The 5-lipoxygenase/cyclooxygenase-2 cross-over metabolite, hemiketal E2, enhances VEGFR2 activation and promotes angiogenesis.

Consecutive oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2 yields the hemiketal eicosanoids, HKE2 and HKD2. Hemiketals stimulate angiogenesis by inducing endothelial cell tubulogenesis in culture; however, how this process is regulated has not been determined. Here, we identify vascular endothelial growth factor receptor 2 (VEGFR2) as a mediator of HKE2-induced angiogenesis in vitro and in vivo. We found that HKE2 treatment of human umbilical vein endothelial cells dose-dependently increased the phosphorylation of VEGFR2 and the downstream kinases ERK and Akt that mediated endothelial cell tubulogenesis. In vivo, HKE2 induced the growth of blood vessels into polyacetal sponges implanted in mice. HKE2-mediated effects in vitro and in vivo were blocked by the VEGFR2 inhibitor vatalanib, indicating that the pro-angiogenic effect of HKE2 was mediated by VEGFR2. HKE2 covalently bound and inhibited PTP1B, a protein tyrosine phosphatase that dephosphorylates VEGFR2, thereby providing a possible molecular mechanism for how HKE2 induced pro-angiogenic signaling. In summary, our studies indicate that biosynthetic cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways gives rise to a potent lipid autacoid that regulates endothelial cell function in vitro and in vivo. These findings suggest that common drugs targeting the arachidonic acid pathway could prove useful in antiangiogenic therapy.

Unlocking the potential: unveiling tyrphostins with Michael-reactive cyanoacrylate motif as promising inhibitors of human 5-lipoxygenase.

Human 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes, mediators of the innate immune system that also play an important role in inflammatory diseases and cancer. In this study, we present compounds, containing a Michael-reactive cyanoacrylate moiety as potent inhibitors of 5-LO. Representatives of the tyrosine kinase inhibitor family called tyrphostins, structurally related to known 5-LO inhibitors, were screened for their 5-LO inhibitory properties using recombinant human 5-LO, intact human PMNL (polymorphonuclear leukocytes), and PMNL homogenates. Their mode of action was characterized by the addition of glutathione, using a fourfold cysteine 5-LO mutant and mass spectrometry analysis. SAR studies revealed several members of the tyrphostin family containing a Michael-reactive cyanoacrylate to efficiently inhibit 5-LO. We identified degrasyn (IC50 0.11 µM), tyrphostin A9 (IC50 0.8 µM), AG879 (IC50 78 nM), and AG556 (IC50 64 nM) as potent 5-LO inhibitors. Mass spectrometry analysis revealed that degrasyn and AG556 covalently bound to up to four cysteines, including C416 and/or C418 which surround the substrate entry site. Furthermore, the 5-LO inhibitory effect of degrasyn was remarkably impaired by the addition of glutathione or by the mutation of cysteines to serines at the surface of 5-LO. We successfully identified several tyrphostins as potent inhibitors of human 5-LO. Degrasyn and AG556 were able to covalently bind to 5-LO via their cyanoacrylate moiety. This provides a promising mechanism for targeting 5-LO by Michael acceptors, leading to new therapeutic opportunities in the field of inflammation and cancer.

Exosomes derived from human adipose-derived stem cells alleviate hepatic ischemia-reperfusion (I/R) injury through the miR-183/ALOX5 axis.

Ischemia-reperfusion (I/R) injury is a crucial factor causing liver injury in the clinic. Recent research has confirmed that human adipose-derived stem cells (ADSCs) can differentiate into functional hepatocytes. However, the mechanism of the effects of ADSCs in the treatment of liver injury remains unclear. The characteristics of ADSCs were first identified, and exosome-derived ADSCs were isolated and characterized. The function and mechanism of action of miR-183 and arachidonate 5-lipoxygenase (ALOX5) were investigated by functional experiments in HL-7702 cells with I/R injury and in I/R rats. Our data disclosed that exosome release from ADSCs induced proliferation and inhibited apoptosis in HL-7702 cells with I/R injury. The effect of miR-183 was similar to that of exosomes derived from ADSCs. In addition, ALOX5, as a target gene of miR-183, was involved in the related functions of miR-183. Moreover, in vivo experiments confirmed that miR-183 and exosomes from ADSCs could improve liver injury in rats and inhibit the MAPK and NF-κB pathways. All of these findings demonstrate that exosomes derived from ADSCs have a significant protective effect on hepatic I/R injury by regulating the miR-183/ALOX5 axis, which might provide a therapeutic strategy for liver injury.

