<p>This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, <i>N</i>-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation.</p><p>Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from <i>Phoradendron tomentosum</i>. In addition, the cardiac glycoside digitoxin was isolated from <i>Digitalis purpurea</i>. All these substances expressed cytotoxicity and a neutrophil challenging activity.</p><p>Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC₅₀: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC₅₀ values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies.</p><p>The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC₅₀ 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.</p>

This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, N-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from Phoradendron tomentosum. In addition, the cardiac glycoside digitoxin was isolated from Digitalis purpurea. All these substances expressed cytotoxicity and a neutrophil challenging activity. Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC50: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC50 values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies. The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC50 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.

Les préparations de Viscum album (VA), connu sous le nom vernaculaire de gui européen, sont fréquemment utilisées en support des traitements anticancéreux, principalement pour améliorer la qualité de vie des malades et réduire la croissance des tumeurs. Elles sont connues pour exercer des effets anti-tumoraux. Il existe de plus en plus de données scientifiques faisant état de liens étroits entre cancer et inflammation. Étant donné que la prostaglandine E2 (PGE2) induite par la cyclo-oxygénase 2 (COX-2) joue un rôle clef dans l’inflammation, j’ai exploré la régulation du système COX-2-PGE2 par VA et ses mécanismes sous-jacents. J’ai montré que VA exerce ses effets anti-inflammatoires en inhibant sélectivement l’expression de COX-2 et en diminuant la production de PGE2 qui en découle, par le biais d’une déstabilisation de l’ARNm de COX-2. En plus de leurs propriétés cytotoxiques, il a été montré que les préparations de VA ont également des effets immunostimulants. Les différentes préparations de VA sont hautement hétérogènes du fait de leurs compositions biochimiques qui varient selon la récolte, l’espèce de l’arbre hôte et les méthodes de préparation qui peuvent influer sur leur efficacité clinique. De ce fait, j’ai réalisé une étude comparative sur cinq préparations de VA dans le but d’analyser leurs capacités de maturation et d’activation des cellules dendritiques (DC) qui peuvent à leur tour présenter une réponse immunitaire anti-tumorale. Les résultats ont montré que parmi les cinq préparations,VA Qu Spez induit de manière significative l’activation des DC et la sécrétion de cytokines pro-inflammatoires telle que l’IL-6, l’IL-8 et le TNF-α qui induisent la production d’IFN-γ,orientant de ce fait la réponse immunitaire vers une réponse Th1. L’orchestration de la11fonction des cellules myélomonocytiques est un élément central à l’interface entre inflammation et cancer. Il constitue un paradigme expliquant la plasticité et la fonction des macrophages. Mon étude met en évidence l’influence de VA Qu Spez sur la polarisation des macrophages qui passent d’un état alternatif (M2) à un état dit classique (ou M1). Les macrophages M2 sont connus pour polariser les réponses immunitaires Th2, pour participer à l’élimination des parasites, pour diminuer l’inflammation, pour promouvoir le remodelage tissulaire et la progression des tumeurs et pour avoir des fonctions immunorégulatrices. Les macrophages M1 sont impliqués dans la réponse Th1, favorisent la résistance aux pathogènes intracellulaires et aux tumeurs et promeuvent des réactions de désagrégation tissulaires. L’ensemble de ces résultats permet de comprendre les propriétés anti-inflammatoires et immunostimulantes des préparations de VA. Des recherches complémentaires permettront d’améliorer les stratégies d’utilisation thérapeutique de VA et son utilisation dans les soins de support aux traitements anticancéreux. / Viscum album (VA) preparations, commonly known as European mistletoe, are frequentlyused as complementary therapy in cancer, mainly to improve quality of life of the patients andto reduce the tumor growth. They are known to exert anti-tumoral effects. There is increasing evidence of the convoluted connection of cancer and inflammation. As cyclooxygenase-2(COX-2)-induced prostaglandin E2 (PGE2) plays a key role in the inflammation, I explored the regulation of COX-2-PGE2 axis by VA and underlying mechanisms. I found that VA exerts anti-inflammatory effects by selectively inhibiting COX-2 expression and ensuing PGE2 production. Inhibition of COX-2 expression implicates COX-2 mRNA destabilisation. In addition to their cytotoxic properties, they have also been shown to have immunostimulatory properties. Each VA preparations are highly heterogeneous because oftheir chemical composition which varies depending on the time of harvest, species of host treeand manufacturing methods, together which might influence clinical efficacy of VA.Therefore I performed a comparative study involving five different preparations of VA concerning maturation and activation of dendritic cells (DCs) which in turn may manifestanti-tumoral immune response. Results showed that among all five preparations, VA Qu Spez significantly induces DC activation, secretion of pro-inflammatory cytokines such as IL-6, Il-8 and TNF-α, enhancing IFN-γ production hence promoting Th1 immune response. The orchestration of myelomonocytic cell function is a key element that links inflammation and cancer and provides a paradigm for macrophage plasticity and function. My study reveals the effect of VA Qu Spez in switching the M2 macrophages which are known to participate inpolarizing Th2 responses, help with parasite clearance, dampen inflammation, promote tissue remodelling and tumor progression and have immunoregulatory functions, towards classicallyactivated M1 macrophages which are part of a polarized Th1 response and mediate resistance13to intracellular pathogens and tumors and elicit tissue-disruptive reactions. These results together should assist in understanding the anti-inflammatory and immunostimulatory properties of VA preparations and further research is warranted to improve the therapeutic strategies of use of VA and their role as complimentary therapy in cancer.