We determined whether resveratrol, a phenolic
antioxidant found in grapes and other food products, inhibited phorbol
ester (PMA)-mediated induction of COX-2 in human mammary and oral
epithelial cells. Treatment of cells with PMA induces COX-2 and causes
a marked increase in the production of prostaglandin
E2. These effects were inhibited by resveratrol.
Resveratrol suppressed PMA-mediated increases in COX-2 mRNA and
protein. Nuclear run-offs revealed increased rates of COX-2
transcription after treatment with PMA, an effect that was inhibited by
resveratrol. PMA caused about a 6-fold increase in COX-2
promoter activity, which was suppressed by resveratrol. Transient
transfections utilizing COX-2 promoter deletion constructs
and COX-2 promoter constructs, in which specific enhancer
elements were mutagenized, indicated that the effects of PMA and
resveratrol were mediated via a cyclic AMP response element.
Resveratrol inhibited PMA-mediated activation of protein kinase C. Overexpressing protein kinase C-
, ERK1, and c-Jun led to 4.7-, 5.1-, and 4-fold increases in COX-2 promoter activity, respectively. These effects also were inhibited by resveratrol. Resveratrol blocked PMA-dependent activation of
AP-1-mediated gene expression. In addition to the above effects on gene
expression, we found that resveratrol also directly inhibited the
activity of COX-2. These data are likely to be important for
understanding the anti-cancer and anti-inflammatory properties of
resveratrol.
 |
INTRODUCTION |
There are two isoforms of cyclooxygenase
(COX)1 that catalyze the
formation of prostaglandins (PGs) from arachidonic acid. COX-1 is a housekeeping gene that is expressed
constitutively (1). COX-2 is an immediate, early response
gene that is highly inducible by mitogenic and inflammatory stimuli
(2-4). The differences in the regulation of COX-1 and
COX-2 gene expression reflect differences in the regulatory
elements in the 5'-flanking regions of the two genes (5).
Considerable evidence has accumulated to suggest that COX-2 is
important for tumorigenesis. For example, COX-2 is up-regulated in
transformed cells (6-8) and various forms of cancer (9-12), whereas
levels of COX-1 remain essentially unchanged. A null mutation for
COX-2 markedly reduced the number and size of intestinal
tumors in a murine model of familial adenomatous polyposis,
i.e. APC
716 knockout mice (13).
COX-2 deficiency also protected against the formation of
extraintestinal tumors. Thus, COX-2 knockout mice developed
approximately 75% fewer chemically induced skin papillomas than
control mice (14). A selective inhibitor of COX-2 caused nearly
complete suppression of azoxymethane-induced colon cancer (15).
There are several possible mechanisms that could account for the link
between COX-2 and cancer. Enhanced synthesis of PGs, which occurs in a
variety of tumors (16-19), can favor the growth of malignant cells by
increasing cell proliferation (20), promoting angiogenesis (21), and
inhibiting immune surveillance (22). Overexpression of COX-2 inhibits
apoptosis and increases the invasiveness of malignant cells (23, 24);
these effects were reversed by the nonsteroidal anti-inflammatory drug,
sulindac sulfide. In combination, these studies suggest that targeted
inhibition of COX-2 is a promising approach to prevent cancer.
Therefore, chemopreventive strategies have focused on inhibitors of COX
enzyme activity. An equally important strategy may be to identify
compounds that suppress the expression of COX-2 (25, 26).
Resveratrol is a phytoalexin found in grapes and other foods that has
anti-cancer and anti-inflammatory effects (27) (Fig. 1). It inhibits the development of
preneoplastic lesions in carcinogen-treated mouse mammary glands, for
example, and it blocks tumorigenesis in a two-stage model of skin
cancer that was promoted by treatment with phorbol ester (27). The
anti-inflammatory properties of resveratrol were demonstrated by
suppression of carrageenan-induced pedal edema (27), an effect
attributed to suppression of PG synthesis via direct, selective
inhibition of COX-1. In the current work, we have extended prior
observations (27) concerning the effects of resveratrol on PG synthesis
by determining if resveratrol modulates the expression of the
COX-2 gene. Our data show that resveratrol suppresses the
activation of COX-2 gene expression by inhibiting the PKC
signal transduction pathway. Contrary to prior results (27), we also
found that resveratrol directly inhibits the activity of COX-2. These
data provide a mechanistic basis for the chemopreventive and
anti-inflammatory properties of resveratrol.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Minimal essential medium, PKC assay kits, and
LipofectAMINE were from Life Technologies, Inc. Keratinocyte basal and
growth media were from Clonetics Corp. (San Diego). Phorbol
12-myristate 13-acetate, sodium arachidonate, resveratrol,
PGE2, indomethacin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(thiazolyl blue), lactate dehydrogenase diagnostic kits,
epinephrine-hydrogentartrate, epidermal growth factor, hydrocortisone,
and o-nitrophenyl-
-D-galactopyranoside were
from Sigma. NS398 was from Biomol Research Labs Inc. (Plymouth Meeting,
PA). Enzyme immunoassay reagents for PGE2 assays were from
Cayman Co. (Ann Arbor, MI). [32P]CTP was from DuPont NEN.
