The Extracts from Two Antarctic Fish Species, Trematomus newnesi and Trematomus bernacchii, Enhance JEG-3 Cell Migration and Invasion via MMP9 Activation Through Akt/Protein Phosphatase1/ß-Catenin Pathway.

SCOPE: This study investigates the impact of extracts derived from Antarctic fish species, Trematomus newnesi and Trematomus bernacchii, on the migration of human placental trophoblast JEG-3 cells, which is a crucial aspect of successful pregnancy. METHODS AND RESULTS: The extracts, obtained from the muscles of these fish, significantly enhance the migration and invasion of JEG-3 cells in in vitro wound healing, Transwell, and collagen invasion assays. These effects are accompanied by an increase in matrix metalloproteinase (MMP) 9 activity, as demonstrated by zymography. Furthermore, the extracts activated Akt and protein phosphatase 1, resulting in the dephosphorylation of ß-catenin at Ser33/37/Thr41, as confirmed by western blot analysis. Consequently, MMP9 is upregulated, while metallopeptidase inhibitor 1/3 is downregulated, as verified by western blot and qRT-PCR analyses. Finally, utilizing ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, followed by matching with the Global Natural Product Social Molecular Networking library, the study annotates the compound responsible for the observed migratory activity as taurocholic acid. Importantly, the study confirms that taurocholic acid enhances cell migration in JEG-3 cells. CONCLUSION: The results of this study emphasize the potential of Antarctic fish extracts in promoting extravillous trophoblast migration and invasion, which are critical for successful pregnancy.

Bisphenol A and its analogue bisphenol S exposure reduce estradiol synthesis via the ROS-mediated PERK/ATF4 signaling pathway.

As a kind of endocrine-disrupting chemicals, BPA may affect the human placenta. Due to consumer unease about BPA, many manufacturers are using alternatives to BPA, such as BPS. However, some reports suggest that BPS may produce similar results to BPA. To understand how BPA/BPS leads to reduced synthesis of placental estradiol (E2), we conducted studies using a human choriocarcinoma cell (JEG-3) model for research. In this study. Elisa assay revealed that both BPA/BPS exposures decreased E2 synthesis in JEG-3 cells. The results of RT-PCR showed that both BPA and BPS could reduce the mRNA expression of CYP19A1, a key enzyme for E2 synthesis in JEG-3 cells. In addition, Western blot assay showed that BPA/BPS-induced ER-stress PERK/eIF2α/ATF4 signaling protein expression was increased. The expression of ROS in cells after exposure to BPA/BPS was detected using the 2,7-dichlorodihydrofluorescein diacetate (DCF-DA) method. The results of this experiment showed that BPA/BPS significantly induced an inhibition of ROS in JEG-3 cells. The present study concluded that, firstly, BPS exposure induced almost the same effect as BPA in reducing E2 synthesis in JEG-3 cells. Second, BPA/BPS exposure may reduce E2 synthesis in JEG-3 cells by increasing ROS levels and thus activating endoplasmic reticulum stress.

Vital role of autophagy flux inhibition of placental trophoblast cells in pregnancy disorders induced by HEV infection.

Hepatitis E virus (HEV) has become one of the important pathogens that threaten the global public health. Type 3 and 4 HEV are zoonotic, which can spread vertically and cause placental damage. At the same time, autophagy plays an important role in the process of embryo development and pregnancy maintenance. However, the relationship between HEV and autophagy, especially in the placenta tissue, has not been clarified. We found lower litter rates in HEV-infected female mice, with significant intrauterine abortion of the embryo (24.19%). To explore the effects of HEV infection on placenta autophagy, chorionic cells (JEG-3) and mice placenta have been employed as research objects, while the expression of autophagy-related proteins (ATGs) has been detected in JEG-3 cells with different times of HEV inoculation. The results demonstrated that the expression of protein LC3 decreased and p62 accumulated, meanwhile ATGs such as ATG4B, ATG5, and ATG9A in JEG-3 cells have decreased significantly. In addition, the maturation of autophagosomes, which referred to the process of the combination of autophagosomes and lysosomes was prevented by HEV infection as well. All processes of autophagic flux, which include the initiation, development, and maturation three stages, were suppressed in JEG-3 cells after HEV infection. Similarly, the protein and gene expression of LC3 were significantly decreased in the placenta of pregnant mice with HEV infection. In summary, our results suggested that HEV inhibited autophagy in JEG-3 cells and placenta of pregnant mice, which might be the important pathogenic mechanisms of HEV infection leading to embryo abortion.

