Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation

Article abstract of DOI:10.1007/s00210-005-1078-y

We investigated the effect of hydroxytyrosol (HT), a phenolic compound from virgin olive oil, on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in J774 murine macrophages stimulated with lipopolysaccharide (LPS). Incubation of cells with LPS caused an increase in iNOS and COX-2 mRNA and protein level as well as ROS generation, which was prevented by HT. In addition, HT blocked the activation of nuclear factor-κB (NF-κB), signal transducer and activator of transcription-1α (STAT-1α) and interferon regulatory factor-1 (IRF-1). These results, showing that HT down-regulates iNOS and COX-2 gene expression by preventing NF-κB, STAT-1α and IRF-1 activation mediated through LPS-induced ROS generation, suggest that it may represent a non-toxic agent for the control of pro-inflammatory genes. Springer-Verlag 2005.

Full text of DOI:10.1007/s00210-005-1078-y

Naunyn-Schmiedeberg’s Arch Pharmacol (2005) 371: 457–465
DOI 10.1007/s00210-005-1078-y
ORIGINAL ARTICLE
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Maria Chiara Maiuri Daniela De Stefano
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.
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Paola Di Meglio Carlo Irace Maria Savarese
Raffaele Sacchi Maria Pia Cinelli Rosa Carnuccio
.
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Hydroxytyrosol, a phenolic compound from virgin olive oil,
prevents macrophage activation
Received: 1 February 2005 / Accepted: 9 June 2005 / Published online: 16 July 2005
# Springer-Verlag 2005
Abstract We investigated the effect of hydroxytyrosol
(HT), a phenolic compound from virgin olive oil, on
inducible nitric oxide synthase (iNOS) and cyclooxygen-
ase-2 (COX-2) expression in J774 murine macrophages
stimulated with lipopolysaccharide (LPS). Incubation of
cells with LPS caused an increase in iNOS and COX-2
mRNA and protein level as well as ROS generation, which
was prevented by HT. In addition, HT blocked the acti-
vation of nuclear factor-κB (NF-κB), signal transducer
and activator of transcription-1α (STAT-1α) and interferon
regulatory factor-1 (IRF-1). These results, showing that
HT down-regulates iNOS and COX-2 gene expression by
preventing NF-κB, STAT-1α and IRF-1 activation me-
diated through LPS-induced ROS generation, suggest that
it may represent a non-toxic agent for the control of pro-
inflammatory genes.
Introduction
Numerous epidemiological data have demonstrated an
association between a diet rich in antioxidants, such as the
“Mediterranean diet,” and a lower incidence of several
pathologies (Keys et al. 1986; De Lorgeril et al. 1999).
Although the protective effect of such a diet is likely to
be multifactorial, there is consistent evidence for an an-
tioxidant activity of some selected polyphenolic com-
pounds from extra virgin olive oil (Owen et al. 2000a,b;
Tuck and Hayball 2002; Visioli et al. 2002). Recently it
has been shown that virgin olive oil with a high content of
polyphenolic compounds has protective effects on carra-
geenin-induced oedema and adjuvant arthritis in rat
(Martínez-Domínguez et al. 2001); nevertheless, the mo-
lecular mechanisms have not been elucidated. Hydro-
xytyrosol (HT), present in the phenolic fraction of virgin
olive oil in a concentration range of 10–300 ppm, is abun-
dant in naturally fermented table olives and can be also
recovered from virgin olive oil by-products (olive pomace,
olive waste water; Boskou 1996). At nutritionally relevant
concentrations, HT possesses a marked antioxidant activity
and is a good radical scavenger (Deiana et al. 1999;
Manna et al. 1999). HT in vitro prevents LDL oxidation
(Salami et al. 1995), platelet aggregation (Petroni et al.
1995) and inhibits 5- and 12-lipoxygenase (Kohyama et al.
