# Apocynin: Molecular Aptitudes

Authors: J. Stefanska; R. Pawliczak
Journal: Mediators of Inflammation
 (2008)

---

## Abstract

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. It can decrease the production of superoxide (O2−) from activated neutrophils and macrophages while the ability of phagocytosis remains unaffected. The anti-inflammatory activity of apocynin has been demonstrated in a variety of cell and animal models of inflammation. Apocynin, after metabolic conversion, inhibits the assembly of  NADPH-oxidase that is responsible for reactive oxygen species (ROS) production. It is, therefore, extensively used to reveal the role of this enzyme in cell and experimental models. Although some of the ROS serve as signaling molecules in the cells, excessive production is damaging and has been implicated to play an important role in the progression of many disease processes. This is why in many studies apocynin presents a promising potential treatment for some disorders; however, its utility with inflammatory diseases remains to be determined. Since its mode of action is not well defined, we tried to get a more precise insight into the mechanisms by which apocynin exerts its activity. Considering the anti-inflammatory activities of apocynin, we may conclude that this compound definitely deserves further study.


---

## Body

## 1. Introduction

Oxidative stress describes an imbalance between reactive oxygen species (ROS) synthesis and antioxidants. The
normal production of oxidants is counteracted by several antioxidative
mechanisms in the body
[1, 2].Normally, cells possess antioxidant
defense systems that include ROS degrading molecules (ROS scavengers), such as
uric acid, ascorbic acid, and sulfhydryl-containing molecules (e.g.,
glutathione), and antioxidant enzymes, such as catalase, glutathione
peroxidase, and superoxide dismutases. In pathologic conditions, in which
excessive production of ROS outstrips endogenous antioxidant defense, oxidative
stress may irreversibly modify (oxidize) biologic macromolecules such as DNA,
protein, carbohydrates, and lipids. Enhanced generation ofO2− also causes loss of NO bioavailability. O2− and NO
undergo a very fast radical-radical termination reaction to yield a secondary
oxidizing species, peroxynitrite anion (ONOO−), and peroxynitrous
acid. ROS induce apoptotic cell death in various cell types, and deregulation
of apoptosis causes clinical disorders [3]. NADPH-oxidase is the enzyme
responsible for ROS production, and inhibition of this enzyme represents an
attractive therapeutic target for the treatment of many diseases. To counteract oxidative
stress, the body produces an armory
of antioxidants to defend itself, which in fact are sometimes insufficient to
effectively defend the organism from ROS [2, 4]. There
are a lot of substances that have been researched in order to find a way to inhibit
production of ROS, and thus protect the body from diseases. Apocynin, NADPH-oxidase
inhibitor, is one of such agents; however, it is considered variously in publications. Some of these were
found to have potential clinical success while others have already been abandoned again, as will be discussed in this short
selective review.


