Drug binding to plasma proteins influences processes such as liberation, adsorption, disposition, metabolism, and elimination of drugs, which are thus one of the key steps of a new drug development. As a result, the characterization of drug-protein interactions is an essential part of these time- and money-consuming processes. It is important to determine not only the binding strength and the stoichiometry of interaction, but also the binding site of a drug on a protein molecule, because two drugs with the same binding site can mutually affect free drug concentration. Capillary electrophoresis-frontal analysis with mobility shift affinity capillary electrophoresis is one of the most used affinity capillary electrophoresis methods for the characterization of these interactions. In this study, a well-known sensitivity problem of most capillary electrophoresis-frontal analyses using ultraviolet detection is solved by its combination with contactless conductivity detection, which provided sixfold lower limits of quantitation and detection. Binding parameters of the human serum albumin-salicylic acid model affinity pair were evaluated by this newly developed approach and by the classical approach with ultraviolet detection primarily used for their mutual comparison. The results of both approaches agreed well and are also in agreement with literature data obtained using different techniques.

• MeSH
• elektrická vodivost MeSH
• elektroforéza kapilární metody   MeSH
• krevní proteiny * MeSH
• lidé MeSH
• lidský sérový albumin * MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

• MeSH
• akutní myeloidní leukemie * MeSH
• elektroforéza kapilární metody   MeSH
• ibuprofen MeSH
• izotachoforéza * MeSH
• lidé MeSH
• lidský sérový albumin metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• tryptofan MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

CE/frontal analysis (CE/FA) is probably one of the most frequently used modes of CE for studying affinity interactions. It is typically performed with classic UV-Vis detection that suffers from low concentration sensitivity. To overcome this limitation, the applicability of CE/FA in combination with ESI-MS detection for the investigation of drug-HSA interactions was demonstrated. The developed new method combines the advantages of CE/FA, such as low sample consumption and no labeling or immobilization of interacting partners, with the benefits of MS detection, such as higher selectivity and sensitivity; moreover, it can be used for molecules lacking a fluorophore or chromophore. The binding parameters of tolbutamide (TL) and glimepiride (GLP), first- and second-generation antidiabetics that differ strongly in their solubility in aqueous solutions, were investigated by this CE/FA-MS method. This method, in contrast to the CE/FA method with the most commonly used UV-Vis detection, is more sensitive; an almost three times lower LOD was reached. The binding parameters of TL and GLP were investigated by this CE/FA-MS method and compared with the literature data. The binding constant value of TL obtained by UV-Vis detection was lower than the value obtained by the method hyphenated with MS detection, which is probably given by the influence of the ESI parameters on the stability of drug-HSA complex. In addition, the ratio of TL and HSA concentrations was divergent in both of the experimental approaches. Finally, it can be concluded that both detection methods have their strengths and weaknesses.

• MeSH
• elektroforéza kapilární * metody   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• hmotnostní spektrometrie metody   MeSH
• krevní proteiny * metabolismus   MeSH
• voda MeSH
• Publikační typ
• časopisecké články MeSH

MicroRNAs (miRNAs) have been proven to be important oncogenes and tumor suppressors in wide range of cancers, including renal cell carcinoma (RCC). In our study, we evaluated miRNA-429 as potential diagnostic/prognostic biomarker in 172 clear cell RCC patients and as a potential regulator of epithelial-mesenchymal transition (EMT) in vitro. We demonstrated that miR-429 is down-regulated in tumor tissue samples (P < 0.0001) and is significantly associated with cancer metastasis (P < 0.0001), shorter disease-free (P = 0.0105), and overall survival (P = 0.0020). In addition, ectopic expression of miR-429 in 786-0 RCC cells followed by TGF-β treatment led to increase in the levels of E-cadherin expression (P < 0.0001) and suppression of cellular migration (P < 0.0001) in comparison to TGF-β-treated controls. Taken together, our findings suggest that miR-429 may serve as promising diagnostic and prognostic biomarker in RCC patients. We further suggest that miR-429 has a capacity to inhibit loss of E-cadherin in RCC cells undergoing EMT and consequently attenuate their motility.

