Placental Choriocarcinoma Research Articles

Alteration in folate carrier expression via histone deacetylase inhibition in BeWo human placental choriocarcinoma cells

Folates are essential nutrients for fetal development during pregnancy. Valproic acid (VPA), an inhibitor of histone deacetylases (HDACs), alters the expression of folate carriers in placental cells; however, the underlying mechanisms remain unclear. Here, we aimed to determine the profiles of folate carriers (folate receptor alpha [FOLR1], solute carrier [SLC]-19A1, and SLC46A1) after inhibition of HDACs, especially class I and IIa HDACs, using different inhibitors and gene knockdown tests. Quantitative polymerase chain reaction revealed that BeWo cells (a trophoblast model) expressed HDACs and folate carriers, similar to human placental villi. FOLR1 expression was upregulated by VPA, apicidin, and trichostatin A, but downregulated by MS-275 after 24 h treatment. VPA and apicidin upregulated the expression of SLC46A1. These inhibitors downregulated SLC19A1 expression. TMP269 (a class IIa inhibitor) did not affect folate carrier levels. HDAC1/2 knockdown upregulated FOLR1 and SLC46A1 levels, whereas HDAC1/3 knockdown downregulated FOLR1 levels. Our findings suggest that the pharmacological inhibition of class I HDACs alters the expression of folate carriers in BeWo cells. By contrast, HDAC inhibitors exert different regulatory effects on folate carriers. Moreover, HDAC1/2 inhibition may be a potential mechanism involved in altering FOLR1 and SLC46A1 levels.

Stimulus-Induced Activation of the Glycoprotein Hormone α-Subunit Promoter in Human Placental Choriocarcinoma Cells: Major Role of a tandem cAMP Response Element.

The glycoprotein hormones LH, FSH, TSH and chorionic gonadotropin consist of a common α-subunit and a hormone-specific β-subunit. The α-subunit is expressed in the pituitary and the placental cells, and its expression is regulated by extracellular signal molecules. Much is known about the regulation of the α-subunit gene in the pituitary, but few studies have addressed the regulation of this gene in trophoblasts. The aim of this study was to characterize the molecular mechanism of stimulus-induced α-subunit gene transcription in JEG-3 cells, a cellular model for human trophoblasts, using chromatin-embedded reporter genes under the control of the α-subunit promoter. The results show that increasing the concentration of the second messengers cAMP or Ca2+, or expressing the catalytic subunit of cAMP-dependent protein kinase in the nucleus activated the α-subunit promoter. Similarly, the stimulation of p38 protein kinase activated the α-subunit promoter, linking α-subunit expression to stress response. The stimulation of a Gαq-coupled designer receptor activated the α-subunit promoter, involving the transcription factor CREB, linking α-subunit expression to hormonal stimulation and an increase in intracellular Ca2+. Deletion mutagenesis underscores the importance of a tandem cAMP response element within the glycoprotein hormone α-subunit promoter, which acts as a point of convergence for a multiple signaling pathway.

Establishment of a 3D multicellular placental microtissues for investigating the effect of antidepressant vortioxetine

The placenta is a unique organ with an active metabolism and dynamically changing physiology throughout pregnancy. It is difficult to elucidate the structure of cell-cell and cell-extracellular matrix interactions of the placenta in in vivo studies due to interspecies differences and ethical constraints. In this study, human umbilical cord vein cells (HUVEC) and human placental choriocarcinoma cells (BeWo) were co-cultured for the first time to form spheroids (microtissues) on a three-dimensional (3D) Petri Dish® mold and compared with a traditional two-dimensional (2D) system. Vortioxetine is an antidepressant with a lack of literature on its use in pregnancy in established cultures, the toxicity of vortioxetine was studied to investigate the response of spheroids representing placental tissue. Spheroids were characterised by morphology and exposed to vortioxetine. Cell viability and barrier integrity were then measured. Intercellular junctions and the localisation of serotonin transporter (SERT) proteins were demonstrated by immunofluorescence (IF) staining in BeWo cells. Human chorionic gonadotropin (beta-hCG) hormone levels were also measured. In the 3D system, cell viability and hormone production were higher than in the 2D system. It was observed that the barrier structure was impaired, the structure of intracellular skeletal elements was altered and SERT expression decreased depending on vortioxetine exposure. These results demonstrate that the multicellular microtissue placenta model can be used to obtain results that more closely resemble in vivo toxicity studies of various xenobiotics than other 2D and mono-culture spheroid models in the literature. It also describes the use of 3D models for soft tissues other than the placenta.

