Faculty of Health Sciences School of Pharmacology 9410866v kermifrum@yahoo.com / ABSTRACT Thirty plant species traditionally used to treat skin pathologies were chosen from the readily available ethnobotanical literature. Four plants (aqueous or methanol extracts) displayed promising 5-lipoxygenase inhibitory activity with IC50 values below 61 ppm. These included Aloe greatheadii, Melianthus comosus, Pentanisia prunelloides and Warburgia solutaris. Essential oils generally displayed superior 5- lipoxygenase inhibitory activity with IC50 values between 22 and 75 ppm. These included the essential oils of Ballota africana, Helichrysum odoratissimum, Heteropyxis natalensis and Lippia javanica. A large proportion of the plants exhibited dose-dependent DPPH anti-oxidant activity with IC50 values between 5 and 94 ppm for the most active. These included Halleria lucida, Croton sylvaticus, Melianthus comosus, Lippia javanica and Pentanisia prunelloides. Aqueous extracts of Melianthus comosus exhibited the most potent anti-inflammatory and anti-oxidant activity. The methanol extract of the leaves of Halleria lucida was subjected to activity guided fractionation and two anti-oxidant molecules were isolated, namely luteolin-5-Oglucoside and verbascoside (acteoside). Isobologram construction resulted in a concentration-dependent additive and antagonistic interaction being recognised between the two isolated compounds. Warburgia salutaris displayed promising 5-lipoxygenase inhibitory activity. Two isolated compounds, mukadiaal and warburganal were found to partially contribute to the anti-inflammatory activity of the plant. The essential oils of Helichrysum odoratissimum, Heteropyxis natalensis and Lippia javanica were subjected to gas chromatography and major compounds contributing to possible anti-inflammatory effects identified. These included β-caryophyllene, 1,8-cineole, limonene and α- humulene. Enantiomers and racemic mixtures of limonene displayed significantly different 5-lipoxygenase inhibitory activity suggesting stereoselectivity of the enzyme-catalysed reaction. The monoterpene 1,8-cineole appeared to cause partial potentiation of the anti-inflammatory activity displayed by limonene. These results provide some in vitro scientific rationale for their traditional use as dermatological agents.

Many studies highlighted the biological potential of vitamin E, especially tocotrienols (T3), a vitamin E subfamily, particularly in the field of cardiovascular diseases and chronic inflammation. A pharmacophore based virtual screening of these substances against various antiinflammatory targets showed that this class could be considered as potential inhibitors of 5- lipoxygenase, a key enzyme in the biosynthesis of chemoattractant and vasoactive leukotrienes. Consequently, this screening was confirmed by in vitro assays. However, usual natural sources of T3 provide complex mixtures involving particularly challenging purification processes. Thus, this work aims at designing and optimizing efficient semisynthesis towards pharmacologically relevant T3 derivatives were developed from δ- tocotrienol, the main T3 isolated from Bixa orellana seeds, a renewable and easily available vegetal source from tropical regions, analyzed mainly by HPLC chromatography. Verification of the most effective reaction conditions of semisynthesis and synthesis another potential inhibitors of 5-LOX based on tocotrienols' structure are the following aims of the work. During this study, the semisynthesis based on δ-tocotrienol was completely optimized and 3 new T3 derivatives were synthesized and fully characterized....

