α,ß-Thujone suppresses human placental choriocarcinoma cells via metabolic disruption.

α,ß-Thujone is a natural terpenoid found in many medicinal herbs, such as Artemisia absinthium (wormwood), that exhibits antioxidant, anti-diabetic, and anti-tumorigenic effects. α,ß-Thujone has numerous functions; it serves as a food ingredient, cosmetic additive, and medicinal remedy. Although the therapeutic properties of α,ß-thujone were previously revealed, a comprehensive description of the mechanisms of its anti-cancer potential in choriocarcinoma is yet to be provided. To our knowledge, this study is the first to demonstrate that α,ß-thujone attenuates JEG3 and JAR choriocarcinoma cells through a caspase-dependent intrinsic apoptotic pathway. Moreover, α,ß-thujone was demonstrated to induce a global mitochondrial defect and ER stress in choriocarcinoma by causing mitochondrial depolarization, calcium overload, and metabolic alterations, thereby leading to energy deprivation, which eventually contributes to the increase in apoptosis of choriocarcinoma cells. Herein, we also revealed the synergistic anti-cancer activity of α,ß-thujone via its sensitization effect on paclitaxel in choriocarcinoma cells. Altogether, our findings suggest that α,ß-thujone is a novel, natural pharmacological compound that can be used to treat human placental choriocarcinoma.

Carvacrol induces mitochondria-mediated apoptosis via disruption of calcium homeostasis in human choriocarcinoma cells.

Carvacrol is a monoterpenoid phenol present in the oils of various plants including Origanum vulgare (oregano) or Origanum majorana (marjoram). For a long time, it has been used as spice in foods because of its antimicrobial properties. Additionally, it appears to have anticancer effects against some cancer but this has not been well studied. Therefore, we conducted various assays to confirm the effects of carvacrol on choriocarcinoma cell lines (JAR and JEG3). Our results indicate that carvacrol has antiproliferative properties and induces apoptosis, resulting in increased expression of proapoptotic proteins. Additionally, carvacrol disrupted the mitochondrial membrane potential and induced calcium ion overload in the mitochondrial matrix in both JAR and JEG3 cells. Furthermore, carvacrol generated oxidative stress and lipid peroxidation in both JAR and JEG3 cells. Moreover, carvacrol-suppressed phosphoinositide 3-kinase-protein kinase B and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (MAPK) signal transduction whereas expression of phosphor-P38 and c-Jun N-terminal kinase MAPK was increased. Together, our results indicate that carvacrol may be a possible new therapeutic agent or supplement for the control of human choriocarcinomas.

Naringenin suppresses growth of human placental choriocarcinoma via reactive oxygen species-mediated P38 and JNK MAPK pathways.

BACKGROUND: Human placental choriocarcinoma is a gestational trophoblastic tumor with high rates of metastasis and reoccurrence. However, some patients with choriocarcinoma are chemoresistance to conventional chemotherapeutic agents. HYPOTHESIS: Naringenin increases apoptosis in human placental choriocarcinoma cells. METHODS: We investigated the effects of naringenin on proliferation and migration of JAR and JEG3 cells, and performed TUNEL and Annexin V/PI staining assays to examine apoptotic effects of naringenin on both cells. In addition, we studied the loss of mitochondrial membrane potential (MMP) and the production of mitochondrial reactive oxygen species (ROS) to determine the specific reason for apoptosis of choriocarcinoma cells being mediated via mitochondria. Consistent with the induction of production of ROS by naringenin in both choriocarcinoma cell lines, we investigated lipid peroxidation and glutathione levels in both JAR and JEG3 cells since both are affected by ROS. We next determined dose-dependent effects of naringenin and its pharmacological inhibitors on signal transduction pathways in JAR and JEG3 cells by western blot analyses. RESULTS: Naringenin reduced viability and migratory functions of both cell lines, and increased mitochondria related apoptosis induced by ROS and lipid peroxidation, decreased glutathione and decreased mitochondrial membrane potential MMP in a dose-dependent manner. We also determined naringenin activated phosphorylation of ERK1/2, P38, JNK and P70S6K in JAR and JEG3 cells in a dose-response manner. Although naringenin induced phosphorylation of AKT proteins in JAR cells, it suppressed phosphorylation of the protein in JEG3 cells. In addition, we confirmed the mechanism of naringenin-induced cell signaling by using a combination of naringenin and pharmacological inhibitors of the PI3K and MAPK pathways, as well as a ROS inhibitor in JAR and JEG3 cell lines. CONCLUSIONS: Collectively, results of this study indicate that naringenin is a potential therapeutic molecule with anti-cancer effects on choriocarcinoma cells by inducing generation of ROS and activation of the MAPK pathways.

