Choriocarcinoma Cell Line Research Articles

PIM kinases 1, 2 and 3 in intracellular LIF signaling, proliferation and apoptosis in trophoblastic cells

Proviral insertion in murine (PIM) lymphoma proteins are mainly regulated by the Janus Kinase/Signal Transducer Activator of Transcription (JAK/STAT) signaling pathway, which can be activated by members of the Interleukin-6 (IL-6) family, including Leukemia Inhibitory Factor (LIF). Aim of the study was to compare PIM1, PIM2 and PIM3 expression and potential cellular functions in human first and third trimester trophoblast cells, the immortalized first trimester extravillous trophoblast cell line HTR8/SVneo and the choriocarcinoma cell line JEG-3. Expression was analyzed by qPCR and immunochemical staining. Functions were evaluated by PIM inhibition followed by analysis of kinetics of cell viability as assessed by MTS assay, proliferation by BrdU assay, and apoptosis by Western blotting for BAD, BCL-XL, (cleaved) PARP, CASP3 and c-MYC. Apoptosis and necrosis were tested by flow cytometry (annexin V/propidium iodide staining). All analyzed PIM kinases are expressed in primary trophoblast cells and both cell lines and are regulated upon stimulation with LIF. Inhibition of PIM kinases significantly reduces viability and proliferation and induces apoptosis. Simultaneously, phosphorylation of c-MYC was reduced. These results demonstrate the involvement of PIM kinases in LIF-induced regulation in different trophoblastic cell lines which may indicate similar functions in primary cells.

Role of SDF-1/CXCR4 axis in the invasion and migration of human choriocarcinoma cells and its significance in preeclampsia

Objective To investigate the effects of stromal cell-derived factor 1 (SDF-1) and an CXC chemokine receptor 4 (CXCR4) antagonist (AMD3100) on the invasion and migration capabilities of the huaman choriocarcinoma cell line JAR for further elucidating the role of SDF-1/CXCR4 axis in the pathogenesis of preeclampsia. Methods JAR cells were divided into four groups: SDF-1 group (treated with 50 ng/ml of SDF-1), SDF-1+ AM3100 mixed group (first treated with 100 ng/ml of AMD3100 for 2 hours and then treated with 50 ng/ml of SDF-1), AMD3100 group (treated with 100 ng/ml of AMD3100) and blank control group (without any treatment). RT-PCR was performed to detect the expression of CXCR4 at mRNA level in JAR cells. Western blot assay was used to measure the expression of CXCR4 and p-AKT at protein level. MTT assay was used to analyze the effects of different concentrations of SDF-1 (10, 30, 50 and 100 ng/ml) on the proliferation of JAR cells at different time points (0, 24, 48, 72 h). Transwell invasion assay and wound-healing assay were used to test the changes in invasion and migration capabilities of JAR cells after different treatments. Results (1) Results of the RT-PCR showed that the expression of CXCR4 at mRNA level in JAR cells was increased in the SDF-1 group (1.839±0.083) as compared with that in the blank control group (1.372±0.086), AMD3100 group (0.694±0.045) or SDF-1+ AM3100 mixed group (0.703±0.093). Moreover, the differences between the SDF-1 group and the other three groups were statistically significant (F=30.67, P<0.05). Compared with the blank control group, the expression of CXCR4 at mRNA level in JAR cells was decreased in the AMD3100 group (P<0.01). (2) Results of the Western blot assay showed that the expression of CXCR4 and p-AKT at protein level in JAR cells were enhanced in the SDF-1 group as compared with that in the blank control group, AMD3100 group or SDF-1+ AM3100 mixed group. Compared with the blank control group, the expression of CXCR4 and p-AKT at protein level in JAR cells were inhibited in the AMD3100 group. (3) Results of the MTT assay showed that SDF-1, especially at the concentration of 50 ng/ml, could enhance the proliferation of JAR cells (P<0.05) and its best effect on proliferation was seen at 48 h. (4) Results of the Transwell invasion assay showed that the number of transmembrane cells in the SDF-1 group (70.49±2.42) was more than that in the blank control group (54.36±2.26), AMD3100 group (21.68±8.31), or SDF-1+ AMD3100 mixed group (28.18±4.61). The differences between the SDF-1 group and the other three groups were statistically significant (F=116.26, P<0.01). Compared with the blank control group, the number of transmembrane cells was reduced in the AMD3100 group (P<0.05). (5) Results of the wound-healing assay showed that the relative migration distance was increased in the SDF-1 group (1.162±0.034) as compared with that in the blank control group (0.823±0.101), AMD3100 group (0.160±0.047), or SDF-1+ AMD3100 mixed group (0.183±0.064). The differences between the SDF-1 group and the other three groups were statistically significant (F=30.500, P<0.05). Compared with the blank control group, the relative migration distance was decreased in the AMD3100 group (P<0.01). Conclusion The invasion and migration of huaman choriocarcinoma JAR cells can be enhanced by SDF-1, but inhibited by AMD3100. This study indicates that the blocked biological axis of SDF-1/CXCR4 may play an important role in the pathogenesis of preeclampsia through inducing abnormal activation of PI3K/AKT pathway, which results in inhibited invasion and migration of trophoblast cells and placenta abnormality. Key words: SDF-1/CXCR4 axis; Invasion; Migration; PI3K/AKT pathway; Trophoblast cell; Preeclampsia

