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Free Radical Biology & Medicine, Vol. 37, No. 2, pp. 156 –165, 2004Copyright D 2004 Elsevier Inc.
Printed in the USA. All rights reserved0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.04.020
Original Contribution
APOCYNIN PREVENTS CYCLOOXYGENASE 2 EXPRESSION IN HUMAN
MONOCYTES THROUGH NADPH OXIDASE AND GLUTATHIONE
REDOX-DEPENDENT MECHANISMS
SILVIA S. BARBIERI,* VIVIANA CAVALCA,y SONIA ELIGINI,* MARTA BRAMBILLA,*
ALESSIA CAIANI,y ELENA TREMOLI,*,y and SUSANNA COLLI*
*E. Grossi Paoletti Center, Department of Pharmacological Sciences, and yDepartment of Cardiac Surgery,Centro Cardiologico Fondazione Monzino IRCCS, University of Milan, Milan, Italy
(Received 7 October 2003; Revised 5 April 2004; Accepted 16 April 2004)
Available online 11 May 2004
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Abstract—In the present study we report the preventive effect of apocynin, an active constituent of the Himalayan herb
Picrorhiza kurrooa, on cyclooxygenase-2 (Cox-2) synthesis and activity in human adherent monocytes exposed to serum
treated zymosan (STZ) and phorbol myristate acetate (PMA). Apocynin markedly decreases the intracellular reduced/
oxidized glutathione ratio (GSH/GSSG) and prevents nuclear factor-nB (NF-nB) activation in stimulated monocytes.
Moreover, it reduces intracellular reactive oxygen species (ROS) generation, NADPH oxidase activity in monocyte
homogenates and translocation of p47phox subunit in monocyte membranes. p47phox levels are also reduced in lysates of
apocynin-treated monocytes. The inhibition of Cox-2 by apocynin is completely abrogated by GSH provision. Results
from this study indicate that apocynin inhibits Cox-2 synthesis and activity induced in monocytes by an increased
oxidative tone and provide an explanation for the protective effect exerted by this compound in numerous cell and animal
models of inflammation. Attenuation of NADPH oxidase derived ROS coupled with GSH/GSSG reduction and
suppression of NF-nB activation are highlighted as the molecular mechanisms responsible for Cox-2 inhibition.
D 2004 Elsevier Inc. All rights reserved.
Keywords—Apocynin, Monocytes, Cyclooxygenase 2, NADPH oxidase, Reactive oxygen species, Reduced/oxidized
glutathione ratio, Prostaglandin E2, Nuclear Factor nB, Free radicals
INTRODUCTION
Picrorhiza kurroa is a well-known Himalayan herb in
Ayurvedic medicine. Current research has focused on the
hepatoprotective, antioxidant, and immune-modulating
activity of its active constituents [1]. Among these,
apocynin (4-hydroxy-3-methoxy-acetophenone) is a cat-
echol that inhibits neutrophil oxidative burst and reduces
neutrophil-mediated oxidative damage [2,3]. The anti-
inflammatory activity of apocynin has been proved in a
variety of cell and animal models of inflammation [4–9],
ress correspondence to: Dr. Susanna Colli, Department of
acological Sciences, via Balzaretti, 9, 20133-Milano-Italy; Fax:
50318250; E-mail: susanna.colli@unimi.it.
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156
in ischemia–reperfusion lung injury [10], and in airway
hyperresponsiveness [11].
Apocynin, after metabolic conversion, inhibits the
assembly of NADPH oxidase [12]. It is therefore exten-
sively used to reveal the role of this enzyme in cell and
experimental models be they characterized or not by an
inflammatory component [13–15].
