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1.
Pharmacol Rep ; 2021 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-34741761

RESUMO

BACKGROUND: Hepatic enzymes involved in drug metabolism vary markedly in expression, abundance and activity, which affects individual susceptibility to drugs and toxicants. The present study aimed to compare mRNA expression and protein abundance of the most pharmacologically relevant drug-metabolizing enzymes in two main sources of the control liver samples that are used as the reference, i.e. organ donor livers and non-tumorous tissue from metastatic livers. An association analysis of the most common genetic variants with mRNA and protein levels was also performed. METHODS: The CYP450 and UGT enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, UGT1A1, UGT1A3, UGT2B7 and UGT2B15) were analyzed for mRNA (qPCR) and protein abundance (LC-MS/MS) in healthy donors (n = 11) and metastatic (n = 13) livers. Genotyping was performed by means of TaqMan assays and pyrosequencing. RESULTS: Significantly higher protein abundance in the metastatic livers was observed in case of CYP2C9, CYP2D6, and UGT2B7, and for UGT1A3 the difference was only significant at mRNA level. For all the enzymes except CYP2E1 some significant correlation between mRNA and protein content was observed, and for UGT1A1 an inverse correlation with age was noted. CYP2C19, CYP3A5 and CYP2D6 were significantly affected by genotype. CONCLUSION: The selection of a control group for the study on drug-metabolizing enzymes (e.g. in pathological states) may possibly affect its conclusions on differences in mRNA and protein content. Genotyping for common functional variants of CYP450 enzymes should be performed in all studies on drug-metabolizing enzymes.

2.
Pharmaceutics ; 13(9)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34575411

RESUMO

Hepatic drug metabolizing enzymes (DMEs) markedly affect drug pharmacokinetics. Because liver diseases may alter enzymatic function and in turn drug handling and clinical efficacy, we investigated DMEs expression in dependence on liver pathology and liver failure state. In 5 liver pathologies (hepatitis C, alcoholic liver disease, autoimmune hepatitis, primary biliary cholangitis and primary sclerosing cholangitis) and for the first time stratified according to the Child-Pugh score, 10 CYPs (CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and 4 UGTs (UGT1A1, UGT1A3, UGT2B7 and UGT2B) enzymes were quantified for protein abundance (LC-MS/MS) and gene expression (qRT-PCR). CYP2E1 was the most vulnerable enzyme, and its protein levels were significantly reduced just in Child-Pugh class A livers. The protein abundance of CYP1A1, CYP2B6, CYP2C19, CYP2D6 as well as UGT1A1, UGT1A3 and UGT2B15 was relatively stable in the course of progression of liver function deterioration. Alcoholic liver disease and primary biliary cholangitis were involved in the most prominent changes in the protein abundances, with downregulation of 6 (CYP1A2, CYP2C8, CYP2D6, CYP2E1, CYP3A4, UGT2B7) and 5 (CYP1A1, CYP2B6, CYP2C8, CYP2E1, CYP3A4) significantly downregulated enzymes, respectively. The results of the study demonstrate that DMEs protein abundance is affected both by the type of liver pathology as well as functional state of the organ.

3.
Pharmacol Rep ; 73(5): 1427-1438, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34117631

RESUMO

BACKGROUND: Wilson's disease is a genetic disorder inherited in a recessive manner, caused by mutations in the copper-transporter ATP7B. Although it is a well-known disease, currently available treatments are far from satisfactory and their efficacy varies in individual patients. Due to the lack of information about drug-metabolizing enzymes and drug transporters profile in Wilson's disease livers, we aimed to evaluate the mRNA expression and protein abundance of selected enzymes and drug transporters in this liver disorder. METHODS: We analyzed gene expression (qPCR) and protein abundance (LC-MS/MS) of 14 drug-metabolizing enzymes and 16 drug transporters in hepatic tissue from Wilson's disease patients with liver failure (n = 7, Child-Pugh class B and C) and metastatic control livers (n = 20). RESULTS: In presented work, we demonstrated a downregulation of majority of CYP450 and UGT enzymes. Gene expression of analyzed enzymes ranged between 18 and 65% compared to control group and significantly lower protein content of CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP3A4 and CYP3A5 enzymes was observed in Wilson's disease. Moreover, a general decrease in hepatocellular uptake carriers from SLC superfamily (significant at protein level for NTCP and OATP2B1) was observed. As for ABC transporters, the protein abundance of BSEP and MRP2 was significantly lower, while levels of P-gp and MRP4 transporters were significantly higher in Wilson's disease. CONCLUSIONS: Altered hepatic expression of drug-metabolizing enzymes and drug transporters in Wilson's disease patients with liver failure may result in changes of drug pharmacokinetics in that group of patients.

