Choriocarcinoma Cell Line Research Articles

Divalent Metal Transporter 1 Expression and Regulation in Human Placenta

Divalent metal transporter 1 (DMT1) is likely responsible for the release of iron from endosomes to the cytoplasm in placental syncytiotrophoblasts (STB). To determine the localization and the regulation of DMT1 expression by iron directly in placenta, the expression of DMT1 in human term placental tissues and BeWo cells (human placental choriocarcinoma cell line) was detected and the change in expression in response to different iron treatments on BeWo cells was observed. DMT1 was shown to be most prominent near the maternal side in human term placenta and predominantly in the cytoplasm of BeWo cells. BeWo cells were treated with desferrioxamine (DFO) and human holotransferrin (hTf-2Fe) and it was found that both DMT1 mRNA and protein increased significantly with DFO treatment and decreased with hTf-2Fe treatment. Further, DMT1 mRNA responded more significantly to treatments if it possessed an iron-responsive element than mRNA without this element. This study indicated that DMT1 is likely involved in endosomal iron transport in placental STB and placental DMT1 + IRE expression was primarily regulated by the IRE/IRP mechanism.

Differential expression and the anti-apoptotic effect of human placental neurotrophins and their receptors

Neurotrophin (NT) is important in the survival, maintenance and differentiation of neuronal tissue, and functions in follicle maturation, tumor growth, angiogenesis and immunomodulation; however, the expression of NT and its receptors (NTR) in human placenta and their influence on fetal growth are unclear. Here we investigated the correlation of NT and NTR in human placenta with uterine environment and fetal growth. TrkB, a NTR, mRNA was expressed on decidual and villous tissue and increased with gestational age, localizing in the trophoblast layer and endothelium by immunohistochemistry. Villous TrkB mRNA was significantly increased in preeclampsia (PE) than in controls and was higher in the normotensive small for gestational age (SGA) placenta, although it was not significant. It was also significantly increased in the small twin of discordant twin pregnancies. Brain-derived neurotrophic factor (BDNF), the main ligand of TrkB, was expressed in membranous chorion and villous tissue and was significantly higher in maternal plasma in normotensive SGA and PE than in controls. TrkB mRNA expression was up-regulated on cultured villous tissue explants and on JEG-3, a choriocarcinoma cell line, by H 2O 2 treatment. BDNF decreased apoptotic cells in H 2O 2-treated JEG-3, indicating that BDNF/TrkB signaling had anti-apoptotic effects against oxidative stress in JEG-3, suggesting a protective role of BDNF/TrkB in human villous tissue under unfavorable conditions in utero.

Effects of Bufalin on the Proliferation of Human Choriocarcinoma Cells

Bufalin is a traditional Chinese medicine, and it induces apoptosis in some lines of human tumor cells. We investigated the effect of bufalin in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of bufalin, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that BeWo cells were sensitive to the growth inhibitory effect of bufalin. Cell cycle analysis indicated that exposure to bufalin decreased the proportion of cells in the synthesis phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and by the loss of mitochondrial transmembrane potential. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that bufalin may serve as a therapeutic agent for the treatment of choriocarcinoma.

Impact of CCN3 (NOV) glycosylation on migration/invasion properties and cell growth of the choriocarcinoma cell line Jeg3

Recently we have shown that the matricellular CCN3 protein expressed in invasive extravillous trophoblast cells (EVTs) is decreased in early-onset pre-eclampsia and is regulated by oxygen tension. Pathogenesis of pre-eclampsia relies on a shallow invasion of EVTs into the spiral arteries, which leads to hypoxia accompanied by uteroplacental insufficiency. Here we investigated the function of glycosylated and non-glycosylated CCN3 protein on cell growth as well as migration and invasion properties of the malignant trophoblast cell line Jeg3 which is a widely used model for the invasive trophoblast. Stable transfection of Jeg3 choriocarcinoma cells with full length CCN3 resulted in high expression of secreted glycosylated and cellular non-glycosylated CCN3. These cells revealed significantly reduced growth in cell numbers combined with a significantly increased migratory and invasive capacity. Matrix metalloprotease (MMP)-2 and MMP-9 activities were enhanced dependent on CCN3 expression, which could be confirmed by CCN3 knockdown studies. Using recombinant glycosylated and non-glycosylated CCN3, we revealed that CCN3 decreased growth in Jeg3 cell numbers independent of its glycosylation status, whereas only non-glycosylated CCN3 was able to enhance migration and invasion properties. The present results suggest that CCN3 protein regulates the decrease in Jeg3 cell numbers independent of its glycosylation status, whereas migratory and invasive properties are influenced only by non-glycosylated CCN3. An impaired balance in the expression of glycosylated and non-glycosylated CCN3 could contribute to the shallow invasion of EVTs observed in pre-eclampsia.

