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Influence of V-ATPase inhibitors on chemoresistant neuroblastoma lines in vitro
Honzejková, Karolína ; Eckschlager, Tomáš (advisor) ; Martínková, Markéta (referee)
Tumor diseases are one of the most common causes of death worldwide. Despite the great advances in therapy in the last fifty years, this is still a serious health problem. Therefore, great efforts are still concentrated on development of new anti-cancer drugs and therapeutic approaches. Neuroblastoma (NBL) is the most common tumor in infants and the fourth most common in children. Successful treatment is greatly complicated by its heterogeneity. Chemoresistance is an undesirable phenomenon of chemotherapy. One of the chemoresistance mechanisms is the accumulation of weakly basic anticancer drugs in lysosomes. This work deals with the measurement of lysosomal uptake of these compounds in neuroblastoma cell lines UKF-NB-4 and derived, ellipticine-resistant, line (UKF-NB- 4ELLI ) under different conditions. A method for determining the cell lysosomal capacity (volume) by measuring fluorescence intensity of lysosome-specific LTR dye was introduced and the ability of bafilomycin A, a V-ATPase inhibitor, to potentiate the effects of an anticancer agent ellipticine by inhibiting its lysosomal accumulation was investigated. Keywords: neuroblastoma, lysosome, vacuolar ATPase, multidrug resistance
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The expression of macrophage migration inhibitory factor in neuroblastoma cell lines
Polatová, Daniela ; Poljaková, Jitka (advisor) ; Groh, Tomáš (referee)
A macrophage migration inhibitory factor (MIF) is a cytokine which participates in immune responses induced by outer stimuli such as lipopolysaccharides of bacteria or glucocorticoids. It is produced mainly by macrophages. It activates other macrophages and T-lymphocytes and allows anti-inflammatory cytokines to be released. It contributes to angiogenesis - endogenously produced MIF is crucial for the proliferation of endothelial cells. The formation of new blood vessels stimulated by MIF was also described in tumors and it leads to tumor growth. The natural receptor for MIF is an integral membrane protein CD74, which could be presented on the cell surface. If it is presented on the surface of T-lymphocytes, it could be modified by chondroitin- sulphate and interact with a protein CD44. This modification and interaction is important for an effective activation of T-cells. MIF then binds to the complex CD74-CD44. The presence of CD74 in human neuroblastoma cell lines UKF-NB-3 and UKF-NB-4 was studied in this bachelor thesis. Using electrophoresis and Western blot CD74 was detected in the UKF-NB-4 neuroblastoma cell line but it wasn't detected in the UKF-NB-3 neuroblastoma cell line. Nevertheless, the presence of this receptor in neuroblastoma cells indicates, that even neuroblastomas could be...
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In silico design and validation of conotoxin-based peptides for neuroblastoma nanotherapy
Mokrý, Michal ; Gumulec, Jaromír (referee) ; Heger, Zbyněk (advisor)
Práca sa zaoberá in silico dizajnom a validáciou peptidov založených na konotoxíne - MrIA, izolovaného z morských slimákov druhu Conus marmoreus a možnosti využitia týchto peptidov v liečbe neuroblastómu pomocou cielenia norepinefrínového transportéru. Päť peptidov založených na tomto konotoxíne bolo simulovaných pomocou simulácii molekulárnej dynamiky, ich trajektórie boli analyzované pre zistenie vlastností týchto peptidov. Dva homologické modely ľudského norepinefrínového transportéru boli vytvorené pre analýzu väzobných vlastností peptidov založených na konotoxíne ku norepinefrínovému transportéru. Peptidy boli následne syntetizované a použité na pokrytie apoferitínových nanočastíc s elipticínom uväzneným vnútri apoferitínu. Vytvorené peptidy a nanočastice boli ďalej skúmané pre objasnenie ich fyzikálo-chemických vlastností. Interakcie a cytotoxicita boli skúmané aplokáciou nanočastíc na bunky neuroblastómu a epitelu. Z in silico a in vitro analýz vyšiel YKL-6 peptid ako najlepší kandidát na ďalší výskum.
