Eun-Wie Cho

α-Melanocyte-stimulating Hormone Inhibits Lipopolysaccharide-induced Tumor Necrosis Factor-α Production in Leukocytes by Modulating Protein Kinase A, p38 Kinase, and Nuclear Factor κB Signaling Pathways

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) in leukocytes via stimulation of α-MSH cell surface receptors. However, the signaling mechanism of α-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which α-MSH inhibits lipopolysaccharide (LPS)-induced TNF-α production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor κB (NFκB) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NFκB to induce TNF-α production. We found that pretreatment of cells with α-MSH blocked LPS-induced p38 kinase and NFκB activation as well as TNF-α production. This response was proportional to α-MSH receptor expression levels, and addition of an α-MSH receptor antagonist abolished the inhibitory effects. In addition, α-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of α-MSH on p38 kinase activation, NFκB activation, and TNF-α production. Taken together, our results indicate that stimulation of PKA by α-MSH causes inhibition of LPS-induced activation of p38 kinase and NFκB to block TNF-α production.

Ganglioside GM3 is involved in neuronal cell death

Gangliosides abundant in the nervous system have been implicated in a broad range of biological functions, including the regulation of cell proliferation and death. Glutamate‐induced cell death, which is accompanied by an accumulation of reactive oxygen species (ROS), is a major contributor to pathological cell death within the nervous system. However, the mechanism underlying this neuronal cell death has not been fully elucidated. In this study, we report that ganglioside GM3 is involved in neuronal cell death. GM3 was up‐regulated in the mouse hippocampal cell line HT22 death caused by glutamate. Increment in GM3 levels by both the exogenous addition of GM3 and the overexpression of the GM3 synthase gene induced neuronal cell death. Overexpression of GM3 synthase by microinjecting mRNA into zebrafish embryos resulted in neuronal cell death in the central nervous system (CNS). Conversely, RNA interferencemediated silencing of GM3 synthase rescued glutamateinduced neuronal death, as evidenced by the inhibition of massive ROS production and intracellular calcium ion influx. 12‐lipoxygenase (12‐lipoxygenase) (12‐LOX) was recruited to glycosphingolipid‐enriched microdomains (GEM) in a GM3‐dependent manner during oxidative glutamate toxicity. Our findings suggest that GM3 acts as not only a mediator of oxidative HT22 death by glutamate but also a modulator of in vivo neuronal cell death.—Sohn, H., Kim, Y.‐S., Kim, H.‐T., Kim, C.‐H., Cho, E.‐W., Kang, H.‐y., Kim, N.‐S., Kim, C.‐H., Ryu, S. E., Lee, J.‐H., Ko, J. H. Ganglioside GM3 is involved in neuronal cell death. FASEB J. 20, E525–E535 (2006)

Tumor-associated autoantibodies as diagnostic and prognostic biomarkers

In the process of tumorigenesis, normal cells are remodeled to cancer cells and protein expression patterns are changed to those of tumor cells. A newly formed tumor microenvironment elicits the immune system and, as a result, a humoral immune response takes place. Although the tumor antigens are undetectable in sera at the early stage of tumorigenesis, the nature of an antibody amplification response to antigens makes tumor-associated autoantibodies as promising early biomarkers in cancer diagnosis. Moreover, the recent development of proteomic techniques that make neo-epitopes of tumor-associated autoantigens discovered concomitantly has opened a new area of ‘immuno-proteomics’, which presents tumor-associated autoantibody signatures and confers information to redefine the process of tumorigenesis. In this article, the strategies recently used to identify and validate serum autoantibodies are outlined and tumor-associated antigens suggested until now as diagnostic/prognostic biomarkers in various tumor types are reviewed. Also, the meaning of autoantibody signatures and their clinical utility in personalized medicine are discussed. [BMB Reports 2012; 45(12): 677-685]

Determination of the Protective Effects of Neutralizing Anti-Hepatitis B Virus(HBV) Immunoglobulins by Epitope Mapping with Recombinant HBV Surface-Antigen Proteins

Anti‐hepatitis B virus (HBV) surface‐antigen immunoglobulins prepared from human sera are clinical reagents which have been approved for prophylactic treatment in HBV‐exposed persons. The passive immunoprophylaxis with immunoglobulins is meant to cross‐link viral particles, which are then further cleared by the hosts own immune system. While antibodies specific for both anti‐S‐ and anti‐preS proteins have been proved to serve as effective anti‐viral agents, so far the fine antigen specificity of clinical immunoglobulin preparations has not been determined. Using recombinant proteins covering the hepatitis B surface antigen, in the present study, the specificity of a commercially available immunoglobulin preparation was determined and immunodominant epitopes were mapped. Here, it is shown that the major reactivity of anti‐HBV immunoglobulins is directed against the S‐protein, and that no reactivity to the preS2 but a weak binding activity to the preS1 region was detectable. The antigen reactivity within the preS1 region was biased to the C‐terminal region, which indicates the presence of a putative B‐cell epitope. The evaluation of the antigen specificity and determination of novel protective epitopes will provide valuable information for the further development and improvement of prophylactic HBV immunoglobulins.

