Effect of supracervical apposition and spontaneous labour on apoptosis and matrix metalloproteinases in human fetal membranes.

BACKGROUND: Apoptosis and matrix metalloproteinase (MMP-9) are capable of hydrolysing components of the extracellular matrix and weakening the fetal membranes which leads to eventual rupture, a key process of human parturition. The aim of this study was to determine the effect of supracervical apposition and spontaneous labour on apoptosis and MMP-9 in human fetal membranes at term. METHODS: Fetal membranes were obtained from term non-labouring supracervical site (SCS) and compared to (i) a paired distal site (DS) or (ii) site of rupture (SOR) after spontaneous labour onset. RESULTS: The expression of the proapoptotic markers Bax, Smac, Fas, FasL, caspase-3, and PARP, was significantly higher in the non-labouring SCS chorion compared to paired DS. Bax, Smac, FasL, caspase-3, and PARP staining was higher in the non-labouring SCS fetal membranes than that in the post-labour SOR. MMP-9 expression and activity were higher in the post-labour SOR fetal membranes compared to non-labouring SCS fetal membranes. CONCLUSION: Components of the apoptotic signalling pathways and MMP-9 may play a role in rupture and labour. Non-labouring SCS fetal membranes display altered morphology and altered apoptotic biochemical characteristics in preparation for labour, while the laboured SOR displays unique MMP characteristics.

Apelin is decreased with human preterm and term labor and regulates prolabor mediators in human primary amnion cells.

A critical role of proinflammatory mediators including cytokines, prostaglandins, and extracellular matrix remodeling enzymes in the processes of human labor and delivery, at term and preterm, has been demonstrated. In nongestational tissues, apelin plays an important role in a number of physiologic processes, including the regulation of inflammation. However, the role and regulation of apelin and the apelin receptor (APJ) in human gestational tissues are not known. The aims of this study were to determine the effect of (i) preterm and term labor on apelin and APJ expression in human gestational tissues and (ii) apelin small interfering RNA (siRNA) knockdown in human primary amnion cells on prolabor mediators. Human placenta and fetal membranes were collected from term nonlaboring women and women after spontaneous labor and delivery. Preterm and term spontaneous labor were associated with significantly lower apelin expression in fetal membranes. On the other hand, there was no effect of labor on APJ expression and no effect of term labor on placental apelin or APJ expression. Transfection of primary amnion cells with apelin siRNA was associated with significantly increased interleukin (IL)-1ß-induced IL-6 and IL-8 release and cyclooxygenase-2 messenger RNA (mRNA) expression and resultant prostaglandin E2 and prostaglandin F2α release. There was no effect of apelin siRNA on matrix metalloproteinase (MMP)-9 mRNA expression and pro MMP-9 release. In summary, human labor downregulates apelin expression in human fetal membranes. Furthermore, a role of apelin in the regulation of proinflammatory and prolabor mediators in human fetal membranes is supported by our studies.

Investigation of human cationic antimicrobial protein-18 (hCAP-18), lactoferrin and CD163 as potential biomarkers for ovarian cancer.

