAGCCATGCCCGGG-3' and 5'-GCTCTAGATATGCAGTGGCAGTCTTTGG-3'. The fragment was digested with EcoRI and XbaI restriction enzymes and cloned into pcDNA4/TO/myc-HisA. The plasmid was sequenced for verification and transfected into T-RexTM-U2OS cells using LipofectAMINETM (Gibco-BRL, Rockville, MD) according to the manufacturer's protocol. Cells were grown with 200 µg/ml of zeocin (Invitrogen) until colonies formed (3–4 weeks). Single colonies were isolated using cloning cylinders, and two clonal cell lines were expanded and identified by western blot analysis to over express NAG-1 protein. For induction, cells were washed in PBS and cultivated in the absence or presence of the indicated amounts of tetracycline. Cells were maintained in complete media with 100 µg/ml zeocin, 50 µg/ml hygromycin (Calbiochem, La Jolla, CA) and tet-free serum.

Colony formation
For colony formation assays of U2OS cells, 1000 cells/10 cm dish were plated and grown until colonies formed. The selected cells were stained with Giemsa and photographed.

Apoptosis assay
The DNA content for sub G1 population was determined by fluorescence-activated cell sorter (FACS). HCT-116 and U2OS cells were plated at 3x105 cells/well in 6 well plates, incubated for 16 h and then treated with different compounds in the presence of serum. The cells (attached and floating cells) were then harvested, washed with PBS, fixed by the slow addition of cold 70% ethanol to a total of 1 ml and stored at 4°C overnight. The fixed cells were pelleted, washed with 50%, 30% ethanol, followed by PBS and stained in 1 ml of 20 µg/ml propidium iodide), 1 mg/ml RNase in PBS for 20 min. 7500 cells were examined by flow cytometry using Becton Dickinson (Franklin Lakes, NJ) FACSort equipped with CellQuest software by gating on an area versus width dot plot to exclude cell debris and cell aggregates. Apoptosis was measured by the level of subdiploid DNA contained in cells following treatment with compounds using CellQuest software.

Results

Effects of resveratrol on growth and apoptosis of HCT-116 cells
To investigate the effects of resveratrol on the growth of colorectal cancer cells in culture, resveratrol at various concentrations was added into the culture medium. In the presence of different concentrations of resveratrol, a significant inhibition of cell growth was observed (Figure 1AGo). Concentrations as low as 50 µM resveratrol resulted in a complete growth arrest of HCT-116 cells, whereas the 10 µM concentration showed ~30% reduction of cell growth compared with vehicle-treated cells observed after growing for 4 days. Subsequently, to determine if resveratrol at these concentrations increased apoptosis in HCT-116 cells, flow cytometry was used to estimate apoptosis under these treatment conditions. As shown in Figure 1BGo, resveratrol also induced apoptosis (sub G1 population, M1) in a concentration-dependent manner. Therefore, in HCT-116 cells, resveratrol treatment resulted in growth arrest and enhanced apoptosis. These results are consistent with previous reports that resveratrol inhibits growth rate and induces apoptosis in cell culture (13,14,19,20).



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Fig. 1. Concentration-dependent growth retardation and apoptosis of HCT-116 cells treated with resveratrol. (A) Effect of resveratrol on HCT-116 cell growth. HCT-116 cells were plated at 500 cells/well in a 96 well plate and incubated with vehicle (DMSO) or various concentrations of resveratrol. Cell growth was measured using PMS cell proliferation kit (Promega). Values are expressed as mean ± SD of four to five replicate experiments. (B) Flow cytometric analysis of resveratrol-treated HCT-116 cells. HCT-116 cells were plated at 5x105 cells/well in 6 well plates and incubated without or with various concentrations of resveratrol for 48 h and analyzed for apoptosis as described in Materials and methods. `M1' indicates apoptotic cell population (sub G1 population), whereas x-axis indicates DNA content.

 
Resveratrol induces NAG-1 expression in a time- and concentration-dependent manner
As resveratrol induces apoptosis in HCT-116 cells (Figure 1BGo), and NAG-1 has pro-apoptotic and/or antitumorigenic properties (15), we measured NAG-1 expression after treatment with resveratrol. Northern and western analyses were performed to estimate the expression of NAG-1 in HCT-116 cells treated with resveratrol. NAG-1 mRNA expression was increased with duration of resveratrol treatment (100 µM), and the highest mRNA expression was observed after 24 h of treatment (Figure 2AGo). The NAG-1 mRNA expression was also dependent on resveratrol concentration, with a significant increase in expression observed at 10 µM, maximum expression at 50 µM and followed by a decrease in expression at 100 µM (Figure 2BGo). In addition, NAG-1 protein also increased in a concentration-dependent manner (Figure 2CGo). NAG-1 protein expression was consistent with northern data, showing a maximum induction at the 50 µM concentration. Thus, resveratrol induced NAG-1 mRNA and protein expression in a concentration- and time-dependent manner.



