Choriocarcinoma Cells Research Articles

α-Solanine Inhibits Proliferation, Invasion, and Migration, and Induces Apoptosis in Human Choriocarcinoma JEG-3 Cells In Vitro and In Vivo.

α-Solanine, a bioactive compound mainly found in potato, exhibits anti-cancer activity towards multiple cancer cells. However, its effects on human choriocarcinoma have not been evaluated. In the present study, we investigated the effect of α-solanine on cell proliferation and apoptosis in human choriocarcinoma in vitro and in vivo. The results showed that α-solanine, at concentrations of 30 μM or below, did not affect the cell viability of the choriocarcinoma cell line JEG-3. However, colony formation was significantly decreased and cell apoptosis was increased in response to 30 μM α-solanine. In addition, α-solanine (30 μM) reduced the migration and invasion abilities of JEG-3 cells, which was associated with a downregulation of matrix metalloproteinases (MMP)-2/9. The in vivo findings provided further evidence of the inhibition of α-solanine on choriocarcinoma tumor growth. α-Solanine suppressed the xenograft tumor growth of JEG-3 cells, resulting in smaller tumor volumes and lower tumor weights. Apoptosis was promoted in xenograft tumors of α-solanine-treated mice. Moreover, α-solanine downregulated proliferative cellular nuclear antigen (PCNA) and Bcl-2 levels and promoted the expression of Bax. Collectively, α-solanine inhibits the growth, migration, and invasion of human JEG-3 choriocarcinoma cells, which may be associated with the induction of apoptosis.

A crossroad between placental and tumor biology: What have we learnt?

Placenta in certain species including the human has evolved as a highly invasive tumor-like organ invading the uterus aned its vasculature to derive oxygen and nutrients for the fetus and exchange waste products. While several excellent reviews have been written comparing hemochorial placentation with tumors, no comprehensive review is available dealing with mechanistic insights into what makes them different, and what tumor biologists can learn from placental biologists, and vice versa. In this review, we analyze the structure-function relationship of the human placenta, emphasizing the functional need of the spatio-temporally orchestrated trophoblast invasiveness for fetal development and growth, and pathological consequences of aberrant invasiveness for fetal and maternal health. We then analyze similarities and differences between the placenta and invasive tumors in terms of hallmarks of cancer, some key molecules regulating their invasive functions, and how placental cancers (choriocarcinomas) or other cancers become refractory or even addicted to these invasion-restraining molecules. We cite in vitro models of human trophoblast and choriocarcinoma cell lines utilized to study mechanisms in normal placental development as well as those responsible for tumor progression. We discuss the pathobiology of hyper-invasive placentas and show thattrophoblastic neoplasias are a unique and heterogeneous class of tumors. We delve into the questions as to why metastasis from other organs rarely occurs at the placental site and whether pregnancy makes the mother more or less vulnerable to cancer-related morbidity/mortality. We attempt to compare trophoblast stem cells and cancer stem cells. Finally, we leave the readers with some thoughts as foods of future investigations.

Combined Effects of Methyldopa and Flavonoids on the Expression of Selected Factors Related to Inflammatory Processes and Vascular Diseases in Human Placenta Cells-An In Vitro Study.

The aim of the study was to investigate combined effects of flavonoids (apigenin, baicalein, chrysin, quercetin, and scutellarin) and methyldopa on the expression of selected proinflammatory and vascular factors in vitro for prediction of their action in pregnancy-induced hypertension. The research was conducted on a trophoblast-derived human choriocarcinoma cell line and a primary human umbilical vein endothelial cell line. Cytotoxicity of compounds in selected concentrations (20, 40, and 100 µmol) was measured using the MTT test and the concentration of 40 µmol was selected for further analysis. Subsequently, their effects with methyldopa on the expression of selected markers responsible for inflammation (TNF-α; IL-1β; IL-6) and vascular effects (hypoxia-inducible factor 1α—HIF-1α; placental growth factor—PIGF; transforming growth factor β—TGF-β; vascular endothelial growth factor—VEGF) at the mRNA and protein levels were assessed. It was found that every combined administration of a flavonoid and methyldopa in these cells induced a down-regulating effect on all tested factors, except PIGF, especially at the mRNA expression level. As hypertension generally raises TNF-α, IL-1β, IL-6, HIF-1α, TGF-β, and VEGF mRNA expression and/or protein levels, the results obtained in the studied model may provide a positive prognostic factor for such activity in vivo.

