The G protein-coupled estrogen receptor 1 (GPER/GPR30) does not predict survival in patients with ovarian cancer

<p>Abstract</p> <p>Background</p> <p>Even though ovarian tumors are not generally considered estrogen-sensitive, estrogens may still have an impact on ovarian tumor progression. The recently identified trans-membrane estrogen receptor GPER is involved in rapid estrogen signaling. Furthermore, it binds selective estrogen receptor modulators with agonistic effect, which could explain tamoxifen controversies.</p> <p>Methods</p> <p>GPER mRNA was assayed with quantitative real-time PCR (qPCR) in 42 primary ovarian tumors and 7 ovarian cancer cell lines. ERα and ERβ mRNA were analyzed for comparison. GPER protein was semi-quantified with densitometric scanning of Western blots and its tissue distribution analyzed with immunohistochemistry (IHC) in 40 ovarian tumors. In addition, IHC was evaluated in a tissue microarray (TMA) of 150 primary malignant ovarian tumors.</p> <p>Results</p> <p>All tumor samples contained GPER mRNA. The content of mRNA was not different between benign and malignant tumors, but one third of malignant samples over-expressed GPER mRNA. The content of ERα mRNA was higher in malignant than in benign tumors, whereas ERβ mRNA was higher in benign than in malignant tumors. GPER mRNA was detected in all seven ovarian cancer cell lines with highest levels in TOV21G and TOV112D cells. Similar expression pattern was seen for ERβ mRNA. Western blot demonstrated GPER protein in all tumor samples. Semi-quantification showed no difference between benign and malignant tumors, but about one third of malignant samples over-expressed GPER protein. GPER staining was localized mainly in epithelial cells. In the TMA study we found no correlation between GPER staining and clinical stage, histological grade or patient survival.</p> <p>Conclusions</p> <p>GPER mRNA as well as GPER protein is present in both benign and malignant ovarian tumor tissue. About one third of malignant tumors over-expressed both GPER mRNA and protein. This, however, correlated neither with histological or clinical parameters nor with patient survival.</p

High preoperative blood levels of HE4 predicts poor prognosis in patients with ovarian cancer

The aim of this study was to assess the clinical value of preoperative blood levels of HE4 as a predictor of overall survival in patients with ovarian cancer and to validate previous data of HE4 and the ROMA algorithm including HE4 and CA125 in discriminating benign and malignant ovarian tumors. Experimental design: The preoperative plasma levels of HE4 and CA125 were analyzed with ELISA in 312 patients with adnexal lesions. Tumors were classified as benign (n= 206), borderline (i.e. low malignant potential tumors) (n= 25), and well (n= 14), moderately (n= 15), and poorly (n= 51) differentiated malignant. Results: In univariate Cox regression analyses high levels (dichotomized at the median) of HE4, CA125, increased age (continuous variable), advanced-stage of disease 2-4, histological grade 3 and non-optimal tumor debulking at primary surgery were all significantly associated with shorter overall survival. A multivariate Cox regression model including pre-operative available covariates HE4 and CA125 both dichotomized at median in addition to age as continuous variable showed that high levels of HE4 was an independent prognostic marker for worse prognosis HR 2.02 (95% CI 1.1-3.8). In postmenopausal women the ROMA algorithm gave the highest AUC of 0.94 (95% CI, 0.90-0.97) which was higher than the separate markers HE4 AUC 0.91 (95% CI 0.86-0.95) and CA125 AUC 0.91(95% CI 0.87-0.96). Conclusions: High concentration of plasma HE4 is an independent preoperative marker of poor prognosis in patients with ovarian cancer. The algorithm ROMA discriminates in postmenopausal women between malignant and benign tumors with an AUC of 0.94

Normalizing to GADPH jeopardises correct quantification of gene expression in ovarian tumours – IPO8 and RPL4 are reliable reference genes

BACKGROUND: To ensure a correct interpretation of results obtained with quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), it is critical to normalize to a reference gene with stable mRNA expression in the tissue of interest. GADPH is widely used as a reference gene in ovarian tumour studies, although lacking tissue-specific stability. The aim of this study was to identify alternative suitable reference genes for RT-qPCR studies on benign, borderline, and malignant ovarian tumours. METHODS: We assayed mRNA levels for 13 potential reference genes – ABL1, ACTB, CDKN1A, GADPH, GUSB, HPRT1, HSP90AB, IPO8, PPIA, RPL30, RPL4, RPLPO, and TBP –with RT-qPCR in 42 primary ovarian tumours, using commercially pre-designed RT-qPCR probes. Expression stability was subsequently analysed with four different statistical programs (GeNorm, NormFinder, BestKeeper, and the Equivalence test). RESULTS: Expression of IPO8, RPL4, TBP, RPLPO, and ACTB had the least variation in expression across the tumour samples according to GeNorm, NormFinder, and BestKeeper. The Equivalence test found variation in expression within a 3-fold expression change between tumour groups for: IPO8, RPL40, RPL30, GUSB, TBP, RPLPO, ACTB, ABL1, and CDKN1A. However, only IPO8 satisfied at a 2-fold change as a cut-off. Overall, IPO8 and RPL4 had the highest, whereas GADPH and HPRT1 the lowest expression stability. Employment of suitable reference genes (IPO8, RPL4) in comparison with unsuitable ones (GADPH, HPRT1), demonstrated divergent influence on the mRNA expression pattern of our target genes − GPER and uPAR. CONCLUSIONS: We found IPO8 and RPL4 to be suitable reference genes for normalization of target gene expression in benign, borderline, and malignant ovarian tumours. Moreover, IPO8 can be recommended as a single reference gene. Neither GADPH nor HPRT1 should be used as reference genes in studies on ovarian tumour tissue

Proteinases and proteinase inhibitors in uterine fluid, with special reference to IUD-users

[abstract missing

Monoamine transporters in human endometrium and decidua.