1997; De la Puerta et al. 1999). A recent report indicates
that HT and other phenolic antioxidants reduce vascular
cell adhesion molecule-1 mRNA expression by blocking
the activation of transcription factors nuclear factor-kappaB
(NF-κB) and activator protein-1 (Carluccio et al. 2003). It
has been demonstrated that natural and synthetic antiox-
idants inhibit pro-inflammatory gene expression regulated
by transcription factors, including NF-κB, signal transduc-
er and activator of transcription-1α (STAT-1α) and inter-
feron regulatory factor-1 (IRF-1; Hecker et al. 1996; Epinat
and Gilmore 1999; Faure et al. 1999; Kim et al. 2003; Lee
et al. 2003). These transcription factors are dependent on
the intracellular redox state (Pahl 1999; Ramana et al.
2000; Kröger et al. 2002) and can cooperate in order to
.
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.
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Keywords Hydroxytyrosol NF-κB IRF-1 STAT-1α
iNOS COX-2
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M. C. Maiuri D. De Stefano P. Di Meglio C. Irace
R. Carnuccio (*)
Dipartimento di Farmacologia Sperimentale,
Università degli Studi di Napoli Federico II,
Via D. Montesano n. 49,
80131 Naples, Italy
e-mail: carnucci@unina.it
Tel.: +39-81-678431
Fax: +39-81-678403
.
M. Savarese R. Sacchi
Dipartimento di Scienza degli Alimenti,
Università degli Studi di Napoli Federico II,
Portici, 80055 Naples, Italy
M. P. Cinelli
Dipartimento di Scienze Biomorfologiche,
Università degli Studi di Napoli Federico II,
Via Pansini 5,
80131 Naples, Italy

458
promote synergistically transcriptional activity (Ohmori sured by an ELISA kit according to the manufacturer’s
and Hamilton 1993; Kinugawa et al. 1997). The inducible instructions (TEMA Ricerche, Milan, Italy). NO genera-
enzymes nitric oxide synthase (iNOS) and cyclooxygen- tion was measured as nitrite (NO2−, nmol/106 cells) accumu-
ase-2 (COX-2) are mediators of the inflammatory re- lated in the incubation medium, 24 h after LPS challenge,
sponse (D’Acquisto et al. 2000a) and implicated in the using a spectrophotometric assay based on Griess reaction
pathogenesis of several diseases, including human gastro- as previously described (D’Acquisto et al. 2000b). The
intestinal cancers (Lala and Chakraborty 2001; Zha et al. nitrite concentration was determined by reference to a
2004). The promoter regions of iNOS and COX-2 contain standard curve of sodium nitrite.
consensus sequences for NF- κB, STAT-1α and IRF-1
(Fletcher et al. 1992; Lowenstein et al. 1993; Sirois et al. Measurement of reactive oxygen species The formation of
1993; Kamijo et al. 1994; Kosaka et al. 1994; Gao et al. ROS was evaluated by means of the probe 2′,7′-dichloro-
1997). In the present study, we show that HT inhibits iNOS fluorescin-diacetate (H2DCF-DA, Sigma) as described
and COX-2 gene expression in lipopolysaccharide (LPS)- elsewhere (Santamaria et al. 2004). Briefly, J774 cells
stimulated J774 macrophages by preventing the activation were grown in DMEM containing 10% (v/v) foetal bovine
of NF-κB, STAT-1α and IRF-1.
serum, then were plated at a density of 1.5×104 cells/well
into 96-well dishes. Cells were allowed to grow for 24 h
Materials and methods
Hydroxytyrosol The (3,4-dihydroxyphenyl) ethanol (hydro-
xytyrosol, HT) was obtained by acid hydrolysis of ole-
uropein glucoside (Extrasynthese Z.I., Lyon Nord, France)
according to the procedure described by Owen et al. (2000a,
b). Acid hydrolysis was achieved by dissolving 100 mg of
the oleuropein in 1 l of 1 mol/l H2SO4 and incubating at
37°C for 3 h. The hydrolysate was extracted twice with
250 ml of ethyl acetate and dried over anhydrous sodium
sulphate; the solvent was removed under reduced pressure.
The purity of HT was checked by HPLC analysis (Sacchi
et al. 2002). The residue was dissolved in ethanol and
stored at 4°C until the tests.