[3H]Arachidonic acid was from American Radiolabeled
Chemicals Inc. Random-priming kits were from Boehringer Mannheim.
Nitrocellulose membranes were from Schleicher & Schuell. Reagents for
the luciferase assay were from Analytical Luminescence (San Diego). The
18 S rRNA cDNA was from Ambion, Inc. Rabbit polyclonal anti-human
COX-2 antiserum and goat polyclonal anti-human COX-1 antiserum were from Santa Cruz Biotechnology, Inc. Western blotting detection reagents
(ECL) were from Amersham Pharmacia Biotech. Plasmid DNA was prepared
using a kit from Promega.
Tissue Culture--
The 184B5/HER cell line has been described
previously (28). Cells were maintained in minimal
essential-keratinocyte basal media mixed in a ratio of 1:1 (basal
medium) containing epidermal growth factor (10 ng/ml), hydrocortisone
(0.5 µg/ml), transferrin (10 µg/ml), gentamicin (5 µg/ml), and
insulin (10 µg/ml) (growth medium). Cells were grown to 60%
confluence, trypsinized with 0.05% trypsin, 2 mM EDTA, and
plated for experimental use. MSK Leuk1 was established from a
dysplastic leukoplakia lesion adjacent to a squamous cell carcinoma of
the tongue in a 46-year-old nonsmoking female (29). Cells were
routinely maintained in keratinocyte growth medium and passaged using
0.125% trypsin, 2 mM EDTA. In all experiments, 184B5/HER
and MSK Leuk1 cells were grown in basal medium for 24 h prior to
treatment. Treatment with vehicle (0.2% Me2SO),
resveratrol, or PMA was always carried out in basal medium. Cellular
cytotoxicity was assessed by measurements of cell number, release of
lactate dehydrogenase, and the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay,
which was performed according to the method of Denizot and Lang (30).
Lactate dehydrogenase assays were performed according to the
manufacturer's instructions. There was no evidence of toxicity in any
of our experiments.
PGE2 Production by Cells--
5 × 104 cells/well were plated in 6-well dishes and grown to
60% confluence in growth medium. The cells were then treated as described below. Levels of PGE2 released by the cells were
measured by enzyme immunoassay. Rates of production of PGE2
were normalized to protein concentrations.
Determination of COX-2 Enzyme Activity--
The effect of
resveratrol, indomethacin, and NS398 on the activity of COX-2 was
measured using baculovirus-expressed recombinant human COX-2 enzyme
(supplied by J. K. Gierse, Monsanto Co., St. Louis, MO) (31).
The activity of COX-2 was determined in a microliter scale by measuring
the synthesis of PGE2. The incubation mixture contained COX-2 protein (0.45 µg), various concentrations of test compounds dissolved in ethanol, 1 mM reduced glutathione, 1 mM epinephrine-hydrogentartrate, and 0.05 mM
sodium-EDTA in 0.1 M Tris buffer (pH 8.0) (32). The
reaction was started by addition of 2.5 µM
[3H]arachidonic acid (0.25 µCi), in a final volume of
100 µl, and incubated for 30 min at 37 °C. The reaction was
terminated by the addition of 5 µl of 10% formic acid. Samples were
extracted with an equal volume of ethyl acetate. After centrifugation,
the ethyl acetate layer was evaporated under N2 and
resuspended in 100 µl of acetonitrile for high pressure liquid
chromatography analysis. An aliquot (10 µl) was applied to a
reverse-phase column (RCM Nova-pak C18, 8 × 100 mm,
Waters Associates, Milford, MA) in conjunction with a C18
guard column, and eluted with an acetonitrile-water (30:70 (A), 80:20
(B)) gradient containing 1% (v/v) 0.1 N phosphoric acid.
Elution conditions were as follows: gradient from 100% A to 100% B in
18 min, then 100% B for 10 min, with a flow rate of 1 ml/min (32). The
separated arachidonic acid and PGE2 were monitored with an
on-line radiochemical detector (
-Ram IN/US System Inc., Tempa, FL),
and the peaks were identified by cochromatography with unlabeled
reference compounds.
