The Journal of Immunology
NCX 4040, a Nitric Oxide-Donating Aspirin, Exerts
Anti-Inflammatory Effects through Inhibition of IkB-a
Degradation in Human Monocytes
Emanuela Ricciotti,*,†,1,2Melania Dovizio,*,†,1Luigia Di Francesco,*,†Paola Anzellotti,*,†
Tania Salvatore,*,†Andrea Di Francesco,*,†Maria G. Sciulli,*,†Giuseppa Pistritto,‡
Angela Monopoli,xand Paola Patrignani*,†
NO-donating aspirins consist of aspirin to which a NO-donating group is covalently linked via a spacer molecule. NCX 4040 and
NCX 4016 are positional isomers with respect to the ‑CH2ONO2group (para and meta, respectively) on the benzene ring of the
spacer. Because positional isomerism is critical for antitumor properties of NO-donating aspirins, we aimed to compare their anti-
inflammatory effects with those of aspirin in vitro. Thus, we assessed their impacts on cyclooxygenase-2 activity (by measuring
PGE2levels), protein expression, and cytokine generation(IL-1b, IL-18, TNF-a, and IL-10) in human whole blood and isolated
human monocytes stimulated with LPS. Interestingly, we found that micromolar concentrations of NCX 4040, but not NCX 4016
or aspirin, affected cyclooxygenase-2 expression and cytokine generation. We compared the effects of NCX 4040 with those of
NCX 4016 or aspirin on IkB-a stabilization and proteasome activity in the LPS-stimulated human monocytic cell line THP1.
Differently from aspirin and NCX 4016, NCX 4040, at a micromolar concentration range, inhibited IkB-a degradation. In fact,
NCX 4040 caused concentration-dependent accumulation of IkB-a and its phosphorylated form. This effect was not reversed by
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase, thus excluding the contribution of NO-dependent
cGMP generation. In contrast, IkB-a accumulation by NCX 4040 may involve an inhibitory effect on proteasome functions.
Indeed, NCX 4040 inhibited 20S proteasome activity when incubated with intact cells but not in the presence of cell lysate
supernatants, thus suggesting an indirect inhibitory effect. In conclusion, NCX 4040 is an inhibitor of IkB-a degradation and
proteasome function, and it should be taken into consideration for the development of novel anti-inflammatory and chemo-
preventive agents. The Journal of Immunology, 2010, 184: 2140–2147.
duction of prostanoid generation by the inhibition of cyclo-
oxygenase (COX)-1 and COX-2 (1). However, aspirin is unique in
the class of nonsteroidal anti-inflammatory drugs because it irre-
versibly inhibits both COX-1 and COX-2 activity by acetylating
a serine residue (Ser529and Ser516, respectively) positioned in the
arachidonic acid-binding channel (2, 3).
spirin (acetylsalicylic acid) is a nonsteroidal anti-
inflammatory drug that shares the same mechanism of
action with other molecules of the same class: the re-
COX-1 and COX-2 have the same catalytic activities: the
cyclooxygenase that oxidizes arachidonic acid to PGG2and the
peroxidase that reduces PGG2to the unstable endoperoxide PGH2
precursor of prostanoids (4, 5). However, the two isoforms of
COX are the products of different genes. COX-1 is considered
a housekeeping gene by virtue of constitutive low levels of ex-
pression in most cell types. In contrast, the gene for COX-2 is
a primary response gene with many regulatory sites; thus, COX-2
expression can be rapidly induced by LPS, cytokines, such as IL-
1b and TNF-a, growth factors, and the tumor promoter PMA (6).
COX-1–dependent prostanoids play an essential homeostatic
role in gastrointestinal cytoprotection (7–9), whereas COX-2–
dependent prostanoids play dominant roles in pathophysiologic
processes such as inflammation and cancer (10, 11).
NO-donating aspirins (NO-aspirins) were originally designed to
reduce gastrotoxicity of the parent aspirin (12, 13). They are hybrid
drugs that combine aspirin properties with the gastroprotection
exerted by NO that they are able to release. These products were
originally obtained by incorporating, through a simple ester
bridge, the carboxylic group of aspirin with moieties containing
NO-donor nitrooxy groups (‑ONO2) (14). Positional isomers of
NO-aspirin can be obtained, such as NCX 4016 and NCX 4040,
which are defined by the position of ‑CH2ONO2on the benzene
ring of the spacer in meta and para with respect to the ester bond
linking the two benzene rings (Supplemental Fig. 1) (15).
NO-aspirins require enzymatic metabolism for NO release (16–
18). In addition to the release of NO, they can be metabolized in
cells to the formation of different compounds that may have bi-
ological activities. The two positional isomers differ in their
*Department of Medicine and Center of Excellence on Aging, School of Medicine,
“G. d’Annunzio” University and†“G. d’Annunzio” University Foundation, Centro
Scienze dell’Invecchiamento, Chieti;
Rome Tor Vergata Medical School, Rome; andxNicOx Research Institute, Bresso,
‡Department of Neuroscience, University of
1E.R. and M.D. contributed equally to this work.