4.
Int J Mol Sci ; 22(5)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33671106

RESUMO

Most anticancer drugs target mitosis as the most crucial and fragile period of rapidly dividing cancer cells. However the limitations of classical chemotherapeutics drive the search for new more effective and selective compounds. For this purpose structural modifications of the previously characterized pyridine aalog (S1) were incorporated aiming to obtain an antimitotic inhibitor of satisfactory and specific anticancer activity. Structure-activity relationship analysis of the compounds against a panel of cancer cell lines allowed to select a compound with a thiophene ring at C5 of a 3,4-dihydropyridine-2(1H)-thione (S22) with promising antiproliferative activity (IC50 equal 1.71 ± 0.58 µM) and selectivity (SI = 21.09) against melanoma A375 cells. Moreover, all three of the most active compounds from the antiproliferative study, namely S1, S19 and S22 showed better selectivity against A375 cells than reference drug, suggesting their possible lower toxicity and wider therapeutic index. As further study revealed, selected compounds inhibited tubulin polymerization via colchicine binding site in dose dependent manner, leading to aberrant mitotic spindle formation, cell cycle arrest and apoptosis. Summarizing, the current study showed that among obtained mitotic-specific inhibitors analogue with thiophene ring showed the highest antiproliferative activity and selectivity against cancer cells.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Di-Hidropiridinas/química , Melanoma/tratamento farmacológico , Tionas/química , Apoptose , Desenho de Fármacos , Humanos , Melanoma/patologia , Mitose , Estrutura Molecular , Relação Estrutura-Atividade , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/farmacologia , Células Tumorais Cultivadas
5.
Pharmacol Rep ; 73(2): 583-593, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33270185

RESUMO

BACKGROUND AND OBJECTIVE: Clopidogrel is frequently used as part of optimal dual antiplatelet therapy in high-bleeding risk patients with the acute coronary syndrome. The concentration of the inactive carboxylic acid metabolite of clopidogrel might be useful to evaluate the response to clopidogrel therapy. Therefore, we sought to correlate the inhibition of platelet aggregation with the plasma level of the inactive metabolite of clopidogrel in patients after percutaneous coronary interventions (PCI) and their associations with the most frequently studied genetic polymorphisms. For this purpose, the fast and simple HPLC method for determining the concentration of the inactive metabolite was developed. METHODS: The effect of CYP2C19, CYP3A4/5, ABCB1 and PON1 genes on the plasma inactive metabolite concentration of clopidogrel and the platelet aggregation was investigated in 155 patients before and after PCI. RESULTS: The concentration of the inactive metabolite of clopidogrel was not significantly different in the intermediate metabolizers (IM) of CYP2C19 compared with extensive metabolizers (EM) both before and after PCI, while inhibition of platelet aggregation was found to be significantly better in EM than in IM. The presence of the A allele at position 2677 in the ABCB1 gene was associated with a significantly lower concentration of inactive metabolite of clopidogrel before PCI. CONCLUSION: The CYP2C19*2 allele was associated with decreased platelet reactivity during clopidogrel therapy before and after PCI. Simultaneous determination of platelet aggregation and concentration of the inactive clopidogrel metabolite may be useful in clinical practice to find the cause of adverse effects or insufficient treatment effect in patients chronically treated with clopidogrel.

6.
Int J Mol Sci ; 21(19)2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33036164

RESUMO

Liver diseases are important causes of morbidity and mortality worldwide. The aim of this study was to identify differentially expressed microRNAs (miRNAs), target genes, and key pathways as innovative diagnostic biomarkers in liver patients with different pathology and functional state. We determined, using RT-qPCR, the expression of 472 miRNAs in 125 explanted livers from subjects with six different liver pathologies and from control livers. ANOVA was employed to obtain differentially expressed miRNAs (DEMs), and miRDB (MicroRNA target prediction database) was used to predict target genes. A miRNA-gene differential regulatory (MGDR) network was constructed for each condition. Key miRNAs were detected using topological analysis. Enrichment analysis for DEMs was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). We identified important DEMs common and specific to the different patient groups and disease progression stages. hsa-miR-1275 was universally downregulated regardless the disease etiology and stage, while hsa-let-7a*, hsa-miR-195, hsa-miR-374, and hsa-miR-378 were deregulated. The most significantly enriched pathways of target genes controlled by these miRNAs comprise p53 tumor suppressor protein (TP53)-regulated metabolic genes, and those involved in regulation of methyl-CpG-binding protein 2 (MECP2) expression, phosphatase and tensin homolog (PTEN) messenger RNA (mRNA) translation and copper homeostasis. Our findings show a novel panel of deregulated miRNAs in the liver tissue from patients with different liver pathologies. These miRNAs hold potential as biomarkers for diagnosis and staging of liver diseases.


Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Hepatopatias/genética , MicroRNAs/metabolismo , Transdução de Sinais , Idoso , Colangite/genética , Colangite/metabolismo , Biologia Computacional , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Perfilação da Expressão Gênica , Hepatite C/genética , Hepatite C/metabolismo , Hepatite Autoimune/genética , Hepatite Autoimune/metabolismo , Degeneração Hepatolenticular/genética , Degeneração Hepatolenticular/metabolismo , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Hepatopatias/metabolismo , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/metabolismo , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade
7.
Biomolecules ; 10(7)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32708725

RESUMO

Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein that is encoded by the AIF1 gene. The main functions of AIF-1 are the activation of macrophages and enhancing the production of pro-inflammatory cytokines. To date, three different AIF-1 isoforms have been identified. In this study, we examined the expression of AIF-1 isoforms on the level of mRNA, and we compared the percentage of AIF-1-positive white blood cells (WBCs) in blood and AIF-1/CD68 cells in the synovial membranes in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). We examined 15 patients with RA and 15 patients with OA who had previously undergone knee arthroplasty. Peripheral blood and synovial membranes (SMs) were collected from these patients during knee arthroplasty. We identified three AIF-1 mRNA expression variants in peripheral mononuclear cells (PBMCs) and SMs from patients in both groups. Spearman's rank correlation coefficient tests showed strong, positive, and significant correlations between the three AIF-1 mRNA expression variants in PBMCs and/or SMs in patients with RA and OA. There were no statistically significant correlations for any of the AIF-1 mRNA expression variants between PBMCs and SMs in patients with RA and OA. We observed a statistically significant increased percentage of AIF-1-positive cells in patients with RA in comparison to patients with OA. The percentage of AIF-1-positive cells in the blood of patients with RA and OA was 1.35 ± 0.81% and 0.71 ± 0.25% (p < 0.01), respectively, whereas the percentage of AIF-1/CD68-positive WBC cells in the SMs was 24.05 ± 7.17% and 4.78 ± 1.52% (p < 0.001), respectively. In conclusion, three AIF-1 mRNA expression variants occurred in PBMCs and SM cells in patients with RA and OA. The AIF-1 mRNA expression levels of the variants correlated with each other in PBMCs and SM cells, but there were no statistically significant correlations for AIF-1 mRNA expression variants between PBMCs and SM cells in patients with RA and OA. Both in the blood and SMs, we observed an increased percentage of AIF-1-positive cells in patients with RA in comparison to patients with OA. The above results suggested that AIF-1 was the cytokine involved in the pathogenesis of RA. The precise knowledge of the role of AIF-1 in RA pathogenesis and the development of inflammatory response requires further investigations.


Assuntos
Artrite Reumatoide/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas dos Microfilamentos/genética , Regulação para Cima , Idoso , Células Cultivadas , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Membrana Sinovial/metabolismo
8.
Int J Mol Sci ; 21(5)2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32111097

RESUMO

Membrane monocarboxylate transporter 1 (SLC16A1/MCT1) plays an important role in hepatocyte homeostasis, as well as drug handling. However, there is no available information about the impact of liver pathology on the transporter levels and function. The study was aimed to quantify SLC16A1 mRNA (qRT-PCR) and MCT1 protein abundance (liquid chromatography-tandem mass spectrometry (LC---MS/MS)) in the livers of patients diagnosed, according to the standard clinical criteria, with hepatitis C, primary biliary cirrhosis, primary sclerosing hepatitis, alcoholic liver disease (ALD), and autoimmune hepatitis. The stage of liver dysfunction was classified according to Child-Pugh score. Downregulation of SLC16A1/MCT1 levels was observed in all liver pathology states, significantly for ALD. The progression of liver dysfunction, from Child-Pugh class A to C, involved the gradual decline in SLC16A1 mRNA and MCT1 protein abundance, reaching a clinically significant decrease in class C livers. Reduced levels of MCT1 were associated with significant intracellular lactate accumulation. The MCT1 transcript and protein did not demonstrate significant correlations regardless of the liver pathology analyzed, as well as the disease stage, suggesting posttranscriptional regulation, and several microRNAs were found as potential regulators of MCT1 abundance. MCT1 membrane immunolocalization without cytoplasmic retention was observed in all studied liver pathologies. Overall, the study demonstrates that SLC16A1/MCT1 is involved in liver pathology, especially in ALD.


