Decorin and biglycan immunolocalization in non-villous structures of healthy and pathological human placentas.

AIMS: Decorin and biglycan are members of the small leucine-rich proteoglycan family, and constituents of both the extracellular matrix (ECM) and the cell surface. They are recognized as important factors in the control of proliferation, migration and invasion in vivo and in vitro. In this study, the localization patterns of decorin and biglycan were determined in healthy placentas and in highly invasive placental pathologies. METHODS AND RESULTS: The study included immunolocalization of decorin and biglycan in samples of first-trimester and term placentas, placenta accreta, invasive mole, and choriocarcinoma. Extravillous cytotrophoblast (EVT) cells were positive for both proteoglycans in all pathologies and in first-trimester placentas, although not in term placentas. Biglycan was immunolocalized in the ECM of all healthy and pathological placentas, whereas decorin was observed only in term placenta ECM. CONCLUSIONS: The expression of both proteoglycans was cell-specific and gestation time-dependent in healthy placentas, and was associated with invasive EVT cells in pathological placentas. In view of the biological properties of these molecules, it is possible that the biglycan pattern found here is intrinsically implicated in the invasive activity of EVT cells in both healthy and disordered placentas.

A translocator protein ligand PK11195 shows antigrowth activity in human choriocarcinoma cells.

The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein ligand (initially described as a ligand for the peripheral benzodiazepine receptor), induces apoptosis in some lines of human tumor cells. We investigated the effect of PK11195 in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of PK11195, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. A WST-1 assay showed that BeWo cells were sensitive to the growth inhibitory effect of PK11195. In contrast, the nonsite selective ligand diazepam has a little effect on these cells. Cell cycle analysis indicated that exposure to PK11195 decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine, by the loss of mitochondrial transmembrane potential, and by antibodies directed against histones from fragmented DNA. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that PK11195 may serve as a therapeutic agent for the treatment of choriocarcinoma.

Proteomic identification of predictive biomarkers for malignant transformation in complete hydatidiform moles.

INTRODUCTION: Protein expression in cells are associated with oncogenesis. This study aims to explore proteomic profiles and discover potential biomarkers that can predict malignant transformation of hydatidiform mole. METHODS: Retrospective analysis was done in 14 cases of remission hydatidiform mole and 14 cases of hydatidiform mole who later developed malignancy (GTN group). Molar tissues were retrieved from -70 °C frozen tissue. Subsequently, a large-scale proteomic analysis was performed to identify proteins and compare their abundance levels in the preserved molar tissues from these two groups using a dimethyl-labeling technique coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 2,153 proteins were identified from all samples. 22 and 10 proteins were significantly up-regulated and down-regulated, respectively, in the GTN group compared with the mole group. These altered proteins were found in several biological groups such as cell-cell adhesion, secreted proteins, and ribonucleoproteins. Several hormone-related proteins were among the most up-regulated proteins in the GTN group including choriogonadotropin subunit beta (ß-hCG) and alpha (α-hCG), growth/differentiation factor 15, as well as both pregnancy-specific beta-1-glycoproteins 2 and 3. In contrast, protein S100-A11 and l-lactate dehydrogenase A chain, were down-regulated in molar tissue from most patients in the GTN group. DISCUSSION: This study identified a set of differentially expressed proteins in molar tissues that could potentially be further examined as predictive biomarkers for the malignant transformation of CHMs. A molar proteome database was constructed and can be accessible online at http://sysbio.chula.ac.th/Database/GTD_DB/Supplementary_Data.xlsx.

DOC-2/hDab2, a candidate tumor suppressor gene involved in the development of gestational trophoblastic diseases.

Gestational trophoblastic diseases comprise a spectrum of interrelated diseases including partial mole, complete mole and gestational choriocarcinoma. Using reverse transcriptase PCR (RT-PCR) analysis, we identified higher levels of DOC-2/hDab2 expression in the normal trophoblast cells in culture than in choriocarcinoma cell lines. Subsequent study using immunohistochemistry showed high levels of DOC-2/hDab2 protein expression in normal trophoblast tissues but significantly lower levels of expression in gestational trophoblastic disease tissues, particularly in complete mole and choriocarcinoma. When DOC-2/hDab2 was transfected into the choriocarcinoma cell lines, Jar, JEG and BeWo, the stable transfectants showed significantly reduced growth rate in culture. These data suggest that down regulation of DOC-2/hDab2 may play an important role in the development of gestational trophoblastic diseases.