5-Lipoxygenase (5LO) is a lipid-peroxidizing enzyme which inserts molecular oxygen into fatty acids leading to the biosynthesis of leukotrienes. This protein is widely expressed in the brain including the cortex and hippocampus regions, where its levels and activity increase in an age-dependent manner. Previous work has shown that 5LO modulates both amyloid beta (A) and tau pathology in Alzheimer's disease (AD) models. However, whether the effect of 5LO on tau is direct or indirect still remains unclear. Tau is a microtubule-associated protein usually found in the axons of neurons where it promotes assembly and stabilization of microtubules. In post-mortem brains of AD patients, tau is hyperphosphorylated and altered conformationally, followed by the formation of intracellular aggregates known as neurofibrillary tangles (NFTs), which are also the major pathological hallmark of another group of neurodegenerative diseases collectively referred to as tauopathies such as Pick's Disease, Progressive Supranuclear Palsy (PSP), Frontotemporal Dementia (FTD) and Parkinsonism linked to chromosome 17. The central hypothesis of the thesis is that 5LO directly influences tau metabolism, the development of related neuropathology and behavioral phenotype. To prove this hypothesis, a comprehensive genetic and pharmacologic experimental approach, combining both in vivo and in vitro experiments, was implemented. We initially showed that human brains from patients with a confirmed diagnosis of PSP, had significantly higher levels of 5LO when compared with brains form healthy controls. Next, we assayed the levels of 5LO in brains from htau (transgenic tau mice) mice at 4 different age time-points and two regions (cortex and cerebellum). Interestingly, compared with wild type controls, cortices from htau mice had a non-significant increase in 5LO protein levels as early as 6 months of age, which became significant by 10 months of age in the cortex only. Taken together, the age-dependent and region-specificity of the 5LO up-regulation supports the hypothesis that this pathway may have a functional role in the development of the tauopathy phenotype. To prove it, we treated tau mice with a selective 5LO inhibitor, zileuton, and explored the effect on learning and memory. Treatment of the htau mice with zileuton restored their short term working memory and spatial memory deficits. Shortly after completion of the behavioral tests, mice were euthanized and brains harvested for biochemistry and immunohistochemistry analyses. In association with the changes in behavior, we observed that pharmacologic inhibition of 5LO had an influence on tau metabolism, more specifically a significant decrease in tau phosphorylation. In search for the molecular mechanism involved in this biological effect, we assayed different kinases and phosphatase which have been implicated in tau metabolism and showed the specific involvement of the cdk5 pathway. This observation was further confirmed by in vitro studies, in which by using primary neuronal cells we showed that zileuton decreased tau phosphorylation via a cdk-5-dependent mechanism. Since the development of tau pathology results in biochemical and functional manifestation of synaptic deficits, next we assessed levels of pre- and post-synaptic protein markers. Compared with wild type, htau mice had significant reduction in the levels of three distinct markers of synaptic integrity (that is synaptophysin, post-synaptic density protein-95 and microtubule associated protein-2). By contrast, the decrease was completely restored to wild type levels by zileuton treatment. To further support the involvement of this pathway in the improvement of the behavioral and cognitive deficits, we explored the effects of its pharmacological blockade on synaptic function by performing electrophysiological studies. As reported previously, there was a significant difference in Long Term Potentiation (LTP) between the wild type and htau mice, with the latter showing significant deficits. However, pharmacologic blockade of 5LO in the htau mice was adequate to restore the LTP responses to a level comparable to those measured in the wild type mice. In the genetic portion of the study, WT and htau pups were intracranially injected with both AAV2/1 control vector and AAV2/1 5LO vector. Compared with the htau control group, the htau mice injected with AAV2/1 5LO displayed a significant deficits in cognition and memory associated with a decline in their synaptic integrity. Also, genetic upregulation of 5LO yielded significant increases in tau phosphorylation associated with an increase in cdk-5 kinase activation both in vivo and in vitro. Taken together these results describe a pluripotent role for 5LO in the context of tauopathy by representing its direct functional role in modulating behavior along with tau phosphorylation, neuroinflammation and synaptic function in a relevant mouse model of the human disease. The demonstration of the pleiotropic role 5LO in tauopathy pathogenesis makes it not only a valid pharmacological target, as 5LO inhibitors are already FDA approved but, more importantly represents a unique therapeutic opportunity with true disease modifying potential for the treatment of these dementing disorders for which there is no cure. / Pharmacology

Cardiovascular disease (CVD) including atherosclerosis is the leading cause of illness and death in the United States. The American Heart Association indicated that an estimated 81,100,000 American adults have one or more types of CVD and the estimated direct and indirect cost of CVD for 2010 is $503.2 billion, which is $27.9 billion more than last year. Although the exact cause of this disease remains unsolved, previous studies have demonstrated that pro-oxidative and pro-inflammatory pathways in vascular endothelium play a critical role in early pathological events of atherosclerosis. However, the detailed molecular signaling mechanisms underlying this process are not yet completely understood. Recently, the role of interleukin-4 (IL-4) in atherogenesis became controversial and gained attention. IL-4 is a pleiotropic immunomodulatory cytokine secreted by T-helper 2 (Th2) lymphocytes, eosinophils, and mast cells. It was traditionally believed to be an anti-inflammatory cytokine. Increasing evidence, however, has suggested that IL-4 contributes to the initiation and progression of atherosclerosis by oxidative stress-mediated up-regulation of pro-inflammatory mediators such as vascular cell adhesion moledule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in vascular endothelium. 5-Lipoxygenase (5-LOX) is one of the key sources that generate reactive oxygen species (ROS) via metabolic pathways of arachidonic acid. Although 5-LOX has been implicated in the development of atherosclerosis, it remains unclear whether 5-LOX-mediated ROS generation is associated with IL-4-induced MCP-1 expression in vascular endothelium. The present study was focused on the oxidative mechanisms by which IL-4 induces vascular inflammation as well as how 5-LOX is involved in this process. The results showed that IL-4 significantly up-regulates mRNA and protein expression of MCP-1 in vascular endothelium. In addition, DHE and DCF fluorescence staining demonstrated that IL-4 increases ROS production in human vascular endothelial cells. We have also provided the first novel evidence that 5-LOX, one of the enzymes associated with arachidonic acid metabolism, is responsible for the IL-4-induced ROS generation and MCP-1 expression in human vascular endothelial cells. / Master of Science