2Current address: Institute for Translational Medicine and Therapeutics, University of
Pennsylvania, Philadelphia, PA.
Received for publication September 22, 2009. Accepted for publication December
This work was supported by grants from NicOx and the European Community’s
Sixth Framework Program (Eicosanox, LSMH-CT-2004-00503) to P.P.
Address correspondence and reprint requests to Prof. Paola Patrignani, Department
of Medicine, Center of Excellence on Aging, School of Medicine, Centro Scienze
dell’Invecchiamento, “G. d’Annunzio” University, Via dei Vestini, 31, 66100 Chieti,
Italy. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this paper: ASA, acetylsalicylic acid; COX, cyclooxygenase;
ICE, IL-1b converting enzyme; NO-aspirin, NO-donating aspirin; ODQ, 1H-[1,2,4]
metabolism in vivo, which likely correlates with their differential
effects on cancer cell growth (15). In fact, NCX 4040, but not NCX
4016, was found to have antitumor activities (19, 20). It was re-
cently found that neither aspirin nor NO contributes to the antitu-
mor effect of NCX 4040, which, in contrast, seems to be due to the
quinone methide formed after carboxylic ester hydrolysis (21, 22).
In the current study, we explored the anti-inflammatory prop-
erties of NCX 4040 versus those of NCX 4016 and aspirin in vitro
by assessing their impact on COX-2 expression and activity and
cytokine generation (IL-1b, IL-18, TNF-a, and IL-10) in human
whole blood and isolated human monocytes stimulated with LPS.
Interestingly, we found that micromolar concentrations of NCX
4040, but not NCX 4016 or aspirin, affected COX-2 expression
and cytokine generation. These results suggest that NCX 4040
may act through the inhibition of NF-kB activation, which is a key
event in the proinflammatory response to LPS (23). Because
degradation of IkB-a due to phosphorylation, ubiquitination, and
ultimately proteolysis is required for the activation of NF-kB (24),
we supposed that the anti-inflammatory effect of NCX 4040 may
involve its activity on this pathway. Thus, we compared the effects
of NCX 4040 versus those of NCX 4016 or aspirin on IkB-a
stabilization and proteasome activity in human monocytic cell line
THP1 stimulated with LPS. Differently from aspirin and NCX
4016, NCX 4040, in the micromolar concentration range, in-
hibited IkB-a degradation. Furthermore, we provide evidence that
NCX 4040 acts through inhibition of proteasome function. Taken
together, our findings describe a novel mechanism of action for
NO-aspirins and have implications for the development of novel
anti-inflammatory and chemopreventive agents.
Materials and Methods
Biosynthesis of PGE2and cytokines in human whole blood
Whole blood was drawn from eight healthy volunteers (age range 23–35 y).
The in vitro study was approved by the local ethics committee, and in-
formed consent was obtained from each subject. One-milliliter aliquots
containing 10 IU sodium heparin were incubated in the absence or pres-
ence of LPS (10 mg/ml) (Sigma-Aldrich, St. Louis, MO) for 24 h at 37˚C,
as previously described (25). The contribution of platelet COX-1 was
suppressed by pretreating the subjects with 300 mg aspirin 48 h before
sampling. Prostanoids were measured in plasma by previously described
and validated RIAs (25), whereas cytokines (IL-1b, IL-18, TNF-a, and IL-
10) were measured by commercially available ELISA kit (Pierce, Rock-
Biosynthesis of thromboxane B2in human whole blood
One-milliliter aliquots of whole blood were allowed to clot at 37˚C for 60
min. Serum was separated by centrifugation (10 min at 3000 rpm) and kept
at 280˚C until assayed for thromboxane B2by RIA (26).
Isolation of human monocytes
Human monocytes were separated from the buffy coat by Ficoll-Paque (GE
Healthcare Life Sciences, Bucks, U.K.), as previously described (25). The
contribution of platelets to prostanoid biosynthesis was minimized by pre-
treating the buffy coat with aspirin (300 mM). Cell suspensions routinely
cultured in RPMI 1640 supplemented with 0.5% (v/v) FCS, 1% (v/v) peni-
cillin/streptomycin,and 2 mM L-glutamine (Invitrogen, Carlsbad, CA), were
incubated with LPS (10 mg/ml) at 37˚C for 24 h. PGE2and cytokine (IL-1b,
IL-18, TNF-a, and IL-10) levels were measured in the culture media by RIA
or ELISA kit and adjusted per million of cells. COX-2 protein levels were
evaluated in cell lysates by Western blot analysis.
Monocytic cell line THP1
The THP1 cell line was obtained from the American Type Culture Col-
lection (LGC Promochem, Milan, Italy) and cultured in RPMI 1640 con-
taining 10% FBS, 1% penicillin/streptomycin, and 2 mM L-glutamine.
