Cyclooxygenase Enzyme Activity Research Articles

Ellagic acid, a plant phenolic compound, activates cyclooxygenase-mediated prostaglandin production.

In recent years, ellagic acid (EA), a naturally-occurring phenolic compound richly contained in some of the human food sources such as Longan and Litchi, was reported to have a number of biological effects. Based on our earlier 3D-QSAR/CoMFA models for cyclooxygenase (COX) I and II, we hypothesize that EA may have the potential to modulate the catalytic activity of COX enzymes, and this hypothesis is examined in the present study. The results from both in vitro and in vivo experiments show that EA is an activator of COX enzyme-catalyzed production of prostaglandin E2, a representative prostaglandin tested. Mechanistically, EA can activate the peroxidase active site of COX enzymes by serving as a co-substrate, presumably for the reduction of protoporphorin IX with FeIV inside. The effect of EA is abrogated by the co-presence of galangin, which is known to bind to COX's peroxidase active site and thereby blocks the effect of the reducing co-substrates. In view of the known physiological functions of COX enzymes in the body, it is suggested that some of the pharmacological and/or toxicological effects of EA may result from an increased production of certain prostaglandins and their related derivatives in the body.

Anti-Inflammatory Effects of 5α,8α-Epidioxycholest-6-en-3β-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis.

Sea hares of Aplysia genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3β-ol (EnP(5,8)) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.

Hindlimb Denervation Alters the Regulation of Autophagy and Mitophagy

During chronic muscle disuse, fibre atrophy occurs concomitantly with reductions in mitochondrial content and function. The reduction in organelle content is due, in part, to depressed mitochondrial biogenesis and elevations in intracellular degradation processes. The regulation of mitochondrial biogenesis has been studied extensively. However, the processes that underlie the degradation of mitochondria have yet to be fully elucidated. The breakdown of the organelle is due to a selective form of autophagy, termed mitophagy. When a mitochondrion becomes dysfunctional it dissociates from the mitochondrial network within the muscle and is subsequently degraded by lysosomes. The objective of this work is to better understand the processes of autophagy and mitophagy in the context of muscle disuse. Accordingly, we employed a hindlimb denervation protocol in which we unilaterally sectioned the peroneal nerve of one hindlimb, using the contralateral limb as a control in Sprague‐Dawley rats. Protein measurements of autophagy and mitochondrial content markers were made at 1, 3 and 7 days post‐denervation. We observed significant 25–30% (p<0.05) reductions in extensor digitorum longus (EDL) and tibialis anterior (TA) muscle mass by 7 days post‐denervation. Significant elevations in the autophagy proteins Tfeb, Beclin1 and ATG‐7 were also measured 7 days post denervation, while mitochondrial protein COX subunit‐IV and COX enzyme activity were reduced by 25% and 40%, respectively. However, SS and IMF mitochondrial respiratory capacity was diminished, accompanied by enhanced reactive oxygen species production, as early as 1–3 days following denervation. To investigate the early changes in autophagy and mitophagy flux that may explain the alterations in mitochondrial content and function, a subgroup of animals was treated with colchicine (4mg/kg/day) for 2 days to inhibit autophagic breakdown in response to 1 and 3 days of denervation. LC3‐II autophagy flux was increased as early as 1 and 3 days post‐denervation, but diminished by 7 days, in part as a result of a denervation‐induced increase in LC3‐II expression. Mitophagy flux was increased in parallel in SS mitochondria, and by 3 days in the IMF subfraction. These changes appeared to coincide with the declines in mitochondrial function, suggesting that although mitophagy is elevated, it is not sufficient to eliminate all of the dysfunctional organelles. Furthermore, the immediate elevations in autophagy/mitophagy flux at 1 and 3 days occurred prior to the upregulation of autophagy and lysosomal markers at 7 days post‐denervation, suggesting that the intrinsic activity of the autophagosomal breakdown pathway is sufficient in the early time course, but is subsequently upregulated to meet the demands of prolonged denervation.Support or Funding InformationSupported by NSERC, CanadaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating the Connection Between Endogenous Heme Accumulation and COX2 Activity in Cancer Cells.

