Specific COX-2 Inhibitor NS-398 Research Articles

Fisetin suppresses cigarette smoke extract-induced epithelial to mesenchymal transition of airway epithelial cells through regulating COX-2/MMPs/β-catenin pathway

Cigarette smoke exposure leads to upregulation of cyclooxygenase‐2 (COX‐2), an inducible enzyme that synthesizes prostaglandin E2 (PGE2) and promotes airway inflammation. COX-2 overexpression is frequently implicated in inflammation, invasion, metastasis, and epithelial-mesenchymal transition (EMT). However, its detailed molecular mechanism in cigarette smoke induced EMT is not clear. Further, fisetin, a bioflavonoid, exhibits antioxidant and anti-inflammatory properties, but its effect in modulating COX-2-mediated inflammation and downstream sequelae remains unexplored. Therefore, we have investigated the mechanism of cigarette smoke-induced COX-2-mediated EMT in airway epithelial cells and examined the role of fisetin in controlling this aberration. MTT, trypan blue staining, gelatin zymography, Western blotting, invasion, wound healing, and tumor sphere formation assays in cigarette smoke extract (CSE) and/or fisetin treated airway epithelial cells, and in-silico molecular docking studies were performed. Results revealed that CSE exposure increased the expression and activity of COX-2, MMP-2/9, and β-catenin and also enhanced expression of EMT markers leading to higher migration and invasion potential of airway epithelial cells. A specific COX-2 inhibitor NS-398 as well as fisetin treatment reversed the expression of EMT biomarkers, reduced the activity of MMP-2/9, and blocked the migration and invasion potential induced by CSE. Further, PGE2 also increased MMPs activity, invasion, and migration potential similar to CSE, which were significantly reversed by fisetin. In-silico studies showed a high binding affinity of fisetin to key EMT associated proteins, validating its anti-EMT potential. Thus, our study firstly unearths the mechanism of CSE-induced EMT in airway epithelial cells via COX-2/MMP/β-catenin pathway, and secondly, it reveals that fisetin could significantly reverse CSE-induced EMT by inhibiting COX-2, indicating that fisetin could be an effective drug candidate for cigarette smoke-induced lung dysfunction.

MicroRNA-214 targets COX-2 to antagonize indoxyl sulfate (IS)-induced endothelial cell apoptosis.

Cardiovascular disease (CVD) serves as the major cause of mortality in chronic kidney disease (CKD) patients. The injury of endothelium associated with the long-term challenge of uremic toxins including the toxic indoxyl sulfate (IS) is one of key pathological factors leading to CVD. However, the mechanisms of uremic toxins, especially the IS, resulting in endothelial injury, remain unclear. miR-214 was reported to contribute to the pathogenesis of cardiovascular diseases, while its role in IS-induced endothelial cell apoptosis is unknown. In this study, we investigated the role of microRNA-214 (miR-214) in IS-induced endothelial cell apoptosis and the underlying mechanisms using mouse aortic endothelial cells (MAECs). Following IS treatment, miR-214 was significantly downregulated in MAECs in line with enhanced cell apoptosis. Meanwhile, COX-2 was upregulated at both mRNA and protein levels along with increased secretion of PGE2 in medium. To define the role of miR-214 in IS-induced endothelial cell apoptosis, we modulated miR-214 level in MAECs and found that overexpression of miR-214 markedly attenuated endothelial cell apoptosis, while antagonism of miR-214 deteriorated cell death after IS challenge. Further analyses confirmed that COX-2 is a target gene of miR-214, and the inhibition of COX-2 by a specific COX-2 inhibitor NS-398 strikingly attenuated IS-induced endothelial cell apoptosis along with a significant blockade of PGE2 secretion. In conclusion, this study demonstrated an important role of miR-214 in protecting against endothelial cell damage induced by IS possibly by direct downregulation of COX-2/PGE2 axis.