Melatonin induces PERK-ATF4 unfolded protein response and apoptosis in human placental choriocarcinoma cell line

Melatonin induces PERK-ATF4 unfolded protein response and apoptosis in human placental choriocarcinoma cell line

PHEMA-based cryogels as 3D placental scaffolds

The placental barrier regulates the passage of nutrients, waste, drugs, and chemicals between maternal and fetal tissues. Mono and co-culture systems were constituted with human placental choriocarcinoma cells (BeWo) and human umbilical cord vein cells (HUVEC) on biocompatible poly (2-hydroxyethyl methacrylate) (PHEMA)-based cryogels. PHEMA cryogels were supported with extracellular matrix (ECM) elements. According to the Fourier transform infrared spectrometry-Attenuated Total Reflectance (FTIR-ATR) results, it was confirmed that gelatin, folic acid (FA), fibronectin (FN), and collagen type IV (COL Type IV) were successfully incorporated into the structure of cryogels. The mean pore size of the cryogels confirmed by micro-computed tomography analysis was in the range of 15–195 µm. The antidepressant active ingredient Vortioxetine was investigated by 3-(4,5-dimethylthiazol2-yl)− 2,5-diphenyltetrazolium-bromide (MTT) viability analysis in the constituted cryogel co-culture system. The cryogels did not cause any toxic effect on the cells. High doses of Vortioxetine showed reduced cell viability in monocultures, but not the expected reduction in cell viability at the same doses of co-culture. The highest amount of human chorionic gonadotropin hormone was in the PHEMA+COL Type IV+FA group. There were no significant changes in matrix metalloproteinase (MMP) 2 and 9 protein levels due to Vortioxetine dose administration.

3βHSD activity saturates at physiological substrate concentrations in intact cells.

Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3β-hydroxysteroid dehydrogenase (3βHSD) or to Δ5 -androstenediol by 17βHSD. To better understand this process, we studied the kinetics of these reactions in cells. Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. The two reactions displayed very different saturation profiles, with only the 3βHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3βHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17βHSD-catalyzed conversion to Δ5 -androstenediol. Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3βHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

Uptake of antiepileptic drugs in forskolin-induced differentiated BeWo cells: alteration of gabapentin transport

Previous studies have indicated that the expression levels of several transporters are altered during placental trophoblast differentiation. However, changes in the transport activities of therapeutic agents during differentiation must be comprehensively characterised. Antiepileptic drugs, including gabapentin (GBP), lamotrigine (LTG), topiramate, and levetiracetam, are increasingly prescribed during pregnancy. The objective of this study was to elucidate differences in the uptake of antiepileptic drugs during the differentiation process. Human placental choriocarcinoma BeWo cells were used as trophoblast models. For differentiation into syncytiotrophoblast-like cells, cells were treated with forskolin. The uptake of GBP and LTG was lower in differentiated BeWo cells than in undifferentiated cells. In particular, the maximum uptake rate of GBP transport was decreased in differentiated BeWo cells. Furthermore, GBP transport was trans-stimulated by the amino acids His and Met. We investigated the profiles of amino acids in undifferentiated and differentiated BeWo cells. Supplementation with His and Met, which demonstrated trans-stimulatory effects on GBP uptake, restored GBP uptake in differentiated cells. The findings of this study suggest that drug transport in BeWo cells can be altered before and after differentiation, and that the altered GBP uptake could be mediated by the intracellular amino acid status.

