Obstetrics and Gynaecology - Research Publications
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ItemProteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer(NATURE PUBLISHING GROUP, 2004-07-05)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.
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ItemDownregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR-β1 integrin complex in colon cancer cells(NATURE PUBLISHING GROUP, 2003-07-21)Cancer invasion is regulated by cell surface proteinases and adhesion molecules. Interaction between specific cell surface molecules such as urokinase plasminogen activator receptor (uPAR) and integrins is crucial for tumour invasion and metastasis. In this study, we examined whether uPAR and beta1 integrin form a functional complex to mediate signalling required for tumour invasion. We assessed the expression of uPAR/beta1 integrin complex, Erk signalling pathway, adhesion, uPA and matrix metalloproteinase (MMP) expression, migration/invasion and matrix degradation in a colon cancer cell line in which uPAR expression was modified. Antisense inhibition of the cell surface expression of uPAR by 50% in human colon carcinoma HCT116 cells (A/S) suppressed Erk-MAP kinase activity by two-fold. Urokinase plasminogen activator receptor antisense treatment of HCT116 cells was associated with a 1.3-fold inhibition of adhesion, approximately four-fold suppression of HMW-uPA secretion and inhibition of pro-MMP-9 secretion. At a functional level, uPAR antisense resulted in a four-fold decline in migration/invasion and abatement of plasmin-mediated matrix degradation. In empty vector-transfected cells (mock), uPA strongly elevated basal Erk activation. In contrast, in A/S cells, uPA induction of Erk activation was not observed. Urokinase plasminogen activator receptor associated with beta1 integrin in mock-transfected cells. Disruption of uPAR-beta1 integrin complex in mock-transfected cells with a specific peptide (P25) inhibited uPA-mediated Erk-MAP kinase pathway and inhibited migration/invasion and plasmin-dependent matrix degradation through suppression of pro-MMP-9/MMP-2 expression. This novel paradigm of uPAR-integrin signalling may afford opportunities for alternative therapeutic strategies for the treatment of cancer.
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ItemRelease of proinflammatory cytokines and 8-isoprostane from placenta, adipose tissue, and skeletal muscle from normal pregnant women and women with gestational diabetes mellitus(ENDOCRINE SOC, 2004-11)The aim of this study was to 1) profile the basal release of TNF-alpha, IL-6, IL-8, and 8-isoprostane (a marker of oxidative stress); and 2) investigate the effect of stimulation on the release of cytokines from sc adipose tissue and skeletal muscle from normal pregnant women and women with gestational diabetes mellitus (GDM). Placenta, sc adipose tissue, and skeletal muscle were incubated in the absence (control) or presence of lipopolysaccharide (LPS; 10 microg/ml), TNF-alpha (10 ng/ml), IL-6 (10 ng/ml), or IL-8 (10 ng/ml). After an 18-h incubation, the medium was collected, and the release of TNF-alpha, IL-6, IL-8, and 8-isoprostane was quantified by ELISA. In all three tissues, 8-isoprostane release was greater in women with GDM, and stimulation with LPS increased 8-isoprostane release from adipose and skeletal muscle, but not placenta, obtained from women with GDM. However, in tissues obtained from normal pregnant women, LPS stimulation increased 8-isoprostane release in placenta and had no effect in adipose tissue and skeletal muscle. Their was no difference in the release of TNF-alpha, IL-6, and IL-8 from placenta, adipose tissue, and skeletal muscle obtained from normal pregnant women and women with GDM. Stimulation of placenta, adipose tissue, and skeletal muscle with LPS and TNF-alpha resulted in greater release of IL-6 and IL-8, whereas only LPS increased TNF-alpha release from all three tissues. The data presented in this study demonstrate that there is a differential release of 8-isoprostane from fetal (placenta) and maternal (adipose tissue and skeletal muscle) tissues obtained from normal pregnant women and women with GDM. These data are consistent with the hypothesis that oxidative stress may be involved in the progression and/or pathogenesis of GDM.
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ItemN-acetyl-cysteine inhibits phospholipid metabolism, proinflammatory cytokine release, protease activity, and nuclear factor-κB deoxyribonucleic acid-binding activity in human fetal membranes in vitro(ENDOCRINE SOC, 2003-04)The production of reactive oxygen species (ROS), prostaglandins (PGs), proinflammatory cytokines, and proteases has been implicated in the pathogenesis of term and preterm labor. The nuclear factor-kappaB (NF-kappaB) transcription pathway is activated by ROS and is a key regulator of PGs, proinflammatory cytokine release, and protease activity. N-Acetyl-cysteine (NAC) is an antioxidant that through its ability to scavenger ROS suppresses NF-kappaB DNA-binding activity and resultant gene expression. The aim of this study was to elucidate the effect of NAC on NF-kappaB DNA-binding activity, phospholipid metabolism, cytokine release, and protease activity from human fetal membranes. Human amnion and choriodecidua (n = 9 separate placentas) were treated with 0 (control), 5, 10, or 15 mM NAC in the presence of 10 micro g/ml lipopolysaccharide. After 6-h incubation, the tissues were collected, NF-kappaB DNA binding activity was assessed by gel shift binding assays, and matrix metalloproteinase-9 and urokinase-type plasminogen activator activity were determined by zymography. The incubation medium was collected and assayed for type II phospholipase A(2) tissue content, IL-6, IL-8, TNFalpha, and 8-isoprostane release by ELISA. The release of PGF(2alpha) was measured by RIA. Treatment of fetal membranes with NAC significantly suppressed lipopolysaccharide-stimulated type II phospholipase A(2) release and content; PGF(2alpha), IL-6, IL-8, TNFalpha, and 8-isoprostane release; and matrix metalloproteinase-9 and urokinase-type plasminogen activator enzyme activity and suppressed NF-kappaB DNA-binding activity (by ANOVA, P < 0.05). The data presented in this study demonstrate that NAC inhibits an NF-kappaB-activated pathway and subsequent phospholipid metabolism, proinflammatory cytokine release, and protease activity in human fetal membranes.