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

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

Insulin-like growth factor 1 signaling in the placenta requires endothelial nitric oxide synthase to support trophoblast function and normal fetal growth.

Currently, there is no effective treatment for placental dysfunction in utero. In a ligated mouse model of fetal growth restriction (FGR), nanoparticle-mediated human insulin-like 1 growth factor (hIGF1) gene delivery (NP-Plac1-hIGF1) increased hIGF1 expression and maintained fetal growth. However, whether it can restore fetal growth remains to be determined. Using the endothelial nitric oxide synthase knockout (eNOS-/-) mouse model, a genetic model of FGR, we found that despite inducing expression of hIGF1 in the placentas treated with NP-Plac1-hIGF1 (P = 0.0425), FGR did not resolve. This was associated with no change to the number of fetal capillaries in the placental labyrinth; an outcome which was increased with NP-Plac1-hIGF1 treatment in the ligated mouse model, despite increased expression of angiopoietin 1 (P = 0.05), and suggested IGF1 signaling in the placenta requires eNOS to modulate placenta angiogenesis. To further assess this hypothesis, BeWo choriocarcinoma cell line and human placental explant cultures were treated with NP-Plac1-hIGF1, oxidative stress was induced with hydrogen peroxide (H2O2), and NOS activity was inhibited using the inhibitor NG-monomethyl-l-arginine (l-NMMA). In both BeWo cells and explants, the protective effect of NP-Plac1-hIGF1 treatment against H2O2-induced cell death/lactate dehydrogenase release was prevented by eNOS inhibition (P = 0.003 and P < 0.0001, respectively). This was associated with an increase in mRNA expression of oxidative stress markers hypoxia inducing factor 1α (HIF1α; P < 0.0001) and ADAM10 (P = 0.0002) in the NP-Plac1-hIGF1 + H2O2 + l-NMMA-treated BeWo cells. These findings show for the first time the requirement of eNOS/NOS in IGF1 signaling in placenta cells that may have implications for placental angiogenesis and fetal growth.

Core 2 β1,6-N-acetylglucosaminyltransferases accelerate the escape of choriocarcinoma from natural killer cell immunity

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted from choriocarcinoma and contains a core2 O-glycan formed by core2 β1,6-N-acetylglucosaminyl transferase (C2GnT). Choriocarcinoma is considered immunogenic as it is gestational and contains paternal chromosomal components. Here we examined the function of C2GnT in the evasion of choriocarcinoma cells from natural killer (NK) cell-mediating killing. We determined that C2GnT is highly expressed in malignant gestational trophoblastic neoplasms. C2GnT KO downregulates core2 O-glycan expression in choriocarcinoma cells, which are more efficiently killed by NK cells than control cells. C2GnT KO cell containing tumor necrosis factor-related apoptosis inducing ligand have lower viability than control cells. Additionally, poly-N-acetyllactosamine in core2 branched oligosaccharides on MHC class I-related chain A (MICA) and mucin1 (MUC1) is significantly reduced in C2GnT KO cells. Meanwhile, the cumulative survival rate of nude mice inoculated with C2GnT KO tumors was higher than that of the control group. These findings suggest that choriocarcinoma cells may escape NK cell-mediated killing via glycosylation of MICA and MUC1.

Human Chorionic Gonadotropin Polypeptide Nanoparticle Drug Delivery System Improves Methotrexate Efficacy in Gestational Trophoblastic Neoplasia in vitro.

