Potential misidentification of cyclooxygenase-2 by western blot analysis and prevention through the inclusion of appropriate controls

Cyclooxygenase (COX)-2 plays an important role in the development of cancer and has been recognized as a potential therapeutic target. Because nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the activity of this enzyme, the potential efficacy of such drugs for purposes of cancer prevention or therapy is an area of intense research. Therefore, it is of critical importance to unequivocally determine the expression levels of COX-2 protein in tumor cells. In this regard, there are several conflicting reports in the literature where the same type of tumor cell lines were reported as COX-2 positive and as COX-2 negative. We found that during Western blot analysis of COX-2 positive and COX-2 negative cells, different antibodies to COX-2 protein are able to generate strong signals, which are false-positives and can be confused with COX-2. Thus, we believe that some of the conflicting reports on COX-2 expression in tumor cell lines could be the result of improper interpretation of the Western blot signals. Here, we present some of these pitfalls and suggest the inclusion of appropriate controls to unequivocally identify COX-2 protein levels.     

Design,synthesis and structure–activity relationship studies of novel and diverse cyclooxygenase-2 inhibitors as anti-inflammatory drugs

Abstract

Because of the pivotal role of cyclooxygenase (COX) in the inflammatory processes, non-steroidal anti-inflammatory drugs (NSAIDs) that suppress COX activities have been used clinically for the treatment of inflammatory diseases/syndromes; however, traditional NSAIDs exhibit serious side-effects such as gastrointestinal damage and hyper sensitivity owing to their COX-1 inhibition. Also, COX-2 inhibition-derived suppressive or preventive effects against initiation/proliferation/invasion/motility/recurrence/metastasis of various cancers/tumours such as colon, gastric, skin, lung, liver, pancreas, breast, prostate, cervical and ovarian cancers are significant. In this study, design, synthesis and structure–activity relationship (SAR) of various novel {2-[(2-, 3- and/or 4-substituted)-benzoyl, (bicyclic heterocycloalkanophenyl)carbonyl or cycloalkanecarbonyl]-(5- or 6-substituted)-1H-indol-3-yl}acetic acid analogues were investigated to seek and identify various chemotypes of potent and selective COX-2 inhibitors for the treatment of inflammatory diseases, resulting in the discovery of orally potent agents in the peripheral-inflammation model rats. The SARs and physicochemical properties for the analogues are described as significant findings. For graphical abstract: see Supplementary Material. (www.informahealthcare.com/enz)     

Design,Synthesis and Biological Evaluation of New N-Acyl Hydrazones with a Methyl Sulfonyl Moiety as Selective COX-2 Inhibitors

The mechanism of action of nonsteroidal anti-inflammatory drugs (NSAIDs) is inhibition of specific prostaglandin (PG) synthesis by inhibition of cyclooxygenase (COX) enzymes. The two COX isoenzymes show 60 % similarity. It is known that the nonspecific side effects of conventional NSAIDs are physiologically caused by inhibition of the COX-1 enzyme. Therefore, the use of COX-2 selective inhibitors is seen to be a more beneficial approach in reducing these negative effects. However, some of the existing COX-2 selective inhibitors show cardiovascular side effects. Therefore, studies on the development of new selective COX-2 inhibitors remain necessary. It is important to develop new COX-2 inhibitors in the field of medicinal chemistry. Accordingly, novel N-acyl hydrazone derivatives were synthesized as new COX-2 inhibitors in this study. The hydrazone structure, also known for its COX activity, is important in terms of many biological activities and was preferred as the main structure in the design of these compounds. A methyl sulfonyl pharmacophore was added to the structure in order to increase the affinity for the polar side pocket present in the COX-2 enzyme. It is known that methyl sulfonyl groups are suitable for polar side pockets. The synthesis of the compounds ( 3a – 3j ) was characterized by spectroscopic methods. Evaluation of in vitro COX-1/COX-2 enzyme inhibition was performed by fluorometric method. According to the enzyme inhibition results, the obtained compounds displayed the predicted selectivity for COX-2 enzyme inhibition. Compound 3j showed important COX-2 inhibition with a value of IC₅₀=0.143 uM. Interaction modes between the COX-2 enzyme and compound 3j were investigated by docking studies.     

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).     