Genistein inhibits placental choriocarcinoma cell line JAR invasion through ERβ/MTA3/Snail/E-cadherin pathway

Genistein, the most abundant phytoestrogen in soybeans, may bind to estrogen receptors and perform anticancer activities. Choriocarcinoma is a malignant, trophoblastic and aggressive cancer of the placenta. Few studies are currently available concerning the effects of genistein on choriocarcinoma. In the present study, we investigated the effect of genistein on the invasive potential of the choriocarcinoma cell line JAR and its underlying mechanism. Our data revealed that genistein inhibited JAR cell invasion in a dose-dependent manner by a matrigel invasion assay. Moreover, genistein was found to have decreased the metastasis-associated gene MTA3 mRNA level, increased the transcriptional suppressor Snail mRNA level and upregulated the protein expression of the cell-cell adhesion molecule E-cadherin by real-time RT-PCR and Western blot analysis, respectively. ERβ siRNA was used to knock down ERβ expression in JAR cells. In the ERβ-knockdown JAR cells, genistein failed to inhibit JAR cell invasion. The effects of genistein on MTA3, Snail and E-cadherin expression were also eradicated following ERβ siRNA transfection. These results demonstrated that genistein triggered the MTA3/Snail/E-cadherin regulatory pathway by binding with ERβ, thereby inhibiting JAR cell invasion. In conclusion, our findings have significant implications for the prevention and therapy of choriocarcinoma.

Reduction in miR‐141 is Induced by Leukemia Inhibitory Factor and Inhibits Proliferation in Choriocarcinoma Cell Line JEG‐3

Starting from the peri-implantation period, leukemia inhibitory factor (LIF) is a major regulator of trophoblast functions. Micro-RNAs (miRNA) are short non-coding RNA sequences, which regulate expression of genes at post-transcriptional level. The influence of LIF on miRNA expression in trophoblastic cells has not yet been analyzed and was focus of this investigation. JEG-3 choriocarcinoma cells have been stimulated with LIF for 1, 2, 4, 6, and 24 hr. The expression of miR-9, miR-141, miR-21, miR-93, and let-7 g has been analyzed by real-time PCR. Subsequently, miR-141 has been silenced and over-expressed to test its role in the proliferation of JEG-3 cells after 24 and 48 hr. MiR-141 has been significantly downregulated by more than 50% after LIF stimulation, while miR-21 and miR-93 expression has been significantly upregulated. Silencing of miR-141 completely inhibited the proliferation of JEG-3 cells, while over-expression had no effect. LIF regulates expression of miRNA in trophoblastic cells, which may be responsible for several functional effects induced by LIF.

Adrenomedullin Stimulates Human Trophoblast Invasion Through the Upregulation of Nitric Oxide and Urokinase Plasminogen Activator Activity.

