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Histopathology and Molecular Biology of Gastrointestinal Tumours
Červenková, Lenka ; Souček, Pavel (advisor) ; Eckschlager, Tomáš (referee) ; Pivovarčíková, Kristýna (referee)
The incidence and mortality of gastrointestinal cancers are at a level that represents a serious problem. These tumours have non-specific clinical symptoms, which often results in late diagnosis. In recent years, there have been significant advances in the identification and monitoring of molecular markers of tumour changes in the diagnosis, prognosis and treatment of these diseases. However, clinical oncology still faces a shortage of such biomarkers. The aim of this study was to find new biomarkers that correlate especially with prognostic indicators and predictors of treatment response or chemoresistance in gastrointestinal tract cancers. This thesis is based on 5 studies addressing ductal adenocarcinoma of the pancreas, colorectal cancer and hepatocellular carcinoma using IHC and molecular methods. All studies were performed using archival formalin-fixed paraffin-embedded samples. The main results include immunohistochemical evidence of protein expression of MRP2, SLC22A3 and SUR1/ABCC8 transporters and its significant association with the prognosis of pancreatic adenocarcinoma. Patients without SLC22A3 protein expression in the apical membrane were found to have significantly shorter disease-free and overall survival. We found longer disease-free survival in patients with positive membrane...
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Differences in histone acetylation in normoxia and hypoxia
Čepek, Pavel ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
Histones and their N and C terminal tails undergo different covalent modifications that regulate gene transcription. Among these histone modifications are methylation, ubiquitinilation, SUMOylation, ADP- ribosylation, phosphorylation, proline izomerization, deimination and acetylation. Histone acetylation is regulated by histonacetyltransferases (HATs) and histondeacetylases (HDACs). The balance between acetylation/deacetylation influences chromatin condensation and thus regulates gene transcription. Acetylation balance is disrupted in many human cancers and this fact can contribute to the development of malignant diseases. Histondeacetylase inhibitors (HDACi) can restore this acetylation imbalance. One of these HDACi is valproic acid (VPA) which has been used in treatment of epilepsy for decades. VPA shows antitumour effect in many studies. Decreased expression of n-myc oncoprotein, inhibition of tumour growth and angiogenesis are one of these anticancer effects observed in neuroblastoma cell lines after treatment with VPA. Despite the fact that exact mechanism of antitumour effect of VPA remains unclear, one of the most important mechanisms is hyperacetylation of histone H3 and H4. It is shown in this work that VPA increases acetylation of histones H3 and H4 in human neuroblastoma cell lines...
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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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Genetic variability in sporadic colorectal cancer: Searching for novel risk, prognostic and predictive biomarkers.
Jirásková, Kateřina ; Vodička, Pavel (advisor) ; Machoň, Ondřej (referee) ; Eckschlager, Tomáš (referee)
Colorectal cancer (CRC) is a major public health problem worldwide. Despite improvements in the diagnostic process and advancement in the treatment methods, the prognosis remains poor. To improve survival rates, it is important to identify people with the predisposition for CRC and to detect the potentially curable early stage of the disease. Furthermore, identifying those who would have an adverse clinical outcome associated with a particular chemotherapy would help to avoid redundant chemotherapy burden in patients and contribute to enhanced therapeutic efficacy, while minimizing treatment-related toxicity. The aim of the Thesis was to search for novel promising diagnostic, prognostic and predictive DNA-based biomarkers of sporadic form of CRC. As each patient is genetically unique, these biomarkers would aid clinicians in better diagnosis and/or in the selection of an optimal type of therapy for an individual CRC patient based on their molecular profile. In order to explore this issue, we investigated several candidate genes in healthy individuals as well as in newly diagnosed cancer patients. The major outcomes of this PhD study, which were fully reported in seven publications included in the present Thesis, are 1) The observation of several candidate single nucleotide polymorphisms in microRNA...