Genotoxicity and endocrine disruption potential of haloacetic acids in human placental and lung cells.

Chlorination of water results in the formation of haloacetic acids (HAAs) as major disinfection byproducts (DBPs). Previous studies have reported some HAAs species to act as cytotoxic, genotoxic, and carcinogenic. This work aimed at further exploring the toxicity potential of the most investigated HAAs (chloroacetic (CAA), bromoacetic (BAA), iodoacetic (IAA) acid) and HAAs species with high content of bromine (tribromoacetic acid (TBAA)), and iodine in their structures (chloroiodoacetic (CIAA) and diiodoacetic acid (DIAA)) to human cells. Novel knowledge was generated regarding cytotoxicity, oxidative stress, endocrine disrupting potential, and genotoxicity of these HAAs by using human placental and lung cells as in vitro models, not previously used for DBP assessment. IAA showed the highest cytotoxicity (EC50: 7.5 µM) and ability to generate ROS (up to 3-fold) in placental cells, followed by BAA (EC50: 20-25 µM and 2.1-fold). TBAA, CAA, DIAA, and CIAA showed no significant cytotoxicity (EC50 > 250 µM). All tested HAAs decreased the expression of the steroidogenic gene hsd17b1 up to 40 % in placental cells, and IAA and BAA (0.01-1 µM) slightly inhibited the aromatase activity. HAAs also induced the formation of micronuclei in A549 lung cells after 48 h of exposure. IAA and BAA showed a non-significant increase in micronuclei formation at low concentrations (1 µM), while BAA, CAA, CIAA and TBAA were genotoxic at exposure concentrations above 10 µM (100 µM in the case of DIAA). These results point to genotoxic and endocrine disruption effects associated with HAA exposure at low concentrations (0.01-1 µM), and the usefulness of the selected bioassays to provide fast and sensitive responses to HAA exposure, particularly in terms of genotoxicity and endocrine disruption effects. Further studies are needed to define thresholds that better protect public health.

Modeling Preeclampsia In Vitro: Polymorphic Variants of STOX1-A/B Genes Can Downregulate CD24 in Trophoblast Cell Lines.

CD24 is a mucin-like immunosuppressing glycoprotein whose levels increase during pregnancy and decrease in the syncytio- and cytotrophoblasts in early and preterm preeclampsia. We used two modified cell lines that mimic in vitro features of preeclampsia to identify if this phenomenon could be reproduced. Our model was the immortalized placental-derived BeWo and JEG-3 cell lines that overexpress the STOX1 A/B transcription factor gene that was discovered in familial forms of preeclampsia. BeWo and JEG-3 cells stably transduced with the two major isoforms of STOX1-A/B or by an empty vector (control), were propagated, harvested, and analyzed. CD24 mRNA expression was determined by quantitative real-time polymerase nuclear chain reaction (qRT-PCR). CD24 protein levels were determined by Western blots. In STOX1-A/B overexpressing in BeWo cells, CD24 mRNA was downregulated by 91 and 85%, respectively, compared to the control, and by 30% and 74%, respectively in JEG-3 cells. A 67% and 82% decrease in CD24 protein level was determined by immunoblot in BeWo overexpressing STOX1-A/B, respectively, while the reduction in JEG-3 cells was between 47 and 62%. The immortalized BeWo and JEG-3 cell lines overexpressing STOX1-A/B had reduced CD24. Although both cell lines were affected, BeWo appears to be more susceptible to downregulation by STOX-1 than JEG-3, potentially because of their different cell origin and properties. These results strengthen the in vivo results of reduced CD24 levels found in early and preterm preeclampsia. Accordingly, it implies the importance of the reduced immune tolerance in preeclampsia, which was already demonstrated in vivo in the STOX1-A/B model of preeclampsia, and is now implied in the in vitro STOX-1 model, a subject that warrants further investigations.