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

4 Abstract Mast cells are involved in variety of immunological processes, but they are mostly known for their role in allergy and asthma. As asthma and allergy are serious diseases with spreading tendency during last decades, mast cells are subject of intensive research. It is expected that studies of mast cell signalling pathways will contribute to our understanding of the nature of these diseases and help to design efficient treatment strategies. In an attempt to identify genes responsible for asthma disease, genome-wide screening methods have been currently applied. Using these methods, mutations in ORMDL3 (Orosomucoid1-like) protein were found out as a high risk asthma factor. ORMDL3 is a member of evolutionary conserved ORMDL family, comprising in mammals also of ORMDL1 and ORMDL2. Physiological function of these proteins is poorly understood and it has not been studied in mast cells. We decided to study the role of ORMDL proteins in mast cells. Lentiviral delivery system was optimised for generation of stable knock-downs (KD) of all three members of the ORMDL family in primary mast cells. The ORMDL gene expression was measured by improved qPCR (quantitative PCR) reaction buffers. We found that all ORMDL genes are expressed in mast cells in order ORMDL3 > ORMDL2 > ORMDL1. Next, we investigated the...

Lipoxygenases convert polyunsaturated fatty acids into biologically active metabolites such as inflammatory mediators—prostaglandins and leukotrienes. The inhibition of lipoxygenases is increasingly employed in the treatment of cancer. We evaluated the anticancer potential of two novel 5-lipoxygenase inhibitors, named CarbZDNaph and CarbZDChin, which are analogues of the commercially available inhibitor Rev-5901. The in vitro segment of this study was conducted on a mouse colorectal carcinoma cell line—CT26CL25. For an in vivo model, we induced tumors in BALB/c mice by the implantation of CT26CL25 cells, and we treated the animals with potential inhibitors. A 48 h treatment resulted in diminished cell viability. Calculated IC50 values (halfmaximal inhibitory concentrations) were 25 µM, 15 µM and 30 µM for CarbZDNaph, CarbZDChin and Rev-5901, respectively. The detailed analysis of mechanism revealed an induction of caspasedependent apoptosis and autophagy. In the presence of chloroquine, an autophagy inhibitor, we observed an increased mortality of cells, implying a cytoprotective role of autophagy. Our in vivo experiment reports tumor growth attenuation in animals treated with CarbZDChin. Compounds CarbZDNaph and Rev-5901 lacked an in vivo efficacy. The results presented in this study display a strong effect of compound CarbZDChin on malignant cell growth. Having in mind the important role of inflammation in cancer development, these results have a significant impact and are worthy of further evaluation.

Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIP2) is a kinase which modulates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. It is known that activation of RIP2 by engaging NOD receptors increases the production of pro-inflammatory cytokine and lipid mediators. We have some data indicating that RIP2 may also be involved in specialized pro-resolution lipid mediator (SPM) production. However, the molecular mechanisms by which RIP2 is involved in lipid mediator biosynthesis, are currently unknown. Understanding this process may have significant implications for RIP2-targeted therapies, which may not only inhibit pro-inflammatory cytokine and lipid mediator production but may also disrupt SPM production and resolution programs. This thesis aims to demonstrate that RIP2 is involved in promoting the activation of ALOX5 in a transient overexpression setting but also in an endogenous setting using relevant bacterial stimuli. These aims were accomplished through the optimization of a fluorescent assay to assess ALOX5 enzymatic activity, by optimization of ALOX5 enzyme purification and through molecular cloning of ALOX5 into a retroviral vector followed by viral transduction of the THP-1 human monocytic cell line. We find that co-expression of RIP2 with ALOX5 significantly enhances the enzymatic activity of ALOX5. We have successfully cloned NTAP-tagged ALOX5 into the pBABE retroviral vector and are currently selecting transduced cells so that we might test if this effect also occurs endogenously. Understanding the mechanisms underlying the production and regulation of SPMs would provide greater insight into potential new therapeutic approaches to promote resolution in chronic inflammatory diseases.

