Specific COX-2 Inhibitor Research Articles

Celecoxib synergizes human pancreatic ductal adenocarcinoma cells to sorafenib-induced growth inhibition

BackgroundPancreatic ductal adenocarcinoma is frequently associated with aberrant activation of the Ras/Raf/MAPK pathway and cyclooxygenase-2 (COX-2) overexpression. This study evaluated the potential for combining the multikinase inhibitor sorafenib and the specific COX-2 inhibitor celecoxib as therapy in pancreatic ductal adenocarcinoma cells. MethodsBxPC-3, MIAPaCa-2, PANC-1 and AsPC-1 pancreatic adenocarcinoma cells were exposed to sorafenib and celecoxib combined treatment in vitro. Cell viability and various growth promoting and survival signaling pathways were monitored by MTT, flow cytometry and Western blotting. ResultsCombined treatment with sorafenib and celecoxib synergistically inhibited pancreatic adenocarcinoma cell proliferation. This regimen produced combination index (CI) values between 0.67 and 0.92 for the various cell lines, indicating significant synergistic interactions between sorafenib and celecoxib, which also markedly inhibited the migratory capacity. The growth inhibition was associated with an accumulation of cells in the G0/G1 phase of the cell cycle and induction of apoptosis. These changes were accompanied by a significant reduction of p21WAF1/Cip1 levels, where celecoxib sensitized the cells to sorafenib-mediated p21WAF1/Cip1 suppression. ConclusionThese results suggest that combined treatment with sorafenib and celecoxib synergistically induce growth inhibition and apoptosis in pancreatic adenocarcinoma cells through a process involving p21WAF1/Cip1 suppression.

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

Cyclooxygenase‐2 inhibition prevent angiotensin II‐induced oxidative stress but not hypertension in humans

Angiotensin II (AngII) is known as an important oxidative stress mediator in hypertension. There is controversy whether cyclooxygenase‐2 (COX‐2) specific inhibitors would be associated with elevations or decrease in blood pressure (BP) despite evidences showing decrease in ROS generation under COX‐2 inhibition. The aim of this study was to determine in healthy humans whether the COX‐2 selective inhibitor celecoxib has antioxidant and hypertensive protective effects against AngII. BP and selected plasma oxidative stress markers (8OHdG; AOPP), peroxynitrite activity (nitrotyrosine), endothelin‐1 (ET1) and nitric oxide metabolism (NOx) were measured at baseline and in response to a graded infusion of AngII before and after 14 days of celecoxib in 14 healthy subjects in high‐salt balance. In response to AngII challenge, all markers of oxidative stress were increased and there was blunting of NOx. Though no effect was observed in terms of BP response to AngII challenge, celecoxib significantly attenuated the increase in oxidative stress as observed by a reduction in the rise of 8OHdG, nitrotyrosine, aldosterone and the NOx inhibition induced by AngII infusion. In conclusion, exposure to AngII increases oxidative stress in humans, though this AngII‐mediated upregulation is attenuated with COX‐2 inhibition. Supported by Alberta Innovates Health Solutions and Canadian Institutes for Health Research.

Celecoxib inhibits growth of human autosomal dominant polycystic kidney cyst-lining epithelial cells through the VEGF/Raf/MAPK/ERK signaling pathway