Extravillous cytotrophoblasts (EVCTs) invasion into the uterine endometrium is important to placentation and successful pregnancy. Dysregulation of the process is associated with a wide range of pregnancy complications, like intrauterine growth restriction, choriocarcinoma and preeclampsia. EVCTs produce urokinase plasminogen activator (uPA) that activates plasmin to degrade the extracellular matrix of the endometrium for invasion. Adrenomedullin is a 52-amino acid polypeptide belonging to the calcitonin gene-related peptide family which has diverse physiological functions including vasodilation, differentiation, hormone secretion and cell invasion. Nitric oxide (NO) signaling has been implicated to play a role in the biological functions of adrenomedullin in different cell types. In pregnant women, adrenomedullin is most abundantly expressed in the first-trimester placenta when the EVCTs exhibit the maximal invasive behavior, suggesting a possible role of the peptide in EVCT invasion during early pregnancy. The objective of this study is to investigate the effect of adrenomedullin on human EVCT invasion. JEG3, a choriocarcinoma cell line derived from first trimester human EVCT, was used as the study model. Adrenomedullin treatment significantly enhanced the invasiveness and uPA expression and activity of the JEG3 cells as demonstrated by transwell invasion assay, RT-PCR and uPA activity assay respectively. Immunostaining, western blotting and PCR demonstrated the presence of adrenomedullin receptor components in JEG3 cells. We further demonstrated that the stimulatory effect of adrenomedullin on trophoblast invasion is mediated by NO signaling pathway. Adrenomedullin treatment was shown to increase the NO production and nitric oxide synthase (NOS) expression in JEG3 cells. NOS inhibitors significantly suppressed the stimulatory effect of adrenomedullin on JEG3 invasion, uPA expression and activity. In contrast, NO donors were able to mimic the biological activities of adrenomedullin. The present results suggest that adrenomedullin enhances EVCT invasion by up-regulating the uPA expression and activity through a NO-dependent manner. This work was supported in part by grants from the Small Project Funding, The University of Hong Kong, Hong Kong (ref: 201007176081). (poster)

Cytotoxicity of tubeimoside I in human choriocarcinoma JEG-3 cells by induction of cytochrome c release and apoptosis via the mitochondrial-related signaling pathway

Mitochondria play important roles in the intrinsic pathways that trigger apoptosis. Anticancer chemotherapies eliminate cancer cells mainly through the induction of apoptosis. In the present study, we investigated the mechanism of the cytotoxic effects of tubeimoside I (TBMS1) on the human choriocarcinoma cell line (JEG-3). Choriocarcinoma is one of the most common malignant tumors in the reproductive system. TBMS1, a triterpenoid saponin, isolated from the tubers of Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), showed potent antitumor effects. However, the potential roles of TBMS1 in the treatment of choriocarcinoma remain unknown. In the present study, we examined the effects of TBMS1 on JEG-3 cells. TBMS1 displayed strong growth inhibitory effects on JEG-3 cell growth. In addition, TBMS1 treatment with TBMS1 led to marked cell apoptosis, significant cell cycle arrest at G2 phase and decrease in mitochondrial transmembrane potential (ΔΨm). Cytochrome c was released from the mitochondria and caspase-3 expression was enhanced. Furthermore, TBMS1 induced the up-regulation of Bcl-2 associated X protein (Bax) expression, down-regulation of Bcl-2 expression, inhibition of nuclear factor-κ-B (NF-κB) function and impacted the phosphorylation of p38 mitogen-activated protein kinase (p38/MAPK), extracellular signal-regulated kinases (ERK)1/2 and protein kinase B (Akt). Taken together, our findings suggest that TBMS1 is an efficient apoptosis-inducing agent for choriocarcinoma cells, which exerts its effects, at least partially, by the induction of mitochondrial dysfunction and regulation of the p38/MAPK, ERK1/2 and PI3K/Akt signaling pathways.

Retinoid X receptor α and retinoids are key regulators in apoptosis of trophoblasts of patients with recurrent miscarriages

The retinoid X receptor α (RXRα) is a nuclear hormone receptor that is able to bind other nuclear receptors in a heterodimeric complex, thereby activating gene transcription. Recently, we identified enhanced expression of RXRα in extravillous trophoblasts (EVT) and villous trophoblasts (VT) of miscarried placentas. In addition, an increased number of apoptotic EVT was present in miscarried placentas. In this study, on the basis of immunocytochemical analysis, western blots, and quantitative real-time reverse transcription PCR, we could demonstrate a reduced expression of RXRα in choriocarcinoma cell lines and in human VTs after stimulation with the retinoids 9-cis-retinoic acid and all-trans-retinoic acid and the prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J(2). Furthermore, a simultaneous expression of RXRα and the apoptotic marker M30 CytoDEATH in EVT of miscarried placentas from the first trimester was shown. In EVT of control placentas from legal termination of pregnancies, no co-expression of RXRα and M30 could be detected. A likely conclusion is that RXRα plays an important role in the induction of apoptosis. Downregulation of RXRα, as observed in the tested choriocarcinoma cells and trophoblasts, might serve as a protection against apoptosis and miscarriage. In conclusion, RXRα represents a potential target in the treatment of recurrent miscarriages.