PurposeTo alleviate the sufferings of the chemotherapy patients, we developed a novel active targeted therapeutic system and showed its potential as a promising drug delivery strategy.MethodsWe utilized the human chorionic gonadotropin (HCG) ligand-receptor mediation to make an actively targeted drug delivery system with optimal HCG polypeptide fragment as target head base, polyethylene glycol–polylactic acid copolymers as nanometer materials to load chemotherapy drug methotrexate (MTX), to highly selectively deliver MTX into choriocarcinoma lesions, and to investigate the efficacy, targeting and tolerability of the complex in vitro experiments.ResultsOur data show that choriocarcinoma cell lines JEG-3 and JAR exhibited high expression levels of HCG receptor, peptide HCGβ81-95 specifically bonded to HCG receptor-positive cells and HCG81-NP efficiently delivered MTX to choriocarcinoma cells. HCG81-NP-MTX inhibited cell proliferation and reduced G0/G1 to S phase transition in JEG-3 and JAR cells.ConclusionWe designed an active targeting therapy system of choriocarcinoma, significantly improved chemotherapy efficacy in vitro, and provided a theoretical basis for the treatment of malignant trophoblastic tumors.

663 Specific polycyclic aromatic hydrocarbons elicit differential expression of CYP1A1 in trophoblast cells

Prenatal exposures to polycyclic aromatic hydrocarbons (PAHs) are associated with adverse outcomes including small for gestational age infants. We have previously reported differential levels of PAHs in the placenta associated with preterm delivery. We found that levels of a specific PAH, dibenzoanthracene (DBA), was significantly higher than levels of either benzo[a]pyrene (BaP) or Benzo[b]fluoranthene (BbF). Furthermore, we reported that placental DBA levels are significantly and negatively correlated with gestational age at delivery. In this study we aimed to determine if there is a differential response in trophoblast cells with distinct PAH treatments. Immortalized choriocarcinoma cells (BeWo) were treated with 10 μM of either BaP, BbF or DBA for 24 hours. DMSO treatment served as a control. Media was collected and measured to determine levels of hCG production using an ELISA assay. RNA was extracted from cells after 24 hours of treatment, and used to make cDNA. TaqMan primers and probes were used to measure expression levels of CYP1A1, a gene involved in the processing of xenobiotic compounds, using RT-qPCR. GAPDH served as a control. While treatment with each PAH increased CYP1A1 expression compared with DMSO, DBA elicited the largest response, with a 54-fold increase in CYP1A1 expression (p<0.001). CYP1A1 expression was increased 16.5-fold with BbF treatment (p=0.001), and 7.4-fold with BaP treatment (p=0.006). Levels of hCG were not altered with any PAH treatment compared with DMSO, consistent with previous publications in trophoblast cells. In placenta tissue, levels of DBA are found at higher levels compared with BaP and BbF, and DBA levels are increased in placentae from preterm birth. In this study we find that DBA elicits a higher upregulation of CYP1A1 compared with BaP and BbF. Further experimentation is necessary using RNA-Seq analyses to help yield insight into pathways differentially responsive to DBA, yielding insight into its association with preterm delivery.

1112HAND1 knockdown disrupts trophoblast but not placental endothelial global gene expression

We previously reported disrupted placental structure, and vascular changes in placentas from fetuses with Congenital Heart Defects. Signaling between trophoblast and villous endothelium is proposed for proper placental development and function. We hypothesize that disruption of genes associated with CHD within the trophoblast impacts fetal endothelium and results in dysregulated placental vasculature in CHD fetuses. A model of human cytotrophoblast, BeWo choriocarcinoma cells and isolated human placental microvascular endothelial cells (HPMVEC) were treated for 48 hrs with HAND1 siRNA/lipofectamine. Additionally, control HPMVECs were exposed to conditioned BeWo media (+/- HAND1) for 48 hrs. Cells were harvested and total RNA isolated. RNA was sequenced on an Illumina NextSeq 550 platform (single end reads 85bp, read depth 25M). The Galaxy Bioinformatic Platform was used for quality control, alignment (Bowtie2) and generation of read counts (Feature Counts). Normalization and differential gene expression analyses were conducted using general linear modeling in edgeR package from Bioconductor. Significance was determined using a fold change cut off of >1.5 and raw p<0.05. Overrepresentation analysis was performed using Panther DB for Reactome pathways and GO terms Direct HAND1 knockdown in the BeWo choriocarcinoma cell line caused downregulation of 632 genes, and upregulation of 102 genes. Overrepresentation analysis identified disruption to pathways including cell differentiation, neurogenesis, localization, and cell projection organization. In contrast, direct HAND1 knockdown in HPMVECs resulted in minimal disruption to global gene expression with differential expression in just 18 genes. Indirect HAND1 disruption did not alter HPMVEC gene expression. Disruption to HAND1 expression significantly alters gene expression profile in trophoblast but not HPMVEC, either directly or indirectly. Disruption to the structure and vasculature of the placenta in cases of CHD is likely primarily due to abnormal trophoblast development.