A drug-drug interaction study and physiologically based pharmacokinetic modelling to assess the effect of an oral 5-lipoxygenase activating protein inhibitor on the pharmacokinetics of oral midazolam.

AIMS: Early clinical studies have indicated that the pharmacokinetics of Atuliflapon (AZD5718) are time and dose dependent. The reason(s) for these findings is(are) not fully understood, but pre-clinical profiling suggests that time-dependent CYP3A4 inhibition cannot be excluded. In clinical practice, Atuliflapon will be co-administered with CYP3A4 substrates; thus, it is important to determine the impact of Atuliflapon on the pharmacokinetics (PK) of CYP3A4 substrates. The aim of this study was to evaluate the effect of Atuliflapon on the pharmacokinetics of a sensitive CYP3A4 substrate, midazolam, and to explore if the time-/dose-dependent effect seen after repeated dosing could be an effect of change in CYP3A4 activity. METHODS: Open-label, fixed-sequence study in healthy volunteers to assess the PK of midazolam alone and in combination with Atuliflapon. Fourteen healthy male subjects received single oral dose of midazolam 2 mg on days 1 and 7 and single oral doses of Atuliflapon (125 mg) from days 2 to 7. A physiologically based pharmacokinetic (PBPK) model was developed to assess this drug-drug interaction. RESULTS: Mean midazolam values of maximum plasma concentration (Cmax) and area under the curve (AUC) to infinity were increased by 39% and 56%, respectively, when co-administered with Atuliflapon vs. midazolam alone. The PBPK model predicted a 27% and 44% increase in AUC and a 23% and 35% increase in Cmax of midazolam following its co-administrations with two predicted therapeutically relevant doses of Atuliflapon. CONCLUSIONS: Atuliflapon is a weak inhibitor of CYP3A4; this was confirmed by the validated PBPK model. This weak inhibition is predicted to have a minor PK effect on CYP3A4 metabolized drugs.

Expression and putative biological roles of lipoxygenases and leukotriene receptors in leukemia and lymphoma.

This mini-review addresses lipoxygenases and receptors for leukotrienes in hematological malignancies. Potential novel biomarkers and drug targets in leukemia and B-cell lymphoma are discussed.

Interactions between Inhibitors and 5-Lipoxygenase: Insights from Gaussian Accelerated Molecular Dynamics and Markov State Models.

Inflammation is a protective stress response triggered by external stimuli, with 5-lipoxygenase (5LOX) playing a pivotal role as a potent mediator of the leukotriene (Lts) inflammatory pathway. Nordihydroguaiaretic acid (NDGA) functions as a natural orthosteric inhibitor of 5LOX, while 3-acetyl-11-keto-ß-boswellic acid (AKBA) acts as a natural allosteric inhibitor targeting 5LOX. However, the precise mechanisms of inhibition have remained unclear. In this study, Gaussian accelerated molecular dynamics (GaMD) simulation was employed to elucidate the inhibitory mechanisms of NDGA and AKBA on 5LOX. It was found that the orthosteric inhibitor NDGA was tightly bound in the protein's active pocket, occupying the active site and inhibiting the catalytic activity of the 5LOX enzyme through competitive inhibition. The binding of the allosteric inhibitor AKBA induced significant changes at the distal active site, leading to a conformational shift of residues 168-173 from a loop to an α-helix and significant negative correlated motions between residues 285-290 and 375-400, reducing the distance between these segments. In the simulation, the volume of the active cavity in the stable conformation of the protein was reduced, hindering the substrate's entry into the active cavity and, thereby, inhibiting protein activity through allosteric effects. Ultimately, Markov state models (MSM) were used to identify and classify the metastable states of proteins, revealing the transition times between different conformational states. In summary, this study provides theoretical insights into the inhibition mechanisms of 5LOX by AKBA and NDGA, offering new perspectives for the development of novel inhibitors specifically targeting 5LOX, with potential implications for anti-inflammatory drug development.