Silibinin stimluates apoptosis by inducing generation of ROS and ER stress in human choriocarcinoma cells.

Silibinin is a flavonolignan extracted from seeds of milk thistles. Traditionally, it has been used as a therapeutic agent for liver disorders, and now it is well-known for its anti-cancer effects. However, studies on anti-cancer effects of silibinin on choriocarcinoma are very limited. Therefore, we performed proliferation and apoptosis assays to determine effects of silibinin on the viability of human choriocarcinoma (JAR and JEG3) cells. Our results showed that silibinin significantly inhibited proliferation and induced apoptosis in both JAR and JEG3 cells, and significantly increased reactive oxygen species (ROS) and lipid peroxidation. Moreover, silibinin disrupted mitochondrial function by inducing permeabilization of mitochondrial membrane potential and calcium ion efflux in JAR and JEG3 cells. Furthermore, silibinin-induced apoptosis in choriocarcinoma cells via AKT, mitogen-activated protein kinases (MAPK) and unfolded protein response (UPR) signal transduction. Collectively, our results suggest that silibinin is a novel therapeutic agent or dietary supplement for management of human placental choriocarcinomas.

In vitro evaluation of combination of EGCG and Erlotinib with classical chemotherapeutics on JAR cells.

Gestational Trophoblastic Neoplasia (GTN) is a term used for a group of malignant gynecological tumors including choriocarcinoma. Low-risk neoplasias can be cured using single agents Methotrexate (MTX) and actinomycin-D (ACD), but in certain cases, decreased responsiveness and serious side effects occur. Therefore, researchers have been attempting to find new treatment modalities. One of the most popular way for increasing cancer patient survival rates is supporting treatment with adjuvant molecules or chemosensitizers. For this purpose, we investigated epigallocatechin-3-gallate (EGCG), a green tea cathecin, and Erlotinib, an EGFR tyrosine kinase inhibitor, as single agents and combined with MTX or ACD. In accordance with this, JAR (human placenta choriocarcinoma) cell line was used as an in vitro model and MTT, LDH, caspase-3 activation, RT-PCR, and Western Blot analyses were performed to investigate the effects of the test materials. Our studies demonstrate that combination of Erlotinib and EGCG with MTX and ACD decreases JAR cell proliferation and metastatic HER2 protein synthesis and increases caspase-3 activation compared to ACD or MTX alone. In addition, significant increase was observed in the apoptotic Bax gene, but no notable protein synthesis occurred in the Western Blot analysis, which suggests that combination of Erlotinib and EGCG with classical chemotherapeutics ACD or MTX may lead the JAR cells to apoptosis, but not by a mitochondrial pathway. All the results indicate that the synergetic effect of Erlotinib and EGCG with classical chemotherapeutics may help to increase patient survival rates of choriocarcinoma, but the detailed mechanism needs further investigation.

Isolation, Culture and Identification of Choriocarcinoma Stem-Like Cells from the Human Choriocarcinoma Cell-Line JEG-3.

