International Conference on Diet, Nutrition, and Cancer

Zingiberaceous and Citrus Constituents, 1ⴕ-Acetoxychavicol Acetate,

Zerumbone, Auraptene, and Nobiletin, Suppress Lipopolysaccharide-
Induced Cyclooxygenase-2 Expression in RAW264.7 Murine Macrophages
through Different Modes of Action1–3
Akira Murakami, Tomohiro Shigemori, and Hajime Ohigashi4

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Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan

ABSTRACT In the present study, we explored the suppressive activities of 1⬘-acetoxychavicol acetate (ACA), au-
raptene, nobiletin, and zerumbone toward LPS-induced cyclooxygenase (COX)-2 mRNA expression in mouse macro-
phages and the underlying molecular mechanisms. Pretreatment of RAW264.7 cells with LPS led to the activation of
mitogen-activated protein kinase (MAPK)s [p38, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal
kinase (JNK)1/2] and Akt, together with degradation of the inhibitor of nuclear factor-␬B (I␬B)-␣ protein and nuclear
translocation of nuclear factor (NF)-␬B p65, and the resultant activation of activator protein (AP)-1, NF-␬B, and
cAMP-responsive element-binding protein (CREB) transcription factors. ACA abrogated ERK1/2 and JNK1/2, but not
p38 MAPK, as well as the activation of those transcription factors. Although it allowed LPS-triggered phosphorylation
of those MAPKs and NF-␬B nuclear translocation, nobiletin suppressed the activation of AP-1, NF-␬B, and CREB.
Zerumbone had no effect on those transcription factors, though it attenuated COX-2 mRNA expression, suggesting that
it disrupts the stabilization of COX-2 mRNA. Conversely, zerumbone significantly accelerated spontaneous COX-2
mRNA decay, the potency of which was comparable with that of SB203580, an inhibitor of p38 MAPK, whose activation
has key roles in the proinflammatory mRNA stabilization processes. Because SB203580 but not zerumbone suppressed
LPS-induced p38 MAPK activation, the molecular targets of zerumbone may be MAPK-activated protein kinase-2 or
located downstream. However, auraptene suppressed the expression of COX-2 protein but not mRNA, implying that it
targets translation. We propose that these phytochemicals are promising chemopreventive agents for inflammation-
associated carcinogenesis. Their use in combination may enhance their efficacy because of their different modes of
action. J. Nutr. 135: 2987S–2992S, 2005.

KEY WORDS: ● cyclooxygenase-2 ● cancer prevention ● anti-inflammation ● macrophage

● dietary factor

Inflammation is a pathophysiological phenomenon that is
involved in numerous diseases, including the development of
neoplasms. Stromal activation of inflammatory cells induces
dormant tumor cells to grow and progress into malignant
tumors. Upon stimulation, those cells produce and secrete a
1
Published in a supplement to The Journal of Nutrition. Presented as part of
the International Research Conference on Food, Nutrition, and Cancer held in
Washington, DC, July 14 –15, 2005. This conference was organized by the Amer-
ican Institute for Cancer Research and the World Cancer Research Fund Inter-
national and sponsored by (in alphabetical order) California Avocado Commis-
sion; California Walnut Commission; Campbell Soup Company; The Cranberry
Institute; Danisco USA, Inc.; The Hormel Institute; National Fisheries Institute; The
Solae Company; and United Soybean Board. Guest editors for this symposium
were Vay Liang W. Go, Ritva R. Butrum, and Helen A. Norman. Guest Editor
Disclosure: R. R. Butrum and H. Norman are employed by conference sponsor
American Institute for Cancer Research; V.L.W. Go, no relationships to disclose.
2
Author Disclosure: No relationships to disclose.
3
Supported by grants-in-aid from the Ministry of Agriculture, Forestry, and
Fisheries Food Research Project, Integrated Research on Safety and Physiolog-
ical Function of Food.
4
To whom correspondence should be addressed.
E-mail: ohigashi@kais.kyoto-u.ac.jp.
cocktail of biochemical mediators, including reactive oxygen
and nitrogen species and proinflammatory chemokines and
cytokines, as well as eicosanoids such as prostaglandins (PGs).5
Cyclooxygenase (COX; PGH2 synthase) donates 2 oxygen
molecules to arachidonic acid to form PGG2 by peroxidation,
which in turn is reduced to PGH2. Nonsteroidal anti-inflam-
matory drugs such as aspirin have received considerable atten-
tion for their ability not only to mitigate inflammatory re-
sponses but also potentially to prevent cancer incidence in the
human colon (1). Conversely, numerous animal experiments
have demonstrated the cancer preventive efficacy of COX
5
Abbreviations used: ACA, 1⬘-acetoxychavicol acetate; AP-1, activator pro-
tein-1; COX, cyclooxygenase; CREB, cAMP-responsive element-binding protein;
DMSO, dimethylsulfoxide; ERK, extracellular signal-regulated kinase; FBS, fetal
bovine serum; GPx, glutathione peroxidase; GST, glutathione S-transferase;
HPRT, hypoxanthine phosphoribosyl transferase; HRP, horseradish peroxidase;
I␬B, inhibitor of NF-␬B; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide;
MAPK, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein
kinase-2; NF-␬B, nuclear factor ␬B; NQO1, NAD(P)H quinone oxidoreductase;
PG, prostaglandin; Pi, proteinase inhibitor.