Leukotriene signaling in neurodegeneration: implications for treatment strategies.

Leukotrienes (LTs) are a group of substances that cause inflammation. They are produced by the enzyme 5-lipoxygenase (5-LOX) from arachidonic acid. Cysteinyl LTs are a group of lipid molecules that have a prominent role in inflammatory signaling in the allergic diseases. Although they are traditionally known for their role in allergic disease, current advancements in bio-medical research have shed light on the involvement of these inflammatory mediators in diseases such as in the inflammation related to central nervous system (CNS) disorders. Among the CNS diseases, LTs, along with 5-LOX and their receptors, have been shown to be associated with multiple sclerosis (MS), Alzheimer's disease (AD), and Parkinson's disease (PD). Through a comprehensive review of current research and experimentation, this investigation provides an insight on the biosynthesis, receptors, and biological effects of LTs in the body. Furthermore, implications of leukotriene signaling in CNS and its intricate role in neurodegeneration are also studied. Through the revelation of these insights, our aim is to establish a foundation for the development of enhanced and focused therapeutic approaches in the continuous endeavor to combat neurodegeneration. Furthermore, the pharmacological inhibition of leukotriene signaling with selective inhibitors offers promising prospects for future interventions and treatments for neurodegenerative diseases.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye.

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes. Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss). We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues. Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

ALOX5 inhibition protects against dopaminergic neurons undergoing ferroptosis.

Oxidative disruption of dopaminergic neurons is regarded as a crucial pathogenesis in Parkinson's disease (PD), eventually causing neurodegenerative progression. (-)-Clausenamide (Clau) is an alkaloid isolated from plant Clausena lansium (Lour.), which is well-known as a scavenger of lipid peroxide products and exhibiting neuroprotective activities both in vivo and in vitro, yet with the in-depth molecular mechanism unrevealed. In this study, we evaluated the protective effects and mechanisms of Clau on dopaminergic neuron. Our results showed that Clau directly interacted with the Ser663 of ALOX5, the PKCα-phosphorylation site, and thus prevented the nuclear translocation of ALOX5, which was essential for catalyzing the production of toxic lipids 5-HETE. LC-MS/MS-based phospholipidomics analysis demonstrated that the oxidized membrane lipids were involved in triggering ferroptotic death in dopaminergic neurons. Furthermore, the inhibition of ALOX5 was found to significantly improving behavioral defects in PD mouse model, which was confirmed associated with the effects of attenuating the accumulation of lipid peroxides and neuronal damages. Collectively, our findings provide an attractive strategy for PD therapy by targeting ALOX5 and preventing ferroptosis in dopaminergic neurons.

Structure and ligand based design for identification of highly potent molecules against 5-LOX.

We report here small molecules consisting of dichlorophenyl substituted oxindole that is further tagged with pyrrole/indole moieties. These molecules were designed on the basis of the analysis of binding mode of 5-LOX with arachidonic acid and zileuton. The molecules traverse the active site pocket of the enzyme that otherwise hosts AA and zileuton. Moreover, with a provision of derivatization at pyrrole/indole-N, the physico-chemical properties of the molecules can be adjusted. Appreciable 5-LOX inhibitory activities of the compounds in sub-micromolar range were observed and their aqueous solubility, binding with human serum albumin and stability in blood plasma and liver microsomes were checked. The Michaelis-Menten constants obtained during the binding of the compounds with 5-LOX indicated competitive binding of the compounds with the enzyme. Overall, the combination of molecular modelling and experimental studies identified promising molecules against inflammatory diseases.

Novel thiazolopyridine derivatives of diflapolin as dual sEH/FLAP inhibitors with improved solubility.