BACKGROUND: Epithelial ovarian cancer is one of the leading causes of gynaecological cancer morbidity and mortality in women. Early stage ovarian cancer is usually asymptomatic, therefore, is often first diagnosed when it is widely disseminated. Currently available diagnostics lack the requisite sensitivity and specificity to be implemented as community-based screening tests. The identification of additional biomarkers may improve the diagnostic efficiency of multivariate index assays. The aims of this study were to characterise and compare the ovarian tissue immunohistochemical localisation and plasma concentrations of three putative ovarian cancer biomarkers: human cationic antimicrobial protein-18 (hCAP-18); lactoferrin; and CD163 in normal healthy women and women with ovarian cancer. METHODS: In this case-control cohort study, ovarian tissue and blood samples were obtained from 164 women (73 controls, including 28 women with benign pelvic masses; 91 cancer, including 21 women with borderline tumours). Localisation of each antigen within the ovary was assessed by immunohistochemistry and serum concentrations determined by ELISA assays. RESULTS: Immunoreactive (ir) hCAP-18 and lactoferrin were identified in epithelial cells, while CD163 was predominately localised in stromal cells. Tissue ir CD163 increased significantly (P<0.05) with disease grade. Median plasma concentrations of soluble (s)CD163 were significantly greater in the cases (3220 ng/ml) than in controls (2488 ng/ml) (P< 0.01). Median plasma concentrations of hCAP-18 and lactoferrin were not significantly different between cases and controls. The classification efficiency of each biomarker (as determined by the area under the receiver operator characteristic curve; AUC) was: 0.67± 0.04; 0.62 ± 0.08 and 0.51 ± 0.07 for sCD163, hCAP-18 and lactoferrin, respectively. When the 3 biomarkers were modelled using stochastic gradient boosted logistic regression, the AUC increased to 0.95 ± 0.03. CONCLUSIONS: The data obtained in this study establishes the localisation and concentrations of CD163, hCAP-18, and lactoferrin in ovarian tumours and peripheral blood. Individually, the 3 biomarkers display only modest diagnostic efficiency as assessed by AUC. When combined in a multivariate index assay, however, diagnostic efficiency increases significantly. As such, the utility of the biomarker panel, as an aid in the diagnosis of cancer in symptomatic women, is worthy of further investigation in a larger phase 2 biomarker trial.

The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry.

Our aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We used two-dimensional difference gel electrophoresis (2D-DIGE), utilising narrow-range immobilised pH gradient strips that encompassed the broad pH range of 4-5 and 5-6, followed by MALDI-TOF mass spectrometry of selected protein spots. Western blot and quantitative RT-PCR (qRT-PCR) analyses were performed to validate representative findings from the 2D-DIGE analysis. Eight proteins were altered (six down-regulated and two up-regulated on obese placentas). Annexin A5 (ANXA5), ATP synthase subunit beta, mitochondria (ATPB), brain acid soluble protein 1 (BASP1), ferritin light chain (FTL), heterogeneous nuclear ribonucleoprotein C (HNRPC) and vimentin (VIME) were all lower in obese patients. Alpha-1-antitrypsin (A1AT) and stress-70 protein, mitochondrial (GRP75) were higher in obese patients. Western blot analysis of ANXA5, ATPB, FTL, VIME, A1AT and GRP75 confirmed the findings from the 2D-DIGE analysis. For brain acid soluble protein 1 and HNRPC, qRT-PCR analysis also confirmed the findings from the 2D-DIGE analysis. Immunohistochemical analysis was also used to determine the localisation of the proteins in human placenta. In conclusion, proteomic analysis of placenta reveals differential expression of several proteins in patients with pre-existing obesity. These proteins are implicated in a variety of cellular functions such as regulation of growth, cytoskeletal structure, oxidative stress, inflammation, coagulation and apoptosis. These disturbances may have significant implications for fetal growth and development.

SIRT1 is a novel regulator of key pathways of human labor.

Human sirtuin (SIRT) 1 and SIRT2, which possess nicotinamide adenosine dinucleotide (NAD(+))-dependent deacetylase activity, exhibit anti-inflammatory actions. However, there are no data available on SIRT1 and SIRT2 expression and regulation in human intrauterine tissues. Thus, the aim of this study was to characterize the localization and expression of SIRT1 and SIRT2 in 1) placenta and fetal membranes before and after term spontaneous labor onset, 2) prelabor fetal membranes from the supracervical site (SCS) and a distal site (DS), and 3) in response to proinflammatory stimuli. Further, the effect of SIRT activation using resveratrol and SRT1720 on prolabor mediators was also assessed. SIRT1 and SIRT2 were localized in the syncytiotrophoblast layer and the cytotrophoblasts of the placenta, amnion epithelium, trophoblast layer of the chorion, and decidual cells. Additionally, SIRT2 was found within the endothelial walls of placental vessels. SIRT2, but not SIRT1, staining was significantly lower in amnion and chorion obtained from the SCS compared to a DS. On the other hand, SIRT1, but not SIRT2, gene and/or protein expression was significantly lower in placenta, amnion, and chorion obtained after labor compared to prelabor. SIRT1 expression, but not SIRT2, was down-regulated by lipopolysaccharide (LPS) and proinflammatory cytokines TNF and IL1B. The SIRT1 activators resveratrol and SRT1720 significantly decreased LPS-induced TNF, IL6, and IL8 gene expression and release and PTGS2 mRNA expression and resultant prostaglandin (PG) E(2) and PGF(2α) release from human gestational tissues. In conclusion, SIRT1 possesses anti-inflammatory actions and thus may play a role in regulating pregnancy and parturition.

