apocynin prevents cyclooxygenase 2 expression in human monocytes through nadph oxidase and...

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: [email protected].

P

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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.


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.


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.


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.


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.


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


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.

REFERENCES

[1] Picrorhiza kurroa (monograph). Altern. Med. Rev. 6:319–321;2001.

[2] Simons, J. M.; t’Hart, B. A.; Ip Vai Ching, T. R. A. M.; Van Dijk,H.; Labadie, R. P. Metabolic activation of natural phenols intoselective oxidative burst agonists by activated human neutrophils.Free Radic. Biol. Med. 8:251–258; 1990.

[3] ’T. Hart, B. A.; Simons, J. M.; Knaan-Shanzer, S.; Bakker, N. P.M.;Labadie, R. P. Antiarthritic activity of the newly developed neutro-phil oxidative burst antagonist apocynin. Free Radic. Biol. Med.9:127–131; 1990.

[4] Wang, W.; Suzuki, Y.; Tanigaki, T.; Rank, D. R.; Raffin, T. A.Effect of the NADPH oxidase inhibitor apocynin on septic lunginjury in guinea pigs. Am. J. Respir. Crit. Care Med. 150:1449–1452; 1994.

[5] Weber, C.; Erl, W.; Pietsch, A.; Strobel, M.; Ziegler-Heitbrock,H. W.; Weber, P. C. Antioxidants inhibit monocyte adhesion bysuppressing nuclear factor-nB mobilization and induction of vas-cular cell adhesion molecule-1 in endothelial cells stimulated togenerate radicals. Arterioscler. Thromb. 14:1665–1673; 1994.

[6] Stolk, J.; Rossie, W.; Dijkman, J. H. Apocynin improves the effi-cacy of secretory leukocyte protease inhibitor in experimental em-physema. Am. J. Respir. Crit. Care Med. 150:1628–1631; 1994.

[7] Lafeber, F. P. J. G.; Beukelman, C. J.; van den Worm, E.; van Roy,J. L. A. M.; Vianen, M. E.; van Roon, J. A. G.; van Dijk, H.;Bijlsma, J. W. J. Apocynin, a plant-derived, cartilage-saving drug,might be useful in the treatment of rheumatoid arthritis.Rheumatology 38:1088–1093; 1999.

[8] Muller, A. A.; Reiter, S. A.; Heider, K. G.; Wagner, H. Plant-derived acetophenones with antiasthmatic and anti-inflammatoryproperties: inhibitory effects on chemotaxis, right angle lightscatter and actin polymerization of polymorphonuclear granulo-cytes. Planta Med. 65:590–594; 1999.

[9] Muijsers, R. B. R.; van den Worm, E.; Folkerts, C. J.; Beukelman,C. J.; Koster, A. S.; Postma, D. S.; Nijkamp, F. P. Apocynininhibits peroxynitrite formation by murine macrophages. Br. J.Pharmacol. 130:932–936; 2000.

[10] Dodd-O, J. M.; Pearse, D. B. Effect of the NADPH oxidase in-hibitor apocynin on ischemia– reperfusion lung injury. Am. J.Physiol. Heart Circ. Physiol. 279:H303–H312; 2000.

[11] Muijsers, R. B.; van Ark, I.; Folkerts, G.; Koster, A. S.; vanOosterhout, A. J.; Postma, D. S.; Nijkamp, F. P. Apocynin and1400W prevents airway hyperresponsiveness during allergic reac-tions in mice. Br. J. Pharmacol. 134:434–440; 2001.

[12] Stolk, J.; Hiltermann, T. J. N.; Dijkman, J. H.; Verhoeven, A. J.Characteristics of the inhibition of NADPH oxidase activation in

neutrophils by Apocynin, a methoxy-substituted catechol. Am. J.Respir. Cell Mol. Biol. 11:95–102; 1994.

[13] Salmon, M.; Koto, H.; Lynch, O. T.; Haddad, E.-B.; Lamb, N. J.;Quinlan, G. I.; Barnes, P. J.; Chung, K. F. Proliferation of airwayepithelium after ozone exposure. Effect of apocynin and dexame-thasone. Am. J. Respir. Crit. Care Med. 157:970–977; 1998.

[14] Javesghani, D.; Magdar, S. A.; Barreiro, E.; Quinn, M. T.; Hus-sain, S. N. Molecular characterization of a superoxide-generatingNADPH oxidase in the ventilatory muscles. Am. J. Respir. Crit.Care Med. 165:412–418; 2002.

[15] Ulker, S.; McKeown, P. P.; Bayraktutan, U. Vitamins reverseendothelial dysfunction through regulation of eNOS andNADP(H) oxidase activities. Hypertension 41:534–539; 2003.

[16] Irani, K. Oxidant signaling in vascular cell growth, death, andsurvival. Circ. Res. 87:179–183; 2000.

