Visfatin increases the invasive potential of ovarian granulosa tumor spheroids by reprogramming glucose metabolism.

In brief: The role of visfatin in ovarian granulosa cell tumor (GCT) invasion and glucose metabolism reprogramming is largely unexplored. These studies imply that visfatin or its inhibitor is involved in regulating ovarian granuloma invasion by reprogramming glucose metabolism and may be a potential candidate for the diagnosis and treatment of ovarian GCT. Abstract: Visfatin is an adipokine with nicotinamide phosphoribosyltransferase (NAMPT) activity, the concentration of which is higher in ascitic fluid than in serum, and is associated with ovarian cancer peritoneal dissemination. Potentially important effects of visfatin on glucose metabolism have been previously reported. However, the mechanism underlying the effects of visfatin on ovarian cancer cell invasion, and whether this involves altered glucose metabolism, has not been elucidated. Here, we tested the hypothesis that visfatin, which can reprogram cancer metabolism, promotes invasion by ovarian cancer spheroids. Visfatin increased glucose transporter (GLUT)1 expression and glucose uptake in adult granulosa cell tumor-derived spheroid cells (KGN) and also increased the activities of hexokinase 2 and lactate dehydrogenase. We showed a visfatin-induced increase in glycolysis in KGN cells. Moreover, visfatin increased the potential invasiveness of KGN spheroid cells by upregulating MMP2 (matrix metalloproteinase 2) and downregulating CLDN3 and CLDN4 (claudin 3 and 4) gene expression. Interestingly, an inhibitor of GLUT1 and lactate dehydrogenase (LDHA) abolished the stimulatory effect of visfatin on the potential invasiveness of KGN cells. More importantly, silencing expression of the NAMPT gene in KGN cells demonstrated its important effect on glycolysis and invasiveness in adult granulosa cell tumor cells (AGCTs). In summary, visfatin appears to increase AGCT invasiveness through effects on glucose metabolism and to be an important regulator of glucose metabolism in these cells.

Persistent organic pollutants affect steroidogenic and apoptotic activities in granulosa cells and reactive oxygen species concentrations in oocytes in the mouse.

CONTEXT: The destruction of granulosa cells (GCs), the main functional cell type in the ovary, prevents steroid hormone production, which in turn may damage oocytes, resulting in ovarian failure. The accumulation of a number of persistent organic pollutants (POPs) in the ovarian follicular fluid (FF) has been documented, which raises serious questions regarding their impact on female fertility. AIMS: We aimed to determine whether a mixture of POPs reflecting the profile found in FF influences mouse GCs or oocyte function and viability. METHODS: A mixture of POPs, comprising perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, polychlorinated biphenyl 153, and hexachlorobenzene, was used. In addition to using the exact concentration of POPs previously measured in human FF, we tested two other mixtures, one with10-fold lower and another with 10-fold higher concentrations of each POP. KEY RESULTS: Steroidogenesis was disrupted in GCs by the POP mixture, as demonstrated by lower oestradiol and progesterone secretion and greater lipid droplet accumulation. Furthermore, the POP mixture reduced GC viability and increased apoptosis, assessed using caspase-3 activity. The POP mixture significantly increased the number of oocytes that successfully progressed to the second meiotic metaphase and the oocyte reactive oxygen species (ROS) concentration. CONCLUSIONS: Thus, a mixture of POPs that are typically present in human FF has detrimental effects on ovarian function: it reduces the viability of GCs, and increases the oocyte concentrations of ROS. IMPLICATIONS: These results indicate that chronic exposure to POPs adversely affects female reproductive health.

Apelin abrogates the stimulatory effects of 17ß-estradiol and insulin-like growth factor-1 on proliferation of epithelial and granulosa ovarian cancer cell lines via crosstalk between APLNR and ERα/IGF1R.

