Isatins inhibit cyclooxygenase-2 and inducible nitric oxide synthase in a mouse macrophage cell line

Article abstract of DOI:10.1016/j.ejphar.2006.10.057

Isatin is a versatile compound with a diversity of effects. We designed to investigate the inhibitory effect of isatin derivatives on lipopolysaccharide/interferon-γ-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, production of prostaglandin E₂ (PGE₂), nitric oxide (NO), tumor necrosis factor (TNF-α), and their capacity to scavenge NO. Isatins inhibit TNF-α production and iNOS and COX-2 protein expression resulting on reduced levels of NO and PGE₂. Our results indicate isatin and it derivatives as inhibitors of iNOS and COX-2 enzymes, which might be used as anti-inflammatory and antitumoral agents.

Full text of DOI:10.1016/j.ejphar.2006.10.057

European Journal of Pharmacology 556 (2007) 200–206
www.elsevier.com/locate/ejphar
Isatins inhibit cyclooxygenase-2 and inducible nitric
oxide synthase in a mouse macrophage cell line
a
b
b
Maria Eline Matheus , Flávio de Almeida Violante , Simon John Garden ,
b
a,
⁎
Angelo C. Pinto , Patricia Dias Fernandes
a
Department of Basic and Clinical Pharmacology, ICB, Federal University of Rio de Janeiro, Brazil
b
Department of Organic Chemistry, Chemistry Institute, CT, Federal University of Rio de Janeiro, Brazil
Received 13 July 2006; received in revised form 27 September 2006; accepted 30 October 2006
Available online 3 November 2006
Abstract
Isatin is a versatile compound with a diversity of effects. We designed to investigate the inhibitory effect of isatin derivatives on
lipopolysaccharide/interferon-γ-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins,
production of prostaglandin E
α production and iNOS and COX-2 protein expression resulting on reduced levels of NO and PGE
inhibitors of iNOS and COX-2 enzymes, which might be used as anti-inflammatory and antitumoral agents.
2006 Published by Elsevier B.V.
2
(PGE
2
), nitric oxide (NO), tumor necrosis factor (TNF-α), and their capacity to scavenge NO. Isatins inhibit TNF-
2
. Our results indicate isatin and it derivatives as
©
Keywords: Inducible nitric oxide synthase; Cyclooxygenase-2; Isatin; Cancer; Chemoprevention
1
. Introduction
been no study so far showing the modulating effects of isatins
on the expression or enzymatic activities of COX-2 or iNOS.
Isatins and its derivatives are heterocyclic compounds with
considerable synthetic versatility (Shvekhgeimer, 1996; Silva
et al., 2001). Isatin has been found to have anticholinesterase,
anticonvulsant, anti-inflammatory, anti-hypertensive, antihi-
poxia, antimicrobial, antineoplasic, antiulcer, antiviral and
central nervous system activities (Silva et al., 2001). Isatin
therefore has a wide spectrum of behavioral and metabolic
effects. Isatin is known to inhibit monoamine oxidase (MAO) B
and improves bradykinesia and striatal dopamine levels in rat
models of Parkinson's disease. The effects of isatin on the
survival of dopaminergic humans neuroblastoma (SH-SY5Y)
cells have been studied whereby it was found that isatin triggers
a dose-and time-dependent switch from apoptosis to necrosis
(Igosheva et al., 2005). Its most potent known in vitro actions
are as an antagonist of atrial natriuretic peptide (ANP) function
and nitric oxide signaling (Medvedev et al., 2005).
Chemoprevention is considered to be one of the most
promising propositions for prevention of human cancers. Based
on this strategy, many probable compounds have been
undergoing clinical trials for prevention of various sites of
malignancy (Turini and DuBois, 2002). The mechanistic
determination as well as entire evaluation of efficacy and safety
of such chemopreventive compounds will facilitate a mecha-
nism-based approach for cancer chemoprevention. Several line
of evidence suggests the critical role of inducible nitric oxide
synthase (iNOS) and cyclooxygenase-2 (COX-2) in tumori-
genesis. In many types of cancers, both enzymes are found in
multiple cells, i.e., epithelial, endothelial, stromal, and inflam-
matory cells (Cao and Prescott, 2002; Evans and Kargman,
2
004; Rigas and Kashfi, 2005; Simmons et al., 2004). There has
There are several reports showing a positive association
between chronic inflammation and carcinogenic risk (Ernst
et al., 2001; Prinz et al., 2001; Shacter and Weitzman, 2002).
