Anti-inflammatory and anti-diabetic properties of indanone derivative isolated from Fernandoa adenophylla in vitro and in silico studies.

Fernandoa adenophylla, due to the presence of phytochemicals, has various beneficial properties and is used in folk medicine to treat many conditions. This study aimed to isolate indanone derivative from F. adenophylla root heartwood and assess in-vitro anti-inflammatory and anti-diabetic characteristics at varying concentrations. Heat-induced hemolysis and glucose uptake by yeast cells assays were conducted to evaluate these properties. Besides, docking analyses were performed on four molecular targets. These studies were combined with molecular dynamics simulations to elucidate the time-evolving inhibitory effect of selected inhibitors within the active pockets of the target proteins (COX-1 and COX-2). Indanone derivative (10-100 µM) inhibited the lysis of human red blood cells from 9.12 ± 0.75 to 72.82 ± 4.36% and, at 5-100 µM concentrations, it significantly increased the yeast cells' glucose uptake (5.16 ± 1.28% to 76.59 ± 1.62%). Concluding, the isolated indanone might act as an anti-diabetic agent by interacting with critical amino acid residues of 5' adenosine monophosphate-activated protein kinase (AMPK), and it showed a binding affinity with anti-inflammatory targets COX-1, COX-2, and TNF-α. Besides, the obtained results may help to consider the indanone derivative isolated from F. adenophylla as a promising candidate for drug delivery, subject to outcomes of further in vivo and clinical studies.

Exploration of isoxazole analogs: Synthesis, COX inhibition, anticancer screening, 3D multicellular tumor spheroids, and molecular modeling.

In this study, a new series of Isoxazole-carboxamide derivatives were synthesized and characterized via HRMS, 1H-, 13CAPT-NMR, and MicroED. The findings revealed that nearly all of the synthesized derivatives exhibited potent inhibitory activities against both COX enzymes, with IC50 values ranging from 4.1 nM to 3.87 µM. Specifically, MYM1 demonstrated the highest efficacy among the compounds tested against the COX-1, displaying an IC50 value of 4.1 nM. The results showed that 5 compounds possess high COX-2 isozyme inhibitory effects with IC50 value in range 0.24-1.30 µM with COX-2 selectivity indexes (2.51-6.13), among these compounds MYM4 has the lowest IC50 value against COX-2, with selectivity index around 4. Intriguingly, this compound displayed significant antiproliferative effects against CaCo-2, Hep3B, and HeLa cancer cell lines, with IC50 values of 10.22, 4.84, and 1.57 µM, respectively, which was nearly comparable to that of doxorubicin. Compound MYM4 showed low cytotoxic activities on normal cell lines LX-2 and Hek293t with IC50 values 20.01 and 216.97 µM respectively, with safer values than doxorubicin. Furthermore, compound MYM4 was able to induce the apoptosis, suppress the colonization of both HeLa and HepG2 cells. Additionally, the induction of Reactive oxygen species (ROS) production could be the mechanism underlying the apoptotic effect and the cytotoxic activity of the compound. In the 3D multicellular tumor spheroid model, results revealed that MYM4 compound hampered the spheroid formation capacity of Hep3B and HeLa cancer cells. Moreover, the molecular docking of MYM4 compound revealed a high affinity for the COX2 enzyme, with energy scores (S) -7.45 kcal/mol, which were comparable to celecoxib (S) -8.40 kcal/mol. Collectively, these findings position MYM4 as a promising pharmacological candidate as COX inhibitor and anticancer agent.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

BACKGROUND: Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. METHODS: To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-; low-density lipoprotein receptor) backgrounds. RESULTS: Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 (prostaglandin E) and PGD2 (prostanglandin D), activation of the inflammasome, elevated plasma levels of IL-1ß (interleukin), reduced plasma levels of HDL-C (high-density lipoprotein receptor-cholesterol), and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. CONCLUSIONS: Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Exposure to nicotine regulates prostaglandin E2 secretion and autophagy of granulosa cells to retard follicular maturation in mammals.

