Trophoblastic neoplasms express fatty acid synthase, which may be a therapeutic target via its inhibitor C93.

Fatty acid synthase (FASN) is an emerging tumor-associated marker and a promising antitumor therapeutic target. In this study, we analyzed the expression of FASN in normal and molar placentas, as well as gestational trophoblastic neoplasia, and assessed the effects of a new FASN inhibitor, C93, on cellular proliferation and apoptosis in choriocarcinoma cells. Using a FASN-specific monoclonal antibody, we found that FASN immunoreactivity was detected in the cytotrophoblast and intermediate (extravillous) trophoblast of normal and molar placentas, as well as in placental site nodules. All choriocarcinomas (n = 33), 90% of epithelioid trophoblastic tumors (n = 20), and 60% of placental site trophoblastic tumors (n = 10) exhibited FASN positivity. FASN expression was further confirmed in vitro by Western blot and real-time PCR. Treatment of JEG3 and JAR cells with C93 induced significant apoptosis through the caspase-3/caspase-9/poly(ADP)ribose polymerase pathway. Cell cycle progression was not affected by the inhibitor. In summary, the data indicate that FASN is expressed in the majority of gestational trophoblastic neoplasias, and is essential for choriocarcinoma cells to survive and escape from apoptosis. FASN inhibitors such as C93 warrant further investigation as targeted therapeutic agents for metastatic and chemoresistant gestational trophoblastic neoplasia.

Signal pathway involved in increased expression of neutral endopeptidase 24.11 by gonadotropin releasing hormone in choriocarcinoma cells.

Neutral endopeptidase 24.11 (NEP) is known to regulate cellular functions by degrading several bioactive peptides, such as gonadotropin-releasing hormone (GnRH). The present study was performed to clarify the mechanisms of NEP expression by GnRH in human choriocarcinoma (BeWo) cells. GnRH increased NEP expression and enzyme activity in a dose- and time-dependent manner in BeWo cells. The phosphorylation levels of protein kinase C (PKC) delta, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK1 and 2) were enhanced after 10 min exposure of 10(-6)m GnRH. The effect of GnRH on both NEP expression and enzyme activity was completely inhibited by inhibitors of PKC, PKC delta, and p38MAPK. Cell number was reduced by 54.4 per cent of the control by culture with 10(-6)m GnRH for 24 h. However, phosphoramidon, a NEP specific inhibitor, inhibited antiproliferative effect of GnRH and reverted to the control level. In conclusion, GnRH induces NEP expression by PKC delta and p38MAPK, and increased NEP expression may be involved in antiproliferative effect in BeWo cells.

The expression and localization of neutral endopeptidase 24.11/CD10 in human gestational trophoblastic diseases.

Neutral endopeptidase 24.11 (NEP)/CD10 is a cell-surface peptidase that hydrolyzes various bioactive peptides. NEP is distributed in both normal and neoplastic cells and plays a functional role by modulating cellular responses to peptide substrates. Recently, NEP has been shown to be expressed in normal placental trophoblasts, suggesting its physiological role during pregnancy. In the present study, we investigated the expression of NEP in hyperplastic and anaplastic trophoblasts in gestational trophoblastic diseases (GTDs). Flow cytometric analysis demonstrated that NEP was expressed in all choriocarcinoma cell lines examined. The NEP enzyme activity in these cell lines correlated with cell-surface protein levels and was abolished by the NEP inhibitor phosphoramidon. On immunoblot analysis, NEP protein was detected in both hydatidiform mole and choriocarcinoma tissues as a double band of 95 and 100 kDa similar to that of the normal placental tissues. Immunohistochemical analysis revealed that NEP was present on syncytiotrophoblasts, while no or very faint NEP immunoreactivity was observed on cytotrophoblasts in the normal placenta. Similarly, NEP in hydatidiform mole and invasive mole was localized on the membrane of syncytiotrophoblasts, but not on hyperplastic cytotrophoblasts. In contrast, in choriocarcinoma, NEP was highly expressed not only on syncytiotrophoblastic cells but also on invading anaplastic cytotrophoblasts. In addition, NEP was also expressed on intermediate trophoblasts in placental site trophoblastic tumors. In summary, this is the first study demonstrating the expression of NEP/CD10 in GTDs. The differential localization of NEP among various trophoblastic tumors suggests that NEP may play a functional role in the regulation of trophoblast transformation and human chorionic gonadotropin secretion.