Autosomal dominant polycystic kidney disease (ADPKD) is a progressive chronic kidney disease. To date there are no effective medicines to halt development and growth of cysts. In the present study, we explored novel effects of celecoxib (CXB), a COX-2 specific inhibitor, on primary cultures of human ADPKD cyst-lining epithelial cells. Primary cultures of ADPKD cyst-lining epithelial cells were obtained from five patients. Effects of CXB were measured by various assays to detect BrdU incorporation, apoptosis and proliferation in vitro. Additionally, effects of CXB on kidney weight, the cyst index, the fibrosis index, blood urea nitrogen (BUN), serum creatinine (SCr), serum 6-keto-PGF-1α, serum thromboxane-2 (TXB2) and renal PCNA expression were assessed in Han:SPRD rat, a well-characterized rodent model of PKD. CXB inhibited proliferation of ADPKD cyst-lining epithelial cells, blocked the release of VEGF from the cells and induced extensive apoptosis in a time- and dose-dependent manner. Moreover, CXB up-regulated the cell cycle negative regulator p21CIP/WAF1 and the cell cycle positive regulator Cyclin A, blocked ERK1/2 phosphorylation, induced apoptotic factors (Bax and caspase-3) and reduced Bcl-2. Furthermore, CXB inhibited the expression of VEGFR-2 and Raf-1 in ADPKD cyst-lining epithelial cells. CXB markedly reduced the cyst index, the fibrosis index, leukocyte infiltration, BUN, SCr, serum 6-keto-PGF-1α, TXB2 and renal PCNA expression in Han:SPRD rat. We demonstrated for the first time that CXB could suppress renal cyst-lining growth both in vitro and in vivo in Han:SPRD rat. CXB can inhibit proliferation, suppress cell cycle progression, and induce apoptosis in ADPKD cyst-lining epithelial cells through the inhibition of the VEGF/VEGFR-2/Raf-1/MAPK/ERK signaling pathway.

Cyclooxygenase-1-Selective Inhibitors Based on the (E)-2′-Des-methyl-sulindac Sulfide Scaffold

Prostaglandins (PGs) are powerful lipid mediators in many physiological and pathophysiological responses. They are produced by oxidation of arachidonic acid (AA) by cyclooxygenases (COX-1 and COX-2) followed by metabolism of endoperoxide intermediates by terminal PG synthases. PG biosynthesis is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). Specific inhibition of COX-2 has been extensively investigated, but relatively few COX-1-selective inhibitors have been described. Recent reports of a possible contribution of COX-1 in analgesia, neuroinflammation, or carcinogenesis suggest that COX-1 is a potential therapeutic target. We designed, synthesized, and evaluated a series of (E)-2′-des-methyl-sulindac sulfide (E-DMSS) analogues for inhibition of COX-1. Several potent and selective inhibitors were discovered, and the most promising compounds were active against COX-1 in intact ovarian carcinoma cells (OVCAR-3). The compounds inhibited tumor cell proliferation but only at concentrations >100-fold higher than the concentrations that inhibit COX-1 activity. E-DMSS analogues may be useful probes of COX-1 biology in vivo and promising leads for COX-1-targeted therapeutic agents.

Etoricoxib: A Probable Safe Alternative For NSAID Intolerant Patients In Asia

NSAID intolerance is not uncommon. Etoricoxib, a specific cox-2 inhibitor NSAID, has been shown to be a safe alternative in patients with this condition. The aim of this study is to determine the rate of NSAID intolerant patients who are able to tolerate etoricoxib without adverse reactions.

A 4-trifluoromethyl analogue of celecoxib inhibits arthritis by suppressing innate immune cell activation

IntroductionCelecoxib, a highly specific cyclooxygenase-2 (COX-2) inhibitor has been reported to have COX-2-independent immunomodulatory effects. However, celecoxib itself has only mild suppressive effects on arthritis. Recently, we reported that a 4-trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2-inhibitory activity inhibits secretion of IL-12 family cytokines through a COX-2-independent mechanism that involves Ca2+-mediated intracellular retention of the IL-12 polypeptide chains. In this study, we explored the capacity of TFM-C as a new therapeutic agent for arthritis.MethodsTo induce collagen-induced arthritis (CIA), DBA1/J mice were immunized with bovine type II collagen (CII) in Freund's adjuvant. Collagen antibody-induced arthritis (CAIA) was induced in C57BL/6 mice by injecting anti-CII antibodies. Mice received 10 μg/g of TFM-C or celecoxib every other day. The effects of TFM-C on clinical and histopathological severities were assessed. The serum levels of CII-specific antibodies were measured by ELISA. The effects of TFM-C on mast cell activation, cytokine producing capacity by macophages, and neutrophil recruitment were also evaluated.ResultsTFM-C inhibited the severity of CIA and CAIA more strongly than celecoxib. TFM-C treatments had little effect on CII-specific antibody levels in serum. TFM-C suppressed the activation of mast cells in arthritic joints. TFM-C also suppressed the production of inflammatory cytokines by macrophages and leukocyte influx in thioglycollate-induced peritonitis.ConclusionThese results indicate that TFM-C may serve as an effective new disease-modifying drug for treatment of arthritis, such as rheumatoid arthritis.