Reactive oxygen species (ROS) are produced nor-
mally during the respiratory burst of phagocytes as a
defense mechanism and regulate multiple cell functions
and gene expression. The mode of action of ROS entails
redox-mediated activation or deactivation of signal-
transducing proteins and transcription factors [16]. Se-
veral intracellular sources contribute to ROS generation
in monocytes, including cyclooxygenases, lipoxyge-
nases, mitochondrial respiration, and NADPH oxidase
Apocynin prevents Cox-2 expression in monocytes 157
[17]. The latter is composed by various subunits that are
localized both in the membrane (gp91phox and p22phox)
and in the cytosol (p47phox, p67phox, and the small GTPase
rac) [18].
In response to inflammatory agents, monocytes syn-
thesize and release eicosanoids that modulate the inflam-
matory response. Cyclooxygenase 2 (Cox-2) synthesis is
an essential step in this event [19]. Cox-2 promoter
contains two putative nuclear factor-nB (NF-nB) motifs,
which render it greatly sensitive to redox imbalance [20].
In fact, modulation of the transcription factor NF-nB by
ROS is recognized as the key event through which
oxidative stress affects eicosanoid biosynthesis [21,22].
Reduced glutathione (GSH) is an important intracel-
lular redox buffer that exists as a reduced predominant
form, as a disulfide form (GSSG) or as mixed disulfide
(GSSR) with protein thiols [23]. The redox status within
the cell, reflected by the ratio GSH/GSSG [24], has been
shown to be relevant for the regulation of pro-inflam-
matory genes [25].
In this study we report the preventive effect of
apocynin on Cox-2 induction by inflammatory agents
in human adherent monocytes. Impairment of intracel-
lular ROS generation and changes in the intracellular
GSH/GSSG, leading to reduced NF-nB activation, are
highlighted as molecular mechanisms responsible for
Cox-2 inhibition.
MATERIALS AND METHODS
Reagents
Culture media were purchased from BioWhittaker
Italia SRL (Bergamo, Italy). M-199 was supplemented
with 2 mM L-glutamine, 100 units/ml penicillin, 100 Ag/ml streptomycin (all from Sigma, Milan, Italy), and 5%
heat-inactivated human AB serum. The following
reagents were purchased from Sigma: phorbol myristate
acetate (PMA), rotenone, diphenyleneiodonium (DPI),
bacterial lipopolysaccharide (LPS), ortho-phenylenedi-
amine (OPD), N-acetylcysteine (NAC), NADPH, hypo-
xanthine, xanthine oxidase, superoxide dismutase, nitro-
blue tetrazolium (NBT), and glutathione reduced form
ethyl ester (GSH-OEt). MK-886 was from Calbiochem
(Inalco SpA, Milan, Italy). Apocynin was from Aldrich,
Milan, Italy. Opsonized zymosan (STZ) was prepared by
mixing boiled saline-washed zymosan A (Sigma, Milan,
Italy) with freshly obtained human AB serum, as de-
scribed [26].
Antibodies
Antibodies against Cox isoforms were gifts from Aida
Habib (American University of Beirut, Lebanon). Anti-
body against p47phox was from Becton Dickinson (Milan,
Italy). Peroxidase-conjugated anti-mouse IgG antibody
was from Jackson ImmunoResearch Labs Inc. (West
Grove, PA, USA).
Culture of human monocytes and treatments
Monocytes were isolated from blood of healthy
donors. Blood sampling was performed in accordance
with the principles outlined in the Declaration of Hel-
sinki. Mononuclear cells were separated by Ficoll–Paque
density gradient (Amersham Pharmacia Biotech.), as
described [27]. Monocytes were isolated by selective
adherence to tissue culture dishes for 90 min at 37jCand incubated with various agents in medium M-199
supplemented with 5% human serum. Cell population
was >90% monocytes, as determined by nonspecific
esterase staining. Cells were exposed to stimuli for
different periods, as indicated. Inhibitors were added to
monocytes 1 h before exposure to stimuli. Cell viability
was determined by trypan blue exclusion. The concen-
trations of PMA and STZ used throughout the study were
selected on the basis of preliminary experiments.
