Recently, several synthetic biologic products have been de- veloped. Despite the biochemical differences of these prod- ucts, they act as a barrier between peritoneal surfaces and in- tra-abdominal organs. In addition, pharmacologic agents that target fibrin, fibroblasts and angiogensis have been ap- plied and assessed. These agents reduce the peritoneal in- flammatory response following the surgical trauma, thereby reducing the fibrous reaction and mature stages of adhesion formation. 4 The cyclo-oxygenase-2 inhibitor (COX-2) in-
Various mechanisms may be responsible for the observed effects of NSAIDs against breast cancer. Inhibition of cyclo- oxygenase (COX), particularly the COX-2 isozyme, and block- ade of the prostaglandin (PG) cascade may have impacts on neoplastic growth and development by inhibiting several key features of mammary carcinogenesis – namely proliferation, angiogenesis and metastasis. Inhibition of COX also causes induction of apoptosis in malignant cells and enhances antin- eoplastic activity of cytotoxic T lymphocytes [5-8]. Our study conducted in newly diagnosed stage I and stage II breast can- cer patients  showed impaired functionality of T cells and dendritic cells, which correlated with COX-2 overexpression in the tumors and increased levels of PGE 2 in the serum and tumor milieu. Therefore, a convincing case has been made for COX-2 being an important target for the antineoplastic action of NSAIDs. Unlike NSAIDs, COX-2 selective inhibitors such as celecoxib and rofecoxib do not inhibit COX-1 and thus show promise as drugs that spare the gastrointestinal system. COX-2 is overexpressed in breast cancer tissues, and greater extent of its expression is associated with poorer prognosis . Various environmental and nutritional risk factors induce COX-2 expression in animal models of breast cancer [11,12]. Moreover, COX-2 selective inhibitors significantly delayed the incidence of mammary tumors in transgenic mice expressing the Her2/Neu, and polyoma-middle T oncogenes [13,14]. Recently, a transgenic mouse model was developed in which the human COX-2 gene was expressed in the mammary gland under the control of the murine mammary tumor virus promoter . That study demonstrated that enhanced COX-2 expres- sion strongly predisposes to transformation of the mammary gland in multiparous animals. These data strongly suggest that local expression of COX-2 is sufficient for in situ tumor initia- tion and/or progression. Another transgenic overexpression study with COX-2 targeted to the epidermis also supports the concept that COX-2 is a critical regulator of tumor progression . Transfections of the breast cancer cell line Hs578T with cDNA for COX-2 led to an increase in expression and activity of matrix metalloproteinase-2, resulting in increasingly invasive behavior of the cells . COX-2 specific inhibitors have the ability to block cell growth, and induce apoptosis and cell cycle arrest in murine mammary tumor cell lines . However, the molecular mechanisms involved are not well understood. If COX-2 inhibitors act only by modulating COX-2 expression, then that would imply that this therapy would be limited to COX-2 overexpressing tumors; hence, this question is of con- siderable clinical importance.
