Upregulation Of Cyclooxygenase-2 Research Articles

Abstract 1333: Dual blockade of the EP2 and EP4 PGE2 receptors with TPST-1495 is an optimal approach for drugging the prostaglandin pathway

Abstract Prostaglandin E2 (PGE2) is a bioactive lipid produced by tumor cells that drives disease progression through stimulating tumor proliferation, enhancing angiogenesis and suppressing immune function in the TME1, 2. PGE2 is also a mediator of adaptive resistance to immune checkpoint inhibitor therapy via the upregulation of cyclooxygenase-2 (COX-2). While the role of PGE2 signaling in cancer is clear, how best to inhibit PGE2 for cancer treatment remains under investigation. Inhibition of COX-1 and/or COX-2 has shown promising results in observational studies and meta-analyses, but inconsistent results in prospective studies. PGE2 signals through four receptors, EP1-4, that are variably expressed on tumor and immune cells and have distinct biological activities. The EP2 and EP4 receptors signal through cAMP and drive pro-tumor activities, while the EP1 and EP3 receptors signal through calcium flux and IP3 and drive immune activation and inflammation. While COX-2 and single EP inhibitors continue to be developed, the nature of PGE2 signaling supports our rationale to inhibit PGE2 by dual antagonism of the pro-tumor EP2/EP4 receptors, while sparing the pro-immune EP1/EP3 receptors. To our knowledge, TPST-1495 is the first clinical-stage dual inhibitor of both the EP2 and EP4 receptors. In mouse and human whole blood assays, dual blockade of EP2 and EP4 receptors with TPST-1495 reversed PGE2-mediated suppression of LPS-induced TNF-α, while single receptor antagonists were unable to block suppression at higher PGE2 concentrations. Similarly, in murine and human T cells in vitro, TPST-1495 inhibited PGE2-mediated suppression, resulting in a significant increase of IFN-γ production in response to stimulation with cognate peptide antigen. In vivo, TPST-1495 monotherapy significantly reduced tumor outgrowth in CT26 tumor-bearing mice and correlated with increased tumor infiltration by NK cells, CD8+ T cells, AH1-specific CD8+ T cells, and other anti-tumor myeloid and adaptive immune cell populations. The relative contribution of increased immune infiltration may be simultaneously dependent on the immunogenicity of the tumor model and on the direct antitumor effect of TPST-1495, because we also observed significant tumor regression in metastatic burden in the LS174T xenograft model in NSG mice as well as CT26 tumors in RAG2-/- animals, both of which are deficient in immune cell development. To that end, TPST- 1495 monotherapy significantly decreased the tumor burden compared to COX2 inhibition and EP2 or EP4 single antagonism in the Adenomatous Polyposis (APCmin/+) model, a model that is hypo-responsive to PD-1 monotherapy. TPST-1495 is currently being evaluated in an ongoing Phase 1 first-in-human study (NCT04344795) to characterize PK, PD, safety, and to identify a recommended phase 2 dose for expansion cohorts in key indications and biomarker-selected patients. Citation Format: Brian Francica, Justine Lopez, Anja Holtz, Dave Freund, Dingzhi Wang, Amanda Enstrom, Raymond Dubois, Francielle Kipper, Dipak Panigrahy, Chan Whiting, Sam Whiting, Thomas W. Dubensky. Dual blockade of the EP2 and EP4 PGE2 receptors with TPST-1495 is an optimal approach for drugging the prostaglandin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1333.

Ferroptosis in brain microvascular endothelial cells mediates blood-brain barrier disruption after traumatic brain injury

