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The mechanism of action of anticancer drug ellipticin in target tissues of its effect
Vranová, Iveta ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Ellipticine is an alkaloid isolated from Apocynaceae plants exhibiting significant antitumor and anti-HIV activities. Cytochromes P450 (CYP) and peroxidases are the enzymes participating in metabolism of ellipticine. This process provides activation and detoxication metabolites of ellipticine. The CYP enzymes, which participate in oxidation of ellipticine in different tissues (liver, lung and kidney) of rat, a model organism simulating the fate of ellipticine in humans have already been identified. In this work, the effects of ellipticine on contents and catalytic activities of CYPs and other components of the mixed-function oxidase (MFO) system in this animal system were studied. For detection of contents of CYPs and other components of the MFO system, spectroscopic and electrochemical methods were used. To determine catalytic activities of CYPs and NADPH:cytochrome P450 reductase, reactions with specific substrates of these enzymes were utilized. The results found in this study demonstrate that expression and catalytic activity of CYP1A is induced by ellipticine in all of the tested organs (liver, kidney and lung) of rats treated with the drug. Moreover in liver, the cytochrome b5 expression is also induced. In addition, in this organ, expression and catalytic activity of CYP3A was increased by...
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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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The comparison of the efficiency of cytochromes P450 expressed in prokaryotic and eukaryotic systems
Kroftová, Natálie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
v anglickém jazyce Cancer disease is a group of clinically diverse diseases characterized by uncontrolled proliferation of cancer cells incapable of differentiation. Tumor therapy consists of a combination of surgery, radiotherapy and chemotherapy utilizing cytostatic agents. The plant alkaloid ellipticine ranks among a group of cytostatic agents. Its mode of action is based on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts. Ellipticine is oxidized through cytochromes P450 catalysis into detoxication metabolites 9-hydroxyellipticine and 7-hydroxyellipticine, and into activation metabolites 12-hydroxyellipticine, 13-hydroxyellipticine and ellipticine N2 -oxide. The aim of practical part of this work was to compare the efficiency of human cytochromes P450 1A1/2 and 3A4 expressed in prokaryotic and eukaryotic systems in the process of ellipticine oxidation. Ellipticine metabolites were analysed using high performance liquid chromatography. It was found that cytochrome P450 1A1 expressed in the prokaryotic system catalyses predominantly the formation of 9-hydroxyellipticine and 7-hydroxy- ellipticine. The difference is minimal in the production of ellipticine metabolites catalysed by cytochromes P450 1A2 expressed in both cellular systems. The formation of...
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Plant alkaloids and their effects on enzymes metabolizing xenobiotics
Višněvská, Kateřina ; Stiborová, Marie (advisor) ; Černá, Věra (referee)
Sanguinarine and chelerythrine are quaternary benzo[c]phenanthridine alkaloids. The first step in sanguinarine metabolism is its reduction to dihydrosanguinarin. Antimicrobial and anti-inflammatory activities of these alkaloids are used in dentistry and as feed additives. Sanguinarine and chelerythrine induce apoptosis of cells. Fluorescence of these alkaloids and intercalation into DNA could be utilized to use the alkaloids as supravital DNA probe. Negative effect of sanguinarine and chelerythrine is their genotoxicity. Cytochrome P450 and peroxidase oxidize ellipticine to detoxication and activation metabolites. Ellipticine is a potent antineoplastic agent exhibiting the multimodal mechanism of its action. Ellipticine intercalates into DNA and inhibits topoisomerase II. Covalent DNA aducts are mediated by CYP or peroxidase oxidation of ellipticine. The anti-tumor activity of ellipticine and its derivatives is caused by a combination mechanism of cell cycle arrest and induction of the apoptotic pathway. Pharmacological efficiencies and geneotoxic side effects of ellipticine is dependent on levels and activities of cytochrome P450 or peroxidase in target tissues. Aristolactams are the major metabolites of biotransformation of aristolochic acid. Nitroreduction is the crucial step in formation of an...