Prostaglandin E2 (PGE2) and thromboxane B2
(TXB2)measurement
PGE2 and TXB2 were measured in monocyte super-
natant by enzyme immunoassay (EIA, Cayman Chemi-
cal, Ann Arbor, MI, USA).
Intracellular ROS formation
Superoxide anion (O2S�) generation was detected by
NBT assay. NBT (1 mg/ml) was added to medium of
adherent monocytes and incubations were carried out at
37jC for 15–60 min. Monocytes were then carefully
washed and lysed in buffer containing 90% dimethylsulf-
oxide, 0.01 N NaOH, and 0.1% SDS. Absorbance of NBT
reduction product formazan was measured at 715 nm
against lysis buffer blank. NBT reduction was correlated
with the amount of O2S� produced by adherent mono-
cytes. Data are expressed as nanomoles per milliliter
(molar extinction coefficient 18,000 M�1 cm�1) [28].
This method was chosen among others suitable to detect
intracellular ROS formation because it detects essentially
O2S�, which is not scavenged by apocynin, as in the case
of H2O2 [12]. Second, apocynin is autofluorescent and
interferes with fluorescent probes [29].
Measurement of NADPH oxidase activity in monocyte
homogenates
Monocytes were harvested in lysis buffer (20 mM
K2HPO4 containing 1 mM EDTA, 5 Ag/ml aprotinin, 2
Ag/ml pepstatin, 2 Ag/ml leupeptin, pH 7.0). Incubations
were carried out for 30 min at room temperature in
phosphate-buffered saline (pH 7.4) containing aliquots
of the sample (100 Al) with or without superoxide
S. S. Barbieri et al.158
dismutase (200 units/ml), NADPH (100 AM), and NBT
(1 mg/ml). NBT reduction was measured as described
above.
p47phox translocation
Monocytes, after exposure to PMA and STZ for 30
and 60 min. respectively, were harvested, sonicated on
ice (2 � 10 s) in relaxation buffer (100 mM KCl, 3 mM
Na Cl, 3.5 mM MgCl2, 10 mM Hepes, 1 mM EGTA, 10
Ag/ml pepstatin, 10 Ag/ml leupeptin, 0.5 mM phenyl-
methylsulfonyl fluoride), and centrifuged (600g for 10
min at 4jC) to remove nuclei and unbroken cells; the
supernatant was then ultracentrifuged (100,000g for 30
min at 4jC). Membranes were washed in relaxation
buffer and dissolved in Laemmli sample buffer.
Western blot analysis
Monocytes were lysed as described [27]. Samples from
whole cell or membrane lysates were separated in 7 or
10% SDS–PAGE, respectively, and transblotted to nitro-
cellulose membrane with a semidry transfer unit (Hoefer
Scientific Instruments). Membranes were incubated for 1
h with antibodies directed against Cox-1 (5 Ag/ml), Cox-2
(1/10,000), and p47phox (1/250), and subsequently with
donkey anti-mouse IgG conjugated with peroxidase.
Equal amounts of protein were analyzed. ECL (Amer-
sham Pharmacia Biotech) substrates were used according
to the manufacturer’s instructions to reveal bands reactive
with specific antibodies.
Assay of myeloperoxidase activity
Adherent monocytes were exposed to stimuli for 1 h
and subsequently lysed in Triton X-100, 0.05%. Myelo-
peroxidase activity was measured in cell lysates [30].
GSH and GSSG measurement
Cellular GSH and GSSG levels were measured by
HPLC. In brief, adherent monocytes were washed twice
in phosphate-buffered saline and lysed in buffer contain-
ing 5% trichloroacetic acid and 0.5 mM EDTA. Samples
were centrifuged after 3 cycles of freezing and thawing.