## 2. Apocynin

Apocynin (4-hydroxy-3-methoxyacetophenone, trivial names: apocynin,
acetovanillone) was first described by Schmiedeberg in 1883 and was isolated
from the roots ofApocynum cannabinum (Canadian hemp), and extracts of it were used
as official remedies for dropsy and heart troubles. In 1971, apocynin was
identified during activity-guided isolation of immunomodulatory constituents
from the root of Picrorhiza kurroa (Scrophulariaceae),
a native plant grown in the mountains of India,
Nepal, Tibet, and Pakistan, well known in traditional
Indian medicine (Ayurveda). Apocynin is an acetophenone with a molecular weight
of 166.17 and forms needles upon crystallization from water. It possesses a
faint vanilla odor and has a melting point of 115°C [4, 5].Apocynin has been used as an efficient inhibitor of the complex NADPH-oxidase
in many experimental models involving phagocytic and nonphagocytic cells [6, 7]. The mechanism of inhibition
is not totally known, but involves the impairment of the translocation to the
membrane of the cytosolic component p47phox of the NADPH-oxidase complex [8, 9].A very important finding concerning this mechanism was the discovery
that apocynin is a prodrug that is converted by peroxidase-mediated oxidation
to a dimer, which has been shown to be more efficient than apocynin itself [10] (Figure 1).Figure 1
Creating active form of apocynin-dimerization.Apocynin is an inhibitor of the intracellular translocation of two
critical cytosolic components of the NADPH-oxidase complex present in the cell
membrane [9].The structure of NADPH-oxidase is quite complex, consisting of two membrane-bounded elements
(gp91phox and p22phox), three cytosolic components (p67phox, p47phox and
p40phox), and a low-molecular-weight G protein (either rac 2 or rac 1). The
racs are kept inactive by binding to a guanine nucleotide dissociation
inhibitor, which prevents the exchange of guanine nucleotides from the rac
proteins [5]. Activation of NADPH-oxidase
is associated with, and probably caused by, the migration of the cytosolic
components to the cell membrane so that the complete oxidase can be
assembled [11].Apocynin is a selective inhibitor
of NADPH-oxidase activity and concomitant ROS production (IC50 value: 10μM) in
activated human neutrophils [12]. Interestingly, it does not
seem to interfere with the PMNs other defense mechanisms, as it does not affect
phagocytosis or intracellular killing [2].Apocynin prevents the translocation of p47phox to Nox2 in leukocytes, monocytes,
and endothelial cells [2, 10] (Figure 2). The inhibitory
action of the compound, however, occurs after a lag time only. The latter
process appears to involve MPO because apocynin does not inhibit the oxidase in
cells devoid or deficient of MPO [13], and agents such as zymosan that promote the
release of MPO enhance the efficacy of apocynin [14].Figure 2
The mechanism of NADPH-oxidase inhibition by apocynin.In the original report of apocynin, as an NADPH-oxidase inhibitor, it
was noted that the compound requires activation by myeloperoxidase (MPO) [12]. It is assumed that apocynin
is activated by H2O2 and MPO to form an apocynin radical,
which then oxidizes thiols in the NADPH-oxidase. Indeed, thiols are critical
for the function of p47phox, and thiol oxidizing agents have been shown to
block NADPH-oxidase activation [10, 15]. In line with this concept,
it was observed that supplementation of thiol provided either as glutathione or
cysteine prevents the inhibitory effect of apocynin on the NAPDH oxidase. An
alternative explanation for the lag time of the inhibitory effect of apocynin
was that through the step of an apocynin radical, an apocynin dimer is formed [16]. In fact, it has been
suggested that this dimer only is the active inhibitory compound that may block
NADPH-oxidase activity [14].The in vitro anti-inflammatory effects of apocynin include the following:(i)
the
reduction of neutrophil oxidative burst,(ii)
neutrophil-mediated
oxidative damage [12],(iii)
the
decreased adhesion of the monocytic cell line U937 to tumor necrosis factor
(TNF) treated human umbilical vein endothelial cells [5],(iv)
a
reduction of polymorphonuclear granulocyte chemotaxis [17],(v)
the
inhibition of peroxynitrite [18],(vi)
the
inhibition of inflammation-mediated cartilage destruction [6]. Not much
is known about the kinetics of apocynin in vivo, but interesting metabolic aspects of apocynin were
described by Daly et al. [19]. He showed that after a period of 20
hours upon IP administration of 120 mg/kg apocynin to rats, 80% of the apocynin was
recovered unchanged in the urine of the animals. Side effects of apocynin are
not known. Apocynin has very low toxicity (LD50: 9 g/kg) after oral
administration in mice [20].