Before every experiment, 1 3 106cells were cultured in 2 ml RPMI 1640
supplemented with 0.5% FBS for 16 h and then stimulated with 10 mg/ml
LPS for 1 h.
The nonselective COXinhibitoraspirin (Sigma-Aldrich),NO-aspirins,such
as NCX 4040 and NCX 4016, kindly provided by NicOx (Sophia Antipolis,
France), an irreversible and cell-permeable inhibitor of caspase-1, IL-1b
converting enzyme (ICE) inhibitor II (Calbiochem, San Diego, CA) (27),
a potent, membrane-permeable proteasome inhibitor, MG-132 (Sigma-
Aldrich) (28), and a selective inhibitor of NO-sensitive guanylyl cyclase
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (Sigma-Aldrich) (29)
were dissolved in DMSO. Two microliters of vehicle (DMSO) or stock
solutions of the different compounds were added to 1 ml human whole
blood or cellular culture medium.
Western blot analysis
Isolated human monocytes (1–2 3 106cells) and THP1 cells (1 3 106
cells) were lysed, and 20 mg total proteins was loaded for 4–9% SDS-
on COX-1 and COX-2 activity in human whole blood. A, Concen-
tration‑response curves for inhibition of whole blood COX-1 activity, as
assessed by measuring serum thromboxane B2levels, by aspirin (0.1–1000
mM), NCX 4016 (0.1–5000 mM), and NCX 4040 (0.1–5000 mM). B, Con-
centration‑response curvesfor inhibitionof whole blood COX-2 induced by
mM), NCX 4016 (0.1–5000 mM), and NCX 4040 (0.1–5000 mM). C, The
inhibitory effect of NCX 4040 on LPS-induced PGE2production was not
reversed by the guanylyl cyclaseinhibitor ODQ; ODQ (500 mM) was added
to 1 ml heparinized human whole blood 10 min before the addition of NCX
4040 (10 mM) and LPS. Concentration‑response curves shown in A and B
were fitted, and IC50values were calculated: IC50values for COX-1 in-
hibitionwere: 7.9 mM for aspirinand 102 mM for NCX 4016; IC50value for
COX-2 inhibition was 0.41 mM for NCX 4040. Data were reported as mean
6 SEM (n = 3–6). pp , 0.01 versus vehicle (DMSO);xp , 0.01 versus
vehicle + LPS;ƒp , 0.05 versus vehicle + LPS. ASA, acetylsalicylic acid.
Effects of aspirin (ASA), NCX 4016, NCX 4040, and ODQ
The Journal of Immunology2141
PAGE and transferred to polyvinylidene fluoride membrane (Bio-Rad,
Hercules, CA). Membranes were saturated with a solution of 5% nonfat
milk in TBST, and then incubated with anti–COX-2 (kindly provided
by Dr. Stacia Kargman from Merck Frosst, Kirkland, Quebec, Canada) and
anti–b-actin (Santa Cruz Biotechnology, Santa Cruz, CA) polyclonal Abs
1 h at room temperature or with anti–IkB-a polyclonal Ab (that recognized
both phosphorylated and nonphosphorylated forms, with a major affinity
for the latter) (Santa Cruz Biotechnology) overnight at 4˚C. To detect the
specifically phosphorylated form of IkB-a, we used an anti–p-IkB-a mAb
(raised against synthetic peptide containing phosphorylated Ser32or Ser36)
(Santa Cruz Biotechnology). Then, the membranes were washed in TBST
and incubated with the secondary antibodies: anti-goat peroxidase-
conjugated IgG (Santa Cruz Biotechnology) for b-actin, anti-rabbit
peroxidase-conjugated IgG (Sigma-Aldrich) for COX-2, and IkB-a or anti-
mouse peroxidase-conjugated IgG (Sigma-Aldrich) for p-IkB-a. Finally,
the membranes were washed in TBST, and then all of the blots were de-
veloped by using ECL plus detection according to the manufacturer’s in-
structions (GE Healthcare Life Sciences). OD quantification of different
specific bands was calculated using laser densitometry (Bio-Rad) and
normalized to the OD of b-actin.
THP1 cells (1 3 105) were cultured overnight in 96-well tissue culture
plates in 100 ml RPMI 1640 supplemented with 0.5% FBS. Then, cells
were treated with NCX 4040 (0.1–100 mM), NCX 4016 (100 mM), aspirin
(100 mM), MG-132 (10 mM), or vehicle (DMSO) and stimulated with LPS
(10 mg/ml) for 60 min. Then, cells were processed as reported in the
manufacturer’s instructions for the 20S Proteasome Assay kit (Cayman
Chemical, Ann Arbor, MI). A Jurkat cell lysate supernatant (included in
the kit as a positive control), which contains a high level of 20S protea-
some activity, was used to evaluate the direct effects of NCX 4040 (10–100
mM), aspirin (100 mM), NCX 4016 (100 mM), and MG-132 (10 mM) on
proteasome activity. The fluorescence intensity was reported as relative
fluorescence unit per wavelength and measured at an excitation of 360 nm
and emission of 480 nm using a fluorescence plate reader.
