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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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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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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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Detekce polymorfismu v genu MDR1 u ovčáckých a honáckých psů
Staroveská, Marieta
This thesis is focused on polymorphism of MDR1 gene and related drug resistance. Resistance is caused by deletion of four nucleotids, that resulting in a frame shift and synthesis of nonfunctional transport of P-glycoprotein. The text describes a polymorphism of MDR1 (ABCB1) gene, which results in reduced resistance to drugs belonging to the group of macrocyclic lactones. It also describes inheritance of this phenomenon and it deals with the detection of mutation using PCR (polymerase chain reaction) and by fragmentation analyses. A review of literature study is a form of research solely from scientific publications. 128 dogs were included into the own analysis. The results confirmed that Collies had the highest presence of deletions (29,73 %) with a high number of carriers in the study population of dogs (54,05 %). The percentage of affected individuals in the breed of Australian Shepherd and Sheltie was significantly lower (7,32 % and 6 %), but the percentage of carriers were also high in both Australian Shepherds (34,14 %) and the breed Sheltie (48 %).
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Proteomic analysis of soluble and transmembrane proteins in human lymphoma cells
Vít, Ondřej ; Petrák, Jiří (advisor) ; Šulc, Miroslav (referee) ; Lenčo, Juraj (referee)
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...
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