0022-3166/05 $8.00 © 2005 American Society for Nutrition.

2987S
2988S SUPPLEMENT
inhibitors. The COX enzyme consists of at least two isoforms,
COX-1 and COX-2. In contrast to COX-1, COX-2 protein is
only slightly expressed in most normal mammalian tissues in
response to physical, chemical, and biological stimuli, includ-
ing UV light exposure, dioxin, and lipopolysaccharide insult.
Recently, COX-2 has received the attention of numerous
researchers regarding the relation of its expression to patho-
genesis. In particular, ample evidence exists of the involve-
ment of COX-2 expression in carcinogenesis in many different
target organs (2).
We screened Japanese (3) and subtropical vegetables and
fruits (4 – 6) for their anti–tumor-promoting activities and
identified some active constituents [reviewed in Nakamura et
al. (7)]. It is notable that 1⬘-acetoxychavicol acetate (ACA,
from Alpinia galanga, Zingiberaceae) and zerumbone (from
Zingiber zerumbet, Zingiberaceae) as well as auraptene and
nobiletin (from citrus fruits), which are readily available from
natural sources and/or chemically synthesized, prevented
chemical carcinogenesis in several organs (8 –16), including
the colon, in mouse and rat experiments. The mechanisms of
action of these agents are not fully understood, though impor-
tant findings based on the results of cellular and animal ex-
periments have accumulated. Those mechanisms include the
induction of phase II enzymes and apoptosis, attenuation of
reactive oxygen and nitrogen species from inflammatory cells,
regulation of proinflammatory cytokines, and inhibition of
mutagenesis. In addition, these dietary factors have shown a
marked ability to suppress COX-2 expression both in vitro and
in vivo (8,10,17,18), though their molecular mechanisms re-
main largely unknown. In the present study, we investigated
the mechanisms by which ACA, auraptene, nobiletin, and
zerumbone attenuate LPS-induced COX-2 mRNA expression
in RAW264.7 mouse macrophages.
Materials and methods
Reagents and cells. ACA (19), auraptene (20), nobiletin (12),
and zerumbone (5) were purified as previously reported. DMEM and
fetal bovine serum (FBS) were purchased from Invitrogen. LPS (Esch-
erichia coli serotype 0127, B8) was purchased from Difco Labs. All
other chemicals were purchased from Wako Pure Chemical Industries
unless specified otherwise. RAW264.7 murine macrophages were
purchased from the American Type Culture Collection.
RT-PCR. RAW264.7 cells (1 ⫻ 106) were grown to confluence
in 5 mL DMEM with 10% FBS on 6-well plates, then incubated in an
atmosphere containing 5% CO2 at 37°C for 13 h. The cells were
washed with PBS twice, after which the medium was replaced with
FBS- and phenol-red-free medium (2.5 mL) containing samples dis-
solved in 25 ␮L dimethylsulfoxide (DMSO). After 30 min of prein-
cubation, the cells were treated with LPS (100 ␮g/L). After a 6-h
incubation, the cells were lysed, and total RNA was extracted using
kits (RNeasy® minikit and QIAshredder®, Qiagen). One microgram
of total RNA was reverse transcribed using an RNA PCR Kit®
(Takara) with an oligo dT-adaptor primer, as recommended by the
supplier. Then, PCR assays were performed using a thermal cycler
(PTC-0100; MJ Research) with hypoxanthine phosphoribosyl trans-
ferase (HPRT) and COX-2 using primers synthesized by Proligo with
1 ␮L of a cDNA preparation, 45 ␮L of Platinum® PCR SuperMix
(Invitrogen), and 2 ␮L of each primer (1 ␮mol/L) as follows: HPRT
(5⬘-gTAATgATCAgTCAACggggAC-3⬘ and 5⬘-CCAgCAAgCTT-
gcAACCTTAACCA-3⬘), 20 cycles at 94°C for 2 min, 58°C for 2
min, and 72°C for 2 min; and COX-2 (5⬘-gCATTCTTTgCCCAg-
CACTT-3⬘ and 5⬘-AgACCAggCACCAgACCAAAgA-3⬘), 20 cy-
cles at 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s. The PCR
products were separated on 2% NuSieve® 3:1 agarose (BioWhittaker
Molecular Applications), and each band was visualized using 0.01%
SYBR Gold® stain (Molecular Probes). The amplified products were
photographed with a digital camera, and the band intensities were
analyzed using NIH Image software. For each target gene, we calcu-
lated the relative expression levels of mRNA in the samples as the
ratio of the amount of each gene mRNA to that of HPRT mRNA.
The number of PCR cycles was optimized so that each band intensity
increased proportionally as the amount of cDNA increased. Each
experiment was done at least 3 times.
Western blotting. RAW264.7 cells (2 ⫻ 106) were grown to
confluence in 4 mL DMEM with 10% FBS on a 60-mm dish, then
incubated in an atmosphere containing 5% CO2 at 37°C for 13 h.