Studies have shown that some enzymes such as COX-2 and
⁎
Corresponding author. Departamento de Farmacologia Básica e Clínica,
ICB, UFRJ, Caixa Postal: 68016, 21944-970, Rio de Janeiro, Brazil. Tel.: +55
2
1 2562 64 42.
E-mail address: patfern@farmaco.ufrj.br (P.D. Fernandes).
0014-2999/$ - see front matter © 2006 Published by Elsevier B.V.
doi:10.1016/j.ejphar.2006.10.057

M.E. Matheus et al. / European Journal of Pharmacology 556 (2007) 200–206
201
Table 1
iNOS, in association with an inflammatory response play a key
role in carcinogenesis (Arbabi et al., 2001; Cao et al., 2002).
Expression of such enzymes occurs in response to agents such
as interleukin 1β, tumor necrosis factor-α, interferon-γ (IFN-γ)
lipopolysaccharide (LPS) in different cells, including macro-
phages, endothelial cells and hepatocytes (Aktan, 2004). It has
been suggested that COX-2-mediated paracrine signaling by
macrophages plays a pivotal role of progression of tumors (Ko
et al., 2002). Expression and activity of iNOS is high in colon
adenomas and adenocarcinomas of humans, suggesting a role in
carcinogenesis (Ambs et al., 1998). There has been no study so
far showing the modulating effects of isatin and its derivatives
on the expression or enzyme activities of COX-2 and iNOS.
In the present study we have investigated the effects of isatins
on the expression and activity of COX-2 and iNOS enzymes
induced by LPS/IFN-γ in the RAW 264.7 cell line as well as the
Compound
Melting
point (°C) point (°C)
Literature melting Reference
Isatin (R=H) 186–188 197
5-Fluoroisatin 198–201 226
Sandmeyer (1919)
Pouchert and Behnke (1993)
Sigma-Aldrich catalogue
Sadler (1956)
(
R=5-F)
-Chloroisatin 241–243 256
R=5-Cl)
5
6
(
-Chloroisatin 249–252 263
(R=6Cl)
7-Chloroisatin 184–186 175
R=7Cl)
-Bromoisatin 253–256 267
R=4-Br)
Sandmeyer (1919)
(
4
5
Pouchert and Behnke (1993)
Sigma-Aldrich catalogue
Sadler (1956)
(
-Iodoisatin
(R=5-I)
254–259 278
5-Methylisatin 169–171 180
R=5-CH
(
3
)
production of their metabolites, PGE and NO, respectively.
2
(Garden et al., 1997). 4-Iodoisatin substituted was prepared by the
2
. Material and methods
iodination of isatin using aqueous methanolic KICl (Garden
2
et al., 1997; Sadler, 1956). The general structure of isatin with
respectively substitutes and the codes of each isatin derivative
used on this study are shown below (Fig. 1 and Table 1).
2
.1. General
Isatin was purchased from Merck and used as received. The
anilines used for the preparation of the substituted isatins were
also commercial reagents. The substituted isatins were character-
2.3. Cell culture
1
13
ized by their melting point and by H NMR (200 MHz) and
C
RAW 264.7 mouse monocyte-macrophages (ATCC TIB-71)
were grown in plastic bottles in an RPMI 1640 medium sup-
plemented with 10% fetal bovine serum, penicillin (100 U/ml),
streptomycin (100 μg/ml), glutamine (2 mM) and HEPES
(15 mM) (from now named RPMI) in a humidified atmosphere
NMR spectra (50 MHz) on a Bruker spectrometer using a mixture
of d-chloroform and d .dimethylsulfoxide. Calculated coupling
6
constants (J) are given in Hz. Infrared spectra were recorded as
−
1
KBr discs and were reported in wavenumbers (cm ).
containing 5% CO and 95% air at 37 °C. When cultures formed
2
2
.2. Synthesis of isatin derivatives
a confluent monolayer cells were scrapped, centrifuged and put
to adhere in 96 or 12 wells plate with RPMI at a density of
2×10 cell/ml (Raschke et al., 1978).
6
Isatin derivatives were prepared using a modified Sandmeyer
methodology (Marvel and Hiers, 1941; Sandmeyer, 1919).
Briefly, the anilines were treated with chloral hydrate, hydrox-
ylamine sulfate, sodium sulfate, HCl, water and ethyl alcohol to
obtain the respective isonitrosoacetanilides (Garden et al., 1997).
