Different expression of cyclooxygenase-2 (COX-2) in selected nonmelanocytic human cutaneous lesions

The aim of our study was to elucidate the possible involvement of COX-2 in the development and/or progression of nonmelanocytic skin lesions. To evaluate the usefulness of that enzyme as a potential molecular marker, we examined the intensity and spatial distribution of COX-2 expression in selected types of such tumors using the same immunohistochemical procedure as in our earlier studies of melanocytic cancers. We examined 20 benign epithelial lesions, 11 precancerous lesions, 21 basal cell carcinomas (BCC), 14 squamous cell carcinomas (SCC) and eight fibromas. The levels of COX-2 expression detected in benign lesions and in normal skin were comparable. Elevated expression of this protein may play a role in the development of SCC, as indicated by strong immunostaining both in SCCs and precancerous lesions. Significantly stronger staining in SCCs compared to BCCs may indicate a role of COX-2 in cancer malignancy and serve as an indicator useful for differential diagnostics of the two types of cancer. Strong staining in all skin layers of SCC may help in detecting cancer cells infiltrating surrounding skin layers.     

The induction of cyclooxygenase-2 (COX-2) in intact human amnion tissue by interleukin-4

Infection is a major cause of preterm labor. Amniotic fluid from women in preterm labor associated with intrauterine infection contains increased concentrations of cytokines. The mechanism underlying this association may be a cytokine-mediated stimulation of amnion cell prostaglandin production. The biosynthesis of prostaglandins from arachidonic acid is regulated by the enzyme cyclooxygenase which exists in two forms; the constitutive form (COX-1) and the other mitogen inducible (COX-2). The purpose of this study was to evaluate the effect of the cytokine interleukin-4 (IL-4) on cyclooxygenase activity and PGE2 production in amnion. Amnion tissue was taken at caesarean section from term women not in labor and immediately incubated for 2 hours in media containing concentrations of IL-4 ranging from 1 to 100 ng/ml. An increase in both COX-2 enzyme and prostaglandin E₂ (PGE₂) production was observed for all concentrations of IL-4 greater than 25 ng/ml (P < 0.05, n = 8). No change in COX-1 was observed. Our data suggest that the cytokine IL-4 may be involved in the pathogenesis of premature labor by inducing COX-2 in amnion tissue resulting in increased production of PGE₂ and subsequent myometrial activity.     

Synthesis and evaluation in vitro and in vivo of a 11C-labeled cyclooxygenase-2 (COX-2) inhibitor

The radiosynthesis and radiopharmacological evaluation of 1-[(11)C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-1-enyl)-benzene [(11)C]5 as novel PET radiotracer for imaging of COX-2 expression is described. The radiotracer was prepared via O-methylation reaction with [(11)C]methyl iodide in 19% decay-corrected radiochemical yield at a specific activity of 20-25GBq/mumol at the end-of-synthesis within 35 min. The radiotracer [(11)C]5 was evaluated in vitro using various pro-inflammatory and tumor cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribution of compound [(11)C]5 was characterized in male Wistar rats. Compound [(11)C]5 was rapidly metabolized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioactivity uptake in the tumor was not COX-2 mediated.     

Synthesis and selective cyclooxygenase-2 (COX-2) inhibitory activity of a series of novel bicyclic pyrazoles

Novel series of pyrazolo[5,1-b]1,3-oxazolidines, pyrazolo[5,1-b]1,3-oxazines and imidazolidino[1,2-d]pyrazoles were synthesized. These compounds were evaluated in vitro for their ability to inhibit cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in human whole blood (HWB). Several of the compounds were found to be novel and selective COX-2 inhibitors, the most potent and selective being 1-(5-cyclohexyl (2H,3H-pyrazolo[5,1-b]-1,3-oxazolidin-6-yl)-4-(methylsulfonyl)benzene, 7a (IC(5o) for COX-1>100 microM; for COX-2=1.3 microM).     

Serotonin-stimulated phospholipase A2 and collagenase activation in chondrocytes from human osteoarthritic articular cartilage.