Apelin and chemerin are adipocytokines that play important roles in many physiological and pathological processes throughout the body. Our previous study demonstrated that these two adipokines are expressed and secreted by epithelial and granulosa cancer cell lines. 17ß-estradiol (E2) and insulin-like growth factor-1 (IGF-1) are important regulators of ovarian functions, and their roles are well known. This study investigated whether apelin and chemerin regulate proliferation and apoptosis of epithelial (OVCAR-3) and granulosa (COV434) ovarian cancer cell lines by interacting with E2 and IGF-1. Apelin and chemerin did not affect caspase-3 activation in either cell line. However, apelin abrogated the stimulatory effects of E2 on proliferation of OVCAR-3 cells and of IGF-1 on proliferation of COV434 cells independently of ERK1/2 and PI3K via crosstalk of apelin receptor with estrogen receptor alpha and IGF-1 receptor, respectively.

Chlorinated biphenyls effect on estrogen-related receptor expression, steroid secretion, mitochondria ultrastructure but not on mitochondrial membrane potential in Leydig cells.

To characterize polychlorinated biphenyls (PCBs) action on Leydig cells, PCBs congeners, low-chlorinated (delor 103; d103) and high-chlorinated ones (delor 106; d106) were selected. The cells were treated according to PCBs dose (d103 or d106 0.2 ng/ml in low doses:, or 2 ng/ml in high doses) and type (d103 + d106 in low doses or 103 + 106 in high doses). After 24 h treatment with PCBs, a distinct increase in estrogen-related receptors (ERRs type α, ß and γ) expression was revealed. However, the dose- and type-dependent PCBs effect was mostly exerted on ERRα expression. A similar increase in ERRs expression was demonstrated by estradiol but not testosterone, which was without an effect on ERRs. PCBs caused no decrease in the membrane potential status of Leydig cells (either in dose or type schedule) but had severe effects on the mitochondria number and structure. Moreover, PCBs markedly increased calcium (Ca2+) concentration and sex steroid secretion (both androgens and estrogens were elevated). These findings suggest a similar estrogenic action of PCBs congeners (d103 and d106) on Leydig cell function. We report dose- and type-specific effects of PCBs only on Leydig cell ERRs expression. Both delors showed common effects on the mitochondria ultrastructural and functional status. Based on our results, ERRα seems to be the most sensitive to hormonal modulation. The increases in Ca2+ and sex steroid secretion may be due to the activation of ERRs by PCBs binding and/or direct effect of PCBs on ERRs mRNA/protein expression. Nevertheless, to confirm the existence of possible relationships between ERRs signaling (including PCBs as ligands) and mitochondria function in Leydig cells, further intensive studies are needed.

Stress differentially affects the systemic and leukocyte estrogen network in common carp.

Both systemic and locally released steroid hormones, such as cortisol and estrogens, show immunomodulatory actions. This research gives evidence that circulating and leukocyte-derived estrogens can be involved in the regulation of the immune response in common carp, during homeostasis and upon restraining stress. It was found that stress reduced level of blood 17ß-estradiol (E2) and down-regulated the gene expression of components of the "classical" estrogen system: the nuclear estrogen receptors and the aromatase CYP19, in the hypothalamus, the pituitary and in the ovaries. In contrast, higher gene expression of the nuclear estrogen receptors and cyp19a was found in the head kidney of stressed animals. Moreover, stress induced changes in the E2 level and in the estrogen sensitivity at local/leukocyte level. For the first time in fish, we showed the presence of physiologically relevant amounts of E2 and the substrates for its conversion (estrone - E1 and testosterone - T) in head kidney monocytes/macrophages and found that its production is modulated upon stress. Moreover, stress reduced the sensitivity of leukocytes towards estrogens, by down-regulation the expression of the erb and cyp19 genes in carp phagocytes. In contrast, era expression was up-regulated in the head kidney monocytes/macrophages and in PBLs derived from stressed animals. We hypothesize that, the increased expression of ERα, that was observed during stress, can be important for the regulation of leukocyte differentiation, maturation and migration. In conclusion, these results indicate that, in fish, the estrogen network can be actively involved in the regulation of the systemic and local stress response and the immune response.

Valproic Acid as a Promising Co-Treatment With Paclitaxel and Doxorubicin in Different Ovarian Carcinoma Cell Lines.

