ABSTRACT
BluePen Biomarkers provides a unique comprehensive multi-omics biomarker discovery and validation platform. We can quantify, integrate and analyze genomics, proteomics, metabolomics and lipidomics biomarkers, alongside clinical data, demographics and other phenotypic data. A unique bio-inspired signal processing analytic approach is used that has the proven ability to identify biomarkers in a wide variety of diseases. The resulting biomarkers can be used for diagnosis, prognosis, mechanistic studies and predicting treatment response, in contexts from core research through clinical trials. BluePen Biomarkers provides an additional groundbreaking research goal: identifying surrogate biomarkers from different modalities. This not only provides new biological insights, but enables least invasive, least-cost tests that meet or exceed the predictive quality of current tests.
ABSTRACT
Cyclooxygenases (COX) metabolize arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETE), which can then be oxidized by dehydrogenases, such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH), to oxo-eicosatetraenoic acids (ETE). We have previously established that 11-oxo-eicosatetraenoic acid (oxo-ETE) and 15-oxo-ETE are COX-2/15-PGDH-derived metabolites. Stable isotope dilution (SID) chiral liquid chromatography coupled with electron capture atmospheric pressure chemical ionization (ECAPCI) single reaction monitoring (SRM) MS has been used to quantify uptake of 11-oxo-ETE and 15-oxo-ETE in both LoVo cells and human umbilical vein endothelial cells (HUVEC). Intracellular 11-oxo- and 15-oxo-ETE concentrations reached maximum levels within 1 h and declined rapidly, with significant quantitative differences in uptake between the LoVo cells and the HUVECs. Maximal intracellular concentrations of 11-oxo-ETE were 0.02 ng/4 × 105 cells in the LoVo cells and 0.58 ng/4 × 105 cells in the HUVECs. Conversely, maximal levels of 15-oxo-ETE were 0.21 ng/4 × 105 in the LoVo cells and 0.01 ng/4 × 105 in the HUVECs. The methyl esters of both 11-oxo- and 15-oxo-ETE increased the intracellular concentrations of the corresponding free oxo-ETEs by 3- to 8-fold. 11-oxo-ETE, 15-oxo-ETE, and their methyl esters inhibited proliferation in both HUVECs and LoVo cells at concentrations of 2-10 µM, with 11-oxo-ETE methyl ester being the most potent inhibitor. Cotreatment with probenecid, an inhibitor of multiple drug resistance transporters (MRP)1 and 4, increased the antiproliferative effect of 11-oxo-ETE methyl ester in LoVo cells and increased the intracellular concentration of 11-oxo-ETE from 0.05 ng/4 × 105 cells to 0.18 ng/4 × 105 cells. Therefore, this study has established that the COX-2/15-PGDH-derived eicosanoids 11-oxo- and 15-oxo-ETE enter target cells, that they inhibit cellular proliferation, and that their inhibitory effects are modulated by MRP exporters.
Subject(s)
Arachidonic Acids/metabolism , Arachidonic Acids/pharmacology , Biological Transport , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Multidrug Resistance-Associated Proteins/metabolismABSTRACT
Intracellular aggregates of alpha-syn (alpha-synuclein) represent pathoanatomical hallmarks of neurodegenerative disorders (synucleinopathies). The molecular mechanisms underlying alpha-syn aggregation into filamentous inclusions may involve oxidation and nitration of the protein. Whereas the effects of oxidants and nitrating species on soluble alpha-syn have been studied in detail, the effect of these reactive species on alpha-syn associated with lipids is still unknown. In the present paper, we report that alpha-syn bound to small unilamellar liposomes composed of phosphatidylcholine/phosphatidic acid is resistant to oxidation and nitration when compared with soluble alpha-syn. Additionally, increasing concentrations of unsaturated fatty acids diminished the oxidation and nitration of alpha-syn upon exposure to fluxes of peroxynitrite (8-20 microM x min(-1)). To investigate the effect of oxidized lipids on alpha-syn, the protein was incubated with the bifunctional electrophile 4-HNE [4-hydroxy-2(E)-nonenal]. MS analysis showed the formation of three major products corresponding to the native protein and alpha-syn plus one or two 4-HNE molecules. Trypsin digestion of the modified protein followed by peptide 'finger-printing' revealed that 4-HNE modified the peptide E46GVVHGVATVAEK58. Further analysis of the peptides with liquid chromatography-tandem MS identified the modified residue as His50. The data indicate that the association of alpha-syn with biological membranes protects the protein from oxidation and nitration and thus diminishes the formation of protein molecules capable of forming aggregates. However, products of lipid peroxidation can also modify alpha-syn, generating novel protein adducts that could serve as biomarkers for documenting oxidative processes in human as well as animal and cellular models of alpha-syn aggregation and pathology.