## 3. Application: Respiratory System  and Asthma

Ischemia-reperusion lung injury is a well-known clinical phenomenon
characterized by increased pulmonary vascular permeability, edema, and resistance
to blood flow [21, 22]. The pathogenesis of this
injury appears to involve the generation of ROS, which can be detected during
both ischemia and reperfusion [23, 24]. The ability of superoxide
dismutase to attenuate ischemia-reperfusion lung ininjury suggests that
superoxide anion production represents a critical step in the process [25, 26]. Apocynin has been shown to
confer protection in animal models of arthritis [27], and in ozone  and endotoxin-induced lung injury [13, 28]. Doddo et al. [21] showed that apocynin prevents
the increased vascular permeability caused by ischemia and reperfusion in
isolated sheep lungs. The effect of apocynin on the changes in vascular
permeability after ischemia-reperfusion was dose-dependent. This suggests that
apocynin is able to maintain normal endothelial albumin permeability during
ischemia and reperfusion.P. kurroa, as
mentioned previously, has been used for ages in the treatment of asthma. Peters et al.
[9] observed a statistically
significant and pronounced effect of apocynin on ozone-induced bronchial
hyperresponsiveness to methacholine measured 16 hours after exposure to ozone
in mild asthmatics. In addition, apocynin did not prevent the decline of FEV1 measured directly after ozone exposure, excluding a possible scavenger effect
by apocynin of ozone. These results suggest that apocynin may have a role in
preventing ozone-induced exacerbations of asthma and extend the results of Kudo
et al. which reported the
effect of apocynin on ozone-induced airway hyperresponsiveness in guinea
pigs [29, 30]. The publication showed that
superoxide dismutase, a scavenger of superoxide, as well as apocynin, reduced O3-induced
airway hyperresponsiveness. It was previously described that peroxidase
activation of apocynin is a prerequisite for inhibition of NADPH-oxidase and
that this activated apocynin is unlikely to work at distances such as in the
airway epithelium [9].A possible explanation for the effectiveness of apocynin in the
treatment of respiratory diseases might be the fact that apocynin inhibits
peroxynitrite (ONOO−) formation [18]. ONOO− is the very
reactive product of the reaction of nitric oxide (NO) and superoxide anion (O•2−).
For many years, much attention has been paid to the effects of NO in
respiratory diseases [31], but recently, the focus has
been shifted toward reactive nitrogen species (RNS) in general, and to
peroxynitrite in particular [32, 33]. Peroxynitrite is suggested
to induce epithelial damage, mediator release, and consequently
hyperresponsiveness [34]. This finding may have
important clinical implications since airway inflammation, epithelial damage,
and hyperresponsiveness are characteristic features in patients suffering from
asthma.


## 4. Application: Neuroprotective Features

NADPH-oxidase-mediated superoxide plays an important role in the pathogenesis
of brain injury and that inhibition of NADPH-oxidase by apocynin can attenuate
brain injury following experimental ischemic stroke [35]. It has been reported that apocynin protects
against global cerebral ischemia-/reperfusion-induced
oxidative stress and injury in the gerbil hippocampus [36], and inhibiting superoxide production by NADPH-oxidase
with apocynin protects blood-brain barrier constituents in ischemia-like injury
in vitro [37]. Wang et al. investigated
that apocynin was able to protect against brain injury in a collagenase-induced
rat model of intracerebral
hemorrhage (ICH). Apocynin reduced cerebral and vascular injury in
experimental stroke models at doses similar to those used in the present study [38].On the other hand, Titova et al. claim that apocynin
should not be considered as a potential therapeutic agent in ICH, as ICH
results in the activation of NADPH-oxidase and brain injury that cannot be
countered by a clinically relevant administration of apocynin. Additionally, it
is reported that apocynin had no effects not only on enhanced NADPH-oxidase
activity but also on lipid peroxidation and brain water content, as well as the
profound neurological dysfunction that occurred
after ICH [39].However, the results of Tang's study suggest that an increase in NADPH-oxidase
activity may contribute to the enhancement of superoxide in the infarct area,
and NADPH oxidase-mediated superoxide production may play a crucial role in the
pathogenesis of ischemic brain injury. Apocynin has been shown to confer
protection in animal models of ischemia-/reperfusion-induced lung injury. It
was administrated intraperitoneally, and treatment with apocynin significantly
decreased NADPH-oxidase activity and reduced the superoxide level, in addition
to attenuating significantly the size of the infarct [35].Inflammation
following ischemic stroke is known to contribute to injury. Apocynin has been
studied as a potential treatment in experimental stroke. Tang et al. [40] explored the effect of
different doses of apocynin in a mouse model of 2-hour transient middle
cerebral artery occlusion followed by 22-hour reperfusion. Apocynin, given at a
dose of 2.5 mg/kg 30 minutes before reperfusion, improved neurological
function, reduced infarct volume, and reduced the incidence of cerebral
hemorrhage. Nevertheless, at higher doses of 3.75 and 5 mg/kg, it increased
brain hemorrhage. Apocynin also tended to reduce mortality at the lower dose,
but not at higher doses. This data suggest that apocynin can protect against
experimental stroke, but with a narrow therapeutic window [40].