Matrix metalloproteinases and their inhibitors in gestational trophoblastic diseases and normal placenta.

OBJECTIVE: Our purpose was to investigate the expression of matrix metalloproteinases (MMPs) in gestational trophoblastic diseases and normal first-trimester placenta. METHODS: Paraffin sections of 16 partial moles, 25 complete moles, 10 gestational choriocarcinomas, and 11 normal first-trimester placentas were studied immunohistochemically for expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Nine (90.0%) of the choriocarcinoma cases showed strong intensity of staining for MMP-1. Choriocarcinoma exhibited significantly stronger staining for MMP-1 than syncytiotrophoblast in normal placenta (P < 0.01), partial mole (P < 0.01), and complete mole (P < 0.01). Choriocarcinoma also showed significantly stronger staining for MMP-1 than the extravillous trophoblast in placenta (P < 0.05). MMP-2 was expressed only in syncytio- and extravillous trophoblasts in normal placenta, partial mole, and complete mole. Choriocarcinoma and the extravillous trophoblast in partial mole and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in placenta (P < 0.05, P < 0.01, P < 0.01, respectively). Choriocarcinoma also exhibited significantly stronger staining for MMP-2 than syncytiotrophoblasts in placenta (P < 0.01), partial mole (P = 0.05), and complete mole (P < 0.01). The expression of MMP-3, MMP-9, and MMP-13 was similar in all four tissues with the predominance of syncytiotrophoblast for MMP-3 and MMP-13 and cytotrophoblast for MMP-9. While 8 (73.0%) placentas, 14 (87.5%) partial moles, and 19 (76.0%) complete moles showed strong immunoreactivity for TIMP-1 in syncytiotrophoblasts, no strong staining was found in choriocarcinomas (P < 0.01, P < 0.01, P < 0.01, respectively). CONCLUSION: The extravillous trophoblast of first-trimester placenta has significantly less expression of MMP-1 than choriocarcinoma and significantly less expression of MMP-2 than choriocarcinoma and extravillous trophoblast of partial and complete mole. The expression of TIMP-1 was significantly less in choriocarcinoma than the syncytiotrophoblast of normal placenta, partial mole, and complete mole. MMPs and their inhibitors may play a role in the pathogenesis of gestational trophoblastic diseases.

Endothelial nitric oxide synthase immunoreactivity in early gestation and in trophoblastic disease.

AIMS: To study the localisation of the endothelial nitric oxide synthase (eNOS) in the normal placenta, with special emphasis on the implantation site in the first trimester of pregnancy, and in the different subtypes of trophoblastic cells in gestational trophoblastic disease. METHODS: The immunoperoxidase technique with an antibody directed against eNOS was applied to paraffin sections from first and second trimester placentas, placenta accreta, partial and complete hydatidiform moles, and choriocarcinoma. Immunoperoxidase staining for human placental lactogen (hPL) was performed on parallel sections. RESULTS: Prominent immunoreactivity for eNOS was found to be present in the intermediate trophoblastic cells of the cell columns of the anchoring villi and in trophoblastic cells at the implantation site. Staining was also present in the syncytiotrophoblast, most conspicuous at the apical cell border. In trophoblastic disease, proliferating large mononuclear cells, which were strongly positive for hPL, were found to be immunoreactive for eNOS. CONCLUSIONS: eNOS immunoreactivity is strongly positive in the extravillous trophoblastic cells and to a lesser extent in the syncytiotrophoblast. In the former it may play a role in implantation and vascular invasion. Cells with differentiation to intermediate trophoblast in complete hydatidiform mole and choriocarcinoma also show high levels of eNOS, which may be associated with the haematogenous mode of spread of trophoblastic disease.