The cells were washed with PBS twice, after which the medium was
exchanged with FBS- and phenol-red-free mediium (10 mL) contain-
ing samples dissolved in 20 ␮L DMSO. After 30 min of preincuba-
tion, the cells were treated with LPS (100 ␮g/L) for various times.
After being washed with PBS twice, the cells were separated into
nuclear and cytosol fractions using a kit (Bio Vision; Research Prod-
ucts). Protein concentrations were determined using a DC Protein
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Assay kit (Bio-Rad), with ␥-globulin employed as the standard. Next,
10 or 20 ␮g of proteins (for the nuclear and cytosol fractions,
respectively) were separated on 10% polyacrylamide gels and elec-
trophoretically transferred onto polyvinylidene difluoride membranes
(Millipore). After blocking, the membranes were incubated with
rabbit anti-proteinase inhibitor (Pi)-extracellular signal-regulated ki-
nase (ERK)1/2, rabbit anti-ERK1/2 (Promega), rabbit anti-Pi-c-Jun
NH2-terminal kinase (JNK)1/2 antibody, rabbit anti-JNK1/2 anti-
body, rabbit anti-Pi-p38 mitogen-activated protein kinase (MAPK)
antibody, rabbit anti-p38 antibody (Cell Signaling), rabbit anti-Pi-
Ser473 Akt1/2/3 antibodies, rabbit anti-Akt1/2/3 antibody, rabbit
anti–inhibitor of nuclear factor ␬B (I␬B)-␣ antibody, rabbit anti-
nuclear factor (NF)-␬B p65 antibody, rabbit anti-MAPK-activated
protein kinase (MAPKAPK)-2 (Santa Cruz Biotechnology), and goat
anti-␤-actin antibody (Biochemical Technologies) (1:1000 dilution
each) and then with the corresponding secondary antibodies [horse-
radish peroxidase (HRP)-conjugated anti-rabbit IgG, 1:1000 dilution
(Dako); or HRP-conjugated anti-goat IgG, 1:1000 dilution (Dako)].
The blots were developed using an ECL detection kit (Amersham
Life Science). Each experiment was done at least 3 times.
Reporter assays. The following reporter assays were performed
using a Mercury® Pathway Profiling System (Clontech Laboratories)
with some modifications. RAW264.7 cells (3 ⫻ 105 cells/mL) were
preincubated on a 24-well plate for 12 h. Next, 625 ␮L OPTI-MEM®
(Invitrogen) and 37.5 ␮L LipofectAMINE Reagent® (Invitrogen)
were mixed in a tube, after which 4 ␮g of either pNF-␬B-, pAP-1, or
pcAMP-responsive element-binding protein (CREB)-luciferase vec-
tor, provided in the kit, and 4 ␮g of pRL-TK vector (Promega), which
served as the internal standard, were added and allowed to stand at
room temperature for 30 min, after which an additional 5 mL OPTI-
MEM was added. After the cells were washed with Hanks’ buffer
twice, 250 ␮L transfection mixture was added to each well, and the
cells were incubated at 37°C for 6 h. After being washed, the cells
were incubated in 1 mL DMEM with 10% FBS for 12 h. The cells
were washed again with Hanks’ buffer twice, then exposed to the
vehicle (0.5% DMSO, v:v) or samples dissolved in DMSO in serum-
free DMEM for 30 min. After stimulation of the cells with LPS (100
␮g/L) for 12 h, luciferase activity in the cell lysate was determined
using a Dual-Luciferase Reporter Assay Kit® (Promega).
COX-2 mRNA decay. RAW264.7 cells (2 ⫻ 106) were grown to
confluence in 3 mL DMEM with 10% FBS on 6-well plates, then
incubated in an atmosphere containing 5% CO2 at 37°C for 13 h.
The cells were washed with PBS twice, after which the medium was
exchanged with FBS- and phenol-red-free medium (3 mL) containing
LPS (100 ␮g/L) and incubated for 4.5 h. Then the cells were treated
with actinomycin D (1 ␮g/mL final concentration) for 30 min,
followed by treatment with the vehicle or sample for 6 h. RT-PCR
was performed as described above.
In vitro kinase assay. A cell-free kinase assay was performed
using a Kinase-Glo® Luminescent Kinase Assay (Promega). To a
disposable culture tube (12 ⫻ 75 mm; Iwaki), 1.5 ␮L recombinant
MAPKAPK-2 (PanVera), 0.2 ␮L recombinant active p38␣ (Pan-
Vera), and 23.3 ␮L kinase buffer (25 mmol/L HEPES, pH 7.6; 2
mmol/L dithiothreitol; 20 mmol/L MgCl2; 0.1 mmol/L NaVO4) were
added. After 1 ␮L of each sample and 25 ␮L ATP solution (4
␮mol/L) were added, the reaction mixture was incubated at 28°C for
3 h. Thereafter, 50 ␮L Kinase-Glo Reagent (Promega) was added,
 ZINGIBERACEOUS AND CITRUS CONSTITUENTS
FIGURE 1 (A) Chemical structure of dietary factors. (B) Suppres-
sive effects of dietary factors on LPS-induced COX-2 mRNA expres-
sion detected by RT-PCR, as described in Materials and Methods.
