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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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The role of asparagine synthetase in leukemic cells
Šafrhansová, Lucie ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
This thesis focuses on the detection of mutations in the enzyme asparagine synthetase on leukemia cell metabolism and the role of this enzyme in the context of L-asparaginase- based chemotherapy. The experimental part of the work is divided into two separate sections. Given the lack of asparagine synthetase gene sequencing data in leukemias, the first objective was to determine whether mutations are present in the leukemia cell line that could affect ASNS function and thus play a role in the resistance of leukemia cells to ASNase therapy. No mutations that could affect the activity of the enzyme were detected by next-generation sequencing. In the second part, a model of RS4;11 that expresses ASNS was established. The effect of ASNS on glycolysis was then studied to sensitize these cells to the effects of L-asparaginase and to the depletion of asparagine and glutamine. It was observed that ASNS expression increased the level of glycolysis and increased the resistance of these cells to asparagine and glutamine depletion and their resistance to asparaginase. Key words: ASNS, aspragine, leukemia, L-asparaginase, chemotherapy, drug resistance
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Resistance mechanisms in therapy of acute myeloid leukemia
Suchá, Simona ; Čečková, Martina (advisor) ; Žák, Pavel (referee) ; Matoušková, Petra (referee)
IN ENGLISH LANGUAGE Candidate: Mgr. Simona Suchá Supervisor: Assoc. Prof. PharmDr. Martina Čečková, PhD. Title of the doctoral thesis: Resistance mechanisms in therapy of acute myeloid leukemia Acute myeloid leukemia (AML) is a hematologic cancer known for its extensive heterogeneity, poor treatment outcomes and high relapse rate. Therapy outcome is often compromised by highly resistant leukemic clones present at diagnosis, which evade chemotherapy and continue to spread the disease. Identification of their cellular features is, therefore, a key in successful targeting and eliminating of these resistant leukemic cells. AML cells can, however, develop drug resistance even overtime due to prolonged drug exposure. Extremely high adaptability of leukemic cells enables them to survive whenever therapeutic stress stimuli occur. Uncovering molecular mechanisms that cells utilize to activate their survival mode is crucial in selection of treatment leading to maximal efficacy. Based on these grounds, two main aims of this thesis were set. First, to determine clinical relevance of ABC efflux transporters in AML and to evaluate the effect of targeted agents on chemotherapy. The focus was put on agents belonging to either FLT3 inhibitors (midostaurin) or CDK4/6 inhibitors (abemaciclib, palbociclib,...
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Study on the effect of selected targeted drugs on drug resistance mediated by ABC drug transporters
Paulusová, Viktória ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Viktória Paulusová Supervisor: doc. RNDr. Jakub Hofman, Ph.D. Title od diploma thesis: Study on the effect of selected targeted drugs on drug resistance mediated by ABC drug transporters. Multidrug resistance (MDR) is one of the major problems associated with cancer treatment. A key determinant of MDR is increased efflux of drugs through membrane ATP- binding cassette (ABC) transporters, which are a family of transporter proteins. Their overexpression contributes to MDR by decreasing intracellular drug concentration due to the efflux of various drugs from cells. Targeting ABC transporters appears to be a promising approach to suppress drug resistance. Therefore, one strategy to reverse the resistance of ABC transporter-expressing tumor cells is the combined use of chemotherapeutic drugs with ABC transporter modulators to enhance therapeutic efficacy. The aim of this work was to investigate the effect of targeted drugs (capmatinib, pralsetinib and tazemetostat) in combination with cytostatics (etoposide and topotecan) on drug resistance mediated by ABC efflux transporters. Results were obtained using MTT assays and caspase activity assays performed on the parental A431 cell line and its...
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Resistance mechanisms in therapy of acute myeloid leukemia
Suchá, Simona ; Čečková, Martina (advisor) ; Žák, Pavel (referee) ; Matoušková, Petra (referee)
IN ENGLISH LANGUAGE Candidate: Mgr. Simona Suchá Supervisor: Assoc. Prof. PharmDr. Martina Čečková, PhD. Title of the doctoral thesis: Resistance mechanisms in therapy of acute myeloid leukemia Acute myeloid leukemia (AML) is a hematologic cancer known for its extensive heterogeneity, poor treatment outcomes and high relapse rate. Therapy outcome is often compromised by highly resistant leukemic clones present at diagnosis, which evade chemotherapy and continue to spread the disease. Identification of their cellular features is, therefore, a key in successful targeting and eliminating of these resistant leukemic cells. AML cells can, however, develop drug resistance even overtime due to prolonged drug exposure. Extremely high adaptability of leukemic cells enables them to survive whenever therapeutic stress stimuli occur. Uncovering molecular mechanisms that cells utilize to activate their survival mode is crucial in selection of treatment leading to maximal efficacy. Based on these grounds, two main aims of this thesis were set. First, to determine clinical relevance of ABC efflux transporters in AML and to evaluate the effect of targeted agents on chemotherapy. The focus was put on agents belonging to either FLT3 inhibitors (midostaurin) or CDK4/6 inhibitors (abemaciclib, palbociclib,...