Cell culture The murine monocyte/macrophage cell line
J774 (American Tissue Culture Catalogue T1B) was cul-
tured at 37°C in humidified 5% CO2 /95% air in Dul-
becco’s Modified Eagle’s Medium (DMEM) containing
10% foetal bovine serum, 2 mM glutamine, 100 UI/ml
penicillin, 100 μg/ml streptomycin, 25 mM Hepes and
5 mM sodium pyruvate. The cells were plated in 24-culture
wells at a density of 2.5×105 cells/ml/well or 10 cm di-
ameter culture dishes at a density of 3×106 cells/ml/dish
and allowed to adhere for 2 h. Thereafter, the medium was
replaced with fresh medium and cells were stimulated with
LPS (1 μg/ml). HT (50, 100 and 200 μM) or pyrrolidine
dithiocarbamate (PDTC; 10 μM), a synthetic antioxidant
used as a reference drug, were added to the cells 10 min
before LPS challenge. In some experiments HT (200 μM)
or PDTC (10 μM) were added 12 h after LPS challenge.
The cell viability was assessed by using 3-(4,5-dimethyl-
thiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
conversion assay (D’Acquisto et al. 2001). Cytosolic and
nuclear extracts of macrophages stimulated for 24 h with
LPS (1 μg/ml) in the presence or absence of HT (200 μM)
or PDTC (10 μM) were prepared as previously described
(D’Acquisto et al. 2001). Protein concentration was deter-
mined by Bio-Rad (Milan, Italy) protein assay kit.
Fig. 1 Effect of hydroxytyrosol (HT; 50, 100 and 200 μM) and
pyrrolidine dithiocarbamate PDTC (10 μM) a on nitrite and b PGE2
production by J774 macrophages stimulated with lipopolysaccharide
(LPS; 1 μg/ml) for 24 h. Data are expressed as mean SEM of three
PGE2 and nitrite determination The accumulation of PGE2
in the culture medium, 24 h after LPS challenge, was mea- experiments. **P<0.01, ***P<0.001 vs. LPS alone

459
and then incubated in the growth medium containing oligonucleotide was added to the binding reaction 15 min
5 μM H2DCF-DA for 2 h at 37°C. H2DCF-DA is a non- before the radiolabelled probe. In supershift assay, anti-
fluorescent permeant molecule that passively diffuses bodies reactive to p50, p65, STAT-1α or IRF-1 proteins
into cells, where the acetates are cleaved by intracellular were added to the reaction mixture 15 min before the ad-
esterases to form H2DCF and thereby traps it within the dition of the radiolabelled probe. Nuclear protein-oligo-
cell. In the presence of intracellular ROS, H2DCF is rapidly nucleotide complexes were resolved by electrophoresis on
oxidised to the highly fluorescent 2′,7′-dichlorofluorescein a 6% non-denaturing polyacrylamide gel in 1× TBE buffer
(DCF). Cells were washed twice with PBS buffer; there- at 150 V for 2 h at 4°C. The gel was dried and auto-
after, the medium was replaced with fresh medium and radiographed with intensifying screen at −80°C for 20 h.
cells were stimulated with LPS (1 μg/ml) for 24 h. HT Subsequently, the relative bands were quantified by den-
(200 μM) or PDTC (10 μM) were added to the cells 10 min sitometric scanning of the X-ray films with a GS-700 Im-
before LPS challenge. After treatment, cells were washed aging Densitometer (Bio-Rad) and a computer program
twice with phosphate buffer saline (PBS) buffer and plates (Molecular Analyst, IBM).
were placed in a fluorescent microplate reader (Perkin
Elmer LS55 Luminescence Spectrometer; Perkin Elmer, Western blot analysis Cytosolic and nuclear fraction pro-
Beaconsfield, UK). Fluorescence was monitored using an teins were mixed with gel loading buffer (50 mM Tris,
excitation wavelength of 485 nm and an emission wave- 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, 2 mg/
length of 538 nm. In each experiment, fluorescence in- ml of bromophenol) in a ratio of 1:1, boiled for 3 min and
crease was measured in ten replicate cultures (n=10) for centrifuged at 10,000×g for 5 min. Protein concentration
each treatment.
