Investigating the Anti-tumor and Apoptosis-inducing Effects of Coumarin Derivatives as Potent 15-Lipoxygenase Inhibitors on PC-3 Prostate Cancer Cells.

INTRODUCTION: Prostate cancer is the second most prevalent cancer among men. Despite different treatments, including surgery, chemotherapy, radiation therapy, hormone therapy and immunotherapy for this disease, patients ultimately progress to advanced states. Thus, there is a need for new treatment options targeting cell growth and apoptosis to better control the proliferation and metastasis of these cells. There are many reports indicating overexpression of the 15-lipoxygenase-1 (15-LOX-1) enzyme in prostate tumors. Studies have also shown that inhibition of this enzyme prevents the progression of prostate cancer. OBJECTIVE: This study was conducted to assess the anti-cancer properties of some coumarin derivatives as possible 15- LOX-1 inhibitors, on PC-3 prostate cancer cells. METHODS: In this study, the activity of 15-LOX-1 was evaluated in PC-3 cells by a spectrophotometric assay. In addition, due to high similarity between the 15-LOX-1 and soybean 15-lipoxygenase (SLO) (L1; EC 1, 13, 11, 12) active sites, the soybean SLO was used to investigate inhibitory effects of synthetic coumarin compounds 8- isopentenyloxycoumarin (8-IC), 8-isopentenyloxy-3-carboxycoumarin (8-ICC), 8-geranyloxycoumarin (8-GC), 8- geranyloxy-3-carboxycoumarin (8-GCC), and 8-farnesyloxy-3-carboxycoumarin (8-FCC) on this enzyme. Moreover, the cytotoxic and anticancer effects of the coumarin compounds were examined on PC-3 (Prostate Cancer) and HDF-1 (Human Dermal Fibroblast) cells by alamarBlue assay. Finally, apoptosis-inducing effects of all synthetic compounds were determined by flow cytometry. RESULTS: The IC50 values obtained by the alamarBlue test revealed that 8-IC, 8-GC and 8-GCC had cytotoxic effects on PC-3 cells. Treating both PC-3 and HDF-1 cells with 8-ICC and 8-FCC did not significantly reduce cell number. Furthermore, the IC50 values of 8-IC on HDF-1 cells showed cytotoxic effects, while treating these cells with 8-GC and 8- GCC did not show any significant cytotoxicity on these normal human fibroblasts. Assessing the ability of 4-MMPB (as a specific inhibitor of 15-LOX-1), 8-GC, and 8-GCC compounds to inhibit SLO revealed that these compounds exerted strong 15-LOX-1 inhibitory activity, while 8-IC and 8-FCC had a weak inhibitory effect and also 8-ICC showed no inhibitory effect on SLO enzyme. In addition, flow cytometric analysis by FITC (fluorescein isothiocyanate)- annexin V and propidium iodide showed that treatment with IC50 values of 8-GC and 8-GCC induced apoptosis in 35.2% and 30.8% of PC-3 cells, respectively. CONCLUSION: Thus, 8-GC and 8-GCC can be introduced as effective anticancer agents with apoptosis-inducing properties. Furthermore, our results suggest that the cytotoxic effects of these compounds might be related to the inhibition of 15-LOX-1 enzyme in PC-3 cells. On the other hand, the cytotoxic effects of 8-IC might be due to the inhibition of other signaling pathways in PC-3 cells. However, further in vivo experiments are required to determine the exact mechanisms involved in the anticancer effects of these coumarin compounds.

8-Geranyloxycarbostyril as a potent 15-LOX-1 inhibitor showed great anti-tumor effects against prostate cancer.

Carbostyrils are quinolone derivatives, with possible growth inhibition properties on cancer cells. Unlike many tumors, 15-Lipoxygenase-1 (15-LOX-1) is highly expressed in prostate cancer (PCa) cells and has oncogenic properties. Here, with the hypothesis that 6-, 7- and 8-geranyloxycarbostyril (GQ) have inhibitory properties on 15-LOX-1, their effects were assessed on PCa cells. Their cytotoxic effects were evaluated by MTT assay and mechanism of cell death was investigated using annexin V/PI staining. Finally, the anti-tumor properties of 8-GQ were assessed in immunocompromised C57BL/6 mice bearing human PCa cells. Accordingly, these compounds could effectively inhibit 15-LOX activity in PCa cells. MTT and flow cytometry tests confirmed their toxic effects on PCa cells, with no significant toxicity on normal cells, and apoptosis was the main mechanism of cell death. In vivo results indicated that use of 8-GQ at 50 mg/kg had stronger anti-tumor effects than 5 mg/kg cisplatin, with fewer side effects on normal tissues. Therefore, 8-GQ can be introduced as a potential drug candidate with 15-LOX-1 inhibitory potency, which can be effective in treatment of prostate cancer, and should be considered for further drug screening investigations.