BACKGROUND/AIMS: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. METHODS: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. RESULTS: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. CONCLUSION: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

Green tea (-)-epigallocatechin gallate induced growth inhibition of human placental choriocarcinoma cells.

This study investigated the pathways involved in the effect of green tea epigallocatechin gallate (EGCG) on mitogenesis in BeWo, JEG-3, and JAR placental choriocarcinoma cells. EGCG inhibited cell proliferation in dose-dependent and time-dependent manners, as indicated by the number of cells and incorporation of bromodeoxyuridine (BrdU). A catechin-specific effect of green tea was evident; EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in suppressing cell growth. When all three of the mitogen-activated protein kinase (MAPK) subfamilies, i.e., ERK, p38, and JNK, were examined, EGCG significantly increased levels of phospho-ERK1/2 (pERK1/2) and phospho-p38 (pp38) and did not alter the total protein levels of ERK1/2, p38 MAPK, JNK, and phospho-JNK. EGCG-induced increases in the levels of pERK1/2 and pp38 proteins were prevented by pre-treatment with specific inhibitors of ERK1/2 MAPK and p38 MAPK, respectively. These inhibitors also suppressed EGCG-induced decreases in both cell number and BrdU incorporation. Moreover, pre-treatment with an AMP-activated protein kinase (AMPK) inhibitor prevented the actions of EGCG on proliferation and AMPK phosphorylation. These data suggest that EGCG mediates choriocarcinoma cell growth via the AMPK, ERK, and p38 pathways, but not JNK pathway.

Altered folate metabolism modifies cell proliferation and progesterone secretion in human placental choriocarcinoma JEG-3 cells.

Folate is an essential B vitamin required for de novo purine and thymidylate synthesis, and for the remethylation of homocysteine to form methionine. Folate deficiency has been associated with placenta-related pregnancy complications, as have SNP in genes of the folate-dependent enzymes, methionine synthase (MTR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). We aimed to determine the effect of altered folate metabolism on placental cell proliferation, viability and invasive capacity and on progesterone and human chorionic gonadotropin (hCG) secretion. Human placental choriocarcinoma (JEG-3) cells cultured in low folic acid (FA) (2 nM) demonstrated 13% (P<0.001) and 26% (P<0.001) lower proliferation, 5.5% (P=0.025) and 7.5% (P=0.004) lower invasion capacity, and 5 to 7.5% (P=0.004-0.025) lower viability compared with control (20 nM) or supplemented (100 nM) cells, respectively. FA concentration had no effect on progesterone or hCG secretion. Small interfering RNA (siRNA) knockdown of MTR gene and protein expression resulted in 17.7% (P<0.0001) lower proliferation and 61% (P=0.014) higher progesterone secretion, but had no effect on cell invasion and hCG secretion. siRNA knockdown of MTHFD1 gene expression in the absence of detectable changes in protein expression resulted in 10.3% (P=0.001) lower cell proliferation, but had no effect on cell invasion and progesterone or hCG secretion. Our data indicate that impaired folate metabolism can result in lower trophoblast proliferation, and could alter viability, invasion capacity and progesterone secretion, which may explain in part the observed associations between folate and placenta-related complications.

The inhibitory effects of the standardized extracts of Ginkgo biloba on Aromatase activity in JEG-3 human choriocarcinoma cells.

Breast cancer is the most common cancer in women worldwide. There are many endocrine adjuvant therapies for breast cancer patients that are categorized according to their mechanisms. Among them, aromatase inhibitors (AIs) that block the synthesis of estrogens have proven superiority compared with tamoxifen and have replaced it as a first-line hormonal therapy. However, AIs also have limitations due to their side effects - increased rate of bone loss and musculoskeletal complaints. We therefore need new candidate AIs with fewer side effects. The extracts of Ginkgo biloba (EGb), which contain phytochemicals from the tree, had biphasic effects for estrogens and osteoporosis-inhibiting activities in our previous experiments. In this study, we explored the possibility of EGb as an AI and their mechanisms. Aromatase activities were inhibited by EGb both in JEG-3 cells and in recombinant CYP19 microsomes. The results of polymerase chain reaction for aromatase from a coding sequence and specific promoter sequences (exon I.a, exon I.c) in JEG-3 cells as well as the results of reporter gene assays showed that EGb dose-dependently decreased the aromatase gene expression. The decreased protein levels were demonstrated by Western blotting. From these results, we concluded that EGb could act as an AI at both the enzyme and transcriptional levels.