GSH and GSSG were separated using a Discovery C18 5
mm RP column (4.6 � 250 mm; Supelco, USA) eluted
(30jC) with a mobile phase containing 50 mM NaH2PO4,
0.05 mM octanesulfonic acid, and 2% acetonitrile, ad-
justed to pH 2.7 with phosphoric acid. The flow rate was 1
ml/min. Analysis was carried out with an ESA CoulArray
detector (5600A) with electrodes settled at +400, +700,
and +800 mV. Peak areas were analyzed using software
(CoulArray for Windows ESA). Under these conditions,
GSH and GSSG elute at 5.08 and 9.3 min respectively.
The detection limit for each metabolite is <100 pg/ml on
column. GSH and GSSG values are expressed as nano-
moles per milligram of protein, calculated from calibra-
tion curves using standard GSH and GSSG solutions
(Sigma).
Nuclear extracts and electrophoretic mobility shift assay
(EMSA) of NF-jB
Nuclear extracts were prepared from cell suspensions
as described [31]. After supernatant collection, the pro-
tein content was determined and EMSAs were performed
as follows: the synthetic single-stranded oligonucleo-
tides (Eurogentec, Herstal, Belgium) containing the
distal NF-nB sites were annealed with the complemen-
tary primers and radiolabeled with [32Pd]CTP (Amer-
sham Pharmacia Biotech). The consensus sequence for
NF-nB is in bold face: distal NF-nB site (upstream,
within �455 to 428 from the transcriptional start site) 5V-GGCGGGAGAGGGGATTCCCTGCGCCCCC-3V.Protein–DNA complexes were separated from free DNA
probe by electrophoresis through 5% nondenaturating
acrylamide gels in 0.5� TBE (45 mM Tris base, 45 mM
boric acid, 1 mM EDTA). NF-nB-specific bands were
confirmed by competition with a 100-fold molar excess
of an unlabeled NF-nB probe.
Statistical analysis
Data are reported as means F SD. Computer-assisted
statistical analyses used the ANOVA or unpaired t-test
program. After ANOVA, probability values were calcu-
lated using Fisher’s protected least significant difference
test. A value of p < .05 was considered significant. n =
number of individual experiments.
RESULTS
Apocynin prevents Cox-2 synthesis and activity in human
adherent monocytes
Monocyte exposure to STZ (0.5–1 mg/ml) or PMA
(40-80 nM) caused immunoreactive Cox-2 expression.
Cox-2, identified as a double band of 72 kDa by Western
blot analysis, was already detectable after 4 h monocyte
exposure to stimuli (Fig. 1). In contrast, Cox-1 levels
remained unchanged (data not shown). The extent of
Cox-2 synthesis induced by STZ and PMA was compa-
rable to that induced by LPS (1 Ag/ml) within the same
time frame (Fig. 1). Apocynin dose-dependently (1–100
Ag/ml) prevented Cox-2 induction by STZ and PMA
(Figs. 1A, 1B). In contrast, it did not inhibit, but even
increased, Cox-2 in LPS-stimulated monocytes (Fig. 1C).
DPI, an inhibitor of flavin-containing enzymes including
the NADPH oxidase [32], also impaired Cox-2 synthesis
(Figs. 2A, 2B) that, conversely, was not affected by other
agents known to affect intracellular ROS generation in
monocytes. Rotenone (50 AM) and MK-886 (1 AM),
which inhibit the mitochondrial respiration chain and
Fig. 1. Cox-2 expression is prevented by apocynin in STZ- and PMA-challenged monocytes. Apocynin was added to adherentmonocytes 1 h before stimuli. STZ, PMA, and LPS were then added and incubation was continued for additional 4 h. Cox-2 protein wasidentified as a double band of 72 kDa by Western blot analysis. Blots are representative of six independent experiments showing similarfindings. Densitometry (means F SD of six independent experiments) is shown at the top of each section. Signals were quantifiedrelative to h-actin. Statistically significantly different (*p < .05; **p < .01) from stimulated monocytes.