## 5. Application: Arteriosclerosis  and Hypertension

Atherosclerosis is one of the most common cardiovascular diseases in developed
countries and is yet another disease in which ROS are thought to play an
important role [41]. Among the main causes of the
development of atherosclerosis is a high serum level of low-density cholesterol-containing
lipoprotein [42].Experiments with apocynin in endothelial cells showed
similar results compared with the effects of apocynin in phagocytes. Holland et
al. reported that endothelial cells, incubated with apocynin (600μM) and stimulated with the phospholipase A2 activator thrombin, showed NADPH-oxidase
inhibition, resulting in a significantly impaired ROS production [43]. The same authors also state
that endothelial cell incubation with apocynin markedly diminished high
LDL-induced increases in cellular H2O2 concentrations [44]. Furthermore, apocynin was
shown to be effective at suppressing atherogenesis in vivo in spite of highly
elevated serum low density lipoprotein (LDL) levels using a rabbit model [45]. So, maybe apocynin has
revealed a new strategy in the treatment of atherosclerosis, and, therefore,
future treatments should also focus on NADPH-oxidase inhibition as an effective
way of preventing the endothelium from the initiating events of
atherosclerosis.Apocynin is a reversible inhibitor of NAD(P)H oxidase activity that
impedes assembly of the p47phox subunit with the membrane complex [46]. It has recently been
proposed as a potential therapeutic agent in the treatment of atherosclerotic
disease by Meyer and Schmitt [47]. Increased levels of reactive
oxygen species, in particular O2−, are a major cause of
endothelial dysfunction in many forms of cardiovascular disease. One of the
most important sources of O2− is NAD(P)H oxidases [48]. Hamilton et al.
[49] have shown that
treatment of isolated rat arteries with apocynin decreased NADH-stimulated O2− generation and increased NO bioavailability. In addition,
in human arteries and veins, NADH- and NADPH-oxidase-mediated O2− generation was inhibited by apocynin; endothelium-dependent vasodilation
was improved; and NO production from human SV endothelial cells was enhanced [49].Nox2 (formerly known as gp91phox) expression is upregulated and is associated
with elevated endothelial and adventitial NADPH-oxidase-dependent superoxide
production in model of vascular remodelling, leading to endothelial nitric
oxide dysfunction [50, 51].Direct inhibition of the source
of superoxide generation with apocynin may well be a useful approach to reduce
the remodelling associated with the early stages of postangioplasty remodelling
or atherosclerosis [47]. Local administration of an NADPH-oxidase
inhibitor apocynin in vivo to the collared arteries attenuates superoxide
production and prevents vascular injury associated with this remodelling [21].Apocynin treatment of the collared arteries did not affect Nox2 mRNA
expression, thus indicating that apocynin reduced superoxide generation in the
collared arteries by suppressing the NADPH-oxidase activity rather than
affecting its gene transcription. Apocynin has been demonstrated to reverse
endothelial NO dysfunction in animals or humans subjected to oxidative stress [49]. Several studies have
reported the protective effect of in vivo treatment with apocynin in
experimental vascular injury models associated with ROS overproduction. Beswick
et al. [52] and Ghosh et al. [53] showed that
extended oral treatment of apocynin not only reduces superoxide generation in
arteries isolated from deoxycorticosterone acetate salt- (DOCA-) induced
hypertensive rats, but also reduces blood pressure associated with hypertension
[52, 53]. Apocynin inhibits the
generation of NADPH-derived superoxide and prevents the damaging interaction
between NO and superoxide to form more potent ROS, thereby maintaining
endothelial function despite the stimulus for arterial remodelling [54].NADPH-oxidase is implicated in vascular remodelling
and superoxide-stimulated cell proliferation in the neointima contributes to
intimal hyperplasia in this collar model. Targeting NADPH-oxidase via
adventitial drug delivery not only reduces superoxide generation, but also
normalises endothelial cell function [54]. Targeting the primary source
of NADPH-oxidase-derived superoxide is an effective approach to prevent
deleterious arterial remodelling, providing a rationale for designing more
efficacious and selective inhibitors of vascular NADPH-oxidase as potential
therapeutics for human vascular disease. Reactive oxygen species (ROS) are
thought to play an important role in atherogenesis. It has been demonstrated
that the Nox2 catalytic component of NADPH-oxidase is upregulated in a
non-hyperlipidemic rabbit model of early stage arterial remodelling [50, 55].Apocynin has shown promising application in animal
studies of hypertension and other cardiovascular diseases. In the Dahl
salt-sensitive rat, apocynin inhibits superoxide production in the renal
medulla and decreases hypertension [56]. DOCA-induced increases in
aortic and renal superoxide production and hypertension are also attenuated by
apocynin treatment [52, 57]. In this model, apocynin
inhibits flow-induced superoxide production in the thick ascending loop of
Henle, which is thought to mediate inappropriate NaCl retention in
salt-sensitive hypertension [58] and prevents endothelial dysfunction
[53]. Apocynin also prevents and
reverses increases in systolic blood pressure (SBP) in dexamethasone-induced
hypertension [59]. Furthermore, apocynin might
be effective in treating humoral forms of hypertension such as those induced by
Ang II and aldosterone.Apocynin decreased p22phox mRNA levels in aortic
segments from aldosterone-salt male Sprague-Dawley (SD) rats and impeded
p47phox subunit assembly within the membrane complex in human endothelial cells
to inhibit the activity of NAD(P)H oxidase and its production of superoxide.
Apocynin prevents and reverses adrenocorticotropic hormone- (ACTH-) induced
hypertension [2, 49], indicating that NAD(P)H
oxidase is a major enzymatic source of superoxide overproduction in rat model
of both naturally occurring and synthetic hypertension. Apocynin prevented and
reversed Dex-induced changes in SBP, suggesting that upregulation of superoxide
production in Dex-hypertension is related to increased NAD(P)H oxidase activity
[35].Apocynin may have potential negative effects, causing a defect in bactericidal phagocytosis which can mimic a chronic
granulomatous disease (CGD) by indirect inhibition of respiratory burst. Nevertheless, Wilkins et al. [60] claim that NADPH-oxidase is
not required for LDL oxidation by human monocyte-derived macrophages because human
monocyte-derived macrophages (HMDMs) from chronic granulomatous disease
patients were able to oxidize LDL as suggested by increased lipoprotein uptake
by mouse macrophages.On the other hand, Aviram et al. [61] state that activation of NADPH-oxidase
is essential for macrophage-mediated oxidation of LDL. However, HMDM from patients with CGD that were shown to lack active NADPH-oxidase, but
to possess almost normal 15-1ipoxygenase activity, failed to oxidize LDL. The study shows that
addition of apocynin to an incubation system completely blocked the release of
superoxides to the medium, suggesting that LDL-induced macrophage release of
superoxides under oxidative stress is indeed associated with lipoprotein
stimulation of cellular NADPH-oxidase activity.Although it can be questionable if apocynin may indeed have potential
negative effect triggering a defect in bactericidal phagocytosis which can
mimic CGD, reversible character of action of apocynin and ambiguity of available data display that this problem still
needs investigation.