was determined and equivalent amounts (30 μg) of each
sample were electrophoresed in a 8–12% discontinuous
Electrophoretic mobility shift assay Double stranded oli- polyacrylamide minigel. The proteins were transferred onto
gonucleotides containing the NF-κB (5′-CAACGGCAG nitro-cellulose membranes, according to the manufac-
GGGAATCTCCCTCTCCTT-3′), STAT-1α (5′-CAT GTT turer’s instructions (Protran; Schleicher & Schuell, Dassel,
ATGCATATTCCTGTAAGTG-3′) and IRF-1 (5′-GG AAG Germany). The membranes were saturated by incubation
CGAAAATGAAATTGACT-3′) recognition sequences were at room temperature for 2 h with 10% non-fat dry milk in
end-labelled with 32P-γ-ATP. Nuclear extracts containing PBS and then incubated with (1:1,000) anti-iNOS, anti-
5 μg protein were incubated for 15 min with radiolabelled COX-2, anti-p50, anti-p65, anti-STAT-1α and anti-IRF- 1
oligonucleotides (2.5–5.0×104 cpm) in 20 μl reaction buf- at 4°C overnight. The membranes were washed three
fer containing 2 μg poly dI-dC, 10 mM Tris-HCl (pH 7.5), times with 0.1% Tween 20 in PBS and then incubated
100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% with anti-rabbit, anti-mouse or anti-goat immunoglobu-
(v/v) glycerol. The specificity of the DNA/protein binding lins coupled to peroxidase (1:1,000; DAKO, Milan, Italy).
was determined by competition reaction in which a 50- The immunocomplexes were visualised by the ECL chemi-
fold molar excess of unlabelled wild-type, mutant or Sp-1 luminescence method (Amersham, Milan, Italy). The
Fig. 2 Representative Western blot of a inducible nitric oxide ulated with LPS (1 μg/ml) for 24 h. β-actin expression is shown as a
synthase (iNOS) and b cyclooxgenase-2 (COX-2) protein expres- control. Data in a and b (upper panel) are from a single experiment
sion (upper panel) as well as the densitometric analysis (lower and are representative of three separate experiments. Data (lower
panel) show the effect of HT (200 μM) and PDTC (10 μM) on panel) are expressed as mean SEM of three separate experiments.
iNOS and COX-2 protein expression in J774 macrophages stim- ***P<0.001 vs. LPS alone. OD optical density

460
membranes were stripped and re-probed with β-actin or
histone 1 antibody to verify equal loading of proteins.
Subsequently, the relative expression of iNOS, COX-2,
p50, p65, STAT-1α and IRF-1 proteins in cytosolic and
nuclear fraction was quantified by densitometric scanning
of the X-ray films with a GS 700 Imaging Densitometer
and a computer program (Molecular Analyst, IBM).