OBJECTIVE: The current preferred treatment of ovarian cancer is combination chemotherapy, usually a platinum-based drug coupled with paclitaxel (PTX). Here, we investigated whether co-treatment with valproic acid (VPA) could increase the efficiency of various ovarian cancer drugs-PTX, doxorubicin (DOX), carboplatin (CBP), and cyclophosphamide (CP)-in different ovarian cancer cell lines. METHODS: Three different ovarian cancer cell lines (OVCAR-3, TOV-21G, and TOV-112D) were treated with chemotherapeutic drugs, alone or in combination with VPA. Cell viability (XTT assay), caspase-3 activity, and the expression of cell cycle- and apoptosis-related genes and proteins were assessed. Furthermore, the effects of these drugs on α-tubulin acetylation and DNA fragmentation were investigated. RESULTS: Paclitaxel and DOX decreased cell viability and increased caspase-3 activity, and co-treatment with VPA enhanced this effect. Carboplatin and CP had no effect. Responses to treatment with PAX and DOX together with VPA on gene expression profile were highly variable and depended on the cell line investigated. However, a common feature in all cell lines was an increased expression of CDKN1A, CCNE1, PARP1, and PARP3. Co-treatment with VPA enhanced the effect of DOX and PAX on most protein expressions investigated in TOV-21G and TOV-112D cell lines, whereas in OVCAR-3, the most effect was seen with DOX with VPA. Valproic acid did not increase PTX-induced α-tubulin acetylation. An additive effect of DOX with VPA on DNA fragmentation was observed in TOV-21G and TOV-112D cell lines but not in the OVCAR-3. CONCLUSIONS: Our results indicate that VPA could be a promising agent in combined anticancer therapy for ovarian cancer, with the combination of VPA and DOX being the most effective. Certainly, additional in vivo and ex vivo experiments are necessary to investigate the molecular mechanisms of action underlying the cellular effects reported here and to study possible clinically relevant effects in ovarian cancer explants.

Effects of bisphenol A and 17ß-estradiol on vascular endothelial growth factor A and its receptor expression in the non-cancer and cancer ovarian cell lines.

Tumours secrete several pro-angiogenic factors, among which vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) are the most extensively studied but not in ovarian cancer cells. The study was designed to investigate the effect of bisphenol A (BPA) (environmental oestrogen) and of 17ß-estradiol (E2) (endogenous estrogen) on the gene (real-time PCR) and protein (Western blotting) expression of VEGF-R2 and VEGF-A in human non-cancer (HOSEpiC) and ovarian cancer cell lines (SKOV-3 and OVCAR-3). In addition, VEGF-A levels were measured in culture supernatants using a colorimetric assay. Cells were exposed to BPA (1, 40 and 100 nM) or 17ß-estradiol (0.1, 10 and 40 nM) for 3 to 48 h. Since differential expression levels of basal oestrogen receptor (ERα and ERß) between non-cancer and cancer cell lines may affect the response to oestrogens, receptor expression was measured both at the gene and protein levels. Basal ERß expression was similar in all cell lines, and ERα expression was significantly higher in the SKOV-3 cell line. Basal VEGF-R2 expression was higher in cancer than non-cancer cell lines, and in contrast, VEGF-A expression was significantly lower in both SKOV-3 and OVCAR-3 cancer cell lines. Exposure of non-cancer cells to BPA and E2 was associated with a significant increase in VEGF-R2 expression but had no effect on VEGF-A expression or secretion. In contrast, exposure of cancer cells to BPA, but not E2, increased VEGF-R2 and VEGF-A expression and secretion. In conclusion, (1) BPA and E2 regulated VEGF-R2 and VEGF-A expression differently in non-cancer and cancer cells, and (2) BPA has a direct stimulatory effect on VEGF-R2 and VEGF-A expression in both, while E2 appears to be uninvolved in the regulation of VEGF-R2 and VEGF-A expression in cancer cells. Graphical Abstract A schematic representation showing BPA and E2 action on VEGF-R2 and VEGF-A expression in non-cancer (HOSEpiC) and cancer cells (SKOV-3, OVCAR-3).

Low androgen/progesterone or high oestrogen/androgen receptors ratio in serous ovarian cancer predicts longer survival.