## 6. Application: Cox-2 and Cartilage

Hougee et al. [5] confirmed two important
features of apocynin in vivo: (1) oral administration of apocynin can partially
reverse the inflammation-induced inhibition of cartilage proteoglycan synthesis,
and (2) oral administration of apocynin has COX inhibitory effects similar to
the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen. Therefore, apocynin
might be of potential use during the treatment of chronic inflammatory joint
diseases like osteoarthritis or rheumatoid arthritis [5, 16].Apocynin has been reported to inhibit the superoxide
generating enzyme NADPH-oxidase [12, 13] present in
chondrocytes [62, 63]. Peroxynitrite is the highly
reactive coupling product of superoxide and nitric oxide and has been suggested
to play a role in the inflammation-mediated inhibition of cartilage
proteoglycan synthesis [43]. By inhibiting either NO or
superoxide production, the amount of concurrently generated peroxynitrite would
be decreased. Moreover, another recent study showed that apocynin prevented
COX-2 expression in stimulated human monocyte [8]. The mechanism of action
involved the inhibition of the NADPH-oxidase-dependent superoxide production,
the reduction of the intracellular GSH/GSSG ratio, and the prevention of the
activation of the nuclear transcription factor NF-κB, which is an important mediator of
inflammation [5, 64].However, the recent finding that apocynin is capable
of preventing COX-2 expression might provide an additional explanation for the
anti-inflammatory effects of apocynin that have been observed in vivo. These in
vivo effects of apocynin include the reduction of arthritis incidence [11], decreased joint swelling in
collagen-induced arthritis in mice [27, 65], as well as the reduction of
ulcerative skin lesions in inflamed skin in rats [66]. Apocynin also prevented
airway hyperresponsiveness during allergic reactions in mice [67] and reduced airway
hyperreactivity to metacholine when inhaled by humans suffering from mild
atopic asthma [9]. The COX-2 enzyme and its
major metabolite prostaglandin E2 (PGE2) play an
important role during inflammatory diseases. For example, in osteoarthritis and
rheumatoid arthritis, COX inhibitors such as NSAIDs are used to treat joint
swelling and pain [68–70]. By inhibiting the formation
of superoxide with apocynin, and hence reducing the amount of peroxynitrite,
the inflammation-mediated reduction of proteoglycan synthesis might be
restored. In vitro, this has been demonstrated in human cartilage [5, 6, 11].