Results are expressed as mean 6 SEM. In LPS-stimulated monocytes and
whole blood experiments, PGE2and thromboxane B2values were sub-
tracted from their content detected in vehicle (DMSO) samples.
Statistical analysis was performed with Student t test or one-way AN-
OVA and Newman-Keuls multiple comparison test, using Instat (version
3.00 for Windows) (GraphPad, San Diego, CA). Values of p , 0.05 were
considered statistically significant. Concentration‑response curves were
fitted (using PRISM, GraphPad, version 5.00 for Windows), and IC50
values were reported.
Studies in human whole blood
Effects of NO-aspirins, NCX 4040 and NCX 4016, and aspirin on
COX-1– and COX-2–dependent prostanoid generation in human
whole blood. In human whole blood, aspirin caused a concentra-
tion-dependent inhibition of platelet COX-1 activity (Fig. 1A) with
an IC50value of 7.9 (95% CI, 4.4–14) mM, without significantly
affecting COX-2 activity (Fig. 1B) up to 5000 mM. NCX 4016
was .10-fold less potent than aspirin in inhibiting COX-1 activity
(Fig. 1A) with an IC50value of 102 (95% CI, 65–159) mM, and
similarly to aspirin, it did not affect inducible PGE2generation
(Fig. 1B). The presence of a NO-donating moiety in the para
position in NCX 4040 caused a reversion of COX selectivity. In
fact, it inhibited LPS-induced PGE2generation (Fig. 1B) with an
IC50value of 0.41 (95% CI, 0.23–0.73) mM associated with only
67% inhibition of COX-1, even at 5000 mM (Fig. 1A).
As reported in Fig. 1C, NCX 4040 inhibited COX-2 activity,
and this effect was not reverted by ODQ, an inhibitor of guanylyl
cyclase, thus excluding the contribution of NO-dependent cGMP
Effects of NCX 4040, NCX 4016, and aspirin on cytokine
generation in LPS-stimulated human whole blood. LPS (10 mg/
ml, for 24 h) induced the generation of cytokines, and the values
were reported in Supplemental Table I. NCX 4040 inhibited
with 1 ml heparinized whole blood samples in the presence of LPS (10 mg/ml) for 24 h, and IL-1b, IL-10, IL-18, TNF-a, and PGE2were assayed in plasma.
B, Concentration‑response curves for inhibition of IL-1b, IL-10, IL-18, and TNF-a generation in LPS-stimulated whole blood by NCX 4040 (shown in A)
were fitted, and IC50values were calculated: 0.56 mM for IL-1b, 0.50 mM for IL-18, 1.15 mM for TNF-a, 0.17 mM for IL-10. Results are depicted as
percentage of control (DMSO + LPS) or inhibition (mean 6 SEM) from three to six separate experiments except for IL-18, which was evaluated in two
separate experiments performed in duplicate. pp , 0.05 versus control (DMSO + LPS); ppp , 0.01 versus control.
Effects of NCX 4040 on cytokine and PGE2generation in LPS-stimulated human whole blood. A, NCX 4040 (0.1–100 mM) were incubated
generation in LPS-stimulated human whole blood. ICE inhibitor II (50 mM)
(A) and MG-132 (80 mM) (B) were incubated in 1 ml heparinized whole
IL-1b, IL-18, TNF-a, and IL-10 were assayed. Data were reported as per-
centage of control (DMSO + LPS), mean 6 SEM, n = 3; pp , 0.05 versus
control (DMSO + LPS) (A). Data shown in B were reported as means of two
separate experiments performed in duplicate.
2142 NITRIC OXIDE-DONATING ASPIRINS AND IkB-a
IL-10, TNF-a, IL-1b, and IL-18 in a concentration-dependent
fashion (Fig. 2A, 2B). The IC50value for inhibition of IL-10 [0.17
(95% CI, 0.09‑0.33) mM] was significantly lower than that of
TNF-a [1.15 (95% CI 0.70–1.87) mM] but comparable to those
of IL-1b, IL-18 [0.56 (95% CI, 0.23–1.40) mM and 0.50 (95% CI,
0.25–0.98) mM, respectively], (Fig. 2B), and PGE2[0.41 (95%
CI, 0.23–0.73) mM] (Figs. 1B, 2A). On the contrary, aspirin
and NCX 4016 did not affect PGE2 (Fig. 1B) and cytokine
biosynthesis in LPS-stimulated human whole blood (data
Effects of the inhibition of ICE and proteasome activity on PGE2
and cytokine generation in LPS-stimulated human whole blood.
