Human Placental Choriocarcinoma Cell Line Research Articles

Melatonin induces PERK-ATF4 unfolded protein response and apoptosis in human placental choriocarcinoma cell line

Melatonin induces PERK-ATF4 unfolded protein response and apoptosis in human placental choriocarcinoma cell line

Disruption of p97/VCP induces autophagosome accumulation, cell cycle arrest and apoptosis in human choriocarcinoma cells.

Gestational choriocarcinoma is aggressive trophoblastic disease. The development, progression and the cure of this disease is not well-established. p97/Valosin containing protein has been shown to play critical roles in many cellular processes. In various cancers, higher expression of p97/VCP has been reported and targeting of p97/VCP with its spesific inhibitors or siRNA's (siVCP) in cancer therapy was suggested. However, no study is avaible about the expression and function of p97/VCP in gestational choriocarcinoma. Hence, the aim of the study was to evaluate effects of p97/VCP inhibitor, DBeQ and siVCP on choriocarcinoma cells. We use human placental choriocarcinoma cell line (Jeg3) as model to find out the effects of DBeQ and VCP siRNA's (siVCP) on apoptotic and autophagic pathway by immunflouroscence staining, Western blotting, qPCR and flow-cytometry. p97/VCP siRNA's and DBeQ induced accumulation of autophagic proteins, LC3II and p62 in the cytoplasm of Jeg3 cells detected. Concurrently, Jeg3 cells treated with DBeQ and siVCP demonstrated G0/G1 cell cycle arrest, accompanied by accumulation of poly-ubiquitinated proteins. Moreover, disruption of p97/VCP by siRNA and DBeQ inhibited cancer cell growth managing the caspases-3 and -7. Our results show that inhibition of p97/VCP activity with DBeQ and depletion of p97/VCP expression with siRNA in Jeg3 cells induce caspase activation, inhibits cell proliferation and leads to a defect in autophagosome maturation, thus providing potential target for the prevention and treatment of choriocarcinoma.

Expression of p57KIP2 reduces growth and invasion, and induces syncytialization in a human placental choriocarcinoma cell line, BeWo

IntroductionSyncytiotrophoblasts are the major components of the human placenta involved in fetal maternal exchange and hormone secretion. The syncytiotrophoblasts arise from the fusion of villous cytotrophoblasts. The cell cycle suppressor p57KIP2 is known to be an essential molecule for proper trophoblast differentiation during placental formation. MethodsWe generated p57KIP2-expressing BeWo transfectant cells. Proliferation assay and matrigel invasion assay were used to characterize p57KIP2-expressing BeWo transfectant cells. To reveal the role of p57KIP2 in syncytialization, we proceeded syncytium formation analysis and qRT-PCR for detection of the expression levels Syncytin-1, Syncytin-2 and their receptors. ResultsThe human choriocarcinoma cell line, BeWo has undetectable levels of p57KIP2 expression. Expression of p57KIP2 reduced cell proliferation rate and extracellular matrix invasion activity. p57KIP2 expressing cells displayed multinucleated cells associated with syncytiotrophoblast differentiation. In the syncytialization event, p57KIP2 was found to potentiate forskolin-induced upregulation of Syncytin-2 in a cAMP-independent manner. DiscussionThese results indicate that the expression of p57KIP2 may act on the proliferation/invasion inhibitory factor and enhance the expression of Syncytin-2, which are associated with syncytialization in cytotrophoblasts.