Heme, an iron-containing porphyrin, is fundamental for a variety of functions in cell homeostasis. Nevertheless, recent data indicate that dysregulation of heme metabolism might promote tumorigenesis. The intracellular heme pool is finely regulated through the control of heme synthesis, degradation, incorporation into hemoproteins and trafficking across membranes. All these processes might be potentially targeted to alter endogenous heme content in order to counteract cancer growth. Nevertheless, these putative therapeutic interventions have to take into account the possibility of undesired side effects, such as the over-activation of heme-dependent enzymes involved in cancer. Among them, cyclooxygenase-2 is a prostaglandin-producing hemoprotein, induced during inflammation and in different types of tumor, particularly in colorectal cancer. The aim of this study was to evaluate whether modulation of endogenous heme may affect cyclooxygenase-2 expression and activity, taking advantage of two different approaches able to alter heme levels: the silencing of the heme exporter Feline Leukemia Virus subgroup C receptor 1 and the induction of heme synthesis by 5-aminolevulinic acid administration. Our data demonstrate that the down-regulation of the heme exporter in colorectal cancer cells does not affect cyclooxygenase-2 expression and activity. Conversely, 5-aminolevulinic acid administration results in decreased cyclooxygenase-2 expression. However, the overall cyclooxygenase-2 enzymatic activity is maintained. The present work sheds light on the complex modulation of cyclooxygenase-2 by endogenous heme and support the idea that targeting heme metabolism could be a valuable therapeutic option against cancer.

Cyclooxygenase inhibitors for treating preterm labour: What is the molecular evidence? 1.

Preterm birth (<37 weeks of gestation) significantly increases the risk of neonatal mortality and morbidity. As many as half of all preterm births occur following spontaneous preterm labour. Since in such cases there are no known reasons for the initiation of labour, treatment of preterm labour (tocolysis) has sought to stop labour contractions and delay delivery. Despite some success, the use of cyclooxygenase (COX) inhibitors is associated with maternal/fetal side effects, and possibly increased risk of preterm birth. Clinical use of these drugs predates the collection of molecular and biochemical evidence in vitro, examining the expression and activity of COX enzymes in pregnant uterine tissues with and without labour. Such evidence is important to the rationale that COX enzymes are, or are not, appropriate targets for the tocolysis. The current study systematically searched existing scientific evidence to address the hypothesis that COX expression/activity is increased with the onset of human labour, in an effort to determine whether there is a rationale for the use of COX inhibitors as tocolytics. Our review identified 44 studies, but determined that there is insufficient evidence to support or refute a role of COX-1/-2 in the onset of preterm labour that supports COX-targeted tocolysis.

The role of Cox-2 and prostaglandin E2 receptor EP3 in pancreatic β-cell death.

Elevated levels of lipids, in particular saturated fatty acids, are known to be associated with type 2 diabetes (T2D) and to have a negative effect on β-cell function and survival. We bring new evidence indicating that palmitate up-regulates cyclooxygenase-2 (COX-2) expression levels in human islets and in MIN6 β cells, and that it is elevated in islets isolated from T2D donors. Both small interfering specific cyclooxygenase-2 small interfering RNA (siRNA) or the COX-2 inhibitor celecoxib significantly inhibited apoptosis induced by palmitate. Prostaglandin E2 (PGE2), the predominant product of COX-2 enzymatic activity, activates membrane receptors, which are members of the GPCR-family (EP1-EP4). In the present study, elevated expression of the PGE2 receptor subtype 3 (EP3) receptor was observed in β cells exposed to palmitate and in islets from individuals with T2D. Down-regulation of the pathway using EP3 siRNA or the specific L-798,106 antagonist markedly decreased the levels of palmitate-induced apoptosis. Altogether, our data put forward the COX-2-PGE2-EP3 pathway as one of the mediators of palmitate-induced apoptosis in β-cells.-Amior, L., Srivastava, R., Nano, R., Bertuzzi, F., Melloul, D. The role of Cox-2 and prostaglandin E2 receptor EP3 in pancreatic β-cell death.

