Lynne Kelly

Pre-Treatment of Platinum Resistant Ovarian Cancer Cells with an MMP-9/MMP-2 Inhibitor Prior to Cisplatin Enhances Cytotoxicity as Determined by High Content Screening

Platinum resistance is a major cause of treatment failure in ovarian cancer. We previously identified matrix metalloproteinase 9 (MMP-9) as a potential therapeutic target of chemoresistant disease. A2780cis (cisplatin-resistant) and A2780 (cisplatin-sensitive) ovarian carcinoma cell lines were used. The cytotoxic effect of MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) alone or in combination with cisplatin was determined using high content screening. Protein expression was examined using immunohistochemistry and ELISA. Co-incubation of cisplatin and an MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) resulted in significantly greater cytotoxicity as compared to either treatment alone in a cisplatin resistant MMP-9 overexpressing cell line; A2780cis. In addition, pre-incubating with MMP-9i prior to cisplatin further enhances the cytotoxic effect. No significant difference was observed in MMP-9 protein in tissue but a trend towards increased MMP-9 was observed in recurrent serum. We propose that MMP-9/MMP-2i may be utilized in the treatment of recurrent/chemoresistant ovarian cancers that overexpress MMP-9 mRNA but its role in vivo remains to be evaluated.

Genistein alters coagulation gene expression in ovariectomised rats treated with phytoestrogens

Recent data has shown that hormone therapy (HT) increases the risk of cardiovascular and thromboembolic disease, particularly in users of oral HT. Phytoestrogens are popular alternatives to oestrogen therapy; however, their effects on cardiovascular risk are unknown. We investigated the effect of the phytoestrogen, genistein on the expression of genes and proteins from the haemostatic system in the liver in an ovariectomised rat model. Fifty-nine virgin female Sprague-Dawley rats were fed with soy-free chow supplemented with 17β estradiol (E2) (daily uptake 0.19 or 0.75 mg/kg body weight), or genistein (daily uptake 6 or 60 mg/kg body weight), for three months and compared to soy-free control rats. Gene expression of prothrombin, factor VII, fibrinogen alpha and fibrinogen beta was increased with E2 and genistein compared to the soy-free control group (p<0.001). Genistein increased factor VII significantly more than E2 (p<0.005). Plasminogen mRNA was increased in both treatment groups compared to the soy-free control, with genistein expression significantly higher than E2 (p<0.001). Tissue plasminogen inhibitor (tPA), plasminogen activator inhibitor-1 (PAI-1) and C-reactive protein (CRP) expression were also increased in both groups relative the soy-free control. Results of protein analysis largely concurred with those of the mRNA. Oestrogen receptor β (ERβ) was undetected while oestrogen receptor α (ERα) was detected in each sample group. Genistein can increase the expression of coagulation and fibrinolytic genes. This effect was similar and in some cases higher than 17β estradiol. These results suggest that genistein may not be neutral with respect to the haemostatic system.

Estrogen receptor alpha augments changes in hemostatic gene expression in HepG2 cells treated with estradiol and phytoestrogens.

Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in post-menopausal women are unknown. The aim of this study was to determine the effect of the phytoestrogens, genistein, daidzein and equol on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen. HepG2 cells and Hep89 cells (expressing estrogen receptor alpha (ERα)) were incubated for 24 h with 50 nM 17β-estradiol, genistein, daidzein or equol. Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), Factor VII, fibrinogen γ, protein C and protein S mRNA expression were determined using TaqMan PCR. Genistein and equol increased tPA and PAI-1 expression in Hep89 cells with fold changes greater than those observed for estradiol. In HepG2 cells (which do not express ERα), PAI-1 and tPA expression were unchanged. Increased expression of Factor VII was observed in phytoestrogen treated Hep89 cells but not in similarly treated HepG2s. Prothrombin gene expression was increased in equol and daidzein treated HepG2 cells in the absence of the classical estrogen receptors. These data suggest that phytoestrogens can regulate the expression of coagulation and fibrinolytic genes in a human hepatocyte cell line; an effect which is augmented by ERα.

