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Modulation of activities and expression of enzymes metabolizing ellipticine by histone deacetylase inhibitor trichostatin A
Kopejtková, Barbora ; Stiborová, Marie (advisor) ; Kubíčková, Božena (referee)
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...
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Construction of vectors for heterologous expression of human cytochrome P450 1B1
Sojka, Pavel ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
The thesis was worked out in Laboratory of Molecular Carcinogenesis and Drug Development, which is focus on study of drug metabolizing enzymes including cytochromes P450. Cytochromes P450 are participating at initial phase of biotransformation of xenobiotics and endogenous substances and metabolism of several endogenous compounds, i.e. steroids. This work is focused on construction of expression vectors, based on the plasmid pET- 22b, suitable for heterologous expression of the human cytochrome P450 1B1. This enzyme is predominantly present in the endoplasmic reticulum of extra-hepatic tissues and its expression is induced by dioxins and polycyclic aromatic hydrocarbons. The human gene for cytochrome P450 1B1 was modified using PCR. The cleavage sites for restriction endonucleases were added to both ends of the gene. Another construct also contained N-terminal histidine tag to facilitate easier purification of the enzyme. Both insert and digested plasmids were verified using the agarose electrophoresis and used for ligation and transformation into competent cells (E. coli DH5. Final steps in construction was, however, not successful, probably due to low yields of DNA fragment extraction from agarose gels. Key words: cytochrome P450 1B1, carcinogenesis, plasmid, heterologous expression [In Czech]
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Molecular mechanism of carcinogenicity of aristolochic acid
Levová, Kateřina ; Stiborová, Marie (advisor) ; Ryšlavá, Helena (referee) ; Souček, Pavel (referee)
Aristolochic acids (AA) are carcinogenic and nephrotoxic alkaloids from Aristolochia species. Aristolochic acid I (AAI), the major component of AA, causes the development of Aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). These two diseases cause total renal failure and urothelial malignancies. The fact that these diseases have not been developed in all persons, who have been exposed to their action, might be causd by different activities and protein levels of the enzymes metabolizing AAI. Thus, the identification of enzymes involved in the metabolism, and detailed knowledge of their expression and catalytic specifities is a major importance. Aristolochic acid I (AAI) can be metabolized by several types of reactions. Like most nitroaromatics, the main activation pathway of AAI is reduction of its nitro group to form a cyclic acylnitrenium ion, which can bind to the purine bases, thereby forming AAI-DNA adducts. The detoxication pathway of AAI is its oxidative demethylation by cytochromes P450 forming detoxication metabolite 8-hydroxyaristolochic acid Ia (AAIa). In the present thesis, using rat and human enzymes and as well as several mice models, the metabolism of AAI in vitro and in vivo was investigated. The first model has deleted gene for NADPH:cytochrome P450...
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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
HAA are compounds which are showing numerous carcinogenic impacts on studied animals even human cells. These carcinogenes arise during the heat processing of meat or during (cigarette) smoking. Activation of these compounds is required to their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP) especially subfamily 1A1 and 1A2. As a consequence of activation with these enzymes are created N-hydroxylamines, which weakly reacting with DNA. For better formation of DNA aducts one more activation is essential. More reactive acetate and sulphate esters arise by second activation from N- hydroxylamines. The esters are produced by sulphotranspherase (SULT) even N- acetotranspherase (NAT). When we affect these enzymes we could positive control the formation of carcinoma. Caffeic acid is considered as a strong inhibitor of one SULT subfamily (phenolic sulfotranspherase P - PST). On the other side as a good inhibitor of NAT is considered (known) quercetin. (in czech) Key words: Heterocyclic amine, biotransformation, cytochrome P450, sulfotransferase, N-acetyltransferase
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Optimalization of expression of photoactivatable cytochrome P450 as a nano-probe for the membrane topology studies of enzymes metabolizing drugs and carcinogens
Smolová, Jana ; Hodek, Petr (advisor) ; Černá, Věra (referee)
The cytochromes P450 are among the most important enzymes involved in the biotransformation of xenobiotics in the body. They are part of the moooxygenase system that interact with other enzymes - NADPH:cytochrome P450 reductase and cytochrome b5. Mutual interaction of enzymes in mooxygenase system are not completely solved. Covalent crosslinking technique could contribute to clarify the possible protein-protein interactions and their consequences. One of the possible realization of this plan is to use photoactivatable cytochrome P450, which after exposure to UV radiation created covalent complex with components of monooxygenase system, with which it is in contact. Therefore, this paper focuses on developing optimal conditions for the production of recombinant cytochrome P450 2B4 in order to gain knowledge for the production of photoactivatable cytochrome P450 with incorporated amino acids L-photomethionine and L-photoleucine. In experiments was cytochrome P450 2B4 expressed in two strains of Escherichia coli, C41 (DE3) and BL21 (DE3) Gold, and two culture flasks, glass Erlenmeyer flask and plastic Fernbach flask. During expression optical density of the bacterial suspension (absorbation at 600 nm) and concentration of cytochrome P450 were measured. Methodology of measuring the concentration of...
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Study of inhibition activity of new antitumor drugs against chosen isoforms of cytochromes P450
Kroulíková, Pavla ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Mgr. Pavla Kroulíková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of Thesis: Study of inhibitory activity of new anticancer drugs toward selected isoforms of cytochromes P450 Cytochromes P450 are important biotransformation enzymes that affect pharmacokinetic behavior of many clinically used drugs and are the cause of many major drug interactions. They may have a role in overcoming multidrug resistance of cancer cells because many cytostatic drugs are deactivated by them. Aim of this thesis was in vitro study of inhibition of selected isoforms (CYP3A4, CYP3A5, CYP1A2, CYP2C8, CYP2D6, CYP2C9, CYP2C19 and CYP2B6) by substances from the group of tyrosine kinase inhibitors - alectinib, brivanib, osimertinib and selumetinib. We used commercially available Vividâ CYP Screening kits for the study. In these kits, human cytochromes P450 are recombinantly inserted into insect microsomes. The advantage of this method is that during the experiment no other reaction catalyzed by a different enzyme occurs simultaneously. The principle of this method is conversion of fluorogenic substrate into fluorescent product by CYP450. We determined the inhibition by measuring fluorescence in the 15th minute from start...