Differential proteomics analysis of JEG-3 and JAR placental cell models and the effect of androgen treatment.

The placenta is a vital fetal organ that plays an important role in maintaining fetal sex hormone homeostasis. Xenobiotics can alter placental sex-steroidogenic enzymes and transporters, including enzymes such as aromatase (CYP19A1) and the hydroxysteroid dehydrogenases (HSDs) but studying how compounds disrupt in vivo placental metabolism is complex. Utilizing high-throughput in vitro models is critical to predict the disruption of placental sex-steroidogenic enzymes and transporters, particularly by drug candidates in the early stages of drug discovery. JAR and JEG-3 cells are the most common, simple, and cost-effective placental cell models that are capable of high-throughput screening, but how well they express the sex-steroidogenic enzymes and transporters is not well known. Here, we compared the proteomes of JAR and JEG-3 cells in the presence and absence of physiologically relevant concentrations of dehydroepiandrosterone (DHEA, 8 µM) and testosterone (15 nM) to aid the characterization of sex-steroidogenic enzymes and transporters in these cell models. Global proteomics analysis detected 2931 and 3449 proteins in JAR cells and JEG-3 cells, respectively. However, dramatic differences in sex-steroidogenic enzymes and transporters were observed between these cells. In particular, the basal expression of steroid sulfatase (STS), HSD17B1, and HSD17B7 were unique to JEG-3 cells. JEG-3 cells also showed significantly higher protein levels of aldo-keto reductase (AKR) 1A1 and AKR1B1, while JAR cells showed significantly higher levels of HSD17B4 and HSDB12. Aldehyde dehydrogenase (ALDH) 3A2 and HSD17B11 enzymes as well as the transporters sterol O-acyltransferase (SOAT) 1 and ATP binding cassette subfamily G2 (ABCG2) were comparable between the cell lines, whereas sulfotransferases (SULTs) were uniquely present within JAR cells. Androgen treatments significantly lowered HSD17B11, HSD17B4, HSD17B12, and ALDH3A2 levels in JAR cells. DHEA treatment significantly raised the level of HSD17B1 by 51 % in JEG-3 cells, whereas CYP19A1 was increased to significant levels in both JAR and JEG-3 cells after androgen treatments. The proteomics data were supported by a complementary targeted metabolomics analysis of culture media in the DHEA (8 µM) and testosterone (15 nM) treated groups. This study has indicated that untreated JEG-3 cells express more sex-steroidogenic enzymes and transporters. Nevertheless, JEG-3 and JAR cells are unique and their respective proteomics data can be used to select the best model depending on the hypothesis.

High-throughput analysis of the steroid profile in placental cell cultures to evaluate endocrine disrupting effects of contaminant exposure.

Human placental JEG-3 cells conserve a high P450 aromatase activity and are therefore suitable to evaluate how contaminants may interfere with the routes involved in estrogen synthesis during pregnancy. This has been traditionally assessed by measuring aromatase activity through the amount of tritiated water (3H2O) formed during the aromatization of 1ß-3H-androst-4-ene-3,17-dione (3H-AD). This work presents a greener and safer analytical approach for this purpose, which consists of the determination of the trace amounts of the steroids (estradiol, estrone, testosterone, and androstenedione) present in the culture medium. Turbulent flow chromatography coupled to liquid chromatography-tandem mass spectrometry (TFC-HPLC-MS/MS) delivered the high selectivity and sensitivity (limits of detection between 2 and 5 pg/mL) required for these measurements. Moreover, its automation allows high-throughput of samples with minimum sample handling and achieves high precision in the analysis (relative standard deviation values <6%). As a proof of concept, the method was applied to evaluate the effect of monohaloacetic acid exposure on the steroid profile of JEG-3 cells. Iodoacetic acid showed an estrogenic effect (statistically significant increase of estradiol levels compared to unexposed cells) at the highest concentration level tested (0.5 µM) that deserves further evaluation.

Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell-Trophoblast Interactions.

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFß and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFß, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFß, their cytotoxicity increased. In the case of adding TGFß to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFß in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

Placenta-Specific miR-125b Overexpression Leads to Increased Rates of Pregnancy Loss in Mice.

