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Study the optimal condition to knockout metallothionein by CRISPR-Cas9 in neuroblastoma
Májková, Klára
Cisplatin-based treatment strategies are commonly employed for the treatment of Neuroblastoma (Nbl), an embryonal tumour that most commonly affects infants and children under the age of five. However, the efficacy of these strategies has been found to be lower than 50 % due to the frequent chemoresistance developed by the tumour cells. Among others, this resistance has been linked to the increased expression of metallothionein (MTs) by the Nbl cells. Human MT-3, a protein primarily expressed in central nervous system cells, plays a vital role in metal detoxification and the maintenance of homeostasis during oxidative stress. Therefore, knockout of MT-3 using CRISPR/Cas9 could make the cells sensitive to cytotoxic drugs. This study is aimed to optimize the conditions for successful knockout of MT-3 from Nbl cells. The MT3-CRISPR/Cas9 plasmid was transfected into Nbl using Lipofectamine 2000 in three different plasmid concentrations (250 ng/μL, 500 ng/μL, and 1000 ng/μL). The optimal conditions were achieved using 0.75 μl of Lipofectamine, 1000 ng/μl of plasmid, and 0.5 ng/μl of puromycin.
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Cell senescence in aging, cell stress and chemoresistance
Andrašková, Tereza ; Vokřál, Ivan (advisor) ; Macháček, Miloslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Tereza Andrašková Supervisor: Prof. Dr. Alexandra K. Kiemer and PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Cell senescence in aging, cell stress, and chemoresistance The process of biological aging is connected with a loss of tissue function leading to the various pathologies, including cardiovascular diseases and cancer. Cellular senescence, the phenomenon characterized by permanent cell cycle arrest triggered by endogenous or exogenous stress plays a significant role in age-related metabolic diseases development. Senescent endothelial cells have been found in atherosclerotic plaque and due to the inflammation-inducing factors participate to its progression. Nevertheless, by triggering antiproliferative reaction, senescence may also have a potential as a cancer suppressor. The aim of this work was to establish assay determining senescence with different origins and subsequently, to analyse compounds with a potential to reduce H2O2-induced senescence as well as factors, which may be altered in the senescent phenotype. Senescence-associated β- galactosidase (SA-β-gal) assay in primary human endothelial cells revealed the senescence-inhibiting ability of statins and curcumin in contrast...
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Cell response to genotoxic stress-based anti-cancer therapies
Imrichová, Terezie ; Hodný, Zdeněk (advisor) ; Rossmeislová, Lenka (referee) ; Rotrekl, Vladimír (referee)
The dissertation deals with a cell response to genotoxic stress, specifically to anti-cancer treatments with a genotoxic mechanism of action. In principle, cells can respond to these perturbing stimuli in several ways: in case of severe DNA damage, they usually undergo apoptosis or enter senescence. In case of minor DNA damage, or upon defective checkpoint mechanisms, they may continue the cell cycle, either with successfully repaired DNA or with mutations of various kind. Thanks to selection pressure, the mutations that provide cells with a certain growth advantage under conditions of continuing genotoxic stress, gradually accumulate and render the tumor treatment-resistant. In my thesis, I focus on several aspects of this whole process. First, I participated in a characterization of a radioresistant and anoikis-resistant population of prostate cancer cells. This population was generated by irradiating cells 35 times by 2 Gy, a regime used in clinics. After this treatment, a population of low-adherent cells emerged that demonstrated increased expression of EMT- and stem cell markers. The low-adherent state of these cells was maintained by Snail signaling and their anoikis resistance by ERK1/2 signaling. Interestingly, after a protracted period of time, these cells were able to re-adhere and...
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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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Biomarkers of prognosis and therapy efficacy in ovarian carcinoma
Cerovská, Ela ; Souček, Pavel (advisor) ; Schierová, Michaela (referee)
Ovarian carcinoma is a serious illness with the highest mortality rate among all female cancers. No suitable methods for early diagnosis, precise determination of prognosis or prediction of therapy efficacy are currently available, which leads to diagnosis in advanced stages of disease and therapy efficacy limitation. Consequently, the development of chemoresistance to conventional drugs and frequent relapse of the disease pose a fundamental complication too. The main goal of the current study was identification of new putative prognostic and therapeutic biomarkers, whose introduction into clinical practice could help to improve the dismal outcome of ovarian carcinoma patients. The present master thesis provides results of expression analysis of genes whose products take part in the transport, metabolism and mechanism of action of platinum based drugs and taxanes, and also the regulation of cell cycle and signaling. Transcript levels of these genes have been assessed in series of tumor and control ovarian tissue samples and the difference between both tissue types was evaluated. Gene expression level in tumors was then compared with patient's clinical data and candidate genes, ABCA2 and PRC1, were selected from the obtained results for more detailed analysis. The protein level of candidate genes...