Bavachin suppresses human placental choriocarcinoma cells by targeting electron transport chain complexes and mitochondrial dysfunction

Phytoestrogens are naturally derived estrogen-like therapeutic compounds that have long been studied for their role as anti-cancer agents and supplements during chemotherapy. Bavachin is a therapeutic phytoestrogen used to treat cancer, inflammation, and diabetes mellitus. Though the therapeutic effects of bavachin on various diseases have been explored, its anti-cancer effects and related mechanisms in human placental choriocarcinoma remain unknown. This is the first study to identify the anti-cancer potential of bavachin on human placental choriocarcinoma cell lines JEG3 and JAR. Placental mitochondria support the elevated energy production required for placental development, through oxidative phosphorylation (OXPHOS). Based on this concept, we hypothesized that mitochondrial targeting by bavachin may contribute to anti-cancer activities in high-OXPHOS subtypes of cancer such as placental choriocarcinoma. Apoptosis and caspase activities were increased by bavachin in placental choriocarcinoma cells. Bavachin altered metabolic phenotypes by regulating electron transport chain complex and OXPHOS to suppress choriocarcinoma cell proliferation. It also led to calcium disruption and endoplasmic reticulum stress accompanied by mitochondrial membrane potential depolarization. It showed synergistic anti-cancer effects with paclitaxel on placental choriocarcinoma cells. Taken together, we suggest that bavachin has therapeutic potential against placental choriocarcinoma and may be used to counter paclitaxel-induced toxicity.

Maternal dexamethasone exposure suppresses placental iron transport through GR‐mediated TFR1 pathway in rats

Iron is one of the essential trace elements for almost all living organisms, and of course it is especially important for pregnancy and fetal development. However, inappropriately low or high levels of iron during pregnancy are detrimental and contribute to serious and lasting effects on the fetus, such as fetal growth and development disorders, thereby increasing the risk of adulthood. Here we stress pregnant rats by drinking water with 10−7 mol/L dexamethasone, and then explore its effects on iron metabolism of placenta and fetal rats. The results showed that dexamethasone treatment inhibited placental development, down‐regulated the expression of placental iron transporter TFR1, and decreased iron levels in fetal rats. Furthermore, we use a human placental choriocarcinoma cell line (BeWo) as model to explore the pathway linking inhibit of TFR1 under Dex challenge, our results demonstrate that the decrease in TFR1 caused by dexamethasone is alleviated by the addition of dexamethasone inhibitor RU486. Taken together, our results show that dexamethasone downregulate TFR1 expression adjust iron metabolism via GR‐mediated.Support or Funding InformationThis work was supported by grants from the National Key Research and Development Program of China (2016YFD0500502), the National Natural Science Foundation of China (31872439), the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control.

Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells.

Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor pathways. Hypoxia/reoxygenation (H/R) induces oxidative damage and apoptosis. These consequences activate autophagy, which degrades damaged cellular content, as well as activates Nrf2 the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor, and thereby the expression of protective genes. Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and Nrf2 pathways. The current study shows melatonin to differentially modulate autophagy and Nrf2 pathways in tumor and normal placental cells exposed to H/R. BeWo, a human placental choriocarcinoma cell line, and primary villous cytotrophoblasts isolated from normal term placenta, were maintained in normoxia (8% O2) for 24 h or exposed to hypoxia (0.5% of O2 for 4 h) followed by 20 h of normoxia, creating a situation of H/R, in the presence or absence of 1 mM melatonin. Melatonin induced a 7-fold increase in the activation of 5' adenosine monophosphate-activated protein kinase (AMPK)α, an upstream modulator of autophagy, rising to a 16-fold increase in BeWo cells co-exposed to H/R and melatonin, compared to controls. H/R induced autophagosome formation via the increased expression of Beclin-1 (by 94%) and ATG7 (by 97%) in BeWo cells. Moreover, H/R also induced autophagic activity, indicated by the by the 630% increase in P62, and increased Nrf2 by 314% in BeWo cells. In H/R conditions, melatonin reduced autophagic activity by 74% and Nrf2 expression activation by 300%, leading to BeWo cell apoptosis. In contrast, In human primary villous cytotrophoblasts, H/R induced autophagy and Nrf2, which melatonin further potentiated, thereby affording protection against H/R. This study demonstrates that melatonin differentially modulates autophagy and the Nrf2 pathway in normal vs. tumor trophoblast cells, being cytoprotective in normal cells whilst increasing apoptosis in tumoral trophoblast cells.