Exposure to nicotine by cigarette smoking have shown strongly defectives on the physiological function of ovaries, which in turn leads to disorders of fertility in women. However, the potential molecular mechanisms remain to be elucidated. In this study, we notably found that nicotine was likely to specifically raise the expression of histone deacetylase 3 (HDAC3) to promote the apoptosis and autophagy of granulosa cells (GCs) and block follicular maturation. Moreover, prostaglandin E2 (PGE2) inhibited the apoptosis of GCs and facilitated follicular maturation, and nicotine appeared to inhibit PGE2 secretion by freezing the expression of cyclooxygenase 1 (COX1), which was the rate-limiting and essential enzyme for PGE2 synthesis. Epigenetically, the nicotine was observed to diminish the histone H3 lysine 9 acetylation (H3K9ac) level and compact the chromatin accessibility in -1776/-1499 bp region of COX1 by evoking the expression of HDAC3, with the deactivated Cas9-HDAC3/sgRNA system. Mechanistically, the COX1 protein was found to pick up and degrade the autophagy related protein beclin 1 (BECN1) to control the autophagy of GCs. These results provided a potential new molecular therapy to recover the damage of female fertility induced by nicotine from cigarette smoking.

Antagonistic effects of a COX1/2 inhibitor drug in human HepG2 cells exposed to an environmental carcinogen.

Understanding interactions between legacy and emerging environmental contaminants has important implications for risk assessment, especially when mutagens and carcinogens are involved, whose critical effects are chronic and therefore difficult to predict. The current work aimed to investigate potential interactions between benzo[a]pyrene (B[a]P), a carcinogenic polycyclic aromatic hydrocarbon and legacy pollutant, and diclofenac (DFC), a non-steroidal anti-inflammatory drug and pollutant of emerging concern, and how DFC affects B[a]P toxicity. Exposure to binary mixtures of these chemicals resulted in substantially reduced cytotoxicity in human HepG2 cells compared to single-chemical exposures. Significant antagonistic effects were observed in response to high concentrations of B[a]P in combination with DFC at IC50 and ⅕ IC50. While additive effects were found for levels of intracellular reactive oxygen species, antagonistic mixture effects were observed for genotoxicity. B[a]P induced DNA strand breaks, γH2AX activation, and micronuclei formation at ½ IC50 concentrations or lower, whereas DFC induced only low levels of DNA strand breaks. Their mixture caused significantly lower levels of genotoxicity by all three endpoints compared to those expected based on concentration additivity. In addition, antagonistic mixture effects on CYP1 enzyme activity suggested that the observed reduced genotoxicity of B[a]P was due to its reduced metabolic activation as a result of enzymatic inhibition by DFC. Overall, the findings further support the growing concern that co-exposure to environmental toxicants and their non-additive interactions may be a confounding factor that should not be neglected in environmental and human health risk assessment.

Dihydroartemisinin, a potential PTGS1 inhibitor, potentiated cisplatin-induced cell death in non-small cell lung cancer through activating ROS-mediated multiple signaling pathways.

Dihydroartemisinin (DHA) exerts an anti-tumor effect in multiple cancers, however, the molecular mechanism of DHA and whether DHA facilitates the anti-tumor efficacy of cisplatin in non-small cell lung cancer (NSCLC) are unclear. Here, we found that DHA potentiated the anti-tumor effects of cisplatin in NSCLC cells by stimulating reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress, C-Jun-amino-terminal kinase (JNK) and p38 MAPK signaling pathways both in vitro and in vivo. Of note, we demonstrated for the first time that DHA inhibits prostaglandin G/H synthase 1 (PTGS1) expression, resulting in enhanced ROS production. Importantly, silencing PTGS1 sensitized DHA-induced cell death by increasing ROS production and activating ER-stress, JNK and p38 MAPK signaling pathways. In summary, our findings provided new experimental basis and therapeutic prospect for the combined therapy with DHA and cisplatin in some NSCLC patients.

The protective effects of Gamma-linolenic acid against indomethacin-induced gastric ulcer in rats.