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Modulation of activities and expression of enzymes metabolizing ellipticine by histone deacetylase inhibitor trichostatin A
Kopejtková, Barbora
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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Mechanisms of anticancer drug action in neuroblastomas
Groh, Tomáš
Cancer cells are able to adapt to different stress factors such as hypoxia, which is caused by insufficient tumor vascularization. An increased acetylation status of histones H3 and H4 in UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines was found to be a mechanism of adaptation of these cells to hypoxia. An increase in acetylation of histones H3 and H4 is suggested to cause changes in the structure of chromatin that lead to activation of gene transcription. In addition, cultivation of tested neuroblastoma cells under hypoxic conditions changes expression of proteins of a transcription factor N-myc, which is essential for development of neuroblastomas. This transcription factor is also responsible for a metabolic adaptation of neuroblastoma cells, increases their aggressiveness and its expression leads to a worse prognosis of the disease. Inhibitors of histone deacetylases (HDAC) are suggested to be the promising agents exhibiting various anticancer effects. They can induce cell cycle arrest, differentiation or programmed cell death in sensitive tumors. In this study, the effect of one of inhibitors of HDACs, valproate, on expression of proteins of transcription factors N-myc and hypoxia inducible factor 1α (HIF-1α) was investigated. Valproate decreases protein levels of both transcription factors in...
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Effects of valproic acid and its combinations with cytostatic agents on tumor cells in vitro
Hinďoš Hřebačková, Jana ; Dyr, Jan (advisor) ; Vávrová, Jiřina (referee) ; Entlicher, Gustav (referee)
Cancer is one of the most challenging problems the modern medicine is facing today. An increasing incidence and a great variability of tumor cells are the main reasons those drive the research to develop better diagnostics and therapeutic protocols. Histone deacetylase inhibitors, a group of epigenetic chemotherapeutics, are able to improve the performance of currently used anticancer agents. Vaplroic acid that is commonly used as antiepileptic drug exhibits a remarkable anticancer activity by itself as well as it is capable of therapy potentiation based on other therapeutic agents. Its effect to inhibit growth of tumor cells and induce apoptotic cell death seems to be even greater under hypoxic conditions (<1% O2). This study is focused on effect of valproic acid on neuroblastoma cell lines in vitro under normoxic and hypoxic conditions. We observed significantly greater efficacy of valproic acid in hypoxia compared to normoxia. The mechanism of induction of apoptotic cell death is based on disruption of the balance between pro- and antiapoptoic proteins. Intrinsic apoptotic pathway is probably initiated by the action of 19 kDa variant of proapoptotic protein Bax on mitochondrial membrane. Moreover, we examined the efficiency of a combined treatment of neuroblastoma cells with valproic acid and...
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V-ATPase expression in hypoxic conditions in neuroblastoma cells
Kittlerová, Kateřina ; Eckschlager, Tomáš (advisor) ; Moserová, Michaela (referee)
Tumor diseases belong to one of the most common death causes all around the world these days, therefore scientists still work on new therapeutic procedures. Tumor diseases are the second most common death causes among kids and juvenile. One of the freqeuently diagnosed tumor among kids and juvenile is neuroblastoma. Neuroblastoma is malignant embryonic tumor of the peripheral nervous system. Chemotherapy is used as tumor treatment by therapeutic procedures such as surgical removal and tumor irradiation. Cisplatin is one of the most applicated cytostatics, however it's efficiency it's lowered despite of creating resistance during the treatment. Vacuolar ATPase (V-ATPase) acidifies some of the cell organelles including lysosomes, which can lead to lysosomal sequestration of some of the substances including cytostatics and therefore the cure can't get to the therapeutic target. Key role in lysosomal activity regulation performes transcription factor EB (TFEB). This study deals with expression of d subunit of vacuolar ATPase (ATP6V0D1) and TFEB in neuroblastoma sensitive cell line UFK-NB-4 and resistant to cisplatin UKF-NB-4CDDP in normoxia and hypoxia. After exposure of neuroblastoma resistant cells to cisplatin the expression of ATP6V0D1 gene raised compared to normoxia. In case of TFEB the...