Assuntos
Fígado/metabolismo , Fígado/patologia , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Adulto , Idoso , Animais , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/genética , RNA Mensageiro , Espectrometria de Massas em Tandem
9.
Clin Pharmacol Ther ; 107(5): 1138-1148, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31697849

RESUMO

Hepatocellular transporter levels were quantified using quantitative reverse transcription polymerase chain reaction and liquid chromatography-tandem mass spectrometry methods. Liver function deterioration (Child-Pugh class C) produced significant protein abundance (mean values) increase (to healthy livers) in P-gp (to 260% (CV (coefficient of variation) 82%)) and MRP4 (CV 230%) (not detected in healthy livers), decrease in MRP2 (to 30% (CV 126%)), NTCP (to 34% (CV 112%)), OCT1 (to 35% (CV 153%)), OATP1B1 (to 46% (CV 73%)), and OATP2B1 (to 27% (CV 230%)), whereas BSEP (CV 99%), MRP3 (CV 106%), OAT2 (CV 97%), OCT3 (CV 113%), and OATP1B3 (CV 144%) remained unchanged. Alcoholic liver disease produced significant protein downregulation of MRP2 (to 30% (CV 134%)), NTCP (to 76% (CV 78%)), OAT2 (to 26% (CV 117%)), OATP1B1 (to 61% (CV 76%)), OATP1B3 (to 79% (CV 160%)), and OATP2B1 (to 73% (CV 90%)) of healthy tissue values. Hepatitis C produced BSEP (to 47% (CV 99%)) and OATP2B1 (to 74% (CV 91%)) protein reduction. Primary biliary cholangitis and primary sclerosing cholangitis demonstrated P-gp and MRP4 protein upregulation (to 350% (CV 47%) and 287% (CV 38%), respectively). Autoimmune hepatitis revealed P-gp (to 410% (CV 49%)) and MRP4 (CV 96%) increase, and MRP2 (to 18% (CV 259%)) protein decrease. Drug transporters' protein abundance depends on liver pathology type and its functional state.


Assuntos
Hepatopatias/fisiopatologia , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas/metabolismo , Idoso , Estudos de Casos e Controles , Cromatografia Líquida , Feminino , Humanos , Fígado/fisiopatologia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem
10.
Pharmacol Rep ; 71(4): 738-745, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31207436

RESUMO

BACKGROUND: Analysis of results and conclusions in studies dedicated to pathology of the liver are usually based on comparison of pathological liver specimens and control/reference (considered as healthy) tissues. There are two main sources of the control liver samples used as the reference livers, i.e. deceased organ donor livers and non-tumorous tissue from metastatic livers, which are also applied for drug transporter investigations. However, no information has yet been published on drug transporters in these two major types of reference livers. METHODS: We explored ABC (P-gp, MRP1, MRP2, MRP3, MRP4, BCRP, BSEP) and SLC (NTCP, MCT1, OCT1, OCT3, OAT2, OATP1B1, OATP1B3, OATP2B1) family transporters expression (qPCR) and protein abundance (LC-MS/MS) in healthy donors (n = 9) and metastatic (n = 13) livers. RESULTS: The analysis of mRNA content revealed significant differences in ABCB11, ABCC1, ABCG2, SLC10A1, SLC16A1, SLCO1B1 and SLCO2B1 gene expression between livers from organ donors and patients who underwent surgical resection of metastatic tumors. The protein abundance of NTCP was significantly higher, whereas of P-gp significantly lower in non-tumorous tissues from metastatic livers. Greater inter-individual variability in protein abundance of all studied transporters in subjects with metastatic colon cancer was also observed. CONCLUSIONS: The results suggest that final conclusions in liver pathology studies may depend on the reference liver tissue used, especially in gene expression studies.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Feminino , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Proteômica , Proteínas Carreadoras de Solutos/genética , Doadores de Tecidos
11.
Scand J Clin Lab Invest ; 79(3): 202-207, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30822160