Neuronal transcription factor Brn-3a(l) is over expressed in high-grade ovarian carcinomas and tumor cells from ascites of patients with advanced-stage ovarian cancer.

OBJECTIVES: In view of the recent association of Brn-3 transcription factors with neuroblastomas, cervical, breast, and prostate cancers we examined the expression of Brn-3a(l) in normal ovaries and in different histological grades of ovarian tumors. The expression of Brn-3a(l) was also evaluated in normal ovarian and cancer cell lines and tumor cells isolated from the ascites of advanced-stage ovarian cancer patients. METHODS: Normal ovaries, benign, borderline, grades 1, 2 and 3 ovarian tumors were analyzed by immunohistochemistry for Brn-3a(l) expression. A total of 46 ovarian specimens were included in the study. Immunofluorescence was used to investigate the expression of Brn-3a in normal ovarian and cancer cell lines. Brn-3a(l) expression was also evaluated by Western blot in tumor cells isolated from ascites of advanced-stage ovarian cancer patients and also in ovarian cancer cell lines. RESULTS: Nearly 12% of normal and benign ovarian tissues and 57% of borderline ovarian tumors were positive for epithelial Brn-3a(l) expression. Stromal staining was higher and it constituted 40% of normal non-cancerous ovaries compared to 50 and 86% in benign and borderline tumors. On the other hand, 85-100% of grades 1, 2 & 3 ovarian tumors demonstrated nuclear and cytoplasmic Brn-3a(l) staining in the epithelium. Stromal staining in grades1, 2 and 3 tumors constituted 71-88% of total staining. Overall, immunoreactive Brn-3a was present in all grades of ovarian tumors. The extent of epithelial and stromal Brn-3a staining was significantly different between the normal and histological grades of tumors (epithelial-chi2 = 41.01, df = 20, P = 0.004, stromal-chi2 = 24.66. df = 15, P = 0.05). The extent of epithelial staining was significantly higher in grades 1 and 2 ovarian tumors compared to normal ovaries and benign ovarian tumors (p < 0.05). In parallel, stromal staining was significantly higher in grade 3 tumors compared to normal ovaries (p < 0.05). In addition, cytoplasmic and nuclear Brn-3a expression was evident in ovarian cancer cell lines while no such expression was observed in SV40 antigen immortalized normal ovarian cell lines. CONCLUSION: These data suggest that like other cancers, Brn-3a(l) expression is enhanced in ovarian tumors and its expression is consistent with its known role in inhibiting apoptosis and enhancing tumorigenesis. Specific targeting of Brn-3a may provide a useful strategy for regulating multiple tumor related genes involved with ovarian carcinomas.

Increased expression of ac-FoxO1 protein in prelabor fetal membranes overlying the cervix: possible role in human fetal membrane rupture.