The treatment of ovarian cancer (OC) remains one of the greatest challenges in gynaecological oncology. The presence of classic steroid receptors in OC makes hormone therapy an attractive option; however, the response of OC to hormone therapy is modest. Here, we compared the expression patterns of progesterone (PGR), androgen (AR) and oestrogen alpha (ERα) receptors between serous OC cell lines and non-cancer ovarian cells. These data were analysed in relation to steroid receptor expression profiles from patient tumour samples and survival outcomes using a bioinformatics approach. The results showed that ERα, PGR and AR were co-expressed in OC cell lines, and patient samples from high-grade and low-grade OC co-expressed at least two steroid receptors. High AR expression was negatively correlated, whereas ERα and PGR expression was positively correlated with patient survival. AR showed the opposite expression pattern to that of ERα and PGR in type 1 (SKOV-3) and 2 (OVCAR-3) OC cell lines compared with non-cancer (HOSEpiC) ovarian cells, with AR downregulated in type 1 and upregulated in type 2 OC. A low AR/PGR ratio and a high ESR1/AR ratio were associated with favourable survival outcomes in OC compared with other receptor ratios. Although the results must be interpreted with caution because of the small number of primary tumour samples analysed, they nevertheless suggest that the evaluation of ERα, AR and PGR by immunohistochemistry should be performed in patient biological material to plan future clinical trials.

Visfatin induces ovarian cancer resistance to anoikis by regulating mitochondrial activity.

PURPOSE: Ovarian cancer is characterized by recurrent peritoneal and distant metastasis. To survive in a non-adherent state, floating ovarian cancer spheroids develop mechanisms to resist anoikis. Moreover, ascitic fluid from ovarian cancer patients contains high levels of visfatin with anti-apoptotic properties. However, the mechanism by which visfatin induces anoikis resistance in ovarian cancer spheroids remains unknown. Here, we aimed to assess wheather visfatin which possess anti-apoptotic properties can induce resistance of anoikis in ovarian cancer spheroids. METHODS: Visfatin synthesis were examined using a commercial human visfatin ELISA Kit. Spheroid were exposed to visfatin and cell viability and caspase 3/7 activity were measured using CellTiter-Glo 3D cell viability assay and Caspase-Glo® 3/7 Assay System. mRNA and protein expression were analyzed by Real-time PCR and Western Blot analysis, respectively. Analysis of mitochondrial activity was estimated by JC-1 staining. RESULTS: First, our results suggested higher expression and secretion of visfatin by epithelial than by granulosa ovarian cells, and in non-cancer tissues versus cancer tissues. Interestingly, visfatin increased the proliferation/apoptosis ratio in ovarian cancer spheroids. Specifically, both the intrinsic and extrinsic pathways of anoikis were regulated by visfatin. Moreover, the effect of the visfatin inhibitor (FK866) was opposite to that of visfatin. Furthermore, both NAMPT and FK866 affected mitochondrial activity in ovarian cancer cells. CONCLUSION: In conclusion, visfatin acts as an anti-apoptotic factor by regulating mitochondrial activity, leading to anoikis resistance in ovarian cancer spheroids. The finding suggest visfatin as a potential novel therapeutic target for the treatment of ovarian carcinoma with peritoneal dissemination.

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells.

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10-250 µM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 µM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.

Bisphenol S and F affect cell cycle distribution and steroidogenic activity of human ovarian granulosa cells, but not primary granulosa tumour cells.

Bisphenol S (BPS) and F (BPF), a new generation of bisphenols (BPs), are the main substitutes for bisphenol A (BPA). Both have been detected in human body fluids. Importantly, bisphenols are structurally similar to oestrogen, the main sex hormone in females. Because bisphenols bind to nuclear oestrogen receptors (ESR1 and ESR2) and to membrane G-coupled receptor 30 (GPR30), they can disrupt ovarian function. Here, we reveal the molecular mechanism underlying the effects of BPS and BPF on the cell cycle and steroidogenesis in the human ovarian granulosa cell (GC) line HGrC1. We show that BPS and BPF arrest GCs at the G0/G1 phase by inducing expression of cyclin D2, an important event that triggers maximal steroid synthesis in response to the BPS and BPF. We used pharmacological inhibitors to show that BPS and BPF, despite acting via already described pathways, also stimulate steroid secretion via IGF1R pathways in HGrC1 cells. Moreover, we identified differences critical to bisphenols response between normal (HGrC1) and primary tumour granulosa (COV434) cells, that enable COV434 cells to be more resistant to bisphenols. Overall, the data suggest that BPS and BPF drive steroidogenesis in human ovarian GCs by affecting the cell cycle. Furthermore, the results indicate that BPS and BPF act not only via the classical and non-classical ESR pathways, but also via the IGF1R pathway.