315 Localization and differential expression of HLA-G in term human fetal membranes

Pregnancy requires immunologic tolerance to support the semiallogenic fetus. HLA-G, a non-classical type I MHC molecule, plays an immunomodulatory role and its expression is reported in placental extravillous trophoblasts. This study localized HLA-G in human fetal membrane cell layers and determined its differential expression in term not in labor (TNIL) or term labor (TL) samples as an indicator of immunologic changes during parturition. Fetal membranes were collected from TNIL (N=6) cesarean and TL (N=6) vaginal deliveries. To localize HLA-G, paraffin embedded sections were immunostained with antibody specific to HLA-G. To determine cell layer and cell type specific HLA-G expression, membranes were divided into amnion and chorion (TNIL [N=4], TL [N=6]) or dispersed and grown as primary cells (amnion epithelial cells [AEC], amnion mesenchymal cells [AMC], chorion trophoblast cells [CTC], decidual cells [DEC]). Protein extracts from layers or cells were analyzed by western blot to determine difference in HLA-G expression between TL and TNIL samples. BeWo (choriocarcinoma cell lines) were used as negative control. Regardless of labor status, amnion and chorion express HLA-G. Although not quantitative, staining intensity was higher in chorion (Fig 1). This was confirmed by western blot. Chorion and CTC had higher HLA-G expression than amnion and AEC; AMC of the membrane matrix were negative for HLA-G (Fig 2a). Quantitative differences were not observed between TNIL and TL in any of the cell types expressing HLA-G (Fig 2b). Decidual layer and BeWo cells were negative for HLA-G. This is the first report to localize HLA-G to the components of the fetal membrane and demonstrates that HLA-G is expressed predominantly in chorion and to a lesser extent in amnion. HLA-G in the membrane likely contributes to immune regulation in pregnancy. Lack of change at term suggests that expression is constitutive in the membrane and differential expression is not a prerequisite for parturition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Hypoxia-induced SPOP attenuates the mobility of trophoblast cells through inhibition of the PI3K/AKT/GSK3β pathway.

Placental hypoxia has been implicated in pregnancy pathologies such as pre-eclampsia and intrauterine growth restriction. However, the underlying mechanism by which the trophoblasts respond to hypoxia remains unclear. Speckle-type POZ protein (SPOP), an E3 ubiquitin ligase adapter, was previously reported to play important roles in various physiological and pathological processes. This study aims to investigate the expression and biological functions of SPOP after exposure to cobalt chloride (CoCl2 )-mimicked hypoxia conditions using human trophoblast-derived choriocarcinoma cell lines and extravillous cytotrophoblast. These data showed that SPOP protein was directly induced by CoCl2 -mimicked hypoxia and regulated by HIF-1α at the posttranscription level. CoCl2 treatment could dramatically influence the localization of SPOP in trophoblasts, especially the accumulation of SPOP into the nucleus. In addition, both CoCl2 -mimicked hypoxia and induction of endogenous SPOP expression by lentivirus transfection attenuated the migration and invasion abilities of trophoblasts. Furthermore, we demonstrated that SPOP was involved in CoCl2 -induced the inhibition of the PI3K/AKT/GSK3β pathway in placental trophoblasts. Taken together, these data indicate that accumulation of HIF-1α augments the expression of SPOP in trophoblasts, which impairs trophoblastic mobility by targeting the PI3K/AKT/GSK3β pathway. This potentially leads to insufficient uterine spiral artery remodeling and suboptimal placental perfusion, and thus the development of pregnancy-related complication.