ACA, 5 ␮mol/L; AUR, auraptene, 20 ␮mol/L; BL, negative control
treated with vehicle; NOB, nobiletin, 200 ␮mol/L; ZER, zerumbone, 20
␮mol/L.
and the culture was incubated at room temperature for 15 min before
the determination of chemiluminescence.
Statistical analysis. Each experiment was done 3 times unless
specified otherwise, with values shown as means ⫾ SD. The signifi-
cance of differences between groups in each assay was assessed using
a Student’s t-test (2-sided) that assumed unequal variance.
Results
Effects on COX-2 mRNA expression. Treatment of
RAW264.7 macrophages with LPS for 6 h caused a marked
2989S
induction of COX-2 mRNA as detected by RT-PCR. The
concentration of each dietary factor (Fig. 1A) was nonlethal
up to the highest concentration tested. Both zerumbone and
ACA (20 ␮mol/L each) abolished LPS-induced COX-2 ex-
pression, whereas nobiletin, but not auraptene, moderately
attenuated that expression (Fig. 1B).
Effects on activation of MAPKs and NF-␬B system. LPS
stimulation induced the time-dependent phosphorylation of
p38 MAPK from 0 to 60 min, whereas that of JNK1/2 and
ERK1/2 peaked at 30 min after the LPS challenge. Activation
of p38 was not attenuated by zerumbone, ACA, or nobiletin
(Fig. 2A). However, ACA substantially blocked both JNK1/2
and ERK1/2 activation, whereas both zerumbone and nobile-
tin allowed LPS-induced activation of those protein kinases.
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Furthermore, the phosphorylation of Akt (protein kinase B) at
Ser473 was detectable in nontreated cells and enhanced by LPS
in a time-dependent manner (Fig. 2B). None of the 3 com-
pounds demonstrated any inhibition. LPS-induced NF-␬B ac-
tivation is mediated through the degradation of I␬B, a sup-
pressive partner of NF-␬B, leading to the nuclear translocation
of NF-␬B for transactivation of numerous proinflammatory
and antiapoptotic genes. As shown in Figure 2B, I␬B protein
was scarcely seen at 30 min after the LPS challenge and was
restored within 60 min. Conversely, NF-␬B p65 was signifi-
cantly translocated into the nucleus from 30 to 60 min. ACA
did not allow those LPS-induced biochemical processes to
occur, whereas both zerumbone and nobiletin were virtually
inactive, except that the nuclear protein level of p65 in the
nobiletin-treated cells was comparable with that in the non-
treated cells at 60 min.
Effects on transcriptional activity of NF-␬B, AP-1, and
CREB. We transfected the NF-␬B-, AP-1-, and CREB-
luciferase vectors to examine the effects of dietary factors on
those transcriptional activities (Fig. 3). LPS treatment mark-
edly elevated the activities of the transcription factors by 2.1-
to 6.8-fold. It is notable that ACA and nobiletin significantly
suppressed the 3 transcription factors, whereas zerumbone had
no effect.
FIGURE 2 Effects of dietary fac-
tors on LPS-induced phosphorylation
of p38, JNK1/2, and ERK1/2 (A); and
Akt (Ser473) activation, I␬B protein deg-
radation, and NF-␬B p65 nuclear
translocation (B); detected by Western
blotting assay, as described in Materi-
als and Methods. ACA, 5 ␮mol/L;
NOB, nobiletin, 200 ␮mol/L; ZER,
zerumbone, 20 ␮mol/L.
2990S SUPPLEMENT

FIGURE 3 Effects of dietary fac-

tors on LPS-induced NF-␬B (A), AP-1
(B), and CREB (C) activation, detected
by luciferase reporter assays, as de-
scribed in Materials and Methods. *Dif-
ferent from BL, P ⬍ 0.01; **different
from BL, P ⬍ 0.05; ***different from
LPS, P ⬍ 0.01; ****different from LPS,
P ⬍ 0.05; Student’s t test. ACA, 5
␮mol/L; BL, negative control treated
with vehicle; NOB, nobiletin, 200
␮mol/L; RLU, relative luciferase unit;
ZER, zerumbone, 20 ␮mol/L.

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Zerumbone promotion of COX-2 mRNA degredation. A
number of proinflammatory genes have highly conserved AU-
rich elements in the proximal regions of the 3⬘-untranslated
region, which play critical roles in their mRNA stabilization
processes via activation of the p38 MAPK pathway (21).
RAW264.7 cells were pretreated with LPS to induce COX-2
mRNA expression and then exposed to actinomycin D to
block gene transcription. The cells were then treated with the
vehicle, zerumbone, or SB203580, an inhibitor of p38 MAPK,
after which total RNA was extracted and RT-PCR was per-
formed. Zerumbone accelerated the vehicle-induced degrada-
tion of COX-2 mRNA, and its potency was comparable with
that of SB203580 (Fig. 4A). Further, the p38-induced phos-
phorylation of MAPKAPK-2 was abolished by SB203580 and
slightly attenuated by zerumbone (Fig. 4B). A cell-free in vitro
kinase assay for p38 MAPK activity also revealed that zerum-
bone did not inhibit the p38 enzyme up to a concentration of
100 ␮mol/L, whereas SB203580 demonstrated complete inhi-
bition at 1 ␮mol/L (Fig. 4C).
oral and topical administration of zerumbone suppressed phor-
bol ester– and azoxymethane-induced COX-2 protein expres-
sion in mouse skin (6) and rat colon (10), respectively, though
the molecular mechanisms were not fully elucidated. The
present results provide some preliminary but useful insights
into the molecular mechanisms of these dietary factors (Fig.