Multiple drug transporters mediate the placental transport of sulpiride

Sulpiride is a typical antipsychotic drug for the treatment of schizophrenia, depression and other psychological disorders. It has been proven that a small amount of sulpiride could cross the human placentausing anex vivoplacental perfusion model. However, the placental transfer mechanism has not been elucidated. Considering the structure of sulpiride, we speculated that the transporters expressed in placenta might be involved in sulpiride uptake across the blood-placenta barrier. The aim of our study was to determine which transporters contributed to the placental transfer of sulpiride. Our results revealed that sulpiride was a substrate of human organic cation transporter (hOCT) 3, human multidrug resistance protein (hMDR) 1 and human breast cancer resistance protein (hBCRP) using transfected cells expressing respective transporters. In addition, the accumulation of sulpiride in BeWo cells (a human choriocarcinoma cell line) was obviously affected by inhibitors of carnitine/organic cation transporter (OCTN) 2, MDR1 and BCRP. The accumulation of sulpiride in primary human trophoblast cells was obviously affected by inhibitors of OCT3, OCTN1 and OCTN2. The above results indicate that hOCTN1 and hOCTN2 likely contribute to the sulpiride uptake from maternal circulation to trophoblast cells, while hMDR1 and hBCRP mediate the efflux from trophoblast cells to maternal circulation, and hOCT3 probably is involved in the bidirectional transport of sulpiride between the placenta and fetal blood.

Effect of microRNA-106b on the invasion and proliferation of trophoblasts through targeting MMP-2