The primary goal of the investigation was to analyse the anti-inflammatory and antioxidant properties of Gamma-linolenic acid (GLA) on rats with indomethacin (IND)-induced gastric ulcers. Thirty rats were divided into five groups: Control, IND (50 mg/kg, p.o.), IND pretreated with GLA 100 mg/kg (p.o. for 14 d), IND pretreated with GLA 150 mg/kg (p.o. for 14 d) and IND pretreated with omeprazole (20 mg/kg, p.o. for 14 d). The stomach tissues were examined to calculate the ulcer index and pH and analyse biochemical markers (prostaglandin E2 (PGE2), cyclooxygenase 1 (COX1), TNF-1, IL-6 and intercellular adhesion molecule-1 (ICAM1)) and oxidative stress parameters (malondialdehyde: (MDA), superoxide dismutase (SOD), glutathione (GSH) and CAT (catalase)) as well as undergo histopathological assessment. GLA 100 and 150 mg/kg showed a protective effect against IND-induced gastric damage. It reduced levels of COX1, TNF-1, IL-6 and ICAM and increased PGE2 levels. GLA also normalised antioxidant function by modulating MDA, SOD, GSH and CAT. GLA intervention protects against IND-induced gastric ulcers by restoring oxidant/antioxidant balance and reducing inflammation.

Neuropeptide W facilitates chronic gastric ulcer healing by the regulation of cyclooxygenase and NF-κB signaling pathways.

AIMS: Putative beneficial effects of neuropeptide W (NPW) in the early phase of gastric ulcer healing process and the involvement of cyclooxygenase (COX) enzymes were investigated in an acetic acid-induced gastric ulcer model. MAIN METHODS: In anesthetized male Sprague-Dawley rats, acetic acid was applied surgically on the serosa and then a COX-inhibitor (COX-2-selective NS-398, COX-1-selective ketorolac, or non-selective indomethacin; 2 mg/kg/day, 3 mg/kg/day or 5 mg/kg/day; respectively) or saline was injected intraperitoneally. One h after ulcer induction, omeprazole (20 mg/kg/day), NPW (0.1 µg/kg/day) or saline was intraperitoneally administered. Injections of NPW, COX-inhibitors, omeprazole or saline were continued for the following 2 days until rats were decapitated at the end of the third day. KEY FINDINGS: NPW treatment depressed gastric prostaglandin (PG) I2 level, but not PGE2 level. Similar to omeprazole, NPW treatment significantly reduced gastric and serum tumor necrosis factor-alpha and interleukin-1 beta levels and depressed the upregulation of nuclear factor kappa B (NF-κB) and COX-2 expressions due to ulcer. In parallel with the histopathological findings, treatment with NPW suppressed ulcer-induced increases in myeloperoxidase activity and malondialdehyde level and replenished glutathione level. However, the inhibitory effect of NPW on myeloperoxidase activity and NPW-induced increase in glutathione were not observed in the presence of COX-1 inhibitor ketorolac or the non-selective COX-inhibitor indomethacin. SIGNIFICANCE: In conclusion, NPW facilitated the healing of gastric injury in rats via the inhibition of pro-inflammatory cytokine production, oxidative stress and neutrophil infiltration as well as the downregulation of COX-2 protein and NF-κB gene expressions.

A further pocket or conformational plasticity by mapping COX-1 catalytic site through modified-mofezolac structure-inhibitory activity relationships and their antiplatelet behavior.

