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

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 Maria Eline Matheus a , Flávio de Almeida Violante b , Simon John Garden b , Angelo C. Pinto b , Patricia Dias Fernandes a,⁎ b

a Department of Basic and Clinical Pharmacology, ICB, Federal University of Rio de Janeiro, Brazil 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 E2 (PGE2), nitric oxide (NO), tumor necrosis factor (TNF-α), and their capacity to scavenge NO. Isatins inhibit TNFα 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. © 2006 Published by Elsevier B.V. Keywords: Inducible nitric oxide synthase; Cyclooxygenase-2; Isatin; Cancer; Chemoprevention

1. Introduction 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 mechanism-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 tumorigenesis. In many types of cancers, both enzymes are found in multiple cells, i.e., epithelial, endothelial, stromal, and inflammatory cells (Cao and Prescott, 2002; Evans and Kargman, 2004; Rigas and Kashfi, 2005; Simmons et al., 2004). There has

⁎ Corresponding author. Departamento de Farmacologia Básica e Clínica, ICB, UFRJ, Caixa Postal: 68016, 21944-970, Rio de Janeiro, Brazil. Tel.: +55 21 2562 64 42. E-mail address: [email protected] (P.D. Fernandes). 0014-2999/$ - see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.ejphar.2006.10.057

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, antihipoxia, 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). 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

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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 macrophages, 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 production of their metabolites, PGE2 and NO, respectively. 2. Material and methods 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 characterized by their melting point and by 1H NMR (200 MHz) and 13C NMR spectra (50 MHz) on a Bruker spectrometer using a mixture of d-chloroform and d6.dimethylsulfoxide. Calculated coupling constants (J) are given in Hz. Infrared spectra were recorded as KBr discs and were reported in wavenumbers (cm− 1). 2.2. Synthesis of isatin derivatives Isatin derivatives were prepared using a modified Sandmeyer methodology (Marvel and Hiers, 1941; Sandmeyer, 1919). Briefly, the anilines were treated with chloral hydrate, hydroxylamine sulfate, sodium sulfate, HCl, water and ethyl alcohol to obtain the respective isonitrosoacetanilides (Garden et al., 1997). Subsequently, the isonitrosoacetanilides were cyclized with concentrated H2SO4 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

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Table 1 Compound

Melting Literature melting Reference point (°C) point (°C)

Isatin (R = H) 5-Fluoroisatin (R = 5-F) 5-Chloroisatin (R = 5-Cl) 6-Chloroisatin (R = 6Cl) 7-Chloroisatin (R = 7Cl) 4-Bromoisatin (R = 4-Br) 5-Iodoisatin (R = 5-I) 5-Methylisatin (R = 5-CH3)

186–188 198–201

197 226

Sandmeyer (1919) Pouchert and Behnke (1993)

241–243

256

Sigma-Aldrich catalogue

249–252

263

Sadler (1956)

184–186

175

Sandmeyer (1919)

253–256

267

Pouchert and Behnke (1993)

254–259

278

Sigma-Aldrich catalogue

169–171

180

Sadler (1956)

(Garden et al., 1997). 4-Iodoisatin substituted was prepared by the iodination of isatin using aqueous methanolic KICl2 (Garden 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). 2.3. Cell culture RAW 264.7 mouse monocyte-macrophages (ATCC TIB-71) were grown in plastic bottles in an RPMI 1640 medium supplemented 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 containing 5% CO2 and 95% air at 37 °C. When cultures formed a confluent monolayer cells were scrapped, centrifuged and put to adhere in 96 or 12 wells plate with RPMI at a density of 2 × 106 cell/ml (Raschke et al., 1978). 2.4. Cytotoxicity assay by MTT The mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-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 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

Fig. 1. General structure 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 (Snitroso-N-acetyl DL-penicillamine) was used, as, when in solution,

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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 25 °C. The optical density of the samples was determined at 412 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 triplicate) of nitrite (in μM). ⁎P b 0.05 compared with LPS/IFN-γ activated cells by one-way analyses of variance ANOVA followed by Bonferroni's test.

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

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 24 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% H3PO4) for 10 min at room 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. 2.7. Quantification of PGE2 production Aliquots of supernatant of cell cultures were collected 24 h after LPS/IFN-γ addition. The concentrations of PGE2 were

Fig. 3. Effect of isatins on PGE2 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 PGE2 was measured by ELISA kit. LPS/IFN-γ activated cells (–●–). Results are expressed as mean ± S.E.M. (n = 6, in triplicate) of PGE2 (in ng/ml). ⁎P b 0.05 compared with LPS/IFN-γ activated cells by one-way analyses of variance ANOVA followed by Bonferroni's test.