Comparison of telomerase activity in normal chorionic villi to trophoblastic diseases.

Trophoblasts are derived from the normal placenta, and they infiltrate into the endometrium and the maternal blood vessels under strict control but, unlike malignant cells, never metastasize. To understand the proliferative characteristics of trophoblasts and its related disorders, we assessed telomerase activity in chorionic villi obtained from 27 normal individuals, 9 hydatidiform moles, and 2 choriocarcinomas. Telomerase activity was detected in 13/27 (48%) normal chorionic villi samples. The detectability and the level of telomerase activity depended on gestational age; 8/10 (80%) villi samples in the first trimester (relative telomerase activity; 1.77 +/- 1.37), whereas 2/8 (25%) villi samples in the second trimester (0.78 +/- 1.52) and 3/9 (33%) in the third trimester (0.28 +/- 0.43) had telomerase activity. Telomerase activity of normal chorionic villi in the first trimester was higher than that of the third trimester (P = 0.0251). In contrast, all mole samples had increased telomerase activity compared to normal villi (3.17 +/- 2.81, P = 0.0152). Thus, a relationship may exist among cell proliferation, telomerase activity, and progression to trophoblastic disease.

Basal and interferon-induced 2',5'-oligoadenylate synthetase activity in human placental trophoblast and trophoblast-derived malignant cell lines.

Human placental trophoblasts produce interferon (tro-IFNs) when stimulated with viral inducers. Since the antiviral and cellular functions of IFNs are partly mediated by the 2',5'-oligoadenylate synthetase (2-5A synthetase) pathway, the aim of the present study was to determine the basal and IFN-induced levels of 2-5A synthetase in villous trophoblast cultures. A considerable basal level of 2-5A synthetase was observed in syncytiotrophoblast cultures from both first and third trimester. In contrast no basal activity was detectable in placental fibroblast- and trophoblast-derived malignant cell lines (Far, FEG-3, and BeWo). Stimulation with tro-IFN-beta, -alpha and leucocyte-IFN (leu-IFN)-alpha increased the enzyme activity in first and third trimester human syncytiotrophoblast cultures. Treatment with recombinant-IFN (rec-IFN)-gamma significantly enhanced 2-5A synthetase activity in first trimester syncytiotrophoblast, but had no effect on third trimester syncytiotrophoblast. Tro-IFN-beta, -alpha and leu-IFN-alpha induced high levels of 2-5A synthetase activity in placental fibroblast, BeWo and FEG-3 cell-lines, whereas rec-IFN-gamma treatment did not induce 2-5A synthetase activity in any of these cells. No detectable 2-5A synthetase activity was found in the Far cell line. The capability of cells deriving from the fetoplacental unit to raise an antiviral response by the induction of 2-5A synthetase may be important in defending the fetus against viral infection from the mother. Furthermore 2-5A synthetase in cells of the fetoplacental unit may play a role in their normal growth and development.

Estrogen synthetase (aromatase) in cultured human term placental cells and neoplastic human trophoblast.

Estrogen synthetase (aromatase) is present in large amounts in human term placenta. However, the localization of aromatase within the cellular structure of the placental villus is obscure. By immunocytochemical techniques using antibodies that separately recognize each component of the aromatase cytochrome P-450 enzyme system, the fraction of term placental trophoblast cells in primary culture expressing each aromatase component antigen increased from 20% in fresh mononucleated cells to about 65% for multinucleated giant cells after 72 h. In contrast, about 80% of human choriocarcinoma cells in continuous culture (JAr line) expressed each aromatase component antigen. The fraction of trophoblast cells in primary culture containing human chorionic gonadotropin increased from about 14% in fresh mononucleated cells to about 45% after 72 h and was about 30% in the choriocarcinoma cells. Fibroblast cells in culture, derived from trypsin-treated placental villi, contained aromatase activity, albeit much lower than term placental trophoblast cells. Aromatase specific activity in these placental fibroblasts did not change following growth with dibutyryl cAMP plus theophylline for 72 h.