Inflammatory responses are orchestrated by a plethora of lipid mediators, and perturbations of their biosynthesis or degradation hinder resolution and lead to uncontrolled inflammation, which contributes to diverse pathologies. Small molecules that induce a switch from pro-inflammatory to anti-inflammatory lipid mediators are considered valuable for the treatment of chronic inflammatory diseases. Commonly used non-steroidal anti-inflammatory drugs (NSAIDs) are afflicted with side effects caused by the inhibition of beneficial prostanoid formation and redirection of arachidonic acid (AA) into alternative pathways. Multi-target inhibitors like diflapolin, the first dual inhibitor of soluble epoxide hydrolase (sEH) and 5-lipoxygenase-activating protein (FLAP), promise improved efficacy and safety but are confronted by poor solubility and bioavailability. Four series of derivatives bearing isomeric thiazolopyridines as bioisosteric replacement of the benzothiazole core and two series additionally containing mono- or diaza-isosteres of the phenylene spacer were designed and synthesized to improve solubility. The combination of thiazolo[5,4-b]pyridine, a pyridinylen spacer and a 3,5-Cl2-substituted terminal phenyl ring (46a) enhances solubility and FLAP antagonism, while preserving sEH inhibition. Moreover, the thiazolo[4,5-c]pyridine derivative 41b, although being a less potent sEH/FLAP inhibitor, additionally decreases thromboxane production in activated human peripheral blood mononuclear cells. We conclude that the introduction of nitrogen, depending on the position, not only enhances solubility and FLAP antagonism (46a), but also represents a valid strategy to expand the scope of application towards inhibition of thromboxane biosynthesis.

Extraction and preparation of 5-lipoxygenase and acetylcholinesterase inhibitors from Astragalus membranaceus stems and leaves.

In this study, an efficient method that employs 5-lipoxygenase and acetylcholinesterase as biological target molecules in receptor-ligand affinity ultrafiltration-liquid chromatography was developed for the screening of enzyme inhibitors derived from the Astragalus membranaceus stems and leaves. The effects of the extraction time, number of extraction cycles, ethanol concentration, and liquid-solid ratio on the total yield of the target compounds were investigated using response surface methodology, and the bioactive components were isolated using a combination of semi-preparative high-performance liquid chromatography and high-speed countercurrent chromatography via a two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (1:6:2:6, v/v/v/v). Subsequently, 10 naturally-occurring bioactive components in the Astragalus membranaceus stems and leaves, including wogonin, ononin, isoquercitrin, calycosin-7-glucoside, 3-hydroxy-9,10-dimethoxyptercarpan, hyperoside, 7,2'-dihydroxy-3',4'-dimethoxyisoflavan, baicalein, calycosin, and soyasaponin, were screened using affinity ultrafiltration to determine their potential effects against Alzheimer's disease. Consequently, all target compounds had purities higher than 95.0%, and the potential anti-Alzheimer's disease effect of the obtained bioactive compounds was verified using molecular docking analysis. Based on the results, the back-to-back screening of complex enzyme inhibitors and separation of the target bioactive compounds using complex chromatography could provide a new approach to the discovery and preparation of natural active ingredients.

The PDK1-FoxO1 signaling in adipocytes controls systemic insulin sensitivity through the 5-lipoxygenase-leukotriene B4 axis.

Although adipocytes are major targets of insulin, the influence of impaired insulin action in adipocytes on metabolic homeostasis remains unclear. We here show that adipocyte-specific PDK1 (3'-phosphoinositide-dependent kinase 1)-deficient (A-PDK1KO) mice manifest impaired metabolic actions of insulin in adipose tissue and reduction of adipose tissue mass. A-PDK1KO mice developed insulin resistance, glucose intolerance, and hepatic steatosis, and this phenotype was suppressed by additional ablation of FoxO1 specifically in adipocytes (A-PDK1/FoxO1KO mice) without an effect on adipose tissue mass. Neither circulating levels of adiponectin and leptin nor inflammatory markers in adipose tissue differed between A-PDK1KO and A-PDK1/FoxO1KO mice. Lipidomics and microarray analyses revealed that leukotriene B4 (LTB4) levels in plasma and in adipose tissue as well as the expression of 5-lipoxygenase (5-LO) in adipose tissue were increased and restored in A-PDK1KO mice and A-PDK1/FoxO1KO mice, respectively. Genetic deletion of the LTB4 receptor BLT1 as well as pharmacological intervention to 5-LO or BLT1 ameliorated insulin resistance in A-PDK1KO mice. Furthermore, insulin was found to inhibit LTB4 production through down-regulation of 5-LO expression via the PDK1-FoxO1 pathway in isolated adipocytes. Our results indicate that insulin signaling in adipocytes negatively regulates the production of LTB4 via the PDK1-FoxO1 pathway and thereby maintains systemic insulin sensitivity.