Previous studies have demonstrated the interaction between the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1 both in vitro and in vivo (C. Subramanian, M. A. Cotter II, and E. S. Robertson, Nat. Med. 7:350–355, 2001). EBNA3C can reverse the ability of Nm23-H1 to suppress migration of Burkitt’s lymphoma and breast carcinoma cell lines in vitro. EBNA3C contributes to EBV- associated human cancers by regulating transcription of a number of cellular and viral promoters and by targeting and altering the transcription activities of the metastasis suppressor Nm23-H1. Cyclo-oxygenase-2 (COX-2), an inducible enzyme important in inflammation, is overexpressed in a variety of cancers and can influence cell migration. In this report we show that Nm23-H1 and EBNA3C can modulate expression of COX-2 in the context of EBV infection and transformation. The levels of COX-2 were consistently higher in EBV- positive cells than in EBV-negative cells. Additionally, we show that Nm23-H1 can upregulate the COX-2 promoter element in luciferase reporter assays, whereas EBNA3C alone did not affect the level of response but clearly contributed to an additive increase when coexpressed with Nm23-H1. The downstream effect of COX-2 expression was also evaluated and showed that prostaglandin E 2 levels increased with Nm23-H1 and that there
Background: Experimental studies have shown that cyclo-oxygenase-2 (Cox2) is related to the development and pro- gression of tumors, since this enzyme is induced and expressed by cells such as macrophages, osteoblasts, “activated” endothelial cells, and tumor cells. The activity in tumors includes proliferation, cell transformation, tumor growth, inva- sion and metastasis and may play an important role in carcinogenesis of the canine osteosarcoma, since it has high ex- pression in tissue fragments. The combination of selective Cox2 inhibitors and other treatment modalities is the basis for a new anti-cancer therapy strategy. This in vitro study exposed primary cells of five different canine osteosarcoma cultures to selective Cox2 inhibitor at increasing concentrations and times. Results: For Cox2 negative cultures, despite the absence of differences, greater sensitivity of cells to treatment was observed. For Cox2 positive cultures, a higher number of necrotic cells were observed (P ≤ 0.05), when compared with negative cultures. For exposure times with Celecoxib doses, no difference (P > 0.05) was found between the three times analyzed for living, apoptotic and apop- totic/necrotic cells. There are similarities in the values of 24 h and 48 h, with slight reduction of living cells, increasing those undergoing apoptosis and apoptosis/necrosis. There was significance for necrosis (P ≤ 0.05). In 72 hours, a sig- nificant difference was observed between the other two previous values (P ≤ 0.05). It was found for the group of 100 µM·L −1 , that there was a numerically greater signaling for apoptosis and lower (P = 0.08) for necrosis, and this point
Overall, these observations indicate a hypothetical mechanism for the activation of iNOS by IL-1β that are, respectively, dependent on p38 MAPK signalling and could involve activa- tion of other MAPKs. In free-swelling conditions the p38 inde- pendent pathway is predominantly activated, as indicated by the partial effect of the p38 inhibitor on iNOS expression at later time points. However, at early time points the p38 inhibi- tor blocked activation of the p38 MAPK. Incorporation of the construct into the bioreactor system, with its inherent diffusional constraints, dramatically enhances iNOS induction, potentially via preferential activation of the p38 MAPK depend- ent pathway and possibly through a hypoxia-driven mecha- nism. Accordingly, the inhibitor has a greater effect on iNOS expression under these conditions. Dynamic compression substantially reduces iNOS expression and co-stimulation with both dynamic compression and the p38 MAPK inhibitor abolished iNOS expression when compared with each in iso- lation, suggesting that dynamic compression targets the p38 MAPK dependent pathway. By contrast, COX-2 activation by IL-1β appears to be primarily mediated by a p38 MAPK dependent pathway that is largely unaffected by transfer to the bioreactor system but is highly susceptible to the p38 MAPK inhibitor or dynamic compression. In addition to the regulation of iNOS and COX-2 expression by dynamic compression, co- stimulation with the inhibitor was important in restoring the cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which we observed no response. It is probable that matrix gene expression is control- led by transient factors and could involve rapid activation of multiple MAPKs and transcription factors. Ultimately, elucida- tion of the intracellular pathways will enable the identification of appropriate pharmacological agents and provide a clinical rationale for promoting the benefits of controlled physical activity to manage and treat OA.
bated for 1 h at room temperature with primary antibody (anti- COX-2, 1/1,000 dilution; anti-total ERK1/2, 1/2,000 dilution; anti-phospho-ERK1/2, 1/2,000 dilution) diluted with 2% BSA in TBST. After washing six times with TBST, the blots were then incubated with HRP-labeled secondary antibody (1/ 1,000 dilution) for 1 h at room temperature. Membranes were rewashed extensively and binding was detected using Enhanced Chemiluminescense western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA), according to the manufacturer's instructions. Anti-ERK1/2 and phospho-ERK1/2 antibodies were from Cell Signaling Tech- nology. Mouse monoclonal antibody tubulin Ab-4 (primary anti- body, 1/5,000 dilution; secondary antibody, 1/20,000 dilution) (Lab Vision) served as internal control. The band was semiquantified by densitometry using systems as described above.