Angtensin II elicits a cAMP-dependent intestinal anion secretion by stimulating PGE2 release through AT1 subtype receptors in rat ileum

A growing literature has demonstrated that the renin-angiotensin system (RAS) involves in gut function. Angiotensin II (AngII) stimulates Cl− secretion in intestine epithelial cells. However, the underlying signal pathway remains unexplored. Here, we explored that serosal application of Ang II (5 × 10−8 M) significantly increased the baseline Isc compared to the control group in rat ileum. Tetrodotoxin (TTX) failed to suppress Isc evoked by Ang II. However, the Ang II-evoked Isc was significantly suppressed by the ATR1 antagonist losartan instead of ATR2 antagonist PD123319. Of interest, both cyclooxygenase (COX)-1 inhibitor SC560 and COX-2 specific inhibitor ns398 blocked the Ang II-evoked Isc. Preincubation of submucosa/mucosa preparations with Ang II for 10 min significantly increased PGE2 production, which was abolished by either COX-1 or COX-2 inhibitor. In addition, the Ang II-induced PGE2 release was also attenuated by ATR1 receptor antagonist rather than selective ATR2 receptor antagonist. Furthermore, preincubation of tissues for 15 min with forskolin, a cAMP activator, markedly blocked the Isc evoked by AngII, while intracellular Ca2+ pump inhibitor thapsigargin, L-type Ca2+ channel blocker nicadipine or the epithelial Na+ channel blocker amiloride didn't show such function. These results suggest that Ang II evokes cAMP-activated intestinal anion secretion by stimulating PGE2 release through activation of ATR1.

Cyclooxygenase 2 contributes to bradykinin-induced microvascular responses in peripheral arterioles after cardiopulmonary bypass

BackgroundDiabetic patients are associated with impaired peripheral microvascular function after cardiopulmonary bypass (CPB) and cardiac surgery. We hypothesized that upregulation of the inducible cyclooxygenase 2 (COX-2) contributes to altered microvascular reactivity of peripheral arterioles in diabetic patients undergoing CPB and cardiac surgery. MethodsSkeletal muscle samples of nondiabetic (ND) patients and patients with diabetes mellitus (DM; n = 8 per group) undergoing cardiac surgery were harvested before and after CPB. The protein expression/localization of COX-2 was assayed by Western blotting and immunohistochemistry. Peripheral arterioles were dissected from the harvested skeletal muscle tissue samples, the isolated arterioles (80-180 μm) were cannulated and pressurized, and changes in diameter were measured with video microscopy. In-vitro relaxation responses of precontracted arterioles were examined in the presence of the endothelium-dependent vasodilator bradykinin (10−10 to 10−6M) and in the presence or absence of the selective COX-2 inhibitor NS398 (10−5M). ResultsThe post-CPB protein levels of the inducible COX-2 were significantly increased compared with pre-CPB values in both the ND and DM groups (P < 0.05), whereas, this increase was higher in DM than that of ND (P < 0.05). In the DM arterioles, not the ND vessels, bradykinin-induced relaxation response was inhibited in the presence of the specific COX-2 inhibitor NS398 at baseline (P < 0.05). After CPB, bradykinin-induced relaxation response of the ND and DM arterioles was inhibited in the presence of the specific COX-2 inhibitor NS398, but this effect was more pronounced in the diabetic patients (P < 0.05). ConclusionsDiabetes and CPB are associated with upregulation in COX-2 expression/activation in human peripheral microvasculature. This alteration may lead to altered peripheral microvascular reactivity in diabetic patients undergoing cardiac surgery.

Activation of COX-2/mPGES-1/PGE2 Cascade via NLRP3 Inflammasome Contributes to Albumin-Induced Proximal Tubule Cell Injury