Ferroptosis is a newly recognized form of regulated cell death. Recently, growing evidence has shown that ferroptosis is involved in the pathogenesis of traumatic brain injury (TBI). However, less attention has been paid to its role in brain microvascular endothelial cells (BMVECs) and blood-brain barrier (BBB) damage, the central pathological process in secondary brain injury of TBI. Here, we established a mechanical stretch injury bEnd.3 model and a Controlled Cortical Impact (CCI) mouse model to explore the ferroptosis-related markers in brain endothelial cells after TBI in vitro and in vivo. From the results of RNA-seq analysis, RT-qPCR and immunostaining, glutathione peroxidase 4 (GPX4) downregulation, Cyclooxygenase-2 (COX-2) upregulation, and iron accumulation were observed in brain endothelial cells after TBI both in vitro and in vivo. Furthermore, we utilized Ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, to investigate the protective effects of ferroptosis inhibition on BBB disruption and neurological deficits. From the results of immunostaining, transmission electron microscopy (TEM), and western blotting, we demonstrated that Fer-1 significantly reduced BMVECs death, BBB permeability, and tight junction loss at 3 days after TBI. The neurological tests including grid walking, rotarod test, and wire-hanging test showed that Fer-1 administration exerted neuroprotective effects in the early stage of TBI. Our findings provided evidences for inhibition of BMVECs ferroptosis as a promising therapeutic target against TBI-induced BBB disruption.

Mechanism of Glucose Water as a Neural Injection: A Perspective on Neuroinflammation.

The entrapment of peripheral nerves is associated with chronic neuroinflammation and neuropathic pain, and perineural injection therapy with glucose is emerging as an effective treatment for peripheral entrapment neuropathy. However, the mechanism underlying the pharmacological effect of glucose on nerves remains unclear. One of the hypothesized mechanisms is that glucose reduces neurogenic inflammation. Therefore, we investigated the effects of high glucose concentrations on cytokine-induced neuroinflammation in vitro. Human SH-SY5Y neuronal cells were challenged with 10 ng/mL TNF-α for 16 h and subsequently treated with different glucose concentrations (0–25 mM) for 24 h. Cell viability was evaluated using the diphenyltetrazolium bromide assay, and proinflammatory cytokine levels were assessed using ELISA and quantitative PCR. In addition, mRNA levels of NF-κB and cyclooxygenase-2 were analyzed using quantitative PCR. Exposure to 10 ng/mL TNF-α resulted in decreased viability of SH-SY5Y cells and significant upregulation of IL-6, IL-1β, NF-κB, and cyclooxygenase-2. Subsequent exposure to high glucose levels (25 mM) markedly reduced the upregulation of IL-6, IL-1β, cyclooxygenase-2, and NF-κB, and restored the functional metabolism of SH-SY5Y cells, compared with that of the normal glucose control. Our findings suggest that high glucose concentrations can mitigate TNF-α-induced NF-κB activation, upregulation of proinflammatory cytokines, and metabolic dysfunction.

A phase 1 study of TPST-1495 as a single agent and in combination with pembrolizumab in subjects with solid tumors.

TPS2696 Background: Prostaglandin E2 (PGE2) is a bioactive lipid that promotes cancer through diverse mechanisms including stimulating tumor proliferation, enhancing angiogenesis and suppressing immune function in the tumor microenvironment. PGE2 produced by tumor cells through upregulation of cyclooxygenase-2 (COX-2) is also a key mediator of adaptive resistance to immune checkpoint inhibitor therapy. While PGE2 signaling is important in cancer, how best to inhibit PGE2 for cancer treatment is under investigation. Inhibition of COX enzymes (e.g., with NSAIDS) has shown promise in large observational studies, but inconsistent results in prospective studies. Importantly, COX inhibition alters multiple prostanoids beyond PGE2, resulting in toxicity that limits therapeutic dosing for cancer. PGE2 signals through four receptors, EP1-4, that are variably expressed and have distinct activities. The tumor promoting and immunosuppressive activities of PGE2 predominantly arise from signaling through the EP2 and EP4 receptors, while signaling through the EP1 and EP3 receptors generally is pro-inflammatory. TPST-1495 is designed to be an oral, highly specific, antagonist of the EP2 and EP4 receptors, sparing the EP1 and EP3 receptors and the COX enzymes. Preclinical studies suggest that blocking EP2 and EP4 with TPST-1495 inhibits tumor proliferation and stimulates anti-cancer immunity better than inhibiting all 4 receptors together, the EP2 or EP4 receptors singly, or the upstream COX-2 enzyme. Methods: TPST-1495-001 is a first-in-human Phase 1 study (NCT04344795). In the Dose and Schedule Optimization Stage, the primary objectives are to characterize the safety and tolerability (including dose limiting toxicities) and determine the recommended phase 2 dose (RP2D) of TPST-1495 as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. Additional objectives include characterization of PK, PD, and evaluation of potential biomarkers, including through paired (pre- and on-treatment) tumor biopsies. Monotherapy dose-finding employs a modified 3+3 design evaluating BID and QD TPST-1495 schedules along with continuous or intermittent (Days 1-5 every 7 days) dosing. For the pembrolizumab combination, the starting dose and schedule of TPST-1495 are determined by safety, PK and PD of monotherapy. Indication-specific Expansion Stage cohorts will evaluate TPST-1495 at the selected RP2D and schedule for both monotherapy and combination in endometrial cancer, squamous cell carcinoma of the head and neck, and microsatellite stable colorectal cancer (combination only), as well as in a biomarker-specific cohort enrolling patients with pathogenic tumor PIK3CA gene mutation. Dose and Schedule Optimization enrollment (monotherapy and combination) is ongoing at abstract submission while Expansion Stages are planned after identification of the RP2Ds. Clinical trial information: NCT04344795.