## 7. Objects

A serious potential problem comes from studies showing that apocynin may
actually increase oxidative stress under some conditions. Glutathione
expression is decreased in response to apocynin treatment in alveolar
epithelial cells [16, 71] and apocynin activation by
myeloperoxidase produces an apocynin-free radical that is able to oxidize
glutathione [16]. Glial cells treated with
apocynin show a dose-dependent increase in oxidative stress markers, including
increases in oxidized glutathione. Unfortunately, apocynin must be administered
at high doses for effectiveness [71].Apocynin evoked, in a significant way, an increase ofH2O2 concentration and a decrease of the intracellular
glutathione/glutathione disulfide ratio, accompanied by augmented efflux of
glutathione and glutathione disulfide. Apocynin induced the activation of both
pentose phosphate pathway (PPP) and tricarboxylic acid cycle, which was blocked
when the cells were incubated with glutathione together with apocynin [36, 47, 72]. The cell incubation with
glutathione prevented also the apocynin-induced increase of malonyldialdehyde
generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant
effect also in a cell-free system: indeed, in aqueous solution, it evoked a
faster oxidation of the thiols glutathione and dithiothreitol, and elicited the
generation of reactive oxygen species, mainly superoxide anions. This suggests that apocynin per se can induce an oxidative
stress and exert a cytotoxic effect in different cell types, and that some
effects of apocynin in experimental
models in vitro and in vivo should be interpreted with caution [16, 47].Also, according to Riganti et al. [73] apocynin is able to increase the H2O2 level in resting monocyte-like cells (i.e., the N11 glial cell line) and can induce, under longer times of
exposure, an oxidative damage and a cytotoxic effect. Therefore, it is
suggested that apocynin is per se an inducer of ROS production, independent of
the cell type. It is conceivable that, when NADPH-oxidase is maximally activated,
the inhibition of the respiratory burst is the prevailing effect of the drug,
but in absence of a stimulation of NADPH-oxidase, the oxidative effect of
apocynin itself could predominate. Additionally, Riganti et al. hypothesize that
since sulfhydryl groups are important for the function of the leukocyte NADPH-oxidase, the oxidant effect of apocynin could
participate to the mechanism of enzyme inhibition
[71, 73].Apocynin induces activation of both PPP and tricarboxylic acid cycle,
which is subsequent to the oxidative stress, because the presence of GSH in the
medium together with apocynin actually blocks the activation of both metabolic
pathways. Riganti et al. [71] hypothesized that apocynin is
able to increase the H2O2 level in resting monocyte-like
cells (i.e., the N11 glial cell line) and can induce under longer times of
exposure an oxidative damage and a cytotoxic effect. Therefore, it is suggested
that apocynin is an inducer of ROS production, independent of the cell type. It
is conceivable that when NADPH-oxidase is maximally activated, the inhibition
of the respiratory burst is the prevailing effect of the drug, as a wide body
of literature has already shown, but in absence of a stimulation of NADPH-oxidase,
the oxidative effect of apocynin itself could predominate. It may be also
hypothesized that since sulfhydryl groups are important for the function of the
leukocyte NADPH-oxidase [13, 16], the oxidant effect of
apocynin could participate to the mechanism of enzyme inhibition. It has been
already reported that apocynin may exert other effects beside its ability to
inhibit NADPH-oxidase: for instance, it inhibits cytochrome P450 activity in
endothelial cells [74], interferes with actin
polymerization and cytoskeletal rearrangement in polymorphonuclear granulocytes
[17], and modulates the
arachidonic acid metabolism through a not-yet-clarified mechanism [55, 71].