Aryl Hydrocarbon Receptor and Aryl Hydrocarbon Nuclear Translocator Expression in Human and Rat Placentas and Transcription Activity in Human Trophoblast Cultures

Aryl hydrocarbon receptor (AHR) and its heterodimer aryl hydrocarbon nuclear translocator (ARNT) form a ligand-activated transcription complex that regulates expression of the AHR battery of target genes that includes the most important placental biotransformation enzyme cytochrome CYP1A1. Expression, placental localization, and ontogeny of AHR/Ahr and ARNT/Arnt have not been systematically studied in either human or rat placentas. Moreover, induction of such AHR target genes as CYP1A1, CYP1A2, CYP1B1, UGT1A1, and breast cancer resistance protein (BCRP), as well as of AHR, ARNT, and aryl hydrocarbon receptor repressor (AHRR) genes, after exposure to AHR ligands have not been studied in human placental trophoblast cultures. In this article, we show that only CYP1A1 messenger RNA (mRNA), but not CYP1A2, CYP1B1, UGT1A1, BCRP, AHR, ARNT, and AHRR mRNAs, is significantly induced in human term placental trophoblast cultures after exposure to prototype AHR ligands/activators 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, omeprazole, and β-naphthoflavone. We localized AHR/Ahr and ARNT/Arnt in rat placental trophoblasts throughout gestation and in first trimester and term human placental trophoblast, which comprise the crucial component of the maternal-fetal barrier. We demonstrate that rat Ahr and Cyp1a1 reached highest expression during gestation days 15 and 18, which might indicate different response to Ahr ligands in placental Cyp1a1 induction during rat gestation. We also propose the JEG3 choriocarcinoma cell line as a cellular model for human trophoblast induction studies through AHR. In conclusion, we describe expression and ontogeny of AHR/Ahr and ARNT/Arnt and systematically characterize induction of major AHR target genes in human placental trophoblast forming the placental maternal-fetal morphological and metabolic barrier.

Pituitary choriocarcinoma in an adolescent male: Tumor-derived CG and GH delay diagnosis

BackgroundPrimary intracranial germ cell tumors usually present in the first two decades of life, often with precocious puberty. The most common location is in the pineal gland; suprasellar germ cell tumors are rare. We present an additional case of a suprasellar choriocarcinoma producing GH, and review the literature. CaseThis French Canadian, 17 year-old male presented to the ER with a history of mild weight loss and an episode of syncope while hiking in Mexico, but with no other neurological symptoms. Puberty began at age 13years (growth spurt: 15–16years), and he attained an adult height within genetic target by age 16years. Past medical history was negative except for myopia diagnosed during childhood. System review revealed increased thirst and nocturia. The mother was treated for an oligo-astrocytoma in 2007. Clinical examination showed a euthyroid, well-looking young man with 20ml testicles. Endocrine evaluation revealed elevated testosterone, mildly elevated PRL, borderline low FT4, and decreased IGF-I, morning cortisol and urine osmolality; tumor markers were positive in serum and CSF (hCG>50IU/L, AFP>10ng/mL). A transphenoidal biopsy of a 4.5cm, homogeneous, non-calcified, suprasellar mass was compatible with the diagnosis of choriocarcinoma and stained intensely for hCG and hGH, presumably the placental variant (GH-V) as previously found in vitro in choriocarcinoma cell lines. Combined chemotherapy and irradiation led to tumor regression and undetectable serum hCG to 36months of follow-up. He is doing well with no evidence of tumor progression and is on complete hormone replacement therapy. ConclusionsChoriocarcinomas can have a hormonal profile that delays the development of symptoms, due to hCG stimulation of both the gonadal and thyroid axes. This report corroborates previous in vitro evidence that choriocarcinoma cells are able to make GH-V. To what extent the patient's tumor-derived GH contributed to his normal growth is not known. Prognosis for this intracranial neoplasm is very reserved, although combined radiotherapy and chemotherapy has been successful in our patient now 36months post-diagnosis.

