this parasite is the presence of drug resistance to almost all administered anthelmintics. Ivermectin (IVM) is one of the anthelmintics used in veterinary medicine to eliminate this endoparasite. It is believed that xenobiotic-metabolizing enzymes such as cytochromes P-450 and membrane efflux transporters P-glycoproteins, play a role in resistance in H. contortus.2,3 As the knowledge of the development and mechanism of drug resistance in nematodes is still insufficient, more research is needed. This study focuses on the effect of low concentrations of IVM, which could occur in environmental circulation, on expression of selected cytochromes P450 and P-glycoproteins. Females and males were separated before our experiment. After 4, 12 and 24-hour incubation of nematodes with three different IVM concentrations the levels of selected mRNAs were analyzed by qPCR. The results obtained in nematodes exposed to IVM were compared to the results in control nematodes (exposed to solvent only). Significant IVM-induced changes were found in expression of several genes. Significant sex-differences were observed in inducibility of tested genes. The induction effect of IVM was most pronounced in P-gp 1 genes, thus P-gp 1 might contribute to development of drug resistance in H. contortus. The study was supported by Czech Science Foundation (Project No. 18-07724S) and from the project of Specific Academic Research (SVV 260 416). References
1. TAK, I. R., DAR, S. A., DAR, J. S. et al.: Global Veterinaria, 13 (6), 2014, 960–965. 2. GODOY, P., CHE, H., BEECH, R. N. et al.: Mol. Biochem. Parasitol., 204, 2015, 11–15. 3. LAING, R., BARTLEY, D. J., MORRISON, A. A. et al.: Int. J. Parasitol., 45 (4), 2015, 243–251.
DNA METHYLATION CHANGES IN OROPHARYNGEAL CARCINOMA BIRKNEROVÁ, N.,1 MATOUŠKOVÁ, P.,1 BARANOVÁ, I.,2 KOVAŘÍKOVÁ, H.,2 LACO, J.,3 VOŠMIK, M.,4 CHMELAŘOVÁ, M.2 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine in Hradec Králové, Charles University, and University Hospital Hradec Králové, Czech Republic 3 The Fingerland Department of Pathology, Faculty of Medicine in Hradec Králové, Charles University and University Hospital Hradec Králové 4 Department of Oncology and Radiotherapy, Faculty of Medicine in Hradec Králové, Charles University and University Hospital Hradec Králové e-mail: birknerovan@faf.cuni.cz 1
Oropharyngeal carcinoma (OP) is a type of head and neck cancer (HNC) emerging in the tissue of base of the tongue, tonsils, soft palate and pharynx. Traditional risk factors include excessive alcohol and tobacco consumption. Recently, human papillomavirus (HPV) has been identified as an additional independent risk factor for the development of these tumors.1 Epigenetic alterations, such as DNA methylation, refer to heritable changes in gene expression that occur without changes in the underlying DNA sequence and can contribute to carcinogenesis.2 The aim of this study was to investigate methylation levels 98
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of selected tumor suppressor genes in oropharyngeal squamous cell carcinoma (OPSCC) and compare methylation levels with HPV status. DNA methylation levels of selected tumor suppressor genes were analysed using Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) in metastatic tumor samples, metastases samples, non-metastatic tumor samples and control tissue samples (non-cancerous palatine tonsils). We considered sample to be methylated above cut-off limit 15%. CADM1 methylation in OPSCC is increased by the presence of HPV infection, which is corresponding to methylation of the CADM1 gene in cervix carcinoma.3 CADM1 gene methylation levels are probably not influenced by metastatic process, because our results show that corresponding metastases has the same methylation status as their primary tumors. Methylation of CADM1 could be used as potential OPSCC biomarker in the future. The study was supported by Ministry of Health, Czech Republic – conceptual development of research organization (UHHK, 00179906) and from the project of Specific Academic Research (SVV 260 416). References
1. ZARAVINOS, A.: Oncotarget, 5 (12), 2014, 3956–3969. 2. Van KEMPEN, P. M., NOORLAG, R., BRAUNIUS, W. W. et al.: Epigenetics, 9 (2), 2014, 194–203. 3. Van KEMPEN, P. M. W., Van BOCKEL, L., BRAUNIUS, W. W. et al.: Cancer Med., 3 (5), 2014, 1185–1196.