Forkhead box O proteins have critical roles in a number of cellular processes, including apoptosis. Acetylation and phosphorylation of forkhead box O proteins are posttranslational modifications that attenuate their transcriptional activity. As supracervical fetal membranes are characterized by increased cell death, the aim of this study was to compare the expression of forkhead box O1, acetylated-forkhead box O1, and Ser-256 phosphorylated forkhead box O1 at supracervical and distal site fetal membrane. Fetal membranes overlying the cervix were identified in situ in women undergoing term elective Caesarean section. Immunohistochemistry (n = 7) was used to analyze the protein expression of forkhead box O1, acetylated-forkhead box O1, and Ser-256 phosphorylated forkhead box O1. There was no difference in forkhead box O1 and Ser-256 phosphorylated forkhead box O1 protein expression between the 2 sites. However, when compared with distal site, the intensity and extent of staining of acetylated-forkhead box O1 were greater in amnion and chorion obtained from the supracervical site. In summary, supracervical fetal membranes are characterized by increased acetylated-forkhead box O1 protein expression. Although the precise role and contribution of acetylated-forkhead box O1 in the process of human fetal membrane rupture are unknown, it has been implicated in apoptosis and/or cell cycle regulation.

Defective insulin signaling in placenta from pregnancies complicated by gestational diabetes mellitus.

OBJECTIVE: Studies in adipose tissue and skeletal muscle suggest that impaired insulin action is due to defects in the insulin signaling pathway and may play a role in the pathophysiology of insulin resistance associated with gestational diabetes mellitus (GDM) and obesity. The present study tested the hypothesis that endogenous expression levels in the human term placenta of insulin signaling components are altered in placental tissue from GDM women in comparison with normal controls and maternal obesity. DESIGN AND METHODS: Placental tissue was collected from normal, diet-controlled GDM, and insulin-controlled GDM in both non-obese and obese women (n=6-7 per group). Western blotting and quantitative RT-PCR was performed to determine the level of expression in the insulin signaling pathway. RESULTS: There was a significant increase in insulin receptor (IR) substrate (IRS)-1 protein expression with a concurrent decrease in IRS-2 protein expression in non-obese women with insulin-controlled GDM compared with diet-controlled GDM and normal controls. Furthermore, a decrease in both protein and mRNA expression of phosphatidyl-inositol-3-kinase (PI3-K) p85alpha and glucose transporter (GLUT)-4 was observed in non-obese and obese women with insulin controlled GDM compared with normal controls. When comparing non-obese to obese patients, significant decreases in mRNA expression of IR-beta, PI3K p85alpha and GLUT-4 was found in obese patients. CONCLUSION: Our results suggest that post receptor defects are present in the insulin signaling pathway in placenta of women with pregnancies complicated by diabetes and obesity. In addition, expression studies demonstrate post receptor alterations in insulin signaling possibly under selective maternal regulation and not fetal regulation.

Alpha2beta1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis.

BACKGROUND: Ovarian cancer is characterized by a wide-spread intra-abdominal metastases which represents a major clinical hurdle in the prognosis and management of the disease. A significant proportion of ovarian cancer cells in peritoneal ascites exist as multicellular aggregates or spheroids. We hypothesize that these cellular aggregates or spheroids are invasive with the capacity to survive and implant on the peritoneal surface. This study was designed to elucidate early inherent mechanism(s) of spheroid survival, growth and disaggregation required for peritoneal metastases METHODS: In this study, we determined the growth pattern and adhesive capacity of ovarian cancer cell lines (HEY and OVHS1) grown as spheroids, using the well established liquid overlay technique, and compared them to a normal ovarian cell line (IOSE29) and cancer cells grown as a monolayer. The proteolytic capacity of these spheroids was compared with cells grown as a monolayer using a gelatin zymography assay to analyze secreted MMP-2/9 in conditioned serum-free medium. The disaggregation of cancer cell line spheroids was determined on extracellular matrices (ECM) such as laminin (LM), fibronectin (FN) and collagen (CI) and the expression of alpha2, alpha3, alphav, alpha6 and beta1 interin was determined by flow cytometric analysis. Neutralizing antibodies against alpha2, beta1 subunits and alpha2beta1 integrin was used to inhibit disaggregation as well as activation of MMPs in spheroids. RESULTS: We demonstrate that ovarian cancer cell lines grown as spheroids can sustain growth for 10 days while the normal ovarian cell line failed to grow beyond 2 days. Compared to cells grown as a monolayer, cancer cells grown as spheroids demonstrated no change in adhesion for up to 4 days, while IOSE29 cells had a 2-4-fold loss of adhesion within 2 days. Cancer cell spheroids disaggregated on extracellular matrices (ECM) and demonstrated enhanced expression of secreted pro-MMP2 as well as activated MMP2/MMP9 with no such activation of MMP's observed in monolayer cells. Flow cytometric analysis demonstrated enhanced expression of alpha2 and diminution of alpha6 integrin subunits in spheroids versus monolayer cells. No change in the expression of alpha3, alphav and beta1 subunits was evident. Conversely, except for alphav integrin, a 1.5-7.5-fold decrease in alpha2, alpha3, alpha6 and beta1 integrin subunit expression was observed in IOSE29 cells within 2 days. Neutralizing antibodies against alpha2, beta1 subunits and alpha2beta1 integrin inhibited disaggregation as well as activation of MMPs in spheroids. CONCLUSION: Our results suggest that enhanced expression of alpha2beta1 integrin may influence spheroid disaggregation and proteolysis responsible for the peritoneal dissemination of ovarian carcinoma. This may indicate a new therapeutic target for the suppression of the peritoneal metastasis associated with advanced ovarian carcinomas.