Lipocalin 2 Deficiency Alters Prostaglandin Biosynthesis and mTOR Signaling Regulation of Thermogenesis and Lipid Metabolism in Adipocytes.

Apart from a well-known role in the innate immune system, lipocalin 2 (Lcn2) has been recently characterized as a critical regulator of thermogenesis and lipid metabolism. However, the physiological mechanism through which Lcn2 regulates cellular metabolism and thermogenesis in adipocytes remains unknown. We found that Lcn2 expression and secretion are significantly upregulated by arachidonic acid (AA) and mTORC1 inhibition in differentiated inguinal adipocytes. AA-induced Lcn2 expression and secretion correlate with the inflammatory NFkB activation. Lcn2 deficiency leads to the upregulation of cyclooxygenase-2 (COX2) expression, as well as increased biosynthesis and secretion of prostaglandins (PGs), particularly PGE2 and PGD2, induced by AA in adipocytes. Furthermore, Lcn2 deficiency affects the mTOR signaling regulation of thermogenic gene expression, lipogenesis, and lipolysis. The loss of Lcn2 dismisses the effect of mTORC1 inhibition by rapamycin on COX2, thermogenesis genes, lipogenesis, and lipolysis, but has no impact on p70 S6Kinase-ULK1 activation in Lcn2-deficient adipocytes. We conclude that Lcn2 converges the COX2-PGE2 and mTOR signaling pathways in the regulation of thermogenesis and lipid metabolism in adipocytes.

COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis.

Giardia duodenalis, the causative agent of giardiasis, is among the most important causes of waterborne diarrheal diseases around the world. Giardia infection may persist over extended periods with intestinal inflammation, although minimal. Cyclooxygenase (COX)-2 is well known as an important inducer of inflammatory response, while the role it played in noninvasive Giardia infection remains elusive. Here we investigated the regulatory function of COX-2 in Giardia-induced pro-inflammatory response and defense-related nitric oxide (NO) generation in macrophage-like cell line, and identified the potential regulators. We initially found that Giardia challenge induced up-regulation of IL-1β, IL-6, TNF-α, prostaglandin (PG) E2, and COX-2 in macrophages, and pretreatment of the cells with COX-2 inhibitor NS398 reduced expressions of those pro-inflammatory factors. It was also observed that COX-2 inhibition could attenuate the up-regulated NO release and inducible NO synthase (iNOS) expression induced by Giardia. We further confirmed that Giardia-induced COX-2 up-regulation was mediated by the phosphorylation of p38 and ERK1/2 MAPKs and NF-κB. In addition, inhibition of reactive oxygen species (ROS) by NAC was shown to repress Giardia-induced activation of MAPK/NF-κB signaling, up-regulation of COX-2 and iNOS, increased levels of PGE2 and NO release, and up-expressions of IL-1β, IL-6, and TNF-α. Collectively, in this study, we revealed a critical role of COX-2 in modulating pro-inflammatory response and defense-related NO production in Giardia-macrophage interactions, and this process was evident to be controlled by ROS-dependent activation of MAPK/NF-κB signaling. The results can deepen our knowledge of anti-Giardia inflammatory response and host defense mechanisms.

