JFS S: Sensory and Nutritive Qualities of Food

Lycopene Inhibits LPS-Induced Proinflammatory

Mediator Inducible Nitric Oxide Synthase
in Mouse Macrophage Cells
MOHAMED M. RAFI, PREM NARAYAN YADAV, AND MARYNELL REYES

ABSTRACT: Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived prod-
ucts, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava,
watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underly-
ing the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with
lycopene (10 µM) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with
the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that
lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in
RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS
proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein
and mRNA expression were not affected by treatment with lycopene.
Keywords: COX-2, inflammation, iNOS, lycopene, macrophages

Introduction
L ycopene is a fat-soluble red-orange carotenoid present in toma-
toes and tomato products. In the United States, dietary sources
of lycopene also include dried apricots, guava, watermelon, papaya,
and pink grapefruit. More than 85% of lycopene is consumed in the
form of tomato products (Klein 2005; Krinsky and Johnson 2005),
and total dietary intake of lycopene is approximately 2 mg/d to 5
mg/d, reflecting a diet high in tomato and tomato products rather
than lycopene supplements (Krinsky and Johnson 2005).
The bioavailability and biotransformation of lycopene are not
well characterized. However, 14 to 18 different isomers of lycopene
are present in prostate tissue, indicating high concentrations in
prostate tissue compared to other tissues (Clinton and others 1996).
Approximately 80% of lycopene is found in the cis-isomer form in
prostate tissue, compared to 50% in blood and 5% to 10% in foods
(Krinsky and Johnson 2005). Therefore, a diet high in lycopene,
which shows a decrease in the prostate-specific antigen (PSA) level in
the serum of prostate cancer patients, indicates a protective effect in
prostate cancer (Giovannucci and others 1995; Klein 2005). Although
many fresh fruits and vegetables are known to be dietary sources
of lycopene, a study conducted by Stahl and Sies (1992) indicates
that the bioavailability of lycopene is much higher in cooked toma-
toes when compared to unprocessed tomato juice. This is further
demonstrated by increased bioavailability of trans and cis-lycopene
in tomato paste than in fresh tomatoes (Gartner and others 1997).
Oxidative stress has been recognized as one of the major causes
for cancer (Di Mascio and others 1989, 1990). An overview of the
singlet oxygen. Among the carotenoids, lycopene was also identi-
fied as the most potent antioxidant when used alone, followed by α-
tocopherol, α-carotene, β-cryptoxanthin, zeaxanthin = β-carotene,
and lutein (Stahl and others 1998).
Nitric oxide (NO) is produced by the activated inflammatory cells
and has demonstrated involvement in the pathogenesis of acute and
chronic inflammatory diseases (Molianen and others 1997). NO is
produced from L-arginine through the action of NO synthase (NOS).
This short-lived radical has beneficial and detrimental effects on
its host. In the biological system NO has many functions such as
vasodilation, neurotransmission, and inflammatory response (Lo
and others 2002). There are 3 forms of NO synthase, including en-
dothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS
(iNOS) (Liang and others 1999). During the inflammatory process,
NO production increases and becomes cytotoxic to the cells by re-
acting with superoxide anions to produce peroxynitrite (ONOO–)
(Sautebin 2000). The peroxynitrites induce oxidative damage in host
tissue and are potentially harmful to the cellular targets (Epe and
others 1996; Sautebin 2000).
The bacterial endotoxin (lipopolysaccharide [LPS]) is able to
induce an inflammatory response by activation of macrophages
through the binding of the LPS-binding protein to CD14,
a membrane glycophosphatidylinositol anchored protein. Pro-
inflammatory genes are activated via signal transduction pathways
that lead to NO production (Lahti and others 2000). However, a sig-
nificant amount of NO can be produced by iNOS, which is regulated
at the transcriptional level by transcription factors such as NF-κB
S: Sensory & Nutritive Qualities of Food