Background/Aims: The activation of NOD-like receptor family, pyrin domain containing3 (NLRP3) inflammasome has been shown to be positively correlated with the severity of proteinuria in chronic kidney disease (CKD) patients. Prostaglandin E2 (PGE2), an important inflammatory mediator, is also involved in various kidney injuries. The aim of the present study was to investigate the involvement of NLRP3 inflammasome and PGE2 synthetic pathway in albumin-induced renal tubular injury. Methods: Murine proximal tubular cells (mPTCs) were treated with albumin to induce cell injury. NLRP3 siRNA and specific COX-2 inhibitor NS398 were used to define their roles in mediating albumin-induced mPTC injury or the activation of COX-2/mPGES-1/PGE2 cascade. Results: In mPCTs, inhibition of NLRP3 by a small interfering RNA (siRNA) blocked albumin-induced kidney injury molecule 1 (KIM-1) upregulation, inflammatory response, and cell apoptosis. Albumin markedly activated cyclooxygenase-2 (COX-2)/ microsomal prostaglandin E synthase-1 (mPGES-1)/PGE2 pathway in this cell line, an effect largely abolished by NLRP3 silencing at both mRNA and protein levels. More interestingly, blockade of COX-2 using a specific COX-2 inhibitor NS398 markedly inhibited the upregulation of KIM-1 and inflammatory cytokines, and attenuated cell apoptosis in line with blunted PGE2 release following albumin treatment. Conclusions: The findings suggest that COX-2/mPGES-1/PGE2 axis could be activated by albumin in the proximal tubular cells via a NLRP3 inflammasome-mediated mechanism and could thus contribute to proteinuria-related renal tubular cell injury.

COX-2/mPGES-1/PGE2 cascade activation mediates uric acid-induced mesangial cell proliferation.

Hyperuricemia is not only the main feature of gout but also a cause of gout-related organ injuries including glomerular hypertrophy and sclerosis. Uric acid (UA) has been proven to directly cause mesangial cell (MC) proliferation with elusive mechanisms. The present study was undertaken to examined the role of inflammatory cascade of COX-2/mPGES-1/PGE2 in UA-induced MC proliferation. In the dose- and time-dependent experiments, UA increased cell proliferation shown by the increased total cell number, DNA synthesis rate, and the number of cells in S and G2 phases in parallel with the upregulation of cyclin A2 and cyclin D1. Interestingly, UA-induced cell proliferation was accompanied with the upregulation of COX-2 and mPGES-1 at both mRNA and protein levels. Strikingly, inhibition of COX-2 via a specific COX-2 inhibitor NS-398 markedly blocked UA-induced MC proliferation. Meanwhile, UA-induced PGE2 production was almost entirely abolished. Furthermore, inhibiting mPGES-1 by a siRNA approach in MCs also ameliorated UA-induced MC proliferation in line with a significant blockade of PGE2 secretion. More importantly, in gout patients, we observed a significant elevation of urinary PGE2 excretion compared with healthy controls, indicating a translational potential of this study to the clinic. In conclusion, our findings indicated that COX-2/mPGES-1/PGE2 cascade activation mediated UA-induced MC proliferation. This study offered new insights into the understanding and the intervention of UA-related glomerular injury.

Immunomodulatory effects of nicotine on interleukin 1β activated human astrocytes and the role of cyclooxygenase 2 in the underlying mechanism.

BackgroundThe cholinergic anti-inflammatory pathway (CAP) primarily functions through acetylcholine (ACh)-alpha7 nicotinic acetylcholine receptor (α7nAChR) interaction on macrophages to control peripheral inflammation. Interestingly, ACh can also bind α7nAChRs on microglia resulting in neuroprotective effects. However, ACh effects on astrocytes remain elusive. Here, we investigated the effects of nicotine, an ACh receptor agonist, on the cytokine and cholinesterase production of immunocompetent human astrocytes stimulated with interleukin 1β (IL-1β) in vitro. In addition, the potential involvement of prostaglandins as mediators of nicotine was studied using cyclooxygenase 2 (COX-2) inhibition.MethodsCultured human fetal astrocytes were stimulated with human recombinant IL-1β and treated simultaneously with nicotine at different concentrations (1, 10, and 100 μM). Cell supernatants were collected for cytokine and cholinesterase profiling using ELISA and MesoScale multiplex assay. α7nAChR expression on activated human astrocytes was studied using immunofluorescence. For the COX-2 inhibition studies, enzyme activity was inhibited using NS-398. One-way ANOVA was used to perform statistical analyses.ResultsNicotine treatment dose dependently limits the production of critical proinflammatory cytokines such as IL-6 (60.5 ± 3.3, %inhibition), IL-1β (42.4 ± 1.7, %inhibition), and TNF-α (68.9 ± 7.7, %inhibition) by activated human astrocytes. Interestingly, it also inhibits IL-8 chemokine (31.4 ± 8.5, %inhibition), IL-13 (34.243 ± 4.9, %inhibition), and butyrylcholinesterase (20.8 ± 2.8, %inhibition) production at 100 μM. Expression of α7nAChR was detected on the activated human astrocytes. Importantly, nicotine’s inhibitory effect on IL-6 production was reversed with the specific COX-2 inhibitor NS-398.ConclusionsActivation of the cholinergic system through α7nAChR agonists has been known to suppress inflammation both in the CNS and periphery. In the CNS, earlier experimental data shows that cholinergic activation through nicotine inhibits microglial activation and proinflammatory cytokine release. Here, we report similar anti-inflammatory effects of cholinergic activation on human astrocytes, at least partly mediated through the COX-2 pathway. These results confirm the potential for cholinergic neuroprotection, which is looked upon as a promising therapy for neuroinflammation as well as neurodegenerative diseases and stroke. Our data implicates an important role for the prostaglandin system in cholinergic regulatory effects.