Western Blotting--
Cell lysates were prepared by treating
cells with lysis buffer (150 mM NaCl, 100 mM
Tris (pH 8.0), 1% Tween 20, 50 mM diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, and 10 µg/ml
leupeptin). Lysates were sonicated for 20 s on ice and centrifuged
at 10,000 × g for 10 min to sediment the particulate
material. The protein concentration of the supernatant was measured by
the method of Lowry et al. (33). SDS/PAGE was performed
under reducing conditions on 10% polyacrylamide gels as described by
Laemmli (34). The resolved proteins were transferred onto
nitrocellulose sheets as detailed by Towbin et al. (35). The
nitrocellulose membrane was then incubated with a rabbit polyclonal
anti-COX-2 antiserum or a polyclonal anti-COX-1 antiserum. Secondary
antibody to IgG conjugated to horseradish peroxidase was used. The
blots were probed with the ECL Western blot detection system according
to the manufacturer's instructions.
Northern Blotting--
Total cellular RNA was isolated from cell
monolayers using an RNA isolation kit from Qiagen Inc. 10 µg of total
cellular RNA per lane were electrophoresed in a formaldehyde-containing
1.2% agarose gel and transferred to nylon-supported membranes. After baking, membranes were prehybridized overnight in a solution containing 50% formamide, 5 × sodium chloride-sodium phosphate-EDTA buffer (SSPE), 5 × Denhardt's solution, 0.1% SDS, and 100 µg/ml
single-stranded salmon sperm DNA and then hybridized for 12 h at
42 °C with radiolabeled cDNA probes for human COX-2
cDNA and 18 S rRNA. After hybridization, membranes were washed
twice for 20 min at room temperature in 2 × SSPE, 0.1% SDS,
twice for 20 min in the same solution at 55 °C and twice for 20 min
in 0.1 × SSPE, 0.1% SDS at 55 °C. Washed membranes were then
subjected to autoradiography. COX-2 and 18 S rRNA probes
were labeled with [32P]CTP by random priming.
Nuclear Run-off Assay--
2.5 × 105 cells
were plated in four T150 dishes for each condition. Cells were grown in
growth medium until approximately 60% confluent. Nuclei were isolated
and stored in liquid nitrogen. For the transcription assay, nuclei
(1.0 × 107) were thawed and incubated in reaction
buffer (10 mM Tris (pH 8), 5 mM
MgCl2, and 0.3 M KCl) containing 100 µCi of
uridine 5'[
-32P]triphosphate and 1 mM
unlabeled nucleotides. After 30 min, labeled nascent RNA transcripts
were isolated. The human COX-2, c-jun, c-myc, and
-actin cDNAs were immobilized
onto nitrocellulose and prehybridized overnight in hybridization
buffer. Hybridization was carried out at 42 °C for 24 h using
equal cpm/ml of labeled nascent RNA transcripts for each treatment
group. The membranes were washed twice with 2 × SSC buffer for
1 h at 55 °C and then treated with 10 mg/ml RNase A in 2 × SSC at 37 °C for 30 min, dried, and autoradiographed.
Plasmids--
The COX-2 promoter constructs
(
1432/+59,
327/+59,
220/+59,
124/+59,
52/+59, ILM, CRM, and
CRM-ILM) have been described previously (5). The human COX-2
cDNA was generously provided by Dr. Stephen M. Prescott (University
of Utah, Salt Lake City, UT). Rous sarcoma virus-c-jun was a
gift from Dr. Tom Curran (Roche Laboratories, Nutley, NJ). The AP-1
reporter plasmid (2 × TRE-luciferase), composed of two copies of
the consensus TRE ligated to luciferase, was kindly provided by Dr.
Joan Heller Brown (University of California, La Jolla, CA) (36). The
ERK1 expression vector was obtained from Dr. Melanie Cobb (Southwestern
Medical Center, Dallas, TX). The c-myc cDNA was a gift
from Dr. Charles Sawyer (University of California, Los Angeles). The
PKC-
expression vector was provided by Dr. Geoffrey Cooper (Harvard
University, Cambridge, MA). pSV-
gal was obtained from Promega.
Transient Transfection Assays--
184B5/HER cells were seeded
at a density of 5 × 104 cells/well in 6-well dishes
and grown to 50-60% confluence. For each well, 2 µg of plasmid DNA
were introduced into cells using 8 µg of LipofectAMINE as per the
manufacturer's instructions. After 7 h of incubation, the medium
was replaced with basal medium. The activities of luciferase and
-galactosidase were measured in cellular extract as described previously (25).