[The role of KiSS-1 and matrix metalloproteinase-9 in regulation of invasion of trophoblasts].

OBJECTIVE: To explore the role of metastasis-related gene KiSS-1 and matrix metalloproteinase (MMP)-9 in regulation of invasion of trophoblasts. METHODS: RT-PCR and Western blotting were used to detect the MMP-9 and KiSS-1 expression levels in the placental tissues obtained from 90 cases of normal pregnant women undergoing artificial abortion, induction of labor with water bag or selective cesarean section among which 30 cases were in the first trimester, 30 in second-trimester and 30 cases of term pregnancy, and in the placental tissues of 40 cases of preeclampsia (15 cases of mild and 25 cases of severe preeclampsia) undergoing cesarean section, and tissues of 90 cases of hydatidiform mole, 9 cases of invasive mole and 8 cases of choriocarcinoma, all undergoing surgery. RESULTS: The expression levels of MMP-9 mRNA and protein were higher in first-trimester [A value 0.391 +/- 0.215, (36 +/- 7) microg/100 microg total protein] and then decreased gradually with the progress of gestation. The expression levels of MMP-9mRNA and protein in the term placental samples were significantly lower than those of first-trimester (both P < 0.01). The expression levels of KiSS-1mRNA and metastin in normal placenta increased along with the progress of gestation (both P < 0.01). The KISS-1 mRNA expression level and MMP-9 protein expression level in the placental tissue of preeclampsia were 0.09 +/- 0.06 (A value) and (9.6 +/- 4.3) microg/100 microg total protein respectively, both significantly lower than those of the term placenta (both P < 0.01). The expression levels of MMP-9 mRNA and protein in the tissues of gestational trophoblastic disease were significantly higher than those in the first-trimester placenta (both P < 0.01). The expression level of KISS-1 mRNA and metastin in the tissues of hyddatidiform mole and invasive mole were both significantly lower than those in the first trimester placenta (P < 0.05, P < 0.01). The expression of KiSS-1mRNA and metastin in the tissues of choriocarcinoma could not be detected. CONCLUSION: The expression of the invasion-related gene, MMP-9, is positively related with, while the invasion suppressor gene, KiSS-1, is negatively related with the invasive ability of trophoblasts. The interaction of these two genes plays an important role in regulation of the invasion of trophoblasts.

[Expression of laminin and laminin receptor in gestational trophoblastic tumor and its significance].

OBJECTIVE: This study was conducted to detect the expression of laminin (LN) and laminin receptor (LN-R) in the specimens of hydatidiform mole (HM), invasive mole (IM) and choriocarcinoma(CC), respectively. METHODS: The immunohistological staining method was used. RESULTS: It was found that the level of the laminin expression was higher in the specimens of choriocarcinoma at more advanced stage. CONCLUSION: The results suggest that there may be relations between the invasion and metastasis of choriocacinoma cell and the malignant degree of this disease, chemotherapy has some effect on the expression of LN and LN-R in gestational trophablastic tumor (GTT), and that LN and LN-R may be of potential value in GTT treatment. However, referring whether LN and LN-R could be used as clinical prognosticators, further studies will be needed.

Transcriptional and posttranscriptional mechanisms in epidermal growth factor regulation of human chorionic gonadotropin (hCG) subunits and hCG receptor gene expression in human choriocarcinoma cells.

Epidermal growth factor (EGF) stimulates the secretion of hCG in choriocarcinoma cells. However, the molecular mechanisms involved in this EGF action have never previously been investigated. The present study investigated them as well as EGF regulation of the hCG/LH (LH) receptor gene in JEG-3 human choriocarcinoma cells. The JEG-3 cells contain multiple EGF receptor messenger RNA (mRNA) transcripts and a single 170-kilodalton immunoreactive receptor protein. The human EGF can bind to the receptor protein and stimulate the receptor autophosphorylation as well as the phosphorylation of four other membrane proteins. Culturing JEG-3 cells with recombinant human EGF resulted in a dose- and time-dependent increase in hCG secretion. The maximal effect was seen at 100 ng/ml EGF, with a time lag of about 5 h. Tyrosine kinase, but not protein kinase-C or protein kinase-A, signaling was involved in the EGF action to increase hCG secretion. The EGF-induced increase in hCG secretion was not due to an increase in cell number or differentiation into multinuclear syncytia. EGF treatment resulted in a dose- and time-dependent increase in steady state levels of hCG alpha and hCG beta mRNAs. This increase was due to the stabilization of subunit mRNA transcripts. The increase in subunit mRNAs preceded the increase in hCG secretion. The EGF treatment resulted in a dose- and time-dependent decrease in steady state levels of the hCG/LH receptor mRNA transcripts. The decrease was due to a transcriptional inhibition of receptor gene. EGF treatment paradoxically stabilized hCG/LH receptor protein. In summary, EGF treatment up-regulates hCG subunits gene expression and down-regulates hCG/LH receptor mRNAs involving transcriptional and posttranscriptional mechanisms in JEG-3 human choriocarcinoma cells.

