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 17ß-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 17ß-estradiol, estrone, 2-hydroxyestradiol (2-OH), NETA and NETA/17ß-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/17ß-estradiol. In HUVECS, TF, TFPI and PAI-1 expression was upregulated by estrone but not by 17ß-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.

Potentially important microRNA cluster on chromosome 17p13.1 in primary peritoneal carcinoma.

MicroRNAs are a group of small non-coding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature microRNAs in human cancers. We characterized the alteration in expression of a select group of microRNAs in primary peritoneal carcinoma relative to matched cases of ovarian serous carcinoma. MicroRNA expression was analysed using semi-quantitative stem-loop RT-PCR on a set of 34 formalin-fixed paraffin-embedded samples. Protein expression of p53 and bcl-2 was quantified in the corresponding tissue microarray. We provide definitive evidence that there is downregulation of a select group of microRNAs in tumours meeting Gynaecological Oncology Group criteria for primary peritoneal carcinoma relative to ovarian serous carcinoma. Specifically, we show decreased p53 expression and downregulation of miR-195 and miR-497 from the microRNA cluster site at chromosome 17p13.1 in primary peritoneal carcinoma relative to ovarian serous carcinoma. miR-195 and miR-497 may have potential roles as tumour-suppressor genes in primary peritoneal tumourigenesis.

Altered eIF6 and Dicer expression is associated with clinicopathological features in ovarian serous carcinoma patients.

MicroRNAs are a group of small non-coding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature microRNAs in human cancers. Production and function of microRNAs require coordinated processing by proteins of the microRNA machinery. Dicer and Drosha (RNase III endonucleases) are essential components of the microRNA machinery. Recently, the ribosome anti-association factor eIF6 has also been found to have a role in microRNA-mediated post-transcriptional silencing. We characterized the alterations in the expression of genes encoding proteins of microRNA machinery in ovarian serous carcinoma. Protein expression of eIF6 and Dicer was quantified in a tissue microarray of 66 ovarian serous carcinomas. Dicer, Drosha and eIF6 mRNA expression was analysed using quantitative reverse transcription-PCR on an independent set of 50 formalin-fixed, paraffin-embedded ovarian serous carcinoma samples. Expression profiles of eIF6 and Dicer were correlated with clinicopathological and patient survival data. We provide definitive evidence that eIF6 and Dicer are both upregulated in a significant proportion of ovarian serous carcinomas and are associated with specific clinicopathological features, most notably low eIF6 expression being associated with reduced disease-free survival. The status of eIF6 and proteins of the microRNA machinery may help predict toxicity and susceptibility to future interfering RNA-based therapy.

Haemostatic activation in post-menopausal women taking low-dose hormone therapy: less effect with transdermal administration?

Hormone therapy (HT) increases the risk of cardiovascular and thromboembolic disease in post-menopausal women. Recent studies have suggested that prothrombotic mechanisms are likely to be involved. Transdermal HT avoids the first-pass effect of oestrogen in the liver and may have a less marked effect on the haemostatic system than equivalent oral preparations. The majority of studies have compared HT preparations that have different formulations as well as routes of administration. We investigated changes in the haemostatic system in post-menopausal women using two pharmacologically similar HT preparations, which differed only in their route of administration. Three hundred forty-four healthy post-menopausal women were randomised to six months treatment with either a transdermal matrix patch containing 25 microg 17beta-estradiol/125 microg norethisterone acetate (NETA) applied every 3-4 days, or an equivalent oral preparation (estradiol 1 mg and NETA 0.5 mg given once daily). Oral treatment significantly reduced fibrinogen (p < 0.003), factor VIIc (FVIIc) (p < 0.00001), and antithrombin (AT) levels (p < 0.005); the effects in the transdermal group were less marked with no reduction in fibrinogen levels and lesser effect on FVIIc (p < 0.03) compared with oral treatment. Treatment type significantly affected fibrinolysis with lower plasmin-anti-plasmin (PAP) levels in the transdermal group (p < 0.003) and lower plasminogen activator inhibitor-1 antigen (PAI-1) (p < 0.012) and tissue plasminogen activator (tPA) antigen levels in the oral group (p < 0.002). Prothrombin fragment 1.2 and activated protein C (APC) resistance were not affected by either treatment. Transdermal HT has a less marked effect on coagulation than an equivalent oral preparation. Randomised trials are required to investigate whether this translates into less risk of cardiovascular and thromboembolic disease.