Apocynin prevents Cox-2 expression in monocytes 159
lipoxygenase pathways, respectively, failed to affect Cox-
2 induction (Figs. 2C, 2D).
Apocynin dose-dependently reduced PGE2 levels in
supernatants of stimulated monocytes (Fig. 3). TXB2
levels were also significantly reduced (from 6.57 F2.8 to 1.15 F 0.6 and from 7.19 F 1.4 to 1.77 F 0.88
in PMA, and STZ-stimulated monocytes respectively,
means F S.D., n = 4). No difference in cell viability,
assessed by neutral red, was observed at any dose
of apocynin until 5 h incubation, compared with
PMA- and STZ-treated monocytes (not shown).
Apocynin reduces intracellular ROS production in
human adherent monocytes
STZ and PMA progressively increased intracellular
ROS production in adherent monocytes to similar extents
(Fig. 4A). In contrast, LPS triggered 4-fold lower
amounts of intracellular ROS with respect to PMA and
STZ (not shown). Apocynin (100 Ag/ml) reduced ROS
generation and this effect was shared by DPI (Fig. 4,
panel B). NADPH oxidase activity measured in homo-
genates of monocytes treated with apocynin was signif-
icantly reduced (�78F 15.5 and �63F 25.8% in PMA-
and STZ-stimulated monocytes respectively, means FS.D., n = 4). No scavenging effect of apocynin at any
dose was detected using the cell-free hypoxanthine/
xanthine oxidase system followed by measurement of
NBT reduction (not shown).
Effect of apocynin on NADPH oxidase subunit p47phox
p47phox was detectable in membranes from resting
adherent monocytes and levels increased on exposure to
PMA and STZ (Figs. 5A, 5B). Reduced p47phox translo-
cation was observed in membranes of apocynin-treated
monocytes (Figs. 5A, 5B). In addition, apocynin con-
centration dependently reduced p47phox levels in lysates
of monocytes exposed to stimuli for 4 h (Figs. 5C, 5D).
Differential effect of STZ and PMA on myeloperoxidase
activity
Previous studies in neutrophils, eosinophils and en-
dothelial cells have disclosed the essential role of per-
oxidases in the metabolic conversion of apocynin to the
active compound [2, 33]. Adherent monocytes possess
basal myeloperoxidase activity, which was increased by
STZ but not by PMA, in accordance with what was
observed in neutrophils [2,12,34] (not shown). This
finding suggests that the increased myeloperoxidase
activity is not relevant to the preventive effect of apo-
cynin on Cox-2 expression.
Effect of apocynin on GSH and GSSG levels in
monocytes
The ability of the intracellular GSH/GSSG ratio to
regulate the expression and activation of redox-sensitive
Fig. 2. Cox-2 expression by STZ and PMA is prevented by DPI (A and B) but not affected by rotenone and MK886 (C and D). Inhibitorswere added to adherent monocytes 1 h before stimuli. STZ and PMAwere then added and incubation was continued for additional 4 h.Cox-2 protein levels were evaluated by Western blot analysis. Blots are representative of three independent experiments showing similarfindings. Signals were quantified relative to h-actin. Densitometry (means F SD of three independent experiments) is shown in the toppart of each section. Statistically significantly different (**p < .01) from stimulated monocytes.
S. S. Barbieri et al.160
transcription factors in inflammatory conditions is estab-
lished [35]. Apocynin has been shown to enhance
intracellular GSH in epithelial cells [36]. Under our
Fig. 3. Apocynin prevents PGE2 formation in STZ- and PMA-stimulatwere detected in monocyte supernatants by EIA. Results are expressedfour independent experiments performed in duplicate. Statisticallymonocytes.