Coumestrol induces mitochondrial dysfunction by stimulating ROS production and calcium ion influx into mitochondria in human placental choriocarcinoma cells.

Does coumestrol inhibit proliferation of human placental choriocarcinoma cells? Coumestrol promotes cell death in the choriocarcinoma cells by regulating ERK1/2 MAPK and JNK MAPK signaling pathways and through disruption of Ca2+ and ROS homeostasis. A number of patients who suffer from choriocarcinomas fail to survive due to delayed diagnosis or a recurrent tumor and resistance to traditional chemotherapy using platinum-based agents and methotrexate. To overcome these limitations, it is important to discover novel compounds which have no adverse effects yet can inhibit the expression of a target molecule to develop, as a novel therapeutic for prevention and/or treatment of choriocarcinomas. Effects of coumestrol on human placental choriocarcinoma cell lines, JAR and JEG3, were assessed in diverse assays in a dose- and time-dependent manner. Effects of coumestrol on cell proliferation, apoptosis (annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of reactive oxygen species (ROS), lipid peroxidation, glutathione levels and endoplasmic reticulum (ER) stress proteins in JAR and JEG3 cells were determined. Signal transduction pathways in JAR and JEG3 cells in response to coumestrol were determined by western blot analyses. Results of the present study indicated that coumestrol suppressed proliferation and increased apoptosis in JAR and JEG3 cells by inducing pro-apoptotic proteins, Bax and Bak. In addition, coumestrol increased ROS production, as well as lipid peroxidation and glutathione levels in JAR and JEG3 cells. Moreover, coumestrol-induced depolarization of mitochondrial membrane potential (MMP) and increased cytosolic and mitochondrial Ca2+ levels in JAR and JEG3 cells. Consistent with those results, treatment of JAR and JEG3 cells with a Ca2+ chelator and an inhibitor of IP3 receptor decreased coumestrol-induced depolarization of MMP and increased proliferation in JAR and JEG3 cells. N/A. A lack of in vivo animal studies is a major limitation of this research. The effectiveness of coumestrol to induce apoptosis of human placental choriocarcinoma cells requires further investigation. Our results indicate that coumestrol induces apoptotic effects on placental choriocarcinoma cells by regulating cell signaling and mitochondrial-mediated functions, with a potential to impair progression of the cancer. This research was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.).

Let-7i-Induced Atg4B Suppression Is Essential for Autophagy of Placental Trophoblast in Preeclampsia.

Autophagy, identified as type II programmed cell death, has already been known to be involved in the pathophysiology of preeclampsia (PE), which is a gestational disease with high morbidity. The present study aims to investigate the functional role of let-7i, a miRNA, in trophoblastic autophagy. Placental tissue used in this study was collected from patients with severe preeclampsia (SPE) or normal pregnant women. A decreased level of let-7i was found in placenta of SPE. In addition, autophagic vacuoles were observed in SPE and the expression of microtubule associated protein 1 light chain 3 (LC3) II/I was elevated. In vitro, let-7i mimics suppressed the autophagic activities in human HTR-8/SVneo trophoblast cell line (HTR-8) and human placental choriocarcinoma cell line JEG-3, whereas let-7i inhibitor enhanced the activities. As a potential target of let-7i, autophagy-related 4B cysteine peptidase (Atg4B) had an increased expression level in SPE. As expected, the increased expression of Atg4B was negatively regulated by let-7i using dual luciferase reporter assay. Furthermore, these trophoblast-like cells transfected with the let-7i mimic or inhibitors resulted in a significant change of Atg4B in both mRNA and protein level. More importantly, Atg4B overexpression could partly reverse let-7i mimic-reduced LC3II/I levels; whereas Atg4B silencing partly attenuated let-7i inhibitor-induced the level of LC3II/I expression. Taken together, these findings suggest that let-7i is able to regulate autophagic activity via regulating Atg4B expression, which might contribute to the pathogenesis of PE. J. Cell. Physiol. 232: 2581-2589, 2017. © 2016 Wiley Periodicals, Inc.