Inhibition of DDX3 and COX-2 by forskolin and evaluation of anti-proliferative, pro-apoptotic effects on cervical cancer cells: molecular modelling and in vitro approaches.

Several studies have reported up-regulation of both cyclooxygenase-2 (COX-2) and DEAD-box RNA helicase3 (DDX3) and have validated their oncogenic role in many cancers. Inhibition of COX-2 and DDX3 offers a potential pharmacological strategy for prevention of cancer progression. The COX-2 isoform is expressed in response to pro-inflammatory stimuli in premalignant lesions, including cervical tissues. This study elucidates the potential role of plant derived compound Forskolin (FSK) in plummeting the expression of COX-2 and DDX3 in cervical cancer. To establish this, the cervical cancer cells were treated with the FSK compound which induced a dose dependent significant inhibition of COX-2 and DDX3 expression. The FSK treatment also significantly induced apoptosis in cancer cells by modulating the expression of apoptotic markers like caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, full length-poly ADP ribose polymerase (PARP), cleaved-poly ADP ribose polymerase (C-PARP) and Bcl2 in dose dependent manner. Further FSK significantly modulated the cell survival pathway Phosphatidylinositol 3-kinase (PI3-K)/Akt signalling pathway upon 24h of incubation in cervical cancer cells. The molecular docking studies revealed that the FSK engaged the active sites of both the targets by interacting with key residues.

Reversal of NADPH Oxidase-Dependent Early Oxidative and Inflammatory Responses in Chronic Obstructive Pulmonary Disease by Puerarin.

In the present study, we investigated effects of Puerarin on the early oxidative and inflammatory responses in the lung triggered by acute cigarette smoking (ACS). C57BL/6 mice were exposed to ACS for 1 hr in the presence or absence of Puerarin and harvested at 2, 6, and 24 hours. ACS induced significant increases in superoxide production in mouse lungs at 2 and 6 hours; and superoxide production was also elevated in a time and concentration dependent manner in cigarette smoke extract (CSE) stimulated human small airway epithelial cells (HSAECs), which was dose-dependently abrogated by Puerarin. ACS exposure upregulated NOX1, NOX2, and NOX4 protein expression in mouse lungs. Likewise, NOX1 and NOX4 were upregulated in CSE-stimulated HSAECs. These responses were significantly or completely attenuated by Puerarin. ACS induced significant infiltrations of neutrophils and macrophages in mouse lung parenchyma and BAL fluid, which were completely or significantly abrogated by Puerarin, so was the activation of the NF-кB pathway and the upregulation in inflammatory mediators including TNF-α, KC (murine homolog of IL-8), COX-2, IL-6 and MCP-1. Nuclear translocation of p65, IL-8 secretion, and upregulation of COX-2 in CSE stimulated HSAECs were also markedly attenuated by Puerarin. Moreover, ACS or CSE stimulated upregulation in reactive oxygen species (ROS) production and expression of inflammatory mediators were alleviated by ROS scavenger TEMPO in vivo and vitro, with no synergy combining with Puerarin, indicating that the effects of Puerarin are redox-sensitive following activation of NOX. In summary, our data for the first time demonstrate that Puerarin robustly attenuates NOX isoform-dependent ROS production and inflammatory activation in ACS exposed mice and CSE treated HSAECs, indicating that Puerarin might be used as a robust therapeutic agent for early or early stage COPD.

Thrombin Induces COX-2 and PGE2 Expression via PAR1/PKCalpha/MAPK-Dependent NF-kappaB Activation in Human Tracheal Smooth Muscle Cells.