5-Oxo-ETE ist ein chemotaktischer Faktor für Granulozyten, der von der NADP+-abhängigen Dehydrogenase 5h-dh aus dem 5-Lipoxygenaseprodukt 5-HETE gebildet wird. Ziel dieser dreiteiligen Studie war es, die der 5-oxo-ETE-Produktion zugrunde liegenden Regulationsmechanismen aufzuklären. I. Einfluß von myeloider Zelldifferenzierung auf die Expression von 5h-dh in HL-60 und U-937 Zellen. Undifferenzierte HL-60 und U-937 Zellen produzieren vergleichbare Mengen von 5-oxo-ETE wie Monozyten oder Granulozyten. Differenzierung von U-937 Zellen mit PMA verdreifacht die Enzymaktivtät von 5h-dh, während die Behandlung von HL-60 Zellen mit dh-VitD3 diese verdoppelte. Der Einfluß von PMA auf 5h-dh wurde darüber hinaus in Mikrosomen von U-937 Zellen untersucht. Die Behandlung PMA verdreifachte Vmax, liess aber KM unbeeinflußt. II. Regulation der 5-oxo-ETE-Produktion durch oxidativen Stress und Glukose. Da der GSH-Redoxzyklus die Produktion von NADP+ zur Folge hat, stimulierten die Hydroperoxide H2O2 und tBOOH die Synthese von 5-oxo-ETE in U-937 Zellen. Aufgrund seiner Verarbeitung durch den Pentosephosphat Zyklus, der NAD+ in NADPH umwandelt, inhibierte Glucose diesen Effekt von H2O2. Die Synthese von 5-oxo-ETE wurde durch H2O2 auch in humanem Monozyten, Lymphocyten und Thrombozyten, aber nicht in Neutrophilen angeregt. Im Gegensatz zu Monozyten zeigten sich Thrombozyten und Lymphozyten allerdings glukose-resistent. T-BOOH ehöhte auch die Produktion von 5-oxo-ETE nach Zugabe von Ionophore und Arachidonsäure zu mononukleären Blutzellen. III. 5h-dh-Expression in human Strukturzellen. Zunächst rasterten wir mehrere sekundäre Epithelzelllinien und fanden 5h-dh in allen Zellen. Drei Indizien lassen vermuten, daß die epithele 5h-dh der myeloiden entspricht: (i) die enzymatische Aktivtät liegt vor allem in der mikrosomalen Fraktion vor, (ii) bei dem Kofaktor handelt es sich um NADP+ und nicht um NAD+, und (iii) 5S-HETE ist das bevorzugte Substrat. Weitere Studien zeigten, daß auch primäre humane Aorta-Endothelzellen 5h-dh expremieren. Vergleichbar zu Entzündungszellen wird die Produktion von 5-oxo-ETE auch in Endothel- und in Epithelzellen durch oxidativen Stress angeregt. / 5-Oxo-ETE is a highly potent granulocyte chemoattractant that is formed by the NADP+-dependent dehydrogenase 5h-dh by oxidation of the 5-lipoxygenase product 5-HETE. The objective of this study was to investigate underlying regulatory mechanisms of 5-oxo-ETE production in human cells. This matter was addressed from three directions. I. Expression of 5h-dh in HL-60 and U-937 cells and its activity changes during myeloid cell differentiation. Undifferentiated U-937 and HL-60 cells produce similar amounts of 5-oxo-ETE compared to monocytes or neutrophils. Differentiation of U-937 cells with PMA resulted in a 3-fold increase in 5-oxo-ETE production. Similarly, incubation of HL-60 cells with dh-VitD3 induced a 2-fold increase in 5-oxo-ETE production. The impact of PMA on 5h-dh was also investigated in the microsomal fraction of U-937 cells and compared to neutrophil microsomes. PMA treatment leads to a increase of Vmax but does not affect KM. II. Regulation of 5-oxo-ETE by oxidative stress and glucose levels. We found that H2O2 and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U-937 cells through the GSH redox cycle by providing NADP+. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, which converts NADP+ back to NADPH. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-BOOH. III. Expression of 5h-dh in human structural cells. We screened several secondary epithelial cell lines and detected 5h-dh in all cell lines. Epithelial 5h-dh and the inflammatory cell 5h-dh are identical: (i) the enzymatic activity is localized in microsomes, (ii) the cofactor is NADP+, and (iii) 5S-HETE is the preferred substrate. We also found that primary human aortic endothelial cells express 5h-dh. 5-oxo-ETE production by both endothelial and epithelial cells is regulated by oxidative stress in a manner similar to inflammatory cells.