Objective: To investigate the expression of microRNA-106b (miR-106b) in the placentas of patients with pre-eclampsia and its relationship with matrix metallopeptidase (MMP) -2, and its effect on the invasion and proliferation of trophoblasts. Methods: (1) Placental tissues were collected from patients with mild pre-eclampsia (mPE, n=30), severe pre-eclampsia (sPE, n=30) and normal pregnant women (n=40). Human choriocarcinoma cell lines JAR and JEG3 were assigned to the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group, respectively. (2) The target gene of miR-106b(such as MMP-2) was predicted by bioinformatics. Dual-luciferase reporting system was used to verify the regulation of miR-106b on the expression of MMP-2. (3) The expressions of miR-106b and MMP-2 were measured by quantitative real-time PCR (qRT-PCR) and western blot. (4) Cell proliferation was determined by MTT assay. (5) Invasive activities in each group were assessed by cell transwell invasion assays. Results: (1) Predicting result of bioinformatics indicated that MMP-2 was one of the target genes of miR-106b. Dual-luciferase activity assay demonstrated that MMP-2 was the direct target of miR-106b (P<0.01) .(2) The results of qRT-PCR.①The expression of miR-106b in the placentas of mPE, sPE, normal pregnant women were 2.89±0.04, 1.96±0.03, 1.01±0.03, respectively (P<0.05). And the expression of MMP-2 mRNA in the placentas of mPE, sPE, normal pregnant women were 1.87±0.05, 0.69±0.03, 2.78±0.03, respectively (P<0.05). ②The expression of miR-106b in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 2.39±0.03, 1.03±0.04, 0.73±0.03, 1.11±0.04, respectively (P<0.05). And its expression in the JEG3 cell line were 2.17±0.04, 1.18±0.04, 0.61±0.03 and 1.22±0.03, respectively (P<0.05). ③The expression of MMP-2 mRNA in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 0.45±0.15, 1.02±0.03, 2.28±0.03, 1.11±0.03, respectively (P<0.05). And its expression in the JEG3 cell line were 0.58±0.03, 1.25±0.15, 2.25±0.03, 1.21±0.03, respectively (P<0.05). (3) The results of western blot. ①The expression of MMP-2 protein in the placentas of mPE, sPE, normal pregnant women were 1.63±0.04, 0.55±0.03, 2.82±0.03, respectively (P<0.05). ②The expression of MMP-2 protein in the JAR cell line in the miR-106b mimics group, the mimics negative control group, the miR-106b inhibitor group and the inhibitor negative control group were 0.41±0.03, 0.97±0.03, 2.25±0.03, 1.01±0.03, respectively (P<0.05). And its expression in the JEG3 cell line were 0.53±0.03, 1.20±0.03, 2.31±0.04, 1.19±0.03, respectively (P<0.05). (4) miR-106b could inhibit the proliferation of JAR and JEG3 cells, cell proliferation rates in the miR-106b mimics group were lower than that in the mimics negative control group (P<0.05). And cell proliferation rate in the miR-106b inhibitor group was higher than the inhibitor negative control group (P<0.05). (5) The numbers of JAR cell that passed the membrane in the miR-106b mimics group, the mimics negative control group. The miR-106b inhibitor group and the inhibitor negative control group were 61±15, 79±13, 134±13, 80±12, respectively(P<0.05). And the numbers of JEG3 cell that passed were 57±12, 71±15, 128±15, 70±14, respectively (P<0.05). Conclusion: The miR-106b could inhibit the invasion and proliferation of JAR and JEG3 cells through targeting MMP-2, and have a relationship with the pathogenesis of pre-eclampsia.

Long Noncoding RNA LINC00261 Suppresses Cell Proliferation and Invasion and Promotes Cell Apoptosis in Human Choriocarcinoma.

Choriocarcinoma is one of the gestational trophoblastic neoplasias (GTNs) that originate in the chorionic villi and the extravillous trophoblast. Long noncoding RNAs (lncRNAs) are a type of non-protein-coding RNAs that have recently been implicated in human tumorigenesis. The present study investigated the role of the lncRNA LINC00261 in cell proliferation, metastasis, and apoptosis in choriocarcinoma cell lines. The transcription level of LINC00261 was significantly lower in choriocarcinoma tissues and in choriocarcinoma cell lines. Overexpression of LINC00261 caused a decrease in cell proliferation and arrested the cell cycle at the G0/G1 phase. Furthermore, overexpression of LINC00261 inhibited cell migration and invasion. Meanwhile, it promoted cell apoptosis and the relative activities of caspase 3 and caspase 9 in choriocarcinoma JEG-3 and JAR cells. These data suggested that LINC00261 promotes cell proliferation and metastasis in choriocarcinoma. Our data might provide novel insight into the early diagnosis and treatment of choriocarcinoma in clinics.

The synthetic cannabinoid WIN-55,212 induced-apoptosis in cytotrophoblasts cells by a mechanism dependent on CB1 receptor.