We have previously described several receptors on the chondrocyte membrane. In an attempt to further characterize the coupling mechanisms of serotoninergic receptors, here we examined the involvement of serotonin in the phospholipase A2 activity. Serotonin dose-dependently stimulated phospholipase A2. This activation enhanced collagenase type II activity and had no effect on proteoglycanase activity.     

Novel 5-substituted 1H-tetrazoles as cyclooxygenase-2 (COX-2) inhibitors

A series of novel 5-substituted 1H-tetrazoles as cyclooxygenase-2 (COX-2) inhibitors was prepared via treatment of various diaryl amides with tetrachlorosilane/sodium azide. All compounds were tested in cyclooxygenase (COX) assays in vitro to determine COX-1 and COX-2 inhibitory potency and selectivity. Tetrazoles contained a methylsulfonyl or sulfonamide group as COX-2 pharmacophore displayed only low inhibitory potency towards COX-2. Most potent compounds showed IC(50) values of 6 and 7 μM for COX-2. All compounds showed IC(50) values greater 100 μM for COX-1 inhibition.     

Peroxidase activity of cyclooxygenase-2 (COX-2) cross-links beta-amyloid (Abeta) and generates Abeta-COX-2 hetero-oligomers that are increased in Alzheimer's disease

Oxidative stress is associated with the neuropathology of Alzheimer's disease. We have previously shown that human Abeta has the ability to reduce Fe(III) and Cu(II) and produce hydrogen peroxide coupled with these metals, which is correlated with toxicity against primary neuronal cells. Cyclooxygenase (COX)-2 expression is linked to the progression and severity of pathology in AD. COX is a heme-containing enzyme that produces prostaglandins, and the enzyme also possesses peroxidase activity. Here we investigated the possibility of direct interaction between human Abeta and COX-2 being mediated by the peroxidase activity. Human Abeta formed dimers when it was reacted with COX-2 and hydrogen peroxide. Moreover, the peptide formed a cross-linked complex directly with COX-2. Such cross-linking was not observed with rat Abeta, and the sole tyrosine residue specific for human Abeta might therefore be the site of cross-linking. Similar complexes of Abeta and COX-2 were detected in post-mortem brain samples in greater amounts in AD tissue than in age-matched controls. COX-2-mediated cross-linking may inhibit Abeta catabolism and possibly generate toxic intracellular forms of oligomeric Abeta.     

Recognition of cyclooxygenase-2 (COX-2) active site by NSAIDs: a computer modelling study

The energetics and models of COX-2 complexed with nonsteroidal anti-inflammatory drugs (NSAIDs) having different degrees of selectivity for two isoforms of COX (COX-2 and COX-1) have been studied using computer modelling approach. The models are obtained for complexes of NS398 (NS), a selective COX-2 inhibitor; indoprofen (Ind), a non-selective inhibitor; di-tert-butylbenzofurans (DHDMBFs) with substituents at the 5th position: CONH(CH2)2OMe (BF1), CONH-c-Pr (BF2), 3-methylene-gamma-butyrolactonyl (BF3) and oxicams namely, meloxicam (Mel), piroxicam (Pir) and tenoxicam (Ten). These were optimized using molecular mechanics (MM) and molecular dynamics (MD) techniques. The binding energies and structures were compared with pharmacological parameters and available results with COX-1. In case of NS a larger difference in the binding energies between COX-2 and COX-1 was noticed as compared to that of Ind. It also had stronger interaction with His90 and Tyr355 which is considered important for COX-2 selectivity. There was a difference in the compactness at the channel entrance between COX-2 selective and non-selective ligands. Models with DHDMBFs and oxicams showed a similar correlation. The results were used to design a peptide inhibitor, Tyr-Arg-Cys-Ala-delta Phe-Cys (Pept) which could fit better in the COX-2 cavity. As per our MD simulation results this peptide inhibitor showed both higher activity and COX-2 selectivity.     