Detection of surface asialoglycoprotein receptor expression in hepatic and extra-hepatic cells using a novel monoclonal antibody

The asialoglycoprotein receptor (ASGPR) is a heterodimeric membrane protein which is involved in the internalization of desialylated glycoproteins and also in the binding and uptake of various pathogenic viruses. To facilitate the analysis of ASGPR expression, we generated a monoclonal antibody, termed ASSA-1, that is specific to the ASGPR H1 subunit based on ELISA and Western blots analysis. ASSA-1 also reacted to surface-displayed ASGPR in live cells thus enabling analysis of ASGPR expression by immunofluorescence flow cytometry, which we used to analyze established human liver cell lines previously confirmed to be positive for ASGPR mRNA expression. In agreement with previous reports, surface ASGPR was also detected in extra-hepatic cells and, surprisingly, even in human T cell lines, which was then further confirmed in activated, but not in resting, primary human peripheral blood lymphocytes. These observations suggest that ASGPR has a broad pattern of expression that even extends into cells from the immune system, which biological meanings still have to be analyzed. We expect that monoclonal antibody ASSA-1 will serve as a new powerful tool in analyzing the biological role of ASGPR in hepatic and extra-hepatic cells.

Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display.

Aberrant glycosylation of human tissue inhibitor of metalloproteinase-1 (TIMP-1) by N-acetylglucosaminyltransferase-V (GnT-V) was previously reported to be related to cancer progression. Here, we report on the antibodies recognizing the structural features initiated by an addition of N-linked β(1,6)-N-acetylglucosamine (GlcNAc) branch by GnT-V on TIMP-1. Two glycoforms of TIMP-1, TIMP1-L produced in Lec4 cells without GnT-V activity and TIMP1-B in GnT-V overexpressing transfectant cells, were purified from culture supernatant and used for generation of antibodies. TIMP1-L was injected in the left hind footpad of mice as decoy and TIMP1-B in the right hind footpad as immunogen. Phage-displayed scFv library was constructed from the B cells retrieved from the right popliteal lymph nodes and subjected to panning and screening. Phage ELISA of individual clones revealed the scFv clones with preferential binding activity to TIMP1-B, and they were converted into an scFv-Fc format for further characterization of binding specificity. Glycan binding assay of an antibody, 1-9F, revealed its differential specificity toward an extension of glycan structure initiated with β(1,6)-GlcNAc linkage and terminally decorated with a sialic acid. This study demonstrates feasibility of a new strategy combining decoy immunization with phage display for the efficient generation of antibodies tracking down structural features of different glycoforms.

Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer

A novel circulating tumor-associated autoantibody, K94, obtained from a hepatocellular carcinoma (HCC) mouse model was characterized. The target antigen of K94 autoanti-body was expressed in various tumor cell lines including liver cancer, and its secretion was detectable using MCF-7 breast carcinoma cells. Proteomic analysis revealed that the protein bands reactive to K94 included cytokeratin (CK) 8 and 18, which are known to be related to tumorigenesis and form a heterotypic complex with each other. However, K94 showed no activity toward CK8 or CK18 separately. The epitope of the K94 antibody was only presented by a complex between CK8 and CK18, which was confirmed by analysis using recombinant CK8 and CK18 proteins. To formulate an assay for anti-CK8/18 complex autoantibody, a mimotope peptide reactive to K94 was selected from loop-constrained heptapeptide (-CX7C-) display phage library, of which sequence was CISPDAHSC (K94p1). A mimotope enzyme-linked immunosorbent assay (ELISA) using phage-displayed K94p1 peptide as a coating antigen was able to discriminate breast cancer (n=30) patients from normal subjects (n=30) with a sensitivity of 50% and a specificity of 82.61%. CA15.3 was detected at very low levels in the same breast cancer subjects and did not discriminate breast cancer patients from normal subjects, although it is a conventional biomarker of breast cancer. These results suggest that a mimotope ELISA composed of K94p1 peptide may be useful for the diagnosis of breast cancer.

Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines

Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.

In Vitro Binding Analysis of Hepatitis B Virus preS-derived Putative Helper T-cell Epitopes to MHC Class II Molecules Using Stable HLA-DRB1*0405/-DRA*0101 Transfected Cells

In designing epitope‐based vaccines, the inclusion of a helper T‐lymphocyte (HTL) epitope is necessary to elicit both humoral and cellular immune responses. Whereas the preS region of the hepatitis B virus (HBV) surface antigen is well‐known to raise protective immunity, the epitopes for activating HTLs are poorly characterized. In an attempt to identify such epitopes, the HBV‐preS region was screened for peptide sequences with HLA‐DR4 binding motifs, and putative HTL candidate peptides were synthesized in a biotinylated form. Using L929 mouse fibroblasts stably transfected with HLA‐DRB1*0405 and HLA‐DRA*0101 cDNA, specific binding of the peptides was then detected using fluorescence‐conjugated streptavidin. The cell‐surface expression of HLA‐DR molecules on transfectants was confirmed by confocal microscopy, and quantitative analysis of candidate peptide binding was performed by fluorescence activated cell sorting. Among eight preS‐derived peptides, three candidate peptides?namely preS1(23‐33), preS1(62‐72), and preS1(76‐86)?showed good binding characteristics to HLA‐DR4 molecules, among which the preS1(23‐33) epitope was regarded as the most promising HTL epitope. Further studies with these candidate HTL stimulatory peptides will show their ability to activate the human immune system against HBV.

Optimization of phage-immobilized ELISA for autoantibody profiling in human sera

Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.