Protein Kinase C Assay--
The activity of PKC was measured
according to directions from Life Technologies, Inc. Briefly, cells
were plated in 10-cm dishes at 106 cells/dish and grown to
60% confluence. Cells were then treated with fresh basal medium
containing vehicle (0.2% Me2SO), PMA (50 ng/ml), or PMA
(50 ng/ml) plus resveratrol (15 µM) for 30 min. Total PKC
activity was measured in cell lysates. To determine cytosolic and
membrane-bound PKC activity, cell lysates were centrifuged at
100,000 × g for 30 min. The resulting supernatant
contains cytosolic PKC; membrane-bound PKC activity is present in the
pellet. Subsequently, DEAE-cellulose columns were used to partially
purify PKC enzymes. Protein kinase C activity was then measured by
incubating partially purified PKC with [
-32P]ATP
(3000-6000 Ci/mmol) and the substrate myelin basic protein for 20 min
at room temperature. The activity of PKC is expressed as cpm
incorporated/µg protein.
Statistics--
Comparisons between groups were made by
the Student's t test. A difference between groups of
p < 0.05 was considered significant.
 |
RESULTS |
Resveratrol Inhibits COX-2 Enzyme Activity--
The data in Fig.
2A show that resveratrol
caused dose-dependent suppression of PGE2
synthesis in human mammary epithelial cells. To evaluate whether the
inhibition of PG synthesis was because of inhibition of COX-2 or COX-1,
we compared the effects of resveratrol and a selective inhibitor of
COX-2 (NS398). We note that NS398 decreased the synthesis of PGs to
less than 10% of the control level (Fig. 2B). This means
that more than 90% of basal COX activity in mammary epithelial cells
was because of the COX-2 isoform; the presence of COX-2 under basal
conditions is consistent with its transformed phenotype (6-8, 28). As resveratrol (30 µM) caused approximately a 70% decrease
in basal production of PGE2 (Fig. 2A), our data
show that the predominant inhibitory effect of resveratrol on synthesis
of PGs was via inhibition of the COX-2 isoform of cyclooxygenase.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
. Basal COX-2
activity is inhibited by resveratrol. 184B5/HER cells
were treated with resveratrol (0-30 µM, panel
A) or NS398 (0-10 µM, panel B) for 30 min. The medium was then replaced with fresh medium containing 10 µM sodium arachidonate. 30 min later, the medium was
collected to determine the rate of synthesis of PGE2.
Production of PGE2 was determined by enzyme immunoassay.
Columns, means; bars, S.D.; n = 6. *, p < 0.001 compared with control.
|
|
A second experiment was conducted to confirm that resveratrol directly
inhibited COX-2 enzyme activity (Fig. 3).
Phorbol esters are potent inducers of COX-2 (25), so we also examined
the effect of resveratrol on the synthesis of PGs by cells in which
COX-2 was induced by PMA. In this experiment, mammary epithelial cells were treated with vehicle or PMA for 4.5 h. Fresh medium
containing either resveratrol or NS398 was then added. 30 min later,
synthesis of PGE2 was measured. Resveratrol caused
concentration-dependent suppression of PMA-stimulated
synthesis of PGE2 with complete inhibition at 30 µM resveratrol (Fig. 3A). As shown in Fig.
3B, 0.1 µM NS398 was required to completely
suppress PMA-mediated induction of PG synthesis. Immunoblot analysis
demonstrated that levels of COX-2 protein did not decrease during
treatment with resveratrol or NS398 (data not shown). These results
strongly suggest that resveratrol inhibited COX-2 activity.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Resveratrol directly inhibits PMA-induced
COX-2 activity. 184B5/HER cells were treated with vehicle
(stippled columns) or PMA (50 ng/ml, black
columns) for 4.5 h. PMA was given to induce COX-2. Fresh
medium containing resveratrol (0-30 µM, panel
A) or NS398 (0-10 µM, panel B) was then
added for 30 min. The medium was then replaced with fresh medium
containing 10 µM sodium arachidonate. 30 min later, the
medium was collected to determine the rate of synthesis of
PGE2. Production of PGE2 was determined by
enzyme immunoassay. Columns, means; bars, S.D.;
n = 6. *, p < 0.001 compared with
PMA.
|
|
To further evaluate the effects of resveratrol on COX-2 activity, we
utilized baculovirus-expressed human recombinant COX-2 in a cell-free
assay. Resveratrol caused dose-dependent inhibition of
PGE2 synthesis (Fig. 4). As
expected from the findings in cell culture (Figs. 2 and 3), resveratrol
(IC50 value, 32.2 µM) was a less potent
inhibitor of COX-2 activity than the synthetic inhibitors NS398
(IC50 value, 3.2 µM) and indomethacin
(IC50 value, 1.9 µM).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Resveratrol causes dose-dependent
inhibition of recombinant human COX-2 enzyme activity.
