Liquid chromatography-tandem mass spectrometry has become the most convenient method to identify and quantify low molecular weight metabolites from various sources. Metabolomics studies of hepatocytes hold promise for the identification of the mechanisms of toxicant-related disease processes. In this chapter, we present a rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantification of intracellular concentrations of nine homocysteine-based metabolites, namely homocysteine, methionine, cysteine, dimethylglycine, cystathionine, S-adenosylmethionine, S-adenosylhomocysteine, choline, and betaine. The method is specifically designed for the analysis of cultured primary hepatocytes.
Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1mM and 2mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine-homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.
- MeSH
- 2D gelová elektroforéza MeSH
- apolipoprotein A-I metabolismus MeSH
- apoptóza MeSH
- betain-homocystein-S-methyltransferasa antagonisté a inhibitory metabolismus MeSH
- fibrinogen metabolismus MeSH
- hepatocyty účinky léků metabolismus MeSH
- homocystein farmakologie MeSH
- hyperhomocysteinemie metabolismus MeSH
- kolorektální nádory farmakoterapie metabolismus patologie MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- metabolomika * MeSH
- multimerizace proteinu MeSH
- nádory jater farmakoterapie metabolismus patologie MeSH
- proliferace buněk MeSH
- proteom analýza metabolismus MeSH
- S-adenosylmethionin metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- western blotting MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Human MDA-MB-231 derived breast cancer cell lines 1833 and 4175 have different metastatic potentials in terms of their tissue tropisms and aggressiveness. Cell line 1833 is specifically metastatic to the bone. The highly aggressive cell line 4175 is specific to the lung. We performed 2-DE analysis of the cell lines. We found 16 significantly changed protein spots, 14 protein spots were identified. Expression of cathepsin D, triosephosphate isomerase, phosphoglycerate kinase 1, heme binding protein 1 and annexin 2 could be correlated with the in vitro aggressiveness of the respective cell lines. Interstitial collagenase and dimethylargininase 2 were exclusive to the cell line 1833 and might contribute to its bone specificity. Serpin B9, cathepsin B chain b, galectin 3 and HSP 27 were changed in the lung specific cell line 4175. The possible contribution of identified proteins to differences in metastatic behavior of the cell lines is discussed.
- MeSH
- 2D gelová elektroforéza metody MeSH
- financování organizované MeSH
- hmotnostní spektrometrie metody MeSH
- invazivní růst nádoru MeSH
- kathepsin D biosyntéza MeSH
- kolagenasy biosyntéza MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- metastázy nádorů MeSH
- nádorové buněčné linie MeSH
- nádory prsu metabolismus patologie MeSH
- peptidy chemie MeSH
- počítačové zpracování obrazu MeSH
- regulace genové exprese u nádorů MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.
- MeSH
- 2D gelová elektroforéza MeSH
- duktální karcinom prsu chemie MeSH
- fenotyp MeSH
- financování organizované MeSH
- kmenové buňky chemie MeSH
- lidé MeSH
- mléčné žlázy lidské cytologie chemie MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny chemie MeSH
- nádory prsu chemie MeSH
- proteom chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