Reverse transcription-polymerase chain reaction J774
cells were plated in a six-well plate at a density of 3×106/
well, where they were allowed to adhere for 2 h. Thereafter,
the cells were treated with 200 μM HT or 10 μM PDTC
and, 10 min after treatment, were stimulated with 1 μg/ml
LPS for 6 h. Total RNA extraction, by using TRIzol (In-
vitrogen, Milan, Italy) was performed as described else-
where (Ialenti at al. 2005). The levels of COX2 and iNOS
mRNA were evaluated by using PCR amplification of re-
verse-transcribed mRNA. The housekeeping gene β-actin
was used as an internal control. Five micrograms of total
RNA was reverse-transcribed into cDNA by using oligo
(dT)12–18 primer (Invitrogen) and MMLV-Reverse Tran-
scriptase (Invitrogen). One microlitre of cDNA was ampli-
fied by PCR using Taq Polymerase (Invitrogen) according
to the manufacturer’s instructions. The primers were: COX-
2: sense 5′-CCGGGTTGCTGGGGGAAGA-3′, antisense
5′-GTGGCTGTTTTGGTAGGCTGTGGA-3′; iNOS: sense
5′-TGGGAATGGAGACTGTCCCAG3′, antisense: 5′-GG
GATCTGAATGTGATGTTTG-3′; β-actin: sense 5′-AT
GAAGATCCTGACCGCGCGT-3′, antisense: 5′-AACGC
AGCTCAGTAACAGTCCG-3′. The amplified fragments
were 479 bp, 305 bp and 584 bp respectively. The PCR
reaction was performed under the following conditions: a
Fig. 3 a Representative PCR of iNOS and COX-2 and b the
densitometric analysis show the effect of HT (200 μM) and PDTC
(10 μM) on iNOS (black bars) and COX-2 (white bars) mRNA
expression in J774 macrophages stimulated with LPS (1 μg/ml) for
6 h. iNOS and COX-2 mRNA levels were normalised to β-actin
mRNA. Data in a are from a single experiment and are repre-
sentative of three separate experiments. Data in b are expressed as
mean SEM of three separate experiments. ***P<0.001 vs. LPS
alone
first cycle of denaturation at 94°C for 1 min 40 s, then 25 Fig. 4 Representative electrophoretic mobility shift assay (EMSA)"
of a nuclear factor-kappaB (NF-κB), c signal transducer and
cycles of denaturation at 94°C for 40 s, annealing at 56°C
for 40 s, extension at 72°C for 1 min and one additional
cycle of extension at 72°C for 8 min. The PCR products
were run on a 1% agarose gel and visualised by ethidium
bromide staining.
activator of transcription-1α (STAT-1α), e interferon regulatory
factor-1 (IRF-1; upper panel) and the densitometric analysis (lower
panel) show the effect of HT (200 μM) and PDTC (10 μM) on
NF-κB, STAT-1α, IRF-1/DNA binding activity in J774 macro-
phages stimulated with LPS (1 μg/ml) for 24 h. Data in a, c, e are
from a single experiment and are representative of three separate
experiments. Data (lower panel) are expressed as mean SEM of
three separate experiments. ***P<0.001 vs. LPS alone. b In com-
petition reaction nuclear extracts from LPS-treated J774 macro-
phages were incubated with radiolabelled NF-κB probe in the
absence or presence of: identical but unlabelled oligonucleotides (W.
T., 50×), mutated non-functional NF-κB probe (Mut., 50×) or
unlabelled oligonucleotide containing the consensus sequence for
Sp-1 (Sp-1, 50×). In supershift experiments nuclear extracts were
Statistics Results were expressed as the mean SEM of n
experiments. Statistical significance was calculated by
one-way analysis of variance (ANOVA) and Bonferroni-
corrected P value for multiple comparison test. The level
of statistical significance was defined as P<0.05.
Reagents Dulbecco’s Modified Eagle’s Medium, foetal incubated with antibodies against p50 and p65 15 min before
incubation with radiolabelled NF-κB probe. d In competition
bovine serum, glutamine, penicillin, streptomycin, Hepes,
reaction nuclear extracts from LPS-treated J774 macrophages were
sodium pyruvate and PBS were from BioWhittaker
incubated with radiolabelled STAT-1α probe in the absence or
presence of: W.T. (50×) or unlabelled oligonucleotide containing
(Caravaggio, Italy). 32P-γATP was from Amersham. Poly
dI-dC was from Boehringer-Mannheim (Milan, Italy).
Anti-iNOS antibody was from Pharmingen (BD Biosci-
ences, Milan, Italy). Anti-COX-2, anti-p50, anti-p65, anti-
STAT-1α, anti-IRF-1 and histone 1 antibodies were from
Santa Cruz (Milan, Italy). Anti-β-actin antibody was from
Oncogene (Milan, Italy). Non-fat dry milk was from Bio-
Rad. Oligonucleotide synthesis was performed to our
specifications by Tib Molbiol (Boehringer-Mannheim,
Genova, Italy). Sulphuric acid was from Carlo Erba Re-
the consensus sequence for Sp-1 (50×). In supershift experiments
nuclear extracts were incubated with antibody against STAT-1α
15 min before incubation with radiolabelled STAT-1α probe. f In
competition reaction nuclear extracts from LPS-treated J774 mac-
rophages were incubated with radiolabelled IRF-1 probe in the
absence or presence of: identical but unlabelled oligonucleotides W.