RESUMO

Pain is one of the most interdisciplinary clinical symptoms of a disease. The aim of the study was to evaluate the association of COMT gene polymorphism with pain perception in patients after total hip replacement (THR). The study included 195 patients qualified for surgical treatment (THR) due to osteoarthritis. Patients previously undergoing hip replacement subsequently underwent multimodal pain management therapy, in accordance with the recommendations for treating postoperative pain. The intensity of pain was measured three times at pre-defined time intervals: 1.5, 6 and 12 months after hip replacement, using the visual analogue scale. Single nucleotide polymorphism (SNP) in the COMT gene rs4680: A>G (Val158Met), rs6269: A>G (promoter region), rs4633: C>T (His62His) and rs4818: C>G (Leu136Leu) was genotyped. COMT SNP frequency distribution was analysed. For rs6269 and rs4818, the minor allele was the G allele (38.7 and 38.5%, respectively). It was also observed that rs4633 (T) allele frequency (50%) equalled that of the rs4680 (A) allele (50%). We assessed COMT haplotype frequency in the patients studied. The most frequent haplotype was haplotype M (ATCA) (50%), the rarest haplotype was haplotype R (ATGG), with a frequency of 0.3%. The most frequent diplotype was H/M, which was found in 37.95% of the patients. The frequency of other diplotypes was: M/M-24.10%, H/H-15.90% and L/M-13.33%. The study showed a significant association of rs4818 G allele and equivalent COMT H haplotype with lower sensitivity to pain after hip replacement.


Assuntos
Artroplastia de Quadril/efeitos adversos , Catecol O-Metiltransferase/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Dor/etiologia , Dor/genética , Polimorfismo de Nucleotídeo Único/genética , Idoso , Alelos , Feminino , Frequência do Gene/genética , Haplótipos/genética , Humanos , Modelos Lineares , Masculino , Análise Multivariada , Escala Visual Analógica
12.
Clin Pharmacol Ther ; 105(5): 1204-1212, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30447067

RESUMO

Bioavailability of orally administered drugs is partly determined by function of drug transporters in the liver and intestine. Therefore, we explored adenosine triphosphate-binding cassette (ABC) and solute carriers family transporters expression (quantitative polymerase chain reaction) and protein abundance (liquid chromatography tandem mass spectrometry (LC-MS/MS)) in human liver and duodenum, jejunum, ileum, and colon in paired tissue specimens from nine organ donors. The transporter proteins were detected in the liver (permeability-glycoprotein (P-gp), multidrug resistance protein (MRP)2, MRP3, breast cancer resistance protein (BCRP), organic anion-transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic cation transporter (OCT)1, OCT3, organic anion transporter 2, Na+-taurocholate cotransporting polypeptide, monocarboxylate transporter (MCT)1, and multidrug and toxin extrusion 1) and the intestine (P-gp, multidrug-resistance protein (MRP)2, MRP3, MRP4, BCRP, OATP2B1, OCT1, apical sodium-bile acid transporter (only ileum), MCT1, and peptide transporter (PEPT1)). Significantly higher hepatic gene expression and protein abundance of ABCC2/MRP2, SLC22A1/OCT1, and SLCO2B1/OATP2B1 were found, as compared to all intestinal segments. No correlations between hepatic and small intestinal protein levels were observed. These observations provide a description of drug transporters distribution without the impact of interindividual variability bias and may help in construction of superior physiologically based pharmacokinetic and humanized animal models.


Assuntos
Disponibilidade Biológica , Hepatócitos/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras , Trifosfato de Adenosina/metabolismo , Administração Oral , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Correlação de Dados , Humanos , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/metabolismo , Taxa de Depuração Metabólica/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos , Distribuição Tecidual
13.
Future Med Chem ; 10(20): 2395-2410, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30325216

RESUMO

AIM: The mitotic spindle plays a key role in cell division which makes it an important target in cancer therapy. In the present study the antiproliferative activity of 4-benzyl-5-phenyl-3,4-dihydropyrimidine-2(1H)-thione (1) and its pyridine bioisoster (2) were evaluated and compared with monastrol (MON), the first known cell-permeable small molecule which disrupts bipolar spindle formation by inhibiting Eg5-kinesin activity. RESULTS: Our data revealed that compound 2 showed higher antiproliferative activity than MON against MCF7 and A375 cell lines and comparable reversible cell cycle inhibition in G2/M phase. However, compound 2 produced distinct phenotype from monoastral spindles, and did not affect Eg5 ATPase activity. CONCLUSION: The activity of compound 2 may suggest its new promising anticancer mechanism (different than MON), targeting other component required for spindle bipolarity.