Platelet-derived growth factor-AA promotes placental choriocarcinoma JAR cell proliferation via primary cilia

ObjectiveDuring early pregnancy, the proliferation placental cells is crucial for proper implantation and formation of maternal–fetal circulation. Platelet-derived growth factor-AA (PDGF-AA) has been detected in placenta during early pregnancy; however, the role of PDGF-AA in placental cell growth has not been studied extensively. Primary cilium, a centrosome-based cellular protrusion, is an signaling hub for regulating development and differentiation. Importantly, the receptor of PDGF-AA (Pdgfr-α) is detected in the primary cilium and primary cilia-mediated PDGF-AA signaling regulates development and differentiation. Here we would like to investigate whether PDGF-AA regulates placental cell growth and whether primary cilia play roles in this process. Materials and methodsHuman placental choriocarcinoma JAR cells were treated with PDGF-AA followed by examining cell growth. Primary cilia and subcellular localization of Pdgfr-α were observed by immunofluorescence staining. Manipulation of primary cilia was performed by treating cells with roscovitine or by transfecting cells with siRNA against IFT88. ResultsHere we showed that PDGF-AA induced JAR cell proliferation. In addition, JAR cells grew primary cilia where Pdgfr-α was detected. More importantly, pharmacological inhibition of primary cilia formation or depletion of cilia-related gene, IFT88, alleviated PDGF-AA induced JAR cell proliferation. ConclusionThus, our study show that PDGF-AA facilitates human placental choriocarcinomaJARcell growth via primary cilia.

Effects of tumor necrosis factor on undifferentiated and syncytialized placental choriocarcinoma BeWo cells

Tumor necrosis factor (TNF) regulates trophoblast turnover during the formation of the placental syncytium and can be a potentially relevant target for adverse effects of xenobiotics. We mimicked syncytialization in vitro by stimulating BeWo cells with 50 μM forskolin. Undifferentiated and syncytialized BeWo cells were exposed to TNF (10 pg/mL–10 ng/mL) for 48 h after which cell viability, progesterone release and gene expression of a selected set of markers representative for placental function were assessed. In undifferentiated BeWo cells, high TNF levels (1–10 ng/mL) increased gene expression of TNF, NF-κB, and TNFRSF1B to maximally 99 ± 17, 2.2 ± 0.2, and 3.0 ± 0.4 of control values, respectively (p < 0.001). These effects were also found in syncytialized BeWo cells but less pronounced. Additionally, TNF may induce syncytialization in BeWo cells as it upregulated ERVW-1 expression by 1.55 ± 0.14-fold (p < 0.05). On the contrary, TNF levels of 10 and 100 pg/mL did not affect gene expression in both undifferentiated and syncytialized BeWo cells, but did enhance cell viability in syncytialised BeWo cells (p < 0.001). In conclusion, we found that high TNF levels (1–10 ng/mL) increased gene expression of TNF, NF-κB, and TNFRSF1B especially in undifferentiated BeWo cells, while physiological TNF concentrations positively affected cell viability and while there was no effect on any of the investigated functional markers.

Disruption of p97/VCP induces autophagosome accumulation, cell cycle arrest and apoptosis in human choriocarcinoma cells.