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yl)-2,5-diphenyl tetrazolium bromide (MTT), RPMI 1640 medium, 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 (P b 0.05) were used as the significant level. 3. Results 3.1. Effects of isatins on NO production by RAW 264.7 cells

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 expressed as mean ± S.E.M. (n = 6, in triplicate) of nitrite (in μM). ⁎P b 0.05 compared with SNAP group by one-way analyses of variance ANOVA followed by Bonferroni's test.

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), suspensions 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). 2.10. Reagents Lipopolysaccharide (from Salmonella thyphimurium), NG monomethyl-L-arginine (L-NMMA), 3-(4,5-dimethylthiazol-2-

In order to investigate the possibility that isatins could inhibit LPS/IFN-γ-induced NO production in RAW 264.7 we preincubated cells with isatins or vehicle alone and activated with LPS/IFN-γ. When incubated with vehicle alone, the cells yielded 5.8 ± 1.4 μM of NO2. Treatment of the cells with LPS/IFN-γ resulted in 77.1± 6.7 μM of NO2, resulting in 13.2-fold increase of 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

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). ⁎P b 0.05 compared with LPS/IFN-γ activated cells by one-way analyses of variance ANOVA followed by Bonferroni's test.

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3.5. Effects of isatins on iNOS and COX-2 enzyme expression

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 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. ⁎P b 0.05 compared with LPS/IFN-γ activated cells by oneway analyses of variance ANOVA followed by Bonferroni's test.

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). 3.2. Effects of isatins on PGE2 production by RAW 264.7 cells Fig. 3 shows that after activation of cells with LPS/IFN-γ PGE2 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 PGE2 levels with the exception of 4-Bromoisatin and 5-Iodoisatin (Fig. 3 A and B). 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 “cellfree” 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). 3.4. Effects of isatins on TNF production by RAW 264.7 cells 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.

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 modifying effects of isatins on PGE2 production. Similar to that observed with iNOS-protein, incubation of cells with isatins reduced COX-2-protein expression. Only 4-Bromoisatin and 5Iodoisatin did not inhibit protein expression (Fig. 6). 4. Discussion 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. 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, anticonvulsant, 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 COX-2 and iNOS, the key enzymes for PGE2 and NO, was upregulated 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 PGE2 and NO production in LPS/IFN-γ-activated RAW 264.7 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

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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 anticarcinogenic activity (Rigas and Kashfi, 2005; Tiano et al., 2002). 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 chemopreventive action through mechanisms other than cell proliferation- and apoptosis-dependent ones. We used RAW cells, which is originated from mouse macrophage (Raschke et al., 1978), to study the modulating effect of isatins. Reports demonstrated that expression of COX-2 and iNOS is localized preferentially in the interstitial cells, including macrophages, of various types of tumorous tissues (Bamba et al., 1999; Klimp et al., 2001). It is suggested that stromal–epithelial interactions possibly mediated by macrophages COX-2 might promote tumorigenesis of intestinal epithelial cells (Hull et al., 1999). Furthermore, there is also evidence indicating that NO, causes induction of vascular endothelial growth factor and generation of angiogenic activity (Leibovich et al., 1994). COX-2 may also be involved in the resistance of tumors to chemotherapeutic drugs (Nardone et al., 2004). Thus, the use of COX-2 and/or iNOS inhibitors might decrease resistance of tumors to chemotherapeutic drugs (Nardone et al., 2004; Rigas and Kashfi, 2005). Those results, together with the present result, suggest that isatins might reduce the expression and/or activity of COX-2 and iNOS in macrophages and inhibit tumor growth partly through modulation of stromal–epithelial interaction or angiogenesis, thus resulting in chemopreventive efficacies. 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 activities (Coussens and Werb, 2002). The levels of inflammatory cytokines were enhanced in lung, ovarian, and bladder cancer patients (Alexandralis et al., 2000). Thus, the inhibitory activity of isatins against TNF-α production indicated that isatins may be a promising candidate for the ameliorating inflammatory response as well as suppressing tumor progression. TNF-α regulates the expression of specific target genes involved in a variety of pathological conditions including inflammation and carcinogenesis. Various extra cellular signals such as mitogen, inflammatory cytokine, or oxidative stress stimulate the TNF-α pathway, leading to the up-regulation of the proinflammatory molecules. The reduction on iNOS and COX-2 levels obtained after incubation of LPS/IFN-γ-activated cells with isatins derivatives correlates directly with the inhibition on NO and PGE2 produced, explaining at least in part the mechanism of these substances. The differences obtained with isatins on iNOS, COX-2 and TNF-α inhibition may be due to differences on site of action of those substances at