Cyclooxygenase enzymes have distinct roles in cardiovascular, neurological, and neurodegenerative disease. They are differently expressed in different type of cancers. Specific and selective COXs inhibitors are needed to be used alone or in combo-therapies. Fully understand the differences at the catalytic site of the two cyclooxygenase (COX) isoforms is still opened to investigation. Thus, two series of novel compounds were designed and synthesized in fair to good yields using the highly selective COX-1 inhibitor mofezolac as the lead compound to explore a COX-1 zone formed by the polar residues Q192, S353, H90 and Y355, as well as hydrophobic amino acids I523, F518 and L352. According to the structure of the COX-1:mofezolac complex, hydrophobic amino acids appear to have free volume eventually accessible to the more sterically hindering groups than the methoxy linked to the phenyl groups of mofezolac, in particular the methoxyphenyl at C4-mofezolac isoxazole. Mofezolac bears two methoxyphenyl groups linked to C3 and C4 of the isoxazole core ring. Thus, in the novel compounds, one or both methoxy groups were replaced by the higher homologous ethoxy, normal and isopropyl, normal and tertiary butyl, and phenyl and benzyl. Furthermore, a major difference between the two sets of compounds is the presence of either a methyl or acetic moiety at the C5 of the isoxazole. Among the C5-methyl series, 12 (direct precursor of mofezolac) (COX-1 IC50 = 0.076 µM and COX-2 IC50 = 0.35 µM) and 15a (ethoxy replacing the two methoxy groups in 12; COX-1 IC50 = 0.23 µM and COX-2 IC50 > 50 µM) were still active and with a Selectivity Index (SI = COX-2 IC50/COX-1 IC50) = 5 and 217, respectively. The other symmetrically substituted alkoxyphenyl moietis were inactive at 50 µM final concentration. Among the asymmetrically substituted, only the 16a (methoxyphenyl on C3-isoxazole and ethoxyphenyl on C4-isoxazole) and 16b (methoxyphenyl on C3-isoxazole and n-propoxyphenyl on C4-isoxazole) were active with SI = 1087 and 38, respectively. Among the set of compounds with the acetic moiety, structurally more similar to mofezolac (SI = 6329), SI ranged between 1.4 and 943. It is noteworthy that 17b (n-propoxyphenyl on both C3- and C4-isoxazole) were found to be a COX-2 slightly selective inhibitor with SI = 0.072 (COX-1 IC50 > 50 µM and COX-2 IC50 = 3.6 µM). Platelet aggregation induced by arachidonic acid (AA) can be in vitro suppressed by the synthesized compounds, without affecting of the secondary hemostasia, confirming the biological effect provided by the selective inhibition of COX-1. A positive profile of hemocompatibility in relation to erythrocyte and platelet toxicity was observed. Additionally, these compounds exhibited a positive profile of hemocompatibility and reduced cytotoxicity. Quantitative structure activity relationship (QSAR) models and molecular modelling (Ligand and Structure based virtual screening procedures) provide key information on the physicochemical and pharmacokinetic properties of the COX-1 inhibitors as well as new insights into the mechanisms of inhibition that will be used to guide the development of more effective and selective compounds. X-ray analysis was used to confirm the chemical structure of 14 (MSA17).

Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

BACKGROUND: Acetaminophen and ibuprofen are widely administered to babies due to their presumed safety as over-the-counter drugs. However, no reports exist on the effects of cyclooxygenase inhibitors on undifferentiated spermatogonia and spermatogonial stem cells. Infancy represents a critical period for spermatogonial stem cell formation and disrupting spermatogonial stem cells or their precursors may be associated with infertility and testicular cancer formation. OBJECTIVES: The goal of this study was to examine the molecular and functional impact of cyclooxygenase inhibition and silencing on early steps of undifferentiated spermatogonia (u spg) and spermatogonial stem cell development, to assess the potential reproductive risk of pharmaceutical cyclooxygenase inhibitors. METHODS: The effects of cyclooxygenase inhibition were assessed using the mouse C18-4 undifferentiated juvenile spermatogonial cell line model, previously shown to include cells with spermatogonial stem cell features, by measuring prostaglandins, cell proliferation, and differentiation, using cyclooxygenase 1- and cyclooxygenase 2-selective inhibitors NS398, celecoxib, and FR122047, acetaminophen, and ibuprofen. Cyclooxygenase 1 gene silencing was achieved using a stable short-hairpin RNA approach and clone selection, then assessing gene and protein expression in RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence studies. RESULTS: Cyclooxygenase 2 inhibitors NS398 and celecoxib, as well as acetaminophen, but not ibuprofen, dose-dependently decreased retinoic acid-induced expression of the spg differentiation gene Stra8, while NS398 decreased the spg differentiation marker Kit, suggesting that cyclooxygenase 2 is positively associated with spg differentiation. In contrast, short-hairpin RNA-based cyclooxygenase 1 silencing in C18-4 cells altered cellular morphology and upregulated Stra8 and Kit, implying that cyclooxygenase 1 prevented spg differentiation. Furthermore, RNA sequencing analysis of cyclooxygenase 1 knockdown cells indicated the activation of several signaling pathways including the TGFb, Wnt, and Notch pathways, compared to control C18-4 cells. Notch pathway genes were upregulated by selective cyclooxygenase inhibitors, acetaminophen and ibuprofen. CONCLUSION: We report that cyclooxygenase 1 and 2 differentially regulate undifferentiated spermatogonia/spermatogonial stem cell differentiation. Cyclooxygenases regulate Notch3 expression, with the Notch pathway targeted by PGD2. These data suggest an interaction between the eicosanoid and Notch signaling pathways that may be critical for the development of spermatogonial stem cells and subsequent spermatogenesis, cautioning about using cyclooxygenase inhibitors in infants.