Indoxyl Sulfate Induces Mesangial Cell Proliferation via the Induction of COX-2.

Indoxyl sulfate (IS) is one of important uremic toxins and is markedly accumulated in the circulation of end stage renal disease (ESRD) patients, which might contribute to the damage of residual nephrons and progressive loss of residual renal function (RRF). Thus this study was undertaken to investigate the role of IS in modulating mesangial cell (MC) proliferation and the underlying mechanism. The proliferation of MCs induced by IS was determined by cell number counting, DNA synthase rate, and cell cycle phase analysis. COX-2 expression was examined by Western blotting and qRT-PCR, and a specific COX-2 inhibitor NS398 was applied to define its role in IS-induced MC proliferation. Following IS treatment, MCs exhibited increased total cell number, DNA synthesis rate, and number of cells in S and G2 phases paralleled with the upregulation of cyclin A2 and cyclin D1. Next, we found an inducible inflammation-related enzyme COX-2 was remarkably enhanced by IS, and the inhibition of COX-2 by NS398 significantly blocked IS-induced MC proliferation in line with a blockade of PGE2 production. These findings indicated that IS could induce MC proliferation via a COX-2-mediated mechanism, providing new insights into the understanding and therapies of progressive loss of RRF in ESRD.

English

Exposure to phthalate esters is associated with changes in steroidogenesis, leading to the hypothesis that this is a primary mechanism of phthalate reproductive toxicity. However, some phthalate-induced male reproductive toxicity has been demonstrated in the absence of changes to testosterone production, suggesting additional mechanisms of action. There is evidence that phthalate exposure increases expression of the inflammatory enzyme cyclooxygenase 2 (cox-2). Furthermore, inhibition of cox-2 enhances expression of the steroidogenic acute regulatory protein (StAR), which mediates the rate-limiting step in steroidogenesis. This study hypothesized that phthalate-induced toxicity and testosterone perturbation are mediated in part by cox-2. A 3D in vitro rat testis co-culture to explore the role of cox-2 in phthalate toxicity was employed. Cells were treated with 100 µM dipentyl phthalate (DPP) with and without pre-treatment with the specific cox-2 inhibitor NS-398. Effects were evaluated after 8, 24, and 72 h. DPP exposure significantly increased cox-2 expression at 8 and 24 h (p<0.01) and resulted in significant, dose-dependent cytotoxicity. Pre-treatment with NS-398 significantly reduced the cytotoxicity of DPP at 8 and 24 h (p<0.01). NS-398 also mitigated the effects of DPP on testosterone regulation. Total testosterone concentrations in cell culture media were significantly increased following 8 and 24 hr of DPP exposure (p<0.001) and NS-398 reduced this effect (p<0.05). Simultaneously, DPP significantly decreased StAR protein expression after 8 h (p<0.01) and this effect was significantly attenuated by the presence of NS-398 (p<0.01). These results suggest that the DPP-induced changes in testosterone regulation observed in this experiment are mediated in part by an inflammatory response that is cox-2 dependent. Key words: dipentyl phthalate, testosterone, cyclooxygenase 2, in vitro toxicology

Interactions between neutrophils and non-small cell lung cancer cells: enhancement of tumor proliferation and inflammatory mediator synthesis.