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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. 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. References Aktan, F., 2004. iNOS-mediated nitric oxide production and its regulation. Life Sci. 75, 639–653. Alexandralis, M.G., Coulocheri, S.A., Bouros, D., Mandalaki, K., Karkavitsas, N., Eliopoulos, G.D., 2000. Evaluation of inflammatory cytokines in malignant and benign pleural effusions. Oncol. Rep. 7, 1327–1332. Ambs, S., Merriam, W.G., Bennet, W.P., Fedley-Bosco, E., Ogunfusika, M.O., Oser, S.M., Klein, S., Shields, P.G., Billiar, T.R., Harris, C.C., 1998. Frequent nitric oxide synthase-2 expression in human colon adenomas: Implication for tumor angiogenesis and colon cancer progression. Cancer Res. 58, 334–341. Arbabi, S., Rosengart, M.R., Garcia, I., Jelacie, S., Maier, R.V., 2001. Epithelial cyclooxygenase-2 expression: a model for pathogenesis of colon cancer. J. Surg. Res. 97, 60–64. Attiga, E.A., Fernandez, P.M., Weeraratna, A.T., Manyak, M.J., Patierno, S.R., Padwa, A., 2000. Inhibition of prostaglandins synthesis inhibits human prostate tumor cell invasiveness and reduces the release of matrix metalloproteinases. Cancer Res. 60, 4629–4637. Bamba, H., Ota, S., Kato, A., Adachi, A., Itoyama, S., Matsuzaki, F., 1999. High expression of cyclooxygenase-2 in macrophages of human colonic adenoma. Int. J. Cancer 83, 470–475. Cao, Y., Prescott, S.M., 2002. Many action of cyclooxygenase-2 in cellular dynamics and cancer. J. Cell. Physiol. 190, 279–286. Cao, Q.J., Einstein, M.H., Anderson, P.S., Ronowicz, C.D., Balan, R., Jones, J.B., 2002. Expression of COX-2, Ki-67, cyclinD1, and P21 in endometrial endometrioid carcinomas. Int. J. Gynecol. Pathol. 21, 147–154. Coussens, L.M., Werb, Z., 2002. Inflammation and cancer. Nature 420, 860–867. Denizot, F., Lang, R., 1986. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. 89, 271–277. Ernst, P.B., Takaishi, H., Crowe, S.E., 2001. Helicobacter pylori infection as a model for gastrointestinal immunity and chronic inflammatory diseases. Dig. Dis. 19, 104–111. Evans, J.F., Kargman, S.L., 2004. Cancer and cyclooxygenase-2 (COX-2) inhibition. Curr. Pharm. Des. 10, 627–634. Field, L., Dilts, R.V., Ravichandran, R., Lenhert, P.G., Carnahan, G.E., 1978. An unusually stable thionitrite from N-acetyl-D,L-penicillamine: X-ray crystal and molecular structure of 2-(acetylamino)-2-carboxy-1,1-dimethylethylthionitrite. J. Chem. Soc., Chem. Commun. 6, 249–250. Flick, D.A., Gifford, G.E., 1984. Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J. Immunol. Methods 68, 167–175. Garden, S.J., Torres, J.C., Ferreira, A.A., Silva, R.B., Pinto, A.C., 1997. A modified Sandmeyer methodology and the synthesis of (±)-convolutamydine A. Tetrahedron Lett. 38, 1501–1504. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wisnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126, 131–138. Hull, M.A., Booth, J.K., Tisbury, A., Scott, N., Bonifer, C., Markham, A.F., Coletta, P.L., 1999. Cyclooxygenase 2 is up-regulated and localized to macrophages in the intestine of mice. Br. J. Cancer 79, 1399–1405. Igosheva, N., Lorz, C., O'Conner, E., Glover, V., Mehmet, H., 2005. Isatin, an endogenous monoamine oxidase inhibitor, triggers a dose- and time-