conditions, the GSH/GSSG ratio in resting monocytes
was not influenced by apocynin. The ratio was, however,
significantly reduced (2- to 3-fold) on monocyte expo-
ed monocytes. Experimental conditions as in Fig. 1. PGE2 levelsas nanograms per milliliter and represent means values F SD ofsignificantly different (*p < .05; **p < .01) from stimulated
Fig. 4. Intracellular ROS generation induced by STZ and PMA in adherent monocytes is prevented by apocynin and DPI. Monocyteswere incubated with stimuli for 1 h (A) or preincubated with apocynin or DPI for 1 h and exposed to stimuli for an additional hour (B).NBT reduction was correlated with the amount of superoxide anion (O2
S�) produced. Data are expressed as nanomoles per milliliter (A)
or as percent change of stimulated monocytes (B). Data represent means F SD of seven independent experiments. Statisticallysignificantly different (**p < .01) from stimulated monocytes.
Fig. 5. Apocynin prevents p47phox translocation to membrane and its upregulation in stimulated monocytes. Apocynin has been added toadherent monocytes 1 h before stimuli. p47phox was detected by Western blot analysis in membranes (A and B) and lysates (C and D) ofmonocytes exposed or not to stimuli for 30–60 min. and 4 h, respectively. Blots are representative of three independent experimentsshowing similar findings. Signals were quantified relative to h-actin. Densitometry (mean F SD of three independent experiments) isshown in the top part of each section. Statistically significantly different (*p < .05; **p < .01) from stimulated monocytes.
Apocynin prevents Cox-2 expression in monocytes 161
Table 1. Effects of Apocynin on Intracellular GSH/GSSG Ratio inHuman Monocytes Exposed to PMA and STZa
Unstimulated PMA
(40 nM)
STZ
(0.5 mg/ml)
Apocynin (100 Ag/ml)� 35.2 F 9.9 19.9 F 9.2+ 11.4 F 3.8+
+ 34.6 F 22.1 1.5 F 1.1** 1.1 F 0.3*Fold reduction 1 13.4 10
Statistically significantly different (+p < .05 and ++p < .01) from
unstimulated monocytes.
Statistically significantly different (*p < .05, **p < .01) from
stimulated monocytes.a Human adherent monocytes were preincubated with medium alone
or medium containing apocynin (100 Ag/ml) for 1 h. PMA and STZ were
added for additional 30 min. Amounts of GSH and GSSG (nmol/mg
protein) were determined by HPLC. Values are expressed as meansF SD
of cells isolated from six individuals.
Fig. 7. Apocynin and DPI prevent NF-nB activation in adherentmonocytes exposed to PMA. NF-nB activation was determined innuclear extracts of monocytes incubated for 1 h with apocynin and DPIand subsequently exposed to PMA for 1 h. The EMSA analysis shownin the figure is representative of two independent experiments.
S. S. Barbieri et al.162
sure to PMA and STZ (Table 1), in agreement with
previous observations [37]. Further reduction (>10-fold,
with values lower than 2), was observed when mono-
cytes were pretreated with 100 Ag/ml apocynin for 1 h
(Table 1).
Provision of GSH restores Cox-2 in apocynin-treated
monocytes
To prove that the decrease in the GSH/GSSG ratio
caused by apocynin is responsible for Cox-2 inhibition,
experiments were carried out in monocytes exposed to
NAC, a GSH precursor, and to GSH-OEt, a mem-
brane-permeable compound that increases intracellular
GSH levels [23]. Results in Fig. 6 show that GSH
fully restores Cox-2 expression in monocytes incubated
with apocynin and subsequently exposed to PMA and
STZ.
