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...

Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the...

In recent years, the inhibiition of tyrosine kinases,which may incorrectly regulate some singaling pathway has been used to treat cancer as so-called biological therapy. An example of such inhibitors are vandetanib and lenvatinib. These two substances are used to treat thyroid gland tumors because they affect vascular growth factor receptor or endothelial growth factor receptor that can regulate tumor growth and metastasis. Ellipticine, which has anti-tumor effects on lots of tumor disease, has been investigated in this study together with vandetanib and lenvatinib. In this diploma thesis, the effect of mentioned tyrosine kinase inhibitors, ellipticine and their combinations on gene and protein expression of CYP1A1, 1A2, 3A1 and 3A2 in rat liver in vivo was determined. Protein expression was studied using Western blot method with imunodetection. Gene expression was assessed by quantitative PCR. Moreover, the effect of tested substances and their combinations on CYP1A activity (measured as 7-ethoxyresorufin O-deethylation), CYP1A2 activity (measured as 7-methoxyresorufin O-demethylation), CYP1A1 activity (measured as Sudan I oxidation), CYP3A specific activity (measured as testosteron 6β-hydroxylation) and ellipticine, vandetanib, lenvatinib metabolism was determined. It has been confirmed that...

Made available in DSpace on 2015-04-22T22:02:07Z (GMT). No. of bitstreams: 1 MARYCLEUMA CAMPOS HENRIQUE.pdf: 2547059 bytes, checksum: e7c319952c5d6f86b9976ddc8a877e33 (MD5) Previous issue date: 2007-06-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The species A. desmanthum and A. vargasii (Apocynaceae), known popularly in the Amazon for amargoso and amarelão , respectively, are used in the folk medicine mainly, its barks, in the form of teas. This work describes the chemical study of the barks of these species that resulted in the isolation and the identification of the alkaloids ellipticine and N-methyltetrahydroellipticine from the A. vargasii, and aspidocarpine, of the A. desmanthum. The identification was made based in spectra of 1H, 13C RMN and bidimensional HSQC, COSY, HMBC, NOESY, high resolution for electron spray impact masses (HR-ESI-MS), UV and IR, accomplishment of the identification of alkaloids ellipticine and aspidocarpine was confirmed by analysis of X-rays and comparison with data of the literature. The ethanol extracts, chloroform fractions, as well as isolated alkaloids presented lethality for Artemia franciscana and has been inactive for Aedes aegypti, behaviors this, observed for the two species in study. The antitumor activity of the ethanol extract, as well as some of its fractions and isolated alkaloids of A. vargasii has been active in test in vitro for tumor cells (breast, colon and nervous system). High antimalarial activity was observed for ellipticine alkaloids in vitro for Plasmodium falciparum. The ethanol extract of A. desmanthum presented low antitumor activity, however, great part of its fractions has been active. Antimalarial activity for aspidocarpine alkaloid isolated was observed for this specie. / As espécies A. desmanthum e A. vargasii (Apocynaceae), conhecidas popularmente na Amazônia por amargoso e amarelão, respectivamente, são muito utilizadas na medicina popular principalmente, suas cascas, na forma de chás. Este trabalho descreve o estudo químico das cascas dessas espécies que resultou no isolamento e na identificação dos alcalóides elipticina e Nmetiltetrahidroelipticina provenientes da A. vargasii, e aspidocarpina, da A. desmanthum. A identificação foi feita baseada em espectros de RMN 1H, 13C e os bidimensionais HSQC, COSY, HMBC, NOESY, massas de alta resolução por elétron spray (HR-ESI-MS), UV e IV, realização da identificação dos alcalóides elipticina e aspidocarpina foi apoiada ainda por análise de difração de raios-X e comparação com os dados da literatura. Os extratos etanólicos, frações clorofórmicas, bem como alcalóides isolados apresentaram letalidade para Artemia franciscana e foram inativos para Aedes aegypti, comportamentos este, verificado para as duas espécies em estudo. A atividade antitumoral do extrato etanólico, bem como algumas de suas frações e alcalóides isolados de A. vargasii foram ativos em teste in vitro para as linhagens de células tumorais (mama, cólon e sistema nervoso). Observou-se para o alcalóide elipticina atividade antimalárica in vitro expressiva frente ao Plasmodium falciparum. O extrato etanólico de A. desmanthum apresentou foi apresentou baixa atividade antitumoral, no entanto, grande parte de suas frações foram ativas. Observou-se ainda para essa espécie atividade antimalárica para o alcalóide isolado aspidocarpina.