Cyclosporine A binding to COX-2 reveals a novel signaling pathway that activates the IRE1\u03b1 unfolded protein response sensor

Cyclosporine, a widely used immunosuppressant in organ transplantation and in treatment of various autoimmune diseases, activates the unfolded protein response (UPR), an ER stress coping response. In this study we discovered a new and unanticipated cyclosporine-dependent signaling pathway, with cyclosporine triggering direct activation of the UPR. COX-2 binds to and activates IRE1α, leading to IRE1α splicing of XBP1 mRNA. Molecular interaction and modeling analyses identified a novel interaction site for cyclosporine with COX-2 which caused enhancement of COX-2 enzymatic activity required for activation of the IRE1α branch of the UPR. Cyclosporine-dependent activation of COX-2 and IRE1α in mice indicated that cyclosporine-COX-2-IRE1α signaling pathway was functional in vivo. These findings identify COX-2 as a new IRE1α binding partner and regulator of the IRE1α branch of the UPR pathway, and establishes the mechanism underlying cytotoxicity associated with chronic cyclosporine exposure.

Substrate-inactivated cyclooxygenase-2 is disposed of by exosomes through the ER-Golgi pathway.

Catalysis of arachidonic acid (AA) by cyclooxygenase-2 (COX-2) gives rise to a single product that serves as a precursor for all prostaglandins, which are central mediators of inflammation. Rapid up-regulation of COX-2 expression in response to pro-inflammatory stimuli is a well-characterized means of generating the large pool of prostaglandins necessary for inflammation. However, an efficient inflammatory process must also terminate rapidly and thus requires cessation of COX-2 enzymatic activity and removal of excess protein from the cell. Previous studies showed that COX-2 that has not been exposed to AA ('naive') degrades in the cellular proteasome. However, continuous exposure to AA induces suicide inactivation of COX-2 and its elimination no longer occurs in neither the proteasomal nor lysosomal machineries. In the present study, we show that either overexpressed or endogenously induced COX-2 is secreted via exosomes through the endoplasmic reticulum-Golgi pathway. We further find that excretion of COX-2 is significantly enhanced by prolonged exposure to AA. Genetic or chemical inhibition of COX-2 enzymatic activity has no effect on its secretion in the absence of substrate, but prevents the additional activity-dependent secretion. Finally, transfer of COX-2 to target cells only occurs in the absence of AA stimulation. Together, these results suggest that exosomal secretion of AA-activated COX-2 constitutes a means to remove damaged inactive COX-2 from the cell.

Molecular docking and pharmacological/toxicological assessment of a new compound designed from celecoxib and paracetamol by molecular hybridization.

Nonsteroidal anti-inflammatory drugs are commonly used worldwide; however, they have several adverse effects, evidencing the need for the development of new, more effective and safe anti-inflammatory and analgesic drugs. This research aimed to design, synthesize and carry out a pharmacological/toxicological investigation of LQFM-102, which was designed from celecoxib and paracetamol by molecular hybridization. To evaluate the analgesic effect of this compound, we performed formalin-induced pain, hot plate and tail flick tests. The anti-inflammatory effect of LQFM-102 was evaluated in carrageenan-induced paw oedema and pleurisy tests. The biochemical markers indicative of toxicity-AST, ALT, GSH, urea and creatinine-as well as the index of gastric lesion after prolonged administration of LQFM-102 were also analyzed. In addition, the interaction of LQFM-102 with COX enzymes was evaluated by molecular docking. In all experimental protocols, celecoxib or paracetamol was used as a positive control at equimolar doses to LQFM-102. LQFM-102 reduced the pain induced by formalin in both phases of the test. However, this compound did not increase the latency to thermal stimuli in the hot plate and tail flick tests, suggesting an involvement of peripheral mechanisms in this effect. Furthermore, LQFM-102 reduced paw oedema, the number of polymorphonuclear cells, myeloperoxidase activity and TNF-α and IL-1β levels. Another interesting finding was the absence of alterations in the markers of hepatic and renal toxicity or lesions of gastric mucosa. In molecular docking simulations, LQFM-102 interacted with the key residues for activity and potency of cyclooxygenase enzymes, suggesting an inhibition of the activity of these enzymes.