[3H]Arachidonic acid was added to a reaction mixture
containing human recombinant COX-2 enzyme, 0.1 M Tris-HCl
(pH 8.0), 0.05 mM EDTA, 1 mM reduced
glutathione, 1 mM epinephrine-hydrogentartrate, and the
indicated concentration of resveratrol (circles), NS398
(squares), or indomethacin (triangles) for 30 min
at 37 °C. High pressure liquid chromatography analysis was performed
as described under "Experimental Procedures" to assess levels of
PGE2 synthesis. Percent activity was determined by
comparing levels of synthesis of PGE2 in control
incubations with levels observed in incubation mixtures containing the
indicated concentrations of test compounds.
|
|
Resveratrol Inhibits the Induction of COX-2 by Phorbol
Esters--
We also investigated the possibility that resveratrol
inhibited PMA-mediated induction of PG synthesis by suppressing the induction of COX-2. In these experiments, cells were cotreated for
4.5 h with PMA and the indicated concentrations of resveratrol. The medium then was replaced, and the synthesis of PGs was measured in
the absence of resveratrol over the next 30 min. PMA in this setting
caused about a 2-fold increase in synthesis of PGE2. This effect was suppressed by resveratrol in a dose-dependent
manner (Fig. 5). To confirm that these
effects of resveratrol were not unique to mammary epithelial cells, we
also determined whether resveratrol inhibited PMA-mediated induction of
PG synthesis in a premalignant, oral leukoplakia cell line. Treatment
of these cells with PMA led to a 2-fold increase in PG synthesis. This effect was inhibited completely by 20 µM resveratrol
(data not shown).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Resveratrol suppresses PMA-mediated increases
in the production of PGE2. 184B5/HER cells were
treated with vehicle (stippled column), PMA (50 ng/ml,
black columns), or PMA (50 ng/ml) and resveratrol for
4.5 h. The medium was then replaced with basal medium and 10 µM sodium arachidonate. 30 min later, the medium was
collected to determine the rate of synthesis of PGE2.
Production of PGE2 was determined by enzyme immunoassay.
Columns, means; bars, S.D.; n = 6. *, p < 0.001 compared with PMA.
|
|
To determine whether the above effects on production of
PGE2 could be related to differences in levels of COX,
Western blotting of cell lysate protein was carried out. Fig.
6A shows that PMA induced
COX-2 in human mammary epithelial cells. Cotreatment with resveratrol
caused a dose-dependent decrease in PMA-mediated induction of COX-2; the maximal drug effect was observed at 15-20
µM. Neither PMA nor resveratrol altered amounts of COX-1
(data not shown). It is noteworthy that the effects of resveratrol on
PGE2 synthesis mediated by PMA (Fig. 5) were greater than
the degree of suppression of amounts of COX-2 protein induced by PMA
(Fig. 6A). For example, 5 µM resveratrol
decreased PG synthesis to levels detected in uninduced cells while only
partially blocking PMA-mediated induction of COX-2 protein. This
finding is consistent with the idea that resveratrol inhibited
PMA-mediated induction of PG synthesis both by suppressing levels of
COX-2 protein and by direct inhibition of COX-2 activity. The
down-regulation of COX-2 expression by resveratrol was not limited to
mammary cells but was also demonstrable in oral epithelial cells (Fig.
6B).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Resveratrol causes dose-dependent
inhibition of PMA-mediated induction of COX-2. Cellular lysate
protein (25 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel,
electrophoresed, and subsequently transferred onto nitrocellulose.
Immunoblots were probed with antibody specific for COX-2. A,
lysate protein was from 184B5/HER cells treated with vehicle
(lane 1), PMA (50 ng/ml, lane 2), or PMA (50 ng/ml) and resveratrol (2.5, 5, 7.5, 10, 15, 30 µM;
lanes 3-8) for 4.5 h. Lane 9 represents an
ovine Cox-2 standard. B, lysates were from premalignant oral
epithelial (MSK Leuk1) cells treated with vehicle (lane 2),
PMA (50 ng/ml, lane 3), or PMA (50 ng/ml) and resveratrol
(10, 20, 30, 40 µM; lanes 4-7) for 4.5 h. Lane 1 represents an ovine Cox-2 standard.
|
|
To further elucidate the mechanism responsible for the changes in
amounts of COX-2 protein, we determined steady-state levels of COX-2
mRNA by Northern blotting. Treatment with PMA resulted in a marked
increase in levels of COX-2 mRNA, an effect that was suppressed by
resveratrol in a concentration-dependent manner (Fig.