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Mechanisms of anticancer drug action in neuroblastomas
Groh, Tomáš
Cancer cells are able to adapt to different stress factors such as hypoxia, which is caused by insufficient tumor vascularization. An increased acetylation status of histones H3 and H4 in UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines was found to be a mechanism of adaptation of these cells to hypoxia. An increase in acetylation of histones H3 and H4 is suggested to cause changes in the structure of chromatin that lead to activation of gene transcription. In addition, cultivation of tested neuroblastoma cells under hypoxic conditions changes expression of proteins of a transcription factor N-myc, which is essential for development of neuroblastomas. This transcription factor is also responsible for a metabolic adaptation of neuroblastoma cells, increases their aggressiveness and its expression leads to a worse prognosis of the disease. Inhibitors of histone deacetylases (HDAC) are suggested to be the promising agents exhibiting various anticancer effects. They can induce cell cycle arrest, differentiation or programmed cell death in sensitive tumors. In this study, the effect of one of inhibitors of HDACs, valproate, on expression of proteins of transcription factors N-myc and hypoxia inducible factor 1α (HIF-1α) was investigated. Valproate decreases protein levels of both transcription factors in...
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Study of the mechanism of anticancer drug action on neuroblastomas
Černá, Tereza ; Stiborová, Marie (advisor) ; Souček, Pavel (referee) ; Mrízová, Iveta (referee)
Despite advances in cancer diagnosis and therapy, cancer is the second leading cause of death globally. The improvements of cancer treatment are the major challenge in this research. The aim of the thesis was studying of effects of two anticancer drugs ellipticine (Elli) and doxorubicin (DOX) on some cancer and healthy cell lines. Specific consideration was given to expand current knowledge about the metabolism and cytostatic effects of Elli in neuroblastoma cell lines. Another part of this study was focused on mechanisms contributing to the development of ellipticine-resistance in cancer cells and influence of histone deacetylase inhibitors on anticancer therapy was investigated. Moreover, the aim was to develop apoferritin (Apo) nanocarrier suitable for the active transport of cytostatics to cancer cells. Several essential data were found in this doctoral thesis. Anticancer efficiency of Elli depends on the CYP3A4-mediated metabolism in cancer. The CYP3A4 enzyme encapsulated into two nanoparticle forms, liposomes and SupersomesTM , was tested to activate ellipticine to its reactive species forming covalent DNA adducts. The formation of adducts seems to be dependent on concentrations of CYP3A4 in nanoparticle systems. A higher effectiveness of CYP3A4 in SupersomesTM than in liposomes to form...
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Epigenetically based chemoresistance of cancer cells
Feriančiková, Barbara ; Eckschlager, Tomáš (advisor) ; Šácha, Pavel (referee)
Cancer, despite significant advances in diagnosis and treatment, is the second most common cause of death in economically advanced countries. The main reason for the failure of anticancer therapy is the development of chemoresistance, which can be either internal or acquired, and is primarily mediated by the activation of various key regulators (eg MDR, PI3K/Akt, etc.). Genetic and epigenetic mechanisms are involved in activating these pathwa- ys. Significant epigenetic mechanisms that can participate in chemoresistance include regula- tion of gene expression by microRNA (miRNA) and long noncoding RNA (lncRNA). Dere- gulated expression of these non-coding RNAs has been observed in many diseases and their involvement in the initiation and progression of malignant tumors has been demonstrated. In this study, we investigated the expression of long non-coding RNA MIAT in hypoxia (1% O2) in chemosensitive and chemoresistant neuroblastoma cell lines (NBL), as hypoxia is a significant negative prognostic factor of many tumors and is involved in chemoresistance. Relative expression of MIAT was influenced by the number of cultured cells, where expression was increased by culturing more cells. MIAT expression was also significantly increased after 6 hours of NBL culture UKF-NB-4 in hypoxic conditions, and...
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