Bisphenols S and F drive ovarian granulosa cell tumor invasion via a metabolic switch.

Alterations in the metabolism of cancer cells are crucial for tumor growth and progression. However, the mechanism whereby environmental pollutants such as bisphenols F (BPF) and S (BPS) affect glucose metabolism through the glycolytic pathway, and therefore influence tumor progression, are unclear. Both bisphenols are endocrine-disrupting molecules that are used in plastics. As a consequence of their widespread use, these compounds have been detected in various human body fluids. Thus, hormone-sensitive cancers, such as ovarian cancers, are exposed to these compounds. In the present study, we aimed to determine the effects of the concentrations of BPS and BPF found in body fluids on the cell viability, glucose uptake, glycolysis, oxygen consumption, and invasion by the adult ovarian granulosa cell tumor (AGCT) cell line. We found that BPS and BPF increased the glucose uptake, hexokinase activity, proliferation, and invasion of the cells at environmentally relevant concentrations. Furthermore, we identified an inhibition of glycolysis in parallel with an increase in oxygen consumption, suggesting a BPS/BPF-induced switch from aerobic glycolysis to mitochondrial respiration. In summary, these findings demonstrate a new mechanism through which BPS and BPF promote ovarian granulosa cell tumor progression by increasing energy production through mitochondrial respiration. Thus, both bisphenols induced a metabolic switch that appears to be a stimulus for AGCT progression.

Comparison of combinatory effects of PCBs (118, 138, 153 and 180) with 17 beta-estradiol on proliferation and apoptosis in MCF-7 breast cancer cells.

We analyzed whether polychlorinated biphenyls (PCBs) interfere with the activity of 17 ß-estradiol in the proliferation and apoptosis of the MCF-7 cell line. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) without phenol red supplemented with 5% charcoal-treated fetal bovine serum (CD-FBS) for 3 days with 10 nM 17 ß-estradiol or 0.1 µM, 0.5 µM and 1 µM of the tested PCB congeners (118, 138, 153 and 180), or both. Cell proliferation was determined by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation, and cell apoptosis was measured by caspase-9 activity. From the PCB congeners tested, PCB138 and 153 had the highest stimulatory effects on basal cell proliferation as well as the highest inhibitory actions on basal caspase-9 activity. The proliferative and anti-apoptotic actions of PCB138 and 153 were still observed in the presence of 17 ß-estradiol, while the actions of PCB118 and 180 were reversed. In conclusion, the results of this study suggest the possibility that PCB138 and 153 contribute to the action of endogenous 17 ß-estradiol on cell proliferation and apoptosis in the breast cancer cell line MCF-7.

Mixtures of persistent organic pollutants increase ovarian granulosa tumor cell line migration and spheroid invasion by upregulating MMP2 expression and activity via IGF1R.

Granulosa cell tumors (GCT) of the ovary have a good prognosis. Recurrence tends to be late; however, > 66 % of patients with recurrent GCT die from the disease. Most recurrences are abdominopelvic, although distant metastases have been documented. Here, we tested the hypothesis that a mixture of persistent endocrine-disrupting chemicals (EDCs) stimulates the invasion of GCT cells. We selected perfluorooctanoate (PFOA, 2 ng/mL), perfluorooctanesulfonate (PFOS, 8 ng/mL), 2,2-dichlorodiphenyldichloroethylene (p,p'-DDE, 1 ng/mL), polychlorinated biphenyl 153 (PCB153, 100 pg/mL), and hexachlorobenzene (HCB, 50 pg/mL), which have the highest measured concentrations in follicular fluid of women undergoing treatment with assisted reproductive technology. The human GCT cell lines COV434 and KGN have been used as in vitro models of juvenile (JGCT) and adult (AGCT) GCT subtypes, respectively. Cells were treated with a mixture of the test compounds for 15 min prior to analysis of protein phosphorylation; for 4 h prior to analysis in a circular chemorepellent-induced defect assay; for 6 h prior to analysis of matrix metalloproteinase 2 (MMP2) activity; for 24 h prior to analysis of migration, invasion, and gene expression; and for 48 h prior to analysis of protein expression. First, we showed that KGN cells migrated and exhibited invasive behavior. By contrast, COV434 cells lacked migration and invasion potential. Moreover, expression of mesenchymal genes and the gene encoding MMP2 was higher in KGN cells, and that of epithelial genes lower, than that in COV434 cells. Treatment of KGN cells with the EDC mixture stimulated cell migration, invasion, and lymphatic dissemination. The results suggest that the role of the EDC mixture in AGCT invasion is not related to changes in expression of epithelial and mesenchymal genes; rather, it is related to increased expression and activity of MMP2. Additionally, silencing insulin-like growth factor 1 (IGF1R) in AGCT abolished the stimulatory effect of the EDC mixture on KGN spheroid invasion. These results demonstrate that the EDC mixture increased KGN spheroid invasion by stimulating expression and activity of MMP2 via IGF1R.