Radioligand receptor assay for urinary and serum hCG.

A radioligand receptor assay system for urinary hCG and serum hCG was developed, using 1100-2000 times g fractions of homogenates of pig testis. This method is specific for hCG and LH, and the limit of detection for hCG was 20 mIU, which could be significantly discriminated from zero at the 95% confidence level. A nonspecific inhibition reaction which exerts its influence on the assay system has been found in the urine and serum, and in performing assays for urinary and serum hCG, it was necessary to remove the inhibition reaction by diluting the urine more than eight fold or subject the serum to an extraction procedure. Since the hCG of normal pregnancy and trophoblastic neoplasia showed a dose response curve similar to that of the International Standard hCG, it became clear that these hCG could be assayed with this method. The potency ratio R/I measured by RRA and RIA of urinary hCG and serum hCG was different from those of normal pregnancy and trophoblastic neoplasia.

Galectin-1 and galectin-3 in the trophoblast of the gestational trophoblastic disease.

While the presence and distribution of galectin-1 and galectin-3 in different normal trophoblast cell populations is known, no information is available regarding their occurrence in malignant trophoblast of gestational trophoblastic disease (GTD). Galectins-1 and -3 have, however, been implicated in malignancies of other tissues. Immunoreactivity for these galectins in the transformed trophoblast of invasive mole (n = 8), choriocarcinoma (n = 7) and one case of placental site trophoblastic tumor (PSTT) was compared to that of the invasive trophoblast of the normal first trimester of pregnancy implantation sites (n = 9). A large proportion of the transformed trophoblast cells of all GTD studied were positive for galectin-1 and galectin-3. Immunoreactivity was scored semiquantitatively to include both the prevalence among the trophoblast cells and the intensity of staining. Immunoreactivity for both galectin-1 and galectin-3 in gestational trophoblastic disease is increased (significant differences at p < 0.05, Mann-Whitney Rank Sum Test). This finding may suggest a possible implication of galectins-1 and -3 in the invasiveness of the transformed trophoblastic cell, although the exact physiological significance of this finding remains to be determined.

Expression of epidermal growth factor in gestational trophoblastic disease (GTD).

Gestational Trophoblastic Disease (GTD) is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of the disease is vague and methods to determine the malignant potentialis limited. Placentae of normal (50) and molar pregnancy (95) including 32 patients showing persistent disease were used in this study. EGF expression was analyzed by immunohistochemistry using monoclonal antibody to EGF and quantitated using isotope labelled antibody to EGF. EGF was expressed more strongly in molar placentae in all gestational ages in comparison with normal placentae of similar gestational age, the expression being restricted mainly to first and second trimesters in normal placentae. Moles with early presenting symptoms (< 12 weeks) were at a higher risk for developing persistent disease. In conclusion, the immunohistochemical study shows high expression of EGF in early normal placentae and moles linking its role to proliferative and differentiating activity of trophoblasts. Tumors with histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGF and thus EGF has the potential of identifying high risk lesions.

Mola invasiva--special form of GTD.

Invasive hydatidiform mole is a relative rare form of gestational trophoblastic disease (GTD). Most of hydatidiform moles remit after evacuation but some of them have the tendency to invade the myometrium. In some rare cases the trophoblastic tissue can be found in other tissues like lungs, vulva, vagina or broad ligament. The aim of the study was to demonstrate some of clinical, immunohistochemical and DNA analysis findings of a patient with a previous diagnosis of a complete hydatidiform mole.

Increased expression of interleukin-1 beta is associated with persistence of the disease and invasion in complete hydatidiform moles (CHM).