• MeSH
• dospělí MeSH
• down regulace MeSH
• epitelo-mezenchymální tranzice * MeSH
• invazivní růst nádoru MeSH
• kadheriny genetika   MeSH
• karcinom z renálních buněk genetika   sekundární   terapie   MeSH
• kombinovaná terapie MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• lokální recidiva nádoru genetika   patologie   terapie   MeSH
• lymfatické metastázy MeSH
• messenger RNA genetika   MeSH
• mikro RNA genetika   MeSH
• míra přežití MeSH
• nádorové biomarkery MeSH
• nádorové buňky kultivované MeSH
• nádory ledvin genetika   patologie   terapie   MeSH
• následné studie MeSH
• pohyb buněk * MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• prognóza MeSH
• proliferace buněk MeSH
• regulace genové exprese u nádorů * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• studie případů a kontrol MeSH
• stupeň nádoru MeSH
• transformující růstový faktor beta genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT) is a crucial process involved in initiation of metastatic cascade. The aim of our study was to identify an integrated miRNA/mRNA signature associated with metastasis and prognosis in ccRCC through targeted approach based on analysis of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our study and further divided into discovery, training and validation cohorts. EMT markers were evaluated in ccRCC tumor samples, which were grouped accordingly to EMT status. By use of large-scale miRNA/mRNA expression profiling, we identified miRNA/mRNA with significantly different expression in EMT-positive tumors and selected 41 miRNAs/mRNAs for training phase of the study to evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were analyzed in the validation phase, where all evaluated miRNA/mRNA candidates were confirmed to be significantly deregulated in tumor tissue. Some of them significantly differed in metastatic tumors, correlated with clinical stage, with Fuhrman grade and with overall survival. Further, we established an EMT-based stage-independent prognostic scoring system enabling identification of ccRCC patients at high-risk of cancer-related death. Finally, we confirmed involvement of miR-429 in EMT regulation in RCC cells in vitro.

• MeSH
• analýza přežití MeSH
• dospělí MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• karcinom z renálních buněk genetika   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• metastázy nádorů MeSH
• mikro RNA genetika   MeSH
• nádory ledvin genetika   patologie   MeSH
• prognóza MeSH
• regulace genové exprese u nádorů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3% of adult malignancies. P-Element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a new class of naturally occurring, short non-coding RNAs involved in silencing of transposable elements and in sequence-specific chromatin modifications. There were preliminary data published indicating that piR-823 expression is deregulated in circulating tumor cells and tumor tissue in gastric and kidney cancer, respectively. PATIENTS AND METHODS: In our study, we analyzed piR-823 levels in 588 biological specimens: tumor tissue (N=153), adjacent renal parenchyma (N=121), blood serum (N=178) and urine (N=20) of patients undergoing nephrectomy for RCC; and in blood serum (N=101) and urine (N=15) of matched healthy controls. Expression levels of piR-823 were determined in all biological specimens by quantitative real-time polymerase chain reaction, compared in patients and controls, and correlated with clinicopathological features of RCC. RESULTS: We identified a significant down-regulation of piR-823 in tumor tissue [p<0.0001, area under the curve (AUC)=0.7945]. On the contrary in blood serum and urine, the expression of piR-823 was significantly higher in patients with RCC compared to healthy individuals (p=0.0005, AUC=0.6264 and p=0.0157, AUC=0.7433, respectively). We further observed higher levels of piR-823 in tumor tissue to be associated with shorter disease-free survival of patients (p=0.0186) and a trend for higher piR-823 levels in serum to be associated with advanced clinical stages of RCC (p=0.0691). There were no other significant associations of piR-823 levels in any type of biological specimen with clinicopathological features of RCC. CONCLUSION: piR-823 is down-regulated in tumor tissue, but positively correlated with worse outcome, indicating its complex role in RCC pathogenesis. In blood serum, piR-823 is up-regulated, but with unsatisfactory analytical performance. Preliminary data indicate the promising diagnostic utility of urinary piR-823 in patients with RCC.

• MeSH
• dospělí MeSH
• karcinom z renálních buněk krev   genetika   moč   MeSH
• lidé středního věku MeSH
• lidé MeSH
• malá interferující RNA krev   metabolismus   moč   MeSH
• mladý dospělý MeSH
• nádorové biomarkery krev   genetika   moč   MeSH
• nádory ledvin krev   genetika   moč   MeSH
• plocha pod křivkou MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH

The EMT status reflecting miRNAs identified by expression profiling analysis using Real-Time PCR miRNA Arrays or their target molecules on protein level will enable to identify RCC patients with increased risk of metastatic disease, and by that will improve the treatment strategy by individualizing RCC patients therapy.

MiRNA, které reflektují statut EMT, získané pomocí analýzy profilů exprese miRNA pomocí metody Real-Time PCR miRNA Arrays, případně jejich cílové molekuly na proteinové úrovni, umožní u pacientů s renálním karcinomem identifikovat jedince s vysokým rizikem metastatického onemocnění, a tak přispějí k individualizaci jejich léčby.

• MeSH
• časná detekce nádoru MeSH
• epitelo-mezenchymální tranzice MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• metastázy nádorů diagnóza   MeSH
• mikro RNA analýza   MeSH
• nádorové biomarkery MeSH
• nádory ledvin diagnóza   MeSH
• prediktivní hodnota testů MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• molekulární biologie, molekulární medicína
• nefrologie
• onkologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH