Forskolin promotes vasculogenic mimicry and invasion via Notch­1­activated epithelial­to­mesenchymal transition in syncytiolization of trophoblast cells in choriocarcinoma.

Choriocarcinoma (CC) is characterized by earlier blood metastasis compared with other female genital tumors and a high incidence of massive hemorrhage. Vasculogenic mimicry (VM) is highly associated with metastasis, and syncytiotrophoblast is involved in the formation of VM in CC. Forskolin is a typical activator of the cAMP pathway, which is involved in the syncytiolization of trophoblastic cells. In the present study, to determine the effects and mechanism of forskolin on cell invasion and VM during syncytiolization in vitro and in vivo, JEG­3 and JAR cell lines were treated with 100 µM forskolin for 48 h, and wound healing and invasion assays were used to verify cell migratory and invasive capacities. A 3D culture and tube formation assays were established to detect VM. Variation of morphology and markers of the epithelial­to­mesenchymal transition (EMT) were assessed, and the role of the Notch signaling pathway was investigated in CC cells treated with forskolin. The results of the present study demonstrated that 100 µM forskolin induced syncytiolization of trophoblastic cells and enhanced the migratory and invasive abilities of JEG­3 and JAR cell lines. In addition, the capacity of VM was significantly increased, whereas tube formation ability was decreased by forskolin in vitro and in vivo compared with the respective control groups. The cellular morphology exhibited EMT during the syncytiolization process, which was further supported by the changes in EMT marker expression, including downregulation of E­cadherin and cytokeratin and upregulation of N­cadherin, vimentin and zinc finger E­box­binding homeobox 1. The Notch­1 signaling pathway was activated to induce EMT in forskolin­induced VM process in CC cells, and VM and EMT could be reversed by using the γ­secretase inhibitor DAPT to block the Notch­1 pathway. Overall, the results of the present study demonstrated that forskolin enhanced the capacity of VM formation and metastasis through Notch­1­activated EMT in the syncytiolization of trophoblastic cells.

The use of a unique co-culture model of fetoplacental steroidogenesis as a screening tool for endocrine disruptors: The effects of neonicotinoids on aromatase activity and hormone production.

Estrogen biosynthesis during pregnancy is dependent on the collaboration between the fetus producing the androgen precursors, and the placenta expressing the enzyme aromatase (CYP19). Disruption of estrogen production by contaminants may result in serious pregnancy outcomes. We used our recently developed in vitro co-culture model of fetoplacental steroidogenesis to screen the effects of three neonicotinoid insecticides on the catalytic activity of aromatase and the production of steroid hormones. A co-culture of H295R human adrenocortical carcinoma cells with fetal characteristics and BeWo human choriocarcinoma cells which display characteristics of the villous cytotrophoblast was exposed for 24h to various concentrations of three neonicotinoids: thiacloprid, thiamethoxam and imidacloprid. Aromatase catalytic activity was determined in both cell lines using the tritiated water-release assay. Hormone production was measured by ELISA. The three neonicotinoids induced aromatase activity in our fetoplacental co-culture and concordingly, estradiol and estrone production were increased. In contrast, estriol production was strongly inhibited by the neonicotinoids. All three pesticides induced the expression of CYP3A7 in H295R cells, and this induction was reversed by co-treatment of H295R cells with exogenous estriol. CYP3A7 is normally expressed in fetal liver and is a key enzyme involved in estriol synthesis. We suggest that neonicotinoids are metabolized by CYP3A7, thus impeding the 16α-hydroxylation of fetal DHEA(-sulfate), which is normally converted to estriol by placental aromatase. We successfully used the fetoplacental co-culture as a physiologically relevant tool to highlight the potential effects of neonicotinoids on estrogen production, aromatase activity and CYP3A7 expression during pregnancy.

Asbestos exposure and gestational trophoblastic disease: a hypothesis.