Insulin reverses choriocarcinoma 5- fluorouracil resistance

ABSTRACT Choriocarcinoma (CC) is a gestational trophoblastic tumor secondary to a gravid or non-gravid pregnancy. It is characterized by rapid growth, high invasion, and high metastatic potential and chemotherapy resistance that significantly affect survival rate of CC patients. Insulin is implicated in alleviation of chemotherapy resistance in CC. However, the mechanism of reversing resistance in CC has not been explored. Our purpose was to explore insulin effect on 5-fluorouracil (5-FU) resistance in CC and elucidate its potential mechanism in vitro and in vivo. CKK-8, colony formation, Transwell, and flow cytometry were used to detect the effect of insulin on 5-FU resistance in CC cells JEG-3 and JARS. Xenograft mice were used to evaluate the effect of insulin on 5-FU resistance. Results showed that insulin combined with 5-FU suppressed cell viability by 30% in JEG-3 and 43% in JAR compared with 5-FU alone in 72 h. What’s more, insulin combined with 5-FU promoted cell apoptosis, inhibited cell proliferation, migration, and phosphorylation of survivin at residue threonine 34 (Thr34) and drug resistance-related proteins, P-GP and MRP1 levels (p < 0.05). In vivo experiment showed Insulin combined with 5-FU suppressed tumor volume by 35% compared with 5-FU alone and 73% compared with control in CC xenograft mice. In summary, the findings of this study show that insulin reversed chemoresistance of CC cells to 5-FU by inhibiting phosphorylation of survivin. Development of a therapeutic strategy that combines insulin with the chemotherapeutic agent 5-FU has a great potential in improving survival of CC patients.

Investigation of the effect of steroidal imidazoheterocycles hormone derivatives on JEG-3 trophoblast cells of choriocarcinoma

This study aimed to examine the biological activities of unique steroid hormone derivatives-steroidal imidazoheterocycles. The activity synthesized steroidal compounds were tested for the viability of JEG-3 trophoblast cells of choriocarcinoma. The results of the MTT cytotoxicity test showed that most of the examined steroidal imidazoheterocycles act proliferatively on the JEG-3 cell line, except (E, F, and J). Antiproliferative activity of these derivates is probably influenced by different substituents at position 17-C atom in the chemical structure of the hormone and by different substituents on the oxygen atom of acetamide group in the hormone molecule. In order to better confirm the antiproliferative activity of these derivatives, it is necessary to performed complex research on a large panel of cancer cell lines and by different assay.

Dexamethasone may inhibit placental growth by blocking glucocorticoid receptors via phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin and reactive oxygen species/AMP-activated protein kinase signalling pathways in human placental JEG-3 cells.

This study explored the molecular mechanism underlying the effects of dexamethasone (DEX, 1 µM) on glucose transporters (GLUT) in JEG-3 human placental choriocarcinoma cells. JEG-3 cells were treated with DEX, an expression plasmid encoding human glucocorticoid receptor α (GRα), pcDNA3.1-GRα, GRα short interference (si) RNA, LY294002, xanthine oxidase (XO)/hypoxanthine (HX), rapamycin, insulin-like growth factor (IGF) 1, N-acetylcysteine (NAC) or phosphatidic acid (PA), and cell proliferation, apoptosis, mitochondrial membrane potential (MMP), human chorionic gonadotrophin (hCG) content, human placental lactogen (hPL) content, glucose uptake, reactive oxygen species levels and signalling pathway modulation were evaluated. Treatment of JEG-3 cells with DEX (1 µM), GRα siRNA, LY294002 (50 µM), XO/HX (7.2 µM/36 nM) or rapamycin (80 nM) inhibited cell proliferation, induced apoptosis, significantly decreased MMP and hCG and hPL content and increased ROS levels. In addition, glucose uptake was decreased through downregulation of the mRNA and protein expression of GRα, GLUT1 and GLUT3. Treatment of JEG-3 cells with GRα siRNA, LY294002, XO/HX or rapamycin inhibited phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3 and mammalian target of rapamycin (mTOR) and induced the phosphorylation of AMP-activated protein kinase (AMPK) and tuberous sclerosis complex 2. The effects of GRα overexpression and IGF1 (100 nM), NAC (5 nM) or PA (100 µM) treatment on JEG-3 cells contrasted with those of DEX treatment. DEX blocked glucose uptake by downregulating GRα expression, which reduced GLUT1 and GLUT3 mRNA and protein expression, which, in turn, may have inhibited the PI3K/AKT/mTOR pathway and activated the ROS/AMPK pathway.