7).

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 7.
PMA-mediated induction of COX-2 mRNA is
suppressed by resveratrol. 184B5/HER cells were treated with
vehicle (lane 1), PMA (50 ng/ml, lane 2), or PMA
(50 ng/ml) and resveratrol (2.5, 5, 10, 15, 20 µM;
lanes 3-7) for 3 h. Total cellular RNA was isolated;
10 µg of RNA was added to each lane. Results of densitometry in
arbitrary units: lane 1, 18; lane 2, 225;
lane 3, 135; lane 4, 72; lane 5, 45;
lane 6, 42; lane 7, 9.
|
|
Resveratrol Inhibits Phorbol Ester-mediated Increases in the
Transcription of COX-2--
Differences in levels of mRNA could
reflect altered rates of transcription or changes in mRNA
stability. Nuclear run-offs were performed to distinguish between these
possibilities. As shown in Fig. 8, we
detected higher rates of synthesis of nascent COX-2 mRNA after
treatment with PMA, consistent with the differences observed by
Northern blotting. This effect was suppressed by resveratrol. We also
investigated whether resveratrol suppressed PMA-mediated induction of
c-jun (Fig. 8B) and c-myc (Fig.
8C) transcription. Resveratrol caused a marked decrease in
the activation of c-jun expression. In contrast, resveratrol
did not significantly inhibit PMA-mediated induction of
c-myc transcription.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 8.
Resveratrol inhibits PMA-mediated induction
of COX-2 transcription. 184B5/HER cells were treated with vehicle
(lane 1), PMA (50 ng/ml, lane 2), or PMA (50 ng/ml) and resveratrol (15 µM, lane 3) for 30 min. Nuclear run-offs were performed as described under "Experimental
Procedures." The COX-2 (A), c-jun
(B), c-myc (C), and
-actin cDNAs were immobilized onto nitrocellulose
membranes and hybridized with labeled nascent RNA transcripts.
|
|
To further investigate the importance of PMA and resveratrol in
modulating the expression of COX-2, transient transfections were performed using a human COX-2 promoter-luciferase
construct. Treatment with PMA increased COX-2 promoter
activity about 6-fold. Resveratrol caused dose-dependent
inhibition of PMA-mediated induction of COX-2 promoter
activity (Fig. 9). We next attempted to
define the region of the COX-2 promoter that responded to
PMA and resveratrol. This was accomplished using a series of human
COX-2 promoter deletion constructs. As shown in Fig.
10A, both PMA-mediated
increases in COX-2 promoter activity and inhibition of
promoter activity by resveratrol were detected with all
COX-2 promoter constructs except the
52/+59 construct. A
CRE is present between nucleotides
59 and
53, suggesting that this
element may be responsible for mediating the effects of PMA and
resveratrol. To test this notion, transient transfections were
performed utilizing COX-2 promoter constructs in which
specific enhancer elements including the CRE were mutagenized. As shown
in Fig. 10B, mutagenizing the CRE site had several effects, including a decrease in basal promoter activity and a loss of responsiveness to both PMA and resveratrol. By contrast, mutagenizing the NF-IL6 site had little effect on COX-2 promoter
function.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
Resveratrol suppresses PMA-mediated induction
of COX-2 promoter activity. 184B5/HER cells were cotransfected
with 1.8 µg of human COX-2 promoter construct ligated to
luciferase ( 1432/+59) and 0.2 µg of pSV gal. After transfection,
cells were treated with vehicle (open column), PMA (50 ng/ml, black column), or PMA (50 ng/ml) and resveratrol
(5-15 µM). Reporter activities were measured in cellular
extract 6 h later. Luciferase activity represents data that have
been normalized with -galactosidase activity. Six wells were used
for each of the conditions. Columns, means; bars,
S.D. *, p < 0.001 compared with PMA treatment.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 10.
Localization of region of COX-2 promoter
that mediates the effects of phorbol ester and resveratrol.