Methotrexate with citrovorum factor rescue in gestational trophoblastic disease.

Thirty-three patients with gestational trophoblastic disease treated with methotrexate alone and 68 similar patients treated with methotrexate and citrovorum factor rescue were analyzed. Both groups showed a comparable sustained biochemical remission rate. The group treated with methotrexate and citrovorum factor however showed a shorter interval to induction of remission (p less than 0.05). Contrary to previous reports, the methotrexate and citrovorum factor regimen failed to protect the patients against the development of hepatic toxicity, the incidence of hepatic toxicity being significantly higher in the group treated with methotrexate and citrovorum factor.

Cell surface antigens of human trophoblast and choriocarcinoma defined by monoclonal antibodies.

Six distinct cell surface antigens of human trophoblast and choriocarcinoma were defined with MAbs. The distribution of the antigens was determined by MHA assays on 150 tumor cell lines and normal cell cultures and by immunofluorescence tests with a wide range of normal adult and fetal tissues and a tumor panel. Antigen LK26 is expressed on all cultured choriocarcinoma, teratocarcinoma and renal cancer lines but is absent from most cell lines derived from other tumor types and from cultures of normal kidney epithelium and fibroblasts. LK26 expression in normal tissues is restricted to the trophoblast. No other adult or fetal tissue was found to express the antigen, but choriocarcinoma and teratocarcinoma tissues were LK26+. SV19 is expressed on cultured choriocarcinomas and teratocarcinomas and on subsets of breast and colon cancer lines, but not on 120 additional cultures tested. In tissues, SV19 is detected in normal placenta, mammary gland and colon epithelium as well as in tumors of breast, colon and lung. Two antibodies, AbSV63 and AbK8, react with PLAP and AbSV63 also reacts with the intestinal form of the enzyme. AbLK24 defines a heat-stable determinant present on choriocarcinoma and breast cancer cell lines but absent from most other cultured cells. It is expressed on a small range of normal and malignant epithelial tissues, including normal trophoblast, normal breast epithelium and urothelium and tumors derived from these tissues. One antigen, K66, showed a wide distribution on cultured epithelial cells but was not found in any normal or malignant tissue. Finally, S4, a previously described marker of normal and malignant kidney epithelial cells, was also expressed on the choriocarcinoma cell lines. Four of the antigens are glycoproteins that could be immunoprecipitated from radiolabelled extracts of choriocarcinoma cells: LK26 (Mr 35,000), SV19 (Mr 40,000), PLAP (Mr 68,000) and S4 (Mr 160,000). The highly restricted distribution of LK26, SV19, S4, and PLAP in normal tissues and their expression in tumors make these antigens potential diagnostic markers of gestational choriocarcinoma and germ-cell tumors and, possibly, targets for immunotherapy.

Plasma cholinesterase and trophoblastic disease. Gestational trophoblastic disease and reduced activity of plasma cholinesterase.

A case of prolonged action of suxamethonium in a patient with gestational trophoblastic disease is reported. Postoperatively the patient was found to have markedly reduced plasma cholinesterase activity (363 IU/litre) with a normal cholinesterase phenotype. Consequently plasma cholinesterase activity and phenotype were measured in six other patients with the condition and these results compared with those of 22 patients with normal first trimester pregnancies undergoing therapeutic abortion. Plasma cholinesterase phenotype was normal in all patients studied. The activity was significantly decreased (p less than 0.05) from the normal range (620-1370 IU/litre) in all patients with trophoblastic disease. In the 22 patients with normal pregnancies, 14 had activity values in the abnormal range (less than 620 IU/litre) while the mean cholinesterase activity of the group as a whole was significantly decreased (561.8 IU/litre, p less than 0.05) below the normal range. These results confirm the presence of a decrease in plasma cholinesterase activity in early pregnancy and provide new evidence for a decrease in activity in a pseudopregnancy state.