Purified vitexin compound 1 suppresses tumor growth and induces cell apoptosis in a mouse model of human choriocarcinoma.

OBJECTIVE: In our previous study, we had isolated a series of lignan compounds, termed vitexins, from the seed of Chinese herb Vitex negundo and found broad antitumor activities of these compounds in many cancer xenograft models and cell lines. This study was aimed to determine the antitumor effect of purified vitexin compound 1 (VB1) on choriocarcinoma in vitro and in vivo. MATERIALS AND METHODS: The severe combined immunodeficiency mouse model of choriocarcinoma was established to investigate the in vivo effect of VB1. Its effect on proliferation and apoptosis in JEG-3 cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay and flow cytometry, respectively. The expression of caspase-3, Bcl-2, and some molecules involved in the mammalian target of rapamycin (mTOR) signaling was detected by Western blot. RESULTS: Vitexin compound 1 significantly inhibited the growth of choriocarcinoma in severe combined immunodeficient mice and reduced the serum ß-human chorionic gonadotropin level. Vitexin compound 1 inhibited cell proliferation, induced apoptosis, and inhibited the mTOR signaling in JEG-3 cell line. CONCLUSION: Vitexin compound 1 could inhibit choriocarcinoma via inducing cell apoptosis and suppressing the mTOR pathway.

Effects of bufalin on the proliferation of human choriocarcinoma cells.

OBJECTIVE: Bufalin is a traditional Chinese medicine, and it induces apoptosis in some lines of human tumor cells. METHODS: We investigated the effect of bufalin in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of bufalin, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. RESULTS: An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that BeWo cells were sensitive to the growth inhibitory effect of bufalin. Cell cycle analysis indicated that exposure to bufalin decreased the proportion of cells in the synthesis 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 and by the loss of mitochondrial transmembrane potential. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. CONCLUSIONS: These results suggest that bufalin may serve as a therapeutic agent for the treatment of choriocarcinoma.

Long-chain polyunsaturated fatty acids stimulate cellular fatty acid uptake in human placental choriocarcinoma (BeWo) cells.

Supplementation of long-chain polyunsaturated fatty acids (LCPUFAs) is advocated during pregnancy in some countries although very little information is available on their effects on placental ability to take up these fatty acids for fetal supply to which the fetal growth and development are critically dependent. To identify the roles of LCPUFAs on placental fatty acid transport function, we examined the effects of LCPUFAs on the uptake of fatty acids and expression of fatty acid transport/metabolic genes using placental trophoblast cells (BeWo). Following 24 h incubation of these cells with 100 microM of LCPUFAs (arachidonic acid, 20:4n-6, eicosapentaenoic acid, 20:5n-3, or docosahexaenoic acid, 22:6n-3), the cellular uptake of [(14)C] fatty acids was increased by 20-50%, and accumulated fatty acids were preferentially incorporated into phospholipid fractions. Oleic acid (OA, 18:1n-9), on the other hand, could not stimulate fatty acid uptake. LCPUFAs and OA increased the gene expression of ADRP whilst decreased the expression of ACSL3, ACSL4, ACSL6, LPIN1, and FABP3 in these cells. However, LCPUFAs but not OA increased expression of ACSL1 and ACSL5. Since acyl-CoA synthetases are involved in cellular uptake of fatty acids via activation for their channelling to lipid metabolism and/or for storage, the increased expression of ACSL1 and ACLS5 by LCPUFAs may be responsible for the increased fatty acid uptake. These findings demonstrate that LCPUFA may function as an important regulator of general fatty acid uptake in trophoblast cells and may thus have impact on fetal growth and development.

