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Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level
Moriová, Magdalena ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradci Králové Departement of Pharmacology & Toxicology Student: Magdalena Moriová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level Cytostatic resistance is one of the most problematic obstacles in oncological treatment. Beside pharmacodynamic mechanisms, pharmacokinetic factors play an important role in drug resistance as well. Enzymatic transformation of active substance to inactive metabolite in tumor cells probably belongs to these mechanisms, however, evidences concerning the relevance of this phenomenon are predominantly either indirect and/or affected by interference elements. Using comparative experiments with HepG2 cell lines with/without CYP3A4 overexpression, we focused on the evaluation of the role of this clinically important enzyme in the resistance against docetaxel. Methodologically, it was the assessment of apoptosis induction (activation of caspases 3/7, 8 and 9) using commercial luminescent kits. Our results suggest significant participation of CYP3A4 enzyme on the reduction of docetaxel anticancer efficacy after 48 h from treatment, whereas this effect was not recorded in earlier intervals. These findings perfectly correlate...
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A role of microRNAs in the regulation of hepatic drug-metabolizing enzymes
Sedlmajer, Štěpán ; Smutný, Tomáš (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Štěpán Sedlmajer Supervisor: PharmDr. Tomáš Smutný, Ph.D. Title of diploma thesis: A role of microRNAs in the regulation of hepatic drug-metabolizing enzymes The liver is the main biotransformation organ in the body. Cytochromes P450 and conjugation enzymes are essential enzymes in the metabolism of drugs and other xenobiotics. Recently, there is growing evidence of post-transcriptional regulation of these enzymes through microRNA molecules. MicroRNAs are epigenetic regulators of expression, which usually bind to fully or partially complementary sequences within the 3'UTR (3' untranslated) regions of mRNA molecules. MicroRNAs have a role in interindividual variability of expression and activity of metabolic enzymes. Understanding their role and mechanisms of action may contribute to the development of new drugs and tailored pharmacotherapy. The work summarizes current selected achievements in the research on regulation of mainly hepatic cytochromes P450 and conjugation enzymes through microRNAs. The impact of RNA editing, single nucleotide polymorphisms and pseudogenes is also described.
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Effect of pH and other factors on the metabolic conversion of tyrosine kinase inhibitors.
Čillíková, Olívia ; Indra, Radek (advisor) ; Čermáková, Michaela (referee)
Targeted anti-tumor therapy is a modern and effective approach to cancer treatment. This type of therapy includes several groups of anticancer drugs. The second most commonly used group of targeted chemotherapeutic substances are tyrosinkinase inhibitors. These are small- molecular agents that target specific signalling pathways of the tumor cell, thereby inhibiting it's growth, proliferation and angiogenesis. Representatives of this drug group include vandetanib and sunitinib. Vandetanib is primarily used for the treatment of medullary thyroid cancer and sunitinib has been approved for the treatment of gastrointestinal stromal tumor and advanced renal cell carcinoma. The diploma thesis focuses on the effect of pH and temperature on the in vitro metabolism of vandetanib and sunitinib using first-phase biotrasnformation enzymes. Differently premedicated and control rat liver microsomes were used in the experiments. Human and rat recombinant cytochromes P450 were used for comparison. The primary purpose of the experiments was to find the experimental pH and temperature optimum of the in vitro metabolism of vandetanib and sunitinib. These experiments also investigated the effect of pH and temperature on the drugs themselves and their stability. The effect of pH and temperature on the metabolism of the...
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Effect of the anticancer drug cabozantinib on cytochrome P450 activity
Slobodníková, Eva ; Dračínská, Helena (advisor) ; Václavíková, Radka (referee)
Cabozantinib is an anti-cancer drug used mainly for the treatment of thyroid and renal cell carcinoma. It is classified as a low molecular weight selective tyrosine kinase inhibitor. Tyrosine kinases play a key role in signal transduction and regulation of many cellular processes such as growth, differentiation, and proliferation. The changes in the tyrosine kinase pathways are associated with the formation and progression of tumors where their growth is uncontrolled. Tyrosine kinase inhibitors act on tyrosine kinase receptors, thereby preventing the spread of cancer cells and slowing down the progression of cancer. Because cabozantinib, like other clinically used drugs, is metabolized by cytochromes P450, adverse drug interactions may occur that result in altered pharmacokinetics of the administered drugs and a consequent decrease in the efficacy of these drugs. In this diploma thesis, the effect of cabozantinib on the activity of the main enzymes of phase I biotransformation of xenobiotics, cytochromes P450, was investigated in vitro. The effect on the activity of both rat and human cytochrome P450 isoforms involved in xenobiotic metabolism was studied. CYP isoforms were predominantly incubated with cabozantinib at two concentrations; 10 µM and a concentration corresponding to the substrate...