Because it was suggested that inhibition of ICE, the key step for
intracellular processing and maturation of IL-1b (30) and IL-18
(31) is a possible COX-independent anti-inflammatory mechanism
of NO-aspirins (32), we assessed the impact of the selective and
irreversible inhibitor of ICE, ICE inhibitor II, on cytokine and
PGE2generation in LPS-stimulated whole blood. As shown in
Fig. 3A, the compound selectively inhibited IL-1b and IL-18
generation without affecting IL-10, TNF-a, and PGE2levels to
any statistically significant extent. This finding excluded that ICE
inhibition was involved in NCX 4040 effects and prompted us to
suppose that the NO-aspirin may affect NF-kB activation. In fact,
NF-kB plays a key role in LPS induction of COX-2 and cytokines
(23). Because IkB-a degradation by the ubiquitin‑proteasome
pathway is a central event in NF-kB‑induced gene transcription
(24), we assessed the effect of the specific proteasome inhibitor
MG-132 on PGE2 and cytokine production in LPS-stimulated
human whole blood. Interestingly, MG-132 completely suppressed
PGE2, IL-1b, TNF-a, and IL-10 generation (Fig. 3B). These re-
sults mirrored those caused by NCX 4040 and led us to reckon
that this was the mechanism involved in the potent inhibitory
effects of NCX 4040 on PGE2and cytokine generation. Thus, the
occurrence of this molecular mechanism in the anti-inflammatory
action of NCX 4040 was assessed in human monocytes.
Studies in isolated human monocytes
Effects of NCX 4040 on PGE2and cytokine generation and COX-2
protein expression in LPS-stimulated human monocytes. LPS (10
mg/ml, for 24 h) induced the generation of PGE2and cytokines,
and the values are reported in Supplemental Table I. NCX 4040
inhibited, in a concentration-dependent fashion, LPS-induced
PGE2, IL-1b, TNF-a, and IL-10 generation with comparable IC50
values [0.13 (95% CI, 0.06–0.3) mM, 0.07 (95% CI, 0.03–0.18)
mM, 0.18 (95% CI, 0.06–0.6) mM, and 0.15 (95% CI, 0.06–0.3)
mM, respectively] (Fig. 4A).
As shown in Fig. 4B, NCX 4040 caused a concentration-
dependent downregulation of COX-2 expression with an IC50
value of 0.13 (95% CI, 0.06–0.25) mM, which was superimpos-
able to that found for the inhibition of PGE2generation (Fig. 4A).
Altogether these findings support that NCX 4040 affected LPS
signaling involved in the expression of inflammatory genes.
NCX 4040 caused a concentration-dependent inhibition of PGE2, IL-1b, TNF-a, and IL-10 in LPS-stimulated human monocytes; isolated human mon-
ocytes (1–2 3 106cells) were treated with vehicle (DMSO) or increasing concentrations of NCX 4040 (0.1–100 mM) in the presence of LPS (10 mg/ml) for
24 h, and PGE2, IL-1b, TNF-a, and IL-10 released into the medium were assayed; concentration‑response curves for inhibition of PGE2, IL-1b, TNF-a,
and IL-10 were fitted, and IC50values were calculated: 0.13, 0.07, 0.18, 0.15 mM, respectively. B, Western blot analysis of COX-2 levels evaluated in
lysates of LPS-stimulated human monocytes incubated without and with increasing concentrations of aspirin and NCX 4040; percentage inhibition of COX-
2 expression was calculated by [1 2 ratio of COX-2 protein levels (analyzed by band OD and normalized to b-actin) in NCX 4040-treated cells to those in
vehicle-treated cells] 3 100; NCX 4040 inhibited COX-2 protein expression with an IC50value of 0.13 mM. C, Aspirin inhibited PGE2(IC509.23 mM) but
not cytokine generation in LPS-stimulated human monocytes. Data were reported as percentage of control (DMSO + LPS) or percentage inhibition (mean
6 SEM, n = 3–9). pp , 0.05; ppp , 0.01 versus control (DMSO + LPS).
Effects of NCX 4040 and aspirin on PGE2and cytokine generation and COX-2 protein expression in LPS-stimulated human monocytes. A,
The Journal of Immunology2143
Aspirin was 70-fold less potent than NCX 4040 in inhibiting
PGE2generation [IC509.23 (95% CI, 4.39–19.40) mM] (Fig. 4C).
The inhibition of PGE2was not associated with any significant
effect on COX-2 expression (Fig. 4B and Supplemental Fig. 2),
thus suggesting that aspirin up to a concentration of 100 mM re-
duced monocyte PGE2generation by affecting COX-2 activity.
Effects of the inhibition of ICE and proteasome activity on PGE2
and cytokine generation and COX-2 expression in LPS-stimulated
human monocytes. As shown in Fig. 5A, ICE inhibitor II selec-
tively inhibited IL-1b and IL-18 generation without affecting
IL-10, TNF-a, and PGE2 levels to any statistically significant
extent. The compound did not affect COX-2 levels (Fig. 5B). In
contrast, the proteasome inhibitor MG-132 completely suppressed
IL-1b, TNF-a, and IL-10 generation (Fig. 6A). PGE2reduction by
MG-132 was associated with downregulation of COX-2 (Fig. 6B).