Pregnancy loss (PL) is one of the common complications that women can experience during pregnancy, with an occurrence rate of 1 to 5%. The potential causes of pregnancy loss are unclear, with no effective treatment modalities being available. It has been previously reported that the level of miR-125b was significantly increased in placentas of PL patients. However, the role of miR-125b in the development of PL still remains unknown. In the current study, an miR-125b placenta-specific over-expression model was constructed by lentiviral transfecting zona-free mouse embryos followed by embryo transfer. On gestation day 15, it was observed that the placenta was significantly smaller in the miR-125b placenta-specific overexpression group than the control group. Additionally, the abortion rate of the miR-125b placenta-specific overexpression group was markedly higher than in the control group. The blood vessel diameter was larger in the miR-125b-overexpressing specific placenta. In addition, miR-125b-overexpressing HTR8 and JEG3 cell lines were also generated to analyze the migration and invasion ability of trophoblasts. The results showed that miR-125b overexpression significantly suppressed the migration and invasion ability of HTR8 and JEG3 cells. Overall, our results demonstrated that miR-125b can affect embryo implantation through modulating placenta angiogenesis and trophoblast cell invasion capacity that can lead to PL.

Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix.

Oxygen tension varies during placental and fetal development. Although hypoxia drives early trophoblast invasion, low placental oxygen levels during pregnancy show association with pregnancy complications including fetal growth restriction and preeclampsia. JEG-3 cells are often used as a trophoblast model. We studied transcriptional changes of JEG-3 cells on a uterine leiomyoma derived matrix Myogel. This might be the closest condition to the real uterine environment that we can get for an in vitro model. We observed that culturing JEG-3 cells on the leiomyoma matrix leads to strong stimulation of ribosomal pathways, energy metabolism, and ATP production. Furthermore, Myogel improved JEG-3 cell adherence in comparison to tissue culture treated plastic. We also included PDMS microchip hypoxia creation, and observed changes in oxidative phosphorylation, oxygen related genes and several hypoxia genes. Our study highlights the effects of Myogel matrix on growing JEG-3 cells, especially on mitochondria, energy metabolism, and protein synthesis.

Appropriate fixative for MEM-G/9 staining of cultured human HLA-G-positive JEG-3 trophoblast tumor cells.

Human leukocyte antigen (HLA-G) participates in immunosuppression and is useful for prenatal diagnostics. Isolation of fetal cells positive for HLA-G by HLA-G antibody conjugated nanoparticles from the cervix of pregnant women is the basis for non-invasive prenatal testing. Endocervical specimens are fixed in transport medium before isolation using antibody conjugated nanoparticles. Staining of HLA-G using MEM-G/9 antibody, however, is restricted to unfixed cells. We investigated the effect of several fixatives on the interaction of HLA-G with MEM-G/9 in the HLA-G-positive cell line, JEG-3. We investigated absolute methanol, 1:1 acetate buffer:methanol, Pap solution and paraformaldehyde. The effects of these fixatives were evaluated using immunofluorescence. We found no MEM-G/9 surface staining of methanol fixed cells. Approximately 40% of JEG-3 cells fixed with paraformaldehyde failed to stain. Nearly all cells were stained with MEM-G/9 following fixation with acetate buffer:methanol or Pap solution. Our findings indicate the importance of using an appropriate fixative for preserving HLA-G cell surface antigen for studies using the MEM-G/9 antibody.

Low Abundance Fusobacterium Nucleatum Supports Early Pregnancy Development - An In Vitro Study.

