Carcinogenesis vol.24 no.9 pp.
1515±1524, 2003
DOI: 10.1093/carcin/bgg107
Curcumin inhibits phorbol ester-induced expression of cyclooxygenase-2 in mouse
skin through suppression of extracellular signal-regulated kinase activity and
NF-kB activation
Kyung-Soo Chun1, Young-Sam Keum1, Seong Su Han1,
Yong-Sang Song2, Su-Hyeong Kim2 and Young-Joon
Surh1,3
1
College of Pharmacy and 2Department of Obstetrics and Gynecology,
College of Medicine, Seoul National University, Seoul 151-741, South Korea
3
To whom correspondence should be addressed
Email: surh@plaza.snu.ac.kr
Recently, there have been considerable efforts to search for
naturally occurring substances for the intervention of
carcinogenesis. Many components derived from dietary
or medicinal plants have been found to possess substantial
chemopreventive properties. Curcumin, a yellow coloring
ingredient of turmeric (Curcuma longa L., Zingiberaceae),
has been shown to inhibit experimental carcinogenesis and
mutagenesis, but molecular mechanisms underlying its
chemopreventive activities remain unclear. In the present
work, we assessed the effects of curcumin on 12-O-
tetradecanoylphorbol-13-acetate (TPA)-induced expres-
sion of cyclooxygenase-2 (COX-2) in female ICR mouse
skin. Topical application of the dorsal skin of female ICR
mice with 10 nmol TPA led to maximal induction of cox-2
mRNA and protein expression at ~1 and 4 h, respectively.
When applied topically onto shaven backs of mice 30 min
prior to TPA, curcumin inhibited the expression of COX-2
protein in a dose-related manner. Immunohistochemical
analysis of TPA-treated mouse skin revealed enhanced
expression of COX-2 localized primarily in epidermal
layer, which was markedly suppressed by curcumin pre-
treatment. Curcumin treatment attenuated TPA-
stimulated NF-kB activation in mouse skin, which was
associated with its blockade of degradation of the inhibi-
tory protein IkBa and also of subsequent translocation of
the p65 subunit to nucleus. TPA treatment resulted in
rapid activation via phosphorylation of extracellular sig-
nal-regulated kinase (ERK)1/2 and p38 mitogen-activated
protein (MAP) kinases, which are upstream of NF-kB. The
MEK1/2 inhibitor U0126 strongly inhibited NF-kB activa-
tion, while p38 inhibitor SB203580 failed to block TPA-
induced NF-kB activation in mouse skin. Furthermore,
U0126 blocked the IkBa phosphorylation by TPA, thereby
blocking the nuclear translocation of NF-kB. Curcumin
inhibited the catalytic activity of ERK1/2 in mouse skin.
Taken together, suppression of COX-2 expression by inhi-
biting ERK activity and NF-kB activation may represent
molecular mechanisms underlying previously reported
Abbreviations: AP-1, activator protin 1; COX, cyclooxygenase; DTT,
dithiothreitol; EMSA, electrophoretic mobility shift assay; ERK, extra-
cellular signal-regulated kinase; MAP kinase, mitogen-activated protein
kinase; MEK, MAP kinase kinase; PG, prostaglandin; SDS, sodium dodecyl
sulfate; TPA, 12-O-tetradecanoylphorbol-13-acetate.
# Oxford University Press. All rights reserved
antitumor promoting effects of this phytochemical in
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mouse skin tumorigenesis.
Introduction
There has been accumulating evidence for the association
between inflammatory tissue damage and the process of
cancer development (1). Cyclooxygenase (COX), an important
enzyme involved in mediating the inflammatory process, cat-
alyzes the rate-limiting step in the synthesis of prostaglandins
(PGs) from arachidonic acid. There are two isoforms of COX,
designated COX-1 and COX-2 (2). COX-1 is expressed con-
stitutively in most tissues and appears to be responsible for
maintaining normal physiological functions. In contrast, COX-2
is detectable in only certain types of tissues and is induced
transiently by growth factors, pro-inflammatory cytokines,
tumor promoters and bacterial toxins (1,3). Expression of
COX-2 has been reported to increase in human colorectal
adenocarcinoma (4) and other malignancies such as breast,
cervical, prostate and lung tumors (5). Genetic knockout or
pharmacological inhibition of COX-2 has been shown to pro-
tect against intestinal polyposis in mice (6). Administration of
the selective COX-2 inhibitor celecoxib at 400 mg twice a day
for 6 months reduced the number of polyps significantly in
patients with familial adenomatous polyposis (7). In addition,
celecoxib has been found to inhibit experimentally induced
colon, breast, bladder and skin carcinogenesis (8±11).