The inflammatory tumor microenvironment plays a crucial role in tumor progression. In lung cancer, both bacterial infections and neutrophilia are associated with a poor prognosis. In this study, we characterized the effect of isolated human neutrophils on proliferation of the non-small cell lung cancer (NSCLC) cell line A549 and analyzed the impact of A549–neutrophil interactions on inflammatory mediator generation in naive and lipopolysaccharide (LPS)-exposed cell cultures. Co-incubation of A549 cells with neutrophils induced proliferation of resting and LPS-exposed A549 cells in a dose-dependent manner. In transwell-experiments, this effect was demonstrated to depend on direct cell-to-cell contact. This pro-proliferative effect of neutrophils on A549 cells could be attenuated by inhibition of neutrophil elastase activity, but not by oxygen radical neutralization. Correspondingly, neutrophil elastase secretion, but not respiratory burst, was specifically enhanced in co-cultures of A549 cells and neutrophils. Moreover, interference with COX-2 activity by indomethacin or the specific COX-2 inhibitor NS-398 also blunted the increased A549 proliferation in the presence of neutrophils. In parallel, a massive amplification of COX-2-dependent prostaglandin E2 synthesis was detected in A549–neutrophil co-cultures. These findings suggest that direct cell–cell interactions between neutrophils and tumor cells cause release of inflammatory mediators which, in turn, may enhance tumor growth in NSCLC.

New aspects of an old drug--diclofenac targets MYC and glucose metabolism in tumor cells.

Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies.

Abstract 3324: Cyclooxygenase-2 mediated breast cancer progression by induction of stem like cells and micro RNA

Abstract We established that elevated COX-2 expression by human and murine breast cancer cells promotes tumor progression and metastasis via multiple mechanisms primarily due to activation of the PGE-2 receptor EP4. COX-2 and HER-2, often co-expressed in human breast cancer, are both major determinants of cancer progression. Many of the HER-2 actions were shown to be COX-2 dependent, but the roles of COX-2 in the absence or presence of HER-2 in breast cancer initiation and sustenance remain unclear. To define these roles, we examined the consequences of introducing COX-2 gene into COX-2-ve, HER-2-ve, ER+ve, non-metastatic MCF-7 and COX-2-ve, ER-ve, HER-2-over-expressing SK-BR-3 human breast cancer cell lines. After stable integration of COX-2 cDNA, these two cell lines were named MCF-7-COX-2 and SKBR3-COX-2. When compared with their empty vector-transfected counterparts, they showed (1) epithelial-mesenchymal transition (down regulation of E Cadherin and up regulation Vimentin by qRT-PCR), (2) higher proliferative activity (BrdU uptake), (3) higher migratory and invasive abilities across microporous membranes (8μM pore size) in Boyden chambers, (4) upregulation of EP4 and angiogenic and lymphangiogenic factors VEGF-A, C and D (qRT PCR and western blot), (5) markedly increased stem like cell populations in vitro (spheroid forming ability of single cells in ultra-low attachment plates for successive generations) and an increase in ALDH activity (flow cytometry). (6) Above changes could be abrogated with specific COX-2 inhibitor NS-398 (Pfizer) or an EP4 antagonist ONO-AE3-208 (ONO Pharmaceuticals, Japan) at non toxic concentrations, indicating that all these functions are dependent on COX-2 and EP4 activity. (7) Intravenous injection of MCF-7-COX2 cells into immuno-deficient mice revealed a dramatic increase in their lung colony forming capacity at 4-6 weeks, as compared to empty vector-transfected cells. (8) Using differential gene and micro-RNA (miRNA) arrays we identified two miRNAs upregulated and their target fourteen tumor-suppressor like genes down-regulated by introducing COX-2 into MCF-7 cells. (9) We observed a positive co-relation between the miRNA and COX-2 expression in multiple COX-2 disparate cell lines, in which expression of miRNAs was inhibited by treatment with COX-2 inhibitors or EP4 antagonists. Current studies are testing the functions of the miRNAs by knockdown and knock-in studies, and utilizing human breast cancer tissues to explore the clinical relevance of these findings. Our model system allowing identification and characterization of COX-2 induced stem like cells in breast cancer should help defining COX-2 and EP4 mediated pathways controlling their biology and utilization of the novel miRNA markers for prognostic and therapeutic applications in the clinic. (Supported by the CBCF, Ontario Chapter and the OICR funds from the Government of Ontario to PKL). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3324. doi:1538-7445.AM2012-3324

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.