T. (50×) or unlabelled oligonucleotide containing the consensus
sequence for Sp-1 (50×). In supershift experiments nuclear extracts
were incubated with antibody against IRF-1 15 min before
incubation with radiolabelled IRF-1 probe. Data in b, d, f are
from a single experiment and are representative of three separate
agents (Milan, Italy). Ethanol and ethyl acetate were from experiments

461

462
Labscan Ltd. (Dublin, Ireland). LPS (from S. Thyphosa) and PGE2 (by 30.7 0.6%, 68.5 0.5% and 84.1 0.5%
and all the other reagents were from Sigma (Milan, Italy). respectively; n=3) production (Fig. 1a,b respectively). The
addition of HT (200 μM) or PDTC (10 μM) to the cells
12 h after LPS challenge did not reduce nitrite (65.3
Results
0.6 nmol/106 cells and 64.9 0.3 nmol/106 cells respec-
tively; n=3) and PGE2 (24.5 0.6 ng/ml and 24.2 0.8 ng/ml
respectively; n=3) production at 24 h. Moreover, upon
stimulation with LPS (1 μg/ml) for 24 h the cells expressed
a significantly high level of iNOS and COX-2 protein
Effect of HT on LPS-induced iNOS and COX-2
expression
The stimulation of J774 macrophages with LPS (1 μg/ml) expression compared with control, untreated cells. Treat-
for 24 h resulted in an accumulation of nitrite (66.7 ment of cells with HT (200 μM) or PDTC (10 μM) reduced
0.8 nmol/106 cells; n=3) and PGE2 (25.1 0.9 ng/ml; n=3) iNOS (by 49.9 0.12% and 63.7 0.1% respectively; n=3)
in the medium, compared with unstimulated cells (0.8
and COX-2 (by 53.5 0.09% and 61.6 0.2% respectively;
0.11 nmol/106 cells, n=3; 1.0 0.7 ng/ml, n=3). Treatment n=3) protein expression (Fig. 2a,b). In addition, to explore
of cells with HT (50, 100, 200 μM) or PDTC (10 μM) whether the reduced level either of iNOS or COX-2 protein
reduced, in a concentration-dependent manner, nitrite (by observed in cells treated with HT or PDTC could be at-
23.9 1.8%, 54.87 1.1% and 76.0 0.8% respectively; n=3) tributed to a reduced gene transcription, we analysed the
Fig. 5 Representative Western blot of a p50, b p65, c STAT-1α, rophages. Data in a–d are from a single experiment and are rep-
d IRF-1 (upper panel) and the densitometric analysis (lower panel) resentative of three separate experiments. Histone 1 expression is
show the effect of HT (200 μM) or PDTC (10 μM) on LPS-induced shown as a control. Data (lower panel) are expressed as mean SEM
p50, p65, STAT-1α and IRF-1 nuclear translocation in J774 mac- of three separate experiments. ***P<0.001 vs. LPS alone

463
expression either of iNOS and COX-2 mRNA. Stimula-
tion of J774 cells with LPS (1 μg/ml) for 6 h resulted in a
significantly higher level of iNOS and COX-2 mRNA
compared with control, untreated cells. Treatment of cells
with HT (200 μM) inhibited significantly both iNOS and
COX-2 mRNA levels by 41% and 60% respectively. Sim-
ilarly, treatment of cells with PDTC (10 μM) inhibited both
iNOS and COX-2 mRNA expression by 70% and 88%
respectively (Fig. 3a,b). HT and PDTC did not affect cell
viability (>90%; data not shown).