The eukarytoic transcription factor NF-kB plays a central role
in general inflammatory as well as immune responses. The 50 -
flanking region of the cox-2 promoter contains NF-kB binding
sites. In line with this notion, NF-kB has been shown to be a
critical regulator of COX-2 expression in many cell lines
(12,13). The intracellular signaling cascades controlling NF-
kB activation are highly complex and involve the distinct set
of kinases. Of the potential protein kinases involved in the
activation of NF-kB, mitogen-activated protein (MAP) kinases
such as extracellular signal-regulated kinase (ERK), p38 and
c-Jun N-terminal kinase/stress-activated protein kinase-
signaling pathways have been well characterized (14,15).
Recently, much attention has been devoted to identifying
cancer chemopreventive phytochemicals of dietary and medi-
cinal origin (16). One such compound is curcumin [1,7-bis(4-
hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], the
major yellow coloring pigment found in turmeric (Curcuma
longa Linn, Zingiberaceae), which has been used for centuries
in traditional oriental medicine to treat mainly inflammatory
disorders. The anti-inflammatory properties of curcumin have
also been verified in experimental studies (16). In addition, the
compound has been shown to exert anticarcinogenic or anti-
mutagenic effects in many animal models and also in cultured
cells (reviewed in 16 and references therein). Thus, curcumin
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K.-S.Chun et al.
inhibits the development of chemically induced tumors of oral
cavity, skin, forestomach, duodenum and colon in rodents
(17±20). Curcumin has a variety of biochemical activities
that are related to its chemopreventive action. These include
antioxidation (21,22), inactivation of activator protin (AP)-1
(23) and NF-kB (24), inhibition of PG biosynthesis (25),
and inhibition of activity and expression of ornithine
decarboxylase (20,26).
The anti-inflammatory properties of curcumin are consid-
ered to contribute to its antitumor promoting activity. In the
present study, we examined the effect of curcumin on COX-2
induction by the tumor promoter 12-O-tetradecanoylphorbol-
13-acetate (TPA) in mouse skin in vivo. To further elucidate
the molecular mechanisms by which curcumin regulates cox-2
gene expression, we also investigated its effect on activation of
upstream signaling enzymes, such as ERK or p38 MAP kinase,
and the transcription factor NF-kB in mouse skin in vivo.
Materials and methods
Chemicals
Curcumin was purchased from Sigma Chemical Co. (St Louis, MO, USA).
TPA was obtained from Alexis Biochemicals (San Diego, CA, USA). All other
chemicals used were in the purest form available commercially.
Animal treatment
Female ICR mice (6±7 weeks of age) were supplied from the Dae-Han/Biolink
Experimental Animal Center (Daejeon, Korea). The animals were housed in
climate-controlled quarters (24  1 C at 50% humidity) with a 12-h light/12-h
dark cycle. The dorsal side of skin was shaved using an electric clipper, and
only those animals in the resting phase of the hair cycle were used in all
experiments. Curcumin and TPA were dissolved in 200 ml of acetone and
applied to the dorsal shaven area.
Western blot analysis
The mice were topically treated on their shaven backs with indicated doses of
curcumin 30 min before 10 nmol TPA treatment and were killed by cervical
dislocation at the indicated times. For isolation of protein from mouse skin, the
dorsal skin was excised, and the fat was removed on ice, immediately placed in
liquid nitrogen and pulverized in mortar. The pulverized skin was homo-
genized on ice for 20 s with a Polytron tissue homogenizer and lysed in 2 ml
ice-cold lysis buffer [150 mM NaCl, 0.5% Triton X-100, 50 mM Tris±HCl
(pH 7.4), 20 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM Na3 VO4 , protease
inhibitor cocktail tablet (Roche Molecular Biochemicals, Mannheim,
Germany)] for 10 min. Lysates were centrifuged at 12 000 g for 20 min, and
supernatant containing 30 mg protein was boiled in sodium dodecyl sulfate
(SDS) sample loading buffer for 10 min before electrophoresis on 12% SDS±
polyacrylamide gel. After electrophoresis for 2 h, proteins in SDS±polyacryl-
amide gel were transferred to PVDF membrane (Gelman Laboratory, Ann
Arbor, MI), and the blots were blocked with 5% non-fat dry milk-PBST buffer
[phosphate-buffered saline (PBS) containing 0.1% Tween-20] for 60 min at
room temperature. The membranes were incubated for 2 h at room temperature
with 1:1000 dilution of COX-2, p65, IkBa and ERK polyclonal antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA), phospho-p65 and phospho-IkBa
polyclonal antibodies (Cell Signaling Technology, Beverly, MA) and p38,
phospho-p38 and phospho-ERK monoclonal antibodies (Santa Cruz Biotech-
nology). Equal lane loading was assessed using actin (Sigma Chemical Co.) or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody
(Trevigen, Gaithersburg, MD). The blots were rinsed three times with PBST
buffer for 5 min each. Washed blots were incubated with 1:5000 dilution of the
horseradish peroxidase conjugated-secondary antibody (Zymed Laboratories,
San Francisco, CA) and then washed again three times with PBST buffer. The
transferred proteins were visualized with an enhanced chemiluminescence
detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Northern blot analysis
The pulverized skin was homogenized on ice for 20 s with a Polytron for
isolation of total RNA by using TRIzolÒ reagent (Roche Molecular Biochem-
icals). For northern blot analysis, 20 mg of total RNA was subjected to electro-
phoresis on 1.2% agarose±formaldehyde gel and transferred to a Hybond-Nylon
membrane (Amersham Pharmacia Biotech). After being fixed by UV irradiation,
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membranes were pre-hybridized for 30 min at 68 C in ExpressHyb
hybridization solution (Clontech Laboratories, Palo Alto, CA). Hybridization
was carried out for 1 h at 68 C with a cox-2 cDNA probe (Cayman Chemical,
Ann Arbor, MI) labeled with [a-32 P]dCTP (NEN Life Science Products,
Boston, MA) using a Rediprime II labeling system (Amersham Pharmacia
Biotech). After hybridization, the membranes were washed twice for 30 min at
room temperature in low-stringency buffer (2 SSC and 0.05% SDS) and
twice for 40 min at 50 C in high-stringency buffer (0.1 SSC and 0.1% SDS).