The inflammation of the airway and lung could be triggered by upregulation cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induced by various proinflammatory factors. COX-2 induction by thrombin has been shown to play a vital role in various inflammatory diseases. However, in human tracheal smooth muscle cells (HTSMCs), how thrombin enhanced the levels of COX-2/PGE2 is not completely characterized. Thus, in this study, the levels of COX-2 expression and PGE2 synthesis induced by thrombin were determined by Western blot, promoter-reporter assay, real-time PCR, and ELISA kit. The various signaling components involved in the thrombin-mediated responses were differentiated by transfection with siRNAs and selective pharmacological inhibitors. The role of NF-κB was assessed by a chromatin immunoprecipitation (ChIP) assay, immunofluorescent staining, as well as Western blot. Our results verified that thrombin markedly triggered PGE2 secretion via COX-2 upregulation which were diminished by the inhibitor of thrombin (PPACK), PAR1 (SCH79797), Gi/o protein (GPA2), Gq protein (GPA2A), PKCα (Gö6976), p38 MAPK (SB202190), JNK1/2 (SP600125), MEK1/2 (U0126), or NF-κB (helenalin) and transfection with siRNA of PAR1, Gqα, Giα, PKCα, JNK2, p38, p42, or p65. Moreover, thrombin induced PAR1-dependent PKCα phosphorylation in HTSMCs. We also observed that thrombin induced p38 MAPK, JNK1/2, and p42/p44 MAPK activation through a PAR1/PKCα pathway. Thrombin promoted phosphorylation of NF-κB p65, leading to nuclear translocation and binding to the COX-2 promoter element to enhance promoter activity, which was reduced by Gö6976, SP600125, SB202190, or U0126. These findings supported that COX-2/PGE2 expression triggered by thrombin was engaged in PAR1/Gq or Gi/o/PKCα/MAPK-dependent NF-κB activation in HTSMCs.

Chemotherapy-induced COX-2 upregulation by cancer cells defines their inflammatory properties and limits the efficacy of chemoimmunotherapy combinations

Cytotoxic therapies, besides directly inducing cancer cell death, can stimulate immune-dependent tumor growth control or paradoxically accelerate tumor progression. The underlying mechanisms dictating these opposing outcomes are poorly defined. Here, we show that cytotoxic therapy acutely upregulates cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) production in cancer cells with pre-existing COX-2 activity. Screening a compound library of 1280 approved drugs, we find that all classes of chemotherapy drugs enhance COX-2 transcription whilst arresting cancer cell proliferation. Genetic manipulation of COX-2 expression or its gene promoter region uncover how augmented COX-2/PGE2 activity post-treatment profoundly alters the inflammatory properties of chemotherapy-treated cancer cells in vivo. Pharmacological COX-2 inhibition boosts the efficacy of the combination of chemotherapy and PD-1 blockade. Crucially, in a poorly immunogenic breast cancer model, only the triple therapy unleashes tumor growth control and significantly reduces relapse and spontaneous metastatic spread in an adjuvant setting. Our findings suggest COX-2/PGE2 upregulation by dying cancer cells acts as a major barrier to cytotoxic therapy-driven tumor immunity and uncover a strategy to improve the outcomes of immunotherapy and chemotherapy combinations.

The hypoxia-inducible factor prolyl hydroxylase inhibitor FG4592 promotes natriuresis through upregulation of COX2 in the renal medulla.

The renal medulla is a key site for the regulation of renal sodium excretion. However, the molecular mechanism remains unclear. Cyclooxygenase 2 (COX2) is specifically expressed in the renal medulla and contributes to the maintenance of the electrolyte/water balance in the body. Hypoxia-inducible factors (HIFs) have also been found to be expressed in the renal medulla, probably owing to the hypoxic conditions in the renal medulla. This study was designed to test the effects of HIF activation on renal sodium handling and renal medullary COX2 expression. Our data showed that HIF activation by the prolyl hydroxylase inhibitor (PHI) FG4592 enhanced natriuresis in mice challenged with a high-salt diet. In addition, FG4592 upregulated the expression of COX2 in the renal medulla. An in vitro study further supported the finding that HIF can induce the expression of COX2 and that this induction is mediated through direct binding to the promoter region of the Cox2 gene, facilitating its transcription. In addition, the COX2 inhibitor celecoxib diminished the natriuretic effect of FG4592. Together, these results suggest that HIF activation promotes sodium excretion through upregulation of COX2 in the renal medulla and therefore maintains sodium homeostasis in the body.