mechanism of action of lycopene as an antioxidant, antiprolifera-
tive, and hypocholesterolemic properties was reviewed, where ly-
copene was recognized as an antioxidant due to its ability to trap
MS 20060414 Submitted 7/26/2006, Accepted 10/23/2006. Authors are with
Dept. of Food Science, Cook College, Rutgers, The State Univ. of New Jersey, prostaglandins and exit in 2 isoforms, COX-1 and COX-2. COX-1
65 Dudley Rd., New Brunswick, NJ 08901, U.S.A. Direct inquiries to author
Rafi (E-mail: rafi@rci.rutgers.edu).
and AP-1 (Conelly and others 2001). Therefore, inhibition of NO pro-
duction in response to inflammatory stimuli is a useful strategy to
prevent inflammatory diseases such as cancer (Hobbs and others
1999; Sautebin 2000).
Cyclooxygenases (COX) convert arachidonic acid to
is constitutively expressed and protects gastrointestinal mucosa
whereas COX-2 is an inducible enzyme, which mediates acute


C 2007 Institute of Food Technologists Vol. 72, Nr. 1, 2007—JOURNAL OF FOOD SCIENCE S69
doi: 10.1111/j.1750-3841.2006.00219.x
Further reproduction without permission is prohibited
 Lycopene inhibits iNOS genes and proteins . . .
and chronic inflammation, pain, and cellular repair mechanisms
(Liang and others 1999). An increased level of COX-2 is associated
with tumor invasiveness in various tumors (Pruthi and others
2005). For example, in comparison to benign prostatic epithelium,
prostate cancers express more COX-2. However, the inhibition of
COX-2 has been shown to induce apoptosis in several prostate
tumor cell lines (Hussain and others 2003; Pruthi and others 2005).
These results suggest that inhibition of COX-2 may be effective
in preventing inflammatory-associated cancers. Therefore, using
lycopene or tomato-based products to regulate the production of
NO and COX-2 may be classified as a therapeutic approach for the
treatment or prevention of chronic inflammatory diseases.
In this study, we attempt to understand the molecular mechanism
of action of trans-lycopene (Figure 1) in inflammation using an in
vitro model system of a mouse macrophage cell (RAW 264.7) induced
with LPS.
Materials and Methods
Reagents and cells
A mouse macrophage cell line (RAW 264.7) was obtained from the
American Type Culture Collection (ATCC, Manassas, Va.) and grown
in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented
with 15% fetal bovine serum (FBS), penicillin (100 U/mL), and
streptomycin (100 µg/mL). DMEM, LPS, Tri-reagent, Griess reagent,
monoclonal anti β-actin, and lycopene were purchased from
Sigma (St. Louis, Mo., U.S.A.). The relative reverse transcription-
polymerase chain reaction (RT-PCR) kit for mouse iNOS and COX-2
was obtained Ambion, Inc. (Austin, Tex., USA) and mouse mono-
clonal anti-iNOS and COX-2 were purchased from BD-Pharmingen.
Anti-mouse and anti-rabbit IgG conjugated with horseradish perox-
idase was purchased from DAKO Corp. (Carpinterios, Calif., USA).
Nitric oxide measurement
The mouse macrophage cells (RAW 264.7) were cultured in
DMEM supplemented with 15% FBS and with antibiotic mixture.
The cell suspension of 50000 cells/well was cultured in flat bot-
tom microtiter plate in quadruplicate for 12 h. Thereafter, 100 µL of
media was replaced with fresh medium containing either LPS (0.5
µg/mL) alone or LPS with various concentrations of lycopene (1.25
to 10 µM) and further cultured for 18 h. The culture supernatant was
collected at the end of culture for nitrite assay, which was used as a
measure of NO production. Culture supernatant (100 µL) was mixed
with equal volume of Griess reagent and the absorbance was mea-
sured at 570 nm. The concentration of nitrite (µM) was calculated
from standard curve drawn with known concentration of sodium
nitrite dissolved in DMEM. The results are presented as mean ± SD
of 4 replicates of 1 representative experiment and this experiment
was repeated 5 times.
MTT assay for cell viability
MTT is a pale yellow substrate that is reduced by living cells to
yield a dark blue formazan product. This process requires active mi-