Influence of Microbiota-Related Metabolites Associated with Inflammation and Sepsis on the Peroxidase Activity of Cyclooxygenase in Healthy Human Monocytes and Acute Monocytic Leukemia Cells.

The human microbiota produces metabolites that can enter the bloodstream and exert systemic effects on various functions in both healthy and pathological states. We have studied the participation of microbiota-related metabolites in bacterial infection by examining their influence on the activity of cyclooxygenase (COX) as a key enzyme of inflammation. The influence of aromatic microbial metabolites, derivatives of phenylalanine (phenylpropionic acid, PPA), tyrosine (4-hydroxyphenyllactic acid, HPLA), and tryptophan (indolacetic acids, IAA), the concentrations of which in the blood change notably during sepsis, was evaluated. Also, the effect of itaconic acid (ITA) was studied, which is formed in macrophages under the action of bacterial lipopolysaccharides (LPS) and appears in the blood in the early stages of infection. Metabiotic acetyl phosphate (AcP) as a strong acetylating agent was also tested. The activity of COX was measured via the TMPD oxidation colorimetric assay using the commercial pure enzyme, cultured healthy monocytes, and the human acute monocytic leukemia cell line THP-1. All metabolites in the concentration range of 100-500 µM lowered the activity of COX. The most pronounced inhibition was observed on the commercial pure enzyme, reaching up to 40% in the presence of AcP and 20-30% in the presence of the other metabolites. On cell lysates, the effect of metabolites was preserved, although it significantly decreased, probably due to their interaction with other targets subject to redox-dependent and acetylation processes. The possible contribution of the redox-dependent action of microbial metabolites was confirmed by assessing the activity of the enzyme in the presence of thiol reagents and in model conditions, when the COX-formed peroxy intermediate was replaced with tert-butyl hydroperoxide (TBH). The data show the involvement of the microbial metabolites in the regulation of COX activity, probably due to their influence on the peroxidase activity of the enzyme.

Defining the Conformational Ensembles Associated with Ligand Binding to Cyclooxygenase-2.

Cyclooxygenases (COX) catalyze the committed step in the production of prostaglandins responsible for the maintenance of physiological homeostasis. While crystal structures of COX in complex with substrates and inhibitors have provided insight into the molecular interactions governing their binding, they have not uncovered specific details related to the protein conformational motions responsible for important aspects of the COX function. We created a cysteine-free COX-2 construct and introduced a free cysteine at position-122 to enable labeling with 3-bromo-1,1,1-trifluoroacetone (BTFA). Placement of the label adjacent to the cyclooxygenase channel entrance permitted the detection of alterations upon ligand binding. 19F-nuclear magnetic resonance spectroscopy (19F-NMR) was then used to probe the conformational ensembles arising from BTFA-labeled COX-2 constructs in the presence and absence of ligands known to allosterically activate or inhibit COX-2. 19F-NMR analyses performed in the presence of the time-dependent inhibitor flurbiprofen, as well as Arg-120, Tyr-355, and Glu-524 mutations, led to the classification of two ensembles as representing the relaxed and tightened states of the cyclooxygenase channel entrance. A third ensemble, generated in the presence of arachidonic acid and the Y355F mutant and modulated by the allosteric potentiators palmitic acid and oleic acid and the nonallosteric substrates 2-arachidonoyl glycerol ether and anandamide, was classified as being related to the allosteric regulation of COX activity. The ensemble-based insight into COX function demonstrated here complements the static information derived from crystal structure analyses, collectively providing a more detailed framework of the dynamics involved in the regulation of COX catalysis and inhibition.

Identification of Novel Cyclooxygenase-1 Selective Inhibitors of Thiadiazole-Based Scaffold as Potent Anti-Inflammatory Agents with Safety Gastric and Cytotoxic Profile.

