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Study of mechanism of action of anticancer drug ellipticine and its metabolism
Vejvodová, Lucie ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
This bachelor thesis is aimed to study the mechanisms of action of anticancer drugs, their side effects, their resistance and pharmacokinetics. Anticancer alkaloid ellipticine was chosen as a model for this work. Bachelor thesis describes the metabolism of this substance in organisms and its potential to induce of drug metabolizing enzymes. An antineoplastic alkaloid ellipticine is a prodrug, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerasa II, and formation of covalent DNA adducts mediated by cytochromes P450 and/or peroxidases in target tissues. A variety cytochromes P450 oxidize ellipticine forming up to five metabolites (7-hydroxyellipticine, 9- hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine and ellipticine N2 - oxide). 7- hydroxy- and 9-hydroxyellipticine metabolites are considered to be mainly the detoxication products of ellipticine, while 12-hydroxyellipticine, 13-hydroxyellipticine and ellipticine N2 - oxide are considered to be mainly the activation products of ellipticine. The major ellipticine- derived DNA adducts are generated from these activation metabolites. These adducts were found in cancer cells in culture, such as human breast adenocarcinoma MCF-7 cells, neuroblastoma IMR-32, UKF-NB-3, and UKF-NB-4 cells and glioblastoma...
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Mechanism of tumor development and its influencing by ellipticine
Parisová, Martina ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
Ellipticine (5.11-dimethyl-6H-pyridate [4,3-b] carbazole) is a powerful anti-cancer agent, exhibiting multiple mechanisms of action. This work describes the causes of cancer processes and summarizes the main pharmacological mechanisms and cytotoxic effects of ellipticine together with the results found in our laboratory indicating, a new mechanism of ellipticine action. Cytotoxic and mutagenic activity of ellipticine is attributed to its two mechanisms of activity ellipticine intercalation into DNA and its effectivity to inhibit topoisomerase II. Ellipticine also forms covalent DNA adducts after its oxidation with cytochromes P450 and peroxidases. Cytochromes P450 oxidize ellipticine up to five metabolites, of which 13- hydroxyellipticin, 12-hydroxyellipticin and N(2)-oxide of ellipticine are responsible for formation of two major DNA adducts. In the case of peroxidases, ellipticine is oxidized to a radical producing the ellipticine dimer and a minor ellipticine metabolite, the N(2)-oxide of ellipticine. Because of the high efficiency of ellipticine and its derivatives against various types of cancer, this coumpound is studied in detail. Its utilization for drug tangeting is a challenge for further study.
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Interaction of proteins with bromphenol blue - possible analytical uses
Vodičková, Kateřina ; Hudeček, Jiří (advisor) ; Moserová, Michaela (referee)
The so called protein error of pH indicators is often used for protein determination in body fluids. A popular dye for this purpose is the bromophenol blue. A most common calibration standard, bovine serum albumin (or human serum albumin) is used. In this Thesis, I examined the influence of urea on the interaction between bovine serum albumin and bromophenol blue at pH 3,23. Addition of urea in amount corresponding to physiological values in urine (0,2-0,4 mol/l) does not cause changes in absorbance exceeding the experimental error (1 %); the same is true up to 4 mol/l of urea. Higher concentration of urea (6 mol/l) causes a considerable decrease of absorbance (up to 29 %), probably as a result of bovine serum albumin denaturation.
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Role of benzo[a]pyrene in cancer development
Vaňátková, Petra ; Moserová, Michaela (advisor) ; Kubíčková, Božena (referee)
4 ABSTRACT Cancer is nowadays one of the most serious diseases. Tumor development is a multistage process in which the effect of internal and external factors lead to failure of regulatory and defense mechanisms of the organism and to the accumulation of mutations which are generated by these organisms. Chemical carcinogens and also biological and physical factors can be regarded as the main external factors. Polycyclic aromatic hydrocarbons are large group of chemical carcinogens. One of them, benzo[a]pyren is the most studied polycyclic aromatic hydrocarbon. Carcinogenic, mutagenic and teratogenic effects of benzo[a]pyrene had been shown on laboratory animals. Benzo[a]pyrene is considered as the main carcinogen in tobacco smoke and is connected with lung cancer development among smokers. Benzo[a]pyrene is metabolized in activation or detoxication pathways by enzymes of mixed function monooxygenase systeme of cytochromes P450. The most important enzymes involved in the activation of these compounds are CYP1A1 and CYP1B1 with cooperation of epoxide hydrolase. The reactive species generated in its activation pathway are able to form covalent adducts with DNA. The most important carcinogenic product of benzo[a]pyrene is benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, which can caused irreversible ganges in...
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Phytohormones produced by Leptosphaeria maculans as effectors manipulating plant signaling pathways
Krutinová, Hana ; Ryšlavá, Helena (advisor) ; Moserová, Michaela (referee)
Leptosphaeria maculans is the causal agent of blackleg disease in Brassica napus. In this thesis the ability of L. maculans to produce certain phytohormones was established. The thesis focused on auxins and brassinosteroids. Most commonly occurring natural auxin, IAA, and its inactive oxidation product, OxIAA, were found in highest concentration. In plants infected with L. maculans the concentration of OxIAA was higher when compared to control water- treated plants. A surprising difference in IAA production between two sister isolates of L. maculans was discovered. In chemically defined cultivation medium Gamborg JN2 isolate did not produce any IAA. JN3 on the other hand produced IAA in concentration around 1000 pmol/g FW. This difference was used for studying L. maculans putative auxin synthesizing genes. The candidate genes were identified as orthologs of Arabidopsis thaliana genes (YUCCA1.1, NIT1.2) and Ustilago maydis (IAD1.2, TAM1-2.1). An increased transcription level of the auxin biosynthesis candidate genes was observed in JN2 treated in vitro with auxin precursors (tryptophan, tryptamine). Increased IAA concentration was observed as well. No such effect was observed in JN3. Surprisingly, an increased candidate gene transcription and IAA concentration was observed also in JN2 treated with...
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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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The role of V-ATPase in resistence to cytostatics and the possibility of its inhibition
Belhajová, Marie ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
Cancers belong among the most serious problems of modern medicine and their occurrence is constantly increasing. Neuroblastoma is a malignant embryonal tumor in children, emerging from the peripheral nervous system and is the most frequent tumor in infants. Despite the significant development of therapeutic methods during recent years, this disease remains difficult to treat. It is treated surgically and also with chemotherapy using cytostatic drugs. The cytostatic drugs such as doxorubicin, ellipticine, cisplatin and vincristine have become very significant in treating cancer. However, they induced drug resistance in these neuroblastoma cells. This study investigates expression of the vacuolar H+ -ATPase (V-ATPase), in neuroblastoma cells and its role in the development of drug resistance. V-ATPase is a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. A sensitive neuroblastoma cell line (UKF-NB-4) and cells resistant to doxorubicin, ellipticine and cisplatin (UKF-NB-4DOX , UKF-NB-4ELLI , UKF-NB-4CDDP ) were exposed to these agents and the expression of the V-ATPase was studied by Western blot analysis and real-time quantitative reverse tarnscription polymerase chain reaction (RT-PCR). Ellipticine induces an increase in expression of the V-ATPase in neuroblastoma...
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Study of mechanism of action of anticancer drug tamoxifen and its toxic side effects
Kylarová, Salome ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
Anti-estrogen therapy is used for treatment of hormone (estrogen) receptor positive breast cancer. The rise of this type of cancer is associated with a prolonged exposure to these hormones throughout life. Tamoxifen is one of the most used hormonal drugs, which blocks the effects of these hormones in breast cancer tissue by competitive binding to hormonal receptors. The affinity of tamoxifen to these receptors is not sufficient, therefore it has to be activated to metabolites having greater affinity, namely 4-hydroxytamoxifen and endoxifen. The formation of these intermediates is catalysed by cytochromes P450. In the second phase of its biotransformation hydroxylated metabolites of tamoxifen are primarily sulphated by sulphotransferases and eliminated from the body. In addition to these active intermediates, which inhibit the growth of breast tumor tissue, there are metabolites causing negative effects in the others. The most important metabolite is α-hydroxytamoxifen, which forms covalent DNA adducts in liver tissue of rats and endometrium of females. Tamoxifen therapy is associated with numerous side effects, but the greatest attention is focused to formation of endometrial cancer and induction of tumor's resistance to this therapy. Effects of tamoxifen therapy are dependent on the activity of...
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Protein determination - Effect of protein composition, application of small-volume spectrophotometer
Vodičková, Kateřina ; Hudeček, Jiří (advisor) ; Moserová, Michaela (referee)
Recently, several spectrometers for small volume measurements in order of microliter have been introduced. They are primarily intended for protein determination (or determination of proteins and nucleic acids in one measurement) by direct spectrophotometry or other spectral methods. One of such instruments is the NanoVueTM Plus (GE Healthcare). In this work, we first tried to characterize the instrument in general terms (stability) and to optimize measurement condititions (sample volume). Proteins have been determined by direct spectrophotometry using internal programs of the instrument, data were controlled by an independent computation. We studied also influence of differences in composition of various proteins on the results. According to the results of this Thesis, the most accurate values could be obtained using the internal program E 1%, using the E 1% value from an experiment. On the other hand, the program Protein UV is producing often inaccurate values, strongly infleunced by the protein amino acid composition. Keywords: protein determination, spectrophotometer NanoVueTM Plus, influence of amino acid composition
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Heterologous expression and isolation of human cytochrome P450 1B1
Sojka, Pavel ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
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...
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