Fatty acid-binding protein3 expression in BeWo cells, a human placental choriocarcinoma cell line

Cellular uptake of long chain fatty acids in human placental trophoblasts is thought to be mediated by several membrane- and cytoplasmic fatty acid-binding proteins (FABP). FABP3 was shown to be involved in long chain polyunsaturated fatty acids (LCPUFA) uptake in human trophoblastic choriocarcinoma cells, BeWo as the uptake of arachidonic acid,20:4n-6 (ARA) was decreased in FABP3-knockdown BeWo cells. However, the regulation of expression of FABP3 in these cells is not yet well known. The aim of the present study was to examine the FABP3 expression by LCPUFAs, insulin and LXR agonists in BeWo cells. Among all these fatty acids tested, only ARA dose-dependently stimulated the expression of FABP3 protein in these cells after 24h incubation while other fatty acids had no such effect. In addition, LXR agonist and insulin dose-dependently increased FABP3 protein expression in these cells after 24h incubation. Insulin-stimulated FABP3 protein expression was accompanied with an increased arachidonic acid uptake. Differentiated BeWo cells had lesser expression of FABP3 protein than in the undifferentiated cells as the cellular differentiation state was measured by hCG production. In preeclamptic placental tissue, lowered expression of FABP3 protein was observed compared with those in normal pregnancy. All these data indicate that FABP3 may in be part involved in ARA uptake in these cells and its expression may be regulated by ARA, insulin, LXR and the state of cellular differentiation.

Dose-dependent and cell type-specific cell death and proliferation following in vitro exposure to radial extracorporeal shock waves.

Radial extracorporeal shock wave (rESW) therapy is widely used in musculoskeletal disorders and wound repair. However, the mechanisms of action are still largely unknown. The current study compared the effects of rESWs on two cell types. Human fetal foreskin fibroblasts (HFFF2) and human placental choriocarcinoma cell line JEG-3 were exposed to 0, 100, 200, 500 or 5000 rESWs generated with a Swiss DolorClast device (2.5 bar, 1 Hz). FACS analysis immediately after rESW exposure showed that initially, rESWs rather induced mechanical cell destruction than regulated or programmed cell death. Cell damage was nearly negated by reducing cavitation. Furthermore, cell viability decreased progressively with higher numbers of rESWs. Exposure to rESWs had no impact on growth potential of JEG-3 cells, but dose-dependently increased growth potential of HFFF2 cells. Cultivation of cells that were initially exposed to sham-rESWs in conditioned media increased the growth potential of HFFF2 cells, nevertheless, an even stronger effect was achieved by direct exposure to rESWs. Additionally, cell cycle distribution analysis demonstrated a shift in proportion from G0/G1 to G2/M phase in HFFF2 cells, but not in JEG-3 cells. These data demonstrate that rESWs leads to initial and subsequent dose-dependent and cell type-specific effects in vitro.

Dexamethasone Downregulates SLC7A5 Expression and Promotes Cell Cycle Arrest, Autophagy and Apoptosis in BeWo Cells.

Synthetic glucocorticoids (GCs) such as dexamethasone (Dex) are widely given to pregnant women to induce maturation and improve viability of preterm infants. Despite the beneficial effects, synthetic GCs have adverse effects on placental growth and nutrient transport system. However, the molecular mechanisms involved in these events remain unknown. Here we use a human placental choriocarcinoma cell line (BeWo) as model to explore the pathway linking amino acids transport with cell viability under Dex challenge. BeWo cells treated with Dex (100 nM) for 24 h demonstrated G1/S cell cycle arrest together with enhanced autophagy and apoptosis. Concurrently, the amino acid carrier SLC7A5 was down-regulated in association with impaired cellular amino acids uptake and inhibition of mammalian target of rapamycin (mTOR) signaling. Similar cellular responses were observed in BeWo cells treated with BCH, a classical System L inhibitor which inactivates SLC7A5. The glucocorticoid receptor (GR) antagonist RU486 was able to diminish Dex-induced translocation of GR into nucleus and to abolish these effects. Furthermore, Dex treatment significantly promoted the binding of GR to the proximal promoter sequence of SLC7A5 gene. Taken together, our results show that Dex downregulates SLC7A5 expression via GR-mediated transrepression. The impaired amino acids uptake leads to inhibition of mTOR signaling which in turn causes inhibited proliferation and enhanced autophagy and apoptosis in BeWo cells. These findings indicate that SLC7A5 mediates the effect of Dex on cell viability, thus providing a novel molecular target for the prevention and treatment of Dex-induced cell cycle arrest and apoptosis in placental cells.