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Fig. 2. Northern and western analysis of NAG-1 expression in resveratrol-treated HCT-116 cells. (A) Northern blot analysis of NAG-1 expression by resveratrol. HCT-116 cells were treated with 100 µM resveratrol for various time points. Total RNA (10 µg) was loaded in each lane and transferred onto nylon membrane. The blot was hybridized with NAG-1 probe and re-probed with ß-actin cDNA. The hybridization signals were quantified using Scion Image software (Scion), and values for the 1.3 kb NAG-1 transcript were normalized to ß-actin transcripts levels. A >3-fold induction of NAG-1 was seen at 24 and 48 h time points. (B) Dose–response of NAG-1 expression. HCT-116 cells were grown in different concentrations of resveratrol for 24 h, and northern blot analysis was performed as described above. (C) Western analysis of NAG-1 expression. HCT-116 cells were treated with different concentrations of resveratrol for 48 h, and western analysis was performed with total cell lysate. The arrow indicates ~35 kDa pro-form of NAG-1.

 
Resveratrol induces NAG-1 expression in different cell lines
Resveratrol-induced apoptosis is not restricted to colorectal cells, as resveratrol is reported to induce apoptosis in lung (21) and osteoblast (22) cell lines. To test for the induction of NAG-1 by resveratrol in different cell lines, we treated A549 lung epithelial cells and U2OS osteosarcoma cells with different concentrations of resveratrol and then measured NAG-1 expression by northern blot analysis. As shown in Figure 3Go, NAG-1 gene expression was induced by resveratrol in A549 and U2OS cell lines, dependent on concentration. Interestingly, U2OS cells did not express basal NAG-1, but resveratrol significantly increased NAG-1 expression. In addition, resveratrol did not induce NAG-1 in any concentrations up to 100 µM in U937 cells (data not shown). It should be noted that HCT-116, A549 and U2OS cells are p53 positive, whereas U937 cells are p53 negative. The ability of resveratrol to increase the expression of NAG-1 is not restricted to HCT-116 colorectal cells, but is also observed in other cell lines.



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Fig. 3. NAG-1 induction in A549 and U2OS cell lines by resveratrol treatment. A549 and U2OS cells were treated with different concentrations of resveratrol for 24 h and northern blot analysis was performed as described in Figure 2AGo. The membrane was stripped and re-probed with a ß-actin probe.

 
NAG-1 has pro-apoptotic activity
To further obtain evidence in support of the hypothesis that NAG-1 expression has pro-apoptoic and/or antitumorigenic activities, we established a U2OS-NAG stable cell line that expresses NAG-1 under the control of an inducible promoter regulated by tetracycline (tet-on system). U2OS cells were chosen as NAG-1 is poorly or not expressed under basal conditions (Figure 3Go). The T-Rex system was used, and several individual clones were isolated. Two individual clones, U2OS-NAG#11 and U2OS-NAG#18, were selected for further study. First, we investigated NAG-1 expression by tetracycline. The cells were grown in the presence or absence of tetracycline and western analysis was performed to determine the expression of NAG-1. Tetracycline induced a dramatic increase in NAG-1 expression as shown in Figure 4AGo, for the two selected clones, U2OS-NAG#11 and U2OS-NAG#18. The ectopic expression of NAG-1 is first synthesized in a pro-form (~35 kDa), cleaved into a short peptide and secreted into media. Thus, the secreted NAG-1 was determined from concentrated media proteins, and the pro-form was measured in total cell lysate. Secondly, in the presence of tetracycline, U2OS-NAG cells showed a slower growth rate compared with cells grown in the absence of tetracycline (Figure 4BGo). Furthermore, in the presence of tetracycline an increase in apoptosis was observed, as measured by flow cytometry analysis (Figure 4CGo). Similar results were observed with the U2OS-NAG#11 clone (data not shown). Therefore, our data suggest that the expression of NAG-1 in U2OS-NAG cells inhibits cellular growth and stimulates apoptosis. Thirdly, to examine NAG-1 function on cell growth, we determined if the expression of NAG-1 altered the colony formation of U2OS cells. In the presence of increasing concentrations of tetracycline, and hence an increase in NAG-1 expression, a significant reduction in the number of colonies was observed in U2OS-NAG#18 cell lines. A reduction in colony forming efficiency was also dependent on the concentration of tetracycline. No significant reduction in the number of colonies was observed in the parent U2OS cell line (T-RexTM-U2OS) when treated with tetracycline (Figure 4DGo). The treatment of tetracycline on U2OS-NAG#11 produced a similar result to U2OS-NAG#18 cell lines (data not shown). Taken together, the data are consistent with the previous report that NAG-1 has pro-apoptotic and/or antitumorigenic activities (15).