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dependent switch from apoptosis to necrosis in human neuroblastoma cells. Neurochem. Int. 47, 216–224. Kang, J.H., Sung, M.K., Kawada, T., Yoo, H., Kim, Y.K., Kim, J.S., Yu, R., 2005. Soybean saponins suppress the release of proinflammatory mediators by LPS-stimulated peritoneal macrophages. Cancer Lett. 230, 219–227. Klimp, A.H., Hollema, H., Kempinga, C., van der Zee, A.Z., de Vries, E.G., Daeman, T., 2001. Expression of cyclooxygenase-2 and inducible nitric oxide synthase in human ovarian tumors and tumor-associated macrophages. Cancer Res. 61, 7305–7309. Ko, S.C., Chapple, K.S., Howcroft, G., Coletta, P.L., Markham, A.F., Hull, M.A., 2002. Paracrine cyclooxygenase-2 mediated signalling by macrophages promotes tumorigenenic progression of intestinal epithelial cells. Oncogene 21, 7175–7186. Leibovich, S.J., Polverini, P.J., Fong, T.W., Harlow, L.A., Koch, A.E., 1994. Production of angiogenic activity by human monocytes requires an Larginine/nitric oxide-synthase-dependent effector mechanism. Proc. Natl. Acad. Sci. U. S. A. 91, 4190–4194. Lin, W.W., Chen, B.C., Hsu, Y.W., Lee, C.M., Shyue, S.K., 1999. Modulation of inducible nitric oxide synthase induction by prostaglandin E2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages. Prostaglandins Other Lipid Mediat. 58, 87–101. Marvel, C.S., Hiers, G.S., 1941. Isatin. Org. Synth. Coll. I 327–330. Matheus, M.E., Fernandes, S.B.O., Silveira, C.S., Rodrigues, V.P., Menezes, F.S., Fernandes, P.D., 2006. Inhibitory effects of Euterpe oleracea Mart. on nitric oxide production and iNOS expression. J. Ethnopharmacol. 107, 291–296. Medvedev, A., Igosheva, N., Crumeyrolle-Arias, M., Glover, V., 2005. Isatin: role in stress and anxiety. Stress 8, 175–183. Nardone, G., Rocco, A., Vaira, D., Staibano, S., Budillon, A., Tatangelo, F., Sciulli, M.G., Perna, F., Salvatore, G., Di Denedetto, M., De Rosa, G., Patrignani, P., 2004. Expression of COX-2, mPGE-synthase. MDR-1 (P-gp), and Bcl-xL: a molecular pathway of H. pylori-related gastric carcinogenesis. J. Pathol. 202, 305–312. Patrignani, P., Tacconelli, S., Sciulli, M.G., Capone, M.L., 2005. New insights into COX-2 biology and inhibition. Brain Res. Rev. 48, 352–359.

Pouchert, C.J., Behnke, J., 1993. The Aldrich Library of 13C and 1H FT NMR Spectra, edition I. Aldrich Chemical Company, New York. Pradelles, P.J., Maclouf, J., 1985. Enzyme immunoassays of eicosanoids using acetylcholinesterase as label: an alternative to radioimmunoassay. Anal. Chem. 57, 1170–1173. Prinz, C., Schoniger, M., Rad, R., Becker, I., Keiditsch, E., Wagenpfeil, S., Classen, M., Rosch, T., Schepp, W., Gerhard, M., 2001. Key importance of the Helicobacter pylori adherence factor blood group antigen binding adhesion during chronic gastric inflammation. Cancer Res. 61, 1903–1909. Raschke, W.C., Baird, S., Ralph, P., Nakoinz, I., 1978. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 15, 261–267. Rigas, B., Kashfi, K., 2005. Cancer prevention: a new era beyond cyclooxygenase-2? J. Pharmacol. Exp. Ther. 314, 1–8. Sadler, P.W., 1956. Separation of isomeric isatins. J. Org. Chem. 21, 169–170. Sandmeyer, T., 1919. Isonitrosoacetanilides and their condensation to form isatin derivatives. Chem. Abstr. 13, 1840–1841. Shacter, E., Weitzman, S.A., 2002. Chronic inflammation and cancer. Oncology 16, 217–226. Shvekhgeimer, M.G., 1996. Synthesis of heterocyclic compounds by the cyclization of isatin and its derivatives (review). Chem. Heterocycl. Compd. 32, 249–276. Silva, J.F.M., Garden, S.J., Pinto, A.C., 2001. The chemistry of isatins: a review from 1975 to 1999. J. Braz. Chem. Soc. 12, 273–324. Simmons, D.L., Botting, R.M., Hla, T., 2004. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol. Rev. 56, 387–437. Tiano, H.F., Loftin, C.D., Akunda, J., Lee, C.A., Spalding, J., Sessoms, A., Dunson, D.B., Rogan, E.G., Morham, S.G., Smart, R.C., Langenbach, R., 2002. Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis. Cancer Res. 62, 3395–3401. Turini, M.E., DuBois, R.N., 2002. Cyclooxygenase-2: a therapeutic target. Annu. Rev. Med. 53, 35–57.