Nimbolide a limonoid from Azadirachta indica inhibits proliferation and induces apoptosis of human choriocarcinoma (BeWo) cells

We investigated the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human choriocarcinoma (BeWo) cells. Treatment with nimbolide resulted in dose- and time-dependent inhibition of growth of BeWo cells with IC50 values of 2.01 and 1.19 μM for 7 and 24 h respectively, accompanied by downregulation of proliferating cell nuclear antigen. Examination of nuclear morphology revealed fragmentation and condensation indicating apoptosis. Increase in the generation of reactive oxygen species (ROS) that was reversed by addition of reduced glutathione suggested ROS involvement in the cytotoxicity of nimbolide. A decrease in Bcl-2/Bax ratio with increased expression of Apaf-1 and caspase-3, and cleavage of poly(ADP-ribose) polymerase provide compelling evidence that nimbolide-induced apoptosis is mediated by the mitochondrial pathway. The results of the present study suggest that nimbolide has immense potential in cancer prevention and therapy based on its antiproliferative and apoptosis inducing effects.

Nimbolide a limonoid from Azadirachta indica inhibits proliferation and induces apoptosis of human choriocarcinoma (BeWo) cells

We investigated the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human choriocarcinoma (BeWo) cells. Treatment with nimbolide resulted in dose- and time-dependent inhibition of growth of BeWo cells with IC50 values of 2.01 and 1.19 μM for 7 and 24 h respectively, accompanied by downregulation of proliferating cell nuclear antigen. Examination of nuclear morphology revealed fragmentation and condensation indicating apoptosis. Increase in the generation of reactive oxygen species (ROS) that was reversed by addition of reduced glutathione suggested ROS involvement in the cytotoxicity of nimbolide. A decrease in Bcl-2/Bax ratio with increased expression of Apaf-1 and caspase-3, and cleavage of poly(ADP-ribose) polymerase provide compelling evidence that nimbolide-induced apoptosis is mediated by the mitochondrial pathway. The results of the present study suggest that nimbolide has immense potential in cancer prevention and therapy based on its antiproliferative and apoptosis inducing effects.

Effect of human telomerase reverse transcriptase transfection on differentiation in BeWo choriocarcinoma cells

Arrest of proliferation is one of the prerequisites for differentiation of cytotrophoblasts into syncytiotrophoblasts, and thus during differentiation telomerase activity, as well as human telomerase reverse transcriptase (hTERT) expression, is down-regulated. Considering this, it is of interest to investigate whether syncytium formation can be delayed by prolonging the expression of telomerase in cytotrophoblasts. BeWo cells were transfected with pLPC-hTERT retroviral vector and the reverse transcription-polymerase chain reaction analysis for hTERT mRNA concentrations in the transfected cells revealed a several-fold increase in hTERT mRNA compared with the cells transfected with empty vector, and this confirmed that the transfection was successful. An increase in the proliferation, as assessed by bromodeoxyuridine incorporation assay, as well as an increase in mRNA and protein concentration of various cyclins and proliferating cell nuclear antigen, was noticed. The effect of hTERT transfection was also assessed after the addition of forskolin to induce differentiation and it was observed that cell–cell fusion was delayed and differentiation did not occur in hTERT-transfected cells. However, the effects seen were only transient as stable transfection was not possible and the cells were undergoing apoptosis after 72 h, which suggested that apart from hTERT other factors might be important for immortalization of BeWo cells.