Other, COX independent effects of NSAIDs might be involved as well, however . Several studies have demonstrated that certain NSAIDs, such as ibuprofen, cause anti-inflammatory effects independent of COX activity and prostaglandin synthe- sis inhibition [45-47]. These effects are mediated through inhi- bition of certain transcription factors such as nuclear factor (NF)- κ B and activator protein-1 [48-50]. The respective NSAIDs might interfere directly with the transcription factors, but their effects are probably mediated predominantly through alterations of the activity of cellular kinases such as IKK β , Erk, p38, or mitogen-activated protein kinase . These effects are not shared by all NSAIDs, because indomethacin failed to inhibit NF-κB and activator protein-1 activation, as well as Erk activity [49,52,53]. In contrast, indomethacin is able to acti- vate peroxisome proliferator-activated receptor- γ , which is not sensitive to sodium salicylate or aspirin . At the concentra- tion tested, celecoxib inhibits NF- κ B, an effect also observed for other NSAIDs but only at higher concentrations . Inhi- bition of NF-κB is related to inhibition of matrix metalloprotein- ases and aggrecanases . These effects may add to the observed differences in direct effects of NSAIDs on cartilage. Importantly, we discussed solely the direct effects of NSAIDs on cartilage. These effects should be seen within the context of the significant anti-inflammatory effects of these NSAIDs. By inhibiting joint inflammation, they may indirectly be benefi- cial to cartilage, specifically when inflammation is primary in the cause of cartilage damage, as is the case for rheumatoid arthritis. However, in OA, in which inflammation may contribute to but is not primarily responsible for cartilage damage, adverse direct effects of NSAIDs on cartilage with long-term treatment may have an important impact on long-term out-
In recent population studies, genome-wide linkage scans have highlighted several specific genes involved in disease risk . Chromosome 2q was positive in several scans, suggesting that this chromosome is likely to harbor one or more susceptibility genes. Two IL-1 genes (IL1 α and IL1 β ) and the gene encoding IL-1 receptor antagonist (IL1RN), located on chromosome 2q13 within a 430-kilobase genomic fragment, have been shown to associate with the development of primary knee, but not hip, OA . IL1RN haplotype variants have also been shown to associate with radiographic severity of the OA . Recently, a genome- wide association scan has identified a cyclo-oxygenase (COX)-2 variant involved in risk for knee OA . These genetic associations of genes such as IL1 α , IL1 β , IL1RN, and COX2 underscore the potential role of inflammatory pathways in the pathogenesis of knee OA.
All of these drugs (aspirin, NSAIDs, and coxibs) may also be associated with increased risk of cardiovascular harm. There appears to be a dose-related effect of aspirin causing myocardial infarction in a randomised trial of patients undergoing endarterectomy  and in patients with colorectal polyps , and of coxibs in colorectal polyp trials  where the annual event rate with placebo was less than 0.5%. In dementia patients with an annual risk of over 2% with placebo, coxibs were not associated with more thrombotic vascular events than placebo over sev- eral years of treatment . In arthritis, the annual risk with placebo is intermediate between these two condi- tions, at almost 1%. Increased cardiovascular effects for coxibs compared with placebo but not NSAIDs have been seen in studies in patients with arthritis [18-20]. Observa- tional studies indicate that while some cyclooxygenase inhibitors (selective and non-selective), including aspirin, have increased risk of cardiovascular adverse events, oth- ers do not .