Among 2,968 women and girls exposed to crocidolite (blue asbestos) at Wittenoom, three cases of choriocarcinoma and three cases of hydatidiform mole have been identified (crude incidence rate of 9.9 per 1000 women and 1.7 per 1000 deliveries for choriocarcinoma and hydatidiform mole, respectively). The women with choriocarcinoma were resident at Wittenoom at the time of disease development, whereas hydatidiform mole occurred much later in women who had first been exposed to asbestos as young girls. Four of the six cases were known to have lived with asbestos company workers who brought their dusty work-clothes home for washing. Asbestos fibers have been reported in the lung, the pleural and peritoneal mesothelium, and the human ovary. They have also been detected in placental digests of live and stillborn infants. This cluster of gestational trophoblastic diseases has some biological plausibility for asbestos causation. Taking an occupational and residential history and examining pathologic specimens for asbestos fibers or bodies may prove useful in patients with gestational trophoblastic disease.

Activation of telomerase in BeWo cells by estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin in co-operation with c-Myc.

Telomerase activation, known to be stimulated by estrogen, is essential for cellular immortalization and trans-formation, both of which play a role in tumorigenesis. Dioxin and dioxin-like compounds have been shown to induce endometriosis and promote estrogen-dependent tumors. In this study, we show that either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or a combination of TCDD and 17-beta estradiol (E2) increase telomerase activity and the expression of the human telomerase catalytic subunit (hTERT) in human choriocarcinoma (BeWo) cells. Compared with estrogen or TCDD alone, the combination treatment did not show an additive effect. Likewise, treatment with either E2 or TCDD increased DNA synthesis and the cell population in S-phase, as detected by FACS analysis. However, following treatment with the E2 and TCDD combination, the proportion of cells in S-phase was actually lower than in cells treated with TCDD alone. These results suggest that TCDD alone mimics estrogenic action in telomerase activation and cell proliferation but, in the presence of estrogen, TCDD-induced actions were partially counteracted. E2 and TCDD also induced c-Myc, which is a transcriptional activator of hTERT in Bewo, but neither of these agents induced telomerase activity in HO15.19 c-myc-null cells. In contrast, only TCDD upregulated telomerase in TGR-1 cells, which are c-Myc expressing but lacking ER expression. The findings suggest that TCDD induces telomerase activity mediated through AhR signaling and/or ER-independent c-Myc signaling. The present study provides insight into the mechanism of promoter activity of TCDD in estrogen-related tumors.

The development and regression of deciduosarcomas and other lesions caused by estrogens and progestins in rabbits.

A series of experiments were conducted to study the histopathological effects of a combination of exogenous estrogens and progestins in mature rabbits. Estradiol (14-45 microg/day) and levonorgestrel (30-233 microg/day) were administered by intravaginal or subdermal Silastic devices for various time intervals to study the development of lesions with time and to determine if lesions regressed following withdrawal of the steroids. The origin of splenic decidual tumors (primary or metastasis from the uterus) was determined by administering the same steroid combination to castrated male rabbits. It was determined that uterine decidualization is present after 7 days of steroid treatment and that neoplasms of decidual cells may appear in the uterus after only 30 days of steroid administration. Decidual changes were observed frequently in uterine arteries, often concurrent with infarct-like areas of necrosis of the uterine wall. Withdrawal of contraceptive steroids for 14-120 days after 60 days' administration resulted in atrophy and disappearance of decidual cells and decidual tumors. Decidual neoplasms developed in the spleen of all castrated male rabbits given subdermal steroids, demonstrating that these tumors can arise as primary neoplasms of the spleen. The foregoing lesions appear to be peculiar to the rabbit and, together with previous data, suggest the rabbit to be a poor model for evaluating the effects of contraceptive steroids in other species.

The roles of estrogen and progestin in producing deciduosarcoma and other lesions in the rabbit.

