All-trans retinoic acid (atra) inhibits telomerase expression of BeWo choriocarcinoma cell (ATCC CCL-98)

@#Introduction: Choriocarcinoma is malignant cancer originating from placental trophoblast. The incidence of this cancer is estimated at 0.57-1.1 per 1000 births in the United States of America, Australia, Europe, and New Zealand. The rate is much higher in South East Asia and Japan with two occurrences per a thousand births. Telomerase activity is an important part of the apoptotic process. Increased telomerase activity will result in cellular immortality and poor prognosis in cancer. Vitamin A possess an essential role in cell proliferation and differentiation. One of the active metabolites of vitamin A is All-Trans Retinoic Acid (ATRA). Methods: In this study, we examined the role of ATRA against telomerase activity in choriocarcinoma cell. This cell was derived from BeWo cell line (ATCC CCL-98) and were given different doses of ATRA. Results: From this study, Choriocarcinoma cell that was given ATRA in dosage of 50μg/ml inhibit telomerase activity by extending the cycle time of 39.51±0.09, compared to the control group with a cycle time of 37.62±0.43. Cycle length change consistently with higher dose of ATRA. Conclusion: This study has proven that ATRA could inhibit telomerase activity by lengthening the cycle. Changes in the increase of ATRA doses in this experimental test need to be studied further on experimental animals, either administered as a single agent or as an addition to standard treatment of trophoblastic disease

Study on in vitro model of hepatitis B virus-infected transwell chambers mediated by peripheral blood mononuclear cells / 中华传染病杂志

Objective To observe the transport of hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC)through placental barrier set up by choriocarcinoma trophoblast cells (Bewo cells),and to explore the biological role of PBMC as a carrier for HBV transport.Methods Bewo cells and PBMC were cultured and their proliferation and activity were detected by cell counting kit (CCK)-8.One hundred μL serum containing 5 ×10 6 copy/mL HBV DNA was used to infect PBMC,and cells infected with HBV were labeled by fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE).A co-culture model of Bewo cells and HBV-infected PBMC was set up by transwell chamber. The migration of HBV-infected PBMC was detected by flow cytometry.Realtime fluorescence quantitative polymerase chain reaction method was used to detect HBV DNA contents of PBMC under transwell chamber.Results PBMC and Bewo cells proliferated at around 24 h and entered into growth stagnation at around 120 h.The contents of PBMC labeled by green fluorescent at 0,12,24 and 48 h during co-culture under chamber were (0.445 ±0.021)%,(21 .180 ± 4.653 )%,(34.830 ± 7.156 )% and (64.185 ± 3.161)%,respectively.The amount of PBMC marked green fluorescence increased over prolonged incubation time (F =68.983,P =0.001 ).PBMC HBV DNA contents at 24 and 48 h of co-culture under chamber were (1.925±0.431)×103 copy/mL and (2.565 ±0.361)×103 copy/mL,respectively,indicating that PBMC under chamber were infected with HBV.Conclusions PBMC may be a target for HBV infection in extrahepatic tissues.Placental trophoblastic barrier built by transwell chambers may provide new ideas to investigate HBV transmission across the placenta in vitro .

Hypericum caprifoliatum and Hypericum connatum affect human trophoblast-like cells differentiation and Ca

Objective: To study the effect of crude methanol and n-hexane extracts of Hypericum connatum (H. connatum) and Hypericum caprifoliatum on trophoblast-like cells. Methods: BeWo and JEG-3 trophoblast-like cells were submitted to different extract concentrations (1, 5, 10 and 15 μg/mL) and evaluated in relation to cell viability and in vitro trophoblast differentiation and function. Cell viability was evaluated using WST-1 reagent. Differentiation was measured by luciferase production, hCG production/release, and mitogen-activated protein kinase signaling pathway activation. The function of the trophoblast-like cells was measured by

Hypericum caprifoliatum and Hypericum connatum affect human trophoblast-like cells differentiation and Ca2+influx

Objective:To study the effect of crude methanol and n-hexane extracts of Hypericum connatum (H. connatum) and Hypericum caprifoliatum on trophoblast-like cells. Methods: BeWo and JEG-3 trophoblast-like cells were submitted to different extract concentrations (1, 5, 10 and 15 μg/mL) and evaluated in relation to cell viability and in vitro trophoblast differentiation and function. Cell viability was evaluated using WST-1 reagent. Differentiation was measured by luciferase production, hCG production/release, and mitogen-activated protein kinase signaling pathway activation. The function of the trophoblast-like cells was measured by 45Ca2+influx evaluation. Results:The results showed a decrease in cell viability/proliferation. Both plants and different extracts induced a significant decrease in hCG production/release and luciferase production. H. connatum did not cause mitogen-activated protein kinase signaling pathway disturbance;however, Hypericum caprifoliatum n-hexane extract at 15 μg/mL inhibited extracellular signal-regulated kinase 1/2 activation. The significant increase in Ca2+influx by JEG-3 cells was seen after short and long incubation times with H. connatum methanolic extract at 15 μg/mL. Conclusions: The results indicated that these two Hypericum species extracts can interfere on trophoblast differentiation and Ca2+influx, according to their molecular diversity. Although in vivo experiments are necessary to establish their action on placental formation and function, this study suggests that attention must be paid to the potential toxic effect of these plants.