Chemometric strategy for untargeted lipidomics: Biomarker detection and identification in stressed human placental cells

A lipidomic study was developed in a human placental choriocarcinoma cell line (JEG-3) exposed to tributyltin (TBT) and to a mixture of perfluorinated chemicals (PFCs). The method was based on the application of multivariate curve resolution-alternating least squares (MCR-ALS) to data sets obtained by ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC–TOF-MS) using an untargeted approach. Lipids from exposed JEG-3 cells were solid–liquid extracted and analyzed by UHPLC–TOF-MS in full scan mode, together with control samples. Raw UHPLC–TOF-MS data of the different cell samples were subdivided into 20 distinct chromatographic windows and each window was further organized in a column-wise augmented data matrix, where data from every sample was in an individual data matrix. Then, the 20 new augmented data matrices were modeled by MCR-ALS. A total number of 86 components were resolved and a statistical comparative study of their elution profiles showed distinct responses for the lipids of exposed versus control cells, evidencing a lipidome disruption attributed to the presence of the xenobiotics. Results from one-way ANOVA followed by a multiple comparisons test and from discriminant partial least squares (PLS-DA) analysis were compared as usual strategies for the determination of potential biomarkers. Identification of 24 out of the 33 proposed biomarkers contributed to the better understanding of the effects of PFCs and TBT in the lipidome of human placental cells. Overall, this study proposes an innovative untargeted LC–MS MCR-ALS approach valid for -omic sciences such as lipidomics.

Perfluorinated chemicals: Differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells

The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs – PFOS, PFDoA, PFNA, PFOA – showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA>PFOS≫PFNA>PFOA>PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57–80μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells.

Effect of triptolide on aromatase activity in human placental microsomes and human placental JEG-3 cells

Triptolide (CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F, engages in multiple pharmacological activities. However, it interferes with the synthesis of sex steroid hormones. This was proven in the present study of male and female subjects with severe reproductive toxicities related to abnormal androgen or estrogen levels. The inhibitory action of triptolide on aromatase, a terminal enzyme responsible for the formation of estrogens from androgens, was determined in vitro to investigate its role in endocrine secretion. Triptolides were incubated with human placental microsomes for 20 min at concentrations of 10, 20, 30, and 40 nM. At 20 nM, triptolide significantly inhibited aromatase, as shown by [3H]2O assay, and its concentration at 50% inhibition (IC50) was determined as approximately 35 nM. Furthermore, the inhibitory effect of triptolide on aromatase was observed in human placental choriocarcinoma (JEG-3) cell lines. Treatment with 10 nM triptolide significantly inhibited the aromatase of JEG-3 cells and gave an IC50 value of approximately 17 nM. Under these concentrations, decreased levels of mRNA and protein expression of aromatase in JEG-3 cells were observed using Western blot analysis and quantitative real-time reverse transcription polymerase chain reaction assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test revealed that treatment with triptolide for 24 h under these concentrations did not trigger evident cell death. These findings characterize the molecular mechanisms of triptolide on the regulation of sex steroid hormones.

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.

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.

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.