tochondria, and even freshly dead cells do not reduce significant
amounts of MTT. Cells from the mouse macrophage cell line RAW
S: Sensory & Nutritive Qualities of Food
264.7 were cultured in a 96-well flat bottom plate at a concentration
Figure 1 --- The chemical structure of trans-lycopene
S70 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 1, 2007
of 0.25 million/mL. After 12 h of preconditioning, cells were treated
with various concentrations of lycopene (1.25 µM to 10 µM) for 18 h.
Thereafter, the culture medium was aspirated and 100 µL of MTT dye
(1 mg/mL in phosphate-buffered saline [PBS]) was added to the cul-
tures and further incubated for 4 h at 37 ◦ C. The formazan crystals
made due to dye reduction by viable cells were dissolved using acidi-
fied isopropanol (0.1 N HCl). An index of cell viability was calculated
by measuring the optical density of color produced by MTT dye re-
duction at 570 nm (BioRad Model 680 Microplate Reader, BioRad
Laboratories, Calif., U.S.A.).
Western blotting
The RAW 264.7 cells were cultured with LPS alone or with various
concentrations of lycopene (1.25 to 10 µM). At the end of incubation,
cells were rapidly washed with ice-cold PBS and solubilized in cold
lysis buffer containing 10-mM Tris-base, 5-mM EDTA, 50-mM NaCl,
1% Triton X-100, 5-mM phenylmethylsulfonyl fluoride (PMSF), 2-
mM sodium orthovanadate, 10-µg/mL leupeptin, 25-µg/mL apro-
tinin, 1-mM sodium pyrophosphate, and 20% glycerol. After incu-
bation for 30 min on ice, lysates were centrifuged (12500 rpm, 15
min) and supernatants were collected and protein concentration in
samples was estimated by BioRad protein assay reagent following
the manufacturer’s instructions.
Equal amounts of protein (40 µg) from each sample were loaded
on SDS-polyacrylamide electrophoresis gel (10% separating gels)
and resolved for 1.5 h at 120 V in buffer containing 95-mM Tris-HCl,
960-mM glycine, and 0.5% SDS. After electrophoresis the proteins
were transferred to Hybond-enhanced chemiluminescence nitro-
cellulose membrane (Amersham-Pharmacia Biotech, N.J., U.S.A.)
at 200 mA for 1 h in a buffer containing 25-mM Tris-base, 192-
mM glycine, and 20% methanol. After transfer, the membrane
was blocked in PBST (20-mM sodium phosphate buffer, pH 7.6,
150-mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk for
1 h at room temperature and incubated with primary antibody in
the blocking solution at 4 ◦ C overnight. Thereafter, the membrane
was washed 4 times with PBST, incubated with secondary antibody
in the blocking solution for 1 h at room temperature, and washed 4
times with PBST for 5 min every time. Specific bands were detected
by an enhanced chemiluminescence detection system (Amersham-
Pharmacia Biotech, N.J., U.S.A.), and the membrane was exposed to
X-ray film.
Determination of iNOS and COX-2 mRNA expression
The RAW 264.7 cells were cultured (106 /well) in a 96-well plate
for 24 h, the medium was replaced by a fresh medium, and different
concentrations of lycopene either alone or in combination with LPS
were added and further cultured for 18 h. Total RNA was isolated
using Tri-reagent (Sigma) and 5 µg of this total RNA was reverse
transcribed to make cDNA using random hexamer and superscript
reverse transcriptase (Invitrogen, Calif., U.S.A.), following the man-
ufacturer’s instructions. Linear range of amplification of iNOS and
COX-2 cDNA (2 µL for COX-2 and 1 µL for iNOS of RT reaction mix-
ture) was determined using gene-specific primers from Ambion Inc.,
following the manufacturer’s instructions. The optimum amount of
18S primer and competitor for iNOS and COX-2 gene was also de-
termined as per the manufacturer’s instructions. The PCR for iNOS
(2-µL cDNA, 30 cycles) and COX-2 (1-µL cDNA, 25 cycles) was per-
formed in a final volume of 50 µL containing dNTPs (each at 2.5
mM), 1× PCR buffer, 5 units of Taq DNA polymerase, 0.4 µM of gene-
specific primer, and optimum ratio of 18S primer and competitor
(3:7). After an initial denaturation for 30 s at 94 ◦ C, aforementioned
Lycopene inhibits iNOS genes and proteins . . .