Why do membranes rupture at term? Evidence of increased cellular apoptosis in the supracervical fetal membranes.

OBJECTIVE: Reduced tensile strength of the human fetal membranes overlying the cervix has previously been identified. We used transcervical application of Bonney's blue dye, before the onset of term labor to identify the supracervical membranes for analysis after elective cesarean section delivery. We hypothesized that pro- and antiapoptotic proteins, which are representative of both the extrinsic and intrinsic pathways, would be expressed differentially in the supracervical membranes compared with membranes taken from distal sites. Membrane apoptosis would provide a mechanism for the reduced tensile strength that presumably precedes spontaneous intrapartum rupture of the membranes. STUDY DESIGN: Bonney's blue dye was applied transcervically to the chorion-facing fetal membrane before elective cesarean delivery at term. After delivery, samples of fetal membranes were obtained from the supracervical site, where the membrane was marked by the dye (approximately 8-cm diameter) and compared with samples from a distal site (2-cm from the placental edge). Samples from the supracervical and distal sites were fixed and paraffin embedded for immunohistochemical analyses and histologic review and stored at -80 degrees C for Western blotting analysis. RESULTS: The supracervical area of fetal membranes exhibited increased markers of apoptosis that included M30 immunohistochemical staining, cleaved-caspase-3, cleaved-caspase-9, and decreased immunoreactive Bcl-2. Histologic sections that were stained with hematoxylin and eosin demonstrated features of degenerative changes and apoptosis that occurred predominantly at the supracervical site. CONCLUSION: There is evidence of increased cellular apoptosis at the supracervical site in fetal membranes at term. Both morphologic and biochemical changes that were observed at the supracervical site suggest that the intrinsic apoptotic pathway plays an important role in spontaneous membrane rupture at term.

Effect of high oxygen on placental function in short-term explant cultures.

Ex situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygen concentrations on human term placental explants over a 24-h time period. Specifically, the effect of incubating placental explants in oxygen concentrations of 8%, 21% or 95% on tissue viability, metabolism and cell death was measured by assessing glucose consumption, lactate production, release of lactate dehydrogenase, parathyroid hormone-related protein (PTHrP), tumour necrosis factor-alpha (TNF-alpha) and 8-isoprostane, immunoreactivity for cleaved-caspase-9 and immunohistochemistry for the caspase-3-cleaved cytokeratin-18 neoepitope, M30. Exposure to higher oxygen concentrations significantly increased the rates of glucose consumption and lactate production. Apoptosis was significantly increased under conditions of higher oxygen as evidenced by increased M30 in placental explant sections. Similarly, hyperoxia significantly increased the releases of PTHrP, TNF-alpha and 8-isoprostane. Thus, incubation of placental explants with oxygen concentrations of 95% and, to a lesser extent, 21% oxygen was associated with the modulation of multiple cellular response pathways including those associated with tissue viability and cell death. These data are consistent with the hypothesis that hyperoxia activates pathways and mechanisms involved in cellular metabolism, necrosis and apoptosis, thereby shifting the balance from a steady state towards cell death.