These findings reinforced our hypothesis that NCX 4040 affected
NF-kB activation. Thus, we aimed to verify the effect of the
compound on IkB-a stabilization. We chose to study this mech-
anism in the monocytic cell line THP1 because we found that NF-
kB activation occurred during the isolation of human monocytes.
Studies in LPS-stimulated monocytic cell line THP1
Effects of NCX 4040, NCX 4016, and aspirin on the stabilization
of IkB-a. The effects of NO-aspirins and aspirin on IkB-a stabi-
lization were studied after stimulation with LPS (10 mg/ml) for
60 min. The treatment of cells with increasing concentrations of
NCX 4040 (1–100 mM) caused the accumulation of IkB-a (Fig.
a levels were significantly increased by 2-fold even in the presence
of NCX 4040 (1 mM) (Fig. 7B). At concentrations of 10 and 100
In contrast, aspirin and NCX 4016 up to a concentration of 100
mM did not cause any effect (Fig. 7A–D). The finding that NCX
4040 caused accumulation of both IkB-a and its phosphorylated
form supports its possible inhibitory effect on proteasome function.
As shown in Fig. 8, NCX 4040, but not aspirin, caused accu-
mulation of a ladder of more slowly migrating forms of p-IkB-a,
with a major form that is 8 kDa larger than p-IkB-a. The ap-
pearance and m.w. of the more slowly migrating forms of p-IkB-a
mM) or DMSO vehicle were added to isolated human monocytes in the
presence of LPS (10 mg/ml) for 24 h. In the culture medium, PGE2, IL-1b,
IL-18, TNF-a, and IL-10 were assessed (A). In the cell lysate, COX-2 and
b-actin protein levels were assessed by Western blot analysis (B). Data are
reported as percentage of control (DMSO + LPS) (mean 6 SEM, n = 3–6).
pp , 0.05; ppp , 0.01 versus control (DMSO + LPS).
COX-2 protein levels in LPS-stimulated monocytes. Isolated human mono-
cytes were treated with MG-132 (10 mM) and then stimulated with LPS (10
mg/ml) for 24 h. PGE2, IL-1b, TNF-a, and IL-10 were measured in the
medium (A) whereas in the cell lysates COX-2 and b-actin protein levels
control (DMSO + LPS) of two separate experiments performed in duplicate.
Effects of MG-132 on PGE2and cytokine biosynthesis and
and p-IkB-a accumulation. NCX 4040, but not NCX 4016 and
aspirin, caused accumulation of IkB-a (A, B) and p-IkB-a (B, C) in LPS-
stimulated THP1 cells. THP1 cells (1 3 106) were pretreated for 3 min
with increasing concentrations (1–100 mM) of NCX 4016, NCX 4040, and
aspirin, and then they were stimulated with LPS (10 mg/ml) for 1 h.
Twenty micrograms of total protein was analyzed by Western blot for IkB-
a (A), p-IkB-a (C), and b-actin. Protein levels (assessed as ratio of protein
OD normalized to the OD of b-actin) were shown as percentage of control
(DMSO + LPS) (mean 6 SEM, n = 3–6) (B, D). pp , 0.05; ppp , 0.01
versus control (DMSO + LPS).
Effects of NCX 4016, NCX 4040, and aspirin on IkB-a
2144NITRIC OXIDE-DONATING ASPIRINS AND IkB-a
detected in the presence of NCX 4040 are highly suggestive of
multiple additions of ubiquitin. Together these results suggest that
a central mechanism of action of NCX 4040 is to cause protea-
Effects of ODQ and MG-132 on IkB-a stabilization in LPS-
stimulated THP1 cells. As shown in Fig. 9A, ODQ did not in-
duce the accumulation of IkB-a and did not reverse the accumu-
lation of IkB-a induced by NCX 4040, thus suggesting that cGMP
generation was not involved in IkB-a stabilization by NCX 4040.
MG-132 (10 mM) prevented the proteolysis of IkB-a induced by
LPS (Fig. 9B). In the presence of the proteasome inhibitor, IkB-a
was detected as a doublet that represents the nonphosphorylated
form (lower band) and the phosphorylated form (higher band).
Effects of NO-aspirins, aspirin, and MG-132 on proteasome
activity in LPS-stimulated THP1 cells. The proteasome is
a large multimeric protease complex, localized in the cellular
cytoplasm, and consists of the 20S proteasome with proteolytic
activity and the 19S regulatory complex (33). As shown in Fig.
10A, the peptide aldehyde MG-132 (10 mM) almost completely
blocked the proteolytic activity of LPS-stimulated THP1 cells
toward a specific 20S proteasome substrate, SUC-LLVY-AMC.