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.

Pharmacological Profile of Celecoxib, a Specific Cyclooxygenase-2 Inhibitor

The pharmacological profile of celecoxib (CAS 169590-42-5, SC-58635), a specific cyclooxygenase-2 (COX-2) inhibitor, was investigated. Celecoxib inhibited COX-2-mediated prostaglandin E2 (PGE2) production in human dermal fibroblasts (IC50 = 91 nmol/l), whereas it was a weak inhibitor of COX-1-mediated PGE2 production in human lymphoma cells (IC50 = 2800 nmol/l). In in vivo studies, the effects of celecoxib were compared with those of nonsteroidal anti-inflammatory drugs (NSAIDs) in acute rat models of hyperalgesia and pyrexia. Celecoxib abrogated carrageenan-induced hyperalgesia in the hind paw accompanied by a decrease in PGE2 content in paw exudates and cerebrospinal fluid in a dose-related manner, with an ED30 = 0.81 mg/kg. Its analgesic potency was comparable to those of NSAIDs. In lipopolysaccharide-induced pyrexia, the anti-pyretic potency of celecoxib was equal to that of NSAIDs. On the other hand, in a gastric toxicity study in rats, single oral administration of celecoxib had no effect on gastric mucosa or mucosal PGE2 content at doses up to 200 mg/kg. Additionally, celecoxib did not inhibit thromboxane B2 production of calcium ionophore-stimulated peripheral blood of rats or arachidonic acid-induced aggregation of human platelets. These findings suggest that celecoxib might be a safe and effective alternative to NSAIDs for clinical use.

Cyclooxygenase inhibitors protect d-galactosamine/lipopolysaccharide induced acute hepatic injury in experimental mice model

We investigated the protective effects of two non-steroid anti-inflammatory drugs, indomethacin (COX-1 and COX-2 inhibitors) and nimesulide (specific COX-2 inhibitor) on the hepatic injury induced by lipopolysaccharide in d-galactosamine sensitized (Gal/LPS) mice. ICR male mice were injected with a single dose of Gal/LPS with or without pre-treatment of 3mg/kg indomethacin or 30mg/kg nimesulide (single i.p. injection). Sixteen hours later, blood and liver tissues of mice were collected for histological, molecular, and biochemical analyses. Our results showed marked reduction of hepatic necrosis, serum ALT, and tissue TBARS levels in both indomethacin- and nimesulide-pre-treated mice when compared with Gal/LPS-treated mice. Western blot and RT-PCR analysis showed decreased levels of iNOS mRNA, iNOS protein, and nitrotyrosine formation in both COX inhibitor pre-treated groups when compared with Gal/LPS-treated group. There was an inverse relationship between COX-1 and COX-2 expressions, as well as between COX-2 and C/EBP-α expressions in COX inhibitors groups, Gal/LPS and control groups. COX inhibitors reduced the expression of TNF-α mRNA and the activity of NF-κB which were elevated by Gal/LPS treatment. We conclude that COX inhibitors protected the liver from Gal/LPS-induced hepatotoxicity. COX inhibitors could be considered as potential agents in the prevention of acute liver failure and sepsis.