cycles of amplification (95 ◦ C for 30 s, 55 ◦ C for 45 s, and 72 ◦ C for RAW 264.7 cells in comparison to control cells that received no treat-
45 s) were performed followed by a 10-min final extension at 72 ◦ C. ment (Figure 3). The highest concentration of lycopene that did not
Finally, PCR products from each sample (10 µL) were resolved in cause more than 10% loss in cell viability was 10 µM. Therefore,
2% agarose gel (Fisher Biotech, N.J., U.S.A.), stained with ethidium 10 µM lycopene was chosen for further studies.
bromide, and an image of gel was captured at appropriate exposure
time and magnification. Inhibition of LPS-induced iNOS and COX-2 protein
expression by lycopene
To confirm whether the inhibition of NO production is due to
Statistical analysis less enzymatic activity or decreased protein expression of NOS, we
Data are expressed as mean ± standard deviation of indicated ex- further studied the effect of lycopene on iNOS protein expression
periments. Statistical significance between 2 groups was determined by Western blotting. In addition to iNOS, we have also studied the
by Student’s t-test. The significance level was set at P < 0.05. effect of lycopene on the expression of COX-2 protein, known to be
activated in LPS-stimulated macrophages. Equal amounts of pro-
Results teins (40 µg) were resolved to detect the expression of iNOS and
COX-2 proteins by Western blot. Our results suggest that lycopene
Inhibition of LPS-induced nitric oxide production significantly inhibits iNOS, in a dose-dependent fashion. However,
from RAW 264.7 cells by lycopene lycopene did not decrease the expression of COX-2 proteins in a
In order to study inflammatory response, which is evident in dose-dependent fashion (Figure 4). Therefore, the slight decrease of
arthritis and related illnesses, LPS was used to stimulate the release COX-2 with 2.5 µM lycopene was not considered a significant result.
of NO from the macrophage cells. Nitrite is used as an indicator of The detection of β-actin was also performed in the same blot as the
NO production, due to the short half-life of NO. The nitrite con- loading control and is noted as a consistent band.
centration (micromolar) was determined in the supernatant after
treatment with LPS (0.5 µg/mL) alone or cotreated with lycopene Effect of lycopene on LPS-induced iNOS and
(2.5, 5.0, and 10 µM) for 18 h using Griess reagent. As shown in COX-2 mRNA expression
Figure 2, lycopene suppressed the LPS-induced NO formation sig- To investigate whether the inhibition of protein expression is due
nificantly (2.5 µM: P < 0.05; 5 µM: P < 0.01; 10 µM: P < 0.001), in to less protein synthesis or due to modulation of post-translational
dose-dependent fashion. The lycopene had no effect on basal level events, we performed the RT-PCR analysis for iNOS and COX-2. Us-
of NO formation in RAW 264.7 cells. ing gene-specific primers, 2 µL of cDNA was amplified for 349 base
pair (bp) of iNOS, 297 bp of COX-2, and 495 bp of 18S ribosomal
MTT assay for cell viability RNA by PCR as described in materials and methods. We have ob-
RAW 264.7 cells were treated with various concentrations of ly- served that various concentrations of lycopene inhibited the LPS
copene (1.25 to 10 µM) for 18 h and cell viability assay was performed (0.5 µg/mL) stimulated mRNA expression of iNOS after 18 h of treat-
using MTT dye. Lycopene (2.5 µM) exhibited no cytotoxic effect on ment with RAW 264.7 cells. This inhibition of mRNA correlates to the

70
*
60
50 **
Nitrite (uM)

40 ***
30
20
10
0
Control LPS LPS+Lycopene LPS+Lycopene LPS+Lycopene
2.5 µM 5 µM 10 µM

Figure 2 --- Effect of lycopene on NO production. RAW 264.7 macrophage cells were treated with LPS (0.5 µg/mL) and/or
various concentration of lycopene for 12 h, and the amount of nitrite in supernatant from each treatment group was
measured using Griess reagent. Each bar represents mean ± SD of 5 replicates of representative experiment from
a total of 4 experiments. ∗ Represents statistical significance of the difference between LPS and LPS + lycopene
treatment groups (∗ P < 0.05; ∗ ∗ P < 0.01; ∗ ∗ ∗ P < 0.001).