Expression, localisation and hormone regulation of the human copper transporter hCTR1 in placenta and choriocarcinoma Jeg-3 cells

Copper is an essential trace element necessary for normal growth and development. During pregnancy, copper is transported from the maternal circulation to the fetus by mechanisms which have not been clearly elucidated. The copper uptake protein, hCTR1 is predicted to play a role in copper transport in human placental cells. This study has examined the expression and localisation of hCTR1 in human placental tissue and Jeg-3 cells. In term placental tissue the hCTR1 protein was detected as a 105 kDa protein, consistent with the size of a trimer which may represent the functional protein. A 95 kDa band, possibly representing the glycosylated protein, was also detected. hCTR1 was localised within the syncytiotrophoblast layer and the fetal vascular endothelial cells in the placental villi and interestingly was found to be localised toward the basal plasma membrane. It did not co-localise with either the Menkes or the Wilson copper transporting ATPases. Using the placental cell line Jeg-3, it was shown that the 35 kDa monomer was absent in the extracts of cells exposed to insulin, estrogen or progesterone and in cells treated with estrogen an additional 65 kDa band was detected which may correspond to a dimeric form of the protein. The 95 kDa band was not detected in the cultured cells. These results provide novel insights indicating that hormones have a role in the formation of the active hCTR1 protein. Furthermore, insulin altered the intracellular localisation of hCTR1, suggesting a previously undescribed role of this hormone in regulating copper uptake through the endocytic pathway.

Primary non-gestational choriocarcinoma of the uterine cervix: A case report

Background. Primary non-gestational choriocarcinoma of the female genital tract has been described in the ovaries and is very unusual in other genital sites.Case. Primary non-gestational uterine cervical choriocarcinoma was diagnosed in a patient, 32, single, without previous sexual contact nor antecedent pregnancy, admitted to the hospital with irregular vaginal hemorrhaging. Pelvic examination realized under anesthetic revealed a tumor mass occupying the uterine cervix. Metastases investigation was realized and the patient was accepted as FIGO IV: risk factor of 13. She was submitted to intensive chemotherapy and hysterectomy, showing general recovery, but died from drug-resistant disease 12 months later. Histological, immunohistochemical, and molecular genetics studies confirmed non-gestational choriocarcinoma.Conclusion. Primary non-gestational uterine cervical choriocarcinoma may arise from germ cell tumor or epithelial tissue. (c) 2005 Elsevier B.V. All rights reserved.

Primary Pulmonary Choriocarcinoma After Human Chorionic Gonadotropin Normalization Following Hydatidiform Mole A Report of Two Cases

BACKGROUND: Primary pulmonary choriocarcinoma (PPC) is rare and frequently leads to death.CASES: Two young patients presented with previous molar pregnancy and spontaneous serum human chorionic gonadotropin (hCG) normalization. Patient 1 was referred to our center after partial response to chemotherapy. Pulmonary lobectomy was performed, and hCG rapidly declined. During further chemotherapy, liver metastasis was detected by positron emission tomography. Right hepatectomy was performed, and hCG declined for 28 days, but increased again despite chemotherapy. This patient died from hepatic failure 3 years after diagnosis. Patient 2 presented with persistently high hCG, though the affected organ was not identified. Chemotherapy was unsuccessful. Patient reevaluation showed an isolated pulmonary mass. Pulmonary lobectomy was performed; 2 weeks later, hCG was normal and consolidation with 2 cycles of chemotherapy was administered. The patient has been in remission for 24 months. PPC was confirmed by histo pathology and immunohistochemistry in both cases. Gestational origin of the tumor was confirmed by molecular genetic analysis (polymorphic microsatellite markers).CONCLUSION: The possibility of choriocarcinoma cannot be overlooked in young women with an isolated pulmonary mass. Early diagnosis, prompt chemotherapy, and surgical resection in a specialized center improves the prognosis. (J Reprod Med 2010;55:311-316)

INCREASED EXPRESSION OF ONE OF TWO ADENOSINE DEAMINASE ALLELES IN A HUMAN CHORIOCARCINOMA CELL LINE FOLLOWING SELECTION WITH ADENINE NUCLEOSIDES