The endocannabinoid system has evolved as a key regulator in several pathological and physiological processes, including placentation, decidualization and implantation. In addition, it is known that Cannabis and cannabinoids negatively affect female reproduction. Although, the biological action of synthetic cannabinoids, such as WIN-55,212, in human fertility and pregnancy outcome remain to be unveiled. A tight balance between proliferation, differentiation and apoptosis of trophoblast cells is required for placental development and pregnancy outcome. Therefore, in this work, the effects of the synthetic cannabinoid WIN-55,212 in placental cytotrophoblast cells were explored. For that, it was used a human choriocarcinoma cell line, BeWo cells, and primary cultures of human cytotrophoblasts isolated from term placentas. Results demonstrate that this synthetic cannabinoid induces cell cycle arrest. We also observed that cell viability loss was associated with a disruption of mitochondrial membrane potential and activation of caspases -9 and -3/-7 independently of reactive oxygen species (ROS) production or recruitment of the endoplasmic reticulum stress marker CHOP. Moreover, these effects were prevented by pre-incubation with a selective cannabinoid receptor 1 (CBR1) antagonist (AM281). Thus, our results provide strong evidences of the apoptotic process induced by WIN-55,212 through the activation of the CBR1, which may reveal the impact of cannabinoids consumption during placental development.

In vitro evaluation of combination of EGCG and Erlotinib with classical chemotherapeutics on JAR cells.

Gestational Trophoblastic Neoplasia (GTN) is a term used for a group of malignant gynecological tumors including choriocarcinoma. Low-risk neoplasias can be cured using single agents Methotrexate (MTX) and actinomycin-D (ACD), but in certain cases, decreased responsiveness and serious side effects occur. Therefore, researchers have been attempting to find new treatment modalities. One of the most popular way for increasing cancer patient survival rates is supporting treatment with adjuvant molecules or chemosensitizers. For this purpose, we investigated epigallocatechin-3-gallate (EGCG), a green tea cathecin, and Erlotinib, an EGFR tyrosine kinase inhibitor, as single agents and combined with MTX or ACD. In accordance with this, JAR (human placenta choriocarcinoma) cell line was used as an in vitro model and MTT, LDH, caspase-3 activation, RT-PCR, and Western Blot analyses were performed to investigate the effects of the test materials. Our studies demonstrate that combination of Erlotinib and EGCG with MTX and ACD decreases JAR cell proliferation and metastatic HER2 protein synthesis and increases caspase-3 activation compared to ACD or MTX alone. In addition, significant increase was observed in the apoptotic Bax gene, but no notable protein synthesis occurred in the Western Blot analysis, which suggests that combination of Erlotinib and EGCG with classical chemotherapeutics ACD or MTX may lead the JAR cells to apoptosis, but not by a mitochondrial pathway. All the results indicate that the synergetic effect of Erlotinib and EGCG with classical chemotherapeutics may help to increase patient survival rates of choriocarcinoma, but the detailed mechanism needs further investigation.

Myricetin suppresses invasion and promotes cell death in human placental choriocarcinoma cells through induction of oxidative stress

Myricetin is a bioactive compound found in a variety of vegetables and fruits, and its anti-cancer effects are well known. In this study, we confirmed that myricetin reduced proliferation of two choriocarcinoma cell lines (JAR and JEG-3) and also promoted apoptosis and regulated cell cycle progression in a dose-dependent manner in JAR and JEG-3 cells. In addition, we found that invasive and pro-angiogenic properties of malignant JAR and JEG-3 trophoblast cells were attenuated by myricetin treatment via MAPK and PI3K/AKT signaling pathways. In addition, we found that ROS production, lipid peroxidation, glutathione depletion, and loss of mitochondrial membrane potentials were enhanced in JAR and JEG-3 cells treated with myricetin. Moreover, myricetin augmented cytosolic Ca2+ release from the endoplasmic reticulum associated with modulation of ER stress in JAR and JEG-3 cells. Our results also revealed that myricetin had synergistic antiproliferative effects with current chemotherapeutics, etoposide and cisplatin, on choriocarcinoma cells. Collectively, results of the present study provide strong evidence for the potential of myricetin to be an effective therapeutic for the prevention of human placental choriocarcinomas.

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.

