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

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Histone deacetylase inhibitor trichostatin A (TSA) increases cytotoxicity of antineoplastic agent ellipticine to human neuroblastoma cells. Its mechanism of action has not yet been explained. One of the possible mode of action is conformational change in chromatin, which leads to changes in DNA that is more accessible to covalent modification and intercalation. The aim of this work is to study another mode of action, which can explain this phenomenon. The question is, if TSA can increase cytotoxicity of ellipticine to human neuroblastoma cells by modulation of activities and expression of cytochromes P450 and peroxidases. These enzymes are responsible for cytotoxicity of ellipticine to human neuroblastoma cells. TSA has no effect on oxidation of ellipticine mediated by cytochromes P450 leading to metabolites responsible for formation of ellipticine-DNA adducts and detoxication metabolites. TSA increases formation of ellipticine dimer, which is a detoxication metabolite, forming during its oxidation by peroxidases. TSA has no effect on activities of CYP1A1, CYP1A2, CYP3A, which significantly participate in oxidation of ellipticine. TSA modulates expression of enzymes oxidizing ellipticin in human neuroblastoma cells. TSA in the presence of ellipticine increases expression of CYP1A1 a CYP3A4 in...

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and inhibition of topoisomerase II. Ellipticine was also found to form covalent DNA adducts mediated by its enzymatic activation with cytochromes P450 (CYP) and peroxidases. The next study demonstrated increasing formation of these ellipticine-DNA adducts by histone deacetylase inhibitor valproate (VPA) in neuroblastoma cells. This phenomenon correlates with increasing cytotoxicity of ellipticine induced by this histone deacetylase inhibitor. This observation can be explained by several mechanisms. One of them can be loosening the structure of chromatine, which leads to accessing DNA for modification. Another one is the effect of VPA on activities and expression of enzymes metabolizing ellipticine. This study was aimed to test the second hypothesis. Since VPA has been shown to be metabolized by similar enzymes as ellipticine is, we have studied the effect of VPA (i) on oxidation of ellipticine by cytochromes P450 and peroxidases, (ii) on activities of the CYP enzymes, which significantly participate in oxidation of ellipticine (CYP1A, CYP3A) and (iii) on expression of enzymes oxidizing ellipticine (CYP1A1, CYP3A4, lactoperoxidase). Oxidation of ellipticine in vitro by model...

Cytochromes P450 are heme enzymes with very broad substrate specificity, they can oxidize tens to hundreds of different substrates including both eobiotics and xenobiotics, but with varying efficiencies and kinetics. They are responsible for the metabolic activation of many carcinogens resulting in formation of reactive intermediates, these reactive species participate in the formation of DNA adducts and also increase of oxidative stress. Eukaryotic cytochromes P450 are membrane bound enzymes found mostly in the endoplasmic reticulum. In vertebrates, they are expressed in a variety of tissues mostly in the liver, but also in kidney, lung, skin and others. The cytochrome P450 1B1 is an inducible enzyme which occurs mainly in the lung and skin. Its expression is induced predominantly by the presence of polycyclic aromatic hydrocarbons, dioxins and heterocyclic amines. The human cytochromes P450 are typically obtained using heterologous expression and isolated as a C-terminally modified hexa-histaq constructs using immobilized metal affinity chromatography. This thesis focuses on effect of C-terminal modifications on activity of human cytochrome P450 1B1. First, the two expression vectors encoding the human form of cytochrome P450 1B1 were prepared, one contained a classical C-terminal hexa-histaq...

Flavonoids are natural compounds commonly ingested in herbs and vegetables. They are believed to have a positive impact on human organism, in particular by their antioxidant, hepatoprotective and anti-cancer effects. In these days, it is possible to consume high concentrations of these compounds in form of dietary supplements. However it is not clear, whether flavonoids in such unnaturally high concentrations are still beneficial or rather harmful. It has already been proven, that flavonoids can influence the activity of biotransformation enzymes and interfere e.g. with the process of carcinogenesis and drug metabolism. For that reason it is important to investigate the impact of an increased intake of flavonoids. The aim of this thesis was to investigate the influence of dihydromyricetin (a potential drug to cure alcohol use disorder) and its structurally similar flavonoid myricetin on the activity of enzymes, cytochrome P450 2E1 (CYP2E1) and N­acetyltransferases 1 and 2 (NAT1/2). The research included the determination of the impact of a premedication by these flavonoids on the expression and activity of CYP2E1 and NAT1/2. The inhibition capacity of myricetin and dihydromyricetin towards the activity of CYP2E1 and NAT1/2 was also investigated. After the flavonoid premedication of rats the...

