In the present research, we aimed to screen for non-small cell lung cancer (NSCLC)-related proteins in urinary exosomes by comparing urinary exosomes proteome of normal controls and NSCLC patients. Urinary exosomes were isolated by ultracentrifugation and identified by electron microscopy. Exosomal proteins were separated by 1-D SDS-PAGE and the differentially expressed bands between healthy controls and NSCLC patients ranging in size from 35 to 45 kD were cut from the gel. After tryptic digestion, 18 proteins were identified by nano-HPLC-chip-MS/MS. The differential expression of leucine-rich α-2-glycoprotein (LRG1) was further validated in urinary exosomes by Western blot and in lung tissue by immunohistochemistry. The LRG1 was found to be expressed at higher levels in urinary exosomes and lung tissue of NSCLC patients. These results suggested that LRG1 may be a candidate biomarker for non-invasive diagnosis of NSCLC in urine.
The level of circulating nucleosomes in the serum has a predictive value for sepsis and organ dysfunction and may serve as a candidate biomarker for the diagnosis/prognosis of sepsis. Further studies are warranted to confirm the present findings.
Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.
Hepatopoietin (HPO) is a novel hepatotrophic growth factor that stimulates hepatocyte proliferation by two pathways. In the first, intracellular HPO specifically modulates the activator protein-1 (AP-1) pathway through JAB1 (Jun activation domain-binding protein 1), whereas in the second, extracellular HPO triggers the mitogen-activated protein kinase pathway by binding its specific receptor on the cell surface. In this report we demonstrate that HPO is a flavin-linked sulfhydryl oxidase, and the invariant CXXC (Cys-Xaa-Xaa-Cys) motif in HPO is essential for the enzyme activity of HPO but not for its dimerization nor for its binding ability with JAB1. Two intramolecular disulfides were identified in HPO by mass spectrometry, one of which is formed by the redox CXXC cysteine residues. HPO site-directed mutants (Cys/Ser) at active sites, which lost sulfhydryl oxidase activity, could not increase c-Jun phosphorylation and failed to potentiate JAB1-mediated AP-1 activation. However, the mutants still have mitogenic stimulation and mitogen-activated protein kinase activation effects on HepG2 cells. Thus, it can be concluded that the potentiation role of HPO on AP-1 is dependent on its sulfhydryl oxidase activity. Hepatopoietin (HPO)1 /augmenter of liver regeneration (ALR) is a novel human hepatotrophic growth factor. Since LaBrecque et al.(1) first reported hepatic stimulator substance in 1975, HPO has recently been the subject of intense investigation (2-10). Recombinant HPO can stimulate proliferation of hepatocytes as well as hepatoma cells in vitro, promote liver regeneration and recovery of damaged hepatocytes, and rescue acute hepatic failure in vivo (6, 7). In 1999 we identified the existence of HPO-specific receptor on the surface of these cells (8). Furthermore, we proposed that extracellular HPO stimulates proliferation of hepatocytes and enhances liver regeneration by activating the MAPK signaling pathway under the mediation of HPO receptor (9). Intriguingly, we further found that intracellular HPO can specifically modulate the AP-1 pathway through JAB1 via a MAPK-independent pathway and that HPO enhances the increased phosphorylation level of cJun through JAB1 but has no effect on the expression of transfected c-Jun or endogenous c-Jun N-terminal kinase nor on phosphorylation of c-Jun N-terminal kinase (10).Cytokines and growth factors stimulate AP-1 activity through several pathways (11), whereas the intracrine HPO regulates AP-1 transcriptional activity by an additional mechanism different from other cytokines and growth factors with mitogenic effects. It seems strange that intracellular and extracellular cytokine HPO have dissimilar actions in signal transduction. Recently, it was reported that the ERV1/HPO family belongs to sulfhydryl oxidase (SOX) participating in disulfide bond formation (12-17). The SOX proteins contain a conserved CXXC motif and a non-covalent FAD adjacent to CXXC, which are vital to their catalytic activity (18,19). Sulfhydryl oxidases generally form dimers in vivo and catalyze t...
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