Major obstacles faced by the use of nonsteroidal anti-inflammatory drugs (NSAID) are their gastrointestinal toxicity induced by non-selective inhibition of both cyclooxygenases (COX) 1 and 2 and their cardiotoxicity associated with a certain class of COX-2 selective inhibitors. Recent studies have demonstrated that selective COX-1 and COX-2 inhibition generates compounds with no gastric damage. The aim of the current study is to develop novel anti-inflammatory agents with a better gastric profile. In our previous paper, we investigated the anti-inflammatory activity of 4-methylthiazole-based thiazolidinones. Thus, based on these observations, herein we report the evaluation of anti-inflammatory activity, drug action, ulcerogenicity and cytotoxicity of a series of 5-adamantylthiadiazole-based thiazolidinone derivatives. The in vivo anti-inflammatory activity revealed that the compounds possessed moderate to excellent anti-inflammatory activity. Four compounds 3, 4, 10 and 11 showed highest potency (62.0, 66.7, 55.8 and 60.0%, respectively), which was higher than the control drug indomethacin (47.0%). To determine their possible mode of action, the enzymatic assay was conducted against COX-1, COX-2 and LOX. The biological results demonstrated that these compounds are effective COX-1 inhibitors. Thus, the IC50 values of the three most active compounds 3, 4 and 14 as COX-1 inhibitors were 1.08, 1.12 and 9.62 µΜ, respectively, compared to ibuprofen (12.7 µΜ) and naproxen (40.10 µΜ) used as control drugs. Moreover, the ulcerogenic effect of the best compounds 3, 4 and 14 were evaluated and revealed that no gastric damage was observed. Furthermore, compounds were found to be nontoxic. A molecular modeling study provided molecular insight to rationalize the COX selectivity. In summary, we discovered a novel class of selective COX-1 inhibitors that could be effectively used as potential anti-inflammatory agents.

Discovery of Small Molecule COX-1 and Akt Inhibitors as Anti-NSCLC Agents Endowed with Anti-Inflammatory Action.

Targeted therapies have come into prominence in the ongoing battle against non-small cell lung cancer (NSCLC) because of the shortcomings of traditional chemotherapy. In this context, indole-based small molecules, which were synthesized efficiently, were subjected to an in vitro colorimetric assay to evaluate their cyclooxygenase (COX) inhibitory profiles. Compounds 3b and 4a were found to be the most selective COX-1 inhibitors in this series with IC50 values of 8.90 µM and 10.00 µM, respectively. In vitro and in vivo assays were performed to evaluate their anti-NSCLC and anti-inflammatory action, respectively. 2-(1H-Indol-3-yl)-N'-(4-morpholinobenzylidene)acetohydrazide (3b) showed selective cytotoxic activity against A549 human lung adenocarcinoma cells through apoptosis induction and Akt inhibition. The in vivo experimental data revealed that compound 3b decreased the serum myeloperoxidase and nitric oxide levels, pointing out its anti-inflammatory action. Moreover, compound 3b diminished the serum aminotransferase (particularly aspartate aminotransferase) levels. Based on the in vitro and in vivo experimental data, compound 3b stands out as a lead anti-NSCLC agent endowed with in vivo anti-inflammatory action, acting as a dual COX-1 and Akt inhibitor.

LDL-Dependent Regulation of TNFα/PGE2 Induced COX-2/mPGES-1 Expression in Human Macrophage Cell Lines.