Neutrophil gelatinase-associated lipocalin (NGAL) an early-screening biomarker for ovarian cancer: NGAL is associated with epidermal growth factor-induced epithelio-mesenchymal transition.

The expression of neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be upregulated in ovarian cancer cells. In this study, we report that the expression of immunoreactive NGAL (irNGAL) in ovarian tumors changes with disease grade and that this change is reflected in the concentration of NGAL in peripheral blood. A total of 59 ovarian tissues including normal, benign, borderline malignant and grades 1, 2 and 3 malignant were analyzed using immunohistochemistry. irNGAL was not present in normal ovaries and the NGAL expression was weak to moderate in benign tissues. Both borderline and grade 1 tumors displayed the highest amount of NGAL expression with moderate to strong staining, whereas in grade 2 and 3 tumors, the extent of staining was significantly less (p < 0.01) and staining intensity was weak to moderate. Staining in all cases was confined to the epithelium. NGAL expression was analyzed by ELISA in 62 serum specimens from normal and different grades of cancer patients. Compared to control samples, the NGAL concentration was 2 and 2.6-fold higher in the serum of patients with benign tumors and cancer patients with grade 1 tumors (p < 0.05) and that result was consistent with the expression of NGAL performed by Western blot. NGAL expression was evaluated by Western blot in an immortalized normal ovarian cell line (IOSE29) as well as ovarian cancer cell lines. Moderate to strong expression of NGAL was observed in epithelial ovarian cancer cell lines SKOV3 and OVCA433 while no expression of NGAL was evident in normal IOSE29 and mesenchyme-like OVHS1, PEO.36 and HEY cell lines. NGAL expression was downregulated in ovarian cancer cell lines undergoing epithelio-mesenchymal transition (EMT) induced by epidermal growth factor (EGF). Downregulation of NGAL expression correlated with the upregulation of vimentin expression, enhanced cell dispersion and downregulation of E-cadherin expression, some of the hallmarks of EMT. EGF-induced EMT phenotypes were inhibited in the presence of AG1478, an inhibitor of EGF receptor tyrosine kinase activity. These data indicate that NGAL may be a good marker to monitor changes of benign to premalignant and malignant ovarian tumors and that the molecule may be involved in the progression of epithelial ovarian malignancies.

Role of integrin receptors for fibronectin, collagen and laminin in the regulation of ovarian carcinoma functions in response to a matrix microenvironment.