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New molecular biomarkers and therapeuticak targets in solid tumors
Voleská, Iveta ; Václavíková, Radka (advisor) ; Moulisová, Vladimíra (referee)
Breast and ovarian cancers are the most serious cancers among women. Relatively high mortality at advanced stages of the disease is often associated with the development of resistance to the cytotoxic agents. Chemoresistance usually develops on the base of different adaptive mechanisms that significantly decrease therapy efficiency. Currently TRIP6, ABCC3 and CPS1 enzyme has been identified based on high-capacity expression profile monitoring in tumor cell and tissue profiles as one such candidate playing a role in taxane resistance. The main goal of this thesis was to clarify the role or possible association of the ABCC3, CPS1 and TRIP6 genes with the development of tumor cell resistance to taxanes in models of sensitive and paclitaxel-resistant ovarian cancer cells and in the cohorts of patients with ovarian and breast cancer. The in vitro part compares the efficacy of paclitaxel and taxane derivatives in the sensitive and resistant ovarian cancer cell lines and clarifies the association between the different structure of taxane derivatives and the change in CPS1 expression after their application. The study in patient's cohorts with ovarian cancer reveals a relationship between higher levels of the CPS1 gene and shorter progression-free survival. The achieved results may serve as a base for data...
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The role of DNA repair pathways in ovarian cancer therapy response
Vallušová, Dominika ; Opattová, Alena (advisor) ; Rössner, Pavel (referee)
Ovarian cancer is serious and one of the most common gynecologic cancers. Carboplatin is the therapeutic agent of the first choice in the ovarian cancer therapy. However, after the primary therapeutic response to carboplatin, the relapse of the disease may occur with developed resistance to carboplatin. Chemoresistance and insufficient therapy response are considered to be the reason of the high mortality rate of ovarian cancer. The DNA damage response pathways play an important role in the therapeutic response and chemoresistance development. Restoration of homologous recombination function in cancers is the key mechanism of resistance development to platinum agents. Based on this knowledge, we formed our hypothesis, that the inhibition of homologous recombination could increase the sensibility to carboplatin. The main goal of this thesis was to define the role of double-strand breaks repair in response to chemotherapy of ovarian cancer. Protein MRE11 is part of the MRN complex, that participates in double-strand breaks repair. Using mirin as a pharmaceutic inhibitor of MRE11 we were aiming to determine the impact of homologous recombination on the effect of carboplatin and its role in resistant development to carboplatin. In the practical part of the thesis, we described the association between...
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The cancer cell proteome and its changes after anti-cancer drug treatment
Tylečková, Jiřina
Cancers represent a group of unprecedented heterogeneous diseases and currently available anti-cancer therapies provide highly variable efficacy with unsatisfactory cure rates. A wide range of proteomic technologies are being used in quest for newer approaches which could significantly contribute to the discovery and development of selective and specific cancer biomarkers for monitoring the disease state and anti-cancer therapy success. Taking into consideration the above aspects, this research was undertaken to study cancer cell proteomes and their changes after anti-cancer treatment with specific focus on: (a) response to conventional anthracycline/anthracenedione drugs with respect to their different clinical efficacy and (b) identification of novel targets for therapy in cancer cells resistant to biological drugs such as inhibitors of (b1) cyclin-dependent kinases and (b2) Aurora kinases. This study identified several interesting key aspects related to the effects of daunorubicin, doxorubicin and mitoxantrone. With the main focus on early time intervals when the influence of apoptosis is minimised, changes common for all three drugs belonging mainly to metabolic and cellular processes were observed. More importantly, significant changes in proteins involved in the generation of precursor...
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Cell response to genotoxic stress-based anti-cancer therapies
Imrichová, Terezie ; Hodný, Zdeněk (advisor) ; Rossmeislová, Lenka (referee) ; Rotrekl, Vladimír (referee)
The dissertation deals with a cell response to genotoxic stress, specifically to anti-cancer treatments with a genotoxic mechanism of action. In principle, cells can respond to these perturbing stimuli in several ways: in case of severe DNA damage, they usually undergo apoptosis or enter senescence. In case of minor DNA damage, or upon defective checkpoint mechanisms, they may continue the cell cycle, either with successfully repaired DNA or with mutations of various kind. Thanks to selection pressure, the mutations that provide cells with a certain growth advantage under conditions of continuing genotoxic stress, gradually accumulate and render the tumor treatment-resistant. In my thesis, I focus on several aspects of this whole process. First, I participated in a characterization of a radioresistant and anoikis-resistant population of prostate cancer cells. This population was generated by irradiating cells 35 times by 2 Gy, a regime used in clinics. After this treatment, a population of low-adherent cells emerged that demonstrated increased expression of EMT- and stem cell markers. The low-adherent state of these cells was maintained by Snail signaling and their anoikis resistance by ERK1/2 signaling. Interestingly, after a protracted period of time, these cells were able to re-adhere 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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