Subsequently, the isonitrosoacetanilides were cyclized with
concentrated H SO to furnish corresponding isatins. The isatin
derivatives were separated from isomeric products by formation
of isatinates with base (NaOH 3M) and acidification with acetic
acid, resulting in the precipitation of the 4-isomer, which was
removed by filtration. Acidification of the mother liquor with
concentrated HCl resulted in the precipitation of the 6-isomer
2.4. Cytotoxicity assay by MTT
Themitochondrial-dependent reductionof3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan
was used to measure cell respiration as an indicator of cell viability
(Denizot and Lang, 1986). Briefly, after 24 h incubation of RAW
264.7 adherent cells with or without isatin (1 to 300 μM),
supernatants were changed by 100 μl of RPMI medium containing
0.5 mg/ml MTT and cells incubated for 1 h at 37 °C in a 5% CO2
atmosphere. After the medium was aspirated, 100 μl of DMSO
was added to the cells to dissolve the formazan. The absorbance
from each group was measured in a Dynatech microplate reader at
2
4
540 nm. The control groups consisted of cells with medium plus
vehicle used to dissolve isatins and was considered as 100% of
viable cells. Results are expressed as percentage of viable cells
when compared with control groups.
2.5. Nitric oxide-trapping capacity of isatins
To test the capacity of isatins in trapping nitric oxide, we used a
cell-free system as described in Matheus et al. (2006). SNAP (S-
nitroso-N-acetyl DL-penicillamine) was used, as, when in solution,
Fig. 1. General structure of isatins.

202
M.E. Matheus et al. / European Journal of Pharmacology 556 (2007) 200–206
determined by using ELISA commercial kits (Cayman
Chemical Co., MI, USA), according to the method of Pradelles
and Maclouf (1985). Briefly, dilutions of the supernatants were
incubated with the conjugated eicosanoid-acetylcholinesterase
and with the specific antiserum in 96-well plates pre-coated
with anti-rabbit immunoglobulin G antibodies. After overnight
incubation at 4 °C, the plates were washed and the enzyme
substrate (Elmman's reagent) was added for 60 to 120 min at
2
4
5 °C. The optical density of the samples was determined at
12 nm on a microplate reader, and the concentration of PGE2
was calculated from a standard curve.
2.8. Quantification of TNF production
After activation of cells with LPS/IFN-γ with or without
isatins, cells were incubated for 8 h. At the end of incubation
100 μl supernatant was collected and stored at −20 °C until the
dosage. TNF activity in the supernatant of cell cultures was
determined by bioassay using L929 cells based on the method
describes by Flick and Gifford (1984).
2.9. Detection of iNOS and COX-2 enzyme expression
Fig. 2. Effect of isatins on nitric oxide production. RAW 264.7 cells were
activated with LPS (100 ng/ml) plus IFN-γ (10 U/ml) and incubated with isatin
or isatin derivatives (10–100 μM). Supernatants were collected after 24 h
incubation and nitrite accumulated was measured by Griess reagent. LPS/IFN-γ
activated cells (–●–). Results are expressed as mean±S.E.M. (n=6, in
After activation of cells with LPS/IFN-γ and addition of
isatins derivatives (100 μM), cultures were incubated for 6 h. At
the end of incubation the cells were washed with cold PBS and
⁎
triplicate) of nitrite (in μM). Pb0.05 compared with LPS/IFN-γ activated cells
by one-way analyses of variance ANOVA followed by Bonferroni's test.
it liberates to the medium nitric oxide that transforms to nitrite
(Field et al., 1978). The addition of an NO scavenger to the SNAP
solution results in a decay in the supernatant nitrite accumulation.
Using this protocol, each isatin (in doses of 100 μg/ml) was
incubated with 1 mM of SNAP. After 6 h of incubation, an aliquot
of supernatant was removed to quantify the nitrite accumulated by
Griess reaction (Green et al., 1982). Results are expressed as μM
of nitrite calculated in comparison with the sodium nitrite
standard curve.
2
.6. Quantification of nitric oxide production
To evaluate NO production, nitrite concentration in the
supernatants of RAW 264.7 adherent cells was measured using
the Griess reaction (Green et al., 1982). Briefly, cells were
activated with LPS (100 ng/ml) plus IFN-γ (10 U/ml). After
2
4 h of incubation with isatins derivatives (1 to 300 μM), 100 μl
of the supernatant was collected and mixed with equal volume
of Griess Reagent (1% sulphanilamide, 0.1% naphthylethylene
diamine dihydrochloride, 10% H PO ) for 10 min at room
3
4
temperature. The absorbance was measured at 540 nm using a
Dynatech microplate reader, and the nitrite concentration was
calculated using a standard curve of sodium nitrite.