Effect of HT on NF-κB, STAT-1α and IRF-1
activation
Fig. 6 Effect of HT (200 μM) and PDTC (10 μM) on intracellular
ROS production in J774 macrophages stimulated with LPS (1 μg/ml)
for 24 h. Data are expressed as means SEM of three independent
experiments (n=10). ***P<0.001 vs. LPS-stimulated J774 macro-
phages. RFU relative fluorescence units
The effects of HT (200 μM) or PDTC (10 μM) on NF-κB,
STAT-1α and IRF-1/DNA binding activity in J774 mac-
rophages stimulated with LPS (1 μg/ml) for 24 h were
tested by electrophoretic mobility shift assay (EMSA). A Effect of HT and PDTC on intracellular
low basal level of NF-κB, STAT-1α and IRF-1/DNA ROS production
binding activity was detected in nuclear proteins from un-
stimulated macrophages. Conversely, a retarded band was To clarify whether the inhibitory effect of HT on NF-κB,
clearly detected following stimulation with LPS (1 μg/ml). STAT-1α and IRF-1 activation as well as iNOS and COX-2
Treatment of cells with HT (200 μM) or PDTC (10 μM) expression was mediated through the inhibition of ROS
caused a significant reduction in LPS-induced activation generation induced by LPS, we measured intracellular
of NF-κB (by 57.09 0.48% and 61.26 0.72%), STAT-1α ROS production in J774 macrophages stimulated with LPS
(by 40.17 1.1% and 51.47 0.38%) and IRF-1 (by 41.56
(1 μg/ml) for 24 h in the presence or absence of HT
0.13% and 58.7 1.04%; Fig. 4a,c,e). The composition of (200 μM) or PDTC (10 μM). Exposure of J774 macro-
NF-κB, STAT-1α and IRF-1 complexes activated by LPS phages to LPS for 24 h resulted in an increased intracellular
was determined by competition and supershift experi- ROS production compared with unstimulated cells. In the
ments. In the competition reaction the specificity of the presence of HT as well as PDTC, the generation of ROS by
NF-κB/DNA binding complex was demonstrated by the macrophages was significantly reduced by 51% and 53%
complete displacement of NF-κB/DNA binding in the pres- respectively (Fig. 6).
ence of a 50-fold molar excess of unlabelled NF-κB. In
contrast a 50-fold molar excess of unlabelled mutated NF-
κB probe (Mut, 50×) or Sp-1 oligonucleotide (Sp-1, 50×) Discussion
had no effect on this DNA-binding activity. Addition of
either anti-p50 and anti-p65 antibodies to the binding re- The results of the present study show that HT, a strong
action clearly gave rise to a characteristic supershift of the antioxidant compound from extra virgin olive oil, inhibits
retarded complex, suggesting that the NF-κB complex iNOS and COX-2 expression in LPS-stimulated J774 cells
contained p50 and p65 dimers (Fig. 4b). In the competition at the transcriptional level by preventing the activation of
reaction the specificity of STAT-1α and IRF-1/DNA bind- NF-κB, STAT-1α and IRF-1. First we found that HT
ing complex was demonstrated by the complete displace- reduced in a concentration-dependent manner nitrite and
ment of protein/DNA binding in the presence of a 50-fold PGE2 production that was not a consequence of direct
molar excess of unlabelled STAT-1α and IRF-1 probe re- inhibition either of iNOS and COX-2 activity. In fact, HT,
spectively. In contrast, a 50-fold molar excess of unlabeled when added to the cells 12 h after LPS challenge, failed to
Sp-1 oligonucleotide had no effect on DNA-binding ac- inhibit nitrite or PGE2 production, suggesting that HT and
tivity. Addition of anti-STAT-1α or anti-IRF-1 antibody to PDTC are not able to inhibit both enzymes once they have
the binding reaction clearly gave rise to a characteristic been expressed. Interestingly, inhibition of LPS-mediated
supershift of the retarded complex respectively (Fig. 4d,f). nitrite and PGE2 production by HT was correlated and
Moreover, in LPS-stimulated cells the nuclear level of quantitatively comparable with a reduction either of iNOS
p50, p65, STAT-1α and IRF-1 was increased compared or COX-2 protein and mRNA levels. It has been reported
with untreated macrophages. Treatment of cells with HT that NF-κB, STAT-1α and IRF-1 are essential in the
(200 μM) or PDTC (10 μM) reduced the band intensity of regulation of iNOS and COX-2 expression after LPS or
p50 (by 50.68 0.7% and 59.29 0.4%), p65 (by 51.82
cytokine challenge (Kamijo et al. 1994; Gao et al. 1997;
0.5% and 59.4 0.3%), STAT-1α (by 45.67 0.4% and Kinugawa et al. 1997; D’Acquisto et al. 2000b; Ohmori
49.71 0.5%) and IRF-1 (by 44.06 0.2% and 51.05 0.3%; and Hamilton 2001; Zhang et al. 2002; Kim et al. 2003).