Prostacyclin Promotes Degenerative Pathology in a Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is the most common form of dementia in aged populations. A substantial amount of data demonstrates that chronic neuroinflammation can accelerate neurodegenerative pathologies. In AD, chronic neuroinflammation results in the upregulation of cyclooxygenase and increased production of prostaglandin H2, a precursor for many vasoactive prostanoids. While it is well-established that many prostaglandins can modulate the progression of neurodegenerative disorders, the role of prostacyclin (PGI2) in the brain is poorly understood. We have conducted studies to assess the effect of elevated prostacyclin biosynthesis in a mouse model of AD. Upregulated prostacyclin expression significantly worsened multiple measures associated with amyloid-β (Aβ) disease pathologies. Mice overexpressing both Aβ and PGI2 exhibited impaired learning and memory and increased anxiety-like behavior compared with non-transgenic and PGI2 control mice. PGI2 overexpression accelerated the development of Aβ accumulation in the brain and selectively increased the production of soluble Aβ42. PGI2 damaged the microvasculature through alterations in vascular length and branching; Aβ expression exacerbated these effects. Our findings demonstrate that chronic prostacyclin expression plays a novel and unexpected role that hastens the development of the AD phenotype.

Mogroside V exerts anti-inflammatory effects on fine particulate matter-induced inflammation in porcine alveolar macrophages

Mogroside V is the main bioactive component of Siraitia grosvenorii (Swingle), and has a potential anti-inflammatory function. However, the effect of mogroside V on fine particulate matter (PM2.5)-induced inflammation has not been reported. In the present study, the biological effect of mogroside V on inflammation was investigated in PM2.5- treated porcine alveolar macrophages (3D4/21). The results showed that mogroside V significantly inhibited PM2.5-induced nitric oxide (NO) production and rescued the arginase activity inhibited by PM2.5. In the presence of mogroside V, the upregulation of IL-18, TNF-α and COX-2 by PM2.5 in 3D4/21 cells was inhibited. Mogroside V attenuated PM2.5-induced phosphorylation of NF-κB p65 and the expression of NLRP3. Mogroside V reduced intracellular ROS levels induced by PM2.5. In the transcriptomic analysis, inflammation-related genes in 3D4/21 cells were not significantly affected after treatment with mogroside V. These results indicated that mogroside V can alleviate the inflammatory response of porcine alveolar macrophages induced by PM2.5 from pig house and that mogroside V may play the role through the antioxidant function of eliminating ROS. Mogroside V has a clear anti-inflammatory function in the presence of inflammation.

Abstract TP119: Celecoxib In Aneurysmal Subarachnoid Hemorrhage: A Single Center Experience

Introduction: Inflammation may play a central role in delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage (aSAH). Prostaglandins are inflammatory mediators that are elevated in aSAH and are synthesized by cyclooxygenase-2 (Cox-2). Celecoxib is a selective Cox-2 inhibitor with anti-inflammatory effects and good central nervous system penetration. In a mouse model of SAH, Cox-2 gene expression was found to be up-regulated in brain endothelial cells and celecoxib was found to limit Cox-2 up-regulation. We sought to evaluate the safety of celecoxib in patients with aSAH for future clinical studies. Methods: We used a matched case-control study to evaluate safety of celecoxib in aSAH patients. Our prospectively collected aSAH database (N=230) was retrospectively reviewed from 1/2010 - 2/2020. Celecoxib cases (N=13) and controls (N=217) were matched by closest Euclidean distance based on age and Glasgow Coma Scale (Figure 1). Each case was matched with 4 controls to maximize power based on closest Euclidean distance. Baseline characteristics for cases and controls were compared using a t-test or signed rank test for pairs. Categorical variables were compared using McNemar’s test for discordant pairs. A p value ≤.05 was considered significant for all univariate testing. Results are listed in Table 1. Discussion: No adverse events were associated with celecoxib use. At our center, celecoxib was primarily used in high grade aSAH for fever or headache. Maximum and mean temperature are statistically higher in the celecoxib group, consistent with the treatment indication. There were no differences in vasospasm prevalence, although our analysis is limited by sample size. Given the favorable safety profile, ease of drug availability, and plausible mechanism of therapeutic effect, celecoxib warrants further study in aSAH.

Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines.

TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). β-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte® system, cell cycle/apoptosis, and clonogenic potential. COX-2, β-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, β-catenin, MGMT, and SOX-2 up-regulation.