2
Fig. 3. Effect of isatins on PGE production. RAW 264.7 cells were activated
with LPS (100 ng/ml) plus IFN-γ (10 U/ml) and incubated with isatin or isatin
derivatives (10–100 μM). Supernatants were collected after 24 h incubation and
2
.7. Quantification of PGE production
2
PGE
2
was measured by ELISA kit. LPS/IFN-γ activated cells (–●–). Results
⁎
(in ng/ml). Pb0.05
are expressed as mean±S.E.M. (n=6, in triplicate) of PGE
2
Aliquots of supernatant of cell cultures were collected 24 h
compared with LPS/IFN-γ activated cells by one-way analyses of variance
after LPS/IFN-γ addition. The concentrations of PGE were
ANOVA followed by Bonferroni's test.
2

M.E. Matheus et al. / European Journal of Pharmacology 556 (2007) 200–206
203
yl)-2,5-diphenyl tetrazolium bromide (MTT), RPMI 1640 me-
dium, fetal calf serum, 96-well microplates were purchased from
Sigma. SNAP (S-nitroso-N-acetyl DL-penicillamine) was from
Cayman Inc. Nitrocellulose membranes (0.25 μm) were from Bio
Rad, anti-mouse iNOS and COX-2 antibody were from Sigma,
anti-mouse IgG antibody conjugated to horseradish peroxidase
and enhanced chemiluminescence (ECL) kit were from
Amersham.
2.11. Statistical analysis
The results are presented as the mean±S.E.M. (n=6).
Statistical significance between groups was performed by the
application of one-way analyses of variance (ANOVA)
followed by Bonferroni's test. P values less than 0.05
(
Pb0.05) were used as the significant level.
3. Results
3.1. Effects of isatins on NO production by RAW 264.7 cells
In order to investigate the possibility that isatins could inhibit
LPS/IFN-γ-induced NO production in RAW 264.7 we pre-
incubated cells with isatins or vehicle alone and activated with
LPS/IFN-γ. When incubated with vehicle alone, the cells yielded
Fig. 4. NO scavenger activity of isatins. SNAP (1 mM) was incubated with isatin
or isatin derivatives (100 μM) for 6 h. Supernatants were collected and nitrite
accumulated was measured by Griess reagent. SNAP (–●–). Results are
5
.8±1.4 μM of NO . Treatment of the cells with LPS/IFN-γ
2
⁎
resulted in 77.1±6.7 μM of NO , resulting in 13.2-fold increase of
expressed as mean±S.E.M. (n=6, in triplicate) of nitrite (in μM). Pb0.05
2
compared with SNAP group by one-way analyses of variance ANOVA followed
by Bonferroni's test.
the NO production compared with vehicle alone. When cells were
activated with LPS/IFN-γ and treated with isatins (10–100 μM),
the reduction on NO produced occurred in a dose-dependent
manner. With exception of 4-Bromoisatin and 5-Iodoisatin, all
others significantly inhibited NO production at 30 and 100 μM
when compared to the LPS/IFN-γ activated cells (Fig. 2A and B).
To certify that the reduction on NO produced was due to
inhibition on cells activity and not to cell death, we performed
MTT reduction method. The results showed that none of the
tested isatins (10–100 μM) were able to reduce cell viability by
lysated with cold lysis buffer (NP40 10%, NaCl 150 mM, Tris
HCl pH 7.6 10 mM, PMSF 2 mM, Leupeptin 5 μM). Cell debris
was removed by centrifugation (12,000 ×g, 4 °C, 10 min). After
determination of protein concentration of each suspension by
the BCA method (BCA™ Protein Assay Kit, Pierce), suspen-
sions were boiled in Laemmli buffer (Dithiothreitol 100 mM,
Bromophenol Blue 0.1%). For sodium dodecyl sulphate (SDS)-
PAGE aliquots of 20 μg of protein of each sample were
subjected to electrophoresis in 8% polyacrylamide gels.