Analysis of DNA in endometrial cancer cells treated with phyto-estrogenic compounds using comparative genomic hybridisation microarrays.

The aim of this study was to identify genomic aberrations in endometrial cancer cells treated with the phyto-estrogenic compounds tectorigenin, irigenin and apigenin and to compare with those treated with beta-estradiol using array-based comparative genomic hybridisation (array CGH). The microarray contains 287 targets and includes telomeres, microdeletions, oncogenes and tumour suppressor genes and has increased mapping resolution compared to conventional CGH. An endometrial cancer cell line (Ishikawa) was cultured and treated with the phyto-estrogens. Treated cells were examined using the CGH microarray. Over 20 % of the array genes were aberrated in the cells treated with beta-estradiol, tectorigenin and irigenin compared to 3 % in those treated with the same concentration of apigenin. Protein kinase c zeta form, insulin, insulin receptor and protein-tyrosine phosphatase non-receptor-type 1 which are involved in insulin metabolism were aberrated by tectorigenin and irigenin. Apigenin may play a role in the treatment of endometrial cancer and in the treatment of postmenopausal women. Further studies in normal endometrium and primary endometrial cancer cells are needed to elucidate the role of the phyto-estrogens.

Oestrogen regulated gene expression in normal and malignant endometrial tissue.

OBJECTIVES: The aim of this study was to examine the expression of oestrogen regulated genes in premenopausal and postmenopausal normal and malignant endometrial specimens. The molecular mechanisms and the role of these genes in endometrial carcinogenesis are poorly understood. METHODS: Normal and malignant endometrial specimens were collected from patients undergoing hysterectomy. Real time TaqMan PCR was used to examine the mRNA expression levels of oestrogen receptor a (ERa) and b (ERb), progesterone receptor (PR), insulin like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF). RESULTS: Expression analysis was carried out on 60 patients. ERa was more predominantly expressed in the endometrial samples than ERb, 28% of the specimens did not express ER. Normal pre and postmenopausal tissue expressed higher levels of ERa, PR and IGF-1 than malignant tissue. ERa and PR expression was significantly higher in the proliferative phase endometrium compared to the secretory phase (P < 0.05). PR mRNA expression was significantly correlated with ERa in all tissue types. CONCLUSIONS: ERa expression may play an important role in the regulation of PR in normal and malignant endometrium. Further work is needed to establish if IGF-1 plays a role in a subset of endometrial cancers and if isoforms of VEGF play a role in endometrial cancer.

The MTS assay as an indicator of chemosensitivity/resistance in malignant gynaecological tumours.

The aim of this study was to use the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay to determine the response of 17 endometrial and 27 cervical tumours to cytotoxic drugs. Tumour samples were taken at surgery and cultured using the explant technique. Cells were incubated with chemotherapy drugs. The MTS cytotoxicity assay was carried out to ascertain the response to the drugs and correlated retrospectively to the clinical outcome. Tumours of similar stage and grade displayed heterogeneity in their responses to the drugs. A total of 88 of 90 tumours (97.8%), including data from an earlier study of 44 ovarian tumour samples yielded chemosensitivity data. A total of 45 specimens were evaluable for in vitro-in vivo correlations. In vitro sensitivity was associated with clinical response in 26 of 30 patients and in vitro resistance with progressive disease or death in 14 of 15 patients. A randomised prospective trial should be carried out to validate chemosensitivity/resistance testing.