Role of Cyclooxygenase Pathway and Risk Associated with Non-Steroidal Anti-inflammatory Drugs Therapy in Cardiovascular Diseases

&lt;p&gt;Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase enzyme activity through different&lt;br /&gt;mechanisms and prevent inflammation. But they all have different risks associated with them. Some are associated with&lt;br /&gt;gastrointestinal bleeding and some are strongly allied with the cardiovascular risks. Cyclooxygenase enzyme regulates&lt;br /&gt;prostaglandin synthesis by converting arachidonic acid present at the sn-2 position of membrane phospholipids to&lt;br /&gt;prostaglandin H2. Prostaglandin H2 is the precursor of all prostaglandins. There are two isoforms of cyclooxygenase&lt;br /&gt;enzyme, cyclooxygenase-1 and cyclooxygenase-2 which differ in their active site due to an isoleucine to valine&lt;br /&gt;substitution at amino acid 523 in cyclooxygenase-2. Cyclooxygenase-1 is constitutively expressed in platelets&lt;br /&gt;where it helps in the formation of thromboxane whereas cyclooxygenase-2 is inductive form and is expressed in&lt;br /&gt;the endothelial cells due to shear stress and forms prostacyclins. Both thromboxanes and prostacyclins maintain&lt;br /&gt;the homeostasis of the vascular wall. During vascular injury prostacyclin production decreases as a result of which&lt;br /&gt;thromboxane synthesis increases in the platelets which leads to platelet aggregation. Although, being strongly&lt;br /&gt;associated with cardiovascular risks, NSAIDs are still prescribed to the patients to prevent pain according to their&lt;br /&gt;condition. So this review aims to summarise the mechanism of cyclooxygenase pathway, possible mechanism of&lt;br /&gt;action of NSAIDs and the risks of cardiovascular events associated with the use of NSAIDs.&lt;/p&gt;

Anti-inflammatory Activity of 8-Hydroxydaidzein in LPS-Stimulated BV2 Microglial Cells via Activation of Nrf2-Antioxidant and Attenuation of Akt/NF-κB-Inflammatory Signaling Pathways, as Well As Inhibition of COX-2 Activity.

It was demonstrated that isoflavones can cross the blood-brain barrier, making them desirable candidate agents for the prevention of neurological symptoms. 8-Hydroxydaidzein (8-OHD, 4',7,8-trihydoxyisoflavone) is an isoflavone found only in fermented soy food. Current results showed that 8-OHD inhibited LPS-stimulated production of nitric oxide (NO) and proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, by inhibiting gene expression in BV2 microglial cells. Moreover, 8-OHD markedly quenched reactive oxygen species (ROS) and activated NF-E2-related factor 2 (Nrf2) so as to upregulate expression of Phase II enzymes, including heme oxygenase (HO)-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and the modifier subunit of glutamate cysteine ligase (GCLM). 8-OHD also suppressed LPS-stimulated phosphorylation of Akt and NF-κB-p65. The anti-inflammatory activity of 8-OHD was attenuated by the HO-1 inhibitor zinc protoporphyrin (Znpp) but augmented by the PI3K/Akt inhibitor LY294002. 8-OHD also diminished LPS-induced prostaglandin E2 (PGE2) production without affecting cyclooxygenase (COX)-2 expression. In vitro assay shows that 8-OHD displayed mixed-type inhibition of COX-2 with an IC50 of 8.9 ± 1.2 μM. These data suggest that the anti-inflammatory activity of 8-OHD may be associated with the activation of Nrf2/HO-1 and attenuation of Akt/NF-κB signaling pathways as well as inhibition of COX-2 enzyme activity. In conclusion, 8-OHD, a potent Nrf2 activator, Akt/NF-κB activation suppressor, and COX-2 enzyme inhibitor, may have health-promoting effects for mitigating microglia activation and preventing neuroinflammation.