Washed membranes were then autoradiographed on the X-ray film using an
intensifying screen at 70 C. Each band was quantified using a BAS2000 image
analyzer (Fuji Photo Film Co., Tokyo, Japan).
Immunohistochemical staining of COX-2
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The dissected skin was prepared for immunohistochemical analysis of COX-2
localization. Four-micrometer sections of formalin-fixed, paraffin-embedded
tissue were cut onto silanized glass slides and deparaffinized three times with
xylene for 10 min each and rehydrated through graded alcohol bath. The
deparaffinized sections were heated and boiled twice for 6 min in 10 mM
citrate buffer, pH 6.0, for antigen retrieval. To diminish non-specific staining,
each section was treated with 3% hydrogen peroxide in methanol for 15 min.
For the detection of COX-2, slides were incubated with 1:50±100 dilutions of
the monoclonal mouse anti-COX-2 antibody (Cayman Chemical) at room temp-
erature for 60 min in Tris-buffered saline containing and 0.05% Tween-20
and then developed using the HPR EnVisionTM System (Dako, Glostrup,
Denmark). The peroxidase binding sites were detected by staining with 3,30 -
diaminobenzidine tetrahydrochloride (Dako). Finally, counterstaining was
performed using Mayer's hematoxylin.
Preparation of nuclear extracts
The nuclear extract from mouse skin was prepared as described previously
(27). Briefly, scraped dorsal skin of mice was homogenized in 1 ml of ice-cold
hypotonic buffer A [10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2 , 1 mM
DTT, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF)]. After
15 min incubation on ice, the nucleoprotein complexes were lysed with 125 ml
of 10% Nonidet P-40 (NP-40) solution, followed by centrifugation for 2 min at
14 800 g. The nuclei were washed once with 400 ml of buffer A plus 25 ml of
10% NP-40, centrifuged, resuspended in 150 ml of buffer C [50 mM HEPES
(pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM
PMSF and 10% glycerol], and centrifuged for 5 min at 14 800 g. The super-
natant containing nuclear proteins was collected and stored at ÿ70 C after
determination of protein concentrations.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed using a DNA±protein binding detection kit (Gibco
BRL, Grand Island, NY) according to the manufacturer's protocol. Briefly,
the NF-kB oligonucleotide probe (50 -AGT TGA GGG GAC TTT CCC AGG
C-30 ) was labeled with [g-32 P]ATP by T4 polynucleotide kinase and purified
on a Nick column (Amersham Pharmacia Biotech). The binding reaction was
carried out in a total volume of 25 ml containing 10 mM Tris±HCl (pH 7.5),
100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% (v/v) glycerol, 0.1 mg/ml
sonicated salmon sperm DNA, 10 mg of nuclear extracts, and 100 000 c.p.m.
of the labeled probe. 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 in a cold room for 2 h.
Finally, the gel was dried and exposed to X-ray film.
MAP kinase assay (non-radioactive)
Kinase assays for determining the catalytic activities of p38 and ERK were
carried out by using a non-radioative MAP kinase assay kit (Cell Signaling
Technology) as described in the protocol provided by the manufacturer.