NCX 4040 caused concentration-dependent inhibition of THP1
cells’ proteasome activity (Fig. 10A). On the contrary, NCX 4016
did not affect proteasome activity, and aspirin had a weak but
significant (p , 0.05) inhibitory effect (30%) at a concentration of
100 mM (Fig. 10A).
Differently from MG-132, which binds and inactivates pro-
teolytic activity of the 20S proteasome (34, 35), NCX 4040 did not
directly inhibit the enzyme activity. In fact, the compound did not
affect 20S proteasome activity in Jurkat cell lysate, which was
efficiently inhibited by MG-132 (Fig. 10B).
Some novel biological effects of the first series of NO-aspirins (i.e.,
been identified. In particular, the antitumor effects seem to be in-
dependent of NO-donor properties but caused by the metabolic
chemistry of the linker fragments influenced by the position of
study, we showed that NCX 4040 (para-NO-aspirin), but not NCX
4016 (meta-NO-aspirin) oraspirin, interferes with LPS signaling of
ranges in human monocytes. Interestingly, we reported that NCX
dation of p-IkB-a. THP1 cells were pretreated with increasing concen-
trations of NCX 4040 and aspirin (1–100 mM) and exposed to LPS (10 mg/
ml) as indicated. Cytoplasmic extracts (30 mg) were fractionated by SDS-
PAGE, and p-IkB-a protein was analyzed by ECL Western blot analysis
using mAb anti–p-IkB-a (raised against synthetic peptide containing
phosphorylated Ser32or Ser36). The positions of prestained m.w. markers
(Bio-Rad) and the phosphorylated form of the protein IkB-a are indicated.
More slowly migrating forms of p-IkB-a are indicated by the brackets.
Effect of NCX 4040 and aspirin on signal-induced degra-
THP1 cells. A, ODQ did not reverse NCX 4040-dependent IkB-a stabi-
lization in THP1 cells; cells (1 3 106cells) were pretreated with ODQ (10
mM) for 10 min, then NCX 4040 (10 mM) was added for another 3 min
before the stimulation with LPS (10 mg/ml) for 1 h. B, Similarly to NCX
4040, MG-132 (10 mM) caused the accumulation of IkB-a in THP1 cells.
Twenty micrograms of total protein was analyzed by Western blot analysis
for IkB-a and b-actin. The results of an experiment representative of three
separate experiments are shown.
Effects of ODQ and MG-132 on IkB-a stabilization in
proteasome activity. The effects of the compounds (NCX 4040, NCX 4016,
aspirin, and MG-132) on 20S proteasome activity were assessed in intact
were treated with NCX 4040 (0.1–100 mM), NCX 4016 (100 mM), aspirin
Cells were than collected and processed for proteasome activity assay using
a specific 20S proteasome substrate, SUC-LLVY-AMC. Jurkat cell lysate
supernatants, which contain a high level of 20S proteasome activity, were
directly incubated with NCX 4040 (10–100 mM), NCX 4016 (100 mM),
aspirin(100mM),and MG-132(10mM). Proteasome activity isexpressed as
control (DMSO + LPS).
Effects of NCX 4040, NCX 4016, aspirin, and MG-132 on
The Journal of Immunology 2145
4040 acts by affecting the intracellular protein stability machinery
of the proteasome system, translating into accumulation of IkB-a,
a key element in the control of NF-kB activation.
We characterized the COX-1/COX-2 biochemical selectivity of
aspirin and NO-aspirins in human whole blood. Aspirin was the
most potent inhibitor of platelet COX-1 but did not significantly
affect LPS-induced whole blood PGE2up to 5 mM. Failure of
aspirin to inhibit LPS-induced whole blood COX-2 activity may
be explained by the fact that it is rapidly hydrolyzed by plasma
esterases to salicylate before COX-2 expression (37).
The therapeutic effects (analgesic, anti-inflammatory, and an-
tipyretic) of aspirin at doses up to 1–2 g/d are mainly caused by
acetylation of COX-2, leading to the irreversible inhibition of PG
synthesis (38). At very high doses up to 3–5 g/d, aspirin and
salicylates may promote anti-inflammatory and antiproliferative
effects independent of COX-1 and COX-2 activity (39, 40). In
fact, at millimolar concentrations, aspirin and sodium salicylate
inhibit NF-kB in vitro (41), and aspirin was reported to inhibit IkB
kinase-b activity by competing with ATP for the ATP binding site
(42). This observation may explain the use of a very high dose of
aspirin and salicylates for inflammatory disorders such as rheu-
matoid arthritis (43). Finally, it has been reported that aspirin, at
this range of concentrations, inhibited proteasome activity in
mouse Neuro-2a cells associated with an increase in the accu-
mulation of ubiquitylated proteins in the cells, which correlated
with its effect on cell death (44).
Aspirin administration is associated with dose-dependent side
as a result of diminished generation of prostanoids (8, 45). These
effects limit its chronic use at high doses and open the way to the
development of NO-aspirins to reduce gastrotoxicity of the parent
aspirin based on the possible cytoprotective role of NO (46).