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Fig. 4. Biological function of NAG-1 in antitumorigenesis. (A) Western analysis of stably transfected U2OS cells. Two individual clones, U2OS-NAG#11 and U2OS-NAG#18, were treated with 2 µg/ml of tetracycline for 48 h in the absence of serum. Total cell lysates were harvested and 30 µg of protein was loaded onto 15% SDS–PAGE. The media was also harvested, concentrated using Centricon and 30 µg of protein was loaded. Arrows indicate ~35 kDa pro-form of NAG-1 and ~12 kDa secreted form of NAG-1. (B) Cell proliferation analysis of U2OS-NAG#18 clone. Cells were grown in the presence or absence of tetracycline for 9 days. The growth rate was measured by PMS cell proliferation kit. The data represent the mean ± SD from five different experiments. (C) Apoptotic analysis of U2OS-NAG#18 clone. Cells were treated with or without 2 µg/ml of tetracycline for 2 days, and the sub G1 population was analyzed by FACS. Tet- indicates uninduced, whereas Tet+ indicates induced NAG-1. The data represent mean ± SD from three independent experiments. (D) Inhibition of colony formation by NAG-1. 1000 cells of clone U2OS-NAG#18 and parent U2OS cells (T-RexTM-U2OS) were seeded onto a 10 cm tissue culture dish and cultured for 3 weeks in the presence of the indicated amounts of tetracycline. The media was changed every 3 days with fresh tetracycline. The outgrowing colonies were fixed and stained using Giemsa.

 
Expression of NAG-1 and p53 proteins by resveratrol
Resveratrol induces apoptosis in a p53-dependent manner (11) and NAG-1 is regulated by p53 (17,18). Therefore, we examined the expression pattern of NAG-1 and p53 in the presence of resveratrol to determine a possible link between resveratrol and NAG-1 expression, mediated by p53 expression. HCT-116 cells were treated with resveratrol (50 µM), etoposide (25 µM) and sulindac sulfide (30 µM) at different time points. Etoposide was used as a known p53 activator (17), and sulindac sulfide, a COX inhibitor, was used to stimulate NAG-1 expression (15). Total proteins were then isolated and subjected to western analysis. As shown in Figure 5Go, the expression of p53 protein is increased as early as 3 h after treatment of cells with resveratrol or etoposide. However, NAG-1 expression was seen only after 24 h in both treatments, suggesting that NAG-1 expression might be mediated by p53 proteins. In contrast, sulindac sulfide did not induce p53 protein expression, but did induce NAG-1, indicating that sulindac sulfide induces NAG-1 in a p53-independent manner. The data are consistent with the previous report that NSAIDs induce NAG-1 in a p53-independent manner (15).



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Fig. 5. Expression of NAG-1 and p53 in the presence of resveratrol, etoposide and sulindac sulfide. HCT-116 cells were treated with indicated compounds, and harvested at different time points. Total protein (30 µg) was subjected to 15% SDS–PAGE. The antibodies for p53 (Santa Cruz), NAG-1 and ß-actin (Santa Cruz) were probed onto membrane as described in Material and methods. The blots are representative of three independent experiments.