C-Rel Promotes Invasion of Choriocarcinoma Cells via PI3K/AKT Signaling

Objective: Choriocarcinoma is the most common epithelial cancer among gestational trophoblastic diseases (GTDs); the mechanism of trophoblastic carcinogenesis is unknown. This study aimed to examine the expression of NF-κB family proteins in GTDs and placental tissues as well as the roles of c-Rel in choriocarcinoma. Methods: We examined the expression of NF-κB family proteins in normal placenta and hydatidiform mole tissues as well as extravillous trophoblast (EVT) and choriocarcinoma cell lines by Western blot and immunohistochemistry. Immunoprecipitation was performed to determine which proteins can bind with c-Rel in choriocarcinoma cells. To investigate the roles of c-Rel in choriocarcinoma, we examined the effects of c-Rel knockdown and overexpression on cell proliferation, migration, and invasion using small interfering RNAs and gene activation plasmid. Results: The expression of c-Rel was strong in choriocarcinoma and EVTs, but very weak in villi of normal placenta and hydatidiform mole. Immunoprecipitation suggested that c-Rel heterodimerizes with p65 in choriocarcinoma. c-Rel knockdown reduced invasion, migration, and AKT phosphorylation in choriocarcinoma cells. c-Rel overexpression in choriocarcinoma increased migratory and invasive abilities, and the effect on invasion was inhibited by a PI3K inhibitor. Conclusion: These findings suggest that c-Rel might play a role in promoting the invasion of choriocarcinoma cells through PI3K/AKT signaling.

N-acetylglucosaminyltransferase IVa promotes invasion of choriocarcinoma.

Gestational trophoblastic neoplasia (GTN) results from the malignant transformation of placental trophoblasts which secrete human chorionic gonadotropin (hCG) as do normal placenta or hydatidiform mole. N-acetylglucosaminyltransferase IV (GnT-IV) is a glycosyltransferase which catalyses the formation of β1,4GlcNAc branches on the mannose core of N-glycans. Previous studies reported that β1,4GlcNAc branches on hCG were detected in GTN but not in normal pregnancy or hydatidiform mole. The aim of the present study was to understand the role of GnT-IVa in choriocarcinoma and find the target proteins for GnT-IVa glycosylation which contribute to the malignancy of choriocarcinoma. Immunohistochemistry showed that Griffonia simplicifolia lectin-II staining and GnT-IVa staining were intense in trophoblastic cells of invasive mole and choriocarcinoma. We established a choriocarcinoma cell line with GnT-IVa overexpression (Jar-GnT4a), and examined its malignant potential and target proteins for GnT-IVa glycosylation. GnT-IVa overexpression increased the cell migration and invasion (2.5- and 1.4-fold) as well as the ability to adhere to the extracellular matrix (ECM) components, including fibronectin and collagen typeI andIV. The tumour formation potential of Jar-GnT4a in mice was significantly higher than that of control (P=0.0407), and the cumulative survival rate of mice with Jar-GnT4a was relatively lower than those with control. Immunoprecipitation studies showed that β1,4GlcNAc branches of N-glycans on integrin β1 in choriocarcinoma cells were increased by GnT-IVa overexpression. Nano-LC/MS/MS analysis suggested that lysosome-associated membrane glycoprotein 2 (LAMP-2) was a target protein for glycosylation by GnT-IVa. The increase in β1,4GlcNAc branches on LAMP-2 by GnT-IVa overexpression was confirmed by lectin blot analysis using whole cell lysate and conditioned medium. Our results suggest that highly branched N-glycans generated by the action of GnT-IVa are present in trophoblastic cells of GTN in proportion to GnT-IVa expression level, and that GnT-IVa may contribute to the malignancy of choriocarcinoma by promoting cell adhesion, migration and invasion through glycosylation of integrin β1 and LAMP-2.

Inhibitory Effects of Quercetin on Progression of Human Choriocarcinoma Cells Are Mediated Through PI3K/AKT and MAPK Signal Transduction Cascades.