Effects of intermittent high glucose on BeWo choriocarcinoma cells in culture

The aim of this study was to investigate the cellular effects of intermittent high glucose on the human BeWo placental choriocarcinoma cell line, used as a model of the effects of glucose fluctuation in diabetic pregnancies. BeWo cells were subjected to three different glucose conditions for 48 h: 7 mmol/L (control), 42 mmol/L (high glucose), or 7 and 42 mmol/L glucose (intermittent, alternated every 6 h). Cell viability was assessed using cell counts, a cell proliferation assay, and a cell viability assay. Apoptosis was also studied using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and by immunocytochemistry of fractin, the N-terminal fragment of actin, which can distinguish apoptotic from necrotic cells. Furthermore, the expression of the well-known survival factors of trophoblast cells, heparin-binding epidermal growth factor-like growth factor and leptin, was evaluated by real-time polymerase chain reaction and Western blot analyses. Intermittent high-glucose conditions significantly decreased cell viability and enhanced apoptosis compared with control or continuous high-glucose conditions. Furthermore, the expression of heparin-binding epidermal growth factor-like growth factor, but not that of leptin, was significantly increased under intermittent high-glucose conditions compared to its expression under either control or continuous high-glucose conditions. These data indicate that intermittent high glucose is more deleterious to BeWo cells than continuous high-glucose conditions. Although further in vitro and in vivo study is necessary, excess fluctuation of glucose levels in the placental circulation might be involved in the impairment of placental development leading to the placental dysfunction.

Effects of anti‐β2‐glycoprotein I antibody on PlGF, VEGF and sVEGFR1 production from cultured choriocarcinoma cell line

Anti-β2-glycoprotein I (anti-β2-GPI) antibody has been detected in cases of recurrent abortion and intrauterine death. Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) are growth factors that accelerate villous proliferation. Soluble VEGF receptor-1 (sVEGFR1) is a soluble receptor for PlGF and suppresses the function of PlGF. In order to study the pathological mechanism of anti-β2-GPI antibody, we evaluated the effects of anti-β2-GPI antibody on PlGF, VEGF and sVEGFR1 production from cultured choriocarcinoma cells. The sera were taken from six anti-β2-GPI antibody-positive and six anti-β2-GPI antibody-negative patients with recurrent miscarriages. Choriocarcinoma cells (JEG-3) were cultured in 24-well plates. After each serum and isolated immunoglobulin G (IgG) were added to the culture medium, the PlGF, VEGF and sVEGFR1 in the culture medium were measured by ELISA 24 h later. When the isolated IgG was added to culture medium, only the levels of PlGF were measured. The anti-β2-GPI antibody-positive sera and isolated IgG significantly suppressed the production of PlGF from JEG-3 cells more strongly than the anti-β2-GPI antibody-negative sera. The suppressive effects were not changed by serum inactivation. There was no significant difference in the values of the XTT assay and the production of VEGF and sVEGFR1 between the antibody-positive and antibody-negative sera. Anti-β2-GPI antibody-positive sera and IgG suppress the production of PlGF from JEG-3 cells. We suggest that the anti-β2-GPI antibodies may suppress PlGF production from trophoblasts and cause the failure of placenta formation and function.

Abstract 1596: Human choriocarcinomas: Placental growth factor-dependent preclinical tumor models