The human choriocarcinoma cell line JEG-3 is heterozygous at the adenosine deaminase (ADA) gene locus. Both allelic genes are under strong but incomplete repression causing a very low level expression of the gene locus. Because cytotoxic adenosine analogues such as 9-(beta)-D arabinofuranosyladenine (ara-A) and 9-(beta)-D xylofuranosyladenine (xyl-A) can be specifically detoxified by the action of ADA, these analogues were used to select for JEG-3 derived cells which had increased ADA expression. When JEG-3 cells were subjected to a multi-step, successively increasing dosage of either ara-A or xyl-A, resistant cells with increased ADA expression were generated. This increased ADA expression in the resistant cells was unstable, so that when the selective pressure was removed, cellular ADA expression would decrease. Subclone analysis of xyl-A resistant cells revealed that compared to parental JEG-3 cells, individual resistant cells had either elevated ADA levels or decreased adenosine kinase (ADK) levels or both. This altered ADA and ADK expression in the resistant cells were found to be independent events. Because of high endogenous tissue conversion factor (TCF) expression in the JEG-3 cells, the allelic nature of the increased ADA expression in most of the resistant cells could not be determined. However, several resistant subcloned cells were found to have lost TCF expression. These TCF('-) cells expressed only the ADA*2 allelic gene product. Cell fusion experiments demonstrated that the ADA*1 allelic gene was intact and functional in the A3-1A7 cell line. Chromosomal analysis of the A3-1A7 cells showed that they had no double-minutes or homogeneously staining chromosomal regions, although a pair of new chromosomes were found in these cells. Segregation analysis of the hybrid cells indicated that an ADA*2 allelic gene was probably located on this new chromosome. The analysis of the A3-1A7 cell line suggested that the expression of only ADA 2 in these cells was the result of possibly a cis-deregulation of the ADA gene locus or more probably an amplification of the ADA*2 allelic gene. Two effective positive selection systems for ADA('+) cells were also developed and tested. These selection systems should eventually lead to the isolation of the ADA gene.^

Human trophoblast and choriocarcinoma expression of the growth factor pleiotrophin attributable to germ-line insertion of an endogenous retrovirus

Retroviral elements are found in abundance throughout the human genome but only rarely have alterations of endogenous genes by retroviral insertions been described. Herein we report that a human endogenous retrovirus (HERV) type C is inserted in the human growth factor gene pleiotrophin (PTN) between the 5′ untranslated and the coding region. This insert in the human genome expands the region relative to the murine gene. Studies with promoter-reporter constructs show that the HERV insert in the human PTN gene generates an additional promoter with trophoblast-specific activity. Due to this promoter function, fusion transcripts between HERV and the open reading frame of PTN (HERV-PTN) were detected in all normal human trophoblast cell cultures as early as 9 weeks after gestation (n = 7) and in all term placenta tissues (n = 5) but not in other normal adult tissues. Furthermore, only trophoblast-derived choriocarcinoma cell lines expressed HERV-PTN mRNA whereas tumor cell lines derived from the embryoblast (teratocarcinoma) or from other lineages failed to do so. We investigated the significance of HERV-PTN mRNA in a choriocarcinoma model by targeting this transcript with ribozymes and found that the depletion of HERV-PTN mRNA prevents human choriocarcinoma growth, invasion, and angiogenesis in mice. This suggests that the tissue-specific expression of PTN due to the HERV insertion in the human genome supports the highly aggressive growth of human choriocarcinoma and possibly of the human trophoblast.

The BeWo choriocarcinoma cell line as a model of iodide transport by placenta

Cultured human choriocarcinoma cells of the BeWo line exhibited saturable accumulation of radioiodide. Inhibition by competing anions followed the affinity series perchlorate >= iodide >= thiocyanate, consistent with uptake through the thyroid iodide transporter, NIS, whose messenger RNA was found in BeWo cells, and whose protein was distributed towards the apical pole of the cells. Efflux obeyed first order kinetics and was inhibited by DIDS, an antagonist of anion exchangers including pendrin, whose messenger RNA was also present. In cultures where iodide uptake through NIS was blocked with excess perchlorate, radiolodide accumulation was stimulated by exposure to medium in which physiological anions were replaced by 2-morpholinoethanesulfonic acid (MES), consistent with the operation of an anion exchange mechanism taking up iodide. Chloride in the medium was more effective than sulfate at inhibiting this uptake, matching the ionic specificity of pendrin. These studies provide evidence that the trophoblast accumulates iodide through NIS and releases it to the fetal compartment through pendrin. (c) 2004 Elsevier Ltd. All rights reserved.