Inflammation is a hallmark in severe diseases such as atherosclerosis and non-alcohol-induced steatohepatitis (NASH). In the development of inflammation, prostaglandins, especially prostaglandin E2 (PGE2), are major players alongside with chemo- and cytokines, like tumor-necrosis-factor alpha (TNFα) and interleukin-1 beta (IL-1ß). During inflammation, PGE2 synthesis can be increased by the transcriptional induction of the two key enzymes: cyclooxygenase 2 (COX-2), which converts arachidonic acid to PGH2, and microsomal prostaglandin E2 synthase 1 (mPGES-1), which synthesizes PGE2 from PGH2. Both COX-2 and mPGES-2 were induced by a dietary intervention where mice were fed a fatty acid-rich and, more importantly, cholesterol-rich diet, leading to the development of NASH. Since macrophages are the main source of PGE2 synthesis and cholesterol is predominantly transported as LDL, the regulation of COX-2 and mPGES-1 expression by native LDL was analyzed in human macrophage cell lines. THP-1 and U937 monocytes were differentiated into macrophages, through which TNFα and PGE-2 induced COX-2 and mPGES-1 expression by LDL could be analyzed on both mRNA and protein levels. In addition, the interaction of LDL- and EP receptor signal chains in COX-2/mPGES-1 expression and PGE2-synthesis were analyzed in more detail using EP receptor specific agonists. Furthermore, the LDL-mediated signal transduction in THP-1 macrophages was analyzed by measuring ERK and Akt phosphorylation as well as transcriptional regulation of transcription factor Egr-1. COX-2 and mPGES-1 were induced in both THP-1 and U937 macrophages by the combination of TNFα and PGE2. Surprisingly, LDL dose-dependently increased the expression of mPGES-1 but repressed the expression of COX-2 on mRNA and protein levels in both cell lines. The interaction of LDL and PGE2 signal chains in mPGES-1 induction as well as PGE2-synthesis could be mimicked by through simultaneous stimulation with EP2 and EP4 agonists. In THP-1 macrophages, LDL induced Akt-phosphorylation, which could be blocked by a PI3 kinase inhibitor. Alongside blocking Akt-phosphorylation, the PI3K inhibitor inhibited LDL-mediated mPGES-1 induction; however, it did not attenuate the repression of COX-2 expression. LDL repressed basal ERK phosphorylation and expression of downstream transcription factor Egr-1, which might lead to inhibition of COX-2 expression. These findings suggest that simultaneous stimulation with a combination of TNFα, PGE2, and native LDL-activated signal chains in macrophage cell lines leads to maximal mPGES-1 activity, as well repression of COX-2 expression, by activating PI3K as well as repression of ERK/Egr-1 signal chains.

Anti-Inflammatory Properties of Metformin During Cultivation of Primary Rat Astrocytes in a Medium with High Glucose Concentration.

Investigation of the relationship between inflammation and energy metabolism is important for understanding biology of chronic noncommunicable diseases. Use of metformin, a drug for treatment of diabetes, is considered as a promising direction for treatment of neurodegenerative diseases and other neuropathologies with an inflammatory component. Astrocytes play an important role in the regulation of energy metabolism and neuroinflammation; therefore, we studied the effect of metformin on the cellular responses of primary rat astrocytes cultured in a medium with high glucose concentration (22.5 mM, 48-h incubation). Lipopolysaccharide (LPS) was used to stimulate inflammation. The effects of metformin were assessed by monitoring changes in the expression of proinflammatory cytokines and synthesis of oxylipins, assayed with ultra-high-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). Changes at the intracellular level were assessed by analyzing phosphorylation of ERK kinase and transcription factor STAT3, as well as enzymes mediating oxylipin synthesis, cyclooxygenase 1 and 2 (COX). It was found that, independent on glucose concentration, metformin reduced the LPS-stimulated release of cytokines IL-1ß and IL-6, decreased activity of the transcription factor STAT3, ERK kinase, synthesis of the derivatives of the cyclooxygenase branch of metabolism of oxylipins and anandamide, and did not affect formation of ROS. The study of energy phenotype of the cells showed that metformin activated glycolysis and inhibited mitochondrial respiration and oxidative phosphorylation, independent on LPS stimulation and cell cultivation at high glucose concentration. Thus, it has been shown that metformin exhibits anti-inflammatory effects, and its effect on the synthesis of cytokines, prostaglandins, and other lipid mediators could determine beneficial effects of metformin in models of neuropathology.

Differential effects of cyclo-oxygenase 1 and 2 inhibition on angiogenesis inhibitor-induced hypertension and kidney damage.

Vascular endothelial growth factor antagonism with angiogenesis inhibitors in cancer patients induces a 'preeclampsia-like' syndrome including hypertension, proteinuria and elevated endothelin (ET)-1. Cyclo-oxygenase (COX) inhibition with aspirin is known to prevent the onset of preeclampsia in high-risk patients. In the present study, we hypothesised that treatment with aspirin would prevent the development of angiogenesis inhibitor-induced hypertension and kidney damage. Our aims were to compare the effects of low-dose (COX-1 inhibition) and high-dose (dual COX-1 and COX-2 inhibition) aspirin on blood pressure, vascular function, oxidative stress, ET-1 and prostanoid levels and kidney damage during angiogenesis-inhibitor therapy in rodents. To this end, Wistar Kyoto rats were treated with vehicle, angiogenesis inhibitor (sunitinib) alone or in combination with low- or high-dose aspirin for 8 days (n=5-7/group). Our results demonstrated that prostacyclin (PGI2) and ET-1 were increased during angiogenesis-inhibitor therapy, while thromboxane (TXA2) was unchanged. Both low- and high-dose aspirin blunted angiogenesis inhibitor-induced hypertension and vascular superoxide production to a similar extent, whereas only high-dose aspirin prevented albuminuria. While circulating TXA2 and prostaglandin F2α levels were reduced by both low- and high-dose aspirin, circulating and urinary levels PGI2 were only reduced by high-dose aspirin. Lastly, treatment with aspirin did not significantly affect ET-1 or vascular function. Collectively our findings suggest that prostanoids contribute to the development of angiogenesis inhibitor-induced hypertension and renal damage and that targeting the prostanoid pathway could be an effective strategy to mitigate the unwanted cardiovascular and renal toxicities associated with angiogenesis inhibitors.