100
Figure 3 --- The effect of lycopene on
cell viability. RAW 264.7 murine
S: Sensory & Nutritive Qualities of Food

80
% Cell viability

macrophage cells were treated with

60 various concentrations of Lycopene
(2.5 µM to 10 µM) for 18 h, and cell
40 viability assay was performed using
MTT dye as mentioned in materials
20 and methods.
0
Control Lycopene 10 µM Lycopene 5 µM Lycopene 2.5 µM
Lycopene concentration (µM)

Vol. 72, Nr. 1, 2007—JOURNAL OF FOOD SCIENCE S71

 Lycopene inhibits iNOS genes and proteins . . .
inhibition of protein expression by lycopene. As an internal con-
trol 18S rRNA was used, which can be seen as constant band size
(Figure 5). Treatment with lycopene resulted in reduced expression
for iNOS. Furthermore, treatment with 10-µM lycopene exhibited
the most markedly response of inhibition when compared to the
other lycopene dosages and in comparison to COX-2 results. COX-2
exhibited no change in mRNA expression at the various treatment
doses of lycopene (2.5 to 10 µM).
Discussion
L ycopene is a fat-soluble red-orange carotenoid present in toma-
toes and tomato products as well as dried apricots, guava, wa-
termelon, papaya, and pink grapefruit (Heber and Lu 2002). Previ-
ous studies have recognized lycopene as an antioxidant due to its
ability to trap singlet oxygen (Di Mascio and others 1989). Stud-
ies on the effect of carotenoids on lipid peroxidation have also
identified lycopene as the most potent antioxidant among other
carotenoids when used alone, followed by α-tocopherol, α-carotene,
β-cryptoxanthin, zeaxanthin = β-carotene, and lutein (Stahl and
others 1998). However, a mixture of lycopene and lutein exhibited
a synergistic effect, indicating that combinations of carotenoids are
more effective in preventing oxidative damage (Stahl and others
1998).
Chronic inflammation is evident in diseases such as asthma,
arthritis, cancer, and inflammatory bowel disease. In inflammatory
diseases, the increased expression of iNOS is responsible for the pro-
duction of NO, whereas COX-2 is responsible for the production of
prostaglandins (Barnes and Karin 1997). The overexpression of iNOS
and/or COX-2 is associated with inflammation and is known as the
cause of various inflammatory diseases (Liang and others 1999).
The onset of the NO production cascade in macrophages requires
a number of steps, including the activation of nuclear factor-κB (NF-
κB) and subsequent iNOS and COX-2 mRNA expression (Connelly
1 2 3 4 5
iNOS
COX-2
Actin
1. Control-RAW 264.7
2. LPS (0.5 ug/ml)
3. LPS+Lycopene 10 uM
S: Sensory & Nutritive Qualities of Food
4. LPS+Lycopene 5 uM
5. LPS+Lycopene 2.5 uM
Figure 4 --- Inhibition of LPS-induced iNOS and COX-2
protein expression. RAW 264.7 macrophage cells were
treated with LPS (0.5 µg/mL) and/or various concentra-
tions of lycopene for 18 h. The detection of B-actin was
done in the same blot as a loading control.
S72 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 1, 2007
and others 2001). Transcription factor NF-κB regulates the expres-
sion of iNOS and COX-2. Therefore, activation of NF-κB is followed
by increased levels of NO and prostaglandins (Barnes and Karin
1997; Liang and others 1999). NO production by macrophages de-
pends upon iNOS, which can be activated by various agents, in-
cluding LPS, IFN (interferon)-gamma, TNF (tumor necrosis factor)-
alpha, and so on (Palmer and others 1993). In this study, LPS was
used to treat macrophage cells (RAW 264.7) and induce NO synthe-
sis, a model used in studies of the mechanism of iNOS induction
(Szabo and others 1993; Swierkosz and others 1995). Activation of
RAW 264.7 macrophages with LPS resulted in accumulation of ni-
trite in culture medium, reflecting increased NO production due
to activation of iNOS. Cell viability was evaluated using MTT assay
and it was observed that lycopene did not affect cell viability at the
concentrations used to inhibit NO production. This result ensures