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 IC(50) values of 2.01 and 1.19 microM 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.

High-risk gestational trophoblastic neoplasia with brain metastases: individualized multidisciplinary therapy in the management of four patients.

PURPOSE: To report our recent experience managing four patients with brain metastases of gestational trophoblastic neoplasia (GTN), coordinating systemic chemotherapy with early neurosurgical intervention or stereotactic radiosurgery and intensive supportive care during initial therapy to prevent early mortality. MATERIALS AND METHODS: A series of four consecutive patients with brain metastases from high-risk Stage IV GTN managed at our institution in 2003 and 2005. Patients were assigned FIGO stage and risk score prospectively. Because of concern for chronic toxicity resulting from concurrent moderate dose methotrexate and whole brain radiation, an individualized multidisciplinary approach was used to manage patients. RESULTS: All four women presented with brain and pulmonary metastases; one had multiple liver metastases. Neurological symptoms at presentation included grand mal seizures in 2 patients, left upper extremity hemiparesis and headache each in 1 patient, while 1 patient was asymptomatic. Index pregnancies were term pregnancies in all patients with interval from prior delivery ranging from 2 weeks to 4 years. Two had received prior chemotherapy for postmolar GTN prior to the index pregnancy with incomplete follow-up. Initial hCG values ranged from 26,400 to 137,751 mIU/ml; FIGO risk scores were > or =16 for all patients. Systemic combination chemotherapy was initiated with etoposide and cisplatin followed by moderate/high-dose (500-1000 mg/m(2)) methotrexate combinations. Craniotomy was used before or during the first chemotherapy cycle to extirpate solitary lesions in 3 patients, while stereotactic radiosurgery was administered after the first cycle to treat two brain lesions in the remaining patient. None received whole brain radiation or intrathecal methotrexate. In one patient, selective angiographic embolization was used to control hemorrhage from multiple liver metastases. Two patients required ventilator support early in treatment to allow stabilization from intrathoracic hemorrhage and neutropenic sepsis with respiratory distress syndrome, respectively. Hysterectomy was performed in one patient after completion of salvage chemotherapy. All have completed maintenance chemotherapy and are in prolonged remission (12-24 months). Neurologic sequelae include persistent left upper extremity dyskinesia and weakness in one patient, and episodic grand mal seizures and pseudoseizures in a second patient with a pre-existing seizure disorder. CONCLUSION: This case series documents the utility for a multidisciplinary approach to the treatment of brain metastases from GTN. Using early craniotomy or stereotactic radiosurgery combined with etoposide-cisplatin and moderate/high-dose methotrexate combination chemotherapy, we were able to stabilize patients early in their treatment and avoid whole brain radiation therapy or intrathecal chemotherapy.

Flax-seed extracts with phytoestrogenic effects on a hormone receptor-positive tumour cell line.