Complete hydatidiform moles (CHM), a post-conceptual pathologic condition of the placenta, have a high prevalence rate (12/1,000 deliveries) in Kerala, India. This study addresses the expression of IL-1 alpha and beta by immunohistochemistry in relation to persistence and invasion of the disease. Mild to moderate expression of IL-1 alpha in the villous cytotrophoblasts, syncytiotrophoblasts and decidua of the first trimester in the normal placenta and all gestational ages in the molar placenta were observed. IL-1 beta expression was observed in the extravillous trophoblasts, syncytiotrophoblasts and decidua in both the normal and molar placentae and also in the villous cytotrophoblasts and the stromal Haufbaur cells in molar placentae. Strong expression of IL-1 beta in the placenta suggests its involvement in placental physiology supporting earlier reports. Higher expression of IL-1 beta correlated well with the invasive and persistent nature of the tumour and holds potential as a marker of persistence and invasion in CHM.

Expression of c-erbB2 in gestational trophoblastic disease and its clinical significance.

In order to explore a potential indicator of predicting the occurrence and development of gestational trophoblastic tumor, the expression of c-erbB2 oncogene in human normal placenta, hydatidiform mole and choriocarcinoma was investigated. The expression of c-erbB2 was detected immunohistochemically by monoclonal antibody against the gene on the formalin-fixed paraffin sections of 21 hydatidiform moles, 21 invasive moles, 20 choriocarcinomas and 30 normal placentas. Results showed that the expression level of c-erbB2 was significantly higher in gestational trophoblastic tumor than in hydatidiform mole and normal placenta of midterm and term pregnancy (P < 0.05), while there was no significant difference between patients with gestational trophoblastic tumor of stage III, IV and those of stage I, II. It was demonstrated that overexpression of c-erbB2 may closely associated with malignant transformation of hydatidiform mole, not only providing important insight into pathogenesis of gestational trophoblastic tumor, but also having an important significance for the early diagnosis and early treatment of gestational trophoblastic tumor.

Differential expression patterns of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -1, -4, -5, and -14 in human placenta and gestational trophoblastic diseases.

CONTEXT: The ability of intermediate trophoblasts to invade maternal tissue during placentation depends on how well they can degrade the extracellular matrix. Invasion into the extracellular matrix requires many complex proteases. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a novel family of secreted metalloproteinases. The ADAMTS-1, -4, -5, and -14 subtypes are known to be expressed in human placenta, but little is understood about their expression patterns. OBJECTIVE: To examine the expression patterns of ADAMTS-1, -4, -5, and -14 in specific human placenta cell types during gestation and in gestational trophoblastic diseases. DESIGN: Placental tissues were obtained from 25 pregnant women and 21 cases of gestational trophoblastic diseases (10 early complete moles, 3 placental site trophoblastic tumors, 4 invasive moles, and 4 choriocarcinomas). The expression of the 4 ADAMTS was analyzed by immunohistochemistry. RESULTS: ADAMTS-1, -4, -5, and -14 were differentially expressed by the human placenta throughout gestation in a time-specific and cell type-specific manner, as well as in gestational trophoblastic diseases. ADAMTS-1 showed gradually strong staining intensity in gestational trophoblastic diseases according to the invasive potential but showed consistent strong intensity throughout normal placenta. ADAMTS-4 and ADAMTS-5 exhibited higher and restricted expression in first-trimester intermediate trophoblasts. They also exhibited comparably strong expression in gestational trophoblastic diseases. However, ADAMTS-14 expression remained unchanged throughout gestation. CONCLUSIONS: The restricted expression pattern of ADAMTS-4 and ADAMTS-5 and their increased expression in gestational trophoblastic diseases suggest that these 2 ADAMTS subtypes are associated with a biological phenotype of trophoblasts involved in human placentation and the development of gestational trophoblastic diseases.

Differential expression of E-cadherin, ß-catenin, and Lewis x between invasive hydatidiform moles and post-molar choriocarcinomas.

Trophoblast cell adhesion and migration are carefully coordinated during normal placental development. We have compared the expression of three adhesion molecules, E-cadherin, ß-catenin, and Lewis x, by immunohistochemistry during normal trophoblast differentiation, and in hydatidiform moles and choriocarcinomas. Both E-cadherin and ß-catenin were expressed in normal placenta cytotrophoblast, and this expression decreased with trophoblast maturation. E-cadherin was mainly localized along the contact between cytotrophoblast and syncytiotrophoblast, which indicates its role in the differentiation of the syncytial layer. Lewis x disappeared progressively during differentiation of normal villous vessels, and was expressed in molar pregnancies. Interestingly, whereas choriocarcinomas were not, or poorly, stained, invasive hydatidiform moles (invHMs) strongly expressed Lewis x in vascular structures. This observation correlated well with E-cadherin and ß-catenin expression and suggests that these three markers are associated with the invasive transformation. The presence of robust endothelial structures in invHMs could also explain their ability to maintain organized villous architecture (contrary to metastatic choriocarcinomas) during their invasion of extrauterine tissues such as the lung or the brain after dissemination through the blood flow. In our hands, Lewis x appeared to be a new, reliable marker that can be used to clearly distinguish invHMs from choriocarcinomas.