C6-ceramide enhances Interleukin-12-mediated T helper type 1 cell responses through a cyclooxygenase-2-dependent pathway

Ceramides, lipid molecules located predominantly within the plasma membrane of a cell, can function as second messengers, and have been known to carry out a number of cellular functions. T helper type 1 (Th1) immune responses are known to be involved in the cellular immunity, which is crucial in the cancer and allergy immunotherapy. This study was designed to evaluate the effects of ceramides on T helper cell responses and their underlying mechanisms. We demonstrated that a cell-permeable C6-ceramide (C6) together with IL-12 enhanced Th1 cell differentiation, whereas C6 alone had no effects, as demonstrated by the increased populations of IFN-γ expressing CD4+ T cells and the up-regulation of IFN-γ production from CD4+ T cells. In contrast, C2-ceramide and long chain ceramides (C16 and C24) did not affect the Th1 responses. C6 treatment was shown to increase the expression of T-bet, a master transcription factor of Th1 responses, in a dose-dependent fashion. Furthermore, C6 increased the expression of cyclooxygenase-2 (COX-2) in CD4+ T cells. The C6-mediated increase of IFN-γ production and IFN-γ expressing CD4+ T cell populations were significantly suppressed by a COX-2 specific inhibitor (NS-398) in a dose-dependent manner. T-bet expression was also decreased by NS-398 treatment, thereby indicating that C6 ceramide enhances Th1 responses via a COX-2 dependent pathway. This result demonstrates that C6 may be utilized in therapies for the treatment of immune diseases such cancer and allergy by enhancing the Th1 activity.

Anti-inflammatory effects of benfotiamine are mediated through the regulation of the arachidonic acid pathway in macrophages

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent antioxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study (U.C. Yadav et al., Free Radic. Biol. Med. 48:1423–1434, 2010) indicates a novel role for benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating arachidonic acid (AA) pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes, prostaglandin E2, thromboxane 2 (TXB2), and prostacyclin (PGI2) in macrophages. Further, LPS-induced expression of AA-metabolizing enzymes such as COX-2, LOX-5, TXB synthase, and PGI2 synthase was significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-κB and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein–HNE adduct formation. Most importantly, compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented LPS-induced macrophage death and monocyte adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of the COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response.

A novel nitro-oxy substituted analogue of rofecoxib reduces human colon cancer cell growth

Rofecoxib is a specific COX-2 inhibitor able to exert antiproliferative activity against colorectal cancer cells. It was withdrawn from the market after the demonstration of an increased risk of cardiovascular complications after prolonged use. Nevertheless, it remains an interesting compound for laboratory research as an experimental COX-2 inhibitor. In this study, the antiproliferative activity of a novel dinitro-oxy-substituted analogue of rofecoxib (NO-rofe), potentially less cardiotoxic, has been investigated in vitro on human colon cancer cells and compared with the action of the parent drug. Due to the fact that COX-2 inhibition is the main characteristic of coxibs, we performed all experiments in COX-2-overexpressing (HT-29) and COX-2-negative (SW-480) human colon cancer cells, to elucidate whether the observed effects were dependent on COX-2 inhibition. Moreover, experiments were performed in order to evaluate whether COX-2 pharmacological inhibition may affect beta-catenin/E-cadherin signaling pathway. NO-rofe exerted a significant antiproliferative activity on COX-2 positive HT-29 human colon cancer cells, being less effective on the COX-2 negative SW-480 human colon cancer cell line. In particular, the rofecoxib analogue retained similar potencies with respect to COX-2 inhibition but was much more active than rofecoxib in inhibiting the growth of human colon cancer cells in vitro. In addition, this novel compound resulted in the induction of membrane β-catenin/E-cadherin expression, a feature that may significantly contribute to its antiproliferative activity.