Reduction of pl-CSA through ChSy-2 knockout inhibits tumorigenesis and metastasis of choriocarcinoma in JEG3 cells.

Background: Placental-like chondroitin sulfate A (pl-CSA) is exclusively expressed in cancerous and placental tissues and is highly correlated with the degree of malignancy. However, the mechanism through which pl-CSA regulates tumorigenesis and metastasis in choriocarcinoma remains unclear.Methods: Stable transfectants of the JEG3 choriocarcinoma cell line, including a negative control (NC) line and a cell line with knockout of the biosynthetic enzyme CS synthase-2 (ChSy-2) (ChSy-2-/-), were obtained using CRISPR/Cas9 systems and identified by immunofluorescence, flow cytometry, western blots and enzyme-linked immunosorbent assays (ELISAs). The proliferation, migration, invasion and colony formation of the cells were determined by a cell counting kit, scratch-wound assays, transwell assays and soft agar colony formation assays in vitro, respectively. The tumorigenesis and metastasis of choriocarcinoma were also investigated through two xenograft models in vivo.Results: The ChSy-2 protein in the ChSy-2-/-group was below the detection threshold, which was accompanied a significant reduction in the pl-CSA level. Reducing pl-CSA through ChSy-2 knockout significantly inhibited cell proliferation, migration, invasion and colony formation in vitro and tumorigenesis and metastasis of choriocarcinoma, with deceases in tumor volume and metastatic foci and a high percent survival compared to the NC in vivo.Conclusion: pl-CSA, as a necessary component of JEG-3 cells, was efficiently reduced through ChSy-2 knockout, which significantly inhibited the tumorigenesis and metastasis of choriocarcinoma. ChSy-2/pl-CSA could be alternative targets for tumor therapy.

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-β 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.

A dynamic approach to gynaecomastia

Introduction: Gynaecomastia is characterized by enlargement of the male breast, caused by glandular proliferation and fat deposition. Although a fairly common presentation in the primary care setting and is mostly of benign etiology, it can cause patient considerable anxiety. Sometimes it could be due to serious underlying illness or a medication or inherited. Breast cancer risk is slightly higher in males with gynaecomastia. Aim: To determine the prevalence, pathophysiology, etiology and therapy of gynaecomastia. A stepwise approach that includes imaging and laboratory testing to exclude neoplasms and endocrinopathies. Materials and Methods: A one year prospective study of twenty four gynaecomastia cases was done. A detailed clinical history taken and physical examination was done. Laboratory investigations like hepatic, renal and thyroid function tests, hormonal levels like serum estradiol, testosterone, follicular stimulating & luteinizing hormone levels, prolactin, human chorionic gonadotropin and dehydroepiandrosterone levels were done. Other investigating aids like MRI and ultrasonography, mammography, cytogenetics were useful in some of them. However, histopathology proved to be gold standard. Results: We had twenty four cases of gynaecomastia of which there were one case of Klinefelter syndrome, five cases each of idiopathic gynaecomastia, two case each of true hermaphroditism, one case each of androgen insensitivity syndrome, Ehlers danlos syndrome, cirrhosis, drug induced, renal failure, hyperthyroidism, testicular torsion, androgen insensitivity syndrome, choriocarcinoma, pituitary adenoma, adrenal neoplasm and Leydig cell tumour. Six cases were those of physiological gynaecomastia. Conclusions: Gynaecomastia is a common condition that may be attributable to an oestrogen /androgen imbalance caused by several etiological factors. After confirming the diagnosis, searching for a specific cause and classifying the case according to severity grade, the therapy

Targeted-regulating of miR-515-5p by LncRNA LOXL1-AS1 on the proliferation and migration of trophoblast cells