A Newly Synthesized Flavone from Luteolin Escapes from COMT-Catalyzed Methylation and Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages via JNK, p38 and NF-κB Signaling Pathways.

Luteolin is a common dietary flavone possessing potent anti-inflammatory activities. However, when administrated in vivo, luteolin becomes methylated by catechol-O-methyltransferases (COMT) owing to the catechol ring in the chemical structure, which largely diminishes its anti-inflammatory effect. In this study, we made a modification on luteolin, named LUA, which was generated by the chemical reaction between luteolin and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Without a catechol ring in the chemical structure, this new flavone could escape from the COMT-catalyzed methylation, thus affording the potential to exert its functions in the original form when administrated in the organism. Moreover, an LPS-stimulated RAW cell model was applied to detect the anti-inflammatory properties. LUA showed much more superior inhibitory effect on LPS-induced production of NO than diosmetin (a major methylated form of luteolin) and significantly suppressed upregulation of iNOS and COX-2 in macrophages. LUA treatment dramatically reduced LPS-stimulated reactive oxygen species (ROS) and mRNA levels of pro-inflammatory mediators such as IL-1β, IL-6, IL-8 and IFN-β. Furthermore, LUA significantly reduced the phosphorylation of JNK and p38 without affecting that of ERK. LUA also inhibited the activation of NF-κB through suppression of p65 phosphorylation and nuclear translocation.

Recent findings on the cellular and molecular mechanisms of action of novel food-derived antihypertensive peptides

Hypertension impacts negatively on the quality of life of sufferers, and complications associated with uncontrolled hypertension are life-threatening. Hence, many research efforts are exploring the antihypertensive properties of bioactive peptides derived from food proteins using in vitro ACE-inhibitory assay, experimentally-induced and spontaneous hypertensive rats, normotensive and hypertensive human models. In this study, the cellular and molecular mechanisms of blood pressure-lowering properties of novel peptides reported in recent studies (2015-July 30, 2021) were discussed. In addition to common mechanisms such as the inhibition of angiotensin I-converting enzyme (ACE) and renin activities, recently recognized mechanisms through which bioactive peptides exert their antihypertensive properties including the induction of vasodilation via upregulation of cyclo-oxygenase (COX) and prostaglandin receptor and endothelial nitric oxide synthase expression and L-type Ca2+ channel blockade were presented. Similarly, emerging mechanisms of blood pressure-lowering by bioactive peptides such as modulation of inflammation (TNF-α, and other cytokines signaling), oxidative stress (Keap-1/Nrf2/ARE/HO-1 and related signaling pathways), PPAR-γ/caspase3/MAPK signaling pathways and inhibition of lipid accumulation were discussed. The review also highlighted factors that influence the antihypertensive properties of peptides such as method of hydrolysis (type and number of enzymes, and chemical used for hydrolysis, and microbial fermentation), and amino acid sequence and chain length of peptides.

Efficacy profile of noninvasive vagus nerve stimulation on cortical spreading depression susceptibility and the tissue response in a rat model

BackgroundNoninvasive vagus nerve stimulation (nVNS) has recently emerged as a promising therapy for migraine. We previously demonstrated that vagus nerve stimulation inhibits cortical spreading depression (CSD), the electrophysiological event underlying migraine aura and triggering headache; however, the optimal nVNS paradigm has not been defined.MethodsVarious intensities and doses of nVNS were tested to improve efficacy on KCl-evoked CSD frequency and electrical threshold of CSD in a validated rat model. Chronic efficacy was evaluated by daily nVNS delivery for four weeks. We also examined the effects of nVNS on neuroinflammation and trigeminovascular activation by western blot and immunohistochemistry.ResultsnVNS suppressed susceptibility to CSD in an intensity-dependent manner. Two 2-minute nVNS 5 min apart afforded the highest efficacy on electrical CSD threshold and frequency of KCl-evoked CSD. Daily nVNS for four weeks did not further enhance efficacy over a single nVNS 20 min prior to CSD. The optimal nVNS also attenuated CSD-induced upregulation of cortical cyclooxygenase-2, calcitonin gene-related peptide in trigeminal ganglia, and c-Fos expression in trigeminal nucleus caudalis.ConclusionsOur study provides insight on optimal nVNS parameters to suppress CSD and suggests its benefit on CSD-induced neuroinflammation and trigeminovascular activation in migraine treatment.