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Activity of cytochromes P450 1A1, 1A2 and 3A4 expressed in eukaryotic and prokaryotic systems
Indra, Radek ; Stiborová, Marie (advisor) ; Mizerovská, Jana (referee)
Cytochromes P450 (CYP) are a superfamily of heme proteins distributed widely throughout nature, involved in metabolism of a broad variety of substrates and catalyzing a variety of interesting chemical reactions. They play a central role in metabolism of chemotherapeutic agents. Several prodrug antitumor agents have been found as CYP substrates. Ellipticine, an alkaloid found in Apocynaceae plants, is an example of such type of pro-drug. Here, we investigate the efficiencies of human recombinant CYPs expressed in eukaryotic and prokaryotic expression systems, namely in SupersomesTM , microsomes isolated from insect cells transfected with baculovirus construct containing cDNA of human CYP1A1, 1A2 and 3A4 with NADPH:CYP reductase or in Bactosomes, the membrane fraction of E. coli transfected with cDNA of the same human CYP enzymes and NADPH:CYP reductase to oxidize their marker substrates and ellipticine. Cytochrome b5, an aditional component of the mixed function oxidase system, which metabolize xenobiotics was also expressed in some of the systems. The results found in this work demonstrate that human CYP1A1, 1A2 or 3A4 expressed in both eukaryotic and procaryotic systems oxidize their marker substrates (EROD for CYP1A1/2, MROD for CYP1A2 and testosterone 6β-hydroxylation for CYP3A4). They also oxidize...
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Study on potentiaion of pharmacological efficiencies of ellipticine
Vranová, Iveta ; Mrázová, Barbora (referee) ; Stiborová, Marie (advisor)
Cytotoxic chemotherapy offers tool for clinical treatment of neoplasia. One of the drugs suitable for chemotherapy is ellipticine. Ellipticine is an alkaloid, which has significant antineoplastic properties. It acts as a DNA intercalator, inhibitor of topoisomerase II and forms also covalent DNA adducts mediated by cytochrome P450 and/or peroxidases. Oxidation of ellipticine by CYP (CYP3A4, CYP1A1/2, CYP2C9, CYP1B1) provides several metabolites (7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13- hydroxyellipticine, and ellipticine N2 -oxide). Metabolites 12-hydroxyellipticine and 13- hydroxyellipticine, formed by CYP3A4, are responsible for formation of two major DNA adducts. Two carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium were proposed as a reactive species binding to DNA. The main metabolites generated by peroxidases are the ellipticine dimer and ellipticine N2 -oxide, which provide the same carbenium ions and same DNA adducts. Modern chemotherapy uses targeting for higher selectivity for malignant cells and lower cytotoxicity for normal cells. Ellipticine-conjugates and his derivates (N-(2-hydroxypropyl)methakrylamid-ellipticine conjugates, vasoactive intestinal peptide-ellipticine conjugates and human serum albumin-ellipticine conjugates) and epidermal...
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The effect of histone deacetylase inhibitor vaplroate on activity and expression of cytochromes P450 and peroxidases oxidizing ellipticine
Göttlicherová, Markéta ; Souček, Pavel (referee) ; Stiborová, Marie (advisor)
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...
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Synthesis of ellipticine and its pharmacologically more efficient derivative 13-hydroxyellipticine
Hájek, Jan ; Černá, Věra (referee) ; Stiborová, Marie (advisor)
Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is plant alcaloid isolated from Ochrosia elliptica1) (Apocyanaceae). Ellipticine's anticancer effect is mainly caused by its intercalation into DNA, formation of covalent adduct with DNA and inhibition of topoisomerase II. Known syntheses of ellipticine are based on chemicals containing two or three aromatic cycles that are used in cyklisation and/or addition reaction to get ellipticine or ellipticine derivates. 13- hydroxyellipticine can be prepared de novo as a product of casual ellipticine synthesis, or as a modification of already synthetised ellipticine. Keywords: ellipticine; synthesis; derivates of ellipticine
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Comparison of efficiency of bibliographic databases for biochemical problems
Frantíková, Dagmar ; Hodek, Petr (referee) ; Hudeček, Jiří (advisor)
The study presents a comparison between four bibliographic databases widely used in biochemistry: paid SCOPUS and Web of Science and free-accessible MEDLINE and Google Scholar. Their efficacy and suitability for solving biochemical problems was evaluated. Three model problems were chosen and entered to databases: "ellipticine", "Lowry method" and "serine racemase". The located articles in each database were then sorted by their relevance. The best results were obtained with the SCOPUS database (highest proportion of relevant results; thus database has at the same time user-friendly interface). Very good was also free MEDLINE database and if paid databases are unavailable, MEDLINE would be the right option. The best results were found with a combination of both above named databases. Also database Web of Science gave good and relevant results but was not as useful as SCOPUS or MEDLINE. The worst results in this study was given by the database Google Scholar. It would be recommended for searches of basic, not so specific problems. This database locates many articles but with questionnable proportion of relevant articles - and these have to be found manually, which would be time-consuming. Thesis in Czech.
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Modulation of activities and expression of enzymes metabolizing ellipticine by histone deacetylase inhibitor trichostatin A
Kopejtková, Barbora ; Kubíčková, Božena (referee) ; Stiborová, Marie (advisor)
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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