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Monitoring of children's cancer chemoresistance molecular cytogenetic methods
Procházka, Pavel ; Eckschlager, Tomáš (advisor) ; Kočárek, Eduard (referee) ; Kuglík, Petr (referee)
Souhrn Disertacni prace se zabyva chemorezistenci detskych nadorovych onemocneni adetekci cytogenetickych zmen, ktere s ni souvisi. Ovlivneni rizikovych forem detskych nadoru vyzaduje pouziti vysokych davek chemoterapie, ktera muze vest ke vzniku chemorezistence. Prukaz chromozomalnich aberaci muze prispet k urceni prognozy apredikce efektu lecbychemorezistentnichnadoru. Vetsinu vysledku jsme ziskalistudiem neuroblastomu (NBL). Ostatni prezentovane vysledky pak predstavuji geneticke zmeny u Ewingova sarkomu a u detskych feochromocytomu. Ke studiu chromozomalnichaberaci bylypouzitymetodykomparativni genomova hybridizace (CGH)a array CGH doplnene o vysetreni mnohobarevnou nebo int fzni fuor c brdi nst. Zmenyv expr naur mRNA byl s rera l escen ni hy i zaci i iu esi ovni yvyeteny expresni cipovou analyzou doplnenou o kvantitativni polymerazovou retezovou reakci, zmeny v expresiproteinu bylyvysetrenypomoci westernblottingu nebo prutokovou cytometrii. Predkladana disertace je komentovany soubor sesti publikaci. Studium chromozomalnich aberaci Ewingovych sarkomu predstavuje literarni resersi doplnenou o vlastni vysledky. Prace tykajici se feochromocytomu je jednim z nejvetsich souboru podrobnegeneticky vysetrenych detskych feochromocytomu. Nejobsahlejsi castse zabyva cytogenetickymizmenamiu NBL. Dlouhodobou...
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Effects of reovirus on cancer cells
Figová, Katarína ; Eckschlager, Tomáš (advisor) ; Mikyšková, Romana (referee) ; Němečková, Šárka (referee)
Oncolytic viruses are examined to serve as anticancer therapeutics. It is expected that in addition to direct oncolytic effect their action will also help eliciting a solid antitumor immunity. In presented series of experiments we have employed two HPV16- transformed mouse cell lines, TC-1 and MK16, and reovirus type 3, strain Dearing (RV). Both cell lines are highly susceptible to RV and produce large amounts of infectious virus in vitro. Still, some differences were encountered. When inoculating high doses of infected cells into syngeneic animals their oncogenic activity was strongly suppressed, nearly completely in the case of MK16 cells but less efficiently in the case of more oncogenic TC-1 cells. When immunized animals were challenged with TC-1 cells, the irradiated cells proved to be a much better immunogen that the infected cells. However, when challenged with MK16 cells the opposite was true. In another study we demonstrate that RV replicates preferentially in tumor cells and that the virus is able to overcome cellular adaptation to hypoxia (1% O2) and infect and kill hypoxic tumor cells. RV can both replicate in a hypoxic tumor microenvironment and can cause cytophatic effect and subsequently induce cell death. We found that a large proportion of cells are in hypoxia killed by caspase independent...
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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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Epigenetic and Cytotoxic Effects of Histone Deacetylase Inhibitors in Combination with Cytostatics on Neuroblasma
Abdel Rahman, Mohamed Ashraf Khalil ; Eckschlager, Tomáš (advisor) ; Mandys, Václav (referee) ; Krsková, Lenka (referee)
The enhanced expression of histone deacetylases (HDACs) in a variety of malignancies drew attention to investigate a new category of anti-cancer drugs that are based on the inhibition of those enzymes. Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through inhibition of HDACs class I and IIa. Cancer stem cells (CSCs) have been recognized to drive the tumor growth and progression hence; attention has been given to target this small subpopulation of CSCs rather than the whole bulk tumor cells. CD133 is considered to be a CSC marker in several tumors and its transcription is strongly influenced by epigenetic changes that will be altered upon administration of histone deacetylase inhibitors (HDACi) in cancer treatment. Therefore, we evaluated the epigenetic and cytotoxic effects of treatment with 1 mM VPA in combination with other chemotherapeutics and its influence on the expression of CD133 in human neuroblastoma (NB) cell lines. Our results revealed that addition of VPA to DNA-damaging chemotherapeutics induced a synergistic anti-tumor effect that was associated with caspase-3 dependent induction of apoptosis in UKF-NB-4 cells. This synergism was related to the increase of the acetylation status of histones H3 and H4 and was only produced either by...
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