Ultrapure water, COX inhibitor screening assay kits and lipoxygenase (LOX) inhibitor screening assay kits were obtained from Cayman Chemicals, USA. All reagents and solvents used were of analytical grade from Sigma-Aldrich, India, and from HiMedia Laboratories, India. 96 well plates were read on the iMark Microplate absorbance reader and washed with the model 1575 immunowash microplate washer from BioRad, India. Spectrophotometric assays were conducted on ultraviolet 1800 spectrophotometer from Shimadzu, Japan. The COX inhibitor screening directly measures prostaglandin F2 alpha by stannous chloride reduction of COX-derived prostaglandin H2 produced in the COX reaction . The reaction system consists of reaction buffer, heme, enzyme and plant extract pre-incubated at 37°C for 20 min with background and enzyme controls. The reaction was started with the incorporation of arachidonic acid and incubated for 2 min at 37°C temperature. The reaction was stopped with add of saturated stannous chloride solution and 5 min at room temperature (25°C). The prostaglandins are quantified by EIA. An aliquot of these reactions was added to the precoated plates in triplicates together with AChE tracer and antiserum and incubated for 18 h at room temperature on an orbital shaker. The plate was finally developed with Ellman’s Reagent and kept on an orbital shaker in the dark at room temperature for 1 h. The absorbance was read at 420 nm. The data were plotted as standard bound/maximum bound versus log concentration using a 4-parameter logistic curve fit. The concentration of each sample was determined from a standard curve with appropriate dilutions and used to calculate the percent inhibition as per the formula is given below:
Cyclo-oxygenase- (COX-) inhibitors are those drugs which have the ability to inhibit the activity of COX enzymes (types 1 and 2) resulting in inhibition of prostaglandin synthesis . Two types of COX- inhibitors are established, the traditional non-selective non-steroidal anti-inflammatory drugs (NSAIDs) which block both types of COX. The second type is the selective COX-2 inhibitors which have no or minimal affinity, and thus, no effect on COX-1. The development of the COX-2 selective inhibitors was intended to provide drugs that would offer the same pain-relieving and anti-inflammatory effects as the traditional NSAIDs without causing the gastric ulcers and nephrotic effects that have been associated with the pioneer drugs .
Cytomation, Glostrup, Denmark) was applied accord- ing to the manufacturer’s directions. The specific immune reaction was visualised using 3’,3-diami- nobenzidine tetrahydrochloride (DakoCytomation) and finally the sections were counterstained with Mayer’s haematoxylin. TBS buffer rinsing was used after each step. The whole procedure was performed at room temperature. The appropriate positive and negative controls were prepared. Sections treated in the same way but with mouse pre-immune serum instead of the primary antibodies examined were used as negative controls. For the positive COX-1 and COX-2 controls, human skin and colonic mucosa were ap- plied, respectively. Before the proper immunohis- tochemical study was begun, cross-reactivity with rat tissues was verified [4, 6, 7]. All the slides were evaluated using a light microscope (Olympus BX45, Tokyo, Japan).
However, prostaglandins also have anti- inflammatory properties. For example PGE2 has anti-inflammatory actions on innate immune cells, such as neutrophils, monocytes, and natural killer cells . Additionally, products of COX-2 have been implicated as having a role in the resolution of inflammation; however all these products have not yet been fully identified [36,37]. It has been noted that PGD2 expression is up-regulated in UC patients in remission, suggesting that this plays a role in long-term resolution and control of inflammation . The research into the anti-inflammatory effects of prostanoids is on-going, and current thinking is that the same prostanoids could have an opposite effect depending on its location, target tissue and the type of prostanoid receptors expressed . The increase in COX-2 and the resulting increased prostaglandins seen in IBD may contribute to down-regulation of inflammation. This could theoretically lead to a relapse in symptoms when NSAIDs are used. Furrer and Moreno speculated that the use of NSAIDs blocks the formation of prostaglandins, forcing arachidonic acid metabolism down the lipo-oxygenase and cytochrome P-450 pathways . They suggested that this could hypothetically cause an increase in eicosanoids such as leukotrienes and 5-HETE which are proinflammatory and also cause impairment of barrier function . This could also be an explanation of how NSAIDs have a harmful effect of IBD.
integral to the management of OA [9-12]. However, the use of these agents has been linked to an increase in the risk of gastrointestinal [13,14], cardiovascular [15,16], and renal [17,18] adverse events (AEs). The safety of oral NSAIDs is further compromised with increasing patient age and by the use of higher doses, extended periods of use, the presence of comorbid medical conditions, and the coadministration of certain medications [14,16,19]. Selec- tive cyclo-oxygenase-2 (COX-2) inhibitors were developed because they were thought to be safe alternatives for patients at a greater risk for peptic ulcer disease or gastro- intestinal bleeding; however, population-based analyses have called their safety into question . Because COX-2 has substantial expression in cardiovascular and renal tissue, concerns have been raised regarding potential adverse cardiovascular and renal effects associated with the use of these agents [21-24].