lycopene’s ability to effectively inhibit LPS-induced NO production
in the murine macrophage cell line model as opposed to inducing
cell death.
The inhibition of iNOS protein and mRNA expression after treat-
ment with lycopene demonstrates the anti-inflammatory effect of
lycopene by its ability to inhibit NO production in the LPS-induced
mouse macrophages.
Numerous epidemiological studies have suggested that
carotenoids may decrease the incidence of major diseases
such as cancer, cardiovascular diseases, and macular degeneration
(Ziegler 1989; Snodderly 1995; Burrie 1997; Ito and others 2003).
Beta-carotene is also a carotenoid found in fruits and vegetables
that has also been targeted as a major antioxidant (Bai and others
2005; Palozza and others 2005). Few epidemiological studies
have failed to show an inverse relationship between carotenoid
intake and breast cancer (Henneckens and others 1996; Kushi and
others 1996). However, in recent years, encouraging results on the
anti-cancer activities of carotenoids other than beta-carotene have
begun to emerge. For instance, carotenoids such as astaxanthin
(Chew and others 1996), canthaxanthin (Palozza and others 2003),
1 2 3 4 5
18S
iNOS
18S
COX-2
1. Control-RAW 264.7
2. LPS (0.5 ug/ml)
3. LPS+Lycopene 10 uM
4. LPS+Lycopene 5 uM
5. LPS+Lycopene 2.5 uM
Figure 5 --- Inhibition of LPS-induced iNOS and COX-2 gene
expression. Various concentrations of lycopene (2.5 µM
to 10 µM) inhibited the LPS-stimulated mRNA expression
of iNOS after 18 h of treatment with RAW 264.7 cells. As
an internal control, 18S rRNA was used, which can be
seen as constant band size.
Lycopene inhibits iNOS genes and proteins . . .
lycopene (Levy and others 1995; Kim and others 2002; Livny and
others 2002), and lutein (Chew and others 1996; Rock and others
1996; Park and others 1999; Krinsky 2002; Mares-Perlman and
others 2002; Rock and others 2002) have been shown to possess
anti-cancer activity. Proposed mechanisms include their role as an-
tioxidants (Byers and Perry 1992) in modulating immune function
(Chew 1993; Jyonouchi and others 1993). However, the mechanism
by which carotenoids inhibit cancer is not yet confirmed and no
clear data are available to demonstrate a possible direct effect of
carotenoids in modulating the protein and mRNA expression of
certain genes in inflammation and immune cells.
In a study conducted by Heber and Lu, beta-carotene was ranked
number 5 in a group of 6 carotenoids for its potency as an antioxi-
dant, while lycopene was ranked number 1 (Heber and Lu 2002). In
a dose-dependent manner, treatment with beta-carotene showed
a decreased expression of iNOS and COX-2 as well as decreased
production of NO and prostaglandins. The highest dose of beta-
carotene, showed almost complete inhibition of protein and mRNA
levels (Bai and others 2005). In vivo results also indicated decreased
NO and prostaglandin serum levels on LPS-administered mice in-
jected with beta-carotene (Bai and others 2005). This study con-
ducted by Bai and others also suggested beta-carotene’s ability to
suppress NF-κB activation in cells stimulated with LPS. Based on
previous research and our current findings, the carotenoid family,
namely, lycopene and beta-carotene, can be considered effective in-
hibitors of NO and also provide possible prevention against chronic
inflammation.
It appeared that treatment with lycopene did not effectively in-
hibit LPS-induced COX-2 protein and gene expression in our current
study. In a study designed to investigate the relationship between
beta-carotene and COX-2 expression in colon cancer cells, very low
concentrations of the carotenoid were able to inhibit COX-2 protein
expression. Beta-carotene demonstrated the ability to modulate the
COX-2 pathway under steady-state conditions as well as after stim-
ulation by the growth factor heregulin-alpha. These results were ac-