Pregnancy success depends greatly on a balanced immune homeostasis. The detection of bacterial components in the upper reproductive tract in non-pregnant and pregnant women raised questions on its possible beneficial role in reproductive health. The local conditions that allow the presence of bacteria to harmonize with the establishment of pregnancy are still unknown. Among the described bacterial species in endometrial and placental samples, Fusobacterium nucleatum was found. It has been observed that F. nucleatum can induce tumorigenesis in colon carcinoma, a process that shares several features with embryo implantation. We propose that low concentrations of F. nucleatum may improve trophoblast function without exerting destructive responses. Inactivated F. nucleatum and E. coli were incubated with the trophoblastic cell lines HTR8/SVneo, BeWo, and JEG-3. Viability, proliferation, migratory capacity, invasiveness and the secretion of chemokines, other cytokines and matrix metalloproteinases were assessed. The presence of F. nucleatum significantly induced HTR8/SVneo invasion, accompanied by the secretion of soluble mediators (CXCL1, IL-6 and IL-8) and metalloproteinases (MMP-2 and MMP-9). However, as concentrations of F. nucleatum increased, these did not improve invasiveness, hindered migration, reduced cell viability and induced alterations in the cell cycle. Part of the F. nucleatum effects on cytokine release were reverted with the addition of a TLR4 blocking antibody. Other effects correlated with the level of expression of E-cadherin on the different cell lines tested. Low amounts of F. nucleatum promote invasion of HTR8/SVneo cells and induce the secretion of important mediators for pregnancy establishment. Some effects were independent of LPS and correlated with the expression of E-cadherin on trophoblasts.

NK-92 cells change their phenotype and function when cocultured with IL-15, IL-18 and trophoblast cells.

NK cell development is affected by their cellular microenvironment and cytokines, including IL-15 and IL-18. NK cells can differentiate in secondary lymphoid organs, liver and within the uterus in close contact with trophoblast cells. The aim was to evaluate changes in the NK cell phenotype and function in the presence of IL-15, IL-18 and JEG-3, a trophoblast cell line. When cocultured with JEG-3 cells, IL-15 caused an increase in the number of NKG2D+ NK-92 cells and the intensity of CD127 expression. IL-18 stimulates an increase in the amount of NKp44+ NK-92 cells and in the intensity of NKp44 expression by pNK in the presence of trophoblast cells. NK-92 cell cytotoxic activity against JEG-3 cells increased only in presence of IL-18. Data on changes in the cytotoxic activity of NK-92 cells against JEG-3 cells in the presence of IL-15 and IL-18 indicate the modulation of NK cell function both by the cytokine microenvironment and directly by target cells. IL-15 and IL-18 were present in conditioned media (CM) from 1st and 3rd trimester placentas. In the presence of 1st trimester CM and JEG-3 cells, NK-92 cells showed an increase in the intensity of NKG2D expression. In the presence of 3rd trimester CM and JEG-3 cells, a decrease in the expression of NKG2D by NK-92 cells was observed. Thus, culturing of NK-92 cells with JEG-3 trophoblast cells stimulated a pronounced change in the NK cell phenotype, bringing it closer to the decidual NK cell-like phenotype.

3-Dimensional JEG-3 choriocarcinoma cell organoids as a model for trophoblast expansion and differentiation.

JEG-3 choriocarcinoma cells have been widely used as a model for placental trophoblast. Herein, 3-dimensional (3D) JEG-3 organoids (JEG-3-ORGs) were established using a protocol that we recently developed for primary cytotrophoblast organoids (CTB-ORGs). 3D JEG-3-ORGs, cultivated in basic culture medium, rapidly divide and spontaneously undergo differentiation. Under stem cell culture conditions (activation of WNT/EGF signalling and inhibition of TGF-ß signalling) smaller organoids with reduced proliferative capacity were generated specifically abolishing expression of extravillous trophoblast (EVT)-specific genes. Similar to CTB-ORGs, removal of the WNT activator CHIR99021 induced re-expression of these genes in JEG-3-ORGs. Hence, JEG-3-ORGs could be used as a model for directed EVT differentiation.

The uteroplacental contact zone cytokine influence on NK cell cytotoxicity to trophoblasts.

OBJECTIVE: The present study was to estimate the role of cytokines for trophoblast death in NK cells presence. METHODS: This study involves assessment of NK-92 line NK cell cytotoxic activity against JEG-3 line cells, in presence of cytokines. We also assessed the effect of secretory placenta products on NK cell cytotoxic activity toward JEG-3 line cells. RESULTS: Uteroplacental contact zone cytokines are able to enhance trophoblast mortality both by themselves in case of IL-1ß, IL-6, IFNγ, IL-4, TGFß, bFGF, and also through increasing the cytotoxic potential of NK cells in case of IL-1ß, IFNγ, IL-8, TGFß, and GM-CSF. PLGF decreases NK cell cytotoxicity for trophoblasts. Secretory products of first trimester placenta enhance NK cell cytotoxic potential for trophoblasts. CONCLUSIONS: Cytokines of the uteroplacental contact zone can appear a mechanism ensuring trophoblast mortality dynamics throughout pregnancy.