Synthetic methodology-enabled discovery of a tunable indole template for COX-1 inhibition and anti-cancer activity.

Establishing structure-activity relationships (SAR) for privileged pharmacophores, such as the indole scaffold, is a key step in the early stages of drug discovery. Herein, we report the synthesis and preliminary SAR studies on substituted 6-hydroxyindole-7-carboxylates as a tunable framework for COX inhibition and anti-cancer activity. To facilitate the SAR discovery, a modular synthetic methodology was employed which enabled the synthesis of the substituted indoles. From the synthesized compounds, five displayed COX-1 inhibition activity in a colorimetric assay with their intracellular activity further confirmed by a cell-based target validation assay. Following molecular docking analyses, key interactions between the active compounds and the COX enzymes were elucidated. In addition to the identified COX inhibitors, two compounds showed selective cytotoxicity against Hep-G2, MCF-7, and LnCaP. The mechanism of cell death was investigated and found to include induction of Caspase-3 activation and cleavage, down-regulation of anti-apoptotic proteins Bcl-xL and Bcl-2, and upregulation of Bax. Finally, two representative compounds were confirmed to induce cell cycle arrest at the G1/G0 stage. In summary, the 6-hydroxyindole-7-carboxylate framework shows promising versatility as a template for the discovery of anti-inflammation or anti-cancer agents, given the evidence of its COX inhibitory and anti-cancer activities herein presented.

Inflammation: Biochemistry, cellular targets, anti-inflammatory agents and challenges with special emphasis on cyclooxygenase-2.

Keeping in view the involvement of inflammation in the pathogenesis of several diseases including cancer, diabetes, neurodegenerative disorders and rheumatoid arthritis, herein, we review the processes for the initiation of inflammation and the treatment measures. While focusing on the cyclooxygenase mediated arachidonic acid metabolic pathways, biochemistry of inflammatory prostaglandins is discussed. The data corresponding to efficacy, pharmacokinetic profile and the side effects of the available natural and synthetic anti-inflammatory drugs is reviewed. Moreover, the given information for the drug-based design of new anti-inflammatory agents may help in the development of more potent and safe molecules.

Flavonoids: Antiplatelet Effect as Inhibitors of COX-1.

Flavonoids are compounds with a benzopyranic structure that exhibits multiple pharmacological activities. They are known for their venotonic activity, but their mechanism of action remains unclear. It is thought that, as this mechanism is mediated by prostaglandins, these compounds may interfere with the arachidonic acid (AA) cascade. These assays are designed to measure the antiplatelet aggregation capacity of quercetin, rutin, diosmetin, diosmin, and hidrosmin, as well as to evaluate a potential structure-activity ratio. In this paper, several studies on platelet aggregation at different concentrations (from 0.33 mM to 1.5 mM) of different flavone compounds are conducted, measuring platelet aggregation by impedance aggregometry, and the cyclooxygenase (COX) activity by metabolites generated, including the activity of the pure recombinant enzyme in the presence of these polyphenols. The results obtained showed that quercetin and diosmetin aglycones have a greater antiplatelet effect and inhibit the COX enzyme activity to a greater extent than their heterosides; however, the fact that greater inhibition of the pure recombinant enzyme was achieved by heterosides suggests that these compounds may have difficulty in crossing biological membranes. In any case, in view of the results obtained, it can be concluded that flavonoids could be useful as coadjuvants in the treatment of cardiovascular pathologies.