Collected tissues were lysed in 200 ml of lysis buffer per sample [20 mM
Tris±HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton
X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerolphosphate, 1 mM
Na3 VO4 , 1 g/ml leupeptin]. The lysates were centrifuged, and the supernatant
was incubated with specific immobilized phospho-p38 and phospho-ERK
monoclonal antibodies with gentle rocking for overnight at 4 C. The beads
were washed twice each with 500 ml of lysis buffer and the same volume of
kinase buffer [25 mM Tris±HCl (pH 7.5), 5 mM glycerolphosphate, 2 mM
DTT, 0.1 mM Na3 VO4 and 10 mM MgCl2 ]. The kinase reactions were carried
out in the presence of 100 mM ATP and 2 mg of ATF-2 or Elk-1 at 30 C for 30
min. Phosphorylation of ATF-2 and Elk-1 was selectively measured by immu-
noblotting with specific antibodies detecting phosphorylation of ATF-2 and
Elk-1 at Thr71 and Ser383, respectively.

Results
Inhibitory effects of curcumin on TPA-induced COX-2
expression in mouse skin
It has been shown previously that TPA is a potent stimulator of
COX-2 expression in various cell lines (28,29). We determined
whether this was also the case in mouse skin. When 10 nmol of
TPA was applied topically to the shaven backs of female ICR
mice, the COX-2 protein level increased in a time-related
manner with maximal expression observed at 4 h (Figure 1A).
Under the same experimental conditions, cox-2 mRNA expres-
sion peaked 1 h after the TPA application (Figure 1B). TPA
application caused dose-dependent increases in both COX-2
protein and mRNA expression (Figure 2). To determine loca-
lization of COX-2 in mouse skin, we conducted immuno-
histochemical analysis. In acetone-treated control skin,
specific COX-2 immunostaining was barely detectable in
dorsal layer. Upon treatment with TPA for 4 h, the expression
of COX-2 increased mainly in the epidermal layer (Figure 3),
which was suppressed by curcumin pre-treatment (Figure 4).
Curcumin-mediated suppression of COX-2 expression was
verified by western blot analysis (Figure 5).
Fig. 1. TPA-induced COX-2 protein (A) and its mRNA (B) expression in
mouse skin. (A) Dorsal skins of female ICR mice were treated topically with
acetone alone or with 10 nmol TPA in acetone for indicated time periods.
Protein extracts (30 mg) were loaded onto a 12% SDS±polyacrylamide gel,
electrophoresed, and subsequently transferred onto PVDF membrane.
Immunoblots were a probed with a goat polyclonal COX-2 antibody. (B) cox-2
mRNA expression was determined by northern blot analysis using the cox-2
cDNA probe labeled with [a-32 P]dCTP. Quantification of cox-2 mRNA signal
intensities was normalized to those of GAPDH mRNA.
Inhibition of COX-2 expression in mouse skin
Inhibition of TPA-induced NF-kB DNA binding activity by
curcumin
Because NF-kB is known to play a critical role in regulating
the induction of COX-2, we have determined whether curcu-
min could suppress activation of this transcription factor in
nuclear extracts obtained from the mouse skin stimulated with
TPA. Our previous studies demonstrated an apparent increase
in epidermal NF-kB DNA binding as early as 10 min after
10 nmol TPA application, which was abolished by the excess
unlabeled probe (27). Effects of curcumin on NF-kB activa-
tion were examined with varying doses of the compound
topically applied 30 min before, simultaneously with or
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30 min after the TPA treatment. Pre-treatment of curcumin
strongly inhibited TPA-induced NF-kB activation, whereas
co- and post-treatment exhibited weaker inhibitory effects
on NF-kB DNA binding in TPA-stimulated mouse skin
(Figure 6A).
Effects of curcumin on TPA-induced phosphorylation and
degradation of IkBa and nuclear translocation of p65
One of the most critical steps in NF-kB activation is the
dissociation of IkB, which is mediated through phosphoryla-
tion and subsequent proteolytic degradation of this inhibitory
subunit. To determine whether the inhibitory effect of curcu-
min on NF-kB DNA binding was due to its suppression of
IkBa degradation via phosphorylation, the cytoplasmic levels
of IkBa and phospho-IkBa were determined by western blot
analysis. Topical application of TPA led to phosphorylation
and degradation of IkBa, which were significantly repressed
Fig. 2. Dose-related expression of COX-2 protein (A) and its mRNA (B) in
TPA-treated mouse skin. Dorsal skins of female ICR mice were treated
topically with acetone alone or with various doses of TPA in acetone. Mice
were killed 4 or 1 h later for immunoblot analysis of COX-2 protein (A) or
northern blot analysis of cox-2 mRNA (B), respectively.
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K.-S.Chun et al.
Fig. 3. COX-2 immunostaining in mouse skin. Paraffin-embedded tissues
from TPA-treated mouse were immunostained for COX-2 and counterstained
with hematoxylin, as described in Materials and methods. COX-2 staining
gives a brown reaction product. (A) COX-2 staining in acetone-treated
control skin. (B) COX-2 staining in 10 nmol TPA-treated mouse. (C)
COX-2 staining in 100 nmol TPA-treated mouse.
by curcumin pre-treatment (Figure 6B and C). This finding is
consistent with NF-kB DNA binding affected by the same
treatment (Figure 6A). We also measured the level of p65,
the functionally active subunit of NF-kB, in nucleus. Upon
TPA treatment, the nuclear translocation of p65 increased,
which was blocked by curcumin (Figure 6B). These results
indicate that curcumin inhibits TPA-induced translocation of
p65 to the nucleus through blockade of IkBa phosphorylation.