5-Lipoxygenase Contributes to Benzo[a]pyrene-Induced Cytotoxicity and DNA Damage in Human Bronchial Epithelial Cells.

Metabolic activation of indirect-acting carcinogens in target organs is a recognized mechanism of carcinogenesis. This study aimed to determine the role of benzo[a]pyrene (BaP) metabolism enzymes lipoxygenase (LOX), cytochrome P4501A1 (CYP1A1), and prostaglandin synthetase (PGS) in the cytotoxicity and DNA damage induced by BaP in the human tracheobronchial epithelial cells (HBECs) using RNA interference strategy and metabolic enzyme inhibitors. Our results showed that in three epithelial cell lines (HBE, HTR-8/SVneo, and HaCat), BaP significantly upregulated 5-LOX protein expression. 15-LOX-2 expression also increased with increasing BaP concentration, but the change was less pronounced than that of 5-LOX. BaP caused significant cytotoxicity, DNA strand breaks, and 8-hydroxy-2'-deoxyguanosine formation in HBE, which was inhibited by 5-LOXshRNA, a specific inhibitor of 5-LOX (AA861), the CYP1A1 inhibitor α-naphthoflavone, and the PGS inhibitor naproxen. The protective effects of 5-LOXshRNA were stronger than AA861, naproxen and α-naphthoflavone. We conclude that BaP may be activated more by 5-LOX than by CYP1A1 and PGS to produce cytotoxicity and DNA damage in HBE.

Modulation of the 5-Lipoxygenase Pathway by Chalcogen-Containing Inhibitors of Leukotriene A4 Hydrolase.

The 5-lipoxygenase (5-LOX) pathway gives rise to bioactive inflammatory lipid mediators, such as leukotrienes (LTs). 5-LOX carries out the oxygenation of arachidonic acid to the 5-hydroperoxy derivative and then to the leukotriene A4 epoxide which is converted to a chemotactic leukotriene B4 (LTB4) by leukotriene A4 hydrolase (LTA4H). In addition, LTA4H possesses aminopeptidase activity to cleave the N-terminal proline of a pro-inflammatory tripeptide, prolyl-glycyl-proline (PGP). Based on the structural characteristics of LTA4H, it is possible to selectively inhibit the epoxide hydrolase activity while sparing the inactivating, peptidolytic, cleavage of PGP. In the current study, chalcogen-containing compounds, 4-(4-benzylphenyl) thiazol-2-amine (ARM1) and its selenazole (TTSe) and oxazole (TTO) derivatives were characterized regarding their inhibitory and binding properties. All three compounds selectively inhibit the epoxide hydrolase activity of LTA4H at low micromolar concentrations, while sparing the aminopeptidase activity. These inhibitors also block the 5-LOX activity in leukocytes and have distinct inhibition constants with recombinant 5-LOX. Furthermore, high-resolution structures of LTA4H with inhibitors were determined and potential binding sites to 5-LOX were proposed. In conclusion, we present chalcogen-containing inhibitors which differentially target essential steps in the biosynthetic route for LTB4 and can potentially be used as modulators of inflammatory response by the 5-LOX pathway.

In Vitro and In Silico Investigation of Polyacetylenes from Launaea capitata (Spreng.) Dandy as Potential COX-2, 5-LOX, and BchE Inhibitors.