A, 184B5/HER cells were transfected with 1.8 µg of a
series of human COX-2 promoter deletion constructs ligated
to luciferase ( 1432/+59, 327/+59, 220/+59, 124/+59, 52/+59),
and 0.2 µg of pSV gal. B, 184B5/HER cells were
transfected with 1.8 µg of a series of human COX-2
promoter-luciferase constructs ( 327/+59; ILM, CRM, CRM-ILM) and 0.2 µg of pSV gal. ILM represents the 327/+59 COX-2
promoter construct in which the NF-IL6 site was mutagenized; CRM refers
to the 327/+59 COX-2 promoter construct in which the CRE
was mutagenized; CRM-ILM represents the 327/+59 COX-2
promoter construct in which both the NF-IL6 and CRE elements were
mutagenized. After transfection, cells were treated with vehicle
(open columns), PMA (50 ng/ml, black columns), or
PMA (50 ng/ml) and resveratrol (15 µM, stippled
columns). Reporter activities were measured in cellular extract
6 h later. Luciferase activity represents data that have been
normalized with -galactosidase. Six wells were used for each of the
conditions. Columns, means; bars, S.D. *,
p < 0.001 compared with control.
|
|
Defining the Mechanism by Which Resveratrol Inhibits PMA-mediated
Induction of COX-2--
One of the ways that PMA regulates gene
expression is by activating the PKC signal transduction pathway (37). A
key feature of this mechanism is the redistribution of PKC activity
from cytosol to membrane. We therefore investigated the possibility
that resveratrol inhibited the redistribution of PKC activity that was
mediated by PMA. As shown in Fig. 11,
resveratrol completely inhibited the translocation of PKC activity from
cytosol to membrane. To further investigate the effects of resveratrol
on the PKC signal transduction pathway, a series of transient
transfections were performed. As shown in Fig.
12, overexpressing PKC-
or ERK1
caused 4.7- and 5.1-fold increases in COX-2 promoter
activity, respectively. These effects were suppressed by resveratrol.
We also determined the effects of resveratrol on c-Jun-mediated
induction of COX-2 promoter activity. As shown in Fig.
13A, c-Jun caused an
approximately 4-fold increase in COX-2 promoter activity.
This effect was also blocked by resveratrol. Resveratrol also
suppressed PMA-mediated activation of an AP-1 reporter plasmid (2 × TRE-luciferase) (Fig. 13B).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 11.
Resveratrol inhibits the redistribution of
PKC activity induced by PMA. 184B5/HER cells were treated with
vehicle (open columns), PMA (50 ng/ml, black
columns), or PMA (50 ng/ml) plus resveratrol (15 µM)
(stippled columns) for 30 min. Total PKC activity, cytosolic
PKC activity, and membrane PKC activity were measured.
Columns, means; bars, S.D. n = 6, *, p < 0.01 versus PMA.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 12.
Resveratrol suppresses PKC- and
ERK1-mediated induction of COX-2 promoter activity. A,
cells were transfected with 0.9 µg of human COX-2 promoter
construct ( 327/+59) (control) or 0.9 µg of COX-2
promoter construct plus 0.9 µg of expression vector for PKC- .
B, cells received 0.9 µg of human COX-2
promoter construct ( 327/+59) (control) or 0.9 µg of
COX-2 promoter construct plus 0.9 µg of expression vector
for ERK1. All cells received 0.2 µg of pSV gal. The total amount of
DNA in each reaction was kept constant at 2 µg by using empty vector.
Immediately after transfection, cells were treated with vehicle (0.2%
Me2SO) or resveratrol (15 µM) for 24 h.
Luciferase activity represents data that have been normalized with
-galactosidase. Columns, means; bars, S.D.
n = 6.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 13.
Resveratrol inhibits AP-1-mediated induction
of COX-2 promoter activity. A, cells were transfected
with 0.9 µg of a human COX-2 promoter construct ligated to
luciferase ( 327/+59) (control) or 0.9 µg of COX-2
promoter construct and 0.9 µg of expression vector for
c-jun. All cells received 0.2 µg of pSV gal. The total
amount of DNA in each reaction was kept constant at 2 µg by using
empty vector. Immediately after transfection, cells were treated with
vehicle or resveratrol (15 µM) for 24 h.
B, cells were cotransfected with 1.8 µg of 2 × TRE-luciferase and 0.2 µg of pSV gal. 24 h after transfection,
cells were treated with vehicle, PMA (50 ng/ml), or PMA (50 ng/ml) and
resveratrol (15 µM) for 6 h. Luciferase activity
represents data that have been normalized with -galactosidase
activity. Six wells were used for each of the conditions.