Effects of curcumin on TPA-induced activation of MAP
kinases
MAP kinases are known to regulate NF-kB activation by
multiple mechanisms. Accumulating evidence indicates that
NF-kB activation is modulated by ERK as well as p38 MAP
kinase. As shown in Figure 7, ERK and p38 in mouse skin
were phosphorylated in response to TPA treatment. Phos-
phorylation of each MAP kinase was evident at 30 min follow-
ing TPA application and was sustained up to 4 h. To confirm
that phosphorylation of ERK and p38 reflected their enhanced
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Fig. 4. Inhibitory effects of curcumin on phorbol ester-induced COX-2
expression by immunohistochemistry staining. Female ICR mice were treated
topically with curcumin (25 mmol) or acetone alone 30 min before TPA
(10 nmol) application. Control mice were treated with acetone in lieu of TPA.
Mice were killed 4 h after the TPA treatment, and skin samples were subjected
to immunohistochemical analysis with COX-2 antibody as described in
Materials and methods. Positive COX-2 staining yielded a brown-colored
product (arrow). (A) Skin treated with acetone alone. (B) Skin treated
with TPA. (C) Skin treated with curcumin 30 min prior to TPA.
catalytic activity, kinase assays were performed. In parallel
with elevated phosphorylation, the activities of ERK and p38
increased rapidly after TPA application (Figure 7). However,
the JNK activity remained unchanged even after TPA treat-
ment (data not shown). After verifying the ERK and p38
activation in TPA-stimulated mouse skin, we examined
whether curcumin could down-regulate the aforementioned
MAP kinases, thereby inactivating NF-kB and further suppres-
sing the COX-2 induction. Curcumin inhibited catalytic activ-
ities of both p38 MAP kinase and ERK1/2. In addition,
curcumin inhibited activation of p38 MAP kinase through
phosphorylation while it did not much influence the phos-
phorylation of ERK1/2 in mouse skin (Figure 8). Under the
same experimental conditions, the level of the total form of
each kinase remained almost constant.
Inhibition of TPA-induced NF-kB DNA binding activity and
COX-2 expression by MAP kinase inhibitors
To determine which of the MAP kinases is involved in activa-
tion of NF-kB in mouse skin, we investigated the inhibitory

Fig. 5. Inhibitory effects of curcumin on phorbol ester-induced COX-2
expression. Female ICR mice were treated topically with 0.2 ml acetone or
curcumin in the same volume of acetone 30 min prior to 10 nmol TPA, and
animals were killed 4 h after the TPA treatment. Protein was analyzed for
COX-2 by immunoblotting. The western blot is representative of two
independent experiments. Quantification of COX-2 expression was
normalized to actin using a densitometer.
effect of the pharmacological inhibitors of MAP kinases on
NF-kB activation by TPA. SB203580 is known to selectively
inhibit p38 MAP kinase and U0126 is an ultrapotent inhibitor
of MAP kinase kinase (MEK)1/2 responsible for activation of
ERK. We have confirmed that SB203580 and U0126 at 4 mmol
each suppressed the activity of p38 MAP kinase and phos-
phorylative activation of ERK1/2, respectively (data not
shown). U0126 blocked the NF-kB DNA binding activity,
whereas the p38 MAP kinase inhibitor SB203580 failed to
(Figure 9A). This result suggests that the activation of NF-
kB occurs via the ERK-dependent pathway in mouse skin.
Many studies have revealed a close association between the
ERK activity and phosphorylation and degradation of IkB
protein, which leads to increased nuclear translocation of
NF-kB and subsequent DNA binding in various cell systems
(30±33). To investigate the possible role of ERK in IkB phos-
phorylation and degradation, we measured the levels of phos-
phorylated IkBa with and without U0126 pre-treatment. As
illustrated in Figure 9B, U0126 blocked the phosphorylation of
IkBa, thereby suppressing degradation of this inhibitory
protein. In order to further investigate the possible involve-
ment of ERK in the signaling pathway mediating COX-2
induction, we examined the effect of U0126 on the TPA-
induced COX-2 expression. As shown in Figure 9C, U0126
at 4 mmol almost completely abrogated COX-2 induction in
TPA-treated mouse skin. These data suggest that ERK plays a
central role in intracellular signaling cascades mediating TPA-
induced COX-2 expression in mouse skin.