The role of bile acids in cellular invasiveness of gastric cancer

BackgroundBile acids have been implicated in the development of digestive tract malignancy by epidemiological, clinical and animal studies. The growth and transformation signaling by most of the bile acids is thought to be related to the induced cyclooxygenase-2 (COX-2) expression and increased production of prostaglandin E2 (PGE2). The highly hydrophobic bile acids such as chenodeoxycholic acid (CD) and deoxycholic acid can promote carcinogenesis and stimulate the invasion of colon cancer cells. On the contrary, ursodeoxycholic acid (UDCA), a less hydrophobic stereoisomer of CD, inhibits proliferation and induces apoptosis in colon cancer cells. We examined the effects of bile acid on human gastric cancer cells MKN-74.MethodsEarly-passage human gastric cancer MKN-74 cells were used for drug treatment, preparation of whole cell lysates, subcellular extracts and Western blot analysis. The levels of PGE2 released by the cells were measured by enzyme inummoassay to indicate COX-2 enzymatic activity. Cellular invasion assay was performed in Boyden chamber.ResultsExposure of CD led to activation of protein kinase C (PKC) alpha, increased COX-2 expression and increased PGE2 synthesis. The induced COX-2 protein expression could be detected within 4 h exposure of 200 μM CD, and it was dose- and time-dependent. PGE2 is the product of COX-2, and has been reported to cause tumor invasion and angiogenesis in animal study. Safingol (SAF), a PKC inhibitor, suppressed the COX-2 protein expression and PGE2 production by CD in MKN-74. Furthermore, UDCA suppressed PGE2 production by CD but did not affect COX-2 protein expression induced by CD. Using a Boyden chamber invasion assay, both SAF and UDCA impeded CD induced tumor invasiveness of MKN-74 by 30–50%.ConclusionsOur results indicate that signaling of hydrophobic bile acid such as CD in gastric cancer cells is through PKC activation and COX-2 induction, which leads to increased cellular invasion. By perturbing the bile acid pool, UDCA attenuates CD-induced PGE2 synthesis and tumor invasiveness.

Studi Insilico Potensi Minyak Astiri dan Ekstrak Etanol Daun Annona muricata Sebagai Calon Herbal Terstandart Untuk Analgesik dan Antiinflamasi

ABSTRACTInflammatory reactions give rise to a series of processes and symptoms that can cause pain and damage to nearby cells. Inflammatory reactions are mediated by inflammatory and proinflammatory cytokines. NSAID class (non-steroidal anti-inflammatory drug) drugs have been developed to inhibit the inflammatory process by suppressing COX-1 and / or COX-2 enzyme activity. However, this drug can cause gastric mucosal irritation. the ethanol extract of Annona muricata leaf has an analgesic and anti-inflammatory effect[3].The purpose of this research is to know analgesic and antiinflamatory activity from ethanol extract and essential oil of Annona muricata leaf.The method used was the study of in silico by molecular docking of active compounds in ethanol extracts and essential oils to the COX-1, COX-2, phospolipase-A, and TNF-alpha receptor molecules as target molecules. The molecular docking results show that Annona muricata ethanol extract can act as an anti-inflammatory and analgesic, especially through strong bonds between "rutine" compounds with COX-2 receptors with affinity -10 kcal / mold and "quercetin" with affinity bindings -9.8 kkal.mol, better than dexametason (binding affinity -9.6 kcal / mol). Keywords:Inflamation, COX-1, COX-2, phospolipase-A, TNF-alfa, docking

Anti-neuroinflammatory effects of Ginkgo biloba extract EGb761 in LPS-activated primary microglial cells