Gestational choriocarcinoma is aggressive trophoblastic disease. The development, progression and the cure of this disease is not well-established. p97/Valosin containing protein has been shown to play critical roles in many cellular processes. In various cancers, higher expression of p97/VCP has been reported and targeting of p97/VCP with its spesific inhibitors or siRNA's (siVCP) in cancer therapy was suggested. However, no study is avaible about the expression and function of p97/VCP in gestational choriocarcinoma. Hence, the aim of the study was to evaluate effects of p97/VCP inhibitor, DBeQ and siVCP on choriocarcinoma cells. We use human placental choriocarcinoma cell line (Jeg3) as model to find out the effects of DBeQ and VCP siRNA's (siVCP) on apoptotic and autophagic pathway by immunflouroscence staining, Western blotting, qPCR and flow-cytometry. p97/VCP siRNA's and DBeQ induced accumulation of autophagic proteins, LC3II and p62 in the cytoplasm of Jeg3 cells detected. Concurrently, Jeg3 cells treated with DBeQ and siVCP demonstrated G0/G1 cell cycle arrest, accompanied by accumulation of poly-ubiquitinated proteins. Moreover, disruption of p97/VCP by siRNA and DBeQ inhibited cancer cell growth managing the caspases-3 and -7. Our results show that inhibition of p97/VCP activity with DBeQ and depletion of p97/VCP expression with siRNA in Jeg3 cells induce caspase activation, inhibits cell proliferation and leads to a defect in autophagosome maturation, thus providing potential target for the prevention and treatment of choriocarcinoma.

Dexamethasone may inhibit placental growth by blocking glucocorticoid receptors via phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin and reactive oxygen species/AMP-activated protein kinase signalling pathways in human placental JEG-3 cells.

This study explored the molecular mechanism underlying the effects of dexamethasone (DEX, 1 µM) on glucose transporters (GLUT) in JEG-3 human placental choriocarcinoma cells. JEG-3 cells were treated with DEX, an expression plasmid encoding human glucocorticoid receptor α (GRα), pcDNA3.1-GRα, GRα short interference (si) RNA, LY294002, xanthine oxidase (XO)/hypoxanthine (HX), rapamycin, insulin-like growth factor (IGF) 1, N-acetylcysteine (NAC) or phosphatidic acid (PA), and cell proliferation, apoptosis, mitochondrial membrane potential (MMP), human chorionic gonadotrophin (hCG) content, human placental lactogen (hPL) content, glucose uptake, reactive oxygen species levels and signalling pathway modulation were evaluated. Treatment of JEG-3 cells with DEX (1 µM), GRα siRNA, LY294002 (50 µM), XO/HX (7.2 µM/36 nM) or rapamycin (80 nM) inhibited cell proliferation, induced apoptosis, significantly decreased MMP and hCG and hPL content and increased ROS levels. In addition, glucose uptake was decreased through downregulation of the mRNA and protein expression of GRα, GLUT1 and GLUT3. Treatment of JEG-3 cells with GRα siRNA, LY294002, XO/HX or rapamycin inhibited phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3 and mammalian target of rapamycin (mTOR) and induced the phosphorylation of AMP-activated protein kinase (AMPK) and tuberous sclerosis complex 2. The effects of GRα overexpression and IGF1 (100 nM), NAC (5 nM) or PA (100 µM) treatment on JEG-3 cells contrasted with those of DEX treatment. DEX blocked glucose uptake by downregulating GRα expression, which reduced GLUT1 and GLUT3 mRNA and protein expression, which, in turn, may have inhibited the PI3K/AKT/mTOR pathway and activated the ROS/AMPK pathway.

Identification and Functional Assessment of the First Placental Adhesin of Treponema pallidum That May Play Critical Role in Congenital Syphilis.