Discussion
In recent years considerable efforts have been made to develop
chemopreventive agents that could inhibit, retard or reverse
Inhibition of COX-2 expression in mouse skin
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Fig. 6. Effect of curcumin on NF-kB activation in mouse skin treated with
TPA. (A) Dorsal skins of mice were treated topically with acetone alone (lane
2), 10 nmol TPA alone (lane 3) or 1 mmol (A), 5 mmol (B), 10 mmol (C) or
25 mmol (D) of curcumin 30 min before, simultaneously with, or 30 min
after TPA treatment. Mice were killed 1 h after the TPA treatment (acetone for
the control), and epidermal nuclear extracts were prepared and incubated
with the radiolabeled oligonucleotides containing the NF-kB consensus
sequence for analysis by the EMSA. Lane 1, free probe alone (no nuclear
extracts). (B and C) Nuclear and cytoplasmic extracts from mouse skin treated
10 nmol TPA for 1 h, with and without curcumin (1, 5 or 25 mmol) pre-
treatment, were assayed for p65, IkBa (B) and phospho-IkBa (C),
respectively, by western blot analysis. NE, nuclear extract; CE, cytoplasmic
extracts. The western blot is representative of two separate experiments.
the multi-stage carcinogenesis (34). Chemoprevention has
become an emerging area of cancer research that, in addition
to providing a practical approach to identifying potentially
useful inhibitors of malignant transformation, affords oppor-
tunities to study the mechanisms of anticarcinogenesis (34).
Chemopreventive agents can act in any stages of carcinogen-
esis, i.e. initiation, promotion or progression. The intervention
of cancer in the promotion stage, however, seems to be most
appropriate and practical since tumor promotion is a reversible
event, which requires repeated and prolonged exposure to
promoting agents (35). Tumor promotion is closely linked to
inflammation and oxidative stress (35,36), and it is hence
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K.-S.Chun et al.
Fig. 7. Time course of activation of p38 MAP kinase and ERK in TPA-treated
mouse skin. TPA-induced activation of p38 MAP kinse and ERK that are
upstream of NF-kB was assessed based on elevated levels of
phosphorylated enzymes as well as enhancement of their catalytic activity.
(A) TPA (10 nmol) was topically applied onto shaven backs of female ICR
mice. Mice were killed at indicated time intervals. Total protein was isolated
from the dorsal skin and quantified. Cell lysates containing 200 mg protein
were treated with a specific immobilized phospho-p38 kinase monoclonal
antibody. The resulting immunoprecipitate was then incubated with ATF-2
fusion protein (substrate for activated p38) in the presence of 100 mM ATP.
ATF-2 phosphorylation was measured by western blotting of non-radioactive
labeled samples using the phospho-ATF-2 antibody. The phosphorylated form
of p38 was detected by immunoblotting using a corresponding phospho-
specific antibody. (B) The protein extracts were prepared at indicated time
points from TPA-stimulated skin, and ERK phosphorylation was
determined by western blot analysis. The kinase activity of ERK was
determined by the immune complex assay in a manner similar to that described
for p38 except that Elk-1 was included as a substrate.
likely that compounds with strong anti-inflammatory and anti-
oxidative activities act as antitumor promoters as well.
Curcumin, a naturally occurring anti-inflammatory and anti-
oxidant compound, has been shown to inhibit experimentally
induced tumorigenesis in several animal models, including
7,12-dimethylbenz[a]anthracene-initiated and TPA-promoted
skin tumors, benzo[a]pyrene-induced forestomach tumors, and
azoxymethane-induced intestinal tumors in mice (18,19,37±39).
There is a growing body of compelling evidence that targeted
inhibition of COX-2 expression or activity is valuable for not
only alleviating inflammation, but preventing cancer. There-
fore, agents that interfere with the intracellular signaling
mechanisms governing the transcription of COX-2 are consid-
ered to be potential chemopreventives. Treatment of several
human gastrointestinal cell lines with curcumin suppressed
expression of COX-2 protein and mRNA as well as PGE2
production induced by TPA (40). Since chronic inflammation
predisposes to malignancy (41), the inhibition of COX-2 by
curcumin is likely to contribute to both anti-inflammatory and
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Fig. 8. Effects of curcumin on TPA-induced activation of p38 MAP kinase
(A) and ERK (B). Female ICR mice were treated topically with acetone or with
1, 5 or 25 mmol of curcumin 30 min prior to TPA. Mice were killed 1 h
after the TPA treatment, and expression of both kinases was measured by
western blot analysis. The kinase activities were determined by the immune
complex assay as described in the legend to Figure 7. Data are representative of
two independent experiments, which showed a similar trend.
chemopreventive effects this phytochemical exerts. While the
majority of non-steroidal anti-inflammatory drugs inhibit the
catalytic activity of COX-2, our results indicate that curcumin
can inhibit expression of the COX-2 induced by the typical
tumor promoter TPA in mouse skin. These data suggest that
previously reported chemopreventive properties of curcumin
against mouse skin carcinognesis are attributable in part to its
inhibition of expression of COX-2 as well as its catalytic
activity. TPA treatment can induce or activate a wide array
of genes and their protein products. Like COX-2, elevated
expression of inducible nitric oxide synthase (iNOS) and/or
its catalytic activity has been observed in several human
malignancies also in chemically induced tumors in experimen-
tal animals (42±44). We have found that topically applied
curcumin abrogated iNOS expression in TPA-stimulated
mouse skin (K.-S.Chun et al., unpublished observation).