Objectives: Osteoarthritis (OA) has a dramatic impact on patients’ health related quality of life (HRQoL). Chronic use of analgesics and anti-inflammatory medications for pain management may improve symptoms but on long term may affect HRQoL negatively. The objective of the present study was to compare the impact of two different classes of analgesics, traditional non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclo-oxygenase-2 (COX-2) inhibitors on HRQoL among osteoarthritis patients using the SF-36 questionnaire. Methods: Clinic based cross-sec- tional study conducted at Al-Qassimi Hospital, Sharjah, United Arab Emirates (UAE), over a period of six months. Ethical Approval was obtained from the ethics committee at Al-Qassimi Clinical Re- search Center. Total of 200 osteoarthritis patients fulfilling the inclusion and exclusion criteria were involved in the study. Patients’ demographics were collected from their medical records. The Medical Outcome Study Short-Form 36 (SF-36) questionnaire was used to measure patients’ HRQoL. SF-36 data were scored using health outcomes scoring software 4.5. Results: Mean age of the subjects was 62.19 ± 9.81 years with females constituting 151 (75.5%) of the patients. In gen- eral, females scored lower in most of the HRQoL domains compared to males and there was sig- nificant difference between the two groups in the mental health (p = 0.005) & mental component (p = 0.042) domains. Compared to selective COX-2 inhibitors, patients on NSAIDs scored higher on all domains of SF-36 except physical functioning. There was significant difference in mental health
polymorphonuclear leukocyte adherence, nor were they detected in significant quantities in supernates of polymorphonuclear leukocytes exposed to lipopolysaccharide. However, inhibitors of both cyclo-oxygenase and thromboxane synthetase reduced increases in adherence induced by arachidonic acid (10 µg/ml), suggesting that lipopolysaccharide- mediated increases in adherence were due to an arachidonic acid product other than prostaglandin E 2 or F 2 a. 8,11,14-Eicosatrienoic acid, a precursor of monoenoic prostaglandins, did not enhance polymorphonuclear leukocyte adhesiveness.
A number of studies have found increased levels of the major proinflammatory cytokines TNFα and IL-1β in ME/CFS (for a review see Maes et al. ). Recent evi- dence has challenged the view that patients with ME/ CFS display an activated Th2 dominated immune system [5,128]. Proinflammatory and anti-inflammatory cyto- kines are known to coexist also in ME/CFS, although in many patients proinflammatory cytokines are dominant [127,129,130]. Studies examining the Th cytokine profiles in people with ME/CFS also show a large number of dif- ferent findings almost certainly for methodological incon- sistencies, including patient selection . Rose et al.  reported that there was a significant upregulation of cyclo-oxygenase2 (COX2), usually accompanied by in- creased iNOS, in MS lesions and opined that COX2 pro- moted excitotoxic death and damage of oligodendrocytes by coupling with iNOS. The involvement of COX2 in oligodendrocyte death was confirmed by Carslon et al.  using histopathological techniques. Upregulation of nuclear factor (NFκB in lesion-based macrophages amplifies the inflammatory reaction by stimulating the production of adhesion molecules and proinflammatory cytokines . Activated NFκB is found at high levels in microglia of active lesions . These authors proposed that high NFκB levels explains the relative rarity of oligodendrocyte death in MS. Generally it seems that upregulation of NFκB in neurons is pro- tective but activation of NFκB in microglia stimulates neuronal degeneration [135,136]. Maes et al.  reported significantly elevated levels of COX2 and NFκB in patients with ME/CFS compared to healthy controls. Moreover, the severity of the illness corre- lated significantly and positively with the elevation in concentrations COX2 and NFκB.