As a major dietary flavonol, quercetin mitigates proliferation and progression of cancer due to its anti-angiogenic, anti-inflammatory, anti-oxidant, and apoptotic biological effects on cells. Although its apoptotic effects have been reported for various cancers, little is known of the functional role of quercetin in gestational choriocarcinoma. Results of the present study indicated that quercetin reduced proliferation and induced cell death in two choriocarcinoma cell lines, JAR and JEG3 cells, with an increase in the sub-G1 phase of the cell cycle. In addition, quercetin induced mitochondrial dysfunction significantly reduced mitochondrial membrane potential (MMP) and increased production of reactive oxygen species (ROS) in both JAR and JEG3 cells. Further, quercetin inhibited phosphorylation of AKT, P70S6K and S6 proteins whereas, it increased phosphorylation of ERK1/2, P38, JNK and P90RSK proteins in JAR and JEG3 cells. The decrease in viability of choriocarcinoma cells treated with quercetin was confirmed by using combinations of quercetin and pharmacological inhibitors of the PI3K and MAPK signaling pathways. Classical chemotherapeutic agents, cisplatin (a platinum-based drug) and paclitaxel (a taxene-based drug), inhibited proliferation of JAR and JEG3 cells, and when combined with quercetin, the antiproliferative effects of cisplatin and paclitaxel were enhanced for both choriocarcinoma cell lines. Collectively, these results suggest that quercetin prevents development of choriocarcinoma and may be a valuable therapeutic agent for treatment of choriocarcinoma through its regulation of PI3K and MAPK signal transduction pathways. J. Cell. Physiol. 232: 1428-1440, 2017. © 2016 Wiley Periodicals, Inc.

The effects of bortezomib alone or in combination with 5-fluorouracil on proliferation and apoptosis of choriocarcinoma cells.

To investigate the effects of bortezomib alone and in combination with 5-fluorouracil (5-FU) on proliferation and apoptosis in the human choriocarcinoma cell line JEG-3. Cells were treated with bortezomib, 5-FU or with a combination. Proliferation and apoptosis were measured. NF-iB protein expression was examined using immunofluorescence. Following treatment with ten nM bortezomib, rates of apoptosis were significantly higher than controls (p < 0.05) and NF-kB expression increased. 5-FU at 0.025 μg/ml or 0.25 μg/ml resulted in 60.1 ± 0.4% and 67.0 ± 0.2% growth inhibition, respectively, an increase compared to individual treatment (p < 0.05). Apoptosis in cells treated with bortezomib +5-FU was significantly higher than either treatment alone (p < 0.05). Inhibition of proliferation by the combination treatment was synergistic. Bortezomib alone or in combination with 5-FU inhibited JEG-3 cell proliferation and induced apoptosis by increasing NF-kB expression. Combination treatment exerted synergistic effects on growth inhibition.

DNA methylation mediated up-regulation of TERRA non-coding RNA is coincident with elongated telomeres in the human placenta.

What factors regulate elongated telomere length in the human placenta? Hypomethylation of TERRA promoters in the human placenta is associated with high TERRA expression, however, no clear mechanistic link between these phenomena and elongated telomere length in the human placenta was found. Human placenta tissue and trophoblasts show longer telomere lengths compared to gestational age-matched somatic cells. However, telomerase (hTERT) expression and activity in the placenta is low, suggesting a role for an alternative lengthening of telomeres (ALT). While ALT is observed in 10-15% of human cancers and in some mouse stem cells, ALT has never been reported in non-cancerous human tissues. Human term placental tissue and matched cord blood mononuclear cells (CBMCs) were collected as part of the Peri/Postnatal Epigenetic Twins study (PETS). In addition, first trimester placental villi, purified cytotrophoblasts, choriocarcinoma cell lines and a panel of ALT-positive cancer cell lines were tested. Telomere length was determined using the Terminal Restriction Fragment (TRF) assay and a relative quantitative PCR method. DNA methylation levels at several CpG rich subtelomeric TERRA promoters were determined using bisulfite conversion and the SEQUENOM EpiTYPER platform. Expression of TERRA and hTERT was determined using quantitative RT-PCR. ALT was assessed using the C-circle assay (CCA). The human placenta tissue and purified first trimester trophoblasts showed low subtelomeric (TERRA) DNA methylation compared to matched CBMCs and other somatic cells. Interestingly placental TERRA methylation was lower than ALT-cancer cell lines, previously reported to be hypomethylated at these loci. Low TERRA methylation was associated with higher expression of TERRA RNA in placenta compared to matched CBMCs. Detectable levels of C-circles were observed in first trimester placental villi, but not term placenta, suggesting that the ALT mechanism may be active in specific placental cells in early gestation. C-circle analysis of purified first trimester trophoblasts and ALT-associated PML bodies (APB) staining of first trimester villi cross-sections failed to identify this specific cell type population. While first trimester villi showed detectable levels of C-circles, these levels were very low compared with those observed in ALT-positive tumours and cell lines. This is consistent with a small sub-population of ALT-positive cells but this requires further investigation. Finally, no mechanistic link was established between TERRA DNA methylation, the presence of C-circles and longer telomere length. Given the previously described role of TERRA ncRNA as a negative regulator of telomerase, the finding of elevated TERRA and long telomeres is counterintutive. ALT as a mechanism for telomere length maintenance has only been reported in certain human cancers, and recently in mouse embryonic stem cells and embryos. As with many aspects of cancer, it appears that ALT activity in tumours may be the inappropriate activation of a pathway found in very specific cell types in human development. Our data are the first supportive evidence for ALT in a non-cancerous human tissue, a result that requires further investigation and replication. The level of TERRA methylation in the human placenta is significantly lower than found in ALT cancer cell lines and somatic cells, raising the possibility of a novel mechanism in maintaining low methylation at subtelomeric regions. Not applicable. This study was supported by NHMRC early career fellowship (B.N.), NHMRC Senior Research Fellowship (R.S.) and the Victoria Government Infrastructure Grant. R.R. holds a patent for the C-circle assay. No other conflicts declared.

Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells.

A4. EGFL7 in placenta trophoblast and endothelial cells: implications in the pathogenesis of pre-eclampsia

Introduction: Placental development requires invasion and differentiation of trophoblast cells, together with coordinated fetal vasculogenesis and maternal vascular remodeling. During placental development, extravillous cytotrophoblasts invade the uterine spiral arteries of the decidua and myometrium. These invasive cytotrophoblasts replace the endothelial layer of the maternal spiral arteries, transforming them from high-resistance vessels into large-caliber capacitance vessels capable of adequately nourishing the fetus. These events are fine-tuned by a variety of factors critical for a successful pregnancy. Inadequate cytotrophoblast invasion and an incomplete uterine spiral artery remodeling may result in disorders of pregnancy, including intrauterine growth restriction, pre-term labor and preeclampsia (PE). Recent studies suggested that epidermal growth factor-like domain 7 (EGFL7) may play a role in this context. EGFL7, originally identified as an endothelial-restricted gene, is critical for embryonic vascular development. We recently demonstrated that EGFL7 in the placenta is expressed not only by endothelial cells of both the maternal and fetal vasculature, but also by the trophoblast cell lineage, throughout placental development. Previous studies showed that EGFL7 is able to interact with and modulate NOTCH signaling. Moreover, EGFL7 is able to activate EGFR, whose pathways play a major role in trophoblast migration.Methods: To investigate the role of EGFL7 in the trophoblast, we generated stable overexpression and downregulation of EGFL7 in both the human choriocarcinoma cell line Jeg3 and primary human trophoblast. The migration and invasion ability of these cells were tested using wounding and transwell assays. Protein extracts from serum starved EGFL7-overexpressing trophoblast cells were analyzed by western blot for EGFR, ERK and AKT phosphorylation. In addition, we evaluated if EGFL7 overexpression in trophoblast cells is associated to altered expression of the NOTCH signaling pathway using immunofluorescence and qRTPCR. By using inhibitors of both pathways, variations in migration/invasion ability induced by EGFL7 overexpression were investigated. Spatial distribution of EGFL7 was studied by immunofluorescence and qRT-PCR analyses in placental tissues of normal and preeclamptic pregnant women.Results: EGFL7 overexpression in Jeg3 and in primary trophoblast cells resulted in significantly increased cell migration and invasiveness, while its knockdown strongly reduced both phenomena. Analysis of EGFR, MAPK and PI3K pathways and the use of specific inhibitors in transwell assay showed that EGFL7 promoted trophoblast cell motility by activating these pathways. In addition, we demonstrated that EGFL7 triggered migration of trophoblast cells involved a concomitant activation of NOTCH signaling. Immunofluorescence analysis of placental tissues from preeclamptic (n = 10) and normal pregnant women (n = 10) showed lower levels of EGFL7 in both endothelial and trophoblast cells. This observation was quantified by qRT-PCR, which revealed that EGFL7 mRNA expression was significantly decreased by more than 10-fold in PE when compared to normal pregnancies.Conclusions: Our data suggest that EGFL7 regulates cell migration and invasion of trophoblast cells by activating MAPK, PI3K and NOTCH signaling pathways. These results suggest a correlation between EGFL7 reduced expression and the inadequate trophoblast invasion observed in PE.