Besides aforementioned pro-inflammatory enzymes, curcumin
may also target other molecules such as NAD(P)H:oxidore-
ductase (45) and ornithine decarboxylase (20,26) in exerting
its chemopreventive activity. Thus, it is unlikely that COX-2 is
the only target for chemoprevention by curcumin in mouse skin.
Control of cox-2 induction involves a complex array of
regulatory factors including NF-kB (12,13). Curcumin
blocked tumor promoter-mediated NF-kB transactivation by
inhibiting the NF-kB-inducing kinase (NIK)/IkB kinase (IKK)
signaling complex, probably at the level of IKKa/b (46).
When the effect of curcumin on NF-kB activation by TPA
was examined in mouse skin, pre-treatment of curcumin was
found to be most effective in terms of inhibiting NF-kB DNA
binding, whereas co- or post-treatment caused a weaker
effect. The inhibitory effect of curcumin on NF-kB activation
by TPA was due to inhibition of IkB degradation and p65
translocation to the nucleus. Our previous study revealed that

Fig. 9. Effect of MEK1/2 inhibitor U0126 on TPA-induced NF-kB DNA
binding activity and COX-2 expression. (A) Dorsal skin of female ICR
mice was treated topically with acetone, U0126, or SB203580 30 min
prior to topical application of 10 nmol TPA. After 1 h, mice were killed and
epidermal nuclear extracts were prepared for EMSA. Lane 1, free probe
alone; lane 2, acetone control; lane 3, TPA alone; lane 4, U0126 (4 mmol) ‡
TPA; lane 5, SB203580 (4 mmol) ‡ TPA. (B) Dorsal skin of female
ICR mice was treated with acetone (lane 1), 10 nmol TPA (lane 2) alone or
4 mmol of U0126 (lane 3). Cytosolic extracts were assayed by western
blot for phospho-IkBa, as described in the text. (C) U0126 was applied
topically 30 min prior to TPA. Mice were killed 4 h after the TPA
treatment. Lane 1, acetone control; lane 2, TPA alone; lane 3, U0126
(4 mmol) ‡ TPA. All experiments were repeated at least twice.
curcumin could also suppress NF-kB activation through direct
interruption of NF-kB DNA binding in TPA-pre-treated
nuclear extract (47). Production of PGE2 and 6-ketoPGF1a in
LPS-stimulated J774 macrophages was reported to be reduced
by the antioxidant pyrrolidine dithiocarbamate (PDTC) and the
serine protease inhibitor, N-a-p-tosyl-L-lysine chloromethyl-
ketone (TPCK), both of which are inhibitors of NF-kB (12).
Suppression of the prostanoid production by PDTC or TPCK
was not mediated through direct inhibition of COX-2 activities
since these NF-kB inhibitors did not influence the catalytic
activity of the enzyme when added to the new media after
bacterial lipopolysaccharide challenge (12). According to
our previous study, topical application of PDTC resulted in
dose-related suppression of TPA-induced activation of NF-kB
Inhibition of COX-2 expression in mouse skin
and also caused reduced COX-2 protein expression in mouse
skin (48). These data also support the notion that the naturally
occurring anti-inflammatory agent curcumin inhibits TPA-
induced COX-2 expression in mouse skin, possibly by
blocking NF-kB activation.
Another transcription factor that plays an important role in
controlling cox-2 gene is AP-1. A role of AP-1 in COX-2
induction has been demonstrated in various cell lines (49±51).
In our previous data, topical application of curcumin sup-
pressed TPA-stimulated AP-1 activation by directly blocking
the binding of the pre-activated nuclear extract to the AP-1
consensus sequence (47). Therefore, it is plausible that curcu-
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min inhibits COX-2 expression through not only inactivation of
NF-kB but also other transcription factors including AP-1 in
mouse skin.
The molecular signaling mechanisms involved in the induc-
tion of COX-2 as well as activation of NF-kB in response to
various external stimuli have not been fully clarified. One of the
most extensively investigated intracellular signaling cascades
involved in pro-inflammatory responses is the MAP kinase
pathway. MAP kinases regulate NF-kB activation via multiple
mechanisms. A substantial body of data indicates that NF-kB
activation is modulated by MAP kinase/ERK kinase kinase-1
(MEKK1), a kinase upstream of MAP kinases (52±54). There is
accumulating evidence indicating that enzymes of the MAP
kinase family play a role in cox-2 gene expression. The Parke-
Davis MEK inhibitor PD98059 partially blocked LPS-induced
COX-2 expression in RAW 264.7 cells and also in lysophos-
phatidic acid-stimulated rat mesangial cells (55), which further
supports the association of ERK activation with COX-2-
mediated PG production. LPS-induced expression of COX-2
was blunted by SB203580, the p38 MAP kinase inhibitor,
which resulted in decreased PGE2 production in RAW 264.7
cells (56). Similar effects were observed in LPS-stimulated
monocytes (57).