UVB radiation‐induced β‐catenin signaling is enhanced by COX‐2 expression in keratinocytes

UVB radiation is the major carcinogen responsible for skin carcinogenesis, thus elucidation of the molecular pathways altered in skin in response to UVB would reveal novel targets for therapeutic intervention. It is well established that UVB leads to upregulation of cyclooxygenase 2 (COX-2) in the skin which contributes to skin carcinogenesis. Overexpression of COX-2 has been shown to promote colon cancer cell growth through β-catenin signaling, however, little is known about the connection between UVB, COX-2, and β-catenin in the skin. In the present study, we have identified a novel pathway in which UVB induces β-catenin signaling in keratinocytes, which is modulated by COX-2 expression. Exposure of the mouse 308 keratinocyte cell line (308 cells) and primary normal human epidermal keratinocytes (NHEKs) to UVB resulted in increased protein levels of both N-terminally unphosphorylated and total β-catenin. In addition, we found that UVB-enhanced β-catenin-dependent TOPflash reporter activity and expression of a downstream β-catenin target gene. We demonstrated that UVB-induced β-catenin signaling is modulated by COX-2, as treatment of keratinocytes with the specific COX-2 inhibitor NS398 blocked UVB induction of β-catenin. Additionally, β-catenin target gene expression was reduced in UVB-treated COX-2 knockout (KO) MEFs compared to wild-type (WT) MEFs. Furthermore, epidermis from UVB-exposed SKH-1 mice exhibited increased N-terminally unphosphorylated and total β-catenin protein levels and increased staining for total β-catenin, and both responses were reduced in COX-2 heterozygous mice. Taken together, these results suggest a novel pathway in which UVB induces β-catenin signaling in keratinocytes which is enhanced by COX-2 expression.

Houttuynia cordata, a novel and selective COX-2 inhibitor with anti-inflammatory activity

Houttuynia cordata Thunb. (Saururaceae; HC) has been long used in traditional oriental medicine for the treatment of inflammation diseases. Modern research has implicated inducible cyclooxygenase-2 (COX-2) as a key regulator of the inflammatory process. In the present study, we aimed to investigate the effect of HC on COX-2. We examined the effects of HC on lipopolysaccharide (LPS)-induced prostaglandin (PG) E(2) production, an indirect indicator of COX-2 activity, and COX-2 gene and protein expression in mouse peritoneal macrophages. LPS-induced mouse peritoneal macrophages were employed as an in vitro model system. LPS-induced PGE(2) production was assessed by enzyme-linked immunosorbant assay and COX-2 protein expression was assessed by Western blot assay. The results showed that HC was able to inhibit the release of LPS-induced PGE(2) from mouse peritoneal macrophages (IC50 value: 44.8 μg/mL). Moreover, the inhibitory activity of HC essential oil elicited a dose-dependent inhibition of COX-2 enzyme activity (IC50 value: 30.9 μg/mL). HC was also found to cause reduction in LPS-induced COX-2 mRNA and protein expression, but did not affect COX-1 expression. The non-steroidal anti-inflammatory drug (NSAID) and specific COX-2 inhibitor NS398 functioned similarly in LPS-induced mouse peritoneal macrophages. Taken together, our data suggest HC mediates inhibition of COX-2 enzyme activity and can affect related gene and protein expression. HC works by a mechanism of action similar to that of NSAIDs. These results add a novel aspect to the biological profile of HC.

Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts

Mechanical stretch of skeletal muscle activates nitric oxide (NO) production and is an important stimulator of satellite cell proliferation. Further, cyclooxygenase (COX) activity has been shown to promote satellite cell proliferation in response to stretch. Since COX-2 expression in skeletal muscle can be regulated by NO we sought to determine if NO is required for stretch-induced myoblast proliferation and whether supplemental NO can counter the effects of COX-2 and NF-kappaB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, L-NAME (200 microM), the COX-2 specific inhibitor NS-398 (100 microM), the NF-kappaB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10-100 microM) or no supplement (control) for 24 h. Subgroups of each treatment were exposed to 1 h of 15% cyclic stretch (1 Hz), and were then allowed to proliferate for 24 h before fixing. Proliferation was measured by BrdU incorporation during the last hour before fixing, and DAPI stain. Stretch induced a twofold increase in nuclear number compared to control, and this effect was completely inhibited by L-NAME, NS-398 or PDTC (P < 0.05). Although DETA-NONOate (10 microM) did not affect basal proliferation, the NO-donor augmented the stretch-induced increase in proliferation and rescued stretch-induced proliferation in NS-398-treated cells, but not in PDTC-treated cells. In conclusion, NO, COX-2, and NF-kappaB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-kappaB are both involved in basal proliferation, NO does not affect basal growth. Thus, NO requires the synergistic effect of stretch in order to induce muscle cell proliferation.

Simultaneous inhibition of TXA2 and PGI2 synthesis increases NO release in mesenteric resistance arteries from cirrhotic rats

Our present study examines, in mesenteric resistance arteries, possible vasodilation alterations, and the role of NO and COX (cyclo-oxygenase) derivatives, in cirrhosis. The vasodilator response to acetylcholine was analysed in segments from control and cirrhotic rats. The effects of the non-specific COX inhibitor indomethacin, the specific COX-1 inhibitor SC-560 and the specific COX-2 inhibitor NS-398 were analysed in segments from both groups of rats. NO release was measured, and eNOS [endothelial NOS (NO synthase)], phospho-eNOS, iNOS (inducible NOS), COX-1 and COX-2 protein expression was also analysed. The effects of the TP receptor [TXA2 (thromboxane A(2)) receptor] antagonist SQ 29548, the TXA(2) synthesis inhibitor furegrelate, the PGI(2) (prostaglandin I(2)) synthesis inhibitor TCP (tranylcypromine) or TCP+furegrelate were only determined in segments from cirrhotic rats. The vasodilator response to acetylcholine was higher in segments from cirrhotic rats. Indomethacin, SC-560 and NS-398 did not modify the vasodilator response in control rats; however, indomethacin, NS-398 and TCP+furegrelate increased, whereas SC-560 did not modify and SQ 29548, furegrelate or TCP decreased, the vasodilator response to acetylcholine in cirrhotic rats. NO release was higher in cirrhotic rats. Furegrelate decreased, whereas TCP+furegrelate increased, the NO release in segments from cirrhotic rats. eNOS and COX-1 protein expression was not modified, whereas phosho-eNOS, iNOS and COX-2 protein expression was higher in cirrhotic rats. Therefore the increase in iNOS expression and eNOS activity may mediate increases in endothelial NO release. The COX-2 derivatives TXA(2) and PGI(2) may act simultaneously, producing a compensatory effect that reduces NO release and may limit the hyperdynamic circulation.

Interactions between neutrophils and NSCLC cells in vitro effects on neutrophil inflammatory mediator generation and tumour cell proliferation

e22148 Background: Growing evidence indicates that interactions between tumour cells and the inflammatory cellular microenvironment are crucial for tumour biology. In NSCLC, both bacterial infections and leukocytosis are associated with a poor prognosis. Against this background, we studied the effect of normal bronchial epithelial cells (B2B) and NSCLC cells (A549) on inflammatory neutrophil behaviour and quantified tumour cell proliferation in the presence of neutrophils. Methods: Isolated human neutrophils were co-cultured with B2B or A549 cells. Neutrophil respiratory burst was quantified by cytochrome C reduction and elastase activity was measured by the kinetics of substrate turnover. Proliferation of A549 cells was determined by MTS- assay. Results: Co-culturing human neutrophils with A549 cells stimulated neutrophil respiratory burst and degranulation in response to the bacterial tripeptide fMLP. In contrast, when co-cultured with B2B cells, these inflammatory neutrophil reactions were blunted. This could be attributed to release of NO and adenosine by the B2B cells. The increase of oxygen radical release and elastase degranulation in the presence of A549 cells was mediated by direct cell-to-cell contact. COX-2 dependent prostanoids were obviously involved in the activation of inflammatory neutrophil behaviour, as shown by the inhibitory capacity of the COX-2 specific inhibitor NS-398 in the co-culture system. Interestingly, activated neutrophils induced proliferation of NSCLC cells in a dose-dependent manner. Conclusions: The reciprocal interactions between neutrophils and NSCLC cells may enhance neutrophil-derived tissue destruction and tumour growth under inflammatory conditions. No significant financial relationships to disclose.