Hypericum caprifoliatum and Hypericum connatum affect human trophoblast-like cells differentiation and Ca(2+) influx

<p><b>OBJECTIVE</b>To study the effect of crude methanol and n-hexane extracts of Hypericum connatum (H. connatum) and Hypericum caprifoliatum on trophoblast-like cells.</p><p><b>METHODS</b>BeWo and JEG-3 trophoblast-like cells were submitted to different extract concentrations (1, 5, 10 and 15 µg/mL) and evaluated in relation to cell viability and in vitro trophoblast differentiation and function. Cell viability was evaluated using WST-1 reagent. Differentiation was measured by luciferase production, hCG production/release, and mitogen-activated protein kinase signaling pathway activation. The function of the trophoblast-like cells was measured by (45)Ca(2+) influx evaluation.</p><p><b>RESULTS</b>The results showed a decrease in cell viability/proliferation. Both plants and different extracts induced a significant decrease in hCG production/release and luciferase production. H. connatum did not cause mitogen-activated protein kinase signaling pathway disturbance; however, Hypericum caprifoliatum n-hexane extract at 15 µg/mL inhibited extracellular signal-regulated kinase 1/2 activation. The significant increase in Ca(2+) influx by JEG-3 cells was seen after short and long incubation times with H. connatum methanolic extract at 15 µg/mL.</p><p><b>CONCLUSIONS</b>The results indicated that these two Hypericum species extracts can interfere on trophoblast differentiation and Ca(2+) influx, according to their molecular diversity. Although in vivo experiments are necessary to establish their action on placental formation and function, this study suggests that attention must be paid to the potential toxic effect of these plants.</p>

A quantitative assessment of the morphological characteristics of BeWo cells as an in vitro model of human trophoblast cells / Una evaluación cuantitativa de las características morfológicas de las células BeWo como un modelo in vitro de las células de trofoblasto humano

In order to study the detailed morphology of trophoblast cells during human implantation, BeWo cells were cultured as spheroids in suspension culture. These cultures were then processed for light and electron microscopical examination. The present study showed that the BeWo spheroids consist of two cell types which are cytotrophoblast-like and syncytiotrophoblast-like. The cells with larger nuclear diameter made up only about 1 percent of the cell population and appear to be those of syncytiotrophoblast. Therefore the predominant cell type of the BeWo spheroids appeared to be relatively undifferentiated and cytotrophoblast-like. About 10 percent of the BeWo cells in the present study were mitotic, indicating a highly proliferative population. Total cell number increased about 12 times during the culture period from 107 +/- 9 on day 1 to 1211 +/- 145 on day 7 whereas the volume per cell increased about 2 times, from 1300 um3 on day 1 to 2400 um3 on day 7. Therefore overall growth of BeWo spheroids is due to both hyperplasia and hypertrophy. However, it appears that cell proliferation outstrips volumetric growth. These quantitative data show that BeWo cells grow mainly by hyperplasia and provide baseline values for further studies. In addition, the results show that BeWo cell morphology has marked similarities to that reported for human trophoblast, making it a useful model for subsequent in vitro studies.

En un cultivo de suspensión se estudió la morfología de las células durante la implantación del trofoblasto humano, células BeWo. Estos cultivos fueron procesados y examinados a través de microscopía de luz y electrónica. El estudio mostró que los esferoides BeWo constan de dos tipos de células, citotrofoblasto y sincitiotrofoblasto. Las células con mayor diámetro nuclear parecen ser los sincitiotrofoblasto que representaban sólo el 1 por ciento de la población celular. Por tanto, el tipo celular predominante de los esferoides BeWo parecían ser relativamente indiferenciados como citotrofoblasto. Alrededor del 10 por ciento de las células BeWo fueron mitóticas, lo que indica una población altamente proliferativa. El número de células totales aumentó alrededor de 12 veces durante el período de cultivo de 107 +/- 9 días en el día 1 a 1211 +/- 145 en el día 7, mientras que el volumen de la célula creció alrededor de 2 veces, desde 1300 mm3 el día 1 hasta 2400 mm3 el día 7. Por lo tanto, el crecimiento global de esferoides BeWo se debe tanto a la hiperplasia como a la hipertrofia. Sin embargo, parece que la proliferación celular supera al crecimiento volumétrico. Estos datos cuantitativos muestran que las células BeWo crecen principalmente por hiperplasia y proporcionan valores de referencia para estudios posteriores. Además, los resultados muestran que la morfología celular BeWo ha marcado similitudes con los reportado para el trofoblasto humano, por lo que es un modelo útil para posteriores estudios in vitro.