Although the MAP kinase signaling pathways have been
extensively investigated in cultured cell lines, much less is
known about the specificity of MAP kinases and extent to
which they are activated during the tumor promotion in
mouse skin in vivo. Topical application of TPA on the ears of
CD1 mice (58) and skin of SENCAR mice (59) induced a rapid
and sustained activation of ERK but not of p38 MAP kinase.
Most importantly, we have found that treatment of dorsal skins
of female ICR mice with TPA significantly enhanced both
catalytic activities and phosphorylation of p38 MAP kinase
and ERK1/2. Topically applied curcumin inhibited activities of
both p38 and ERK1/2 MAP kinases in mouse skin.
To determine which MAP kinase play an important role in
activation of NF-kB in mouse skin, we investigated the inhi-
bitory effects of the ultrapotent MEK1/2 inhibitor U0126 and
the p38 inhibitor SB203580 on NF-kB activation. Interest-
ingly, only U0126 abolished the NF-kB binding activity. In
another experiment, we examined an inhibitory effect of
U0126 on TPA-induced COX-2 expression. U0126 almost
completely abrogated TPA-induced COX-2 protein expres-
sion, which supports the idea that ERK may play an important
role in the signaling pathway of TPA-induced COX-2 expres-
sion and NF-kB activation in mouse skin.
Dhawan and Richmond (30) showed that in Hs294T cells,
ERK regulation of NF-kB activation involves increased IkB
phosphorylation with concomitant elevation in the NF-kB
DNA binding activity. An increase in phosphorylation of
IkB is responsible for enhanced degradation of this inhibitory
1521
K.-S.Chun et al.
Fig. 10. Hypothetical mechanism underlying suppression of TPA-induced COX-2 expression by curcumin in mouse skin. Note that curcumin can suppress
the activation of ERK1/2 or degradation of IkB (present study). Curcumin can also directly interfere with NF-kB DNA binding (47).
subunit, which leads to increased nuclear localization of NF-
kB and subsequent DNA binding. In our present study, U0126
inhibited the TPA-induced phosphorylation of IkBa in mouse
skin. Most inhibitors of NF-kB activation, such as silymarin
(60) and oleandrin (61), exert their anti-inflammatory effects
through suppression of phosphorylation and degradation of
IkBa. Thus, we examined whether curcumin could also
block NF-kB activation by inhibiting IkBa phosphorylation.
Topical application of curcumin suppressed TPA-induced
IkBa phosphorylation in a dose-dependent manner. Based on
these findings, we suggest that the ERK signaling pathway
regulates NF-kB activation through inhibition of IkB phos-
phorylation. These results may explain the molecular mechan-
ism responsible for the inhibitory effect of curcumin on NF-kB
activation in TPA-treated mouse skin (Figure 10).
In conclusion, the present study demonstrates that curcumin
inhibits induction of COX-2 in TPA-treated mouse skin
in vivo. Since improper and abnormal over-expression of
COX-2 is implicated in the pathogenesis of various types of
human cancers, assessment of the effects of curcumin on cox-2
gene expression may represent a useful surrogate biomarker
for the evaluation of its chemopreventive potential. Our
1522
Downloaded from https://academic.oup.com/carcin/article/24/9/1515/2390540 by Dongguk University user on 28 April 2021
findings that curcumin inhibits TPA-induced COX-2 expres-
sion by blocking the ERK and NF-kB signaling cascades may
provide molecular basis for suppression of tumor promotion as
well as inflammation exerted by this chemopreventive phyto-
chemical in mouse skin. Although chemopreventive and che-
moprotective properties of curcumin have been extensively
investigated and well documented, the compound, under
certain pathophysiologic conditions, may exert detrimental
effects. According to a recent study by Frank and colleagues,
0.5% curcumin in the diet failed to protect Long-Evans
Cinnamon rats against hepatic and renal carcinogenesis, but
rather shortened the median survival time, probably due to
enhanced oxidative stress induced by this phenolic in the
presence of excess copper (62). Therefore, a caution should
be made for intake of curcumin by patients suffering from
metal storage disorders, such as Wilson's disease or hepatitis C
viral infection.
Acknowledgement
This work was supported by the grant (00PJ1-PG3-21400-0052) from the
Ministry and Welfare, Republic of Korea.

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Received April 29, 2003; revised June 15, 2003; accepted June 17, 2003