Abstract CN07-01: Modulation of proinflammatory and anti-inflammatory signaling for cancer chemoprevention

Abstract A new horizon in chemoprevention research is the recent discovery of molecular links between inflammation and cancer. The implication of inflammatory tissue damage in pathophysiology of human maligancy is under intense investigation both at the research level and in clinical practice. Numerous studies have been reported with the global ‘omics’ profiling technologies, such as DNA microarray, proteomics, metabolomics, lipidomics, etc., to identify and characterize a series of critical molecules/changes in the inflammatory signaling. It is by gaining this type of mechanistic understanding of a disease that researchers will unlock the keys to discovering new diagnostics and therapeutic strategies for the management of inflammation-associated cancer. Aberrant upregulation of cyclooxygenase-2 (COX-2), a key enzyme in the arachidonic acid cascade, has been frequently observed in various precancerous and malignant tissues. Therefore, the normalization of inappropriately overamplified signaling cascades implicated in chronic inflammation-associated carcinogenesis by use of COX-2 specific inhibitors has been recognized as a rational and pragmatic strategy in molecular target-based chemoprevention. Proinflammatory stimuli trigger the activation of intracellular signal transduction network comprising proline-directed serine/threonine kinases, and their downstream transcription factors, such as NF-κB, resulting in an overexpression of COX-2. Cyclopentenone prostaglandins of J series as products of cyclooxygenase-2 (COX-2)-mediated arachidonic acid cascades have been reported to possess multifaceted cellular functions, including antiinflammatory and cytoprotective effects. A typical example is 15-deoxy-Δ 12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor-γ (PPARγ), that modulates the activities of various intracellular signaling molecules. Because of the electrophilic α,β-unsaturated carbonyl moiety present in its cyclopentenone ring structure, 15d-PGJ2 acts as a Michael addition acceptor and can readily interact with critical cellular nucleophiles, such as cysteine thiol groups of proteins. Many of the biological effects induced by 15d-PGJ2 are mediated by targeting redox-sensitive transcription factors and their regulators, including IκB kinase-NF-κB, AP-1, Nrf2-Keap1, HIF-1α, STAT3and p53 tumor suppressor. The regulation of the aforementioned signaling molecules by 15d-PGJ2 is not likely to be dependent on PPARγ activation, but rather involves direct modification or oxidation of their critical cysteine residues acting as a redox-sensor. Though 15d-PGJ2 exerts cytoprotective effects in normal cells, elevated production of this cyclopentenone prostaglandin has been implicated in tumorigenesis through induction of angiogenesis and metastasis. As one of the terminal products of COX-2-mediated arachidonic acid metabolism, 15d-PGJ2 upregulates the expression of COX-2 in the human breast cancer MCF-7 cell line by activation of Akt and subsequently AP-1. Some malignant cells might exploit this visious cycle of COX-2 up-expression for maintaining invasiveness and taking survival advantage. Cysteine thiols present in various transcription factors and their regulators function as redox sensors in fine-tuning of transcriptional regulation. Thus, oxidation or covalent modification of thiol groups present in the redox-sensitive transcription factors and their regulating molecules can provide a unique strategy for molecular target-based chemoprevention. Citation Information: Cancer Prev Res 2011;4(10 Suppl):CN07-01.

Delayed Administration of Parecoxib, a Specific COX-2 Inhibitor, Attenuated Postischemic Neuronal Apoptosis by Phosphorylation Akt and GSK-3β

Parecoxib is a recently described novel COX-2 inhibitor whose functional significance and neuroprotective mechanisms remain elusive. Therefore, in this study, we aimed to investigate whether delayed administration of parecoxib inhibited mitochondria-mediated neuronal apoptosis induced by ischemic reperfusion injury via phosphorylating Akt and its downstream target protein, glycogen synthase kinase 3β (GSK-3β). Adult male Sprague-Dawley rats were administered parecoxib (10 or 30 mg kg(-1), IP) or isotonic saline twice a day starting 24 h after middle cerebral artery occlusion (MCAO) for three consecutive days. Cerebral infarct volume, apoptotic neuron, caspase-3 immunoreactivity and the protein expression of p-Akt, p-GSK-3β and Cytochrome C in cerebral ischemic cortex were evaluated at 96 h after reperfusion. Parecoxib significantly diminished infarct volume and attenuated neuron apoptosis in a dose-independent manner, compared with MCAO group alone. Increased p-Akt and p-GSK-3β was observed in the ischemic penumbra of parecoxib group after stroke. Moreover, parecoxib also reduced the release of Cytochrome C from mitochondrial into cytosol and attenuated the caspase-3 immunoreactivity in the penumbra. Taken together, these results suggested that parecoxib ameliorated postischemic mitochondria-mediated neuronal apoptosis induced by focal cerebral ischemia in rats and this neuroprotective potential is involved in phosphorylation of Akt and GSK-3β.

Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target

Medulloblastomas are the most common malignant brain tumors in children. They express high levels of COX-2 and produce PGE2, which stimulates tumor cell proliferation. Human cytomegalovirus (HCMV) is prevalent in the human population and encodes proteins that provide immune evasion strategies and promote oncogenic transformation and oncomodulation. In particular, HCMV induces COX-2 expression; STAT3 phosphorylation; production of PGE2, vascular endothelial growth factor, and IL-6; and tumor formation in vivo. Here, we show that a large proportion of primary medulloblastomas and medulloblastoma cell lines are infected with HCMV and that COX-2 expression, along with PGE2 levels, in tumors is directly modulated by the virus. Our analysis indicated that both HCMV immediate-early proteins and late proteins are expressed in the majority of primary medulloblastomas. Remarkably, all of the human medulloblastoma cell lines that we analyzed contained HCMV DNA and RNA and expressed HCMV proteins at various levels in vitro. When engrafted into immunocompromised mice, human medulloblastoma cells induced expression of HCMV proteins. HCMV and COX-2 expression correlated in primary tumors, cell lines, and medulloblastoma xenografts. The antiviral drug valganciclovir and the specific COX-2 inhibitor celecoxib prevented HCMV replication in vitro and inhibited PGE2 production and reduced medulloblastoma tumor cell growth both in vitro and in vivo. Ganciclovir did not affect the growth of HCMV-negative tumor cell lines. These findings imply an important role for HCMV in medulloblastoma and suggest HCMV as a novel therapeutic target for this tumor.

Chebulagic Acid Synergizes the Cytotoxicity of Doxorubicin in Human Hepatocellular Carcinoma Through COX-2 Dependant Modulation of MDR-1

Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) specific inhibitors are anti-inflammatory agents that have also shown to be useful in anticancer therapy. The effects of chebulagic acid (CA), a benzopyran tannin from Terminalia chebula having COX-2/5-LOX dual inhibitory properties, on the sensitivity of doxorubicin (Dox) in human hepatocellular carcinoma cell line HepG2 were studied in the present investigation. CA increased the accumulation of Dox in a concentration dependant manner and also enhanced the cytotoxicity of Dox in HepG2 cells by 20 folds. Quantitation of interaction by calculating Combination Index (CI) showed a strong synergistic interaction between CA and Dox in terms of cell growth inhibition. Calculation of dose reduction index (DRI) for CA-Dox combinations also showed a significant decrease in the dosage of Dox in the presence of CA. The induction of MDR1 expression by PGE(2), a metabolite of COX-2, and its downregulation by COX-2 knockdown or CA implies that the enhanced sensitivity of HepG2 cells to doxorubicin by CA is mediated by the downregulation of MDR1 expression, via COX-2-dependent mechanism. Further studies reveal the inactivation of signal transduction pathways involving Akt, ERK, JNK and p38 and the transcription factor NF-κB in the CA induced down regulation of MDR1. The present study shows the efficacy of CA to overcome MDR-1 mediated drug resistance in HepG2 cells through COX-2 dependant modulation of MDR-1.