ALOX5-5-HETE promotes gastric cancer growth and alleviates chemotherapy toxicity via MEK/ERK activation.

BACKGROUND: Recent studies highlight the regulatory role of arachidonate lipoxygenase5 (Alox5) and its metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) in cancer tumorigenesis and progression. In this study, we analyzed the expression, biological function and the downstream signaling of Alox5 in gastric cancer. METHODS: Alox5 protein levels were measured using IHC and ELISA. Growth, migration and survival assays were performed. Phosphorylation of molecules involved in growth and survival signaling were analyzed by WB. Analysis of variance and t-test were used for statistic analysis. RESULTS: Alox5 and 5-HETE levels were upregulated in gastric cancer patients. ALOX5 overexpression or 5-HETE addition activates gastric cancer cells and reduces chemotherapy's efficacy. In contrast, ALOX5 inhibition via genetic and pharmacological approaches suppresses gastric cancer cells and enhances chemotherapy's efficacy. In addition, Alox5 inhibition led to suppression of ERK-mediated signaling pathways whereas ALOX5-5-HETE activates ERK-mediated signaling in gastric cancer cells. CONCLUSIONS: Our work demonstrates the critical role of ALOX5-5-HETE in gastric cancer and provides pre-clinical evidence to initialize clinical trial using zileuton in combination with chemotherapy for treating gastric cancer.

Lipoxygenase inhibitor ML351 dysregulated an innate inflammatory response leading to impaired cardiac repair in acute heart failure.

The presistent increase of 12/15 lipoxygenase enzyme activity is correlated with uncontrolled inflammation, leading to organ dysfunction. ML351, a potent 12/15 lipoxygenase (12/15LOX) inhibitor, was reported to reduce infarct size and inflammation in a murine ischemic stroke model. In the presented work, we have applied three complementary experimental approaches, in-vitro, ex-vivo, and in-vivo, to determine whether pharmacological inhibition of 12/15LOX could dampen the inflammatory response in adult mice after Kdo2-Lipid A (KLA) as an endotoxin stimulator or post myocardial infarction (MI). Male C57BL/6 (8-12 weeks) mice were subjected to permanent coronary ligation thereby inducing acute heart failure (MI-d1 and MI-d5) for in-vivo studies. 12/15LOX antagonist ML351 (50 mg/kg) was subcutaneously injected 2 h post-MI, while MI-controls received saline. For ex-vivo experiments, ML351 (25 mg/kg) was injected as bolus after 5 min of inflammatory stimulus (KLA 1 µg/g) injection. Peritoneal macrophages (PMɸ) were harvested after 4 h post KLA. For in-vitro studies, PMɸ were treated with KLA (100 ng/mL), ML351 (10 µM), or KLA + ML351 for 4 h, and inflammatory response was evaluated. In-vivo, 5LOX expression was reduced after ML351 administration, inducing a compensatory increase of 12LOX that sensitized PMɸ toward a proinflammatory state. This was marked by higher inflammatory cytokines and dysregulation of the splenocardiac axis post-MI. ML351 treatment increased CD11b+ and Ly6Chigh populations in spleen and Ly6G+ population in heart, with a decrease in F4/80+ macrophage population at MI-d1. In-vitro results indicated that ML351 suppressed initiation of inflammation while ex-vivo results suggested ML351 overactivated inflammation consequently delaying the resolution process. Collectively, in-vitro, ex-vivo, and in-vivo results indicated that pharmacological blockade of lipoxygenases using ML351 impaired initiation of inflammation thereby dysregulated acute immune response in cardiac repair.

Novel 1,2,4-triazine-quinoline hybrids: The privileged scaffolds as potent multi-target inhibitors of LPS-induced inflammatory response via dual COX-2 and 15-LOX inhibition.

Based on the observed pharmacophoric structural features for the reported dual COX/15-LOX inhibitors and inspired by the abundance of COX/LOX inhibitory activities reported for the 1,2,4-triazine and quinoline scaffolds, we designed and synthesized novel 1,2,4-triazine-quinoline hybrids (8a-n). The synthesized hybrids were evaluated in vitro as dual COXs/15-LOX inhibitors. The new triazine-quinoline hybrids (8a-n) exhibited potent COX-2 inhibitory profiles (IC50 = 0.047-0.32 µM, SI âˆ¼ 20.6-265.9) compared to celecoxib (IC50 = 0.045 µM, SI âˆ¼ 326). Moreover, they revealed potent inhibitory activities against 15-LOX enzyme compared to reference quercetin (IC50 = 1.81-3.60 vs. 3.34 µM). Hybrid 8e was the most potent and selective dual COX-2/15-LOX inhibitor (COX-2 IC50 = 0.047 µM, SI = 265.9, 15-LOX IC50 = 1.81 µM). These hybrids were further challenged by their ability to inhibit NO, ROS, TNF-α, IL-6 inflammatory mediators, and 15-LOX product, 15-HETE, production in LPS-activated RAW 264.7 macrophages cells. Compound 8e was the most potent hybrid in reducing ROS and 15-HETE levels showing IC50 values of 1.02 µM (11-fold more potent than that of celecoxib, IC50 = 11.75 µM) and 0.17 µM (about 43 times more potent than celecoxib, IC50 = 7.46 µM), respectively. Hybrid 8h exhibited an outstanding TNF-α inhibition with IC50 value of 0.40 µM which was about 25 times more potent than that of celecoxib and diclofenac (IC50 = 10.69 and 10.27 µM, respectively). Docking study of the synthesized hybrids into the active sites of COX-2 and 15-LOX enzymes ensures their favored binding affinity. To our knowledge, herein we reported the first 1,2,4-triazine-quinoline hybrids as dual COX/15-LOX inhibitors.

Red-kerneled rice proanthocyanidin inhibits arachidonate 5-lipoxygenase and decreases psoriasis-like skin inflammation.

5-lipoxygenase is a key enzyme in the synthesis of leukotrienes from arachidonic acid. The produced leukotrienes are involved in inflammatory diseases including psoriasis, asthma, and atherosclerosis. A suitable 5-lipoxygenase inhibitor might be useful for preventing and improving the symptoms of leukotriene-related inflammatory diseases. Here, we investigate the mechanism underlying the anti-inflammatory effect of a proanthocyanidin found in red-kerneled rice. Red-kerneled rice proanthocyanidin exhibited potent mixed noncompetitive inhibition of human and rat 5-lipoxygenases, with an IC50 values of 15.1 µM against human enzyme, and 7.0 µM against rat enzyme, respectively. This compound decreased leukotriene B4 production in rat basophilic leukemia-2H3 cells. In imiquimod-induced psoriasis-like mouse skin, topical application of the proanthocyanidin suppressed hyperplasia, decreased inflammatory cell infiltration, and down-regulated expression of the psoriasis-associated genes Il17a, Il22, S100a9, and Krt1. Lipid metabolome analysis by electrospray ionization mass spectrometry showed that red-kerneled rice proanthocyanidin treatment of psoriasis-like mouse skin dose-dependently decreased the production of leukotriene B4 but no other arachidonate metabolites. Red-kerneled rice proanthocyanidin inhibits 5-lipoxygenase, resulting in a decrease in leukotriene B4 production and psoriasis-like mouse skin inflammation. These results suggest that this proanthocyanidin may be therapeutically effective for treating leukotriene-related diseases.

MicroRNA-193b-3p alleviates focal cerebral ischemia and reperfusion-induced injury in rats by inhibiting 5-lipoxygenase expression.