BackgroundNeuroinflammation is a key factor of Alzheimer's disease (AD) and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system (CNS). They play an essential role in the maintenance of homeostasis and responses to neuroinflammation. Ginkgo biloba extract EGb 761 is one of the most commonly used natural medicines owing to its established efficacy and remarkable biological activities especially in respect to CNS diseases. However, only few studies have addressed the effects and mechanisms of Ginkgo biloba extract in microglia activation. MethodsWe measured the production of pro-inflammatory mediators and cytokines by ELISA and analyzed gene expressions by qRT-PCR and Western Blot in LPS treated cultured primary rat microglia. ResultsThe Ginkgo biloba extract EGb 761 significantly inhibited the release of prostaglandin E2 (PGE2) and differentially regulated the levels of pro-inflammatory cytokines. The inhibition of LPS-induced PGE2 release in primary microglia was partially dependent on reduced protein synthesis of mPGES-1 and the reduction in the activation of cytosolic phospholipase A2 (cPLA2) without altering COX-2 enzymatic activity, inhibitor of kappa B alpha (IkappaBalpha) degradation, and the activation of multiple mitogen activated protein kinases (MAPKs). Altogether, we showed that EGb 761 reduces neuro-inflammatory activation in primary microglial cells by targeting PGE2 release and cytokines. ConclusionGinkgo biloba extract EGb 761 displayed anti-neuroinflammatory activity in LPS-activated primary microglia cells. EGb 761 was able to reduce neuroinflammatory activation by targeting the COX/PGE2 pathway. This effect might contribute to the established clinical cognitive efficacy in Alzheimer's disease, vascular and mixed dementia.

Cyclooxygenase inhibitors for treating preterm labour? What is the evidence?

BackgroundPreterm labour, a condition characterized by regular uterine contractions and cervical dilation prior to 37 weeks of pregnancy can result in premature birth of babies that may not be fully developed and/or possess serious health problems such as cerebral palsy and learning disabilities. Is associated with an increase in uterine prostaglandins (PGs) synthesis by cyclooxygenase (COX) enzymes which promotes inflammatory and contractile processes that drive labour. For this reason, COX inhibitors (e.g. indomethacin) have been used to treat/prevent preterm labour. Despite limited success, tocolysis by COX‐inhibition carries serious adverse fetal effects due to inhibition of prostaglandin‐dependent developmental homeostatic functions. The more recent discovery of a second, inducible COX isoform, COX‐2, presented an opportunity to spare homeostatic prostaglandin by selective inhibition of COX‐2. However, subsequent clinical studies have called into question the appropriateness of COX‐2 selective inhibition. This study reviewed the molecular evidence concerning the role of COX‐2 in human parturition that underpins the rationale of use of COX‐2 inhibitors in the treatment of preterm labour and presents evidence of COX‐2 gene expression in term and preterm human myometrium.MethodsFor our systematic literature review, we searched MEDLINE and EMBASE databases to identify papers reporting molecular evidence of COX enzyme expression and activity in gestational tissues at term and preterm. Two independent reviewers evaluated abstracts, and qualifying references were reviewed in full text. Data were extracted using a standardized questionnaire. COX‐2 gene expression analysis was conducted on samples of myometrium and decidua from women at term (after 37 weeks) and preterm (below 37 weeks) of gestation, with and without labour using reverse transcriptase‐polymerase chain reaction technique (RT‐PCR).ResultsThirty‐five studies were included in the review. Eight studies on fetal membranes (amnion and chorion) reported significantly higher COX expression or activity with labour. However, in the myometrium and decidua, which are the most relevant therapeutic target tissues for uterine contraction, nine reported no labour‐associated differences COX expression. In the myometrium, four studies reported higher levels of COX mRNA with labour, but did not consider protein expression. Our data show spatial (upper versus lower segment) and temporal (labour versus non‐labour) demonstrate differences in COX‐2 expression; at term, expression of COX‐2 is higher in lower segment myometrium and decidua samples from women in term labour (n=16) compared to term non‐labour (n=18), but no significant differences were observed for upper segment samples. Similarly, COX‐2 expression was higher in lower segment preterm labourThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The overexpression of cytochrome c oxidase subunit 6C activated by Kras mutation is related to energy metabolism in pancreatic cancer