 
Resveratrol induces NAG-1 expression through p53 protein
To investigate whether NAG-1 expression is dependent on p53, NAG-1 promoter activity was examined. We cloned the NAG-1 promoter (-966 to +70) by PCR as described in Material and methods. The construct, pNAG966/+70, containing two p53 binding sites (p53-A and p53-B), was linked to the luciferase gene and transfected into U2OS cells (Figure 6AGo). After 48 h treatment with different concentrations of resveratrol or etoposide, luciferase activity was measured. Etoposide treatment increased promoter activity by ~10-fold, whereas resveratrol increased promoter activity by 3–4-fold, compared with vehicle-treated U2OS cells. The concentration of resveratrol at 50 µM gave the highest luciferase activity. Next, NAG-1 deletion clones, pNAG966/+41, pNAG133/+41 and pNAG133/+70 were further generated by PCR. The pNAG966/+41 clone contained a p53-A site, whereas the pNAG133/+70 clone contained the p53-B site. The pNAG133/+41 clone contained no p53 binding sites (Figure 6BGo). Transient transfection was performed in U2OS cells (p53 wild-type) and HCT-15 cells (p53 negative) using the above constructs and treated with vehicle or 50 µM resveratrol. As shown in Figure 6BGo, the constructs lacking two p53 binding sites or having only one p53 site (p53-A) did not significantly induce NAG-1 promoter activity in the presence of resveratrol. The constructs containing the p53-B site (pNAG966/+70 and pNAG133/+70) had significantly enhanced luciferase activity compared with vehicle-treated cells, suggesting that the p53-B site has a more critical role for resveratrol-induced NAG-1 expression. The data are consistent with previous data showing that the p53-B site is more critical for etoposide-induced NAG-1 expression (17). In contrast, the same constructs were transfected into HCT-15 cells (p53 negative cell lines) and showed no induction of luciferase activity with resveratrol treatment.



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Fig. 6. NAG-1 induction by resveratrol is mediated by p53 protein. (A) Effect of resveratrol on NAG-1 promoter. The pNAG966/+70 clone linked to luciferase was transfected into U2OS cells and treated with different concentrations of resveratrol for 48 h. Etoposide was used as a positive control for NAG-1 promoter activity. The construct (1 µg) was co-transfected with 0.1 µg of pRL-null (Promega) vector, using LipofectAMINETM (Gibco-BRL), and 48 h later the promoter activities were measured by luciferase activity. Transfection efficiency for luciferase activity was normalized to the Renilla luciferase (pRL-null vector) activity. The y-axis shows fold induction (over relative luciferase activity of vehicle-treated cells as 1.0). The results show the mean ± SD of three independent transfections. (B) Each construct was transiently transfected into HCT-15 cells (left panel) or U2OS cells (right panel), and cell lysates were harvested after 48 h of treatment. The middle panel indicates a schematic diagram of the NAG-1 promoter region. Fold induction refers to ratio of luciferase activity of 50 µM resveratrol-treated cells to vehicle-treated cells. Values are mean ± SD of three independent experiments. (C) Activation of luciferase activity in p53-dependent manner. Each construct was transiently co-transfected with either empty vector (control) or p53 expression vector (p53) into HCT-15, p53-negative cells. The cells were grown for 2 days and harvested for luciferase activity. The y-axis shows fold induction (over relative luciferase activity of empty vector transfected cells as 1.0). Values are mean ± SD of four independent transfections.

 
Finally, to examine whether the transactivation of the NAG-1 promoter by p53 depends on p53 binding sites, we performed co-transfection experiments using p53 cDNA in an expression vector in HCT-15, p53 negative cells. The construct, pNAG133/+70 (p53-B site) and pNAG133/+41 (no p53 sites) clones, were transfected with either empty vector or p53 expression vector. As shown in Figure 6CGo, the ectopic expression of p53 with the pNAG133/+70 clone in p53 negative cells, enhance NAG-1 promoter activity compared with empty vector transfected cells. In contrast, no induction was observed in the pNAG133/+41 clone, suggesting that NAG-1 expression is mediated by p53 protein expression. In addition, gel-shift assay was performed using oligonucleotide corresponding p53 binding site and shift bands were identified in resveratrol-treated nuclear extracts (data not shown). Taken together, resveratrol activates NAG-1 promoter activity through the p53 binding sites and the activation of NAG-1 promoter activity was observed in HCT-15 cells by co-transfection with wild-type p53 cDNA. These results suggest that p53 has an important role in transactivating the NAG-1 promoter by resveratrol.