5). Auraptene may disrupt the translation step of COX-2
mRNA, because it was reported to suppress expression of the
protein in LPS-treated RAW264.7 cells (18), whereas it did
not suppress mRNA expression in our experiment (Fig. 1). In
addition, we recently found that auraptene suppresses the
expression of protein but not mRNA of matrix metallopro-

Discussion
The dietary factors examined in the present study were
shown to be cancer preventive in mouse and rat models
(8 –16). Although the mechanisms of action have not been
fully elucidated, some of our previous findings in regard to
their biological and biochemical activities may be helpful. For
example, oral feeding of ACA and auraptene led to a marked
elevation of phase II drug metabolizing enzymes, including
glutathione S-transferase (GST) and NAD(P)H quinone ox-
idoreductase (NQO1), in the colon and liver of rats (14,22).
Similarly, zerumbone induced the activation of transcription
factor Nrf2 in RL34 rat hepatocytes for upregulating the an-
tioxidative and self-protecting enzymes ␥-glutamylcysteine
synthetase, glutathione peroxidase (GPx), and hemeoxygen-
ase-1 (23). These results are consistent with our other in vivo
observations that a topical application of zerumbone enhanced
or induced mRNA expression by the manganese superoxide
dismutase gene as well as the GPx, GST-P1, and NQO1 genes
without affecting the expression levels of cytochrome P450
1A1 and 1B1 mRNA in mouse epidermis (8). Furthermore, FIGURE 4 (A) Blots for RAW 264.7 cells treated with LPS and
the antioxidative and antinitrosative properties of that com- incubated for 4.5 h, then treated with actinomycin D (1 ␮g/mL) for 30
min, followed by treatment with the vehicle or sample for 6 h. RT-PCR
pound, which are related to the attenuation of endotoxin- or
was performed as described in Materials and Methods. (B) Blots for
phorbol ester–induced superoxide anion and nitric oxide gen- RAW 264.7 cells treated with either zerumbone (ZER; 20 ␮mol/L) or
eration in inflammatory cells, were described in other cell SB203580 (SB; 20 ␮mol/L) for 30 min, then exposed to LPS for 30 or 60
culture and animal model studies (7,12,17–20). min, followed by Western blotting assay, as described in Materials and
ACA, auraptene, nobiletin, and zerumbone were found to Methods. (C) Graphed results of cell-free kinase assay, performed as
suppress COX-2 protein production in macrophages, whereas described in Materials and Methods.
 ZINGIBERACEOUS AND CITRUS CONSTITUENTS
teinase-7 in HT-29 human colon cancer cells (K. Kawabata et
al., unpublished data, 2005).
In the present study, ACA substantially suppressed LPS-
induced activation of both ERK1/2 and JNK1/2, which is
probably associated with its ability to abrogate COX-2 mRNA
via repression of AP-1, NF-␬B, and CREB, as shown previ-
ously for the involvement of ERK1/2 and JNK1/2 with those
transcriptional factors. For example, ERK activation is in-
volved in the phosphorylation of p65, thereby activating
NF-␬B (24); a similar observation was made for RAW264.7
cells (25). Furthermore, the importance of Ser536 as the phos-
phorylation site of p65 was proposed (26). Chen et al. (27)
reported that ERK activation led to NF-␬B activation that was
related to COX-2 expression, and other lines of evidence exist
for crosstalk activity between the ERK and CREB pathways
(28 –30); the effects of ACA on protein kinase A activation
should be explored in the future. Ample evidence shows that
activation of the JNK pathway leads to AP-1 transcriptional
activation, because phosphorylated c-Jun, one of the direct
targets of JNK, is a component of the AP-1 complex (31),
suggesting that ACA suppresses AP-1 activation by blocking
JNK activation, as shown in the present study (Fig. 5). The
direct targets of ACA, which are probably located upstream of
ERK/JNK, should be investigated in the future.
Transcription coactivators, including CREB-binding pro-
tein and its homologue p300, are known to play major roles in
various stimuli-induced or -repressed intracellular signaling
pathways (32) and were shown to potentiate the transcrip-
tional activities of AP-1 (33), NF-␬B (34), and CREB (35). In
addition, ASC-2 has emerged as a novel coactivator partici-
pating in the transcriptional activation of NF-␬B and AP-1
(36). Thus, nobiletin may disrupt the binding of those coac-
tivators with their corresponding partner transcription factors,
because we found that it suppressed NF-␬B, AP-1, and CREB
activation, whereas it was virtually inactive in suppression of
the activation of MAPKs and the Akt-NF-␬B pathway (Figs.
2, 3, and 5).