A mixture of persistent organic pollutants detected in human follicular fluid increases progesterone secretion and mitochondrial activity in human granulosa HGrC1 cells.

Disruption of granulosa cells (GCs), the main functional cells in the ovary, is associated with impaired female fertility. Epidemiological studies demonstrated that women have detectable levels of organic pollutants (e.g., perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, polychlorinated biphenyl 153, and hexachlorobenzene) in their follicular fluid (FF), and thus these compounds may directly affect the function of GCs in the ovary. Considering that humans are exposed to multiple pollutants simultaneously, we elucidated the effects of a mixture of endocrine-disrupting chemicals (EDCs) on human granulosa HGrC1 cells. The EDC mixture directly increased progesterone secretion by upregulating 3ß-hydroxysteroid dehydrogenase (3ßHSD) expression. Furthermore, the EDC mixture increased activity of mitochondria, which are the central sites for steroid hormone biosynthesis, and the ATP content. Unexpectedly, the EDC mixture reduced glucose transporter 4 (GLUT4) expression and perturbed glucose uptake; however, this did not affect the glycolytic rate. Moreover, inhibition of GLUT1 by STF-31 did not alter the effects of the EDC mixture on steroid secretion but decreased basal estradiol secretion. Taken together, our results demonstrate that the mixture of EDCs present in FF can alter the functions of human GCs by disrupting steroidogenesis and may thus adversely affect female reproductive health. This study highlights that the EDC mixture elicits its effects by targeting mitochondria and increases mitochondrial network formation, mitochondrial activity, and expression of 3ßHSD, which is associated with the inner mitochondrial membrane.

Persistent endocrine-disrupting chemicals found in human follicular fluid stimulate IGF1 secretion by adult ovarian granulosa cell tumor spheroids and thereby increase proliferation of non-cancer ovarian granulosa cells.

Insulin-like growth factor-1 (IGF1) is a hormone involved in cell proliferation. We previously showed that IGF1 directly stimulates cell proliferation in granulosa cell tumors (GCTs). Estrogen regulates IGF1 expression in several reproductive organs including the uterus and ovaries. This study aimed to investigate the effects of a mixture of endocrine-disrupting chemicals (EDCs) on secretion of IGF1 by COV434 and KGN cells, which have been used as in vitro models of juvenile and adult GCTs, respectively. The EDC mixture contained perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, hexachlorobenzene, and polychlorinated biphenyl 153, which are persistent hormonally active environmental toxicants present in ovarian follicular fluid (FF). Expression and secretion of IGF1 were significantly higher in GTCs than in HGrC1 human non-cancer granulosa cells (with the profile HGrC1 < COV434 < KGN). Treatment with the EDC mixture as well as individual test compounds significantly increased IGF1 secretion in KGN cells. Moreover, IGFBP3 gene expression in KGN cells was downregulated after treatment with the EDC mixtures. The estrogen receptor alpha pathway was involved in this effect. Conditioned medium of KGN cells treated with the EDC mixture increased proliferation of HGrC1 human non-cancer granulosa cells. These results indicate that the mixture of EDCs found in FF increases secretion of IGF1 by KGN cells and thus indirectly contributes to progression of adult GCTs, and increases proliferation of non-cancer granulosa cells.