Structural analysis of resveratrol responsible for NAG-1 and p53 induction
The stilbenic double bonds and the position of hydroxyl groups are important factors to determine the anti-oxidant activity of resveratrol (23), and are associated with the apoptotic gene induction and COX-2 promoter activity (19,24). To study the structural characteristics of resveratrol-induced NAG-1 and p53, several resveratrol derivatives (50 µM each) were used to treat HCT-116 cells for 36 h and western analysis was performed. Changing the para hydroxyl group of resveratrol to a methoxy (MeO) group (SY-002 and SY-040) that greatly diminishes its anti-oxidant activity did not alter NAG-1 or p53 induction, as shown in Table IGo. However, the removal of the substitutions at the Ring2 para position and/or conversion to the Ring2 meta MeO (SY-001 and SY-006) eliminate activity responsible for NAG-1 or p53 induction. Interestingly, the substitution of the para hydroxyl group to a bromide group resulted in the greatest induction of NAG-1 expression. Resveratrol showed the highest induction of p53. The results suggest that the para position at Ring2 is an important factor in NAG-1 and p53 expression. In addition, the data suggest that the anti-oxidant activity of resveratrol is not directly linked to NAG-1 expression as all the resveratrol derivatives had lost their anti-oxidant activities.


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Table I. HCT-116 cells were treated with 50 µM of each compound for36 h and western analysis was performed using NAG-1 and p53 antibodies

 
Discussion

A numerous variety of naturally occurring substances have been shown to protect against experimental carcinogenesis, and it is becoming increasingly evident that certain phytochemicals, particularly those included in our daily diet, have marked cancer chemopreventive properties. Resveratrol is such a dietary chemopreventive phytochemical compound that has recently attracted considerable interest due to its remarkable multi-functional inhibitory effects on multi-stage carcinogenesis. Resveratrol has chemopreventive effects on several cancers (25,26). The mechanism of action of resveratrol has not been elucidated, but may be related to its ability to inhibit ribonucleotide reductase (7), DNA polymerase (8), COX-2 transcription (6) and activate the MAP kinase pathway (27) and the estrogen receptor (7). The molecular mechanism of resveratrol on antitumorigenesis is not clear, but is probably due to multiple actions. The present study was designed to test whether resveratrol may exert antitumorigenic effects through an antitumorigenic protein, NAG-1, and provide a molecular mechanism for regulation of NAG-1 expression. Our results show that resveratrol is an effective inhibitor of cell growth, and induces apoptosis in HCT-116 cells (Figure 1Go). Furthermore, resveratrol treatment resulted in an increased S phase in a concentration-dependent manner (data not shown), indicating a growth arrest at the S/G2 phase transition. Resveratrol increases the expression of NAG-1 in colorectal and lung cancer cells as well as in osteosarcoma cells, and NAG-1 has pro-apoptotic and antitumorigenic activities, which may provide, in part, a novel mechanism to explain its antitumorigenic activity.

Recently, we reported that the NAG-1 expression is enhanced by some NSAIDs, in a COX- and p53-independent manner (15). However, as reported here, resveratrol induces NAG-1 expression in a p53-dependent manner. As shown in Figure 5Go, resveratrol and etoposide induce p53 protein prior to the NAG-1 protein expression. The COX inhibitor, sulindac sulfide, did not induce p53 protein expression, but enhanced NAG-1 expression. Indeed, the biochemical pathway for resveratrol-induced apoptosis appears to require p53 induction (11). In contrast, NSAID-induced apoptosis does not require p53 induction (28). Our data indicate that NAG-1 induction by resveratrol is mediated by p53, and NAG-1 induction by NSAIDs does not require p53 induction. According to previous reports (17,18), NAG-1 contains two p53 binding sites in the promoter region. The second p53 binding site (p53-B) is the more functionally active of the two p53 binding sites, and consistent with the data, resveratrol-dependent NAG-1 induction is mediated by the second p53 binding site (Figure 6BGo). Interestingly, etoposide is more effective than resveratrol in stimulating NAG-1 promoter activity (Figure 6AGo), although both compounds induce p53 expression at almost the same magnitude. Recently, She et al. reported that resveratrol affects ERKs and p38 kinase activity, and thus increases p53 phosphorylation within 3 h of resveratrol treatment (27). Thus, it is possible that resveratrol may regulate two different mechanisms: one is an early response (within 3 h) resulting in the phosphorylation of p53, followed by a late response (after 3 h) resulting in p53 protein induction. Further investigations are required to elucidate the detailed mechanism of NAG-1 induction with respect to the involvement of p53 protein. In addition to resveratrol mediation, NAG-1 is induced by several resveratrol derivatives (Table IGo). The anti-oxidant activity of resveratrol has been linked to apoptotic gene induction and COX-2 promoter activity (19,23,24). We have shown in this report that resveratrol derivatives with weak anti-oxidant activity increase NAG-1 and/or p53 expression. This suggests that resveratrol-induced NAG-1 expression is not associated with the anti-oxidant activity of resveratrol. Furthermore, the substitution at the para position (Ring2) appears to be a critical determinant of NAG-1 expression.