[17] Bonizzi, G.; Piette, J.; Schoonbroodt, S.; Greimers, R.; Havard,L.; Merville, M.-P.; Bours, V. Reactive oxygen intermediate-dependent NF-nB activation by interleukin-1h requires 5-lipoxygenase or NADPH oxidase activity. Mol. Cell. Biol. 19:1950–1960; 1999.

[18] Segal, A. W.; Abo, A. The biochemical basis of the NADPHoxidase of phagocytes. Trends Biochem. Sci. 18:43–47; 1993.

[19] Dubois, R. N.; Abramson, S. B.; Crofford, L.; Gupta, R. A.;Simon, L.; Van De Putte, L. B.; Lipsky, P. E. Cyclooxygenasein biology and disease. FASEB J. 12:1063–1073; 1998.

[20] Newton, R.; Kuitert, L. M. E.; Bergmann, M.; Adcock, I. M.;Barnes, P. J. Evidence for involvement of NF-nB in the transcrip-tional control of COX-2 gene expression by IL-1h. Biochem.Biophys. Res. Comm. 237:28–32; 1997.

[21] von Knethen, A.; Callsen, D.; Brune, B. Superoxide attenuatesmacrophage apoptosis byNF-nBandAP-1 activation that promotescyclooxygenase-2 expression. J. Immunol. 163:2858–2866; 1999.

[22] Feng, L.; Xia, Y.; Garcia, G. E.; Hwang, D.; Wilson, C. B. In-volvement of reactive oxygen intermediates in cyclooxygenase-2expression induced by interleukin-1, tumor necrosis factor-a, andlipopolysaccharide. J. Clin. Invest. 95:1669–1675; 1995.

[23] Deneke, S. M.; Fanburg, B. L. Regulation of cellular glutathione.Am. J. Physiol. 257:L163–L173; 1989.

[24] Cotgreave, I. A.; Gerdes, R. G. Recent trends in gluthatione bio-chemistry–gluthatione–protein interactions: a molecular link bet-ween oxidative stress and cell proliferation? Biochem. Biophys.Res. Commun. 242:1–9; 1998.

[25] Rahman, I.; MacNee, W. Regulation of redox glutathione levelsand gene transcription in lung inflammation: therapeutic ap-proaches. Free Radic. Biol. Med. 28:1405–1420; 2000.

[26] Colli, S.; Eligini, S.; Lalli, M.; Tremoli, E. Platelet–neutrophilinteraction and superoxide anion generation: involvement of pu-rine nucleotides. Free Radic. Biol. Med. 20:271–278; 1996.

[27] Eligini, S.; Colli, S.; Basso, F.; Sironi, L.; Tremoli, E. Oxidizedlow density lipoprotein suppresses expression of inducible cyclo-oxygenase in human macrophages. Arterioscler. Thromb. Vasc.Biol. 19:1719–1725; 1999.

[28] Yamazaki, A.; Birnboim, C. Potentiation of retinoic acid-inducedU-937 differentiation into respiratory burst-competent cells bynitric oxide donors. Leukemia Res. 19:325–335; 1995.

[29] Gu, Y.; Xu, Y.-C.; Wu, R.-F.; Souza, R. F.; Nwariaku, F. E.;Terada, L. S. TNFa activates c-jun amino terminal kinase throughp47phox. Exp. Cell Res. 272:62–74; 2002.

[30] Kumar, P.; Pai, K.; Pandey, H. P.; Sundar, S. NADH-oxidase,NADPH-oxidase and myeloperoxidase activity of visceral leish-maniasis patients. J. Med. Microbiol. 51:832–836; 2002.

[31] Eligini, S.; Brambilla, M.; Banfi, C.; Camera, M.; Sironi, L.;Barbieri, S. S.; Auwerx, J.; Tremoli, E.; Colli, S. Oxidized phos-pholipids inhibit cyclooxygenase-2 in human macrophages vianuclear factor-nB/InB- andERK2-dependentmechanisms.Cardio-vasc. Res. 55:406–415; 2002.

[32] O’Donnell, B. V.; Tew, D. G.; Jones, O. T.; England, P. J. Studieson the inhibitory mechanism of iodonium compounds with specialreference to neutrophil NADPH oxidase. Biochem. J. 290:41–49;1998.

[33] Johnson, D. K.; Schillinger, K. J.; Kwait, D. M.; Hughes, C. V.;McNamara, E. J.; Ishmael, F.; O’Donnell, R. W.; Chang, M.-M.;


Cohn, Z. A.; Dordick, J. S.; Santhanam, L.; Ziegler, L. M.; Hol-land, J. A. Inhibition of NADPH oxidase activation in endothelialcells by ortho-methoxy-substituted catechols. Endothelium9:191–203; 2002.

