PGF2α-F-prostanoid receptor signalling via ADAMTS1 modulates epithelial cell invasion and endothelial cell function in endometrial cancer

<p>Abstract</p> <p>Background</p> <p>An increase in cancer cell invasion and microvascular density is associated with a poorer prognosis for patients with endometrial cancer. In endometrial adenocarcinoma F-prostanoid (FP) receptor expression is elevated, along with its ligand prostaglandin (PG)F_2α, where it regulates expression and secretion of a host of growth factors and chemokines involved in tumorigenesis. This study investigates the expression, regulation and role of a disintegrin and metalloproteinase with thrombospondin repeat 1 (ADAMTS1) in endometrial adenocarcinoma cells by PGF_2αvia the FP receptor.</p> <p>Methods</p> <p>Human endometrium and adenocarcinoma tissues were obtained in accordance with Lothian Research Ethics Committee guidance with informed patient consent. Expression of ADAMTS1 mRNA and protein in tissues was determined by quantitative RT-PCR analysis and immunohistochemistry. Signal transduction pathways regulating ADAMTS1 expression in Ishikawa cells stably expressing the FP receptor to levels seen in endometrial cancer (FPS cells) were determined by quantitative RT-PCR analysis. In vitro invasion and proliferation assays were performed with FPS cells and human umbilical vein endothelial cells (HUVECs) using conditioned medium (CM) from PGF_2α-treated FPS cells from which ADAMTS1 was immunoneutralised and/or recombinant ADAMTS1. The role of endothelial ADAMTS1 in endothelial cell proliferation was confirmed with RNA interference. The data in this study were analysed by T-test or ANOVA.</p> <p>Results</p> <p>ADAMTS1 mRNA and protein expression is elevated in endometrial adenocarcinoma tissues compared with normal proliferative phase endometrium and is localised to the glandular and vascular cells. Using FPS cells, we show that PGF2α-FP signalling upregulates ADAMTS1 expression via a calmodulin-NFAT-dependent pathway and this promotes epithelial cell invasion through ECM and inhibits endothelial cell proliferation. Furthermore, we show that CM from FPS cells regulates endothelial cell ADAMTS1 expression in a rapid biphasic manner. Using RNA interference we show that endothelial cell ADAMTS1 also negatively regulates cellular proliferation.</p> <p>Conclusions</p> <p>These data demonstrate elevated ADAMTS1 expression in endometrial adenocarcinoma. Furthermore we have highlighted a mechanism whereby FP receptor signalling regulates epithelial cell invasion and endothelial cell function via the PGF_2α-FP receptor mediated induction of ADAMTS1.</p

Seminal Plasma Enhances Cervical Adenocarcinoma Cell Proliferation and Tumour Growth In Vivo

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A(VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway

Hypoxia and Prostaglandin E Receptor 4 Signalling Pathways Synergise to Promote Endometrial Adenocarcinoma Cell Proliferation and Tumour Growth

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E2. PTGS2 expression and PGE2 biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE2 regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1–4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells

Expression of oestrogen receptors, ERα, ERβ, and ERβ variants, in endometrial cancers and evidence that prostaglandin F may play a role in regulating expression of ERα