Interleukin-17F affects cartilage matrix turnover by increasing the expression of collagenases and stromelysin-1 and by decreasing the expression of their inhibitors and extracellular matrix components in chondrocytes

Interleukin (IL)-17, a proinflammatory cytokine, is produced primarily by activated Th17 cells. IL-17 consists of six ligands that signal through five receptors (IL-17Rs); IL-17A and IL-17F share the highest homology in the family. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during cartilage remodeling whereas tissue inhibitor of metalloproteinases (TIMPs) inhibit the action of MMPs. In the present study, we examined the effect of IL-17F on the degradation and synthesis of the extracellular matrix in cartilage using human articular chondrocytes. We examined the effect of IL-17F on the expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and cyclooxygenases (COXs), as well as on prostaglandin E2 (PGE2) production. We also examined the indirect effect of PGE2 on the above IL-17F-induced/reduced components using NS-398, a specific inhibitor of COX-2. Cells were cultured with or without IL-17F in the presence or absence of either an IL-17R antibody or NS-398 for up to 28days. Expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and COXs at mRNA and protein levels was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. PGE2 production was determined by ELISA. The expression of all types of IL-17Rs was detected in chondrocytes. However, IL-17RE expression was extremely low, compared with other IL-17Rs. The expression of MMP-1, MMP-3, MMP-13, and COX-2 as well as PGE2 production were increased by addition of IL-17F, whereas the expression of IL-17RD, TIMP-2, TIMP-4, type II collagen, aggrecan, link protein, and COX-1 was decreased. The expression of IL-17RA, IL-17RB, IL-17RC, MMP-2, MMP-14, TIMP-1, and TIMP-3 was unaffected by addition of IL-17F. The IL-17R antibody blocked the stimulating/reducing effect of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, aggrecan, and link protein. NS-398 blocked the reducing effect of IL-17F on aggrecan expression, whereas it did not completely block the stimulating/reducing effects of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, and link protein. Our results suggest that IL-17F stimulates cartilage degradation by increasing the expression of collagenases (MMP-1 and -13) and stromelysin-1 (MMP-3) and by decreasing expression of their inhibitors (TIMP-2 and -4), type II collagen, aggrecan, and link protein in chondrocytes. Furthermore, our results suggest that the expression of aggrecan, link protein, and TIMP-4 decrease through the autocrine action of PGE2 in chondrocytes.

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.

Radiation-enhancement of MDA-MB-231 breast cancer cell invasion prevented by a cyclooxygenase-2 inhibitor.

Background:Recent evidences support that radiation can promote the invasion of cancer cells. As interactions between cancer cells and surrounding stromal cells can have an important role in tumour progression, we determined whether an irradiation to fibroblasts can enhance the invasiveness of breast cancer cells. The role of cyclooxygenase-2 (COX-2), an inflammatory enzyme frequently induced by radiotherapy, was investigated.Methods:Irradiated 3T3 fibroblasts were plated in the lower compartment of invasion chambers and used as chemoattractant for non-irradiated human breast cancer cell MDA-MB-231, which are oestrogen receptor negative (ER(−)) and the oestrogen receptor positive (ER(+)) MCF-7 cells. Stimulation of COX-2 expression in irradiated 3T3 cells was measured by a semi-quantitative qPCR and western blot. Capacity of the major product of COX-2, the prostaglandin E2 (PGE2), to stimulate the production of the matrix metalloproteinase-2 (MMP-2) and cancer cell invasion were assessed with a zymography gel and invasion chambers.Results:Irradiation (5 Gy) of 3T3 fibroblasts increased COX-2 expression and enhanced by 5.8-fold the invasiveness of non-irradiated MDA-MB-231 cells, while their migration was not modified. Addition of the COX-2 inhibitor NS-398 completely prevented radiation-enhancement of cancer cell invasion. Further supporting the potential role of COX-2, addition of PGE2 has increased cancer cell invasion and release of MMP-2 from the MDA-MB-231 cells. This effect of radiation was dependant on the expression of membrane type 1 (MT1)–MMP, which is required to activate the MMP-2, but was not associated with the ER status. Although irradiated fibroblasts stimulated the invasiveness of MDA-MB-231 ER(−) cells, no enhancement was measured with the ER(+) cell line MCF-7.Conclusions:Radiation-enhancement of breast cancer cell invasion induced by irradiated 3T3 fibroblasts is not dependant on the ER status, but rather the expression of MT1–MMP. This adverse effect of radiation can be prevented by a specific COX-2 inhibitor.