Celecoxib: considerations regarding its potential disease-modifying properties in osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive loss of articular cartilage, subchondral bone sclerosis, osteophyte formation, and synovial inflammation, causing substantial physical disability, impaired quality of life, and significant health care utilization. Traditionally, non-steroidal anti-inflammatory drugs (NSAIDs), including selective cyclooxygenase (COX)-2 inhibitors, have been used to treat pain and inflammation in OA. Besides its anti-inflammatory properties, evidence is accumulating that celecoxib, one of the selective COX-2 inhibitors, has additional disease-modifying effects. Celecoxib was shown to affect all structures involved in OA pathogenesis: cartilage, bone, and synovium. As well as COX-2 inhibition, evidence indicates that celecoxib also modulates COX-2-independent signal transduction pathways. These findings raise the question of whether celecoxib, and potentially other coxibs, is more than just an anti-inflammatory and analgesic drug. Can celecoxib be considered a disease-modifying osteoarthritic drug? In this review, these direct effects of celecoxib on cartilage, bone, and synoviocytes in OA treatment are discussed.     

New phenolic cinnamic acid derivatives as selective COX-2 inhibitors. Design,synthesis, biological activity and structure-activity relationships

Selective inhibition of cyclooxygenase (COX)-2 enzyme is an important achievement when looking for potent anti-inflammatory agents, with fewer gastrointestinal side effects. In this work, a new series of cinnamic acid derivatives, namely hexylamides, have been designed, synthesized and evaluated in human blood for their inhibitory activity of COX-1 and COX-2 enzymes. From this, new structure-activity relationships were built, showing that phenolic hydroxyl groups are essential for both COX-1 and COX-2 inhibition. Furthermore, the presence of bulky hydrophobic di-tert-butyl groups in the phenyl ring strongly contributes for selective COX-2 inhibition. In addition, a correlation with the theoretical log P has been carried out, showing that lipophilicity is particularly important for COX-2 inhibition. Further, a plasma protein binding (PPB) prediction has been performed revealing that PPB seems to have no influence in the activity of the studied compounds. From the whole study, effective selective inhibitors of COX-2 were found, namely compound 9 (IC₅₀ = 3.0 ± 0.3 μM), 10 (IC₅₀ = 2.4 ± 0.6 μM) and 23 (IC₅₀ = 1.09 ± 0.09 μM). Those can be considered starting point hit compounds for further optimization as potential non-steroidal anti-inflammatory drugs.     

Changes in gene expression contribute to cancer prevention by COX inhibitors

Non-steroidal anti-inflammatory drugs (NSAIDs) are used primarily for the treatment of inflammatory diseases. However, certain NSAIDs also have a chemopreventive effect on the development of human colorectal and other cancers. NSAIDs inhibit cyclooxygenase-1 (COX-1) and/or cyclooxygenase-2 (COX-2) activity and considerable evidence supports a role for prostaglandins in cancer development. However, the chemopreventive effect of NSAIDs on colorectal and other cancers appears also to be partially independent of COX activity. COX inhibitors also alter the expression of a number of genes that influence cancer development. One such gene is NAG-1 (NSAID-Activated Gene), a critical gene regulated by a number of COX inhibitors and chemopreventive chemicals. Therefore, this article will discuss the evidence supporting the conclusion that the chemo-preventive activity of COX inhibitors is mediated, in part, by altered gene expression with an emphasis on NAG-1 studies. This review may also provide new insights into how chemicals and environmental factors influence cancer development. In view of the cardiovascular and gastrointestinal toxic side effects of COX-2 inhibitors and non-selective COX inhibitors, respectively, the results presented here may provide the basis for the development of a new family of anti-tumorigenic compounds acting independent of COX inhibition.     

Cyclooxygenase-3 Gene Expression in Alzheimer Hippocampus and in Stressed Human Neural Cells

The cyclooxygenase (COX) superfamily of prostaglandin synthase genes encode a constitutively expressed COX-1, an inducible, highly regulated COX-2, and a COX-3 isoform whose RNA is derived through the retention of a highly structured, G + C-rich intron 1 of the COX-1 gene. As generators of oxygen radicals, lipid mediators, and the pharmacological targets of nonsteroidal anti-inflammatory drugs (NSAIDs), COX enzymes potentiate inflammatory neuropathology in Alzheimer's disease (AD) brain. Because COX-2 is elevated in AD and COX-3 is enriched in the mammalian CNS, these studies were undertaken to examine the expression of COX-3 in AD and in [IL-1beta + Abeta42]-triggered human neural (HN) cells in primary culture. The results indicate that while COX-2 remains a major player in propagating inflammmation in AD and in stressed HN cells, COX-3 may play ancillary roles in membrane-based COX signaling or when basal levels of COX-1 or COX-2 expression persist.     