ObjectiveThis study aims to elucidate the molecular mechanism of LOXL1-AS1 in proliferation and migration of trophoblast cells. MethodsWe have used specific small interfering RNAs (si-RNA) and microRNA (mi-RNA) to knock down the target gene or m-RNA. In this regard we used following siRNAs: si-NC, si-LOXL1-AS1, pcDNA-NC, pcDNA-LOXL1-AS1, miR-NC, miR-515-5p. These si-RNA and miRNA were transfected into JeG-3 cells. Real-time quantitative PCR (RT-qPCR) was used to detect the expressions of LOXL1-AS1 and miR-515-5p. Western blot was used to detect Cyclin D1, matrix metalloproteinase 2 (MMP2), matrix metalloproteinases 9 (MMP9), phosphorylated p65 (p-p65), profilin of phosphorylated nuclear transcription factor κB (p-IκBα). MTT assay was used to detect cell survival rate, and the luciferase assay was used to detected the targeting relationship of LOXL1-AS1 and miR-515-5p. ResultsOur results showed that human placental trophoblast cells had higher level of LOXL1-AS1 in comparison to human choriocarcinoma cells, however, human placental trophoblast cells had lower level of miR-515-5p. In addition, the expression of CyclinD1, MMP2, MMP9 were significantly decreased in JeG-3 cell lines. We observed lower cell survival rate and lower cell migration number in JeG-3 cell lines. Our results demonstrated that LOXL1-AS1 could target miR-515-5p, and subsequently reverse the inhibitory effect of LOXL1-AS1 on proliferation and migration in JEG-3 cells. Also, lower expressions of p-p65 and p-IкBα in JeG-3 cells showed that miR-515-5p could reversed the inhibitory effect of LOXL1-AS1 on NF-κB signaling pathway. ConclusionsThe low expression of LOXL1-AS1 inhibits the proliferation and migration of human choriocarcinoma cells, which might be related to miR-515-5p and NF-κB signaling pathways.

SLAMF1 Promotes Methotrexate Resistance via Activating Autophagy in Choriocarcinoma Cells.

ObjectiveThe acquisition of chemoresistance to methotrexate (MTX) still remains one of the major challenges for choriocarcinoma treatment. Herein, we aimed to evaluate the potential role of Signaling Lymphocytic Activation Molecule Family Member 1 (SLAMF1) as a possible regulator of chemoresistance to MTX in choriocarcinoma.Material and MethodsMTX-resistant JEG3 and JAR sublines (JEG3/MTX, JAR/MTX) were used to study SLAMF1 function. CCK8 assay and soft agar assay were conducted to measure the cell viability and clonogenesis of choriocarcinoma cells, respectively; MDC incorporation assay was conducted for the quantification of intracellular autophagy; BrdU labeling was used to assess the proliferative potential of choriocarcinoma cells; SLAMF1 protein expression was analyzed by Western blotting.ResultsUpregulation of SLAMF1 expression was observed in MTX-resistant JEG3/MTX and JAR/MTX sublines compared to their parental JEG3 and JAR cell lines, respectively. Knockdown of SLAMF1 markedly attenuated cell viability and soft agar clonogenesis after incubation with MTX in JEG3/MTX and JAR/MTX cells. In contrast, constitutive expression of SLAMF1 rescued cell survival soft agar clonogenesis in JEG3 and JAR cells treated with MTX. Moreover, autophagy is apparently activated in MTX-resistant JEG3/MTX and JAR/MTX sublines compared to their parental cell lines. Autophagy inhibitor 3-methyladenine and bafilomycin A1 enhanced MTX-induced cytotoxicity in MTX-resistant JEG3 and JAR sublines. Further, SLAMF1 might activate autophagy-related mechanism to promote resistance to MTX in choriocarcinoma cells. Depletion of SLAMF1 suppressed autophagy and induced apoptosis in MTX-treated JEG3/MTX and JAR/MTX cells.ConclusionSLAMF1 might promote MTX resistance via activating protective autophagy in choriocarcinoma cell lines. Targeting SLAMF1 might be a useful therapeutic strategy to sensitize choriocarcinoma cells to MTX-based regimens.

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

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