Diverse secondary metabolites are biosynthesized by plants via various enzymatic cascades. These have the capacity to interact with various human receptors, particularly enzymes implicated in the etiology of several diseases. The n-hexane fraction of the whole plant extract of the wild edible plant, Launaea capitata (Spreng.) Dandy was purified by column chromatography. Five polyacetylene derivatives were identified, including (3S,8E)-deca-8-en-4,6-diyne-1,3-diol (1A), (3S)-deca-4,6,8-triyne-1,3-diol (1B), (3S)-(6E,12E)-tetradecadiene-8,10-diyne-1,3-diol (2), bidensyneoside (3), and (3S)-(6E,12E)-tetradecadiene-8,10-diyne-1-ol-3-O-ß-D-glucopyranoside (4). These compounds were investigated for their in vitro inhibitory activity against enzymes involved in neuroinflammatory disorders, including cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and butyrylcholinesterase (BchE) enzymes. All isolates recorded weak-moderate activities against COX-2. However, the polyacetylene glycoside (4) showed dual inhibition against BchE (IC50 14.77 ± 1.55 µM) and 5-LOX (IC50 34.59 ± 4.26 µM). Molecular docking experiments were conducted to explain these results, which showed that compound 4 exhibited greater binding affinity to 5-LOX (-8.132 kcal/mol) compared to the cocrystallized ligand (-6.218 kcal/mol). Similarly, 4 showed a good binding affinity to BchE (-7.305 kcal/mol), which was comparable to the cocrystallized ligand (-8.049 kcal/mol). Simultaneous docking was used to study the combinatorial affinity of the unresolved mixture 1A/1B to the active sites of the tested enzymes. Generally, the individual molecules showed lower docking scores against all the investigated targets compared to their combination, which was consistent with the in vitro results. This study demonstrated that the presence of a sugar moiety (in 3 and 4) resulted in dual inhibition of 5-LOX and BchE enzymes compared to their free polyacetylenes analogs. Thus, polyacetylene glycosides could be suggested as potential leads for developing new inhibitors against the enzymes involved in neuroinflammation.

Systemic inhibition of 5-lipoxygenase by MK-886 exacerbates apical periodontitis bone loss in a mouse model.

BACKGROUND: To investigate if 5-LO selective inhibitor (MK-886) could be used for systemic treatment of experimentally induced apical periodontitis in a mouse model. METHODS: Twenty-four C57BL/6 mice were used. After coronal opening, a solution containing Escherichia coli LPS (1.0 µg/µL) was inoculated into the root canals of the lower and upper right first molars (n = 72 teeth). After 30 days apical periodontitis was established, and the animals were treated with MK-886 (5 mg/kg), a 5-LO inhibitor, for 7 and 14 days. The tissues were removed for histopathological and histometric analyses, evaluation of osteoclast number and gene expression for receptor activator of nuclear factor kappa-B (Tnfrsf11a), receptor activator of nuclear factor kappa-B ligand (Tnfsf11), osteoprotegerin (Tnfrsf11b), tartrate-resistant acid phosphatase (Acp5), matrix metalloproteinase-9 (Mmp9), cathepsin K (Ctsk) and calcitonin receptor (Calcr). Statistical data analysis was performed using Kruskal Wallis followed by Dunn's tests (α = 0.05). RESULTS: Administration of MK-886 for 7 days exerted no effect on apical periodontitis progression compared to LPS inoculation without treatment (p = 0.3549), while treatment for 14 days exacerbated bone loss (p < 0.0001). Administration of MK-886 enhanced osteoclastogenesis signaling and osteoclast formation within 7 days (p = 0.0005), but exerted no effect at 14 days (p > 0.9999). After 7 days of treatment, MK-886 induced mRNA expression for Acp5 (p = 0.0001), Calcr (p = 0.0003), Mmp9 (p = 0.0005) and Ctsk (p = 0.0008), however no effect in those gene expression was observed after 14 days (p > 0.05). CONCLUSION: Systemic treatment with MK-886 exacerbated LPS-induced apical periodontitis in a mouse model.

Role of ALOX5 in non-small cell lung cancer: A potential therapeutic target associated with immune cell infiltration. / ALOX5å¯ä½œä¸ºä¸Žå ç–«ç»†èƒžæµ¸æ¶¦ç›¸å ³çš„éžå°ç»†èƒžè‚ºç™Œé¢„åŽç”Ÿç‰©æ ‡å¿—ç‰©.

OBJECTIVES: The efficacy of immunotherapy for lung cancer is closely related to immune cell infiltration. Arachidonic acid 5-lipoxygenase (ALOX5) can activate inflammatory responses and trigger various cell death patterns; however, the relevance of ALOX5 to immune cell infiltration in lung cancer is unclear. The expression of ALOX5 in non-small cell lung cancer (NSCLC) is analyzed using an online database to explore the correlation between ALOX5 and immune cell infiltration in NSCLC and its relationship with prognosis. METHODS: Differences in ALOX5 expression in NSCLC and normal lung tissues were analyzed by online databases such as TIMER, GEPIA and HPA; the UALCAN database was used to reveal the relationship between ALOX5 and clinical features; Kaplan-Meier database was applied to explore the prognostic value of ALOX5; GeneMANIA and String Website was used to explore genes and proteins associated with ALOX5 expression, respectively; the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to analyze ALOX5 differential genes which were picked up through the TCGA database; GSEA software was applied to predict the signal pathways that ALOX5 may be involved in; and the TIMER database was used to analyze the effect of ALOX5 expression on the level of immune cell infiltration. RESULTS: Compared with the normal lung tissues, the ALOX5 expression was low in NSCLC tissues (P<0.05), and which affected the prognosis of lung cancer patients. The expression level of ALOX5 was related to clinical features such as sex, age, metastasis, and pathological staging in NSCLC patients (all P<0.05). The gene interaction network analysis found that the genes interacting with ALOX5 mainly included the genes related to lipid oxidation and pro-inflammatory mediators such as coactosin like protein 1 (COTL1), leukotriene C4 synthase (LTC4S), and prostaglandin endoperoxide synthase 2 (PTGS2), and the protein-protein interaction analysis results were consistent. GO and KEGG analysis found that ALOX5 was involved in the biological process of activation of immune cell function and was involved in immune response function pathways. The GSEA analysis showed that ALOX5 may activate immune responses and mediate immune-related prognosis by affecting the cytokine-cytokine receptor interactions, natural killer-mediated cytotoxicity, and T cell receptor signaling pathways. The ALOX5 mRNA expressions in lung adenocarcinoma and lung squamous cell carcinoma were positively correlated with the tumor infiltration immune cells (B cells, CD8+ T cells, CD4+ T cells, etc.) (all P<0.05), and the ALOX5 mRNA expression was positively correlated with the expression of classic T cell immune checkpoint inhibitor genes (P<0.001). CONCLUSIONS: The ALOX5 gene expression in NSCLC is significantly downregulated, and which can affect NSCLC prognosis and immune cell infiltration levels. ALOX5 gene may be a potential biomarker of NSCLC prognosis associated with immune cell infiltration.

MicroRNA-146a promotes proliferation, migration, and invasion of HepG2 via regulating FLAP.

Abnormal expression of 5-Lipoxygenase Activating Protein (FLAP) has been detected in many tumor cells. MicroRNAs (miRNAs) negatively regulate gene expression post-transcriptionally by binding to the 3'-untranslated region (3'-UTR) of the target mRNA sequences and have been shown to be involved in various types of cancers. Herein, we aimed to demonstrate the expression of miR-146a and FLAP in human HCC tissues and liver cancer cell lines. We demonstrated that miR-146a expression is overexpressed, while FLAP protein and mRNA are suppressed in hepatocellular carcinoma tissues and HepG2 cells compared to para-carcinoma tissues and HL-7702 cells. Dual luciferase reporter gene assay showed that miR-146a-5p can directly target FLAP mRNA. Knockdown of miR-146a also resulted in increased FLAP expression of cancer cells. Additionally, miR-146a silencing or restoration of FLAP led to a reduction of HepG2 cell proliferation, cell cycle progression, migration, and invasion. This study showed that miR-146a has a stimulatory role in HepG2 cells and promotes HepG2 cell migration and invasion by targeting FLAP mRNA. Thus, miR-146a may be a tumor promoter and a potential therapeutic target for the treatment of HCC patients.

Modulation of microRNA processing by 5-lipoxygenase.

The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.