Syphilis is a global, re-emerging sexually transmitted infection and congenital syphilis remains a major cause of adverse pregnancy outcomes due to bacterial infection in developing nations with a high rate of fetus loss. The molecular mechanisms involved in pathogenesis of the causative agent, Treponema pallidum subsp. pallidum remain poorly understood due to the difficulties of working with this pathogen, including the inability to grow it in pure culture. To reduce the spread of syphilis, we must first increase our knowledge of the virulence factors of T. pallidum and their contribution to syphilis manifestations. Tp0954 was predicted to be a surface lipoprotein of T. pallidum. Therefore, we experimentally demonstrated that Tp0954 is indeed a surface protein and further investigated its role in mediating bacterial attachment to various mammalian host cells. We found that expression of Tp0954 in a poorly adherent, but physiologically related derivative strain of the Lyme disease causing spirochete Borrelia burgdorferi B314 strain promotes its binding to epithelial as well as non-epithelial cells including glioma and placental cell lines. We also found that Tp0954 expression facilitates binding of this strain to purified dermatan sulfate and heparin, and also that bacterial binding to mammalian cell lines is mediated by the presence of heparan sulfate and dermatan sulfate in the extracellular matrix of the specific cell lines. These results suggest that Tp0954 may be involved not only in initiating T. pallidum infection by colonizing skin epithelium, but it may also contribute to disseminated infection and colonization of distal tissues. Significantly, we found that Tp0954 promotes binding to the human placental choriocarcinoma BeWo cell line, which is of trophoblastic endocrine cell type, as well as human placental tissue sections, suggesting its role in placental colonization and possible contribution to transplacental transmission of T. pallidum. Altogether, these novel findings offer an important step toward unraveling syphilis pathogenesis, including placental colonization and T. pallidum vertical transmission from mother to fetus during pregnancy.

Expression of p57KIP2 reduces growth and invasion, and induces syncytialization in a human placental choriocarcinoma cell line, BeWo

IntroductionSyncytiotrophoblasts are the major components of the human placenta involved in fetal maternal exchange and hormone secretion. The syncytiotrophoblasts arise from the fusion of villous cytotrophoblasts. The cell cycle suppressor p57KIP2 is known to be an essential molecule for proper trophoblast differentiation during placental formation. MethodsWe generated p57KIP2-expressing BeWo transfectant cells. Proliferation assay and matrigel invasion assay were used to characterize p57KIP2-expressing BeWo transfectant cells. To reveal the role of p57KIP2 in syncytialization, we proceeded syncytium formation analysis and qRT-PCR for detection of the expression levels Syncytin-1, Syncytin-2 and their receptors. ResultsThe human choriocarcinoma cell line, BeWo has undetectable levels of p57KIP2 expression. Expression of p57KIP2 reduced cell proliferation rate and extracellular matrix invasion activity. p57KIP2 expressing cells displayed multinucleated cells associated with syncytiotrophoblast differentiation. In the syncytialization event, p57KIP2 was found to potentiate forskolin-induced upregulation of Syncytin-2 in a cAMP-independent manner. DiscussionThese results indicate that the expression of p57KIP2 may act on the proliferation/invasion inhibitory factor and enhance the expression of Syncytin-2, which are associated with syncytialization in cytotrophoblasts.

Tumor-suppressive function of methiothepin in human placental choriocarcinoma cells.

Placental choriocarcinoma is a malignant trophoblastic tumor associated with placentation. During placentation, complicated molecular networks are mediated by endocrine and paracrine signals. Serotonin neurotransmitters have been identified in the transmembrane region of human placental choriocarcinoma (HPC) cells as tumor promoters; therefore, their antagonists have anti-cancer properties. Although methiothepin, a serotonin receptor antagonist and FDA-approved psychotropic agent, has shown multi-pharmacological functions in various disease models, its anti-tumorigenic activity and mechanisms underlying its action against HPC are unknown. Therefore, we identified the anti-cancer effects of methiothepin in JEG3 and JAR HPC cells. Methiothepin attenuated mitochondrial function and induced endoplasmic reticular stress, reducing oxidative phosphorylation and causing metabolic shifting in HPC cells. Furthermore, methiothepin showed synergistic pharmacological effects with paclitaxel in HPC cells. Our results highlight the robust tumor-suppressive function of methiothepin in HPC. Our findings provide new insights into the repositioning of methiothepin from a psychotropic agent to novel anti-cancer agents, especially against HPC.