<p>Abstract</p> <p>Background</p> <p>Endometrial cancer is the most common gynaecological malignancy; risk factors include exposure to oestrogens and high body mass index. Expression of enzymes involved in biosynthesis of oestrogens and prostaglandins (PG) is often higher in endometrial cancers when compared with levels detected in normal endometrium. Oestrogens bind one of two receptors (ERα and ERβ) encoded by separate genes. The full-length receptors function as ligand-activated transcription factors; splice variant isoforms of ERβ lacking a ligand-binding domain have also been described. PGs act in an autocrine or paracrine manner by binding to specific G-protein coupled receptors.</p> <p>Methods</p> <p>We compared expression of ERs, progesterone receptor (PR) and cyclooxygenase-2 (COX-2) in stage 1 endometrial adenocarcinomas graded as well (G1), moderately (G2) or poorly (G3) differentiated (n ≥ 10 each group) using qRTPCR, single and double immunohistochemistry. We used endometrial adenocarcinoma cell lines to investigate the impact of PGF2α on expression of ERs and PR.</p> <p>Results</p> <p>Full length ERβ (ERβ1) and two ERβ variants (ERβ2, ERβ5) were expressed in endometrial cancers regardless of grade and the proteins were immunolocalised to the nuclei of cells in both epithelial and stromal compartments. Immunoexpression of COX-2 was most intense in cells that were ERα^neg/low. Expression of PR in endometrial adenocarcinoma (Ishikawa) cell lines and tissues broadly paralleled that of ERα. Treatment of adenocarcinoma cells with PGF2α reduced expression of ERα but had no impact on ERβ1. Cells incubated with PGF2α were unable to increase expression of PR mRNA when they were incubated with E2.</p> <p>Conclusion</p> <p>We have demonstrated that ERβ5 protein is expressed in stage 1 endometrial adenocarcinomas. Expression of three ERβ variants, including the full-length protein is not grade-dependent and most cells in poorly differentiated cancers are ERβ^pos/ERα^neg. We found evidence of a link between COX-2, its product PGF2α, and expression of ERα and PR that sheds new light on the cross talk between steroid and PG signalling pathways in this disease.</p

Comparative study of single and multislice computed tomography for assessment of the mandibular canal

OBJECTIVE: The purpose of this study was to evaluate the accuracy of relative measurements from the roof of the mandibular canal to the alveolar crest in multislice (multidetector) computed tomography (MDCT) and single-slice computed tomography (SSCT). MATERIAL AND METHODS: The sample consisted of 26 printed CT films (7 SSCT and 19 MDCT) from the files of the LABI-3D (3D Imaging Laboratory) of the School of Dentistry of the University of São Paulo (FOUSP), which had been acquired using different protocols. Two observers analyzed in a randomized and independent order a series of 22 oblique CT reconstructions of each patient. Each observer analyzed the CT scans twice. The length of the mandibular canal and the distance between the mandibular canal roof and the crest of the alveolar ridge were obtained. Dahlberg test was used for statistical analysis. RESULTS: The mean error found for the mandibular canal length measurements obtained from SSCT was 0.53 mm in the interobserver analysis, and 0.38 mm for both observers. On MDCT images, the mean error was 0.0 mm in the interobserver analysis, and 0.0 and 0.23 mm in the intraobserver analysis. Regarding the distance between the mandibular canal roof and the alveolar bone crest, the SSCT images showed a mean error of 1.16 mm in the interobserver analysis and 0.66 and 0.59 mm in the intraobserver analysis. In the MDCT images, the mean error was 0.72 mm in the interobserver analysis and 0.50 and 0.54 mm in the intraobserver analysis. CONCLUSION: Multislice CT was demonstrated a more accurate method and demonstrated high reproducibility in the analysis of important anatomical landmarks for planning of mandibular dental implants, namely the mandibular canal pathway and alveolar crest height

RNA profiling of cyclooxygenases 1 and 2 in colorectal cancer

Cyclooxygenases (particularily Cox-2) are involved in carcinogenesis and metastatic cancer progression. The expression profiles of the cyclooxygenases and the roles they play in established tumours of similar stage remains unclear. We report that Cox-1 and Cox-2 expression is highly variable in Dukes' C tumours, and changes in Cox-1 expression may be of importance

Angiotensin II Activates the Calcineurin/NFAT Signaling Pathway and Induces Cyclooxygenase-2 Expression in Rat Endometrial Stromal Cells

Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca2+ concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca2+ signals is the activity of the Ca2+- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression -both mRNA and protein- was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression -both mRNA and protein- was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal cells of non-pregnant but not of early-pregnant rats. These results might be related to differential roles that COX-2 plays in the endometrium