## 8. Scavenger Properties

According to Heumüller et al. [75], apocynin predominantly acts
as an antioxidant but not as an inhibitor of NADPH-oxidases in nonphagocytic
cells in culture. It failed to block the O2− production
of Nox1, Nox2, or Nox4 when overexpressed in endothelial cells. Moreover, in
vascular cells, a similar activation of apocynin, as seen in leukocytes, was
not observed. Most importantly, however, apocynin turned out to be a scavenger of
radicals and directly inhibited the ROS-induced signaling in vascular cells [54, 76].Titova et al. [39] found that apocynin and its
oxidation products do not react with GSH. However, this thiol compound was
efficiently oxidized by the apocynin radical during the MPO-catalyzed
oxidation. The strong inhibitor effect of apocynin on the production of
hypochlorous acid by stimulated neutrophils might be the result of an additive
effect of NADPH-oxidase inhibition and, to a lesser extent in less extension,
due to competition with chloride for the catalytic active site of MPO. This is
a further evidence of the importance of apocynin as an anti-inflammatory drug [10]. Titova et al. [39] verified that
neither apocynin nor the dimer and trimer derivatives were able to conjugate
with GSH, which is a common representative of thiol compounds. However, they
obtained strong evidence that GSH is able to react with apocynin radical and/or
its dimer radical, which are formed during MPO-catalyzed oxidation.Stolk et al. [13] showed that apocynin did not
scavenge superoxide anion generated by xanthine oxidase. They concluded,
however, that apocynin scavenged hydrogen peroxide because of a dose-response
inhibition of luminol-enhanced chemiluminescence generated by hydrogen
peroxide. The protective effect of apocynin may not be limited to NADPH-oxidase
inhibition. Apocynin has been shown to inhibit cytochrome P450 [11] and thromboxane synthase [55]. Interestingly, both
cytochrome P450 [77] and thromboxane [78] have been implicated as
mediators of ischemia-reperfusion lung injury.


## 9. Apocynin and Cancer

Klees et al. [79] reported that while apocynin
itself is not effective, its derivatives inhibit migration of the breast cancer
cell line MDAMB-435 at subtoxic concentrations and the migration of
nonmalignant MCF10A breast cells is unaffected. These compounds also cause a
significant rearrangement of the actin cytoskeleton, cell rounding, and
decreased levels of active Rac1 and its related G protein Cdc42. The possible
link between apocynin and Rac1 inhibition suggests that apocynin may be a
source for inhibitors of Rac1-mediated tumor cell migration. Klees et al.
reports the application of an in vitro screening assay to identify
apocynin-derived inhibitors of Rac1-based tumor cell migration. According to
this study, apocynin, upon peroxidase-catalyzed metabolic activation, interferes with NADPH-oxidase
and inhibits lymphocyte migration through a G-protein-regulated pathway without
affecting adhesion. Reactive oxygen species generated by NADPH-oxidase also
control actin structure [80]. Apocynin or its metabolites
have also been shown to affect the migration of polymorphonuclear granulocytes,
suggesting that its
mechanism of action is conserved throughout cell types [4, 79]. Active Rac1 is necessary for
the translocation of p47-phox and p67-phox though it does not mediate it
directly. Rac1's role in NADPH-oxidase activation is not well understood, but
it is able to bind p67-phox, and this binding may be what causes the final
formation of the active NADPH-oxidase complex. When Rac1 is in its inactive
form, there is a decreased level of O2−, signifying
inactive NADPH-oxidase [8]. NADPH-oxidase has also been
shown to associate with the actin cytoskeleton, implicating another mode, by
which Rac1 may manage cytoskeletal structure [49, 79].


## 10. Conclusion

Taken together, there is increasing support to consider using apocynin
as a therapeutic agent for treatment of inflammatory diseases. The data of many
experiments show that NADPH-oxidase is an important contributor to elevated
levels of ROS. The property of apocynin as an inhibitor of NADPH-oxidase and
its dependence on MPO-catalyzed oxidation might be linked to the reaction of
apocynin radical and/or its dimer radical with intracellular GSH or directly
with essential thiols of the cytosolic factor p47phox and this fact might be a
key to further experiments, exploring wider application of apocynin [16, 81, 82].Summarizing
all the applications of apocynin and taking into account its low toxicity, selectivity, and lack of
known side effects, it can be concluded that apocynin deserves further
attention and that studies to elucidate its mode of action may contribute to
the development of safe and selective anti-inflammatory drugs which lack the
often serious side effects of steroids.



---

*Source: 106507-2008-12-02.xml*