A Case Report of Medical Management Prior to Surgical Intervention in Molar Pregnancy with SVT

Gestational trophoblastic diseases comprise a spectrum of premalignant and malignant diseases, including hydatidiform mole (complete or partial), placental site trophoblastic tumor, choriocarcinoma and gestational trophoblastic neoplasia. Hydatidiform mole is the most common form of these (1). We report a case of 35-year-old women in her 5th para with the POA of 12 weeks presented with the features of thyrotoxicosis complicated with multiple episodes of SVT found to have molar pregnancy. She underwent medical cardioversion and thyroid suppressive treatment in order to stabilize and prevent perioperative complications. She underwent surgery and complete recovery was noted. Even though the definitive treatment is surgical its essential to consider the medical management first to stabilize the patient prior to the surgery.

Abstract 2988: PLAC8 confers the chemoresistance of placental site trophoblastic tumor and choriocarcinoma to etoposide in an autophagy-dependent manner

Abstract Chemotherapy is commonly used to treat gestational trophoblastic neoplasia (GTN), but placental site trophoblastic tumor (PSTT) and a small proportion of choriocarcinoma demonstrate chemotherapy resistance, which becomes the major obstacle for the treatment. However, the underlying mechanism remains elusive. In the present study, we found that PLAC8 expression in PSTT tissues was higher than that in placental site intermediate trophoblast cells. In addition, PLAC8 was also highly expressed in human choriocarcinoma tissues compared with normal placental villi. Over-expression of PLAC8 inhibited p53 protein stability and induced the autophagic flux in HTR-8 and JAR cells. PLAC8-induced autophagic activity could be partially recovered by p53 re-expression. Further, PLAC8 over-expression conferred chemoresistance of trophoblast cells to etoposide, which can be effectively abrogated by autophagic inhibitors 3-methyladenine (3-MA) or chloroquine (CQ). In conclusion, over-expression of PLAC8 in PSTT and choriocarcinoma tissues may endow the trophoblast cells with enhanced chemoresistance via inducing autophagy activity. Therefore, PLAC8 may serve as a potential therapeutic target for the treatment of PSTT and choriocarcinoma. Citation Format: Xuan Feng, Huandi Yu, Xiang Tao, Yan Du, Jing Wu, Yinhua Yu, Congjian Xu, Hongbo Zhao. PLAC8 confers the chemoresistance of placental site trophoblastic tumor and choriocarcinoma to etoposide in an autophagy-dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2988.

Bavachin suppresses human placental choriocarcinoma cells by targeting electron transport chain complexes and mitochondrial dysfunction

Phytoestrogens are naturally derived estrogen-like therapeutic compounds that have long been studied for their role as anti-cancer agents and supplements during chemotherapy. Bavachin is a therapeutic phytoestrogen used to treat cancer, inflammation, and diabetes mellitus. Though the therapeutic effects of bavachin on various diseases have been explored, its anti-cancer effects and related mechanisms in human placental choriocarcinoma remain unknown. This is the first study to identify the anti-cancer potential of bavachin on human placental choriocarcinoma cell lines JEG3 and JAR. Placental mitochondria support the elevated energy production required for placental development, through oxidative phosphorylation (OXPHOS). Based on this concept, we hypothesized that mitochondrial targeting by bavachin may contribute to anti-cancer activities in high-OXPHOS subtypes of cancer such as placental choriocarcinoma. Apoptosis and caspase activities were increased by bavachin in placental choriocarcinoma cells. Bavachin altered metabolic phenotypes by regulating electron transport chain complex and OXPHOS to suppress choriocarcinoma cell proliferation. It also led to calcium disruption and endoplasmic reticulum stress accompanied by mitochondrial membrane potential depolarization. It showed synergistic anti-cancer effects with paclitaxel on placental choriocarcinoma cells. Taken together, we suggest that bavachin has therapeutic potential against placental choriocarcinoma and may be used to counter paclitaxel-induced toxicity.