COX‐2 is induced by HGF stimulation in Met‐positive thyroid papillary carcinoma cells and is involved in tumour invasiveness

Thyroid papillary carcinoma (TPC) cells express high levels of cytoplasmic cyclo-oxygenase 2 protein. Analysis of microdissected samples of the tumour and of the paired normal thyroid tissue confirmed that mRNA transcripts for cyclo-oxygenase 2 (COX-2) were significantly more numerous in the tumour (7.6 +/- 13-fold; p = 0.01). High levels of COX-2 mRNA were not associated with age, sex, tumour size or lymph node metastasis. COX-2 was not homogeneously expressed throughout the tumour, but was significantly higher at the tumour invasion front. Hepatocyte growth factor (HGF) can up-regulate the expression of COX-2 mRNA. A marked increase in COX-2 mRNA levels was observed in 8/8 primary TPC cultures after HGF stimulation (6.3 +/- 6-fold) and in two papillary carcinoma cell lines (TPC-1 and NPA). Specific involvement of the high-affinity HGF receptor (Met protein) was suggested by the observation that PHA-665752, an inhibitor of the catalytic activity of c-Met kinase, caused a 54% reduction of the hepatocyte growth factor-induced COX-2 up-regulation. The possibility that HGF-Met interactions also had a causative role in the up-regulation of COX-2 in vivo was investigated in 30 tumour samples, where it was found that there was a statistically significant correlation (p = 0.001, r = 0.85) in the levels of expression of MET and COX-2 RNAs. The biological role of COX-2 in TPC cells was investigated by treating the TPC cell lines with the specific COX-2 inhibitor NS-398. It was found that NS-398 treatment significantly reduced the migration (50-75%) and invasiveness (47-92%) of tumour cells, but did not alter cell proliferation. Our data suggest that the increased expression of Met protein in TPC cells has a role in up-regulating the expression of COX-2, which in turn contributes to the invasive capacity of TPC cells.

COX-2 inhibitor reduces skeletal muscle hypertrophy in mice

Anti-inflammatory strategies are often used to reduce muscle pain and soreness that can result from high-intensity muscular activity. However, studies indicate that components of the acute inflammatory response may be required for muscle repair and growth. The hypothesis of this study was that cyclooxygenase (COX)-2 activity is required for compensatory hypertrophy of skeletal muscle. We used the synergist ablation model of skeletal muscle hypertrophy, along with the specific COX-2 inhibitor NS-398, to investigate the role of COX-2 in overload-induced muscle growth in mice. COX-2 was expressed in plantaris muscles during compensatory hypertrophy and was localized mainly in or near muscle cell nuclei. Treatment with NS-398 blunted the increases in mass and protein content in overloaded muscles compared with vehicle-treated controls. Additionally, the COX-2 inhibitor decreased activity of the urokinase type plasminogen activator, macrophage accumulation, and cell proliferation, all of which are required for hypertrophy after synergist ablation. Expression of insulin-like growth factor-1 and phosphorylation of Akt, mammalian target of rapamycin, and p70S6K were increased following synergist ablation, but were not affected by NS-398. Additionally, expression of atrogin-1 was reduced during hypertrophy, but was also not affected by NS-398. These results demonstrate that COX-2 activity is required for skeletal muscle hypertrophy, possibly through facilitation of extracellular protease activity, macrophage accumulation, and cell proliferation.