UNLABELLED: The higher soy intake in the Asian population compared to Europeans is believed to be an essential factor for the lower incidence of hormone-dependent tumours in Asia. It has already been shown that soya beans, with their ingredients genistein and daidzein from the isoflavonoid group, have protective effects on hormone-caused diseases. Lignans are another, less investigated, group of phytoestrogens. The aim of this study was to investigate the effects of flax-seed, which is typically found in Northern European diets, on the proliferation and hormone production of an estrogen receptor (ER)-positive trophoblast tumour cell line. MATERIALS AND METHODS: Trophoblast tumour cells of the cell line Jeg3 were incubated with 2 different concentrations of the isolated crude extract of flax-seed and 7 chemically partitioned extract fractions. Untreated cells were used as controls. After 48 h of stimulation, cell proliferation was measured using the BrdU method. The concentrations of hCG and progesterone produced by the trophoblast tumour cells were measured 48 h after stimulation. Extract fractions with antiproliferative effects in the BrdU- test were analysed by HPLC-MS. RESULTS: Our study showed an inhibitory influence of some of the isolated flax-seed fractions on the Jeg3 tumour cells. Proliferation of the Jeg3 cells was decreased by flax-seed fractions I, V, VI and VII in a dose-dependent manner. Inhibition of hCG production by flax-seed extracts III, V, VI and VII was also dose-dependent. Extract fractions V and VI decreased the production of progesterone by 58% to 86%. Some extract fractions showed a stimulating effect on hormone production and cell proliferation. HPLC-MS analysis showed the presence of matairesinol and biochanin A in flax-seed fraction VI. DISCUSSION: Flax-seed seems to have similar inhibitory effects to soya on hormone production and proliferation of hormone-sensitive tumour cells. Our results showed a dose-dependent inhibition by isolated flax-seed extracts on the Jeg3 cell line. Matairesinol and biochanin A seem to be useful candidates for extended tests on other tumour cell lines and normal tissues to evaluate the potential benefit of a lignan-containing therapy in hormone-dependent diseases.

mRNA instability in the nucleus due to a novel open reading frame element is a major determinant of the narrow tissue specificity of folate receptor alpha.

The folate receptor type alpha (FR-alpha) is a promising tumor marker and target. Here, we investigate the mechanistic basis for the tumor specificity and vast overexpression of FR-alpha. Among representative FR-alpha-positive (HeLa and JAR) and FR-alpha-negative (MG63, Caki1, and HT3) cell lines, the transcription rates of the endogenous FR-alpha gene, as well as the FR-alpha promoter activity, were relatively weak and comparable, but the FR-alpha transcript was abundant only in total RNA and nuclear RNA from the FR-alpha-positive cells. Rous sarcoma virus (RSV) promoter-driven expression of the FR-alpha gene was 7 to 30 times greater in the FR-alpha-positive than in FR-alpha-negative cells, both at the protein and mRNA levels, independently of intron sequences. Through the use of chimeric FR-alpha/FR-beta cDNAs, the above pattern of FR-alpha expression was attributed to a 60-bp sequence in the FR-alpha open reading frame. This sequence element, when placed in the 5' untranslated region of RSV promoter-luciferase, decreased the reporter expression approximately 7- to 20-fold in FR-alpha-negative cells (MG63, Caki1, HT3, BG1, and MCF7) relative to FR-alpha-positive cells (HeLa, JAR, and JEG3). Substitution of this FR-alpha element in FR-beta increased the in vivo degradation rate of the transcript in the nuclei of MG63 cells but not in the nuclei of HeLa cells or in the cytosol of MG63 or HeLa cells. The results reveal an efficient mechanism by which a novel sequence element causes differential transcript degradation in the nucleus to ensure narrow tissue specificity for a gene (e.g., that for FR-alpha) whose transcription is weak and relatively nonselective. FR-alpha exhibited constitutive mRNA and protein synthesis during the cell cycle and a slow protein turnover, presumably ensuring a high steady-state level of the receptor in cells that could override the nuclear mRNA instability determinant.

Effects of all-trans retinoic acid on choriocarcinoma cells in vitro.

OBJECTIVES: To determine the activities of all-trans retinoic acid (RA) on choriocarcinoma cells in vitro. METHODS: The antiproliferative effect of all-trans RA on 4 choriocarcinoma cell lines was measured by the MTT assay. The effect of all-trans RA combined with methotrexate or actinomycin-D was then examined. The effect of all-trans RA on hCG secretion was also studied. The gene expression of retinoic acid receptors (RARs) was examined by RT-PCR. RESULTS: All-trans RA inhibited cell proliferation dose- and time-dependently; a 6-day exposure to 1 microM all-trans RA suppressed the cell growth by 67.8%-82.0% compared to the controls. An enhanced effect was observed in the combined administration of all-trans RA and methotrexate or actinomycin-D. The secretion of hCG increased 4-fold to 9-fold by the addition of 1 microM all-trans RA. RARs genes were expressed in all cell lines. CONCLUSION: The anticancer activity presented here appears to warrant further evaluation of all-trans RA as adjuvant therapy for choriocarcinoma.