Fig. 6. Provision of GSH restores Cox-2 expression in apocynin-treaadherent monocytes 1 h before stimuli. STZ and PMA were then adderepresentative of three independent experiments showing similar find(means F SD of three independent experiments) is shown in the top pafrom stimulated monocytes or monocytes preincubated with GSH-OE
Apocynin impairs NF-jB activation in stimulated
monocytes
NF-nB activation is critical for Cox-2 induction in
monocytes [38]. To examine whether apocynin affects
NF-nB activation, EMSAs were carried out on nuclear
extracts obtained from control and PMA-treated mono-
cytes (1 h incubation). DNA binding activity of NF-nBwas increased by PMA (40 nM), and the effect was
completely blocked by the addition of a 100-fold molar
excess of unlabeled oligonucleotide, indicating the spec-
ificity of the binding reaction (Fig. 7). Apocynin pre-
vented DNA binding of NF-nB in monocytes exposed to
PMA. This effect was shared by DPI (Fig. 7).
ted monocytes. GSH-OEt, NAC, and apocynin were added tod and incubation was continued for an additional 4 h. Blots areings. Signals were quantified relative to h-actin. Densitometryrt of each section. Statistically significantly different (**p < .01)t or NAC.
Apocynin prevents Cox-2 expression in monocytes 163
DISCUSSION
This study shows that apocynin reduces Cox-2 pro-
tein synthesis and activity induced by stimuli that trigger
ROS generation in human adherent monocytes. The
mechanism through which this effect occurs involves
(1) inhibition of NADPH oxidase-dependent intracellu-
lar ROS production, (2) reduction of the intracellular
GSH/GSSG ratio, and (3) prevention of NF-nB activa-
tion. In contrast, apocynin did not influence Cox-2
induction by LPS. The amount of intracellular ROS
produced by LPS was 4-fold less compared with the
amount produced by PMA and STZ, and it is known
that an oxidative environment is required to convert
apocynin into the active compound [2, 33]. The failure
of apocynin to affect LPS-induced Cox-2 may therefore
be explained by the inability of LPS to trigger intense
intracellular ROS generation.
In agreement with what was previously reported for
neutrophils [12], apocynin prevents NADPH oxidase
activity and ROS formation in monocytes, and this effect
is shared by DPI. The latter observation leads to the
conclusion that intracellular ROS from NADPH oxidase
are involved in Cox-2 synthesis. In contrast, agents
affecting other ROS-generating pathways in monocytes
fail to affect Cox-2.
The contribution of ROS to Cox-2 induction has
recently been recognized in the protection of macrophages
against apoptosis [21] and in the differentiation of mono-
cytes into macrophages [39]. Although a good correlation
between Cox-2 inhibition and reduction of NADPH oxi-
dase activity in homogenates of apocynin-treated mono-
cytes has been reported, this weakens when intact
monocytes are considered. This finding suggests that
additional mechanisms operate to impair Cox-2 expres-
sion. Alternatively, the subcellular localization of the
active compound might be relevant. Cox-2 is, indeed,
localized to the endoplasmic reticulum and nuclear enve-
lope, whereas NADPH oxidase becomes active at the cell
membrane. The impaired NADPH oxidase activity and
consequent ROS formation by apocynin may be respon-
sible, at least in part, for the prevention of NF-nBactivation. Parallel results obtained with DPI reinforce
this conclusion.
In addition to reducing p47phox translocation in mono-
cyte membranes, apocynin downregulates p47phox ex-
pression in lysates of monocytes exposed to stimuli over
a longer period (4 h). Of interest, the effects on p47phox
and Cox-2 go hand in hand. Impaired NF-nB activation
detected in apocynin-treated monocytes may account for
p47phox downregulation and reduction of Cox-2 protein
levels. A link between p47phox upregulation and NF-nBactivation has been highlighted in THP-1 cells differen-
tiated into monocytes [40] and in endothelial cells [41].
In the latter, p47phox participates in NF-nB activation
through association of its SH3 domains with the NF-nBsubunit RelA [41].
Apocynin requires the metabolic conversion by per-
oxidases to prevent NADPH oxidase assembly by con-
jugation to essential thiol groups [12]. The extent of Cox-
2 inhibition by apocynin was similar in STZ- and PMA-
stimulated monocytes, even though PMA and STZ affect
myeloperoxidase activity in different ways. The possi-
bility exists that basal myeloperoxidase activity detected
in resting adherent monocytes is sufficient for the met-
abolic conversion of apocynin.