Evidence suggests that the BBB disruption contributes to astrocyte activation and gliosis. Within 24–48 h of the induction of SE, there is activation of glial fibrillary acidic protein-positive astrocytes (GFAP). The reactive gliosis may be detectable for over 3 to 4 months . These reactive astrocytes may have difficulty handling the extracellular glutamate, leading to neuronal hyper excitability and damage. Reactive astrocytes also serve to further release pro-inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) and recruit more inflammatory cells by secreting C-C motif chemokine ligands 2, 3, and 5 . As a result, there is increased neuronal excitability, development of seizures, cell death, and subsequent neuro-inflammation . The leakage of serum proteins such as albumin through a disrupted BBB may be a key factor in the initiation of specific signaling cascades within neurovascular cells, specifically astrocytes. Albu- min is hypothesized to bind to the astrocytic TGF- βR2, leading to the activation of the TGF-β signaling pathway, production of TGF-β, and astrocyte activation, which causes impaired buffering of potassium and glutamate at the cellular level . ALK5/TGF-β-pathway induces ex- citatory synaptogenesis whereas SJN2511, a specific ALK5/TGF- β inhibitor, prevents synaptogenesis and epi- lepsy . Hence, TGF-β pathway inhibition prevents the activation of astrocytes during epileptogenesis, lead- ing to a reduction in spontaneous seizure activity and brain inflammation [85, 87]. The TGF-Β pathway may serve as a therapeutic target to prevent seizure develop- ment in individuals with brain injury .
ABSTRACT: Gomphostemma Wall. ex Benth. belongs to the family Lamiaceae. The present study evaluated selected species of Gomphostemma endemic to the Western Ghats for their anti-inflammatory property. Traditionally, many species of Gomphostemma are used to treat malarial fever and inflammations caused by insect stings. Methanol extracts of Gomphostemma heyneanum var. heyneanum, G. heyneanum var. rottleri and G. eriocarpum were prepared and subjected to various in-vitro assays to examine their anti-inflammatory potential. Anti-inflammatory activity was initially screened with protein denaturation and proteinase inhibitory assays. Among the three extracts screened, G. heyneanum var. heyneanum showed significant activity comparable to the standard diclofenac sodium. Further, the effective extract of G. heyneanum var. heyneanum was evaluated for its effect on LPS induced RAW 264.7 cells. The effect of the extract on various inflammatory mediators like cyclooxygenase (COX), cellular nitrite and inducible nitric oxide synthase (iNOS) were analyzed. The extract inhibited cyclooxygenase and inducible nitric oxide synthase activity in a dose- dependent manner projecting the anti-inflammatory potential of the plant. The potent phytochemicals present in the plants may be attributed to the activity shown by the plants.
There is a large body of epidemiologic evidence sug- gesting that regular users of aspirin and nonaspirin NSAIDs have a reduced risk of colorectal cancer (21). This is complemented by murine studies of chemical- ly induced intestinal carcinogenesis and genetically engineered models of intestinal polyposis, both of which are sensitive to changes in COX-isozyme expres- sion or activity (ref. 22; see also Fitzpatrick and Sober- man, this Perspective series, ref. 4). One proposed mechanism for this type of chemoprevention is the inhibition of COX-2 in intestinal epithelial cells, stro- mal cells, or endothelial cells of newly formed blood vessels (22). COX-2 is overexpressed in most colonic cancers and in polyps of patients with familial adeno- matous polyposis (FAP). Subsequently, COX-2 has been found to be expressed in a wide variety of epithe- lial tumors. It is unclear how suppression of COX-2 expression or activity might restore antitumor reac- tivity, but the ability of prostanoids to influence cel- lular proliferation and apoptosis and modulate cytokine synthesis suggests some testable hypotheses. Prostanoids may also modulate the nuclear transloca- tion and function of tumor suppressor gene products. In the early stages of intestinal carcinogenesis, COX-2 appears to be expressed predominantly in stromal cells, but it is later detectable in surrounding inflam- matory cells, in the carcinomatous epithelial cells, and in the endothelial cells of new blood vessels, where it may play an additional role in angiogenesis (23).