The mRNA expression of many, if not all, proinflamma-
tory genes is regulated by posttranscriptional mechanisms
2991S
FIGURE 5 Proposed molecular
mechanisms by which dietary factors
attenuate the expression of LPS-in-
duced COX-2 protein production in
RAW264.7 mouse macrophages. ACA
targets both JNK1/2 and ERK1/2, and
nobiletin may interfere with coactiva-
tors such as CBP/p300 that suppress
the transactivation of NF␬B, AP-1, and
CREB. Because zerumbone allowed
LPS-induced MAPKs/Akt activation
and transcriptional activation of those
transcription factors while it abrogated
COX-2 mRNA induction, it may target
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MK-2 or downstream molecules for
destabilizing mRNA. Auraptene may
disturb the translation process of
COX-2 mRNA, because it suppressed
COX-2 protein but not mRNA produc-
tion. AUR, auraptene; CBP, CREB-
binding protein; IKK, I␬B kinase; MK-2,
MAPK-activated protein kinase-2; NOB,
nobiletin; PKA, protein kinase A; SB,
SB203580; TLR, toll-like receptor; ZER,
zerumbone.
(37). Those genes, including the COX-2 gene, contain
highly conserved AU-rich elements in the proximal regions
of the 3⬘-untranslated region (23). Some binding proteins
for AU-rich elements were identified and shown to highly
affect COX-2 mRNA stability in RAW264.7 macrophages
(38). Of importance, the binding abilities of those proteins
are regulated by the p38 MAPK pathway (39). We found
that zerumbone (20 ␮mol/L) abrogated LPS-induced COX-2
mRNA expression (Fig. 1B), whereas it did not show any
suppression of the transcriptional activation of NF-␬B,
AP-1, and CREB. It is likely that CCAAT/enhancer bind-
ing protein ␤ and ␦ also may not be suppressed, because
their production depends on NF-␬B transcriptional activity
(40). This raises the possibility that zerumbone suppresses
COX-2 mRNA expression via a posttranscriptional mech-
anism. In the present study, zerumbone accelerated sponta-
neous degradation of COX-2 mRNA (Fig. 4A), was virtu-
ally inactive for suppressing p38 MAPK activation (Fig. 2),
and slightly attenuated MK2 phosphorylation (Fig. 4B)
whereas our in vitro kinase assay revealed that it was
definitely inactive regarding inhibition of p38 MAPK ac-
tivity (Fig. 4C). Based on these findings, we hypothesize
that zerumbone inhibits MK2 activity or targets unidenti-
fied molecules located downstream of MK2 (Fig. 5).
In conclusion, ACA targets both JNK1/2 and ERK1/2, and
nobiletin may interfere with coactivators, such as CREB-
binding protein/p300, that suppress the transactivation of NF-
␬B, AP-1, and CREB. Zerumbone allowed LPS-induced
MAPKs/Akt activation and transcriptional activation of those
transcription factors, whereas it abrogated COX-2 mRNA
induction. Thus, zerumbone may target MK-2 or downstream
molecules for destabilizing mRNA. In addition, auraptene may
disturb the translation process of COX-2 mRNA. because it
suppressed COX-2 protein but not mRNA production. These
results indicate that the dietary factors studied here have
distinct molecular mechanisms for COX-2 protein suppression
and that their use in combination may lead to synergistic
results with higher efficacy and lower toxicity.
2992S SUPPLEMENT
LITERATURE CITED
1. Kune GA, Kune S, Watson LF. Colorectal cancer risk, chronic illnesses,
operations, and medications: case control results from the Melbourne Colorectal
Cancer Study. Cancer Res. 1998;48:4399 – 404.
2. Subbaramaiah K, Dannenberg AJ. Cyclooxygenase 2: a molecular target
for cancer prevention and treatment. Trends Pharmacol Sci. 2003;24:96 –102.
3. Koshimizu K, Ohigashi H, Tokuda H, Kondo A, Yamaguchi K. Screening
of edible plants against possible anti-tumor promoting activity. Cancer Lett.
1988;39:247–57.
4. Murakami A, Morita H, Safitri R, Ramlan A, Koshimizu K, Ohigashi H.
Screening for in vitro anti-tumor-promoting activities of edible plants from Indo-
nesia. Cancer Detect Prev. 1998;22:516 –25.
5. Murakami A, Takahashi M, Jiwajinda S, Koshimizu K, Ohigashi H. Iden-
tification of zerumbone in Zingiber zerumbet Smith as a potent inhibitor of
12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation.
Biosci Biotechnol Biochem. 1999;63:1811–2.
6. Murakami A, Jiwajinda S, Koshimizu K, Ohigashi H. Screening for in vitro
anti-tumor promoting activities of edible plants from Thailand. Cancer Lett. 1995;
95:139 – 46.
7. Nakamura Y, Murakami A, Ohigashi H. Search for naturally-occurring
antioxidative chemopreventors on the basis of the involvement of leukocyte-
derived reactive oxygen species in carcinogenesis. Asian Pac J Cancer Prev.
2000;1:115–20.
8. Murakami A, Tanaka T, Lee JY, Surh YJ, Kim HW, Kawabata K, Nakamura
Y, Jiwajinda S, Ohigashi H. Zerumbone, a sesquiterpene in subtropical ginger,
suppresses skin tumor initiation and promotion stages in ICR mice. Int J Cancer.