[34] Van den Worm, E.; Beukelman, C. J.; Van den Berg, A. J. J.;Kroes, B. H.; Labadie, R. P.; Van Dijk, H. V. Effect of metho-xylation of apocynin and analogs on the inhibition of reactiveoxygen species production by stimulated human neutrophils.Eur. J. Pharmacol. 433:225–230; 2001.

[35] Droge, W.; Schulze-Osthoff, K.; Mihm, S.; Galter, D.; Schenk, H.;Eck, H.; Roth, S.; Gmunder, H. Functions of glutathione andglutathione disulfide in immunology and immunopathology.FASEB J. 8:1131–1138; 1994.

[36] Lapperre, T. S.; Jimenez, L. A.; Antonicelli, F.; Drost, E. M.;Hiemstra, P. S.; Stolk, J.; MacNee, W.; Rahman, I. Apocyninincreases glutathione synthesis and activates AP-1 in alveolarepithelial cells. FEBS Lett. 443:235–239; 1999.

[37] Seres, T.; Ravichandran, V.; Moriguchi, T.; Rokutan, K.; Tho-mas, J. A.; Johnston, R. B., Jr. Protein S-thiolation and dethio-lation during the respiratory burst in human monocytes: areversible post-translational modification with potential for buf-fering the effects of oxidant stress. J. Immunol. 156:1973–1980;1996.

[38] Inoue, H.; Tanabe, T. Transcriptional role of the nuclear factorkappaB site in the induction by lipopolysaccharide and suppressionby dexamethasone of cyclooxygenase-2 in U937 cells. Biochem.Biophys. Res. Commun. 244:143–148; 1998.

[39] Barbieri, S. S.; Eligini, S.; Brambilla, M.; Tremoli, E.; Colli, S.Reactive oxygen species mediate cyclooxygenase-2 inductionduring monocyte to macrophage differentiation: critical role ofNADPH oxidase. Cardiovasc. Res. 60:187–197; 2003.

[40] Sumi, D.; Hayashi, T.; Matsui-Hirai, H.; Jacobs, A. T.; Ignarro,L. J.; Iguchi, A. 17h-estradiol inhibits NADPH oxidase activitythrough the regulation of mRNA and protein expression inTHP-1 cells. Biochim. Biophys. Acta 1640:113–118; 2003.

[41] Gu, Y.; Xu, Y.-C.; Wu, R.-F.; Nwariaku, F. E.; Souza, R. F.;Flores, S. C.; Terada, L. S. p47phox participates in activation ofRelA in endothelial cells. J. Biol. Chem. 278:17210–17217;2003.

[42] Chen, G.; Wang, S.-H.; Warner, T. D. Regulation of iNOS mRNAlevels in endothelial cells by glutathione, a double-edged sword.Free Radic. Res. 32:223–234; 2000.

[43] Haddad, J. J. E.; Olver, R. E.; Land, S. C. Antioxidant/pro-oxidantequilibrium regulates HIF-1a and NF-nB redox-sensitive: evi-dence for inhibition by glutathione oxidation in alveolar epithelialcells. J. Biol. Chem. 275:21130–21139; 2000.

[44] Pineda-Molina, E.; Klatt, P.; Vazquez, J.; Marina, A.; Garcia deLacoba, M.; Perez-Sala, D.; Lamas, S. Glutathionylation of thep50 subunit of NF-nB: a mechanism for redoxinduced inhibitionof DNA-binding. Biochemistry 40:14134–14142; 2001.

[45] Awad, H. M.; Boersma, M. G.; Boeren, S.; van der Woude, H.; vanZanden, J.; van Bladeren, P. J.; Vervoort, J.; Rietjens, I. M. C. M.Identification of o-quinone/quinone methide metabolites of quer-cetin in a cellular in vitro system. FEBS Lett. 520:30–34; 2002.

[46] Bolton, J. L.; Turnipseed, S. B.; Thompson, J. A. Influence ofquinone methide reactivity on the alkylation of thiol and aminogroups in proteins: studies utilizing aminoacid and peptide models.Chem. Biol. Interact. 28:185–200; 1997.

[47] Perez Sala, D.; Cernuda-Morollon, E.; Pineda-Molina, E.; Cana-da, F. J. Contribution of covalent protein modification to theantiinflammatory effects of cyclopentenone prostaglandins. Ann.NY Acad. Sci. 973:533–536; 2002.

[48] Pullar, J. M.; Hampton, M. B. Diphenyleneiodonium triggers theefflux of glutathione from cultured cells. J. Biol. Chem.277:19402–19407; 2002.

[49] Rouzer, C. A.; Scott, V. A.; Griffith, O. W.; Hamill, A. L.;Cohn, Z. A. Arachidonic acid metabolism in gluthatione-defi-cient macrophages. Proc. Natl. Acad. Sci. USA 79:1621–1625;1982.

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

apocynin prevents cyclooxygenase 2 expression in human monocytes through nadph oxidase and...

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