Columns, means; bars, S.D.
|
|
 |
DISCUSSION |
An expanding body of evidence indicates that inhibitors of COX-2
are useful for treating inflammation and preventing cancer (13, 15,
38). Drugs that interfere with the signaling mechanisms that
up-regulate COX-2 should also be useful in this regard because they too
decrease total COX-2 activity (25, 26). We have shown in the present
experiments that resveratrol suppressed PMA-mediated induction of PG
synthesis by inhibiting COX-2 gene expression and the enzyme
activity of COX-2. This is the first report of a compound that inhibits
COX-2 by both mechanisms. Because effects on gene transcription and
enzyme activity were observed over a similar range of concentrations of
resveratrol, it will be of interest to determine whether the
anti-inflammatory effects of resveratrol relate to one or both of these
mechanisms.
In regard to the mechanism by which resveratrol modulates gene
expression, it suppressed PMA-mediated activation of COX-2 transcription in human mammary epithelial cells by inhibiting the PKC
signal transduction pathway at multiple levels. It blocked both
PMA-induced translocation of PKC activity from cytosol to membrane
(Fig. 11) and the 4.7-fold increase in COX-2 promoter activity mediated by PKC-
(Fig. 12A). Resveratrol also
blocked the induction of COX-2 promoter activity by ERK1
(Fig. 12B) and c-Jun (Fig. 13A); PMA-mediated
induction of c-jun (Fig. 8B) and AP-1 activity
(Fig. 13B) were suppressed by resveratrol. These inhibitory
effects could be explained, in part, by the antioxidant properties of
resveratrol as other phenolic antioxidants inhibit both phorbol
ester-mediated activation of PKC (39) and AP-1 (40). These results are
significant because PKC activity is up-regulated in some cancers (41,
42) and is considered a potential target for anti-cancer therapy (43).
Additionally, as AP-1 has been implicated in promoting carcinogenesis,
these effects are likely to contribute to the anti-tumor activity of resveratrol.
The inductive effects of PMA and suppressive effects of resveratrol on
COX-2 expression are mediated via the CRE (Fig. 10). Xie and Herschman
(44) showed that c-Jun, a component of the AP-1 transcription factor
complex, activated the murine Cox-2 promoter via the CRE
(44). Thus, it seems likely that resveratrol blocks PMA-mediated
induction of COX-2 by suppressing AP-1-dependent transactivation via the CRE (45). The finding that resveratrol inhibited PMA-mediated induction of c-Jun expression (Fig.
8B) is consistent with this idea. Another possibility is
that resveratrol will induce Fra expression like other phenolic
antioxidants (46). Heterodimers of c-Jun and Fra do not activate
AP-1-mediated gene expression as effectively as c-Jun homodimers or
c-Jun/c-Fos heterodimers (47).
We reported previously that retinoids blocked PMA-mediated induction of
COX-2 in oral epithelial cells (25). The same effect of retinoids was
observed in the human mammary epithelial cells used in this study.
However, whereas resveratrol and retinoids both block PMA-mediated
induction of COX-2 transcription, they seem to do so via
different mechanisms. Thus, in contrast to resveratrol, retinoids did
not block the PMA-induced redistribution of PKC activity from cytosol
to membrane (data not shown). Additionally, resveratrol and retinoids
antagonize AP-1 activity via different mechanisms. Retinoids antagonize
AP-1 activity via a receptor-dependent mechanism (48),
whereas our data suggest that resveratrol blocks PMA-mediated
stimulation of AP-1-activity by inhibiting the PKC signaling cascade.
This distinction between resveratrol and retinoids is important for the
design of chemopreventive strategies utilizing combinations of drugs
that act via different mechanisms.
Based on the results of an oxygen consumption assay, Jang et
al. (27) reported that resveratrol did not inhibit the
cyclooxygenase activity of COX-2. However, the results of the present
study clearly show that resveratrol suppressed the synthesis of
PGE2 by inhibiting COX-2 enzyme activity (Figs. 2-4). One
possible explanation for these apparently contradictory results is the
difference in assays used to measure COX-2 activity.
Resveratrol was found recently to be a phytoestrogen that stimulates
the growth of estrogen-dependent breast cancer cells (49).
The mammary cell line used in our work was derived from normal human
breast tissue and was insensitive to estrogen (28). Therefore, we do
not know yet whether resveratrol will have similar effects on COX-2 in
estrogen-dependent and -independent mammary cell lines.
Another interesting but unanswered question is whether the same
structural properties of resveratrol account for inhibition of COX-2
enzyme activity and COX-2 transcription. Analogues of resveratrol are needed to determine the relationship between its structure and these different functions. Finally, based on the finding
that resveratrol inhibited COX-2, further studies are warranted to
determine how effective this compound or its analogues will be in
preventing or treating inflammation and cancer.
We thank Drs. Babette B. Weksler and David
Zakim for reviewing this manuscript. We also acknowledge the helpful
suggestions of Dr. Jaime Masferrer.