2004;110:481–90.
9. Suzuki R, Kohno H, Murakami A, Koshimizu K, Ohigashi H, Yano M,
Tokuda H, Nishino H, Tanaka T. Citrus nobiletin inhibits azoxymethane-induced
large bowel carcinogenesis in rats. Biofactors 2004;22:111– 4.
10. Tanaka T, Shimizu M, Kohno H, Yoshitani S, Tsukio Y, Murakami A, Safitri
R, Takahashi D, Yamamoto K, et al. Chemoprevention of azoxymethane-induced
rat aberrant crypt foci by dietary zerumbone isolated from Zingiber zerumbet. Life
Sci. 2001;69:1935– 45.
11. Kohno H, Yoshitani S, Tsukio Y, Murakami A, Koshimizu K, Yano M,
Tokuda H, Nishino H, Ohigashi H, et al. Dietary administration of citrus nobiletin
inhibits azoxymethane-induced colonic aberrant crypt foci in rats. Life Sci. 2001;
69:901–13.
12. Murakami A, Nakamura Y, Torikai K, Tanaka T, Koshiba T, Koshimizu K,
Kuwahara S, Takahashi Y, Ogawa K, et al. Inhibitory effect of citrus nobiletin on
phorbol ester-induced skin inflammation oxidative stress and tumor promotion in
mice. Cancer Res. 2000;60:5059 – 66.
13. Kobayashi Y, Nakae D, Akai H, Kishida H, Okajima E, Kitayama W, Denda
A, Tsujiuchi T, Murakami A, et al. Prevention by 1⬘-acetoxychavicol acetate of the
induction but not growth of putative preneoplastic glutathione S-transferase
placental form-positive focal lesions in the livers of rats fed a choline-deficient
L-amino acid-defined diet. Carcinogenesis. 1998;19:1809 –14.
14. Tanaka T, Kawabata K, Kakumoto M, Hara A, Murakami A, Kuki W,
Takahashi Y, Yonei H, Maeda M, et al. Citrus auraptene exerts dose-dependent
chemopreventive activity in rat large bowel tumorigenesis: the inhibition corre-
lates with suppression of cell proliferation and lipid peroxidation and with induc-
tion of phase II drug-metabolizing enzymes. Cancer Res. 1998;58:2550 – 6.
15. Tanaka T, Kawabata K, Kakumoto M, Matsunaga K, Mori H, Murakami A,
Kuki W, Takahashi Y, Yonei H, et al. Chemoprevention of 4-nitroquinoline 1-ox-
ide-induced oral carcinogenesis by citrus auraptene in rats. Carcinogenesis.
1998;19:425–31.
16. Tanaka T, Kawabata K, Kakumoto M, Makita H, Hara A, Mori H, Satoh K,
Hara A, Murakami A, et al. Citrus auraptene inhibits chemically induced colonic
aberrant crypt foci in male F344 rats. Carcinogenesis. 1997;18:2155– 61.
17. Murakami A, Takahashi D, Kinoshita T, Koshimizu K, Kim HW, Yoshihiro
A, Nakamura Y, Jiwajinda S, Terao J, et al. Zerumbone, a Southeast Asian ginger
sesquiterpene, markedly suppresses free radical generation, proinflammatory
protein production, and cancer cell proliferation accompanied by apoptosis: the
alpha,beta-unsaturated carbonyl group is a prerequisite. Carcinogenesis. 2002;
23:795– 802.
18. Murakami A, Nakamura Y, Tanaka T, Kawabata K, Takahashi D, Ko-
shimizu K, Ohigashi H. Suppression by citrus auraptene of phorbol ester-and
endotoxin-induced inflammatory responses: role of attenuation of leukocyte ac-
tivation. Carcinogenesis. 2000;21:1843–50.
19. Murakami A, Ohura S, Nakamura Y, Koshimizu K, Ohigashi H. 1⬘-Ace-
toxychavicol acetate, a superoxide anion generation inhibitor, potently inhibits
tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in ICR mouse skin.
Oncology. 1996;53:386 –91.
20. Murakami A, Kuki W, Takahashi Y, Yonei H, Nakamura Y, Ohto Y, Ohi-
gashi H, Koshimizu K. Auraptene, a citrus coumarin, inhibits 12-O-tetradecanoyl-
phorbol-13-acetate-induced tumor promotion in ICR mouse skin, possibly
through suppression of superoxide generation in leukocytes. Jpn J Cancer Res.
1997;88:443–52.
21. Lasa M, Mahtani KR, Finch A, Brewer G, Saklatvala J, Clark AR. Regu-
lation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein
kinase p38 signaling cascade. Mol Cell Biol. 2000;20:4265–74.
22. Tanaka T, Kawabata K, Kakumoto M, Makita H, Matsunaga K, Mori H,
Satoh K, Hara A, Murakami A, et al. Chemoprevention of azoxymethane-induced
rat colon carcinogenesis by a xanthine oxidase inhibitor, 1⬘-acetoxychavicol
acetate. Jpn J Cancer Res. 1997;88:821–30.