Primary gastric choriocarcinoma: A rare case

Introduction Primary gastric choriocarcinoma accounts for 0.08% of all gastric cancers. It is a rapidly growing, widely metastatic and β-HCG-producing tumour of trophoblastic cells. Presentation of case A 69-year-old white man presented to the hospital with symptomatic anaemia. An upper gastrointestinal endoscopy showed an ulcer of the cardia and lesser curvature, whose biopsy specimens proved to be malignant (carcinoma cells, non-specified). The patient underwent total gastrectomy with D2 lymphadenectomy. A histologic evaluation revealed a choriocarcinoma admixed with adenocarcinoma cells without lymph node metastases. The patient died from haemorrhagic shock, due to rupture of liver metastases and a massive haemoperitoneum, within 2 months of the initial presentation. Discussion Primary gastric choriocarcinoma characteristics resemble those of gastric primary adenocarcinoma. The dedifferentiation theory is the most widely accepted theory to explain the pathogenesis of PGC. It is essential to rule out other possible primary lesions such as testicular tumour. The optimal treatment is not yet well established due to very few reported cases. Conclusion Primary gastric choriocarcinoma is a rare tumour with an aggressive behaviour and very poor prognosis.

Gene expression profiling during Forskolin induced differentiation of BeWo cells by differential display RT-PCR

The differentiation of cytotrophoblasts into syncytiotrophoblasts in the placenta has been employed as a model to investigate stage specific expression as well as regulation of genes during this process. While the cytotrophoblasts are highly invasive and proliferative with relatively less capacity to synthesize pregnancy related proteins, the multinucleated syncytiotrophoblasts are non-proliferative and non-invasive. However, syncytiotrophoblasts are the site of synthesis of a variety of protein, peptide and steroid hormones as well as several growth factors. Both the freshly isolated cytotrophoblasts from human placenta as well as the BeWo cell, a choriocarcinoma cell line model which retain several characteristic of cytotrophoblasts has been employed by us to study regulation of differentiation. In the present study, we have employed the differential display RT-PCR analysis (DD-RT-PCR) to evaluate gene expression changes during Forskolin induced in vitro differentiation of BeWo cells. We have identified several genes which are differentially expressed during differentiation and the differential expression of 10 transcripts was confirmed by Northern blot analysis. Based on the identity of the transcripts an attempt has been made to relate the known function of the gene products, to changes observed during differentiation. Of the several transcripts, one of the transcripts, namely Secretory Leukocyte Protease Inhibitor (SLPI) which is known to have multiple functions was found to increase 15-fold in the syntiotrophoblast.

Proteomic profiling of forskolin-induced differentiated BeWo cells: an in-vitro model of cytotrophoblast differentiation

Placental trophoblastic differentiation is characterized by the fusion of monolayer cytotrophoblasts into syncytiotrophoblasts. During this process of differentiation, several morphological and biochemical changes are known to occur, and this model has been employed to investigate the changes that occur at the gene and protein level during differentiation. Using the sensitive technique of proteomics [two-dimensional gel electrophoresis (2DGE)], changes in protein profile were evaluated in the control and forskolin-induced differentiated cells of trophoblastic choriocarcinoma BeWo cell line. Several proteins were differentially expressed in control and differentiated cells. Four major proteins were up-regulated as assessed by silver staining, and were further characterized as c-h-ras p 21 (phosphorylated), retinoblastoma susceptibility protein I and integrase interactor protein 1. These proteins are known to play an important role in growth arrest of cells, and thus may play a role in initiating the process of differentiation.