METABOLISM OF FLUBENDAZOL IN HUMAN LIVER SUBCELLULAR FRACTIONS HOLUBOVÁ, J.,1 KUBÍČEK, V.,2 SKÁLOVÁ, L.1 1
Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: holuboja@faf.cuni.cz
Flubendazole (FLU), a benzimidazole anthelmintic drug with a broad spectrum of activity and low toxicity, has been used in veterinary as well as human medicine for a long time.1 Owing its mechanism of action, based on the specific binding to tubulin, FLU is now considered a promising anti-cancer agent.2 However, we do not have enough information about its biotransformation in human.3 In our project, subcellular fractions from the liver of 12 human patients (6 males and 6 females) were used to study the stereospecificity, cellular localization, coenzyme preference and possible inter-individual or sex differences in FLU reduction. In addition, the risk of FLU interaction with other drugs was evaluated. Results showed that FLU is predominantly reduced in cytosol and the NADPH coenzyme is preferred. The strict stereospecificity of FLU carbonyl reduction was proven and carbonyl reductase 1 was identified as the main enzyme of FLU reduction in the human liver. A higher reduction of FLU and a higher level of carbonyl reductase 1 protein was found in males than in females, but overall inter-individual variability was relatively low. Hepatic 99
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intrinsic clearance of FLU is very low, and FLU had no effect on doxorubicin carbonyl reduction in the liver and in cancer cells. For these reasons, interactions of FLU with other carbonyl bearing drugs are not expected. The obtained results demonstrate the safety of FLU use in human and support FLU repurposing for cancer treatment. The study was supported from the project of Specific Academic Research (SVV 260 416). References
1. MATÉ, M. L., GEARY, T., MACKENZIE, C. et al.: J. Vet. Pharmacol. Therap., 40 (5), 2017, 493–499. 2. ČÁŇOVÁ, K., ROZKYDALOVÁ, L., RUDOLF, E.: Acta Medica (Hradec Králové, Czech Republic), 60 (1), 2017, 5–11. 3. STUCHLÍKOVÁ, L., KRÁLOVÁ, V., LNĚNIČKOVÁ, K. et al.: Drug Test. Anal., 10 (7), 2018, 1139–1146.
NEW MODEL OF MUTATED TGR5 AND ITS TREATMENT BY BILE ACIDS DERIVATIVES IN SILICO MOTAMEDI, N.,1 HORVÁTOVÁ, A.,2 KAŠPAR, M.,3 KUDOVÁ, E.,3 DRASTÍK, M.,1 PÁVEK, P.2 Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 3 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic e-mail: motamedn@faf.cuni.cz 1
TGR5 is a member of the G-protein-coupled receptors family that represents a broad group of transmembrane receptors. It plays a role in regulation of cholesterol homeostasis, lipid, and carbohydrate metabolism. That makes TGR5 an interesting target in the pharmacotherapy of diabetes type 2 and obesity. In this study, a homology model of TGR51 was used since no crystal structure has been hitherto available. All observed orientations of cholic acid derivatives in several studies1,2 are consistent and very similar. The 3-α hydroxyl group on the A-ring is oriented towards a polar part of the cavity, forming a hydrogen bond with Glu169. The carboxylic group is facing another polar subdomain on the extracellular opening of the binding pocket probing for Ser270. These observations are in perfect line with our docking results received by employing Auto-Dock Vina software. Based on this we can conclude that Glu169 together with Ser270 are the crucial amino acids determining the proper orientation of a ligand in the pocket. Hydrophobic interaction between the rather nonpolar central part of the binding pocket and the polycyclic skeleton provides further stabilization. Another small nonpolar subdomain (Leu84, Leu87, Leu160, Phe125) hosts ethyl or ethylidene groups bound to the C6. Macchiarulo et al.3 who propose orientation of ligands towards Tyr89 and Asn93 suggest a different model. This orientation is perpendicular to what we are observing and does not involve any connection of the ligand to Ser270. The goal of this study is to shed light on this discrepancy by preparing series of mutants. Utili zation of molecular docking or use of mutants should help us in further study of novel ligands. 100
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