A decreasing GSH/GSSG ratio inhibits the binding
activity of NF-nB in endothelial and alveolar epithelial
cells [42, 43], with complete abrogation observed at a
GSH/GSSG ratio of 1.25 in the latter cells [43]. More-
over, GSH/GSSG ratios from 1 to 0.1 inhibit DNA
binding of the p50 NF-nB subunit in a cell-free system
[44]. Our finding that the GSH/GSSG ratio is <2 in
apocynin-treated monocytes stimulated with PMA or
STZ brings us to the conclusion that the decrease in
GSH/GSSG ratio represents an additional mechanism
through which apocynin prevents Cox-2 expression and
NF-nB activation in monocytes. The finding that GSH
replenishment fully abrogates the inhibitory effect of
apocynin on Cox-2 supports this conclusion. The GSH/
GSSG equilibrium, therefore, finely tunes Cox-2 tran-
scription in monocytes. Two- to three fold reduction
induces NF-nB activity and Cox-2 expression, whereas
a more pronounced reduction (10-fold, with ratios <2)
prevents NF-nB activation and Cox-2 expression.
A low GSH/GSSG ratio reflects a marked reduction
in intracellular GSH levels, a possible result of the
formation of glutathionyl adducts between GSH and
quinone/quinone methides, metabolic products of apocy-
nin [45]. These compounds alkylate thiol groups, mainly
through the formation of thioether derivatives of cysteine
[46]. The cysteine residue within NF-nB subunit p50 is
essential for DNA binding [47]. Direct interference of
quinone/quinone methides with NF-nB activation, there-
fore, cannot be excluded. Alteration of the GSH/GSSG
ratio may also account for the inhibition of Cox-2
expression by DPI. An impressive GSH-depleting effect
of DPI in cultured cells has recently been reported [48].
Apocynin and DPI thus share the common ability to
reduce NADPH oxidase activity and to interfere with
the GSH/GSSG equilibrium, albeit through different
mechanisms.
In conclusion, apocynin prevents Cox-2 synthesis
and activity induced by STZ and PMA in adherent
monocytes. Alterations of both NADPH oxidase activity
and the GSH/GSSG imbalance concur to prevent NF-
nB activation and Cox-2 expression. This finding might
explain the previous observation on reduced PGE2
S. S. Barbieri et al.164
levels in glutathione-deficient mouse peritoneal macro-
phages challenged with zymosan [49]. To discriminate
the relative role of NADPH oxidase inhibition and
alteration of GSH/GSSG in the observed inhibition of
Cox-2 synthesis is, however, difficult as one might be
expected to influence the other.
Results from this study provide a mechanistic rationale
for the therapeutic significance of apocynin in a wide
range of cell and experimental models of acute and
chronic inflammatory disorders. Appreciation of this
mechanism may represent the starting point toward de-
velopment of the pharmacological potential of this com-
pound in the treatment of redox-linked disease states.
Acknowledgments—This work was supported by grants from the ItalianMinistry of University and Scientific Research and University of Milan(FIRB 2001-RBNE01BNFK and FIRST 2002, to S.C.). The technicalassistance of Michela De Franceschi and Loredana Boccotti is gratefullyacknowledged.
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ABBREVIATIONS
Cox-2—cyclooxygenase 2
DPI—diphenyleneiodonium
EMSA—electrophoretic mobility shift assay
GSH/GSSG—reduced/oxidized glutathione ratio
LPS—bacterial lipopolysaccharide
NAC—N-acetylcysteine
NBT—nitroblue tetrazolium
OPD—ortho-phenylenediamine
NF-nB—nuclear factor nBPGE2—prostaglandin E2
PMA—phorbol myristate acetate
ROS—reactive oxygen species
STZ—serum-treated zymosan
TXB2—thromboxane B2
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