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.

Sera of patients with recurrent miscarriages containing anti-trophoblast antibodies (ATAB) reduce hCG and progesterone production in trophoblast cells in vitro

ProblemReproductive failure including RM has been suggested to correlate with antibodies that cross react with HLA-negative syncytiotrophoblasts and we have reported that 17% of women with 2 or more miscarriages and 34% of women with 3 or more miscarriages express anti-trophoblast antibodies (ATAB). Until now, the mechanism, how ATAB interfere with pregnancy success is not known. HCG and progesterone both play fundamental roles in supporting human pregnancy. Therefore we investigated the effects of sera of RM patients containing ATAB on the hCG and progesterone production of cells of the choriocarcinoma cell line JEG-3. Method of studyIn vitro study to investigate effects of patient sera with and without ATAB on hCG and progesterone secretion of JEG-3 cells. The presence of ATAB was detected as described earlier. Effects of sera from ATAB positive and ATAB negative RM patients on hCG and progesterone secretion by JEG-3 cells were analysed 12 and 24h after plating. Sera of women without pregnancy pathologies served as controls. ResultsSera of ATAB-positive RM patients significantly inhibit hCG secretion of JEG-3 cells for 12h after plating compared to sera of healthy controls (p=0.019) and significantly reduce progesterone production for 12h (p=0.046) and 24h (p=0.027) of co-culture. Sera of ATAB-negative RM patient show no significant effect on progesterone secretion. ConclusionsInhibition of hCG and progesterone production might point to a mechanism, how ATAB interfere with early pregnancies.

Regulation of hypoxia-inducible factor-1α, regulated in development and DNA damage response-1 and mammalian target of rapamycin in human placental BeWo cells under hypoxia

IntroductionHypoxia-inducible factor-1 (HIF-1), regulated in development and DNA damage response-1 (REDD1) and mammalian target of rapamycin (mTOR) are crucial mediators of many metabolic processes in various cell types under hypoxia. The involvement and regulation of these three factors underlying trophoblasts' response to hypoxia remains to be determined. MethodsSpecific siRNAs were applied to inhibit the expression of the corresponding genes and to investigate the roles of HIF-1α in modulating REDD1/mTOR and REDD1 in regulating mTOR/HIF-1α in the human choriocarcinoma cell line BeWo under normoxia and hypoxia. ResultsExposure of BeWo cells to 1% oxygen (compared with 21% oxygen) led to a remarkable increase of both HIF-1α and REDD1 and an obvious decrease of mTOR at both the mRNA and protein levels. Interference of HIF-1α expression by siRNA resulted in an apparent reduction of REDD1 parallel with that of HIF-1α during normoxia and hypoxia. Additionally, the hypoxia-induced REDD1 increase was blocked through loss of HIF-1α, and the downregulation of mTOR in hypoxia could be partly obstructed by HIF-1α-siRNA transfection. Separately, the modulation effect of REDD1 was confirmed in an experiment demonstrating that the hypoxia-induced decrease of mTOR was inhibited by REDD1 knockdown, which was measured by changes in both the mRNA and protein levels. The disruption of REDD1 expression also led to increased accumulation of HIF-1α under both normoxia and hypoxia. DiscussionThe HIF-1α-REDD1-mTOR pathway was involved in the response to hypoxia in BeWo cells. Hypoxia-induced REDD1 upregulation is mediated by a HIF-1α-dependent pathway. Disruption of REDD1 blocked the effects of hypoxia on suppressing mTOR and resulted in additional accumulation of HIF-1α in BeWo cells.