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Cooperativity of cytochrome P450 monooxygenase system in view of the modulation of drug and carcinogen metabolism
Dědič, Jan ; Hodek, Petr (advisor) ; Ječmen, Tomáš (referee)
System of mixed function oxidases and oxygenases is very much involved in metabolism of xenobiotics and endogenous compounds. System consists of several components: cytochrome P450, NADPH: cytochrome P450 oxidoreductase (CPR), cytochrome b5 and NADH: cytochrome b5 oxidoreductase. It was found, that all these components interact with each other, thus ensuring operation of the entire system. Cooperativity of the system is then dependent on many factors, notably the nature of the interactions between the components. Apparently the most frequently discussed are the interactions between CPR, cytochrome b5 and cytochrome P450. The main redox partner of cytochrome P450 is CPR that during the electron transport undergoes a significant conformational change. Cytochrome b5 may have both inhibitory and stimulatory effects on the enzymatic reaction and its mechanism of action has not been fully elucidated. Cytochrome P450 can interact among themselves to create complexes which presence may have significant influence on enzymatic reaction. However cooperativity of the system in terms of character of enzymatic reaction does not depend only on quantitative effects, such as inhibition and stimulation. It turns out, that also depends on qualitative effects, because it has been shown, that certain changes in the...
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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The Bachelor Thesis deals with an activation of heterocyclic aromatic amines (HAA) which have numerous carcinogenic effects on studied animals and human cells. These carcinogens are formed during the heat processing of meat and during the smoking. However, further transformation of the compounds is required to gather their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP), especially its forms 1A1 and 1A2. The products of this activation - N-hydroxylamines - are further activated in conjugation reactions. In this work, we were focused on the transformation of N-hydroxylamines to more reactive acetate esters and sulphate esters, which is catalyzed by sulphotranspherase (SULT) and N-acetyltranspherase (NAT), respectively. The affection of these enzymes can control the formation of carcinoma. For example, some dietary compounds, such as caffeic acid and quercetin, are the most common inhibitors of these enzymes: caffeic acid is considered as a strong inhibitor of phenolic sulphotranspherase (P-PST), whereas quercetin is a good inhibitor of NAT. On the other hand, some dietary compounds can also induce an opposite effect: for instance, phenol acids induce the P-PST. (in Czech) Key words: Heterocyclic aromatic amines, biotransformation, cytochrome P450,...
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Mechanism of enzymatic activation of carcinogens and drugs by the system of cytochrome P450
Indra, Radek ; Stiborová, Marie (advisor) ; Souček, Pavel (referee) ; Koblihová, Jitka (referee)
13 Abstract An environmental pollutant and a human carcinogen benzo[a]pyrene (BaP) is after its activation with cytochrome P450 (CYP) able to covalently bind to DNA. In the thesis, one of the target was to investigate an influence of individual components of mixed function monooxygenase (MFO) system on metabolism of benzo[a]pyrene and generation of adducts of activated BaP with DNA. The study was particularly focused to increase our knowledge on the effect of cyt b5 on metabolism of BaP by cytochrome P450 1A1 (CYP1A1) and its potential to serve as a donor of electrons during the reaction cycle of this cytochrome P450. The effect of cyt b5 on generation of BaP metabolites and adducts of BaP with DNA was investigated. In addition the effect of two different expression systems for cytochrome P450 1A1 (prokaryotic and eukaryotic) was also studied. The influence of cyt b5 on oxidation another xenobiotic compound, a plant alkaloid ellipticine that exhibit antitumor activities, was also investigated. Its pharmacological efficiency, as well as side effects depends on its metabolic activation by cytochrome P450. CYP3A4 is very important for ellipticine activation and therefore this enzyme was used in our experiments. Furthermore, a suitability of rat as a model organism mimicking the metabolic fate of BaP...
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Inhibitors of tyrosine kinases as anticancer drugs of a new generation
Hromek, Vlastimil ; Stiborová, Marie (advisor) ; Bárta, František (referee)
At the present time many types of treatment are used for curing of different cancer diseases. Among the most common types of such treatment belong a surgery, radiotherapy, chemotherapy, and immunotherapy. In the case of chemotherapy, there is used a wide (broad) spectrum of chemotherapeutics such as alkylating agents, platinum compounds, antimetabolites, anthracyclines and, at the present time, also inhibitors of tyrosine kinases. The bachelor thesis describes different types of tyrosine kinase inhibitors and their use in treatment of several cancers. They become popular because of their high specifity and minimal side efects. The first successful use of a tyrosine kinase inhibitor was treatment of the patients suffering from chronic myelogenous leukemia (CML) with imatinib. Vandetanib is another inhibitor of tyrosine kinases that is now used for treatment of another cancer, the medullary thyroid cancer. During treatment, vandetanib is biotransformed with cytochromes P450, which are the terminal oxidases of a mixed function oxidase (MFO) system, into the less efficient metabolites. In the practical part of the bachelor thesis we isolated enzymes, which metabolize xenobiotics, including vandetanib. Rat liver tissue was used for isolation of NADPH:cytochrome P450 reductase, which was isolated as a...
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