Coffee Induces 11.BETA.-Hydroxysteroid Dehydrogenase Type 2 Activity in Human Placental Choriocarcinoma Cells, JEG-3

Coffee is the beverage consumed during pregnancy that is most commonly associated with adversely affecting the developing fetoplacental unit. Since placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2) is an important determinant for maintenance of fetal glucocorticoid levels during pregnancy, we investigated the effect of coffee on 11β-HSD-2 expression in the human placental choriocarcinoma cell line, JEG-3. Addition of coffee to the culture medium of JEG-3 cells increased 11β-HSD-2 gene expression after 24hr in a dose-dependent manner. Cellular 11β-HSD-2 activity, which was calculated by the amount of cortisol that was converted to cortisone in the culture medium, also increased in the coffee-treated JEG-3 cells. None of the major constituents of coffee, such as caffeine, caffeic acid or chlorogenic acid, showed any ability to induce 11β-HSD-2 gene expression. The 11β-HSD-2-inducing activity in coffee was extractable with ethyl acetate. Increasing the degree of roasting improved the ability of the coffee extract to induce 11β-HSD-2 gene expression indicating that the active constituent(s) is produced during the roasting process.

HGH-V gene expression and promoter activity under glucose and 5-azacytidine (5azaC) effects

The metabolic conditions affecting placental development depend on nutritional state, genetic constitution and other external factors. The secretion of human placental growth hormone (hGH-V) had shown to be dependent of glucose, but the regulatory effects of this metabolite on hGH-V promoter activity and gene expression in presence of 5-azacytidine had not been studied. In this work we compared the hGH-V promoter activity and the endogenous mRNA expression in human placental choriocarcinoma cell line JAR in the presence of glucose and demethylating genome conditions. High glucose concentration in culture medium diminished hGH-V mRNA endogenous levels in JAR cells whereas the expression of hGH-V from transfected PACs was slightly higher; but in the presence of 5azaC a higher hGH-V gene expression from both the endogenous and the transfected ones was obtained. A drastic diminution of promoter analysis was shown when cells had no glucose (J0 cells) or in presence of 5azaC; the placental transcription factors that showed modified binding capacity were HES-2, PPAR-γ, H4TF-1 and OCT-1. Our results suggest that in vitro suppressive glucose effect dictates a metabolic context to hGH-V gene expression and promoter regulation whereas a genomic methylation-dependent background is necessary to maintain placental transcription factors able to bind and regulate proximal promoter region of hGH-V in placental cells.

Expression of mTOR and downstream signalling components in the JEG-3 and BeWo human placental choriocarcinoma cell lines

Emerging data suggest that nutritional status and body weight are related to reproductive function, and nutrient imbalances during pregnancy lead to changes in the expression of fetal genes. Recent studies show that the mTOR acts as a placental growth signalling sensor and its expression is down-regulated in intrauterine growth restriction. To date, very little is known about the expression of this signalling pathway in choriocarcinoma, one of the most lethal germ cell cancers. In this study, cultures of fusigenic (BeWo) and non-fusigenic (JEG-3) human choriocarcinoma cell lines were used to investigate the expression of mTOR and its downstream signalling components. The effects of an inducer of syncytialisation (forskolin) on mTOR, eIF4E binding proteins (4EBPs) and ribosomal protein S6 kinases (S6Ks) in BeWo cells were also assessed. RT-PCR studies revealed that mTOR, 4EBP and S6Ks are expressed at mRNA level in both JEG-3 and BeWo cells. Semi-quantitative RT-PCR analysis revealed that in early stages of syncytialisation (50 microM forskolin for 48 h), the expression of mTOR and 4EBP was down-regulated when compared to unstimulated cells. In fully syncytialised cells (50 microM forskolin for 72 h) the expression of both genes was similar to basal levels. Interestingly, the phosphorylation (Ser371, Thr389) status of p70S6K remained unaltered upon forskolin treatment. These data validate BeWo cells as an experimental model to study the effects of forskolin-induced syncytialisation on mTOR signalling.