Assuntos
Di-Hidropiridinas/farmacologia , Pirimidinas/farmacologia , Fuso Acromático/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidropiridinas/síntese química , Di-Hidropiridinas/química , Desenho de Fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Cinesina/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Microscopia Confocal , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/metabolismo , Fuso Acromático/metabolismo , Tionas/metabolismo , Tubulina (Proteína)/metabolismo
14.
Pharmacol Rep ; 70(5): 875-880, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30092417

RESUMO

BACKGROUND: Vascular endothelial cells (EC) are constantly exposed to endo- and exogenous compounds, which may disturb EC function. One of the protecting mechanisms against chemicals consists of drug metabolizing enzymes and transporter proteins regulated by nuclear receptors and transcription factors. Therefore, the aim of the current study was to assess the regulation of nuclear receptors and their coordinated genes in Human Umbilical Vein Endothelial Cells (HUVEC). METHODS: HUVEC were exposed to TCDD (10nM), oltipraz (100µM) and simvastatin (1µM) for 24h. Gene expressions were evaluated using quantitative real-time PCR. The protein expression levels were determined by Western blotting. Enzymatic activity of CYP1A1/CYP1B1 was assessed by luciferin-labelled CYPs substrate. RESULTS: Our study confirmed that nuclear receptor AhR and nuclear factor Nrf2 are highly expressed in HUVECs. Treatment of HUVECs with TCDD (AhR inducer) resulted in a significant induction of AHR target genes CYP1A1, CYP1B1 and NQO1. Oltipraz (Nrf2 inducer) also markedly increased expression of NQO1 but did not affect Nrf2 mRNA nor protein levels. Under simvastatin stimulation PXR and NRF2 target transcripts were not altered, however AHR-regulated genes: CYP1A1, CYP1B1 and MDR1 were significantly induced. Western blot analysis confirmed CYP1B1 induction in TCDD-treated HUVECs, but not in the simvastatin group. Moreover, HUVEC exposure to TCDD resulted in induction of CYP1A1/CYP1B1 enzymatic activity. CONCLUSIONS: This study revealed functional expression of AhR and Nrf2 in HUVECs. Moreover, it was defined that simvastatin induced AhR and its related genes.


Assuntos
Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1B1/biossíntese , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Receptores de Hidrocarboneto Arílico/biossíntese , Sinvastatina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Receptor 1 de Sinal de Orientação para Peroxissomos/biossíntese , Dibenzodioxinas Policloradas/farmacologia , Pirazinas/farmacologia , Tionas , Tiofenos
15.
Neurol Neurochir Pol ; 52(4): 477-482, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29776682

RESUMO

Apolipoprotein E (ApoE) is a vital component of several lipoproteins and plays a major role in lipid metabolism. APOE gene comprises of three alleles determined by two single nucleotide polymorphisms (rs429358 and rs7412) resulting in the protein isoforms, among which ApoE4 is a confirmed risk factor for Alzheimer's Disease. However, the impact of APOE genotypes on Parkinson's Disease Dementia (PDD) is still inconclusive. The PDD diagnostic criteria are very inconsistent, and could be complemented with genetic factors. Our study covers a total of 237 patients diagnosed with Parkinson's Disease (PD) according to UK PD Brain Bank criteria, who were classified as subjects with (PDD, n equals 73) and without (nPDD, n equals 164) dementia, using neuropsychological assessment tests. TaqMan real-time PCR assays were used to determine APOE allele. No statistically significant differences in APOE alleles frequencies between nPDD and PDD patients have been observed. The study results revealed that the APOE polymorphism is not associated with cognitive status in PD patients.