BACKGROUND Monoamines play important roles in decidualization, implantation, immune modulation and inflammation. Furthermore, monoamines are potent vasoactive mediators that regulate blood flow and capillary permeability. Regulation of the uterine blood flow is important both during menstruation and pregnancy. Adequate monoamine concentrations are essential for a proper implantation and physiological development of pregnancy. Unlike most transmitter substances, monoamines are recycled by monoamine transporters rather than enzymatically inactivated. Their intracellular fate is influenced by their lower affinity for inactivating enzymes than for vesicular transporters located in intracellular vesicles. Thus, cells are capable not only of recapturizing and degrading monoamines, but also of storing and releasing them in a controlled fashion. METHODS The general objective of the present review is to summarize the role of the monoamine transporters in the female human reproduction. Since the transporter proteins critically regulate extracellular monoamine concentrations, knowledge of their distribution and cyclic variation is of great importance for a deeper understanding of the contribution of monoaminergic mechanisms in the reproductive process. MEDLINE was searched for relevant publications from 1950 to 2007. RESULTS Two families of monoamine transporters, neuronal and extraneuronal monoamine transporters, are present in the human endometrium and deciduas. CONCLUSIONS New knowledge about monoamine metabolism in the endometrium during menstruation and pregnancy will increase understanding of infertility problems and may offer new pharmacological approaches to optimize assisted reproduction

Ovarian cancer cells stimulate uPA gene expression in fibroblastic stromal cells via multiple paracrine and autocrine mechanisms.

OBJECTIVES: Expression of uPA mRNA is massively up-regulated in the stroma of poorly differentiated ovarian tumors. We hypothesized that this expression was induced by paracrine signals from the epithelial tumor cells, and established an in vitro model of ovarian cancer microenvironment to study intercellular cross-talk. METHODS: ES-2 clear cell carcinoma cells were grown in tissue culture inserts in a double-chamber system with fibroblastic stromal LEP cells embedded in Matrigel. Binding-site directed antibodies were used to neutralize soluble cytokines in ES-2 conditioned medium (CM) before incubation with LEP cells. Real time PCR measured uPA mRNA in LEP cells, as well as mRNA for cytokines in both cell types. RESULTS: Co-culture with ES-2 cells as well as incubation with ES-2 CM induced uPA mRNA in LEP cells about two-fold. In short time (12 h) incubation of LEP cells with CM, antibodies to EGF and bFGF reduced induction of uPA mRNA, suggesting that these cytokines function as paracrine signals. EGF mRNA and bFGF mRNA were also found in ES-2 cells. At longer incubation (24 h) antibodies to bFGF, HB-EGF, HGF, IGF-1, and IL-1alpha reduced uPA mRNA induction, suggesting an autocrine function for these cytokines in LEP cells. In fact, expression of the same five cytokines was up-regulated in LEP cells exposed to CM. CONCLUSION: We identified two cytokines as paracrine signals, and five cytokines as autocrine signals in ovarian cancer cell induced up-regulation of uPA mRNA in stromal fibroblastic cells. It is crucial to understand intra-tumoral cross-talk, since it can offer new therapeutic approaches

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua.

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focusses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, Western blotting, in situ hybridization, and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase , and even lower in the decidua. The expression pattern was similar to that of ERalpha mRNA, but different from that of ERss mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid and late proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at midcycle as well as decidualization and blastocyst implantation in the mid-secretory phase

Paracrine stimulation of capillary endothelial cell migration by endometrial tissue involves epidermal growth factor and is mediated via up-regulation of the urokinase plasminogen activator receptor

Endometrial angiogenesis is not well studied, but has potential as a model for studies of physiological angiogenesis. Migration as well as proliferation of vascular endothelial cells are modulated by other endometrial cells. This study analyzes the chemotactic signal released from endometrial tissue in a wound assay using human microvascular endothelial cells. Endometrial tissue explants stimulate migration, and this effect is significantly weaker with explants taken at midcycle than those obtained earlier or later in the cycle. Migration is inhibited more than 50% by either blocking antibodies to the urokinase plasminogen activator receptor (uPAR) or enzymatic removal of uPAR from the cell surface. Also, migration is inhibited more than 50% by antibodies to epidermal growth factor (EGF), but not by antibodies to vascular endothelial growth factor or basic fibroblast growth factor. The combination, of anti-EGF and anti-uPAR antibodies does not further reduce the response, suggesting that these antibodies target a common pathway. Conditioned medium from endometrial explants contains EGF, and EGF stimulates the migration of endothelial cells in a dose-dependent way. This effect is completely blocked by antibodies to uPAR. These data suggest up-regulation of the uPA system by EGF. Conditioned medium from EGF-treated cells contains less uPA than medium from control cells. In contrast, binding of radiolabeled uPA reveals an increased number of uPA-binding sites in EGF-treated cells. Increased expression of uPAR potentially increases the activation and assembly of focal adhesion sites, a prerequisite for cell migration. We conclude that the endometrial migratory signal has two components. The major part of the signal is blocked by antibodies to EGF, and the response is mediated via upregulation of uPAR in the endothelial cells. The other part of the signal is unknown, and the response does not involve uPAR. Decreased endometrial chemotactic signal at midcycle is probably related to decreased release of EGF, which is secondary to increased binding to endometrial cell membranes