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Metabolism of the mitotic inhibitor BTB-1 by first phase biotransformation enzymes
Vančurová, Kateřina ; Indra, Radek (advisor) ; Martináková, Lenka (referee)
Cancer is still one of the most common deaths in the world and therefore new drugs are needed to be developed to stop or slow down this disease. Recently, there has been a huge expansion in drug development. Cytostatics that are still widely used include a group of mitotic inhibitors aimed at inhibiting mitosis. A representative of a mitotic inhibitor is the small newly discovered molecule BTB-1. This molecule mediates reversible inhibition of the molecular motor Kif18A, which plays an important role in cell division. In the first part of this presented bachelor thesis, a suitable method for the determination of BTB-1 using high performance liquid chromatography was developed and subsequently its sensitivity was verified. Furthermore, a suitable extracting reagent was found. In the second part of the bachelor thesis, the metabolism of BTB-1 was studied by the microsomal system of non-premedicated rats and rats premedicated with various cytochrome P450 inducers. Subsequently, the time dependence of BTB-1 conversion was studied by the microsomal system of premedicated rats. The effect of different concentrations of BTB-1 on its metabolism was also studied using the microsomal system of premedicated rats. Furthermore, the metabolism of this new molecule was studied using cytosolar systems isolated...
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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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Heterologous expression and purification of human cytochrome b5
Kostelanská, Marie ; Černá, Věra (advisor) ; Bořek Dohalská, Lucie (referee)
The metabolism of xenobiotics and endogenous substances is mediated by a mixed function oxidase system which includes cytochrome b5 participating in catalytic activities of CYP. The mechanism of action of the cytochrome b5 has not been fully elucidated yet. But it is assumed that cytochrome b5 is involved either in direct electron transfer within the mixed function oxidase system or in induction of conformational changes in CYPs. So it is important to gain the pure form of apo-cytochrome b5, devoid of heme, which is not capable of electron transfer and further study the effect of this form on CYP-catalyzed reactions. The obtained results can contribute to understanding the mechanism of cytochrome b5 effects. The transformation of bacterial cells of Escherichia coli BL-21 (DE3) Gold was performed by expression vector pET22b which contained genes for microsomal and erythrocyte cytochrome b5. In order to produce a high level of apoprotein form, the heterologous expression of cytochrome b5 was induced by addition of higher amount of IPTG. Expression was performed at 37řC. This bachelor thesis is primarily engaged in purification of both microsomal and erythrocyte form of cytotochrom b5, especially in its apo-form. However, the productions of holo-cytochrome b5 form always occur in a greater or lesser...
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Isolation and characterization of microsomal fraction of fungus Pleurotus ostreatus and its role in the degradation of 17α-ethinylestradiol
Valášková, Petra ; Černá, Věra (advisor) ; Hodek, Petr (referee)
A synthetic hormone 17α-ethinylestradiol (EE2) which is a component of hormonal contraception pills has been identified as a main component of the endocrine-disrupting compounds (EDc). EDc are substances that mimic natural hormones in their action. Recently their amount especially in the groundwater and the surface water has been increased, which results in a negative impact on the hormonal system especially of aquatic organisms. Since it is not easy to replace these substances from the environment by conventional techniques other possibilities of their biodegradation are examined. White rot fungi, which are able to degrade lignin in nature, have promising biodegradation abilities towards many pollutants. These fungi contain a wide range of non-specific extracellular and intracellular enzymes that play an important role in the degradation. This bachelor thesis was targeted on the study of a white rot fungus, Pleurotus ostreatus, and especially on the degradation potential of its intracellular enzymes in the biodegradation of EE2. Initially, the ability of fungi Pleurotus ostreatus to degrade EE2 in vivo was tested. During the 48 hour incubation there was replaced 95,5 % of EE2. However, the role of cytochromes P450 (CYPs) in a metabolism of EE2 was not confirmed in this experiment by reason that an...
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The comparison of properties of cell lines resistant to ellipticine, doxorubicin, and cisplatin
Černá, Tereza ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....
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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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