Abstract Choriocarcinomas are an aggressive form of cancer that develops in the uterus from tissue that would normally become the placenta. Choriocarcinomas are a trophoblastic gestational disease and the tumors are highly angiogenic. Human placental growth factor (hPlGF) is an angiogenic growth factor that promotes the growth of blood vessels in the placenta. hPlGF levels can be higher under pathological conditions such as cancer. Some human tumor specimens have higher PlGF levels than normal tissue, thus hPlGF is a target for anti-angiogenic therapy. The human choriocarcinoma cell lines BeWo, JAr, and JEG-3 were evaluated as preclinical models of hPlGF overexpression. The levels of hVEGF-A and hPlGF secreted by the choriocarcinomas and 11 human and mouse cancer cell lines in culture were measured by ELISA. The choriocarcinomas secreted higher levels of hPlGF and lower levels of hVEGF-A than 10/11 other cancer cell lines. The hPlGF serum levels in mice bearing subcutaneous choriocarcinoma tumors correlated with tumor volume and reached >2,000 pg/ml. By comparison, no hPlGF was detected in the serum of mice bearing H460 non-small lung carcinoma, HT29 colon carcinoma or MOLM-13 acute myeloid leukemia xenograft tumors. Although choriocarcinoma cells secreted both hPlGF and hVEGF-A in culture, serum levels of hVEGF-A in mice bearing choriocarcinoma xenografts were undetectable or <50 pg/ml at the time points serum was collected indicating that angiogenic factor secretion by the cells in culture was not the same in vivo. JEG-3 tumors grew faster than JAr tumors following the subcutaneous implantantion of 1×106 cells into nude mice. BeWo tumors grew the slowest and had the highest serum PlGF levels. Immunohistochemical methods confirmed expression of hPlGF in the choriocarcinoma xenografts. Studies involving double immunofluorescence on the xenograft tumors are ongoing to investigate endothelial cell and pericyte interactions and stromal involvement in the tumors. sFLT01, a novel fusion protein that neutralizes PlGF and VEGF-A, was added to choriocarcinoma cells in culture and administered to mice bearing choriocarcinoma xenografts in efficacy studies. Exposure of JAr and JEG-3 cells in culture to sFLT01 resulted in a phenotypic change whereby the cells produced less hPlGF and no significant hVEGF-A. In vivo, sFLT01 (25 mg/kg, IP injection, 2x per week) slowed tumor growth in JAr and JEG-3 tumors and increased median survival by 5-30 days compared to vehicle controls. These results indicate that human choriocarcinoma xenograft models are a valuable research tool for evaluating anti-angiogenic agents that target hPlGF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1596. doi:10.1158/1538-7445.AM2011-1596

Abstract 191: Dysregulated FBI-1 in gestational trophoblastic neoplasms

Abstract Background: Human placental trophoblast is considered as a pseudo-malignant tissue. Gestational trophoblastic disease (GTD) includes true malignancies such as choriocarcinomas and potentially malignant hydatidiform moles, which may develop into persistent gestational trophoblastic neoplasms requiring chemotherapy. FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor which can inactivate p53 through the ARF-MDM2-p53 pathway. This study investigated the expression and functional effects of FBI-1 in choriocarcinomas as compared with normal placenta and hydatidiform moles. Methods and Results: Twelve choriocarcinomas, 33 placentas and 53 hydatidiform moles were studied. Quantitative PCR and immunohistochemical analysis demonstrated higher FBI-1 mRNA and protein expression in choriocarcinoma and hydatidiform moles when compared with normal placentas (p<0.05). Significantly higher FBI-1 expression was found in hydatidiform moles that progressed to persistent gestational trophoblastic neoplasms than in those regressed (P=0.032). FBI-1 immunoreactivity correlated significantly with that of p53, p21WAF1/CIP1 and PTEN (P<0.05). p21WAF1/CIP1 is a cyclin dependent kinase and downstream effector of p53 while PTEN is another tumour suppressor that physically and genetically interacts with p53. Ectopic expression of FBI-1 in choriocarcinoma cells, JEG-3 and JAR, inhibited apoptosis in vitro as revealed by TUNEL assays and repressed mRNA expressions of apoptotic genes in both intrinsic and extrinsic pathways including Bak, Fas and caspase-8. Proliferation and cell invasion/migration enhancing effects was found by MTT assay and transwell assays after FBI-1 transfection. The mechanisms governing increased expression of FBI-1 in choriocarcinoma were investigated. Histone deacetylase inhibitor Trichostatin A was found to induce FBI-1 mRNA expression in both JEG-3 and JAR choriocarcinoma cell lines. A borderline correlation between RNA level and DNA copy of FBI-1 expression was also detected while FBI-1 expression was not affected by treatment with MG132, an ubiquitination inhibitor. Due to the observed inhibitory effect of FBI-1 on apoptosis, possible interaction between FBI-1 and other p53 related proteins was further investigated. iASPP is a recently identified wild-type p53 binding protein that inhibits p53-dependent apoptosis. Anti-FBI-1 was able to pull-down iASPP in JEG-3 as demonstrated by co-immunoprecipitation study, indicating possible interaction between these two transcriptional regulators. Conclusions: FBI-1 upregulation was associated with frankly malignant choriocarcinoma and hydatidiform moles of aggressive behaviour. Aberrant FBI-1 expression, related to histone acetylation and gene amplification, displays diverse effects on cell proliferation, apoptosis and invasion probably thorough interaction in transcriptional complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 191. doi:10.1158/1538-7445.AM2011-191

