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Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer.

ABSTRACT: Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P<0.05), 31 in grade 2 (P<0.05) and 25 in grade 3 (P<0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.

SUBMITTER: Ahmed N

PROVIDER: S-EPMC2364749 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer.

Ahmed N N Barker G G Oliva K T KT Hoffmann P P Riley C C Reeve S S Smith A I AI Kemp B E BE Quinn M A MA Rice G E GE

British journal of cancer 20040701 1

Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) w ...[more]

PMID: 15199385

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Project description:Ovarian cancer is the most lethal gynecologic disease due to delayed diagnosis, and ascites production is a characteristic of patients in advanced stages. The aim of this study was to perform the proteomic analysis of ascitic fluids of Mexican patients with ovarian carcinoma, in order to detect proteins with a differential expression pattern in the continuing search to identify biomarkers for this disease.Samples were collected from 50 patients from the Instituto Nacional de Cancerología of México under informed consent and with approval of the bioethics and scientific committees. After elimination of abundant proteins (Albumin/IgGs) samples were processed for 2D electrophoresis and further protein identification by Mass Spectrometry (MALDI-TOF). Molecules of interest were followed by western blot and lectin binding assays, and their tissue location by histo-immunofluorescence and confocal analysis.An area with a differential expression pattern among samples was located in the 2D gels. Identified proteins were 6 alpha 1 isoforms and 1 alpha 2 isoform of Haptoglobin, and 2 isoforms of Transthyretin. While Transthyretin isoforms were constitutively expressed in all samples, clear differences in the expression pattern of Haptoglobin alpha isoforms were found. Moreover, increased levels of fucosylation of Haptoglobin alpha isoforms analyzed in 40 samples by Aleuria aurantia lectin binding by 1D overlay assay showed a positive correlation with advanced stages of the disease. Tissue detection of Haptoglobin and its fucosylated form, by histo-immunofluorescence in biopsies of ovarian cancer, also showed a correlation with ovarian cancer progression. Moreover, results show that fucosylated Haptoglobin is produced by tumor cells.Increased numbers of highly fucosylated Haptoglobin alpha isoforms in ascitic fluids and the presence of fucosylated Haptoglobin in tumor tissues of ovarian cancer Mexican patients associated with advanced stages of the disease, reinforce the potential of fucosylated Haptoglobin alpha isoforms to be characterized as biomarkers for disease progression.

Project description:While ovarian cancer remains the most lethal gynecological malignancy in the United States, there are no biomarkers available that are able to predict therapeutic responses to ovarian malignancies. One major hurdle in the identification of useful biomarkers has been the ability to obtain enough ovarian cancer cells from primary tissues diagnosed in the early stages of serous carcinomas, the most deadly subtype of ovarian tumor. In order to detect ovarian cancer in a state of hyperproliferation, we analyzed the implications of molecular signaling cascades in the ovarian cancer cell line OVCAR3 in a temporal manner, using a mass-spectrometry-based proteomics approach. OVCAR3 cells were treated with EGF(1), and the time course of cell progression was monitored based on Akt phosphorylation and growth dynamics. EGF-stimulated Akt phosphorylation was detected at 12 h post-treatment, but an effect on proliferation was not observed until 48 h post-exposure. Growth-stimulated cellular lysates were analyzed for protein profiles between treatment groups and across time points using iTRAQ labeling and mass spectrometry. The protein response to EGF treatment was identified via iTRAQ analysis in EGF-stimulated lysates relative to vehicle-treated specimens across the treatment time course. Validation studies were performed on one of the differentially regulated proteins, lysosomal-associated membrane protein 1 (LAMP-1), in human tissue lysates and ovarian tumor tissue sections. Further, tissue microarray analysis was performed to demarcate LAMP-1 expression across different stages of epithelial ovarian cancers. These data support the use of this approach for the efficient identification of tissue-based markers in tumor development related to specific signaling pathways. LAMP-1 is a promising biomarker for studies of the progression of EGF-stimulated ovarian cancers and might be useful in predicting treatment responses involving tyrosine kinase inhibitors or EGF receptor monoclonal antibodies.

Project description:Haptoglobin (Hp) is a glycoprotein that binds free hemoglobin (Hb) in plasma and plays a critical role in tissue protection and prevention of oxidative damage. Besides, it has some regulatory functions. Haptoglobin is an acute-phase protein, its concentration in plasma changes in pathology, and the test for its concentration is part of normal clinical practice. Haptoglobin is a conservative protein synthesized mainly in the liver and lungs and is the subject of research as a potential biomarker of many diseases, including various forms of malignant neoplasms. Haptoglobin has several unique biophysical characteristics. The human Нр gene is polymorphic, has three structural alleles that control the synthesis of three major phenotypes of haptoglobin: homozygous Нр1-1 and Нр2-2, and heterozygous Нр2-1, determined by a combination of allelic variants that are inherited. Numerous studies indicate that the phenotype of haptoglobin can be used to judge the individual predisposition of a person to various diseases. In addition, Hp undergoes various post-translational modifications (PTMs). These are structural transformations (removal of the signal peptide, cutting off the Pre-Hp precursor molecule into two subunits, α and β, limited proteolysis of α-chains, formation of disulfide bonds, multimerization), as well as chemical modifications of α-chains and glycosylation of the β-chain. Glycosylation of the β-chain of haptoglobin at four Asn sites is the most important variable PTM that regulates the structure and function of the glycoprotein. The study of modified oligosaccharides of the β-chain of Hp has become the main direction in the study of pathological processes, including malignant neoplasms. These characteristics indicate the possibility of the existence of Hp in the form of a multitude of proteoforms, probably performing different functions. This review is devoted to the description of the structural and functional diversity and the potential use of Hp as a biomarker of various pathologies.

Dataset Information

Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer.

Publications

Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer.

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