The interactions of estrogens and progestins in producing decidualization, deciduosarcoma. and other lesions in the rabbit were explored. Steroids were delivered by silicone elastomer implants placed subdermally except for oral dosing in 1 experiment. Varying doses of levonorgestrel (LNG) were given with and without estradiol (E2) and varying doses of E2 with and without LNG. LNG alone delivered at an estimated mean dose of 233 microg/day did not result in endometrial decidualization or deciduosarcoma. Both conditions occurred when E2 was added to the regimen and increased as the dose of E2 was increased. Sixty microg of E2 per day produced endometrial decidualization in all test animals in a 2-month exposure, but deciduosarcoma occurred only when LNG was also supplied and increased as the LNG dose was increased. Progesterone given with E2 resulted in deciduosarcoma in most rabbits. Ethynylestradiol alone at 30 microg/day delivered by implants produced splenic and ovarian deciduosarcomas in 1 of 5 test animals. Adding LNG resulted in more numerous and widespread deciduosarcomas. These experiments indicate that exogenous estrogen is necessary for decidualization of the endometrium and to production of deciduosarcoma in the nonpregnant rabbit. Exogenous progestin promotes the process. Necrosis of the uterine wall tended to increase with increasing dose of estrogens.

Respiratory failure from metastatic choriocarcinoma: a survivor of mechanical ventilation.

A patient with respiratory failure from metastatic choriocarcinoma was treated with mechanical ventilation while receiving chemotherapy with etoposide, methotrexate, actinomycin D, cyclophosphamide, and vincristine. The patient recovered from respiratory failure with the assistance of standard mechanical ventilation using low tidal volumes. The patient has sustained clinical remission with normal respiratory function. Mechanical ventilation with low tidal volumes and a pressure-targeted approach should be considered in the patient who develops early respiratory failure from metastatic choriocarcinoma.

A uterine choriocarcinoma in a virgin Donryu rat.

A uterine choriocarcinoma was found in a 49-wk-old virgin Donryu rat given intrauterine administration of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). The tumor was macroscopically present as a bloody cystic mass and microscopically composed of 2 kinds of cells: small basophilic cells similar to cytotrophoblasts in the rat placenta and large cells with big nuclei resembling giant trophoblasts. The giant cells were positive for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), although immunohistochemical examination was negative or nonspecific for chorionic gonadotrophins, including beta human chorionic gonadotrophin (beta hCG), human placental lactogen (hPL), and placental glycoprotein (SP-1), or for placental alkaline phosphatase (ALP). However, the animal exhibited a slight mammotropic status. The results thus suggest a choriocarcinoma nature for this uterine tumor and that the tumor cells secrete a weakly mammotrophic hormone.

Comparative transplacental carcinogenesis by directly acting and metabolism-dependent alkylating agents in rodents and nonhuman primates.

Transplacental carcinogenesis by N-ethyl-N-nitrosourea (ENU) was studied in patas (Erythrocebus patas) and rhesus (Macaca mulatta) monkeys. Repeated intravenous injections throughout pregnancy caused gestational choriocarcinoma in female patas monkeys and a variety of non-trophoblastic neoplasms in their offspring. Latent periods for transplacentally induced tumours varied from less than one month to more than ten years. One case of congenital neoplasm was observed. Certain kinds of neoplasms were observed only in transplacentally exposed offspring, and not in monkeys given the same carcinogen during juvenile or adult life. These included a variety of embryonal tumours, especially nephroblastoma, and tumours of the brain, mostly gliomas. Schwannomas of the peripheral nervous system were not observed in patas monkeys given ENU. One embryonal tumour yielded DNA that transformed NIH 3T3 cells in a transfection assay. Similar protocols performed in rhesus monkeys also yielded a variety of tumours in the offspring, including brain tumours; but in this species the most common embryonal tumour was pulmonary blastoma, and no choriocarcinoma was seen in adult females given ENU during pregnancy. Administration of N-nitrosodiethylamine to patas monkeys during pregnancy at first appeared to have had no effect on the offspring, but administration of phenobarbital beginning at four years of age resulted in the rapid appearance of multiple hepatocellular tumours. With regard to potential human risk from prenatal exposure to carcinogens, three conclusions deserve special emphasis: (1) the extreme susceptibility of the fetal primate central nervous system to certain chemical carcinogens, which confirms and reinforces what was previously known from studies in rodents; (2) the prolonged latency of transplacentally initiated epithelial tumours and the importance of subsequent postnatal exposure to promoting agents; and (3) the concurrent risk of transplacental chemical carcinogenesis in offspring and gestational choriocarcinoma in the mother, suggesting that gestational choriocarcinoma with its short latent period may serve as an epidemiologically exploitable marker for human populations in which transplacental carcinogenesis is likely.