Following electrophoresis the proteins were electrophoretically
transferred onto nitrocellulose membrane. Membranes were
blocked with 5% nonfat dried milk in Tris buffered saline-tween
(
TBS-T, Tris HCl 10 mM, NaCl 150 mM, tween 20 0.1%) at
room temperature for 2 h. After washing with TBS-T primary
antibody solution, mouse monoclonal IgG was applied
overnight at 4 °C against iNOS or COX-2 at dilutions of
1
:2000. Membranes were washed with TBS-T, secondary
antibody solution, anti-mouse IgG antibody conjugated to
horseradish peroxidase at a dilution of 1:10,000 and then
applied for 1 h at room temperature. The blots were washed with
TBS-T, incubated in enhanced chemiluminescence (ECL)
reagent and exposed to photographic film (Kodak, Brazil).
Fig. 5. Effect of isatins on TNF-α production. RAW 264.7 cells were activated
with LPS (100 ng/ml) plus IFN-γ (10 U/ml) and incubated with isatin or isatin
derivatives (100 μM). Supernatants were collected after 8 h incubation and
TNF-α activity was measured by bioassay using L929 cells. Results are
expressed as mean±S.E.M. (n=6, in triplicate) of TNF-α (in U/ml). Pb0.05
compared with LPS/IFN-γ activated cells by one-way analyses of variance
2
.10. Reagents
⁎
G
Lipopolysaccharide (from Salmonella thyphimurium), N -
monomethyl-L-arginine (L-NMMA), 3-(4,5-dimethylthiazol-2-
ANOVA followed by Bonferroni's test.

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M.E. Matheus et al. / European Journal of Pharmacology 556 (2007) 200–206
3.5. Effects of isatins on iNOS and COX-2 enzyme expression
Trying to elucidate the mechanism of action of isatins
responsible for NO and PGE2 production, we performed
western blot analysis on lysate protein extracted from isatins
and LPS/IFN-γ-treated cells. A representative western blot
analysis of iNOS and COX-2 expression is shown in Fig. 6.
LPS/IFN-γ induced high levels of iNOS and COX-2 protein
when compared with vehicle treated groups (169.3±9.9 and
144.1±9.9, respectively vs 5.8±3.3 in control group). When the
LPS/IFN-γ activated cells were treated with isatins (100 μM),
iNOS-protein levels were significantly reduced with all isatins
(exception to 4-Bromoisatin and 5-Iodoisatin). On the same
membrane, we then determined whether the expression of
COX-2 protein, induced by LPS/IFN-γ, would reflect the
Fig. 6. Effect of isatins on iNOS and COX-2 protein expression. RAW 264.7
cells were activated with LPS (100 ng/ml) plus IFN-γ (10 U/ml) and incubated
with isatin or isatin derivative (100 μM). Cell lysates were extracted after 6 h
incubation for western blot analysis. Membrane was incubated with anti-COX-2
modifying effects of isatins on PGE production. Similar to that
2
observed with iNOS-protein, incubation of cells with isatins
reduced COX-2-protein expression. Only 4-Bromoisatin and 5-
Iodoisatin did not inhibit protein expression (Fig. 6).
and anti-iNOS antibodies. Results are the densitometric analysis of COX-2 (
)
and iNOS (□) protein expression performed using a laser scanner and processed
by AlphaEaseFc™ software. The results are expressed as mean±S.E.M. (n=6)
of arbitrary units. Pb0.05 compared with LPS/IFN-γ activated cells by one-
4. Discussion
⁎
way analyses of variance ANOVA followed by Bonferroni's test.
In this study, we showed that isatin derivatives inhibited the
activity of inducible isoforms of nitric oxide synthase (iNOS) and
cyclooxygenase (COX-2) enzymes in RAW 264.7 activated cells,
suggesting isatins as a new synthetic iNOS and COX-2 inhibitors.
These inhibitions were partly due to the inhibition at protein
expression levels, because iNOS and COX-2-protein expression
was significantly reduced by the treatment with isatins.
more than 20%. Doses of isatins that were greater than 300 μM
reduced cell viability more than 30% and were not used in this
study (data not shown).
Isatin is an endogenous compound that is widely distributed in
mammalian tissues and fluids. Its concentration increases under
conditions of stress and exhibits a variety of biological and
pharmacologic activities, such as anti-inflammatory, anticonvul-
sant, and antineoplasic (Silva et al., 2001). Medvedev et al. (2005)
had shown the effect of isatin on nitric oxide-stimulated soluble
guanylate cyclase from human platelets. However, the exact
mechanism by which isatin develops these activities is still
controversial. Also, the anti-inflammatory properties of isatin in
relation to its chemopreventive potential have not been
demonstrated. In this paper the synthesis of new isatins
derivatives with substitutions at the basic nucleus was done
with the objective of finding new substances with more stability
and lipossolubility that could suggest a more pronounced activity.