AIMS: Ischemic stroke has become one of the main causes of death worldwide. MicroRNAs (miRNAs) have been implicated in cerebral ischemia-reperfusion (I/R) injury and could serve as therapeutic targets. 5-Lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of leukotrienes and has been implicated in inflammatory central nerve system disorders. The objective of this study was to explore the neuroprotective effects of miR-193b-3p against focal cerebral I/R injury in rats by regulating 5-LOX expression. METHODS AND MATERIALS: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion injury. The level of miR-193b-3p expression was observed in the rat cortical peri-infarct region after focal cerebral I/R injury. Bioinformatics analysis was used to predict the binding sites of miR-193b-3p, and a dual-luciferase reporter gene assay was applied to verify the potential interaction between 5-LOX mRNA and miR-193b-3p. Then, rats were injected with a miR-193b-3p agomir (modified and enhanced mimic) or antagomir (modified and enhanced inhibitor) in the right lateral ventricle of the brain. Neurological deficit scores, infarct volumes, neuron damage and 5-LOX enzymatic activity and expression were measured. In an in vitro experiment, cultured PC12 cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R). OGD/R-induced cells were treated with a miR-193b-3p mimic or inhibitor and 5-LOX siRNA. Cell viability, lactate dehydrogenase release, apoptosis rate and 5-LOX expression were evaluated. RESULTS: The level of miR-193b-3p expression was increased in the cortical peri-infarct region of rats with cerebral focal I/R injury. The results of the dual-luciferase reporter gene assay showed that a miR-193b-3p binding site was located in the 3' untranslated region (3'UTR) of 5-LOX mRNA. Neurological deficit scores, infarct volumes and neuronal injury were alleviated by miR-193b-3p agomir treatment but aggravated by miR-193b-3p antagomir. Furthermore, leukotriene B4, cysteinyl-leukotrienes and 5-LOX expression in the cortical peri-infarct region of rats with focal cerebral I/R injury were also downregulated by miR-193b-3p agomir treatment but upregulated by miR-193b-3p antagomir. In PC12 cells, miR-193b-3p mimic significantly decreased OGD/R-induced cell death and reduced lactate dehydrogenase release and 5-LOX expression. In contrast, miR-193b-3p inhibitor exacerbated OGD/R-induced injury in PC12 cells. Additionally, the in vitro effects of miR-193b-3p inhibitor on OGD/R-induced cell injury were partially reversed by 5-LOX siRNA treatment. CONCLUSION: MiR-193b-3p has a potentially neuroprotective effect on focal cerebral I/R-induced injury by inhibiting 5-LOX expression.

Effect of 5-lipoxygenase inhibitor, VIA-2291 (Atreleuton), on epicardial fat volume in patients with recent acute coronary syndrome.

BACKGROUND AND AIMS: The association between inflammation and atherosclerosis has been well described in the literature. There is now mounting evidence in support of adipose tissue as a reservoir for inflammatory markers. Epicardial adipose tissue (EAT) has been shown to associate with coronary atherosclerosis and found to be a predictor of future adverse cardiovascular events. This study, VIA-EAT, assesses the change in epicardial fat volume (EFV) after treatment with an investigational anti-inflammatory agent (VIA-2291) in a cohort of post-acute coronary syndrome (ACS) patients. METHODS: This study is derived from a post-hoc analysis of a previously conducted randomized clinical trial (NCT00358826). Patients were recruited for a prospective, double-blind, multi-center randomized trial of a 5-lipooxygenase inhibitor or placebo in a 3:1 randomization, including doses of placebo, and 25 mg, 50 mg and 100 mg of active treatment. Cardiac computed tomography was performed at baseline and at 24 weeks after treatment with VIA-2291. EAT and pericardial adipose tissue (PAT) were measured using previously published methodology. A Pearson correlation test was used to determine the relationship between change in epicardial fat and change in plaque composition. RESULTS: We analyzed 54 pre- and post-treatment scans. There were no major differences between traditional cardiovascular risk factors among the 4 randomized study arms. There was a significant decrease in EAT and PAT in patients in the treatment arms vs. placebo, -3.0 ± 8.2mm3 and -3.9 ± 10.9mm3 vs. 1.7 ± 7.5mm3 and 1.4 ± 10.7mm3 (p = 0.001), respectively. The changes in EAT and PAT were more pronounced in patients taking 100 mg of the drug vs. placebo: 4.2 ± 9.6mm3, -7.6 ± 8.5mm3, p = 0.0001, respectively. In a subgroup analysis, reduction in epicardial fat volume correlated with reduction in total atherosclerotic plaque volume across all VIA treatment groups, r = 0.52 (p = 0.004). CONCLUSIONS: After adjustment for traditional cardiovascular risk factors including age, gender, body mass index, dyslipidemia and smoking, VIA-2291 decreases EAT and PAT in individuals with recent ACS. Treatment with the drug also appears to alter plaque volume and composition.