The comparison of pegylated liposomal doxorubicin and beta-carotene effects on JAR and JEG-3 choriocarcinoma human cell culture models

Objective: The aim was to investigate the effectiveness of pegylated liposomal doxorubicin (PLD), beta-carotene, and a combination of PLD and beta-carotene on JAR and JEG-3 human choriocarcinoma (CC) cell lines for the treatment of CC. Material and Methods: JAR and JEG-3 cells were cultured. PLD and beta-carotene trial groups were determined with different doses (for single drug trial; PLD 1, 2, 5 µg/mL and beta-carotene 1, 5, 10 µg/mL, and for combined drug trial; all PLD doses combined with beta-carotene 5 µg/mL). Drugs were administered to cultures simultaneously, and 72 hours later the cells were detached using trypsin-ethylenediamine tetraacetic acid solution. The percentage of apoptotic cells was determined by flow cytometry after annexin V staining. One set of the supernatant was collected before trypsin application to investigate beta-human chorionic gonadotropin (ß-hCG) and hyperglycosylated hCG (H-hCG) levels. Statistical analyses of the apoptotic ratios were performed using Shapiro-Wilk, Kruskal-Wallis and Mann-Whitney U tests. Results: Apoptosis increased in JAR and JEG-3 cultures after treatment with all doses of PLD (p<0.05). A single application of each betacarotene dose increased apoptosis in JAR cells (p<0.05) but had no apoptotic effects on JEG-3 cells. In the PLD and beta-carotene combination group, apoptosis increased in both JAR and JEG-3 cells (p<0.05). Conclusion: To our knowledge, this is the first investigation of the effectiveness of PLD, beta-carotene, and PLD + beta-carotene combination therapy in two different CC cell lines. PLD is a promising chemotherapeutic drug, and beta-carotene can be used as a novel non-chemotherapeutic agent for treatment of CC. Based on the results of this study, vitamin A supplementation may have promise as a preventive measure. However, these data need support from animal experiments and clinical trials.

Increased NDRG1 expression suppresses angiogenesis via PI3K/AKT pathway in human placental cells.

OBJECTIVE: To observe whether and how N-myc downstream-regulated gene 1 (NDRG1) regulates placental angiogenesis via JEG-3 placental-derived cells. METHODS: Expression of NDRG1 in stably transfected JEG-3 cells was detected using western blot and real-time quantitative polymerase chain reaction. Angiogenesis was examined by tube formation assay. The levels of placental growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) were examined using enzyme-linked immunosorbent assay. The expression of vascular endothelial growth factor (VEGF), PI3K, and AKT was examined by western blot. The relationship between PI3K and NDRG1 was detected by co-immunoprecipitation. RESULTS: NDRG1 was significantly down-regulated at both the mRNA and protein level by lentivirus (Lv)-NDRG1-shRNA (P < 0.001), whereas it was significantly up-regulated by Lv-NDRG1 (P < 0.001). NDRG1 knockdown significantly increase the expression of PLGF and VEGF in JEG-3 cells (P < 0.001), while NDRG1 knockdown significantly reduced the secretion of sFlt-1 (P < 0.001). NDRG1 was specific bound to PI3K, and NDRG1 knockdown significantly up-regulated the expressions of PI3K and AKT in JEG-3 cells (P < 0.001). CONCLUSION: NDRG1 suppresses angiogenesis in preeclampsia, and the PI3K/AKT signaling pathway may be involved in the regulation of angiogenesis by NDRG1.

Cellular uptake properties of lamotrigine in human placental cell lines: Investigation of involvement of organic cation transporters (SLC22A1-5).