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Identification of expression profile changes of resistance-associated genes in experimental in vivo model of ovarian carcinoma
Bryndová, Barbora ; Václavíková, Radka (advisor) ; Dračínská, Helena (referee)
Ovarian carcinoma is one of the most serious gynecological malignancies in the world. High mortality of this cancer is mainly due to the development of multiple drug resistance to conventional treatments involving taxanes. Therefore, current research is focused on the study of new taxane-based derivatives capable to overcome known mechanisms of drug resistance. At present, there are several promising Stony Brook taxane derivatives (SB-T-taxanes), investigated in frame of efficacy, toxicity and mechanism of action. The aim of this bachelor thesis was to study changes in the expression of genes involved in the development of resistance in tissues and tumor obtained from in vivo mo- dels after the administration of derivatives SB-T-121605 compared to conventional taxane paclitaxel and non-treated models. Expression of selected ABC transporter genes (ABCB1, ABCC1 and ABCG2), taxane metabolizing enzyme genes (CYP3A11, CYP3A13 and CYP2C29) and their mouse orthologs was estimated in liver, intestinal and tumor tissue samples taken from in vivo mouse xenograft models. Tumor tissue was derivated from the highly resistant NCI/ADR-RES ovarian cancer cell line. Gene expression was determined by real-time quantitative polymerase chain reaction. Administration of the SB-T-121605 derivative led to several...
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Evolution of mixed cultures of the pathogenic yeasts Candida albicans and Candida guilliermondii
Hirko, Dominik ; Heidingsfeld, Olga (advisor) ; Schierová, Michaela (referee)
The objective of this thesis was to investigate how cocultures of Candida albicans and Candida guilliermondii change overtime under control conditions and under the influence of fluconazole. These species are opportunistic fungal pathogens but widely differ in their susceptibility to antimycotic of interest - fluconazole. After a brief introduction to special commonalities, the mechanism of pathogenesis, and the treatment of infection, this work explores each organism's growth curves under selected conditions and the process of artificial evolution using the model of passaging of cocultures. Afterwards, these populations of C. albicans and C. guillieromondii were investigated using qPCR and chromogenic media. qPCR analysis revealed that under control conditions, C. albicans (CA) prevails; the possible reason behind this is a 20% shorter generation time, as revealed by the growth curve. In the presence of fluconazole, two trends occurred. One is related to the innate resistance of C. guilliermondii (CG), where CG was dominant by the end of passaging. The second trend led to CA being the dominant one, despite its susceptibility. This is a result of a heightened resistance, where minimal inhibitory concentration 50 (MIC50) increased almost 10-fold, possibly due to mutations. The change in populations...
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Proteomic analysis of soluble and transmembrane proteins in human lymphoma cells
Vít, Ondřej
In the works presented here, we studied molecular changes associated with drug resistance in human mantle cell lymphoma (MCL) cells using proteomics. Our analyses allowed us to identify causal and/or secondary changes in protein expression associated with the development of resistance to the experimental drug TRAIL and the clinically used antimetabolites cytarabine and fludarabine. Resistance of MCL cells to the recombinant proapoptotic cytokine TRAIL was associated with downregulation of key enzymes of purine metabolism. This pathway potentially represents a molecular weakness , which could be used as a therapeutic target for selective elimination of such resistant cells. Resistance to the pyrimidine analog drug cytarabine was associated with cross-resistance to other antinucleosides. Proteomic and transcriptomic analyses showed pronounced downregulation of deoxycytidine kinase (dCK), which activates both purine and pyrimidine antinucleosides. This change explains the cross-resistance and is the causal mechanism of resistance to cytarabine. Our observations suggest that MCL patients, who do not respond to cytarabine-based therapy, should be treated with non-nucleoside drugs. MCL cells resistant to purine-derived antinucleoside fludarabine were cross-resistant to all tested antinucleosides and also...
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Study on impact of selected protein kinase inhibitors on drug resistance mediated by cytochromes P450
Janoušková, Adéla ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Adéla Janoušková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected protein kinase inhibitors on drug resistance mediated by cytochromes P450 Pharmacokinetic drug resistance often leads to failure of an anticancer therapy. One of the mechanisms is increased efflux of drugs from tumour cells, whereas some studies suggest that increased drug conversion to an inactive metabolite might be another contributing mechanism. The aim of this work was to define the possible role of CYP3A4 and CYP2C8 enzymes in the phenomenon of pharmacokinetic resistance and to investigate the possibility of its modulation by new targeted drugs. In the first part, we used the MTT proliferation method together with HepG2 cells stably transduced with particular human enzymes and demonstrated significant involvement of CYP3A4 in docetaxel resistance. In the following part, we examined the inhibitory effects of four selected tyrosine kinase inhibitors on the CYP3A4 activity in intact cells using a commercial kit. Cobimetinib and dabrafenib showed significant inhibitory activity, while osimertinib and brivanib did not. In the final part, we demonstrated the ability of the first two...
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Polymorfismus v genu MDR1 u border kolií
Trýznová, Alena
This thesis is focused on polymorphism of MDR1 gene c.-6-180T>G, which causes phenobarbital resistance. Phenobarbital is one of the most commonly used drug against epilepsy, which is one of the most common neurological disease in dogs with frequency between 0,5-5 % according to breed. This mutation has so far been detected only in the border collie breed, which manifests up to 30 percent resistance to the treatment of epilepsy. In the text is subscribed border collie breed, dog epilepsy and gene MDR1. Methodical part is focused on detection of the polymorphism in model population of 82 dogs. The results agree with previous studies and confirm frequency of mutant alleles 32 % within the population.
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