14-3-3 tau regulates invasiveness of BeWo cells via ERK1/2 pathway / 中国病理生理杂志

AIM: To investigate the potential role of 14-3-3 tau in trophoblast cells on invasiveness. METHODS: 14-3-3 tau expression was detected in first-trimester villi, deciduas and human trophoblastic cell line (BeWo) by immunohistochemistry. Small interference RNA (siRNA) targeting 14-3-3 tau was transfected into BeWo cells. The effects of down-regulated 14-3-3 tau on invasion of human trophoblasts cell line BeWo were examined by matrigel invasion assay, and the transcription, translation of E-cadherin and snail were estimated by RT-PCR or Western blotting. U0126 was used to detect the extracellular-signal related kinase 1/2 (ERK1/2) function on down-regulation of 14-3-3 tau induced cell invasion. RESULTS: 14-3-3 tau was detected in the invasive trophoblastic cells in the first trimester villi and that invaded to the deciduas. BeWo cells also expressed 14-3-3 tau. Down-regulation of 14-3-3 tau increased the invasive cell-number of BeWo, as well as the expression of snail, and inhibited E-cadherin. U0126 inhibited the enhanced invasiveness in these cells induced by the down-regulation of 14-3-3 tau. CONCLUSION: 14-3-3 tau may regulate the invasiveness of human trophoblastic cells through ERK1/2 signaling pathway.

Estandarización del aislamiento y caracterizaciónde células estromales de decidua y útero murinos / Estandardização do isolamento e caracterização de células estromales de decídua eútero murinos / Standardization of isolation and characterization of decidual stromal cells and murine uterus

Las células estromales son las células más abundantes presentes en la decidua y juegan un papelmuy importante durante la implantación, la nutrición fetal y el mantenimiento del embarazo. Losprocedimientos que se han descrito para el aislamiento de células estromales requieren el uso de muchosanticuerpos monoclonales ya que hay contaminación con otros tipos celulares en la decidua y ademásalgunos marcadores características de células estromales, muestran variabilidad en los diferentes díasde la gestación. En este estudio se estandarizó un procedimiento de aislamiento por digestión enzimática,gradiente de densidad y adherencia al plástico y se caracterizaron las células estromales murinas porexclusión de marcadores que se expresan en macrófagos (F4-80), células epiteliales y trofoblasto(citoqueratina-7), obteniéndose un 98% de células negativas para estos marcadores que correspondería alas células estromales. Esta técnica de aislamiento permite obtener células estromales con métodos menoscostosos y altamente eficientes que facilita el acceso a un modelo celular de gran utilidad en el estudio dela fisiología de la gestación en diferentes especies.

Stromal cells are the most abundant cell population present in decidual tissue; they are involved inkey processes during embryo implantation, fetal nutrition and the pregnancy maintenance. Described procedures for stromal cells isolation require the use of many monoclonal antibodies due to contaminationwith another cell types in the decidua; besides, some markers of stromal cells show variability during thedays of gestation. In this study, we standardized a procedure for isolation by enzymatic digestion, densitygradient and adherence to plastic. Murine stromal cells were characterized by exclusion of markers thatare expressed in macrophages (F4-80), epithelial cells and trophoblast (cytokeratin-7), yielding a 98% ofnegative cells for these markers that correspond to stromal cells. This isolation procedure permits to obtainstromal cells with less expensive and high efficiency methods that provide a useful cellular model to studythe physiology of gestation in different species.

As células estromales são as células mais abundantes presentes na decídua e tem um papel muitoimportante durante a implementação, a nutrição fetal e a manutenção da gravidez. Os procedimentosdescritos para o isolamento das células estromales necessitam o uso de muitos anticorpos monoclonais jáque há contaminação com outros tipos celulares na decídua e demais alguns marcadores característicosdas células estromales que tem variabilidade nos diferentes dias de gestação. No presente trabalho foiestandardizado um procedimento de isolamento por digestão enzimática, gradiente de densidade eaderência ao plástico e foram caracterizados as células estromales murinas por exclusão de marcadoresque expressão em macrófagos (F4-80), células epiteliais e trofoblasto (citoqueratina-7), obtendo-se um98% de células negativas para estes marcadores que corresponderia às células estromales. Esta técnicade isolamento permite obter células estromales com métodos menos custosos e altamente eficientes quefacilita o aceso a um modelo celular de grande utilidade no estudo da fisiologia da gestação em diferentesespécies.