Oxidative stress and inflammatory markers in patients with COVID-19: Potential role of RAGE, HMGB1, GFAP and COX-2 in disease severity

BackgroundSARS-CoV-2 infection can lead to the abnormal induction of cytokines and a dysregulated hyperinflammatory state that is implicated in disease severity and risk of death. There are several molecules present in blood associated with immune cellular response, inflammation, and oxidative stress that could be used as severity markers in respiratory viral infections such as COVID-19. However, there is a lack of clinical studies evaluating the role of oxidative stress-related molecules including glial fibrillary acidic protein (GFAP), the receptor for advanced glycation end products (RAGE), high mobility group box-1 protein (HMGB1) and cyclo-oxygenase-2 (COX-2) in COVID-19 pathogenesis. AimTo evaluate the role of oxidative stress-related molecules in COVID-19. MethodAn observational study with 93 Brazilian participants from September 2020 to April 2021, comprising 23 patients with COVID-19 admitted to intensive care unit (ICU), 19 outpatients with COVID-19 with mild to moderate symptoms, 17 individuals reporting a COVID-19 history, and 34 healthy controls. Blood samples were taken from all participants and western blot assay was used to determine the RAGE, HMGB1, GFAP, and COX-2 immunocontent. ResultsWe found that GFAP levels were higher in patients with severe or critical COVID-19 compared to outpatients (p = 0.030) and controls (p < 0.001). A significant increase in immunocontents of RAGE (p < 0.001) and HMGB1 (p < 0.001) were also found among patients admitted to the ICU compared to healthy controls, as well as an overexpression of the inducible COX-2 (p < 0.001). In addition, we found a moderate to strong correlation between RAGE, GFAP and HMGB1 proteins. ConclusionSARS-CoV-2 infection induces the upregulation of GFAP, RAGE, HMGB1, and COX-2 in patients with the most severe forms of COVID-19.

Dapansutrile ameliorated chondrocyte inflammation and osteoarthritis through suppression of MAPK signaling pathway.

Osteoarthritis (OA) is one of the most common joint diseases in the elderly population. Proinflammatory cytokines, such as Interleukin-1β (IL-1β), play an important role in the development and progression of OA. Dapansutrile is a specific inhibitor of the NOD-like receptor protein 3 (NLRP3) inflammasome and exhibits anti-inflammatory properties. In this study, we investigated the protective effect and the underlying mechanism of dapansutrile on cartilage degeneration in vitro and in vivo. In the present study, chondrocytes were isolated from rats and then were treated with dapansutrile. After that, the expression of (Cox-2, inducible nitric oxide synthase (iNOS), Mmp-3, Mmp-9, Mmp-13 and IL-10) were evaluated at RNA level, then the expression of (COX-2, MMP-3, MMP-9, MMP-13, SOX-9 and COL2) were evaluated at protein level. Subsequently, the activation of the mitogen-activated protein kinase (MAPK) pathway was tested using western blotting (WB). Additionally, the rat OA model was developed to evaluate the protective effects of dapansutrile in vivo. The results showed that dapansutrile had no obvious cytotoxicity on rat chondrocytes at 24h (0, 1, 2, 5 and 10μM). Dapansutrile significantly decreased IL-1β-induced upregulation of COX2, iNOS, matrix metalloproteinase 3 (MMP3), 9 (MMP9) and 13 (MMP13), and reversed IL-1β-induced the downregulation of IL-10, SOX9 and COL2. Dapansutrile also inhibited IL-1β-induced upregulation of the MAPK signaling pathway by downregulating the expression levels of phospho-ERK, and phospho-P38 in a concentration dependent manner. In addition, dapansutrile exhibited protective effects in rat OA model with lower Mankin's score and Osteoarthritis Research Society International (OARSI) score. Our study suggested that dapansutrile effectively inhibited chondrocyte inflammation by suppressing MAPK signaling pathway in vitro, and ameliorated cartilage degeneration in vivo, indicating an anti-inflammatory effect in OA treatment.