Background: Kras mutation is frequently detected in pancreatic cancers and leads to altered energy metabolite. Here we investigated molecule markers related with Kras mutation, which could be used as developing new target for Kras mutant driven cancer. Methods: A knockin BxPC-3/ Kras G12D cell line was constructed by CRISPR/Cas9 system. Proliferation and metabolite characterization in BxPC-3/ Kras G12D was compared with wild type BxPC-3 by using colony formation assay and mitochondrial dyes. The differential genes were screened using mitochondrial metabolite-related genes PCR array. The expression of COX6C was confirmed by real time polymerase chain reaction (RT-PCR) and western blot. COX6C expression in 30 pairs of tissue microarray of pancreatic carcinoma and matched adjacent tissues was analyzed by immunohistochemistry. ATP production stimulated by metabolites substrate assay was carried out to investigate whether the decreased COX6C by siRNA transfection affected the metabolite characterization of pancreatic cancer cells. Results: The BxPC-3/ Kras G12D displayed faster proliferation, increased mitochondrial mass and ATP, elevated mitochondrial membrane potential than BxPC-3. Using mitochondrial metabolite-related genes array, we identified COX6C was an up-regulated gene driven by Kras G12D . There was a notable difference of COX enzyme activity between BxPC-3 and BxPC-3/ Kras G12D . The overexpression of COX6C was found in pancreatic cancer tissues. Using COX6C siRNA-mediated knockdown, the cell viability, COX enzyme activity and ATP production of BxPC-3/ Kras G12D cells significantly decreased. Conclusions: These results suggested that activation of Kras G12D in pancreatic cancer cells increased the COX activity and ATP production of mitochondrial via up-regulation of COX6C. This regulatory subunit of COX may have utility as a Ras effector target for development of anti-pancreatic cancer therapeutics.

Anti-neuroinflammatory effects of cudraflavanone A isolated from the chloroform fraction of Cudrania tricuspidata root bark

Context: Cudrania tricuspidata Bureau (Moraceae) is an important source of traditional Korean and Chinese medicines used to treat neuritis and inflammation.Objective: The anti-neuroinflammatory effects of cudraflavanone A isolated from a chloroform fraction of C. tricuspidata were investigated in LPS-induced BV2 cells.Materials and methods: Cudraflavanone A was isolated from the root of C. tricuspidata, and its structure was determined by MS and NMR data. Cytotoxicity of the compound was examined by MTT assay, indicating no cytotoxicity at 5–40 μM of cudraflavanone A. NO concentration was measured by the Griess reaction, and the levels of PGE2, cytokines and COX-2 enzyme activity were measured by each ELISA kit. The mRNA levels of cytokines were analysed by quantitative-PCR. The expression of iNOS, COX-2, HO-1, NF-κB, MAPKs and Nrf2 was detected by Western blot.Results: Cudraflavanone A had no major effect on cell viability at 40 μM indicating 91.5% viability. It reduced the production of NO (IC50 = 22.2 μM), PGE2 (IC50 = 20.6 μM), IL-1β (IC50 = 24.7 μM) and TNF-α (IC50 = 33.0 μM) in LPS-stimulated BV2 cells. It also suppressed iNOS protein, IL-1β and TNF-α mRNA expression. These effects were associated with the inactivation of NF-κB, JNK and p38 MAPK pathways. This compound mediated its anti-neuroinflammatory effects by inducing HO-1 protein expression via increased nuclear translocation of Nrf2.Discussion and conclusions: The present study suggests a potent effect of cudraflavanone A to prevent neuroinflammatory diseases. Further investigation is necessary to elucidate specific molecular mechanism of cudraflavanone A.

The Effect of Sodium Metabisulphite on Locomotor Activity in the Experimental Model of Parkinson’s Disease: The Role of Cyclooxygenase