Cytochrome P450 3A4 is integral membrane protein residing in endoplasmic reticular membrane. In human the highest concentration cytochrome P450 3A4 is expressed in liver, where it plays a major role in metabolism of many drugs and xenobiotics. The main aim of the thesis was to evaluate the effect of gene optimization on heterologous expression of human cytochrome P450 3A4. At first expression constructs based on vectors pET22b were prepared. Then the efficiency of heterologous expression of optimized vs. natural gene sequence encoding truncated form of the human cytochrome P450 3A4 compared. The results show that the gen sequence optimized for E. coli strains K12 was expressed in significantly higher efficiency than the original human gene based on cDNA sequence. Another aim was to evaluate the suitability of pET22b based expression vectors for recombinant production of native (complete) form of human cytochrome P450 3A4. The amount of native form of the protein found in bacterial membrane was however substantially lower then that of the truncated form. Keywords: cytochrome P450 3A4, heterologous expression, pET22b, gene synthesis

The use of food supplements containing natural chemopreventive compounds increased in recent years. Some of the most popular chemopreventive compounds are flavonoids. Due to their natural origin, flavonoids are generally accepted as safe compounds. They exert antioxidant, anti-cancer and anti-inflammatory properties. However, flavonoids should be considered as foreign compounds (xenobiotics). Flavonoids interact with many enzymes, among the most important belong cytochromes P450 (CYPs), key enzymes of the first phase of biotransformation of xenobiotics (e.g. drugs, carcinogens). CYPs catalyze reactions leading mainly to detoxification of xenobiotics. However, some CYPs are involved in the activation of carcinogens, particularly CYP1A1 and CYP1A2 activate e.g. heterocyclic amines. Flavonoids might enhance the activation of carcinogens via induction of these CYPs or stimulation of their activities and hence, increase the risk of a cancer development. The thesis is focused on the influence of flavonoids and food carcinogens on the induction and activity of CYP1A1 and CYP1A2 in liver and small intestine of rats. For the purpose of this study, the small intestine was dissected into three parts: proximal (nearest to stomach), middle and distal. Western blotting was used for the evaluation of CYP...

Tyrosine kinase inhibitors (TKI) are small organic molecules designed for the targeted cancer therapy. They perform the inhibition of activated receptor tyrosine kinases in tumor cells, that defeats tumor growth, proliferation, metastasis and angiogenesis in tumor tissue. Two TKI, lenvatinib and vandetanib, are used in thyroid cancer treatment. This thesis investigates the ways leading to enhancement of efficiency of these anticancer drugs for therapy. One of the studied anticancer drug - lenvatinib - was investigated to be prepared in a nanoform. Nanoparticles were based on protein apoferritin as well as on lipids. Theoretical model of lenvatinib interaction with an apoferritin cavity, as well as the model of its encapsulation obtained by computer modeling indicated that lenvatinib seems not to be suitable for preparation of apoferritin nanoparticles. Since lenvatinib occurs in its neutral form during preparation of nanoparticles, it does not interact with nanoparticle. The unsuccessful experimental preparation of lenvatinib-loaded apoferritin nanoparticles confirmed that lenvatinib is not suitable for its preparation. However, the theoretical model can serve for screening of other potentially suitable drugs before the experimental nanoparticle preparation. Since the experimental preparation of...

CHARLES UNIVERSITY IN PRAGUE Faculty of Science Department of Biochemistry Effects of chemopreventive compounds on cytochrome P450s Summary of Ph.D. Thesis RNDr. Jitka Křížková Supervisor: prof. RNDr. Petr Hodek, CSc. Prague 2010 Introduction 1 Introduction According to the World Health Organization statistics, cancer is one of the leading causes of death in the human population worldwide for more than 50 years. Moreover, colorectal and gastrointestinal tract cancers are one of the main types of cancer leading to overall cancer mortality. Prevention consisting in a healthy lifestyle and a natural diet is suggested to be one of the main approaches to reduce cancer risk. In recent years, the consumption and use of dietary supplements containing concentrated chemopreventive phytochemicals increased dramatically. Flavonoids, as the most popular representatives of chemopreventive compounds, present in foods (fruits, vegetables, herbs, beverages) and dietary supplements have the potential to modulate the activity of xenobiotic-metabolizing enzymes [Hodek et al., 2002]. Among proteins interacting with flavonoids, cytochrome P450s (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens), play the most prominent role. The two members of CYP1A subfamily, CYP1A1 and CYP1A2, are involved in the...