Terphenyl cyclooxygenase-2 (COX-2) inhibitors: optimization of the central ring and o-biphenyl analogs

The discovery of terphenyl derivatives as highly selective COX-2 inhibitors resulted from our efforts to overcome poor pharmacokinetics demonstrated by the COX-2 selective diarylthiophene DuP 697 [2-bromo-4-(4'-sulfonylmethyl)phenyl-5-(4'-fluoro)phenylthiophe ne]. Detailed SAR related to the ortho-biphenyls and variants of the central ring are described herein.     

原发性翼状胬肉中COX-2与iNOS的表达及相关研究

目的:探讨环氧合酶2(COX-2)与诱导型一氧化氮合酶(iNOS)在原发性翼状胬肉中的表达及其在发生发展过程中的作用.方法:原发性翼状胬肉组织与对照组的正常结膜组织标本均取自石河子大学第一附属医院眼科行手术治疗的患者,采用免疫组织化学Elivision法分别检测56例原发性翼状胬内、20例正常结膜中COX-2、iNOS的表达;脱氧核苷酸末端转移酶介导的脱氧尿苷三磷酸末端标记法(terminal deoxynucleotidyl transferase mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling,TUNEL),检测不同时期原发性翼状胬肉细胞中凋亡细胞的表达.结果:56例原发性翼状胬肉中COX-2的阳性表达率,静止期为50%,进行期为87.5%,正常结膜为0(0/20).iNOS的阳性表达率,静止期为50%,进行期为92.5%,正常结膜50%.COX-2、iNOS阳性表达在原发性翼状胬肉与正常结膜两组间差异有显著意义,P＜0.001.正常结膜对照组无凋亡细胞表达,静止期和进行期胬肉组织中均出现凋亡细胞,凋亡细胞的表达在静止期和进行期2组间的表达有明显差异(P＜0.05)).结论:COX-2、iNOS在翼状胬肉中的表达,提示COX-2、iNOS可促进新生血管形成,可能与翼状胬肉的发生、发展以及术后复发有关,细胞凋亡在翼状胬肉的发生中起重要作用,COX-2、iNOS抑制剂以及细胞凋亡的调控可望成为降低翼状胬肉复发率新的依据.     

The expression of cyclooxygenase-2 (COX-2) in amnion and decidua following spontaneous labor

Objective: Prostaglandins production rises dramatically during term and preterm labor. The source of this production is thought to be the fetal membranes and maternal decidua. The enzyme responsible for the conversion of arachidonic acid to the prostaglandins and related endoperoxides is variously known as prostaglandin synthase or cyclooxygenase (COX). An inducible form of this enzyme, COX-2, has been described in several tissues. The purpose of this study was to investigate a possible role for COX-2 in labor by comparing the COX-2 content in amnion and decidua from laboring and non-laboring patients. Study Design: Fetal membranes from seven normal labor and ten elective cesarean sections at term were collected immediately following delivery. The maternal age and gravity were similar between the groups. The amnion and decidua were identified, washed in sterile saline, frozen in liquid nitrogen and stored in −70°C. COX-2 expression was determined using Western Blot analysis with a purified COX-2 antibody. A scanning densitometer was used to quantify the bands. Results were expressed as mean ±S.D. ng/l50μg protein. Results: The concentration of COX-2 in amnion of laboring women showed a twofold increase ( 240.0 ± 17.6 vs. 120.7 ± 5.1) compared to the non-labored group (p<0.05). The concentration in the decidua showed no significant increase during labor (38.1 ± 7.5 vs. 26.4 ± 2.1, p > 0.05).Conclusion: We evaluated the role of COX-2 in normal labor. Our study demonstrate that COX-2 is significantly induced in the amnion following spontaneous labor. These findings suggest that the induction of amnion COX-2 may be involved in the process of human labor.     

Evaluation of glycolamide esters of indomethacin as potential cyclooxygenase-2 (COX-2) inhibitors

A number of novel indomethacin glycolamide esters were synthesized and tested for their cyclooxygenase (COX-1 and COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle structural changes in the substituents on the glycolamide ester moiety altered the inhibitory properties as well as potencies significantly. Their in vitro data were rationalized through molecular modeling studies. Few of them displayed anti-inflammatory activity in vivo. Compound 32, [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester, was identified as a promising compound in this class and its good anti-inflammatory activity was demonstrated in the in vivo model.     