Currently available anticancer therapies are inadequate and spur demand for improved technologies. Among others, the utilization of nanocarriers for anticancer drug delivery has shown great potential in cancer treatment. Nanocarriers can improve the therapeutic efficiency of the drugs with minimization of the undesirable side effects. To evaluate potential application of this technology, two forms of nanocarriers have been studied: multi-walled carbon nanotubes (MWCNTs) and apoferritin. The aim of this study was to determine, whether given cytostatics (ellipticine, etoposide and doxorubicin) are bound to these nanotransporters and how are they released from them, especially depending on pH. Since the pH of the tumor cells is lower than the pH of healthy cells it would be preferred that the drugs would release from nanocarriers at the lower pH while at the physiological pH the release of the drug would be eliminated. The results found show that ellipticine is actually released from its MWCNT- and apoferrtin-encapsulated form at acidic pH (5.0), while at pH 7.4 its interaction with nanocarriers is stable. Ellipticine released from MWCNT is activated by microsomal enzymes to reactive metabolites (13- hydroxyellipticine and 12-hydroxyellipticine) forming DNA adducts. The results indicate that both...

In recent years, tyrosine kinase inhibitors have been more and more used for the targeted cancer therapy, due to their ability to disrupt intracellular signalling pathways associated with the development of tumours. Cabozantinib is the tyrosine kinase inhibitor which has been approved for the treatment of thyroid cancer and it is also effective against several other types of cancer. However, multiple drugs combination is often used in anticancer therapy, which may result in their cytochrome P450-mediated interactions. Although this may affect the therapeutic effect of the drugs and cause adverse effects on the organism, very little is known about the effect of cabozantinib on biotransformation enzymes. Therefore, the effect of cabozantinib not only alone but also in combination with the known cytostatic ellipticine on the expression and the activity of cytochromes P450 1A1, 1A2 and 1B1 in rat liver and kidney in vivo was studied in this work. The gene expression was determined by quantitative PCR, the amount of protein was studied by Western blotting and consecutive immunodetection. The enzyme activity was studied using specific marker reactions, 7-ethoxyresorufin O-deethylation for CYP1A1, 7-methoxyresorufin O-demethylation for CYP1A2 and 17β-estradiol 4-hydroxylation for CYP1B1. Our results...

2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven strong mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans, while 2- NBA shows genotoxic properties lower by 3-4 orders of magnitude. Here we consider the possibility that large difference in the solubility, and consequently also the difference in bioavailability of these isomers could be the factor partially explaining this phenomenon. One goal was to determine solubility of 2-NBA in water and compare it with 3-NBA and also with other carcinogens studied in our laboratory (Sudan I, ellipticin). Another objective was to evaluate the effect of model proteins (bovine serum albumin, lysozyme) on solubility of Sudan I and ellipticine. The last aim was to determine extinction coefficients of these compounds in water and in methanol. Two different methods were employed to determine the solubility of the model compounds. The first method was based on spectrophotometric verification of the Lambert- Beer law. The results were than compared with other method measuring concentration of a compound in saturated solution (In Czech) Key...

2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven strong mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans, while 2- NBA shows genotoxic properties lower by 3-4 orders of magnitude. Here we consider the possibility that large difference in the solubility, and consequently also the difference in bioavailability of these isomers could be the factor partially explaining this phenomenon. One of our goals was to determine the solubility of 2-NBA in water and compare it with 3-NBA and also with other carcinogens studied in our laboratory (Sudan I, ellipticin). The second aim was to determine extinction coefficients of these compounds in water and in methanol. Two different methods were employed to determine the solubility of the model compounds. The first method was based on spectrophotometric verification of the Lambert- Beer law. The results were than compared with other method utilizing determination of concentration of a compound in equilibrium with solid phase. (In Czech) Key words: solubility, UV-VIS spectroscopy, 3-nitrobenzanthrone, 2-nitrobenzanthrone, ellipticine, Sudan I

In recent years, tyrosine kinase inhibitors have been widely used for the treatment of certain tumors as so-called targeted therapy. Many studies are concerned with their metabolism and the role of enzymes in the biotransformation process, but very little is known about the impact of tyrosine kinase inhibitors on the expression and activity of biotransformation enzymes. Nevertheless modification of the expression and activity of enzymes may cause adverse interactions of co-administered drugs and their negative impact on the human body. This diploma thesis studies the effect of tyrosine kinase inhibitors vandetanib and lenvatinib and cytotoxic alkaloid ellipticine on biotransformation enzymes in a rat model organism in vivo. The aim was to characterize the effect of the investigated compounds on gene expression, protein expression and activity of cytochromes P450 (CYP) 1A1, 1A2 and 1B1 and flavin-containing monooxygenases FMO1 and FMO3 in renal and hepatic microsomes. Microsomes and RNA were isolated from kidneys of control rats and the pretreated rats. Western blot and immunodetection was used to compare the protein expression levels of studied enzymes in kidney and liver. By reverse transcription, cDNA was prepared from isolated RNA and used as a template for quantitative PCR to compare the...