The Value of a Novel Panel of Cervical Cancer Biomarkers for Triage of HPV Positive Patients and for Detecting Disease Progression

In the era of primary vaccination against HPV and at the beginning of the low prevalence of cervical lesions, introduction of screening methods that can distinguish between low- and high-grade lesions is necessary in order to maintain the positive predictive value of screening. This case-control study included 562 women who attended cervical screening or were referred for colposcopy and 140 disease free controls, confirmed by histology and/or cytology. The cases were stratified by age. Using routine exfoliated liquid based cytological samples RT-PCR measurements of biomarker genes, high-risk HPV testing and liquid based cytology were performed and used to evaluate different testing protocols including sets of genes/tests with different test cut-offs for the diagnostic panels. Three new panels of cellular biomarkers for improved triage of hrHPV positive women (diagnostic panel) and for prognostic assessment of CIN lesions were proposed. The diagnostic panel (PIK3AP1, TP63 and DSG3) has the potential to distinguish cytologically normal hrHPV+ women from hrHPV+ women with CIN2+. The prognostic gene panels (KRT78, MUC5AC, BPIFB1 and CXCL13, TP63, DSG3) have the ability to differentiate hrHPV+ CIN1 and carcinoma cases. The diagnostic triage panel showed good likelihood ratios for all age groups. The panel showed age-unrelated performance and even better diagnostic value under age 30, a unique feature among the established cervical triage tests. The prognostic gene-panels demonstrated good discriminatory power and oncogenic, anti-oncogenic grouping of genes. The study highlights the potential for the gene expression panels to be used for diagnostic triage and lesion prognostics in cervical cancer screening.

Phytoestrogens Activate the Estrogen Receptor in HepG2 Cells.

Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in postmenopausal women are unknown. This chapter describes a protocol to determine the effect of the phytoestrogens genistein, daidzein and equol, on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen using in vitro culture systems and Taqman(®) gene expression analysis.

Norethisterone acetate alters coagulation gene expression in vitro in human cell culture

INTRODUCTION Both oestrogen and progestin and the route of administration have been implicated in cardiovascular and thromboembolic risk in post menopausal hormone users. Transdermal preparations have been reported as safer indicating that liver derived metabolites of oestrogen may be important. The aim of our study was to investigate the in vitro effects of 17β-estradiol, its metabolites, and norethisterone acetate (NETA) on the expression of coagulation genes in cultured human cells. METHODS Human hepatocytes and human umbilical vein endothelial cells(HUVECS) were treated with 17β-estradiol, estrone, 2-hydroxyestradiol (2-OH), NETA and NETA/17β-estradiol (10nM) for 24hours. Fibrinogen, factor VII, prothrombin and plasminogen activator inhibitor -1 (PAI-1) mRNA expression was determined in hepatocyte cultures using TaqMan PCR. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (tPA) and PAI-1 expression was determined in HUVECS. Expression of estrogen receptors was also determined. RESULTS Fibrinogen and factor VII mRNA expression was upregulated 2-4 fold by estradiol and estrone. Addition of NETA downregulated fibrinogen and prothrombin. PAI-1 expression in hepatocytes was upregulated by estrone, 2-OH, NETA and NETA/17β-estradiol. In HUVECS, TF, TFPI and PAI-1 expression was upregulated by estrone but not by 17β-estradiol. NETA upregulated TF, TFPI and tPA expression. Estrogen receptor status was unaffected by the addition of NETA. CONCLUSIONS This data suggests a role for progestins in modifying the effects of oestrogen and its metabolites on coagulation gene expression which may contribute to the reduced thrombotic risk associated with transdermal preparations.

Clinical performance of the Cobas 4800 HPV test and the Aptima HPV assay in the management of women referred to colposcopy with minor cytological abnormalities

The aim of this study was to evaluate the clinical performance of the Cobas 4800 HPV test and the Aptima HPV assay for the detection of CIN2+ disease in women referred to colposcopy with minor cytological abnormalities.