Coumarins scaffolds as COX inhibitors

The discovery of COX enzymes has led to a better understanding of inflammation and its related biological pathways. Apart from being related to inflammation and pain, it has also been associated with cancer and neuropsychiatric diseases such as schizophrenia. Proverbially speaking, study of these enzymes has been crucial as they happen to “have fingers in many pies”. Non-steroidal anti-inflammatory drugs (NSAID) that act specifically as COX-2 inhibitors have been known for a while; however these are also associated with severe side effects such as cardiac problems. Several heterocylic molecules have been tested for their anti-inflammatory activity specifically as COX-inhibitors. Coumarins also known as benzopyrans are widely found in nature, and are routinely employed as herbal remedies since early days. Over 1300 coumarins have been identified, principally as secondary metabolites in green plants, fungi and bacteria. Recently the use of natural and synthetic coumarins has garnered a lot of attention for their anti-inflammatory activities. In this review we delve further into the study of natural and synthetic coumarins as COX-inhibitors. Although the study is still in its nascent stage, we believe there is scope for a lot of development.     

During a systemic inflammatory response, the effect of non-steroidal anti-inflammatory drugs on seizure susceptibility in the immature brain may depend on the proconvulsant and anticonvulsant mechanisms simultaneously induced by the elevation of parenchymal prostaglandin E2 levels

Clinical evidence from paediatric neurology supports the possibility that a protracted inflammatory state in the central nervous system (CNS) may enhance the predisposition of brain tissue to develop seizures. Consequently, non-steroidal anti-inflammatory drugs (NSAIDs) as well as selective cyclooxygenase-2 (COX-2) inhibitors were expected to positively modulate seizure susceptibility during a systemic inflammatory response. Nevertheless, experimental findings and clinical evidence provide controversial results. As a possible explanation for these apparent discrepancies, it is hypothesised that the amount of prostaglandin E₂ (PGE₂) induced in the immature brain parenchyma during systemic inflammatory response is crucial since PGE₂ plays a dual role. Indeed, on the one hand, this prostaglandin increases seizure susceptibility by stimulation of glutamate release from neurons and astrocytes. On the other hand, however, the same prostaglandin induces a massive release of corticosterone, being this hormone known to inhibit efficiently the seizure susceptibility of the immature brain. Hence, the dose-response curve of any given NSAID/COX-2 inhibitor on seizure susceptibility is expected to show different patterns, depending on the amount of PGE₂ levels produced in the brain parenchyma during the effect of drug. The proposed hypothesis also suggests that mild to moderate increase of PGE₂ levels in the immature brain parenchyma may act as a ‘preconditioning’ stimulus, i.e., it may confer a transient resistance to develop seizure-induced brain injury, besides to efficiently counteract seizure susceptibility.     

Computer-aided, rational design of a potent and selective small peptide inhibitor of cyclooxygenase 2 (COX2)

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors. Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX1 and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

Identification of small molecule inhibitors of neurite loss induced by Aβ peptide using high content screening

Multiple lines of evidence indicate a strong relationship between Αβ peptide-induced neurite degeneration and the progressive loss of cognitive functions in Alzheimer disease (AD) patients and in AD animal models. This prompted us to develop a high content screening assay (HCS) and Neurite Image Quantitator (NeuriteIQ) software to quantify the loss of neuronal projections induced by Aβ peptide neurons and enable us to identify new classes of neurite-protective small molecules, which may represent new leads for AD drug discovery. We identified thirty-six inhibitors of Aβ-induced neurite loss in the 1,040-compound National Institute of Neurological Disorders and Stroke (NINDS) custom collection of known bioactives and FDA approved drugs. Activity clustering showed that non-steroidal anti-inflammatory drugs (NSAIDs) were significantly enriched among the hits. Notably, NSAIDs have previously attracted significant attention as potential drugs for AD; however their mechanism of action remains controversial. Our data revealed that cyclooxygenase-2 (COX-2) expression was increased following Aβ treatment. Furthermore, multiple distinct classes of COX inhibitors efficiently blocked neurite loss in primary neurons, suggesting that increased COX activity contributes to Aβ peptide-induced neurite loss. Finally, we discovered that the detrimental effect of COX activity on neurite integrity may be mediated through the inhibition of peroxisome proliferator-activated receptor γ (PPARγ) activity. Overall, our work establishes the feasibility of identifying small molecule inhibitors of Aβ-induced neurite loss using the NeuriteIQ pipeline and provides novel insights into the mechanisms of neuroprotection by NSAIDs.     