23. Nakamura Y, Yoshida C, Murakami A, Ohigashi H, Osawa T, Uchida K.
Zerumbone, a tropical ginger sesquiterpene, activates phase II drug metabolizing
enzymes. FEBS Lett. 2004;572:245–50.
24. Zhang L, Ma Y, Zhang J, Cheng J, Du J. A new cellular signaling
mechanism for angiotensin II activation of NF-␬B: an I␬B-independent RSK-
mediated phosphorylation of p65. Arterioscler Thromb Vasc Biol. 2005;25:1148 –
Downloaded from https://academic.oup.com/jn/article-abstract/135/12/2987S/4669952 by guest on 13 June 2020
53.
25. Jung SW, Lee MH, Kim YC, Paik SG, Choi YH, Kim YS, Kang KI.
Modulation of the transactivation function of nuclear factor-␬B by lipopolysac-
charide in RAW264.7 macrophages. Int J Oncol. 2004;25:1081–7.
26. Hu J, Nakano H, Sakurai H, Colburn N.H. Insufficient p65 phosphorylation
at S536 specifically contributes to the lack of NF-␬B activation and transformation
in resistant JB6 cells. Carcinogenesis. 2004;25:1991–2003.
27. Chen BC, Yu CC, Lei HC, Chang MS, Hsu MJ, Huang CL, Chen MC, Sheu
JR, Chen TF, et al. Bradykinin B2 receptor mediates NF-␬B activation and
cyclooxygenase-2 expression via the Ras/Raf-1/ERK pathway in human airway
epithelial cells. J Immunol. 2004;173:5219 –28.
28. Impey S, Obrietan K, Wong ST, Poser S, Yano S, Wayman G, Deloulme
JC, Chan G, Storm DR. Cross talk between ERK and PKA is required for Ca2⫹
stimulation of CREB-dependent transcription and ERK nuclear translocation.
Neuron. 1998;21:869 – 83.
29. Davis S, Vanhoutte P, Pages C, Caboche J, Laroche S. The MAPK/ERK
cascade targets both Elk-1 and cAMP response element-binding protein to
control long-term potentiation-dependent gene expression in the dentate gyrus in
vivo. J. Neurosci. 2000;20:4563–72.
30. Chen JC, Huang KC, Wingerd B, Wu WT, Lin WW. HMG-CoA reductase
inhibitors induce COX-2 gene expression in murine macrophages: role of MAPK
cascades and promoter elements for CREB and C/EBP␤. Exp. Cell Res. 2004;
301:305–19.
31. Eferl R, Wagner EF. AP-1: a double-edged sword in tumorigenesis. Nat.
Rev. Cancer 2003;3:859 – 68.
32. Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG, Ju-
guilon H, Montminy M, Evans RM. Role of CBP/P300 in nuclear receptor signal-
ling. Nature 1996;383:99 –103.
33. Horvai AE, Xu L, Korzus E, Brard G, Kalafus D, Mullen TM, Rose DW,
Rosenfeld MG, Glass CK. Nuclear integration of JAK/STAT and Ras/AP-1 signal-
ing by CBP and p300. Proc Natl. Acad. Sci. U S A. 1997;94:1074 –9.
34. Ravi R, Mookerjee B, van Hensbergen Y, Bedi GC, Giordano A, El-Deiry
WS, Fuchs EJ, Bedi A. p53-Mediated repression of nuclear factor-␬B RelA via the
transcriptional integrator p300. Cancer Res. 1998;58:4531–36.
35. Lundblad JR, Kwok RP, Laurance ME, Harter ML, Goodman RH. Adeno-
viral E1A-associated protein p300 as a functional homologue of the transcrip-
tional co-activator CBP. Nature 1995;374:85– 8.
36. Lee SK, Na SY, Jung SY, Choi JE, Jhun BH, Cheong J, Meltzer PS, Lee
YC, Lee JW. Activating protein-1 nuclear factor-␬B and serum response factor as
novel target molecules of the cancer-amplified transcription co-activator ASC-2.
Mol. Endocrinol. 2000;14:915–25.
37. Bakheet T, Frevel M, Williams BR, Greer W, Khabar KS. ARED: human
AU-rich element-containing mRNA database reveals an unexpectedly diverse
functional repertoire of encoded proteins. Nucleic Acids Res. 2001;29:246 –54.
38. Cok SJ, Acton SJ, Sexton AE, Morrison AR. Identification of RNA-binding
proteins in RAW 264.7 cells that recognize a lipopolysaccharide-responsive ele-
ment in the 3-untranslated region of the murine cyclooxygenase-2 mRNA. J. Biol.
Chem. 2004;279:8196 –205.
39. Winzen R, Kracht M, Ritter B, Wilhelm A, Chen CY, Shyu AB, Muller M,
Gaestel M, Resch K, et al. The p38 MAP kinase pathway signals for cytokine-
induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an
AU-rich region-targeted mechanism. EMBO J. 1999;18:4969 – 80.
40. Wadleigh DJ, Reddy ST, Kopp E, Ghosh S, Herschman HR. Transcrip-
tional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7
macrophages. J. Biol. Chem. 2000;275:6259 – 66.