Parkinson’s disease (PD) is characterized by a progressive and selective loss of dopaminergic neurons in substantia nigra (SN). PD is the second most common neurodegenerative disease after Alzheimer disease. Although the exact cause of disease is not known, oxidative stress, neuroinflammation, mitochondrial dysfunction and microglial activation are observed in PD. The aim of this study was to investigate the mechanism underlying possible toxic effects of sulphite on the experimental model of PD. Male Wistar rats were assigned into one of four groups Control (Control), Sulphite-treated (Sulphite), 6-hydroxydopamine (6-OHDA)-injected (6-OHDA) and sulphite-treated and 6-OHDA-injected (6-OHDA+Sulphite). Sodium metabisulphite was administered at a dose of 100 mg/kg/day for 45 days by gavage. Experimental PD was created stereotactically via the unilateral infusion of 6-OHDA into the medial forebrain bundle (MFB). 6-OHDA-injected rats exhibited reduced locomotor activity compared to control. A significant increase in catalepsy was found in the 6-OHDA-injected group as compared to the the control group. Plasma levels of S-sulfonate increased in Sulphite and 6-OHDA+Sulphite groups as compared to their respective controls. Cyclooxygenase (COX) enzyme activity, prostaglandin E2 (PGE2) and nuclear factor kappa B (NF-κB) levels increased in the 6-OHDA group as compared to control. The tyrosine hydroxylase (TH)-positive immunostainingdecreased significantly in the 6-OHDA-injected group where the sulphite and control groups had almost the same immunoreaction for the dopaminergic neurons. In conclusion, sulphite is not a potentially aggravating factor for the activity of COX and the levels of PGE2 or NF-κB in a 6-OHDA-induced experimental model of Parkinson’s disease.

Metamizol - aktuelno stanje bezbednosti i efikasnosti

Metamizole is a nonsteroidal anti-inflammatory drug, with analgesic, antipyretic, spasmolytic, and weak anti-inflammatory properties. The analgesic effect of metamizole seems to be based on the inhibition of cyclooxygenase enzyme activity and stimulation of cannabinoid receptors. Its use is still controversial, mainly due to agranulocytosis, metamizole-induced serious adverse reaction. While in many countries it is withdrawn from the market, in some countries metamizole is available as a medication prescribed for strictly defined indications, while in the others like Mexico, Brazil and China it can be obtained as an Over-The-Counter drug. The most common adverse effects of metamizole are the skin and subcutaneous tissue disorders. Metamizole appears to be of similar efficacy to analgesics which are frequently used in the treatment of moderate to severe postoperative pain.

Protective role of β-patchoulene from Pogostemon cablin against indomethacin-induced gastric ulcer in rats: Involvement of anti-inflammation and angiogenesis

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) are most widely used as effective anti-inflammatory agents. However, their clinical application brings about inevasible gastrointestinal side effects. Pogostemon cablin is a traditional herbal medicine used for the treatment of gastrointestinal diseases in China. One of its representative components, the tricyclic triterpenoid β-patchoulone (β-PAE) has demonstrated great anti-inflammatory activity and gastroprotective effect against ethanol-induced gastric injury, but its protective effect against gastric ulcer induced by indomethacin is still unknown. PurposeTo assess the protective effect of β-PAE against ulcer produced by indomethacin and reveal the underlying pharmacological mechanism. Study designWe used an indomethacin-induced gastric ulcer model of rats in vivo. MethodsGastroprotective activity of β-PAE (10, 20, 40 mg/kg, i.g.) was estimated via indomethacin-induced gastric ulcer model in rats. Histopathological and histochemical assessment of ulcerated tissues were performed. Protein and mRNA expression were determined by Elisa, Western blotting and qRT-PCR. Resultsβ-PAE could inhibit ulcer formation. Histopathological and histochemical assessment macroscopically demonstrated that β-PAE alleviates indomethacin-induced gastric ulceration in dose-dependent manner. After administration of β-PAE, elevated tumor necrosis factor -α level was significantly decreased and the phosphorylation of JNK and IκB was markedly inhibited. β-PAE suppressed the levels of E-selectin, P-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule and monocyte chemoattractant protein 1, as well as myeloperoxidase. Meanwhile, β-PAE increased cyclooxygenase enzyme activities (COX-1 and COX-2) to enhance the production of prostaglandin E2. Proangiogenic protein, vascular endothelial growth factor and its receptor fms-like tyrosine kinase-1 mRNA expression were promoted while anti-angiogenic protein, endostatin-1 and its receptor ETAR mRNA expression were decreased. Conclusionβ-PAE may provide gastroprotection in indomethacin-induced gastric ulcer in rats by reducing inflammatory response and improving angiogenesis.