Fig. 5a–d).
Our data also show that HT inhibited the LPS-induced

464
NF-κB, STAT-1α and IRF-1/DNA binding activity in J774 inflammation and ultimately play a key role as a compo-
cells. Moreover, PDTC, a synthetic antioxidant able to nent of virgin olive oil in cancer-preventing action.
inhibit NF-κB, STAT-1α and IRF-1 activation (D’Acquisto
et al. 2000b; Faure et al. 1999; Lee et al. 2003) and a
reference drug used in this study, showed the same profile
of activity as HT. Growing bodies of evidence report that
the inflammation is a critical component of tumour de-
velopment and progression (Coussens and Werb 2002;
Marx 2004). In particular, it has been shown that iNOS and
COX-2 induction contribute to promoting the neoplastic
process (Lala and Chakraborty 2001; Zha et al. 2004);
nevertheless, the mechanisms are not clear. Several studies
have been devoted to the development of new molecules
that are inhibitors of the enzymatic activity of either iNOS
or COX-2. However, an alternative approach is to find new
agents that can prevent expression of the respective gene
coding for these enzymes. Furthermore, it is now becom-
ing clear that many of the important anti-inflammatory
agents, including salicylates and glucocorticoids, share the
ability to inhibit transcription factor activation and thus a
large variety of pro-inflammatory genes (Kopp and Ghosh
1994; Hu et al. 2003). Natural and synthetic antioxidants
have been reported to have anti-inflammatory properties; a
likely target for these compounds seems to be the signal
transduction cascade leading to the activation of tran-
scription factors (Hecker et al. 1996; Epinat and Gilmore
1999; Faure et al. 1999; D’Acquisto et al. 2000b;
Carluccio et al. 2003; Kim et al. 2003; Lee et al. 2003).
Our findings indicate that HT inhibits iNOS and COX-2
gene expression by preventing NF-κB, STAT-1α and IRF-
1 activation. LPS is a potent macrophage-activating stim-
ulus that appears to initiate an ordered recruitment of
adaptor molecules and tyrosine, serine/threonine kinases
leading to the transcriptional activation of many genes
(Lowenstein et al. 1993; Ohmori and Hamilton 1993;
Kinugawa et al. 1997; Zhang et al. 2002; Kim et al. 2003).
Indeed, NF-κB, STAT-1α and IRF-1 activation are depen-
dent on the intracellular redox state (Pahl 1999; Ramana et
al. 2000; Kröger et al. 2002). In our study, HT effectively
reduced the LPS-induced ROS formation that was corre-
lated to the inhibition of NF-κB, STAT-1α and IRF-1 acti-
vation at 24 h. Although we did not investigate the precise
mechanism by which HT prevents the activation of three
transcription factors in LPS-stimulated J774 macrophages,
this effect is likely to be correlated to its antioxidant prop-
erty. Furthermore, since the inhibitory effects elicited by
HT were observed when it was added to cells prior to LPS
exposure, but not after LPS challenge, it is possible to
suggest that antioxidant HT acts at an early step of the LPS-
induced signalling cascade leading to NF-κB, STAT-1α
and IRF-1 activation. Further studies are necessary to ver-
ify this hypothesis and evaluate the potential anti-inflam-
matory activity of HT. Our results suggest that HT, by
preventing NF-κB, STAT-1α and IRF-1 activation, may
represent a potential non-toxic agent for the control of
Acknowledgements This work was supported in part by Misura
3.17 P.O.R Campania and by the MURST-Cluster C08-A program.
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