Assuntos
Apolipoproteínas E/genética , Doença de Parkinson , Alelos , Cognição , Genótipo , Humanos , Doença de Parkinson/genética
16.
Hum Immunol ; 79(4): 213-217, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29454820

RESUMO

The role of interleukin-23 is crucial in the pathogenesis of psoriasis, and IL23A, IL12B and IL23R genetic variants have been associated with the disease in genome-wide association studies. In the current paper we have conducted a confirmation study of the abovementioned genetic factors in a case-control analysis of 507 psoriatic patients and 396 controls from a Polish population, and subsequently analyzed the impact of genetic variants on response to topical and NB-UVB therapy in a subset of 306 patients. Case-control analysis revealed an association of IL12B rs3212227 and IL23R rs11209026 minor allele carrier status with reduced odds for psoriasis (OR = 0.66, 95%CI: 0.50-0.87, and OR = 0.41, 95%CI: 0.26-0.67, respectively), while HLA-C*06 allele carriers were more frequent in patients group (OR = 4.56, 95%CI: 3.41-6.10). The studied polymorphic variants of IL12B, IL23A, and IL23R genes did not influence therapy outcome, i.e. there were no significant differences in PASI reduction between patients with different genotypes. However, HLA-C*06 carriers showed poorer response to the applied treatment, when compared to non-carriers. The results of the current study confirm an association between IL12B and IL23R genetic polymorphism and psoriasis vulgaris (with a protective effect of minor alleles). HLA-C*06 carriers show reduced effectiveness of topical/NB-UVB therapy, and that observation could be potentially used in treatment personalization.


Assuntos
Antígenos HLA-C/genética , Subunidade p40 da Interleucina-12/genética , Subunidade p19 da Interleucina-23/genética , Psoríase/genética , Receptores de Interleucina/genética , Adulto , Alelos , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Psoríase/terapia , Resultado do Tratamento , Terapia Ultravioleta/métodos
17.
Pain Med ; 19(5): 1010-1014, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29106681

RESUMO

Objective: Lumbar intervertebral disk herniation (LDH) is considered one of the major risk factors for lower back pain, mainly caused by irritation of a spinal nerve or its root. One of the genes related to pain perception is SCN9A, which encodes the voltage gated sodium channel NaV1.7, a key molecule involved in peripheral pain processing. It had been presented before that a common polymorphism within SCN9A (rs6746030: G > A, R1150W) might influence nociception in the general population. Hence, the present study was aimed at investigating the association between SCN9A polymorphism and pain sensitivity. Methods: Pain intensity was measured by means of the visual analog pain scale (VAS) in 176 Caucasian patients with a history of leg and back pain who had been diagnosed with LDH and underwent lumbar discectomy. SCN9A polymorphism was determined by means of TaqMan assay. Results: A significantly higher preoperative back pain intensity was observed among rs6746030 A minor allele carriers, compared with GG homozygotes (VAS = 7.5 ± 2.4 vs 6.5 ± 2.7, P = 0.012). Similarly, A allele carriers were characterized by higher values of leg pain prior to surgery (VAS = 7.8 ± 2.3 vs 6.8 ± 2.6, P = 0.013). However, postoperative improvement in pain reduction was similar in both groups. Conclusions: Our results suggest that the SCN9A rs6746030 polymorphism may be associated with pain intensity in patients suffering from symptomatic disc herniation.


Assuntos
Dor Crônica/genética , Degeneração do Disco Intervertebral/genética , Deslocamento do Disco Intervertebral/genética , Mutação de Sentido Incorreto/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor Crônica/complicações , Feminino , Humanos , Deslocamento do Disco Intervertebral/complicações , Dor Lombar/complicações , Dor Lombar/genética , Vértebras Lombares/cirurgia , Masculino , Pessoa de Meia-Idade , Medição da Dor/métodos , Limiar da Dor/fisiologia , Polimorfismo de Nucleotídeo Único
18.
Clin Pharmacol Ther ; 104(3): 515-524, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29205295

RESUMO

This work revises and complements existing findings on the distribution of drug-metabolizing enzymes in the first-pass effect organs. We explored gene expression (quantitative polymerase chain reaction) and protein abundance (liquid chromatography/ tandem mass spectrometry) of CYP1A2, CYP2B6, CYP2C8/9/19, CYP2D6, CYP2E1, CYP3A4/5, UGT1A1/3, UGT2B7/15 in the liver, duodenum, jejunum, ileum, and colon in paired tissues from nine organ donors. All proteins were detected in the liver, but in the intestine CYP2C9/19, CYP2D6, CYP3A4/5, UGT1A1/3, and UGT2B7 were found. CYP3A4 showed comparable abundance in the liver and jejunum, whereas other enzymes were markedly higher in the hepatic tissue. Nearly all detected enzymes showed their highest abundance in the jejunum. Significant correlations between mRNA and protein levels in liver or intestine were found for most enzymes. CYP3A4 and CYP3A5 protein abundance, but not other enzymes, were significantly correlated in the liver and the small intestine. Our data may contribute to an improved understanding of hepatic and intestinal drug metabolism.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Intestinos/enzimologia , Fígado/enzimologia , Adulto , Biotransformação , Cromatografia Líquida , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Espectrometria de Massas em Tandem , Adulto Jovem
19.
Pharmacol Rep ; 70(5): 875-880, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32002969