Integrins play an important role in cellular matrix interactions requisite for cancer cell adhesion, growth, migration and invasion. In this study, we have investigated the expression of integrin subunits alpha3, alpha6, alphav and beta1 in normal ovaries, benign ovarian tumors and ovarian carcinomas of different pathological grades. The expression of these integrins in ovarian cancer cell lines was also investigated, and their role in sustaining proliferation, adhesion, migration and invasion in cohort with the activation of signaling pathways in response to extracellular matrices (ECM) was evaluated. We demonstrate a differential expression pattern of alpha3, alpha6, alphav and beta1 integrin subunits in ovarian carcinomas compared to normal ovaries and benign ovarian tumors. Ovarian cancer cell lines (Hey, Ovcar3 and Peo.36) demonstrated significantly high expression of alpha3, alpha6, alphav and beta1 integrin subunits. A significant increase in proliferation and adhesion (P<0.05) in response to collagen 1 (Coll) and laminin (LM), ligands for integrin receptor alpha3beta1 and alpha6beta1 was observed in ovarian cancer cell lines. On the other hand, fibronectin (FN), a receptor for alphavbeta1 integrin, increased proliferation in all ovarian cancer cell lines studied but only enhanced adhesion in Hey cell line (P<0.05). Neutralizing antibodies against alpha3, alpha6, alphav and beta1 integrin subunits inhibited ECM-induced proliferation, but increased adhesion to ECM was inhibited by beta1 integrin subunit antibody. No suppression of Coll, LM and FN-induced (Hey cells only) adhesion was observed in the presence of alpha3 or alphav subunit antibodies but LM-induced adhesion was inhibited by blocking alpha6 subunit functions. LM, FN and Coll enhanced chemotactic migration in Hey cells, but direct invasion across ECM was observed only in the presence of LM and Coll. Blocking antibodies against alpha3, alpha6 and beta1 integrin subunits inhibited both chemotactic migration and invasion of Hey cells in response to respective ECM. Adhesion of ovarian cancer cells to FN, Coll and LM activated Ras, Erk and Akt pathways. Neutralizing alphav and beta1 functions did not inhibit FN-induced activation of Ras and Erk pathways but inhibited the Akt pathway. On the other hand, antibodies against alpha6 and beta1 subunits, but not alpha3 subunit, inhibited LM-induced activation of Ras but did not inhibit the downstream Akt pathway. Neutralizing beta1 subunit function however, inhibited LM-induced Erk activation. Coll-induced activation of Ras, Erk and Akt pathways was inhibited by alpha3 and beta1 integrin subunit antibodies. These results indicate that alpha3beta1, alphavbeta1 and alpha6beta1 integrin mediate proliferation, adhesion, migration and invasion of ovarian cancer cells in response to ECM and targeting these integrins to modulate integrin-ECM interactions in tumor cells may be a promising tool to reduce the dissemination of ovarian carcinoma in vivo.

Expression and localization of alphavbeta6 integrin in extraplacental fetal membranes: possible role in human parturition.

Successful outcome of human parturition is dependent upon extensive remodelling of the extracellular matrix (ECM) of the cervix, uterus and fetal membranes, a process that involves adhesion molecules and is also common in tumour invasion and metastasis. To elucidate the role of integrins in human parturition, this study characterizes the expression of the tumour-associated alpha(v)beta(6) integrin in human placenta and extraplacental membranes. Immunohistochemical analysis of the placenta and fetal membranes from normal vaginal deliveries (NVD) (n = 10) exhibited strong intensity of staining for alpha(v)beta(6) integrin (3 = dark brown) in the epithelial layer of the amnion. Weak immunohistochemical staining of alpha(v)beta(6) integrin (1 = pale brown) was detected in the chorion and at the decidual edge. These results were consistent with the immunodetection of alpha(v)beta(6) integrin by western blot analysis that showed 4-fold enhanced expression in the amnion compared to chorion of both NVD and term elective caesarean section (CS) deliveries. Even though there was no difference in the extent of immunohistochemical staining of alpha(v)beta(6) integrin between the amnion of NVD and CS groups, significantly higher intensity of staining was observed in the NVD amniotic epithelium compared to that of CS (n = 10) (chi(2) = 10.25, P = 0.0059). Western blot analysis of the fetal membranes showed no differences in the expression of alpha(v)beta(6) integrin between the NVD and CS groups. Gelatin zymography demonstrated the presence of pro-matrix metalloprotein-9 (MMP-9) and pro-MMP-2 in the amnion and chorion of NVD, whereas in CS only the presence of pro-MMP-2 was observed. These results suggest that in term pregnancy, human fetal membranes express alpha(v)beta(6) integrin and that the expression is significantly higher in amnion compared to chorion. The fact that enhanced expression of alpha(v)beta(6) integrin in fetal membranes correlates with the expression of pro-MMP-9 in NVD is consistent with the invasive role of the integrin in cancer and suggests that the molecule may have a proteolytic role in the initiation and progression of labour.

Integrin-linked kinase expression increases with ovarian tumour grade and is sustained by peritoneal tumour fluid.