Design and synthesis of (E)-1,1,2-triarylethenes: novel inhibitors of the cyclooxygenase-2 (COX-2) isozyme

A novel class of acyclic 1,1,2-triaryl (E)-ethenes was designed that were synthesized via an (E)-selective Takeda olefination reaction. Among the group of compounds evaluated, (E)-2-(4-fluorophenyl)-1-(4-methylsulfonylphenyl)-1-phenylethene (10c) emerged as the most potent (COX-2 IC(50)=0.0316 microM), and selective (selectivity index>3164), COX-2 inhibitor.     

Design and synthesis of acyclic triaryl (Z)-olefins: a novel class of cyclooxygenase-2 (COX-2) inhibitors

A group of acyclic 2-alkyl-1,1-diphenyl-2-(4-methylsulfonylphenyl)ethenes was designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 isozyme inhibition structure-activity studies identified 1,1-diphenyl-2-(4-methylsulfonylphenyl)hex-1-ene as a highly potent (IC(50) = 0.014 microM), and an extremely selective [COX-2 selectivity index (SI) > 7142], COX-2 inhibitor that showed superior anti-inflammatory (AI) activity (ID(50) = 2.5 mg/kg) relative to celecoxib (ID(50) = 10.8 mg/kg). This initial study was extended to include the design of a structurally related group of acyclic triaryl (Z)-olefins possessing an acetoxy (OAc) substituent at the para-position of the C-1 phenyl ring that is cis to a C-2 4-methylsulfonylphenyl substituent. COX-1 and COX-2 inhibition studies showed that (Z)-1-(4-acetoxyphenyl)-1-phenyl-2-(4-methylsulfonylphenyl)but-1-ene [(Z)-13b] is a potent (COX-1 IC(50) = 2.4 microM; COX-2 IC(50) = 0.03 microM), and selective (COX-2 SI = 81), COX-2 inhibitor which is a potent AI agent (ID(50) = 4.1mg/kg) with equipotent analgesic activity to celecoxib. A molecular modeling (docking) study showed that the SO(2)Me substituent of (Z)-13b inserts deep inside the 2 degrees -pocket of the COX-2 active site, where one of the O-atoms of SO(2) group undergoes a H-bonding interaction with Phe(518). The p-OAc substituent on the C-1 phenyl ring is oriented in a hydrophobic pocket comprised of Met(522), Gly(526), Trp(387), Tyr(348), and Tyr(385), and the C-2 ethyl substituent is oriented towards the mouth of the COX-2 channel in the vicinity of amino acid residues Arg(120), Leu(531), and Val(349). Structure-activity data acquired indicate that a (Z)-olefin having cis C-1 4-acetoxyphenyl (phenyl) and C-2 4-methylsulfonylphenyl substituents, and a C-1 phenyl substituent in conjunction with either a C-2 hydrogen or short alkyl substituent provides a novel template to design acyclic olefinic COX-2 inhibitors that, like aspirin, have the potential to acetylate COX-2.     

PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE(2) production. PGE(2) production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE(2) increased VEGF synthesis via the EP(2) receptor. Whereas PGE(2) stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP(2) receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE(2) generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.     

3-(2-Methoxytetrahydrofuran-2-yl)pyrazoles: a novel class of potent, selective cyclooxygenase-2 (COX-2) inhibitors

A series of 3-(2-methoxytetrahydrofuran-2-yl)pyrazoles (4–10) was synthesized. The compounds were evaluated for their ability to inhibit cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) activity in human whole blood (HWB). The compound, 5-(4-methanesulfonylphenyl)-3-(2-methoxytetrahydrofuran-2-yl)-1-p-tolyl-1H-pyrazole 5 showed potent and selective COX-2 inhibition (IC₅₀ for COX-1: >100 μM and COX-2: 1.2 μM).