Genipa americana(Rubiaceae) Fruit Extract Affects Mitogen-Activated Protein Kinase Cell Pathways in Human Trophoblast–Derived BeWo Cells: Implications for Placental Development

Genipa americana L. (Rubiaceae) is a fruit tree and a traditional medicine used to treat anemia, icterus, asthma, and liver and spleen problems. The aim of the present study was to verify the effect of G. americana fruit ethanolic extract on the mechanism for proliferation and differentiation of trophoblast-like cells. Qualitative analysis of G. americana fruit extract was performed, and BeWo cells, a well-established placental choriocarcinoma cell line that can undergo differentiation, were used to analyze cell viability and proliferation. Methods consisted of cytotoxic and proliferation measurement, detection of release of human chorionic gonadotrophins, cell fusion observation, and evaluation of cell-signaling pathways (production of cyclic adenosine monophosphate and phosphorylation of mitogen-activated protein kinases [MAPKs]). A stock solution of the extract was diluted in Ham's F-12 medium with 10% fetal bovine serum at concentrations ranging from 50 to 1000 μg/mL. Cells treated with dimethylsulfoxide, forskoline, and MAPK inhibitors (PD98059 or SB203580) were used as a control. Forskoline was used to induce the differentiation state in BeWo cells. Phytoanalysis indicated the presence of steroids only. Results showed that the G. americana fruit extract did not cause any cytotoxicity or interference in cell differentiation. However, a significant antiproliferative state related to inhibition and reactivation of ERK1/2 and p38 MAPK in BeWo cells was seen. These results suggest that steroids from G. americana may affect placental cell regulation.

Protein trafficking‐ a road map for embryogenesis

Transmembrane Emp24 domain trafficking protein (TMED) 2, a member of the p24 family of chaperones is involved in protein transport between the ER and Golgi. TMED2 complexes with TMED7, TMED9 and TMED10 and is required for normal levels of these proteins. Loss of Tmed2 results in delayed and abnormal development of homozygous mutant mouse embryos and placentas, loss of TMED7 and TMED10, and decreased expression of TMED9 protein. In Tmed2 mutant embryos, the labyrinth layer of the placenta - the site of gas and nutrient exchanges between the mother and the fetus - fails to form. Formation of the labyrinth layer depends on proper differentiation of the syncytiotrophoblast cells that separate the maternal and fetal environment. The choriocarcinoma cell lines BeWo and JEG-3 were used to examine TMED2 expression and function during trophoblast differentiation. These cells share some properties in terms of biochemical markers and hormone secretion. However, only BeWo cells fuse to form syncytiotrophoblasts. The aim of this study was to determine if TMED2 was expressed in human placenta and was required during trophoblast differentiation. In 1st trimester placentas, TMED2 was expressed in cytotrophoblast stem cells and in differentiated syncytiotrophoblasts. In the choriocarcinoma cell lines, we observed 60-fold more TMED2 mRNA and protein levels in BeWo when compared with JEG-3 cells. BeWo cells also expressed significantly higher levels of TMED7 and TMED10 mRNA. TMED2 was localized to the ER-Golgi intermediate compartment and was upregulated during differentiation of BeWo cells into syncytiotrophoblasts. Our results show that TMED2, TMED7, and TMED10 are highly expressed in human placentas and suggest a role for these proteins during syncytiotrophoblast differentiation. This work was supported by operating grants from NSERC and CIHR.

Downregulation of ASPP1 in gestational trophoblastic disease: correlation with hypermethylation, apoptotic activity and clinical outcome