We have shown that the expression of NAG-1 resulted in the induction of apoptosis in vitro and the reduction of tumor size in vivo (15). In this study, we further analyzed the effects of its antitumorigenic activity by an inducible expression system. Our findings demonstrate that functional NAG-1 can be expressed (Figure 4AGo) and that this protein inhibits cell proliferation (Figure 4BGo) and induces apoptosis (Figure 4CGo). However, the induction of apoptosis by NAG-1 is not robust (Figure 4CGo), suggesting that some additional triggers for resveratrol-induced apoptosis may be required. Furthermore, an unexpected finding was that the effect of NAG-1 was a dramatic inhibition of colony formation, while the growth of an exponential culture was only reduced ~30% (Figure 4BGo). One explanation for this difference might be that NAG-1 exerts its function predominantly under restrictive cell growth conditions. Indeed, we have reported that the over expression of NAG-1 resulted in the reduction of tumor size in nude mice experiments (15). These findings are consistent with studies published previously on the function of Mad1. The induction of Mad1 reduced cellular growth but, more profoundly, inhibited colony formation (29).

The biological significance of our findings is implicated in the linkage between resveratrol, NAG-1 expression, mediated by p53 protein and apoptosis. NAG-1 is the most notable p53-induced gene, as determined in a recent investigation using cDNA array technology (30). Resveratrol has well established antitumorigenic and pro-apoptotic activities. Our data shown here indicate that these effects are mediated by p53, and NAG-1 is a critical down stream protein. Although p53 has been well documented as a tumor suppressor, the p53 target protein, produced as a secreted form, has not been studied in detail. NAG-1 is secreted into the media and has specific antitumorigenic and pro-apoptotic activity, although the induction of p21/WAF-1/CIP1 by resveratrol is also mediated by p53 in HCT-116 cells (data not shown). In addition, p53 expression results in the decrease of COX-2 expression (31). Thus, resveratrol treatment followed by the induction of the p53 protein enhances apoptosis by up regulating apoptotic proteins including NAG-1, and down regulating anti-apoptotic proteins like COX-2. Although the mechanisms by which resveratrol regulate apoptosis are complex, the data will provide a new pathway to understand how phenolic compounds affect antitumorigenic activity.

Resveratrol concentrations used in this study are comparable with those occurring in wine and grapes (32). These concentrations cause an arrest of HCT-116 cell proliferation and an induction of NAG-1 antitumorigenic protein. It is believed that two glasses of red wine could provide a 10–30 µM concentration of resveratrol in vivo, where most of the pharmacological effects of resveratrol are observed (2). In addition, NAG-1 expression by resveratrol is not restricted to colorectal cells, as resveratrol can induce NAG-1 in A549, and U2OS cells. NAG-1 induction by resveratrol in different cell lines supports the evidence that resveratrol has an antitumorigenic effect in several cancers.

In summary, we have been able to document resveratrol as a potent inducer of NAG-1, a TGF-ß superfamily protein that has antitumorigenic activity. Furthermore, we have shown that the expression of NAG-1 results in the reduction of colony formation, induction of apoptosis and is mediated in a p53-dependent manner. NAG-1 expression is correlated with pro-apoptosis and antitumorigenesis in cell culture systems, and thus resveratrol effects on antitumorigenesis are mediated, in part, by NAG-1. The detailed analysis of biological functions and p53 regulation of NAG-1 expression should shed light on how dietary compounds alter tumorigenesis.

Notes

1 To whom correspondence should be addressed Email: eling{at}niehs.nih.gov Back

Acknowledgments

The authors would like to thank Dr Carl Bortner (NIEHS) for helping with flow cytometry analysis and Dr Sang Kook Lee (Ewha Womans University, Korea) for providing resveratrol derivatives. We thank Drs Yuji Mishina, Linda Hsi, Hiroo Kawajiri and Alex Merrick of NIEHS for their comments and suggestions, and also thank Allison Call for preparation of the manuscript.

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Received September 18, 2001; revised December 6, 2001; accepted December 12, 2001.