companied by a decreased production of prostaglandins, indicating
the possibility of beta-carotene as an antitumor agent in colon can-
cer (Palozza and others 2005). Due to inconsistent results in our lab-
oratory study, further research on the effects of lycopene on COX-2
modulation is necessary. We could not demonstrate modulation of
COX-2 proteins and genes using lycopene in contrast to the previous
study with beta-carotene.
Several nutraceuticals such as garcinol and curcurmin have also
shown to suppress the activation of NF-κB, thereby inhibiting COX-2
and iNOS genes (Liao and others 2004; Bor and others 2006). Results
involving treatment with garcinol showed a marked reduction in
the expression of iNOS in LPS-induced RAW264.7 macrophage cells.
Curcurmin and garcinol also exhibited similar findings at the mRNA
level, inhibiting iNOS expression as well as COX-2 expression (Liao
and others 2004). Results indicate garcinol’s potential as a chemo-
preventative agent due to its ability to inhibit LPS-induced iNOS
and COX-2 expression, including NF-kB signaling (Liao and oth-
ers 2004). Data from Lo and others correlate with garcinol findings,
where carnosol, a phytopolyphenol found in rosemary, suggests a
protective effect against inflammation by blocking NF-κB through
inhibition of iNOS mRNA and protein expression in LPS-induced
mouse macrophages (Lo and others 2002).
Several flavonoids, which act as antioxidants protecting bio-
logical systems against oxidative stress, are also reported to have
antiinflammatory, anticarcinogenic, and antioxidative properties
(Liang and others 1999). Apigenin, genistein, and kaempferol were
all flavonoids found to be inhibitors of COX-2 protein expression.
However, apigenin was found to be the most potent inhibitor of
prostaglandin and nitrite production along with COX-2 and iNOS
protein expression in LPS-induced mouse macrophage cells (Liang
and others 1999). Liang and others also demonstrated that the in-
duction of NF-κB by LPS was also inhibited by apigenin in a dose-
dependent manner (Liang and others 1999). Quercetin, another
flavonoid present in many fruits and vegetables, has been shown to
exhibit anti-inflammatory properties by reducing LPS-induced NO
production in mouse microglia cells (Chen and others 2005). Chen
and others were successful in demonstrating the inhibited forma-
tion of NO in a concentration-dependent manner with the addition
of quercetin. Results remained consistent with a reduction in iNOS
mRNA level when quercetin was present (Chen and others 2005). In
animal models, further confirmation was given with the inhibitory
effect of quercetin on LPS-induced NO production (Shen and others
2002).
Present studies involving treatment with various carotenoids,
flavonoids, and nutraceuticals indicate an inhibitory effect on LPS-
induced COX-2 and iNOS activity in RAW macrophages. Their results
also support our findings on lycopene as an anti-inflammatory com-
pound.
Conclusions
O ur results suggest that lycopene has anti-inflammatory activity
by inhibiting iNOS proteins and mRNA expressions in mouse
macrophage cell lines. Furthermore, COX-2 protein and mRNA ex-
pression were not affected by treatment with lycopene.
This is the 1st in vitro study using mouse cell lines demonstrat-
ing lycopene as an effective inhibitor of NO production and iNOS
expression. These findings await confirmation in human cell lines,
animal models, and clinical trials. Thus, the inhibition of iNOS ex-
pression, along with decreased production of NO by lycopene, will
serve as a basis on how dietary carotenoids are able to prevent in-
flammatory diseases.
Acknowledgments
This work was supported by the New Jersey Agricultural Experiment
Stations Hatch project (Cook College, Rutgers, The State Univ. of
New Jersey). We are grateful to Dr. Paul Lachance for reading and
commenting on the manuscript.
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