Phytochemical study, molecular docking, genotoxicity and therapeutic efficacy of the aqueous extract of the stem bark of Ximenia americana L. in the treatment of experimental COPD in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ximenia americana L. is popularly known as yellow plum, brave plum or tallow wood. All the parts of this plant are used in popular medicine. Its reddish and smooth bark are used to treat skin infections, inflammation of the mucous membranes and in the wound healing process. OBJECTIVE: Verification of phytochemical profile, the molecular interaction between flavonoid, (-) epi-catechin and 5-LOX enzyme, by means of in silico study, the genotoxic effect and to investigate the pharmacological action of the aqueous extract of the stem bark of X. americana in pulmonary alterations caused by experimental COPD in Rattus norvegicus. MATERIALS AND METHODS: The identification of secondary metabolites was carried out by TLC and HPLC chromatographic methods, molecular anchoring tests were applied to analyze the interaction of flavonoid present in the extract with the enzyme involved in pulmonary inflammation process and the genotoxic effect was assessed by comet assay and micronucleus test. For induction of COPD, male rats were distributed in seven groups. The control group was exposed only to ambient air and six were subjected to passive smoke inhalations for 20 min/day for 60 days. One of the groups exposed to cigarette smoke did not receive treatment. The others were treated by inhalation with beclomethasone dipropionate (400 mcg/kg) and aqueous and lyophilized extracts of X. americana (500 mg/kg) separately or in combination for a period of 15 days. The structural and inflammatory pulmonary alterations were evaluated by histological examination. Additional morphometric analyses were performed, including the alveolar diameter and the thickness of the right ventricle wall. RESULTS: The results showed that the aqueous extract of the bark of X. americana possesses (-) epi -catechin, in silico studies with 5-LOX indicate that the EpiC ligand showed better affinity parameters than the AracA ligand, which is in accordance with the results obtained in vivo studies. Genotoxity was not observed at the dose tested and the extract was able to stagnate the alveolar enlargement caused by the destruction of the interalveolar septa, attenuation of mucus production and decrease the presence of collagen fibers in the bronchi of animals submitted to cigarette smoke. CONCLUSION: Altogether, the results proved that the aqueous extract of X. americana presents itself as a new option of therapeutic approach in the treatment of COPD.

hucMSCs Attenuate IBD through Releasing miR148b-5p to Inhibit the Expression of 15-lox-1 in Macrophages.

Mesenchymal stem cells (MSCs) exert powerful immunosuppression in inflammatory bowel disease (IBD). Macrophages are the dominant inflammatory cells in enteritis regulated via MSCs. However, the roles of macrophages in the process of MSCs attenuating IBD and the mechanisms of MSCs regulating macrophages are largely unknown. In this study, DSS- (dextran sulfate sodium salt-) induced IBD in macrophage-depleted models of CD11b-DTR mice was used to study the relationship between hucMSCs (human umbilical cord mesenchymal stromal cells) and macrophage. Body weights, disease activities, and pathological changes were documented to assess the therapeutic effects of hucMSCs. Furthermore, hucMSCs transfected with miR148b-5p mimics and miR148b-5p inhibitors were cocultured with LPS-induced RAW264.7 cells to investigate the role of miR148b-5p in hucMSC-regulated colitis. The outcome indicated that hucMSCs attenuated the IBD by downregulating 15-lox-1 expression in macrophages. Further findings pointed out that hucMSCs transfected with miR148b-5p mimics could be elevated to promote the tissue repair and inhibit the expression of 15-lox-1 but failed to perform the function of easing enteritis when treated with miR148b-5p inhibitors. In conclusions, we propose that hucMSCs attenuate IBD by releasing miR148b-5p to inhibit the expression of 15-lox-1 in macrophages.

Inhibition of 5-lipoxygenase decreases renal fibrosis and progression of chronic kidney disease.