Altered p16 and Bcl-2 expression reflects pathologic development in hydatidiform moles and choriocarcinoma.

Abnormal trophoblast differentiation is the main cause of gestational trophoblast diseases in the case of hydatidiform moles and choriocarcinomas. Here we investigated the expression patterns of two gene products, p16 and Bcl-2, implicated in cell cycle regulation and apoptosis, respectively, using immunohistochemistry during normal placenta differentiation, hydatidiform moles (partial, complete and invasive) and post-molar choriocarcinomas. The p16 protein shows a gradual expression in cytotrophoblast of normal villous, from a p16 weak proliferative phenotype to a p16 strong invasive phenotype reaching a maximum around 17 weeks of gestation. The expression pattern in cytotrophoblast was similar in moles in contrast to the villous mesenchyme of invasive moles where p16 was strongly expressed. Bcl-2 expression was syncytiotrophoblast specific in normal placenta and moles and increased gradually during normal differentiation. The results explain the persistence of normal and molar villous fragments during their development and their dramatic invasion in the uterine arteries in case of invasive moles. In choriocarcinomas the weak Bcl-2 expression is associated with weak p16 expression indicating a great apoptotic and proliferative potentials. The results suggest that strong p16 expression in the villous mesenchyme may be responsible in part of the morbidity of the moles, and the key of cancer progression in the choriocarcinomas would be a fast cell-cycle turnover.

Molecular alterations in epidermal growth factor receptors as potential predictors of invasion in complete hydatidiform moles (CHM).

Molecular alterations in Epidermal growth factor receptor (EGFR) were investigated for the first time in molar placenta using protein expression, activation status, differential amplification status and mutational analysis. Invasive lesions showed upregulation of internal domain and downregulation of external domain with concomitantly high gene amplification and phosphorylation. Mutations distributed across different exons in non-invasive cases in contrast to single mutations restricted to exons 4 and 6 in invasive cases displayed a strong correlation to overexpression and phosphorylation status suggesting that higher copies of EGFR gene and mutations in exon 4&6 influence the invasive capacity of trophoblasts and can be used as a biomarker of invasion.

Invasive hydatidiform mole: immunohistochemical labelling of inhibin/activin subunits, Ki67, p53 and glycodelin A in a rare case.

Invasive trophoblastic mole is an extremely rare condition. Its early recognition is essential since it can transform into an invasive type of tumour. Immunohistochemistry was performed with monoclonal antibodies against inhibin-alpha, -betaA and -betaB, Ki67, p53 and glycodelin A in a rare case of accidentally diagnosed invasive trophoblastic mole. There was labelling of the inhibin/activin subunits, Ki67 and p53, while glycodelin A showed minimal immunopositivity. Therefore, since the pathological diagnosis of an invasive mole is difficult, the immunohistochemical detection of inhibin/activin subunits, Ki67, p53 and glycodelin A might be additional useful tumour markers.

[Expression of TM4SF9 in human trophoblasts].

OBJECTIVE: To investigate the expression of TM4SF9 in the villi of early pregnancy, hydatidiform mole, invasive hydatidiform mole and chorionic carcinoma tissue. METHODS: Immunohistochemistry was used to detect the expression of TM4SF9 in normal villi of early pregnancy, hydatidiform mole, invasive hydatidiform mole and chorionic carcinoma tissues. RESULTS: TM4SF9 was expressed in the cytotroblasts but not in the syncytiotrophoblast of normal villi. The intensity of TM4SF9 expression increased in the order of normal villi, hydatidiform mole, invasive hydatidiform mole and chorionic carcinoma, with strong positivity rates of 0, 10%, 36.4% and 100%, respectively, showing significant differences between the samples (P<0.001). CONCLUSION: TM4SF9 expression in the trophoblasts may relate to their invasiveness and play an important role in the metastasis of trophoblastic tumor.