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

α,β-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,β-Thujone has numerous functions; it serves as a food ingredient, cosmetic additive, and medicinal remedy. Although the therapeutic properties of α,β-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,β-thujone attenuates JEG3 and JAR choriocarcinoma cells through a caspase-dependent intrinsic apoptotic pathway. Moreover, α,β-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,β-thujone via its sensitization effect on paclitaxel in choriocarcinoma cells. Altogether, our findings suggest that α,β-thujone is a novel, natural pharmacological compound that can be used to treat human placental choriocarcinoma.

Toxicity of diuron metabolites in human cells

Diuron, a highly used herbicide worldwide, is metabolized into several toxic metabolites. DCA (3,4-dichloroaniline), DCPU [3-(3, 4-dichlorophenyl)urea] and DCPMU [3-(3,4-dichlorophenyl)-1-methyl urea] reduced viability of human placental choriocarcinoma BeWo, human breast adenocarcinoma MCF-7 and human colon adenocarcinoma Caco-2 cells as judged by the MTT assay, where color formation is dependent on functional mitochondria in viable cells. Based on the IC50 values in BeWo cells the order of cytotoxicity was DCA > DCPU > diuron > DCPMU, and in Caco-2 cells DCPMU > DCPU > DCA, diuron. In MCF-7 cells, only DCPU had an IC50 within the range of the concentrations used. In the PI-digitonin viability assay, only the highest concentration (200 μM) of DCPU caused a statistically significant decrease in viability in any cell line. There was no correlation between cytotoxicity and ROS production. This indicates that diuron metabolites are toxic in cells of human origin with mitochondria as the target, but ROS not the likely mechanism.

SAT-734 HSD3B1 Expression in the Human Immune System

3β-hydroxysteroid dehydrogenase-1 (3βHSD1), catalyzing conversion of dehydroepiandrosterone (DHEA) to Δ 4-androstenedione, is an essential enzyme in the pathway toward production of biologically active androgens such as dihydrotestosterone from the adrenally produced precursor DHEA sulfate, the most predominant steroid hormone in circulation. We previously identified, in the gene (HSD3B1) that encodes 3βHSD1, a germline gain-of-function missense-encoding variant that has now been validated in several studies as predicting more rapid progression in prostate cancer patients treated with gonadal testosterone deprivation. Production of androgens from adrenal precursors is important not just in the context of prostate cancer but in other physiologic and pathophysiologic processes, which could include asthma. Androgens are associated with better lung function in both asthma and healthy cohorts, and increasing circulating androgen levels in males help explain the switchover in asthma being more common in boys than girls but then more common in women than men. A main treatment for asthma, as well as other inflammatory processes, is administration of glucocorticoids, yet unresponsiveness to glucocorticoids in a subset of patients remains a major problem. Systemic glucocorticoid administration suppresses adrenally produced DHEA and DHEA-S, suggesting a depleted pool for biologically active androgen production as a mechanism for glucocorticoid resistance. Our surprising preliminary data support a link between glucocorticoid responsiveness and the more active HSD3B1 allele: patients homozygous for the adrenal-permissive HSD3B1(1245C) allele exhibit better response to oral glucocorticoids than those homozygous for the adrenal-restrictive HSD3B1(1245A), with heterozygous patients falling in the middle. This suggests a model in which patients with the more active (adrenal-permissive) form of 3βHSD1 produce sufficient androgens despite the depleted pool of precursor hormones whereas patients with the less active (adrenal-restrictive) form cannot. To further elucidate the link between 3βHSD1 activity and immune response, we assayed HSD3B1 expression in different types of white blood cells. Leukocyte subsets from asthma patients and healthy controls were purified using fluorescence-activated cell sorting, and HSD3B1 expression was analyzed using qPCR. White blood cells of several types expressed HSD3B1 at levels comparable to or greater than both prostate cancer and placental choriocarcinoma cell lines with very robust 3βHSD1 activity. Further determining the cell type specific expression and activity of this key enzyme is an important step in unraveling the link between the HSD3B1 polymorphism and asthma along with potentially many other immune processes.