Cyclooxygenase-2 modulates cellular growth and promotes tumorigenesis

Cyclooxygenase (COX)-2 and the prostaglandins resulting from its enzymatic activity have been shown to play a role in modulating cell growth and development of human neoplasia. Evidence includes a direct relationship between COX-2 expression and cancer incidence in humans and animal models, increased tumorigenesis after genetic manipulation of COX-2, and significant anti-tumor properties of non-steroidal anti-inflammatory drugs in animal models and in some human cancers. Recent data showed that COX-2 and the derived prostaglandins are involved in control of cellular growth, apoptosis, and signal through a group of nuclear receptors named peroxisome proliferator-activated receptors (PPARs). In this article we will review some of the findings suggesting that COX-2 is involved in multiple cellular mechanisms that lead to tumorigenesis.     

Cyclooxygenase-2: a therapeutic target in angiogenesis

Angiogenesis has a role in the pathogenesis of several disorders, including cancer, chronic inflammatory diseases and retinopathies. Recent evidence demonstrates that the production of prostanoids by cyclooxygenase-2 (COX-2) promotes the expression of pro-angiogenic factors. Furthermore, inhibition of COX-2 by non-steroidal anti-inflammatory drugs leads to restricted angiogenesis and downregulated production of pro-angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor. These findings suggest that COX enzymes could be important therapeutic targets in the treatment of pathological angiogenesis.     

Computer-Aided,Rational Design of a Potent and Selective Small Peptide Inhibitor of Cyclooxygenase 2 (COX2)

Abstract

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors.

Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX' and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

Short-term administration of non-selective and selective cox-2 nsaids do not interfere with bone repair in rats

Selective cyclooxygenase-2 non-steroidal anti-inflammatory drugs are known to inhibit bone repair, especially when long-term administration is required due to chronicle inflammatory diseases. In order to evaluate the action of this drug in bone repair during short-term administration, 48 rats underwent surgical bone defects in their tibias, being randomly distributed into three groups: (Group 1) negative control; (Group 2) animals treated with celecoxib, and (Group 3) animals treated with ketoprofen, both experimental groups at 1 mg/kg dose, beginning 1 h before the surgical procedure and after every 12 h for the following 3 days, or until the day of sacrifice. The animals were killed after 48 h, 7, 14, and 21 days. The tibias were removed for morphological, morphometric, and immunohistochemistry analysis for COX-2. No statistical significant differences were observed in the quality of bone repair and quantity of formed bone among the groups. COX-2 immunoreactivity of the celecoxib treated specimens was more intense in the first analyzed period, and no longer observed in the periods of 14 and 21 days. Such results suggest that the administration of the analyzed drugs in short periods does not interfere with the process of bone repair in the tibia of rats.     

Regulation of inflammation in cancer by eicosanoids

Inflammation in the tumor microenvironment is now recognized as one of the hallmarks of cancer. Endogenously produced lipid autacoids, locally acting small molecule lipid mediators, play a central role in inflammation and tissue homeostasis, and have recently been implicated in cancer. A well-studied group of autacoid mediators that are the products of arachidonic acid metabolism include: the prostaglandins, leukotrienes, lipoxins and cytochrome P450 (CYP) derived bioactive products. These lipid mediators are collectively referred to as eicosanoids and are generated by distinct enzymatic systems initiated by cyclooxygenases (COX 1 and 2), lipoxygenases (5-LOX, 12-LOX, 15-LOXa, 15-LOXb), and cytochrome P450s, respectively. These pathways are the target of approved drugs for the treatment of inflammation, pain, asthma, allergies, and cardiovascular disorders. Beyond their potent anti-inflammatory and anti-cancer effects, non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 specific inhibitors have been evaluated in both preclinical tumor models and clinical trials. Eicosanoid biosynthesis and actions can also be directly influenced by nutrients in the diet, as evidenced by the emerging role of omega-3 fatty acids in cancer prevention and treatment. Most research dedicated to using eicosanoids to inhibit tumor-associated inflammation has focused on the COX and LOX pathways. Novel experimental approaches that demonstrate the anti-tumor effects of inhibiting cancer-associated inflammation currently include: eicosanoid receptor antagonism, overexpression of eicosanoid metabolizing enzymes, and the use of endogenous anti-inflammatory lipid mediators. Here we review the actions of eicosanoids on inflammation in the context of tumorigenesis. Eicosanoids may represent a missing link between inflammation and cancer and thus could serve as therapeutic target(s) for inhibiting tumor growth.