Lamotrigine (LTG) is an important antiepileptic drug for the treatment of seizures in pregnant women with epilepsy. However, it is not known if the transport of LTG into placental cells occurs via a carrier-mediated pathway. The aim of this study was to investigate the uptake properties of LTG into placental cell lines (BeWo and JEG-3), and to determine the involvement of organic cation transporters (OCTs, SLC22A1-3) and organic cation/carnitine transporter (OCTNs, SLC22A4-5) in the uptake process. The uptake of LTG at 37 °C was higher than that at 4 °C. OCT1 and OCTNs were detected in both cell lines. The uptake of LTG was not greatly affected by the extracellular pH, Na+-free conditions, or the presence of l-carnitine, suggesting that OCTNs were not involved. Although several potent inhibitors of OCTs (chloroquine, imipramine, quinidine, and verapamil) inhibited LTG uptake, other typical inhibitors had no effect. In addition, siRNA targeted to OCT1 had no significant effect on LTG uptake. The mRNA expression in human term placenta followed the order OCTN2 > OCT3 > OCTN1 > OCT1 ≈ OCT2. These observations suggested that LTG uptake into placental cells was carrier-mediated, but that OCTs and OCTNs were not responsible for the placental transport process.

A new series of aryl sulfamate derivatives: Design, synthesis, and biological evaluation.

Steroid sulfatase (STS) has recently emerged as a drug target for management of hormone-dependent malignancies. In the present study, a new series of twenty-one aryl amido-linked sulfamate derivatives 1a-u was designed and synthesized, based upon a cyclohexyl lead compound. All members were evaluated as STS inhibitors in a cell-free assay. Adamantyl derivatives 1h and 1p-r were the most active with more than 90% inhibition at 10 µM concentration and, for those with the greatest inhibitory activity, IC50 values were determined. These compounds exhibited STS inhibition within the range of ca 25-110 nM. Amongst them, compound 1q possessing a o-chlorobenzene sulfamate moiety exhibited the most potent STS inhibitory activity with an IC50 of 26 nM. Furthermore, to assure capability to pass through the cell lipid bilayer, compounds with low IC50 values were tested against STS activity in JEG-3 whole-cell assays. Consequently, 1h and 1q demonstrated IC50 values of ca 14 and 150 nM, respectively. Thus, compound 1h is 31 times more potent than the corresponding cyclohexyl lead (IC50 value = 421 nM in a JEG-3 whole-cell assay). Furthermore, the most potent STS inhibitors (1h and 1p-r) were evaluated for their antiproliferative activity against the estrogen-dependent breast cancer cell line T-47D. They showed promising activity with single digit micromolar IC50 values (ca 1-6 µM) and their potency against T-47D cells was comparable to that against STS enzyme. In conclusion, this new class of adamantyl-containing aryl sulfamate inhibitor has potential for further development against hormone-dependent tumours.

Di-(2-ethylhexyl) phthalate inhibits expression and internalization of transthyretin in human placental trophoblastic cells.

During pregnancy, fetal thyroid hormones (THs) are dependent on maternal placental transport and their physiological level is crucial for normal fetal neurodevelopment. Earlier research has shown that Di-(2-ethylhexyl) phthalate (DEHP) disrupts thyroid function and THs homeostasis in pregnant women and fetuses, and affects placental THs transport. However, the underlying mechanisms are poorly understood. The present study, therefore, aimed to systematically investigate the potential mechanisms of DEHP-induced disruption in the placental THs transport using two human placental trophoblastic cells, HTR-8/SVneo cells and JEG-3 cells. While the exposure of DEHP at the doses of 0-400 µM for 24 h did not affect cell viability, we found reduced consumption of T3 and T4 in the culture medium of HTR-8/Svneo cells treated with DEHP at 400 µM. DEHP treatment did not affect T3 uptake and the expression of monocarboxylate transporters 8 (MCT8) and organic anion transporters 1C1 (OATP1C1). However, DEHP significantly inhibited transthyretin (TTR) internalization, down-regulated TTR, deiodinase 2 (DIO2), and thyroid hormone receptors mRNA expression and protein levels, and up-regulated deiodinase 3 (DIO3) protein levels in a dose-dependent manner. These results indicate that DEHP acts on placental trophoblast cells, inhibits its TTR internalization, down-regulates TTR expression and affects the expression of DIO2, DIO3, and thyroid hormone receptor. These may be the mechanisms by which PAEs affects THs transport through placental.