Preeclampsia, migración celular y canales iónicos / Preeclampsia, cellular migration and ion channels

En la placenta humana, el sinciciotrofoblasto es la barrera que regula el transporte de nutrientes, solutos y agua entre la sangre materna y fetal. Dentro de este movimiento transepitelial se encuentra el del Na+, su contribución a la presión osmótica es fundamental en la regulación del volumen de líquido extracelular. El canal epitelial de sodio sensible al amiloride (ENaC) media el transporte de Na+ desde el lumen hacia el interior celular en numerosos epitelios absortivos. Está regulado por la aldosterona, vasopresina, catecolaminas, estrógenos y progesterona. Es bloqueado por el amiloride y sus análogos. Para su activación, diversas proteasas lo escinden en la membrana plasmática y esto a su vez es regulado por la aldosterona. El ENaC está expresado también en la placenta humana y aunque su función no es conocida, podría participar en la homeostasis de agua y electrolitos. El ENaC también es influenciado por el estado de las proteínas del citoesqueleto y los cambios en el volumen celular alteran a su vez a éste. De esta manera existe una relación entre el ENaC y el citoesqueleto. Además, las corrientes de Na+ por el ENaC y otros canales de sodio participan en la migración celular en células normales y cancerosas. Aquí presentamos evidencias que avalan la hipótesis que el ENaC es necesario para la migración celular en células BeWo, derivadas del trofoblasto humano, que sintetizan hormonas y expresan el ENaC. Las células BeWO han sido utilizadas como modelo experimental para estudiar el transporte en células de placenta.

The syncytiotrophoblast acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. This transepithelial transport involves movement of Na+ and its contribution to the osmotic pressure is an important determinant of the extracellular fluid volume. ENaC is a channel that mediates entry of Na+ from the luminal fluid into the cells in many reabsorbing epithelia; it is aldosterone, vasopressin, insulin and catecholamine-inducible, modulated by estrogens and progesterone and blocked by amiloride and its analogs. Multiple proteases are involved in the proteolytic processing and activation of ENaC subunits and aldosterone alters the protease-protease inhibitors balance. ENaC is also expressed in human placenta; although its function is not well known, the Na+ conductive properties may participate in electrolyte and extracellular volume homeostasis. The activity of ENaC channels and other ion channels and transporters is regulated by the state of actin filaments; on the other hand, changes in volume influence the actin cytoskeleton. Thus, there is an interaction between ENaC and components of the apical membrane cytoskeleton. In addition to their role in cellular homeostasis and electrical properties, Na+ currents through ENaC and other sodium channels are involved in cell migration, well documented in normal and cancer cells. In this work we presented evidences supporting the hypothesis that ENaC channels are required for the migration of BeWo cells, a human hormone-synthesizing trophoblastic cell line that express the three subunits of the ENaC channels. BeWo cell line has also been used as a model to investigate the placental transport mechanisms.

The Influence of Human Chorionic Gonadotropin and Human Placental Lactogen on Growth and Differentiation of Placental Trophoblast / 대한산부인과학회잡지

BACKGROUND: There are many factors that influence the differentiation and growth of trophoblasts. During differentiation of trophoblasts, two major hormones are secreted ; human chorionic gonadotropin (hCG) and human placental lactogen (hPL). These two hormones are secreted in a peculiar pattern during pregnancy and function of these hormones is not yet fully understood. Also, it is not known whether these hormones directly influence the differentiation and growth of trophoblasts. On the other hand, it is known that choriocarcinoma cells are undifferentiated, so they are unable to form syncytiotrophblasts. And many factors may be associated with this inhibitory potential. OBJECTIVE: The purpose of this study was to observe whether the hCG and hPL are associated with differentiation and growth of early placental trophoblasts and becoming malignant. METHOD: The hCG, hPL, IL-6 and insulin were added to cytotrophoblasts isolated from normal 8 to 10 gestational weeks' placental tissues by a degree of concentration, and observed the secreted hPL concentration and morphological change to syncytiotrophoblasts daily. And it was performed in Bewo cells in same manner. RESULT: The increased hPL secretion was noted in hCG, hPL, IL-6 and insulin were added normal trophoblasts and this may result from differentiation of cytotrophoblasts to syncytiotrophoblasts. Also, morphological changes to syncytiotrohoblasts was observed at the same time. But, Increased hPL secretion and syncytiotrophoblasts formation was not detected in Bewo cells. CONCLUSION: In this study, it seems that hCG, hPL, IL-6 and insulin had an influence on differentiation and growth of normal trophoblasts. On the other hand, no changes in hPL secretion and morphology at the choriocarcinoma cell line tells us that defects of differentiation in choriocarcinoma is due to abnormalities of the receptors on hCG and hPL or a differentiation associated gene, not a defect of these hormones themselves.