Gestational trophoblastic disease encompasses a spectrum of trophoblastic lesions including true neoplasms such as choriocarcinomas and the potentially malignant hydatidiform moles, which may develop persistent disease requiring chemotherapy. ASPP1, a member of apoptosis-stimulating proteins of p53 (ASPPs), is a proapoptotic protein that can stimulate apoptosis through its interaction with p53. We evaluated the promoter methylation and expression profiles of ASPP1 in different trophoblastic tissues and its in vitro functional effect on two choriocarcinoma cell lines, namely JEG-3 and JAR. Significant downregulation of ASPP1 mRNA and protein levels was demonstrated in hydatidiform moles and choriocarcinomas, when compared with normal placentas by quantitative-PCR and immunohistochemistry. The ASPP1 mRNA level was significantly correlated with its hypermethylation status, evaluated with methylation-specific PCR, in placenta and gestational trophoblastic disease samples (P=0.024). Moreover, lower ASPP1 immunoreactivity was shown in hydatidiform moles that progressed to persistent gestational trophoblastic neoplasms than in those that regressed (P=0.045). A significant correlation was also found between expression of ASPP1 and proliferative indices (assessed by Ki67 and MCM7), apoptotic activity (M30 CytoDeath antibody), p53 and caspase-8 immunoreactivities. An in vitro study showed that ectopic expression of ASPP1 could trigger apoptosis through intrinsic and extrinsic pathways as indicated by an increase in cleaved caspase-9 and Fas ligand protein expression. The latter suggests a hitherto unreported novel link between ASPP1 and the extrinsic pathway of apoptosis. Our findings suggest that downregulation of ASPP1 by hypermethylation may be involved in the pathogenesis and progress of gestational trophoblastic disease, probably through its effect on apoptosis.

Expression of membrane and nuclear progesterone receptors in two human placental choriocarcinoma cell lines (JEG-3 and BeWo): Effects of syncytialization

A vital function of the human placenta is to produce steroid hormones such as progesterone, which are essential for the maintenance of pregnancy and the onset of parturition. Although choriocarcinoma cell lines are valuable placental models for investigations of steroid hormone actions, little is known about the expression of progesterone receptors (PRs) in these cell lines. Therefore, in this study, the expression of membrane and nuclear PRs was investigated in cultures of fusigenic (BeWo) and non-fusigenic (JEG-3) human choriocarcinoma cell lines. In addition, the effects of an inducer of syncytialization (forskolin) on the PR expression in BeWo cells were assessed. Quantitative RT-PCR revealed that in fully syncytialized BeWo cells (treated with 50 µM forskolin for 72 h) there was a significant down-regulation of mPRα and up-regulation of mPRβ and of the progesterone membrane component-1 (PGRMC1) when compared with non-syncytialized BeWo cells. Expression of all the mPR and PGRMC1 mRNAs was significantly lower in JEG-3 cells compared to non-syncytialized BeWo cells. Interestingly, expression of PR-B was unaltered between the two BeWo states but was significantly higher in JEG-3 cells. Immunofluorescence analysis revealed that mPR proteins are differentially expressed in these choriocarcinoma cell lines as well as in the human placenta. The data demonstrate that human choriocarcinoma cell lines have a complex system of progesterone signalling involving multiple classes of PRs. The finding that syncytialization is accompanied by changes in the expression of these receptors may suggest that this process influences progesterone signalling.

Molecular Regulation of Human Placental Growth Factor (PlGF) Gene Expression in Placental Villi and Trophoblast Cells is Mediated via the Protein Kinase A Pathway

Cyclic 3',5'-adenosine monophosphate (cAMP) is a critical second messenger for human trophoblasts and regulates the expression of numerous genes. It is known to stimulate in vitro the fusion and differentiation of BeWo choriocarcinoma cells, which acquire characteristics of syncytiotrophoblasts. A DNA microarray analysis of BeWo cells undergoing forskolin-induced syncytialization revealed that among the induced genes, placental growth factor (PlGF) was 10-fold upregulated. We verified this result in two choriocarcinoma cell lines, BeWo and JEG-3, and also in first trimester placental villous explants by quantifying PlGF mRNA (real time PCR) and PlGF protein secreted into the supernatant (ELISA). Similar effects were noted for vascular endothelial growth factor (VEGF) mRNA and protein expression. Treatment with cholera toxin and the use of a specific inhibitor of protein kinase A (PKA) blocked these effects, indicating that the cAMP/PKA pathway is responsible for the cAMP-induced upregulation of PlGF and that one or more G protein coupled receptor(s) was involved. We identified two functional cAMP responsive elements (CRE) in the PlGF promoter and demonstrated that the CRE binding protein, CREB, contributes to the regulation of PlGF gene expression. We speculate that defects in this signaling pathway may lead to abnormal secretion of PlGF protein as observed in the pregnancy-related diseases preeclampsia and intrauterine growth restriction.