Several lines of evidence have shown that the expression of
3
.2. Effects of isatins on PGE production by RAW 264.7 cells
2
Fig. 3 shows that after activation of cells with LPS/IFN-γ
2
PGE levels reached values of 118.3±19.4 pg/ml (vs 9.3±5.6 pg/
ml for the control group). Similar to that observed with NO
production, all isatins significantly reduced PGE levels with the
exception of 4-Bromoisatin and 5-Iodoisatin (Fig. 3 A and B).
2
3
.3. NO-trapping capacity of isatins
To determine whether the reduction on NO production could
be related to scavenger activity of isatins, we developed a “cell-
free” system. Using this protocol SNAP (1 mM) was incubated
with each isatin derivative for 6 h. Fig. 4 shows that none of the
isatins tested reduced levels of nitrite accumulated in the
supernatant indicating that these substances do not have
capacity to scavenge NO (Fig. 4A and B).
COX-2 and iNOS, the key enzymes for PGE and NO, was up-
2
regulated in the inflammatory process, as well as in transformed
cells and malignant tissue of lung or colorectal cancer (Attiga
et al., 2000; Kang et al., 2005). In this study isatins inhibited
3
.4. Effects of isatins on TNF production by RAW 264.7 cells
PGE and NO production in LPS/IFN-γ-activated RAW 264.7
2
cells. The inhibitory action of isatins further down-regulated the
expression of COX-2 and iNOS, indicating the action of isatins
occurs to inhibit enzyme expression. Our current data support
the idea that isatins may have chemopreventive activity against
the malignant process by down-regulating COX-2 and/or iNOS.
Attenuation of expression and/or activity of iNOS and/or COX-2
leads to anti-inflammatory actions both in localized and systemic
conditions (Lin et al., 1999; Patrignani et al., 2005). There are
RAW 264.7 cells were activated with LPS/IFN-γ and 8 h later
supernatant was collected to measure TNF levels. Results showed
in Fig. 5 reveal that activation of RAW 264.7 cells with LPS/IFN-γ
leads to 698±51.1 U/ml TNF-α (vs 21±12 U/ml in non-activated
cells). Incubation of LPS/IFN-γ-activated cells with isatins resulted
in a significant decrease on TNF levels. The most pronounced
effect was observed to 6-Chloroisatin and 5-Iodoisatin.

M.E. Matheus et al. / European Journal of Pharmacology 556 (2007) 200–206
205
also many reports showing the involvement of iNOS and COX-2
in tumorigenesis (Cao et al., 2002; Evans and Kargman, 2004;
Rigas and Kashfi, 2005). Many other reports indicate that
inhibition of the COX-2 and iNOS enzymes causes anti-
carcinogenic activity (Rigas and Kashfi, 2005; Tiano et al.,
signal transduction cascade. The fact that 5-iodoisatin had a
significant activity against TNF-α production and did not
inhibit iNOS and COX-2 expression and/or activity could be
explained at least in part by differences on its site of action. In
may be that reduction on TNF-α levels by this isatin derivative
was not enough to inhibit iNOS/COX-2 expression.
2
002). These results together with ours, therefore, suggest that
inhibitory effects of isatins on COX-2 and iNOS activities might
be related at least in part to the chemopreventive activity of
chemically-induced carcinogenesis. Isatins may cause chemo-
preventive action through mechanisms other than cell prolifer-
ation- and apoptosis-dependent ones. We used RAW cells,
which is originated from mouse macrophage (Raschke et al.,
In summary, our results suggest that isatins derivatives inhibit
expression and activity of inducible isoforms of NOS and COX
in macrophage cells. The anti-inflammatory action of isatins
derivatives may be useful for developing preventive agents
against inflammatory processes as well as anti-carcinogenesis.
1
978), to study the modulating effect of isatins. Reports
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Inflammatory cytokines also play a major role in regulating
inflammation and tumor progression. Our data showed that isatins
suppressed the production of TNF-α, indicating that isatins
inhibited the production of proinflammatory cytokine. TNF-α
produced by inflammatory cells can influence neoplasic growth
and metastasis by regulating a cascade of cytokines, adhesion
molecules, matrix metalloproteinases and pro-angiogenic activ-
ities (Coussens and Werb, 2002). The levels of inflammatory
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