PM.06 Low Molecular Heparin Within the Uteroplacental Unit

Background Perturbation of the uteroplacental haemostasis has been implicated in placenta mediated pregnancy complications in thrombophilic women. LMWH may be effective in altering local thrombin production in the uteroplacental compartment. Aim We determined the effects of LMWH (tinzaparin) on the peripheral, uteroplacental and fetal circulation and on haemostatic gene and antigen expression in placental tissue. Method Eight women on antenatal LMWH prophylaxis (tinzaparin 75 IU/kg) due to moderate risk of VTE undergoing caesarean section (CS) and a control group of 15 healthy pregnant women undergoing CS had venous blood taken from the peripheral and uterine vein before delivery of placenta. Simultaneously, cord venous blood and placental biopsy was collected. Tissue factor pathway inhibitor (TFPI), thrombin antithrombin (TAT) and endogenous thrombin potential (ETP) were measured. Real-time PCR and ELISA were used to quantify mRNA and protein expression of TFPI and TF in placental tissue. Results TAT levels within uterine vein are significantly higher compared to maternal peripheral circulation in both the control group (P < 0.0001) and LMWH group (P < 0.02). In the LMWH group, TAT is reduced compared with controls in the uterine vein (P < 0.001). ETP and TFPI within uterine circulation is reduced significantly in the LMWH group (P < 0.05) and (P < 0.02) respectively. Down-regulation of placental TFPI and TFPI2 mRNA expression was also found (p < 0.05). Placental TF mRNA expression in LMWH group showed a non significant increase compared to control and this is replicated in placental TF antigen expression. Conclusion TAT is reduced in uteroplacental circulation in thrombophilic women on LMWH prophylaxis and this is mirrored by decreased ETP in uteroplacental circulation. LMWH may be effective in reducing in- vivo thrombin production in the uteroplacental circulation of thrombophilic women.

P10. Placental expression of haemostatic and angiogenic markers in preeclampsia

cantly higher than in late onset PE (497 ± 81 vs. 13 ± 4, p < 0,001 and 131 ± 15 vs. 29 ± 5, p < 0.001; p for early vs. late PE <0.001). Again ROC curves and their AUC showed best performance for the sFlt1/PlGF ratio, being 98.6% (vs. 97.6% for sFlt1 and 97.4% for PlGF) in early onset PE vs. controls and 90.8% (vs. 82.1% for sFlt1 and 88.4% for PlGF) in late onset PE. Patients with superimposed, mild or severe PE and HELLP each showed higher ratios than controls (202 ± 110; 137 ± 27; 497 ± 91 and 254 ± 72 vs. 16 ± 2, each p < 0.001); in calculated ROC curves, their AUC were 93.6%, 94.8%, 99.4% and 98.6%, respectively. Whereas sFlt1/PlGF values compared to controls were significantly higher in patients with PIH and GP, no significant difference was observed in patients with cHT (PIH 65 ± 18 vs. 16 ± 2, p < 0.001; GP 62 ± 25 vs. 16 ± 2, p = 0.01 and cHT 13 ± 8 vs. 16 ± 2, p = 0.6). Ratio values of patients with each PIH and GP were significantly lower than in patients with mild PE (p < 0.007), severe PE (p < 0.001) and HELLP (p < 0.003). Compared to patients with superimposed PE a significant difference was observed only vs. GP (p = 0.015) whereas vs. PIH no significant difference was observed (p = 0.178). Conclusion: The automated measurement of sFlt1/PlGF is a reliable tool to discriminate between different types of pregnancy-related hypertensive disorders, particularly in early onset PE, severe PE and HELLP, being the most severe variations of PE. Thus, sFlt1/PlGF gives additional valuable information and can be used as an aid in diagnosis and therefore adapt clinical management. First Author < 35 years: Yes.