RESUMO

BACKGROUND: Vascular endothelial cells (EC) are constantly exposed to endo- and exogenous compounds, which may disturb EC function. One of the protecting mechanisms against chemicals consists of drug metabolizing enzymes and transporter proteins regulated by nuclear receptors and transcription factors. Therefore, the aim of the current study was to assess the regulation of nuclear receptors and their coordinated genes in Human Umbilical Vein Endothelial Cells (HUVEC). METHODS: HUVEC were exposed to TCDD (10 nM), oltipraz (100 µM) and simvastatin (1 µM) for 24 h. Gene expressions were evaluated using quantitative real-time PCR. The protein expression levels were determined by Western blotting. Enzymatic activity of CYP1A1/CYP1B1 was assessed by luciferin-labelled CYPs substrate. RESULTS: Our study confirmed that nuclear receptor AhR and nuclear factor Nrf2 are highly expressed in HUVECs. Treatment of HUVECs with TCDD (AhR inducer) resulted in a significant induction of AHR target genes CYP1A1, CYP1B1 and NQO1. Oltipraz (Nrf2 inducer) also markedly increased expression of NQO1 but did not affect Nrf2 mRNA nor protein levels. Under simvastatin stimulation PXR and NRF2 target transcripts were not altered, however AHR-regulated genes: CYP1A1, CYP1B1 and MDR1 were significantly induced. Western blot analysis confirmed CYP1B1 induction in TCDD-treated HUVECs, but not in the simvastatin group. Moreover, HUVEC exposure to TCDD resulted in induction of CYP1A1/CYP1B1 enzymatic activity. CONCLUSIONS: This study revealed functional expression of AhR and Nrf2 in HUVECs. Moreover, it was defined that simvastatin induced AhR and its related genes.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Sinvastatina/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Membrana Transportadoras/genética
20.
Pharmacogenet Genomics ; 27(10): 372-377, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28777242

RESUMO

OBJECTIVES: Several genetic factors were identified to be responsible for interidividual variability in tacrolimus (TAC) pharmacokinetics, with the predominant role of CYP3A5 and CYP3A4 polymorphisms. In this study, genetic variants of NR1I2 and NR1I3 nuclear receptors (responsible for the regulation of drug-metabolizing enzymes and transporters at the transcriptional level) were evaluated for their potential association with altered TAC concentrations. MATERIALS AND METHODS: Two hundred and forty White kidney transplant patients were genotyped for five single-nucleotide polymorphisms (rs3814055, rs6785049, rs2276707, rs2307424, and rs2307418) in NR1I2 and NR1I3 genes. Genetic data were analyzed in relation to TAC dose-adjusted trough concentration measured 6 months after transplantation (unadjusted and adjusted for patient's CYP3A5 expresser status). RESULTS: There were significant differences in TAC concentrations between patients with different NR1I2 rs3814055:C>T genotypes (mean values: 121.3 ng/ml mg/kg in major CC homozygotes, 169.6 ng/ml mg/kg in CT heterozygotes, and 186.0 ng/ml mg/kg in patients homozygous for the minor T allele) that remained significant after excluding CYP3A5 expressers from analysis. The TAC dose administered to minor T allele carriers (CT or TT genotype) was significantly lower (~22%) compared with CC homozygotes. For all the other loci analyzed, no significant associations were noted. CONCLUSION: Our results support the previous data on the functionality of NR1I2 rs3814055 single-nucleotide polymorphism that points to its association with interindividual differences in activity and inducibility of a broad range of drug-metabolizing enzymes and drug transporters.


Assuntos
Imunossupressores/administração & dosagem , Transplante de Rim , Polimorfismo de Nucleotídeo Único , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Tacrolimo/administração & dosagem , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Pregnano X , Adulto Jovem
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