ABSTRACT
During metastatic growth, cells of solid tumors undergo phenotypical changes related to epithelial-mesenchymal transition. Epithelial-mesenchymal transition is regarded as a potential target for prospective antitumor drugs. Fluorescent reporter systems for evaluation of the expression of markers of epithelial and mesenchymal status (E- and N-cadherins) were created. The described approaches can be used for creation of analogous reporter systems.
Subject(s)
Cadherins/metabolism , Epithelial-Mesenchymal Transition/genetics , Biomarkers, Tumor/genetics , Cadherins/genetics , Cell Line, Tumor , Epithelial-Mesenchymal Transition/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Prospective StudiesABSTRACT
The possibility of interactions between warfarin and dasatinib and their interactions with other drugs metabolized by cytochrome P450 isoform CYP3A4 was demonstrated using a previously created cytochrome P450 substrate-inhibitor panel for preclinical in vitro studies of drug biotransformation on a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). Dasatinib and warfarin are inhibitors of CYP2C19 isoform and hence, can interfere the drugs metabolized by this isoform. Our findings are in line with the data obtained on primary culture of human hepatocytes and suggest that the model can be used in preclinical in vitro studies of drugs.
Subject(s)
Anticoagulants/metabolism , Antineoplastic Agents/metabolism , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Dasatinib/metabolism , Inactivation, Metabolic/drug effects , Models, Biological , Warfarin/metabolism , Anticoagulants/pharmacology , Antineoplastic Agents/pharmacology , Cytochrome P-450 CYP2B6/genetics , Cytochrome P-450 CYP2B6/metabolism , Cytochrome P-450 CYP2C19/genetics , Cytochrome P-450 CYP2C19/metabolism , Cytochrome P-450 CYP2C9/genetics , Cytochrome P-450 CYP2C9/metabolism , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Dasatinib/pharmacology , Drug Evaluation, Preclinical/instrumentation , Drug Evaluation, Preclinical/methods , Drug Interactions , Gene Expression , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Lab-On-A-Chip Devices , Liver/cytology , Liver/drug effects , Liver/metabolism , Substrate Specificity , Warfarin/pharmacologyABSTRACT
We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.
Subject(s)
Cytochrome P-450 CYP2B6/metabolism , Cytochrome P-450 CYP2C19/metabolism , Cytochrome P-450 CYP2C9/metabolism , Cytochrome P-450 CYP3A/metabolism , Lab-On-A-Chip Devices , Bupropion/metabolism , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP2B6/analysis , Cytochrome P-450 CYP2C19/analysis , Cytochrome P-450 CYP2C9/analysis , Cytochrome P-450 CYP3A/analysis , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Humans , Ketoconazole/pharmacology , Liver/drug effects , Liver/enzymology , Mass Spectrometry , Mephenytoin/analogs & derivatives , Mephenytoin/pharmacology , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Omeprazole/metabolism , Phencyclidine/analogs & derivatives , Phencyclidine/pharmacology , Substrate Specificity , Sulfaphenazole/pharmacology , Testosterone/metabolism , Tolbutamide/metabolismABSTRACT
We studied the relationship between microcirculation parameters and functional status of HepaRG cells in spheroids and chose an optimal regimen within the physiologically permissible limits of mechanical impact for the cells that maintains the expression of functional genes of the liver.
Subject(s)
Liver/cytology , Bioreactors , Cell Survival/physiology , Hepatocytes/cytology , Humans , Microcirculation/physiologyABSTRACT
We studied the effects of DMSO and fibroblasts during HepaRG cell spheroid formation and conditions of their subsequent culturing on the levels of mRNA of the major cytochromes P450. A protocol of spheroid formation from differentiated HepaRG cells and their culturing in serum- and DMSO-free medium is developed.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Hepatocytes/enzymology , Spheroids, Cellular/enzymology , Cell Line , Culture Media/chemistry , Cytochrome P-450 Enzyme System/metabolism , Dimethyl Sulfoxide/chemistry , Gene Expression , Gene Expression Regulation, Enzymologic , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
A new cysteine protease (SpCP) with a molecular mass of about 50 kDa and optimal functioning at pH 8.0 was isolated from the culture medium of a Serratia proteamaculans 94 psychrotolerant strain using affinity and gel permeation chromatography. The enzyme N terminal amino acid sequence (SPVEEAEGDGIVLDV-) exhibits a reliable similarity to N terminal sequences of gingipains R, cysteine proteases from Polphyromonas gingivalis. Unlike gingipains R, SpCP displays a double substrate specificity and cleaves bonds formed by carboxylic groups of Arg, hydrophobic amino acid residues (Val, Leu, Ala, Tyr, and Phe), Pro, and Gly. SpCP can also hydrolyze native collagen. The enzyme catalysis is effective in a wide range of temperatures. Kinetic studies of Z-Ala-Phe-Arg-pNA hydrolysis catalyzed by the protease at 4 and 37 degrees C showed that a decrease in temperature by more than 30 degrees C causes a 1.3-fold increase in the kcat/Km ratio. Thus, SpCP is an enzyme adapted to low positive temperatures. A protease displaying such properties was found in microorganisms of the Serratia genus for the first time and may serve as a virulent factor for these bacteria.
Subject(s)
Cysteine Endopeptidases/isolation & purification , Serratia/enzymology , Amino Acid Sequence , Aniline Compounds/chemistry , Cysteine Endopeptidases/chemistry , Hydrogen-Ion Concentration , Hydrolysis , Insulin/chemistry , Melitten/chemistry , Molecular Sequence Data , Serine Proteinase Inhibitors/chemistry , Substrate SpecificityABSTRACT
MicroRNAs (miRNAs) are a class of small regulatory RNAs that control a level of expression of protein encoding genes. Their role in brain pathologies is unknown. We made a first attempt to carry out a genetic study coupled with gene expression analysis of microRNA in human neuropsychiatric pathology. Presumably, at least one third of known miRNA genes are expressed in the brain. Mutations disrupting MECP2 protein lead to abnormal development of the brain and resulting behavior. MiR-130b expressed in the brain and potentially targeting MECP2 is located in the susceptibility locus for schizophrenia (22q11). We performed a comparative analysis of the expression of miR-130b in 24 brain neocortex samples from schizophrenic and normal individuals. The stability and effective detection of mature microRNA in postmortem tissues using Real-time PCR have been shown. Screening for mutations has identified a population polymorphism in the 5 -upstream miR-130b gene region containing DNA elements for putative transcription factors. Genetic association analysis of 300 schizophrenia and 316 normal control individuals revealed no statistically significant association of any of the miR-130b allelic variants with schizophrenia. The data demonstrated feasibility and perspective of convergent genetic and expression analysis of human microRNA genes in testing their role in human diseases.