In inflammatory diseases, the 5-lipoxygenase (5-LO) pathway contributes to epithelial damage and fibrosis by catalyzing the production of leukotrienes (LTs). Antagonists of the 5-LO pathway are currently approved for use in patients and are well tolerated. We found that expression of 5-LO is strongly induced in three models of chronic kidney disease: unilateral ureteral obstruction (UUO), folate nephropathy, and an orthologous mouse model of polycystic kidney disease. Immunohistochemistry showed that macrophages are the dominant source of 5-LO. Zileuton, a US Food and Drug Administration-approved antagonist of 5-LO, significantly reduced fibrosis at 7 and 14 days after UUO; these findings were confirmed using a genetically modified [5-LO-associated protein-knockout ( Alox5ap-/-)] mouse strain. Inhibition of 5-LO did not appear to change infiltration of leukocytes after UUO as measured by flow cytometry. However, fluorescence-lifetime imaging microscopy showed that 5-LO inhibitors reversed the glycolytic switch in renal tubular epithelial cells after UUO. Two downstream enzymes of 5-LO, LTA4 hydrolase (LTA4H) and LTC4 synthase (LTC4S), are responsible for the synthesis of LTB4 and cysteinyl LTs, respectively. Fibrosis was reduced after UUO in Ltc4s-/-, but not Lta4h-/-, mice. In contrast, using the folate nephropathy model, we found reduced fibrosis and improved renal function in both Ltc4s-/- and Lta4h-/- mice. In summary, our studies suggest that manipulation of the 5-LO pathway may represent a novel treatment approach for chronic kidney disease.

The importance of 15-lipoxygenase inhibitors in cancer treatment.

Cancer-targeted therapy is an expanding and successful approach in treatment of many types of cancers. One of the main categories of targeted therapy is use of small molecule inhibitors. 15-Lipoxygenase (15-LOX) is an enzyme which reacts with polyunsaturated fatty acids and produces metabolites that are implicated in many important human diseases, such as cancer. Considering the role of 15-LOX (mainly 15-LOX-1) in the progression of some cancers, the discovery of 15-LOX inhibitors could potentially lead to development of novel cancer therapeutics and it can be claimed that 15-LOX inhibitors might be suitable as chemotherapy agents in the near future. This article reviews relevant publications on 15-LOX inhibitors with focus on their anticancer activities in vitro and in vivo. Many 15-LOX inhibitors have been reported for which separate studies have shown their anticancer activities. This review paves the way to further explore the mechanism of their antiproliferative effects via 15-LOX inhibition.

Benzimidazole scaffold based hybrid molecules for various inflammatory targets: Synthesis and evaluation.

Designing of hybrid drugs with specific multitarget profile is a promising line of attack against inflammation. In light of this, a series of benzimidazole scaffold based hybrid molecules were designed by integrating benzimidazoles (containing pharmacophoric elements for COXs and LOXs inhibitors) with phthalimide subunit of thalidomide (pharmacophore element for TNF-α inhibitor) under one construct via molecular hybridization strategy. The designed molecules were synthesized and evaluated for their inhibitory activity against COXs (COX-1, COX-2), LOXs (5-LOX, 15-LOX) enzymes as well as TNF-α inhibitory effect. The results revealed that, compounds (3a-l) obtained showed inhibition in submicromolar range against COXs and LOXs targets whereas milder inhibitory activity was obtained against lipopolysaccharides (LPS)-induced TNF-α secretion by murine macrophage-like cells (RAW264.7). Within this class of compounds, 3j emerged as having alluring multiple inhibitory effects on set of COX-1/2 and 5-/15-LOX enzymes (COX-1 IC50 = 9.85 µM; COX-2 IC50 = 1.00 µM; SI = 9.85; 5-LOX IC50 = 0.32 µM; 15-LOX IC50 = 1.02 µM) in conjunction with a good anti-inflammatory and analgesic activities. Additionally, compound 3j showed gastrointestinal safety with reduced lipid peroxidation. Docking results of compound 3j with COX-2 and 5-LOX were also consistent with the in vivo anti-inflammatory results.

Effects of phenidone (DuCLOX-2/5 inhibitor) against N-methyl-N-nitrosourea induced mammary gland carcinoma in albino rats.

The present study was designed to evaluate the effects of phenidone (Dual inhibitor of COX-2 and 5-LOX, DuCLOX-2/5 inhibitor) on various aspects of cancer chemoprevention. Treatment with the phenidone was inquested to validate the implications of dual inhibition of arachidonic acid (AA) metabolism against MNU induced mammary gland carcinogenesis. MNU treated rat showed altered hemodynamic profile, distorted cellular architecture, upregulated inflammatory enzyme markers (COX, LOX, Nitric oxide and hydrogen sulfide) and distorted oxidative stress markers (thiobarbituric acid reactive substances, protein carbonyl, superoxide dismutase, catalase and glutathione). Phenidone treatment regulated histological architecture in the experimental animals similar to control. The treatment with phenidone favorably regulated the levels of inflammatory markers, and oxidative stress markers against toxic treatment. Our findings emphasize the potential role of phenidone in suppression of mammary gland carcinoma against the deleterious effects of MNU.

5-LOX in Alzheimer's Disease: Potential Serum Marker and In Vitro Evidences for Rescue of Neurotoxicity by Its Inhibitor YWCS.

The inflammatory process plays a key role in neurodegenerative disorder. The inflammatory molecule, 5-lipooxygenase (5-LOX), protein is involved in the pathologic phenotype of Alzheimer's disease (AD) which includes Aß amyloid deposition and tau hyperphosphorylation. This study determined the level of 5-LOX in serum of AD patients, mild cognitive impairment (MCI) patients, and the normal elderly, and the rescue effect by YWCS, a peptide inhibitor of 5-LOX on neurotoxicity by Aß amyloid25-35 (Aß25-35) in neuroblastoma cells. The concentration of serum 5-LOX was estimated by surface plasmon resonance and western blot. The neuroprotective effect of 5-LOX peptide inhibitor YWCS in Aß25-35-induced neurotoxicity was analyzed by MTT assay and western blotting. We found significant upregulated serum 5-LOX in AD patients and also in MCI patients compared to the normal control group. The peptide inhibitor of 5-LOX, YWCS, prevented the neurotoxic effect of Aß25-35 by reducing the expression of γ-secretase as well as p-Tau181 in SH-SY5Y cells. However, YWCS was nontoxic towards normal HEK cells. The differential expression of serum 5-LOX among the study groups suggests it can be one of potential serum protein marker and a therapeutic regimen for AD and MCI. The negative correlation with neuropsychological parameters, i.e., MoCA and HMSE, increases its importance and makes it useful during the clinical setup which is very needful in developing countries. Peptide YWCS can serve as a new platform as a 5-LOX inhibitor which can prevent neurotoxicity developed in AD.

Optimization of benzoquinone and hydroquinone derivatives as potent inhibitors of human 5-lipoxygenase.

Aiming to assess the biological activities of synthetic 1,4-benzoquinones, we previously synthesized different libraries of benzoquinones with lipophilic and bulky alkyl- or aryl-substituents that inhibited 5-lipoxygenase (5-LO). The high potency of 4,5-dimethoxy-3-alkyl-1,2-benzoquinones on 5-LO led to the idea to further modify the structures and thus to improve the inhibitory potential in vitro and in vivo as well as to investigate SARs. Systematic structural optimization through accurate structure-based design resulted in compound 30 (3-tridecyl-4,5-dimethoxybenzene-1,2-diol), an ubiquinol derivative that exhibited the strongest anti-inflammatory effect, with a 10-fold improved 5-LO inhibitory activity (IC50 = 28 nM) in activated neutrophils. Moreover, 30 significantly reduced inflammatory reactions in the carrageenan-induced mouse paw oedema and in zymosan-induced peritonitis in mice. Compound 30 (1 mg/kg, i.p.) potently suppressed the levels of cysteinyl-LTs 30 min after zymosan, outperforming zileuton at a dose of 10 mg/kg. The binding patterns of the quinone- and hydroquinone-based 5-LO inhibitors were analyzed by molecular docking. Together, we elucidated the optimal alkyl chain pattern of quinones and corresponding hydroquinones and reveal a series of highly potent 5-LO inhibitors with effectiveness in vivo that might be useful as anti-inflammatory drugs.

Use of a balanced dual cyclooxygenase-1/2 and 5-lypoxygenase inhibitor in experimental colitis.

Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) play an important role in inflammatory bowel diseases (IBDs). We investigated the effects of flavocoxid, a dual COX/LOX inhibitor, in experimental colitis induced with either dinitrobenzenesulfonic acid (DNBS) or dextrane sulphate sodium (DSS) In the first model, colitis was induced in rats by a single intra-colonic instillation (25mg in 0.8ml 50% ethanol) of DNBS; after 24h animals were randomized to receive orally twice a day, flavocoxid (10mg/kg), zileuton (50mg/kg), or celecoxib (5mg/kg). Sham animals received 0.8ml of saline by a single intra-colonic instillation. Rats were killed 4 days after induction and samples were collected for analysis. In the second model, colitis was induced in rats by the administration of 8% DSS dissolved in drinking water; after 24h animals were randomized to the same above reported treatments. Sham animals received standard drinking water. Rats were killed 5 days after induction and samples were collected for analysis. Flavocoxid, zileuton and celecoxib improved weight loss, reduced colonic myeloperoxydase activity, macroscopic and microscopic damage, and TNF-α serum levels. Flavocoxid and celecoxib also reduced malondialdheyde, 6-keto PGF1α and PGE-2 levels while flavocoxid and zileuton decreased LTB-4 levels. In addition, flavocoxid treatment improved histological features and apoptosis as compared to zileuton and celecoxib; moreover only flavocoxid reduced TXB2, thus avoiding an imbalance in eicosanoids production. Our results show that flavocoxid has protective effect in IBDs and may represents a future safe treatment for inflammatory bowel diseases.

Serum 5-LOX: a progressive protein marker for breast cancer and new approach for therapeutic target.

Lipoxygenase (LOX) pathway has emerged to have a role in carcinogenesis. There is an evidence that both 12-LOX and 5-LOX have procarcinogenic role. We have previously reported the elevated level of serum 12-LOX in breast cancer patients. This study evaluated the serum level of 5-LOX in breast cancer patients and its in vitro inhibition assessment with peptide inhibitor YWCS. The level of 5-LOX was determined by surface plasmon resonance (SPR). The peptide inhibitor of 5-LOX was designed by molecular modeling and kinetic assay was performed by spectrophotometry. The siRNA mediated 5-LOX gene silencing was performed to investigate the effect on proliferation of MDA-MB-231, breast cancer cell line. The serum 5-LOX level in breast cancer (5.69±1.97ng/µl) was almost 2-fold elevated compared to control (3.53±1.0ng/µl) (P < 0.0001). The peptide YWCS had shown competitive inhibitory effects with IC50, 2.2 µM and dissociation constant (K D), 4.92×10(-8) M. The siRNA mediated knockdown of 5-LOX, resulted in the decreased gene expression for 5-LOX and increased cell death in MDA-MB-231 cell line and thereby play a key role in reducing tumor proliferation. Thus, it can be concluded that 5-LOX is one of the potential serum protein marker for breast cancer and a promising therapeutic target for the same.

Co-administration of 3-Acetyl-11-Keto-Beta-Boswellic Acid Potentiates the Protective Effect of Celecoxib in Lipopolysaccharide-Induced Cognitive Impairment in Mice: Possible Implication of Anti-inflammatory and Antiglutamatergic Pathways.

Neuro-inflammation is known to initiate the underlying pathogenesis of several neurodegenerative disorders which may progress to cognitive, behavioral, and functional impairment. Boswellia serrata is a well-known powerful anti-inflammatory agent used to treat several inflammatory diseases. The aim of the current study is to investigate the effect of the combination therapy of 3-acetyl-11-keto-ß-boswellic acid (AKBA), a 5-lipoxygenase (5-LOX) inhibitor and celecoxib, and a selective cyclooxygenase-2 (COX-2) inhibitor as dual enzyme inhibitors compared to monotherapies with celecoxib and AKBA. Cognitive dysfunction is induced by intraperational injection of lipopolysaccharide (LPS) in mice. Radial maze, Y maze, and novel object recognition (NOR) were performed to evaluate the possible behavioral changes. Moreover, estimation of glutamate and tumor necrosis factor-alpha (TNF-α), as well as an immunohistochemical investigation of amyloid beta peptide (Aß) was performed to evaluate the molecular changes that followed the LPS or drug treatment. The results showed that the combination therapy of AKBA and celecoxib reversed the behavioral and molecular changes caused by LPS cognitive dysfunction model that predispose cognitive dysfunction in mice. This study showed the effectiveness of the dual therapy with AKBA and celecoxib as anti-inflammatory, antiglutamatergic, and anti-amyloidogenic agents in the management of cognitive dysfunction.