Hormonal contraception and cancer.

The natural menstrual cycle can influence the development of some cancers. Combined oral contraceptives, which replace normal hormonal fluctuations with steadier levels of artificial sex hormones, appear to protect against cancers of the endometrium and ovary, but their effect on carcinomas of the breast and cervix remains uncertain.

PIP: This article examines the realtionship between the menstrual cycle and cancer and between oral contraceptive (OC) use and cancer. It is noted that the natural menstrual cycle can influence the development of some cancers: ovarian, endometrial, and breast cancer. Thus, suppression of the menstrual cycle theoretically could protect against these diseases. Conversely, hormonal contraceptive replace the natural cycle with relatively steady levels of artificial hormones, and the possibility that these hormones could cause malignancies must be examined critically. The prevention of ovulation with combined OCs appears to offer some protection against ovarian and endometrial cancer, but their effect on both breast cancer and cervical cancer remains uncertain. Since few women have taken progestogen-only OCs for extended periods, little is known about their longterm effects. Further epidemiologic studies of combined OCs, especially regarding breast cancer risks, are in progress. However, because of the latent period between OC exposure and clinical disease, current studies will reveal the effects of higher-dose formulations used 10-20 years ago. In response to questions from patients about whether OCs cause cancer, physicians can point out that: 1) the effects of combined OCs are not yet clear, 2) 2 cancers are definitely les frequent among OC users combined and 3) there is as yet no cause to believe that the overall risk of cancer is greater in OC users than in nonusers.

Neoplasia and hormonal contraception.

PIP: There are an estimated 8-10 million oral contraceptive (OC) users in the U.S. Investigation of the effects of OCs on neoplasia is not easy; currently 4 investigative methods are used: 1) case reports, 2) disease rate and trends, 3) case-control studies, which are the main source of careful retrospective information, and 4) cohort studies, which compare the incidence of disease in patients exposed to suspected environmental factors, and in those who are not exposed. Major risk factors for carcinoma of the breast are female sex, age, genetic predisposition, previous benign breast disease, and previous cancer of one breast; undetected breast cancer may be present for many years before diagnosis, and risk is increased in patients with chronic cystic mastitis or fibrocystic disease of the breast. Clinical observations have suggested a strong association between endocrine influence and the incidence or progression of breast cancer; current evidence tends to support the role of estrogens in the etiology of carcinoma of the breast with respect to long-term estrogen administration, but this evidence is not valid for young patients who are on combined OCs. Most studies have documented a decreased risk of benign breast disease with length of OC use persisting for 4 years; these studies, however, did not analyze lesions by histologic type. Studies that show a protective effect on benign disease do not show the same protective effect for breast cancer. Data from cohort studies show no association of OCs with breast cancer. Since 1972 a number of reports have associated OCs with liver tumors, stating that risk increases with duration of use. A national survey revealed that the frequency of malignant tumors increased with age, but that the frequency of benign lesions had a peak in the 26-30 age group which corresponds to increased use of OCs. Benign tumors are dangerous because they tend to rupture spontaneously. The association between pituitary adenoma, causing postpill amenorrhea, and OC use is very controversial. OC use may also cause endometrial hyperplasia; postmenopausal estrogen use has also been associated with endometrial carcinoma, although the causal relationship has never been proven; progestogens may be useful in the therapy of some endometrial carcinomas. Carcinoma of the cervix seems to be more influenced by age at 1st intercourse and by multiple sexual partners than by OC use; several case-control studies have shown that there is no significant difference between incidence among OC users and nonusers. Data about the association between OCs and ovarian carcinoma are reassuring but incomplete. OCs should not be used in patients with positive chorionic gonadotropin titers who have been treated for hydatidiform mole.^ieng

Effect of whole-body x-irradiation on the morphogenesis of rat deciduoma.