Integrin-linked kinase (ILK) is a serine threonine kinase, overexpression of which promotes tumour growth and invasion through deregulation of the cell cycle. This study demonstrates the relative expression of ILK in normal, benign, low-grade, and high-grade (borderline, grade I/II, and grade III) ovarian tumours of serous, mucinous, endometrioid, and clear cell types in order to assess its potential as a marker for epithelial ovarian cancer progression. Seventy-three specimens including ten normal, ten benign, 14 borderline, 17 grade I/II, and 22 grade III were evaluated by immunohistochemistry. Immunoreactive ILK was not detectable in normal ovarian surface epithelium. All 53 carcinomas studied were positive and the staining intensity correlated significantly with the grade of the tumour. Ovarian cancer cell lines had high expression of ILK, while immortalized normal ovarian surface epithelial cell lines (HOSE) showed low basal expression of ILK by western blotting. Peritoneal tumour fluid (PTF) upregulated ILK expression in ovarian cancer cell lines but had no effect on HOSE cells. PTF-induced up-regulation of ILK expression in ovarian cancer cell lines correlated with the activation of the downstream protein kinase B (PKB/Akt) pathway. Collectively, these data demonstrate that ILK expression increases with ovarian cancer progression and that soluble factors in PTF mediate sustained overexpression of ILK in ovarian cancer cells. Suppression of ILK expression may therefore represent a novel and an efficient mechanism for controlling ovarian tumour growth.

Alpha(v)beta(6) integrin-A marker for the malignant potential of epithelial ovarian cancer.

The mechanism(s) responsible for the progression of non-metastatic or borderline ovarian cancer to invasive Grade I/III ovarian cancer is still unknown. An epithelium-restricted integrin, alpha(v)beta(6), is present in malignant epithelia but not in normal epithelia. We studied the relative expression and distribution of alpha(v)beta(6) integrin in early and late-stage invasive (Grade I and Grade III) and non-invasive (benign and borderline) ovarian tumors of serous, mucinous, endometrioid, and clear-cell carcinoma subtypes, to assess its potential as a marker for epithelial ovarian cancer progression. Sixty-six specimens, including eight normal, 13 benign, 14 borderline, 13 Grade I, and 18 Grade III tumors were evaluated by immunohistochemistry (IHC) using a monoclonal antibody (MAb) against alpha(v)beta(6) integrin. Normal ovarian surface epithelium was negative for alpha(v)beta(6) integrin expression. All 45 carcinomas studied were positive, and the staining intensity significantly correlated with the grade of the tumor. The Grade III carcinomas of all types showed strong staining intensity. Only mucinous benign tissues were positive, and no reactivity was observed in benign serous neoplasms. On the basis of these observations, we hypothesize that the expression of alpha(v)beta(6) integrin is associated with epithelial ovarian cancer and that a gradual increase in the expression of the molecule may be a correlative index of the progression of this disease.

FTIR/ATR study of protein adsorption and brushite transformation to hydroxyapatite.

Previous study has demonstrated that brushite (CaHPO4 x 2H2O), modified by partial potassium substitution for calcium, can transform quickly into hydroxyapatite (HA, Ca5(PO4)3OH) when exposed to aqueous salt solutions at room temperature. Analyses techniques used in those studies required sample retrieval from solution, which may alter the sample surface. In this work FTIR/ ATR was used in analysis, enabling in situ study of the transformation within the aqueous environment. To test the biocompatibility of this brushite, cellular response to the transformation needs to be understood. Cellular response was initiated by bovine serum albumin adsorption on the brushite surface. The response was studied by monitoring the conformation of the adsorbed protein, which is critical to cellular reaction. This required monitoring the brushite transformation and surface adsorbed protein conformation simultaneously which can be realized using FTIR/ATR. Based on band fitting and second derivative results from the spectra it was found that the conformation of the adsorbed BSA changes during the brushite transformation to HA. This study also demonstrated that the deposition of the brushite could be monitored in real time which offers the possibility for studying surface bonding during electrodeposition.