Interestingly, NCX 4040, but not NCX 4016, was a very potent
inhibitor of LPS-induced whole blood PGE2generation (IC500.41
mM), and this effect was coincident with the inhibition of cytokine
inhibitory effect of aspirin toward COX-2 activity was found.
However, aspirin was 70-fold less potent than NCX 4040 and did
not affect COX-2 expression and cytokine generation.
Due to the central role played by NF-kΒ activation in LPS-
dependent responses (23), we hypothesized that NCX 4040 acted
by interfering with this pathway in human monocytes. This was
further sustained by the finding that the proteasome inhibitor MG-
132, a known inhibitor of NF-kB activation (34, 35), mimicked
the effects of NCX 4040 on prostanoid and cytokine generation in
both human whole blood and isolated monocytes.
NF-kB is a transcription factor inactivated in the cytoplasm by
association with members of the IkB family of proteins in qui-
escent cells (47). Under inflammatory conditions and during ma-
lignant transformation, IkB is degraded, which then allows
nuclear translocation and DNA binding of NF-kB (24). The most
prominent member of the IkB family is IkB-a, the degradation of
which is usually initiated by phosphorylation on Ser32and Ser36
by the IkB kinase complex (24). This phosphorylation of IkB-a is
followed by ubiquitination and degradation by the 26S proteasome
complex, which can be blocked by a variety of proteasome in-
hibitors such as MG-132 (34, 35). Similarly to MG-132, NCX
4040 caused accumulation of IkB-a and its phosphorylated form
in the human monocyte-derived cell line THP1 at micromolar
concentrations. NCX 4040 (10 mM) completely suppressed
monocyte COX-2–dependent PGE2and cytokine generation, and
it was able to cause a dramatic accumulation of IkB-a and its
phosphorylated form in THP1 cells. Interestingly, Western blot
analysis showed that NCX 4040 at these concentrations, but not
aspirin, induced the accumulation of a ladder of more slowly
migrating forms of p-IkB-a suggestive of multiple additions of
ubiquitin. Together these results suggest that NCX 4040 caused
Interestingly, we found that NCX 4040 affected proteasomal
activity when incubated for60 min with LPS-stimulated THP1 cells
but not in Jurkat cell lysate supernatant that contains a high level of
the core 20S particle of the proteasomal system. Differently, MG-
132, which binds and inactivates proteolytic activity of the 20S
proteasome, inhibited proteasome activity in both THP1 cultures
and Jurkat cell lysate supernatant. These results suggest that NCX
4040 did not directly inhibit enzyme activity, but it probably needs
to be transformed to an active metabolite (such as quinone methide)
in cells to exert its inhibitor activity toward the proteasome (22).
Aspirin (100 mM) caused an incomplete (∼30%) but significant
reduction of proteasomal activity without inducing IkB-a accu-
mulation. This degree of inhibition of proteasomal activity was
comparable to that observed with NCX 4040 (10 mM), which,
however, differently from aspirin, was associated with dramatic
accumulation of IkB-a and its phosphorylated form. These find-
ings suggest that in addition to an inhibitory effect on proteolytic
activity NCX 4040 may interfere with the function of other pro-
teasome components, such as the receptors that recognize ubiq-
uitin chains attached to substrates (48). Thus, NCX 4040 may
impair proteasome function indirectly by inhibiting the expression
of various subunits of the proteasome or by inducing oxidative
stress, which has been reported to induce proteasomal malfunction
(49). The molecular mechanism of proteasome inhibition by NCX
4040 will be determined in a specific study.
In LPS-stimulated RAW 264.7 cells, endogenous NO was shown
to downregulate inducible NO synthase and COX-2 mRNA and
we did not study the effect of NCX 4040 on the activity of the
transcription factor NF-kB, our data indirectly exclude the occur-
rence of this mechanism. In fact, the inhibitor of guanylyl cyclase
ODQ did not reverse the inhibitory effects of NCX 4040 on PGE2
generation. In contrast, it is plausible that NCX 4040 exerts its ef-
fects through the formation of a quinone methide that might react
with glutathione or with ‑SH-bearing molecules, such as various
proteins and among them possibly the proteasome (18, 21, 22).
In summary, NCX 4040 reduced COX-2 and cytokine production
of IkB-a. This mechanism did not involve NO-dependent cGMP
generation, but presumably it was due to an inhibitory effect on
proteasome function by NCX 4040 metabolite(s). In conclusion,
NCX 4040 is an inhibitor of IkB-a degradation and proteasome
function, and it should be taken into consideration for the de-
velopment of novel anti-inflammatory and chemopreventive agents.
A.M. is an employee of NicOx. P.P. received grants from NicOx and from
the European Community’s Sixth Framework Program (Eicosanox, LSMH-
CT-2004-00503) to perform the study.
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