Exposure of rats to whole-body X-irradiation (600 R) within 30 minutes of artificial induction of deciduoma resulted in impaired growth of deciduoma involving both endometrium and myometrium. Protein synthesis in endometrium was low 2 days after X-irradiation when compared with unirradiated controls. In addition, levels of RNA and DNA were low in the endometrium of irradiated rats. Development of metrial gland was inhibited in the irradiated rats. The number and morphology of antimesometrial and mesometrial decidual cells were altered in the irradiated animals. Endometrial and myometrial soluble proteins of irradiated rats resolved into fewer bands than those of unirradiated animals.

Pathology of gestational choriocarcinoma induced in patas monkeys by ethylnitrosourea given during pregnancy.

A rapidly fatal neoplastic disease with histological and clinical features resembling gestational choriocarcinoma in humans has been observed in patas monkeys. Timed pregnant females were given ethylnitrosourea (ENU) intravenously at doses of 0.1 to 0.4 mmol/kg body weight, beginning on day 30 of gestation and continuing weekly for a total of 12 injections. Of 59 monkeys given ENU during pregnancy, four of 12 subjected to the highest dose and three of the remaining 47 given lower doses died of choriocarcinoma within six months of cessation of ENU exposure. Death was usually caused by exsanguinating haemorrhage. At necropsy, tumour deposits were always numerous in the lungs and were frequently observed in abdominal viscera. An obvious primary uterine tumour was never found, and only one small primary was detected grossly. Sub-endometrial masses of tumour cells were generally observed microscopically, invading the endometrial stroma and forming endovascular tumour deposits in the veins. Both uterine and extrauterine tumour deposits were highly haemorrhagic, often partially necrotic, and consisted of cytotrophoblast-like cells with frequent mitoses, a high degree of cellular pleomorphism and variable but often prominent cytoplasmic glycogen. This tumour was never seen in males or non-gravid adult females. Chorionic gonadotrophin assays conventionally used for human and macaque samples were negative in both normally pregnant and tumour-bearing patas, and did not contribute to the diagnosis. Trophoblast of patas monkeys appears highly susceptible to the carcinogenic effects of ENU and provides an animal model for gestational choriocarcinoma.

Oral contraceptives and neoplasia.

PIP: Some of the available data concerning the suspected association between oral contraceptive (OC) use and the development of cancer is surveyed, and the attempt is made to evaluate possible associations between OCs and human neoplasia in light of pregnancy risk or benefit of oral contraception. The principal investigative methods in humans include various epidemiologic approaches, and the methodologies most often used are case reports (tumor registries), disease rates and trends, case-control studies, and cohort studies. These methods cannot prove a causal relationship between exposure to a possible carcinogen and the occurrence of disease. Consistent positive or negative evidence, confirmed by multiple epidemiologic approaches, can be used to guide physicians and regulatory agencies in formulating policy for the clinical use of OCs. Both the progestogen-only and the combined OCs have been shown to have a protective effect on the development of benign breast disease with this protective effect not appearing until 2 years of use. Long-term combined OC use appears to be related to the development of benign liver neoplasia, and this risk increases with the dose of the steroid and the age of the user. These lesions are quite rare but may be life threatening because of potential spontaneous rupture and hemorrhage. Long-term postmenopausal use of estrogens appears to increase significantly the risk of developing endometrial hyperplasia and adenocarcinoma of the endometrium. Estrogens appear to be related to the growth of pre-existing uterine leiomyomas. Endocervical cells under the influence of progestogens may develop adenomatous changes, and these benign changes have on occasion been misinterpreted as carcinoma.^ieng

Electron microscopic studies of placental and uterine tumors induced in rats.

The ultrastructure of eight 7,12-dimethylbenz (a) anthracene (DMBA) or 4-nitroquinoline-N-oxide (4-NQO) induced malignant tumors of placental and uterine origin in the rat were studied. Three of eight tumors had the ultrastructural characteristics of choriocarcinoma. The other five tumors had different fine structures. Two were classified as squamous cell carcinoma, one was as a leiomyosarcoma and the other two were undifferentiated sarcoma.