Clinicopathologic analysis of gestational trophoblastic disease--report of 158 cases.

Gestational trophoblastic disease (GTD) forms a heterogeneous pool of clinically and histopathologically defined entities with different malignant potential. The clinicopathologic characteristics of 158 cases, including 110 complete hydatidiform moles (CHM), 13 invasive moles, 32 choriocarcinomas, two placental site nodules and one placental site trophoblastic tumor are reported. Of all cases, 63.9% showed spontaneous regression after D&C. 36.1% resulted in a persistent or metastatic (11.4%) disease, including 12 CHM. Lung is found to be the most common site of metastasis (61%). The median time between antecedent pregnancy and GTD was 4.4 months. 44% had an antecedent CHM, 16% a term pregnancy. The median complete remission rate was 91.2% with 5.3% recurrent disease. Three women died. Eight patients received adjuvant surgical therapy for chemoresistant foci. In general, management of GTD is interdisciplinary with an emphasis placed on individualized treatment. In most cases, exact histopathologic diagnosis of the trophoblastic lesion remains the gold standard for guiding clinical therapy. Currently, there are no reliable genetic or molecular biologic markers predicting an aggressive behavior of CHM. Thus, all lesions should be followed by serial measurements of serum-HCG. All cases of persistent GTD should be treated in specialized centers.

Inhibition of cyclic AMP-triggered aromatase gene expression in human choriocarcinoma cells by antisense oligodeoxynucleotide.

Aromatase, an endomembrane-bound cytochrome P450, is the key enzyme of estrogen biosynthesis. Aromatase inhibitors, therefore, are clinically important tools in the treatment of estrogen-dependent tumor growth. To improve the specificity of these tools, inhibition at the nucleic acid level was examined. An antisense oligodeoxynucleotide complementary to the translation start region of human aromatase transcripts (antisense-arom) was synthesized and used to inhibit cyclic AMP-triggered aromatase gene expression in a human choriocarcinoma cell line (JEG-3), both as occurring in an autocrine fashion by secreted human chorionic gonadotropin or as induced by application of the membrane-permeating dibutyryl cyclic AMP. Significant inhibition was obtained in both cases, reaching 70% and 60%, respectively. In addition, the antisense-arom treatment led to accelerated mRNA degradation. The inhibition at the nucleic acid level was accompanied by a decrease of both the aromatase protein and microsomal aromatase activity. The data appear to indicate the antisense strategy to be a most promising approach for the development of a novel type of specific aromatase inhibitor.

Estrogen negatively regulates rat gonadotropin releasing hormone (rGnRH) promoter activity in transfected placental cells.

To dissect the functional architecture of the rat gonadotropin releasing hormone (GnRH) gene promoter and its regulation by estrogen, gene transfer studies were performed in a placental cell line. 5'-Deletional constructs demonstrated that cis-acting elements important for rGnRH promoter activity in placenta were contained within a region of the proximal promoter between -73 and -16 bp upstream of the transcription initiation site. In addition, an inhibitory region was found from -903 to -424. Deletion of sequences to -424 which removed the inhibitory region, produced a promoter fragment which exhibited a 50% inhibition of GnRH promoter activity in the presence of estrogen (E) and cotransfected estrogen receptor. Negative regulation by E was retained in constructs deleted to -73 bp. We conclude that sequences important for placental cell expression and estrogen regulation of rGnRH lie within the region from -73 to -16. In addition, potential repressor sequences active in placental cells are present between -903 to 424.