Disruption of 17ß-estradiol secretion by persistent organic pollutants present in human follicular fluid is dependent on the potential of ovarian granulosa tumor cell lines to metabolize estrogen.

Endocrine-disrupting chemicals (EDCs), such as perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, hexachlorobenzene, and polychlorinated biphenyl 153 are persistent pollutants that are found in human follicular fluid (FF). These compounds may affect endocrine function, disrupt steroid secretion by granulosa cells, and play a role in granulosa cell tumor (GCT) development. GCTs demonstrate endocrine activity, expressing aromatase and secreting 17ß-estradiol (E2). We aimed to determine the effects of a mixture of EDCs, similar to that found in human FF, on human granulosa tumor cell lines representing the juvenile (JGCT) and adult (AGCT) forms (COV434 and KGN cells, respectively). We found that all the individual compounds and mixtures tested altered granulosa tumor cell function by disrupting E2 secretion. In KGN cells, which possess significantly higher basal aromatase gene expression, and therefore secrete more E2 than JGCT cells, EDC mixtures activated estrogen receptors (ERs) and G protein-coupled receptor-30 signaling, thereby stimulating E2 secretion, without affecting aromatase expression. By contrast, in COV434 cells, which demonstrate higher CYP1A1 expression, a key mediator of estrogen metabolism, than KGN cells, EDC mixtures reduced E2 secretion in parallel with increases in the 2-hydroxyestrogen 1/E2 ratio and CYP1A1 expression, implying an upregulation of E2 metabolism. These results indicate that the EDC mixture present in FF disrupts E2 secretion in JGCT and AGCT cells according to the estrogen metabolic potential of the cell type, involving both classical and non-classical ER pathways.

Persistent endocrine-disrupting chemicals found in human follicular fluid stimulate the proliferation of granulosa tumor spheroids via GPR30 and IGF1R but not via the classic estrogen receptors.

Epidemiological studies have found that women have detectable levels of organic pollutants such as hexachlorobenzene (HCB), 2,2-dichlorodiphenyldichloroethylene (p,p'-DDE), polychlorinated biphenyl 153 (PCB153), perfluorooctanoate (PFOA), and perfluorooctane sulfonate (PFOS) in their follicular fluid. Thus, these compounds may directly affect the function of granulosa cells within the ovary and may promote granulosa cell tumor (GCT) progression. Two human GCT cell lines, COV434 and KGN, have been used as in vitro model systems to represent juvenile (JGCT) and adult (AGCT) GCT subtypes, respectively. In this study, we found that basal expression of estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), and insulin-like growth factor 1 receptor (IGF1R) was higher in the AGCT subtype than in the JGCT subtype. All of the compounds acted as mitogenic factors at low nanomolar concentrations in the JGCT and AGCT forms of GCT. Interestingly, PFOA, PFOS, and HCB stimulated cell proliferation through IGF1R, whereas p,p'-DDE acted through GPR30. Moreover, a mixture of the five compounds also significantly stimulated granulosa cell proliferation; however, the observed effect was lower than predicted. Interestingly, the proliferative effect of a mixture of these compounds was dependent on IGF1R and GPR30 but independent of the classic estrogen receptors. Taken together, our results demonstrate for the first time that mixtures of persistent organic pollutants present in follicular fluids may induce granulosa tumor progression through IGF1R and GPR30 by acting as mitogenic factors in granulosa cells.

Action of defined mixtures of PCBs, p,p'-DDT and its metabolite p,p'-DDE, on co-culture of porcine theca and granulosa cells: steroid secretion, cell proliferation and apoptosis.

We recently reported on a stimulatory effect of a mixture of persistent organic pollutants (POPs) extracted from the liver oil of Atlantic cod (Gadus morhua) on estradiol (E2) secretion by porcine ovarian follicular cells [Gregoraszczuk EL, Milczarek K, Wójtowicz A, Berg V, Skaare JU, Ropstad E. Steroid secretion following exposure of ovarian follicular cells to three different natural mixtures of persistent organic pollutants (POPs). Reproductive Toxicol 2007;25:58-66]. The objective of the present study was to identify what compound or compounds within this natural mixture could be responsible for the stimulatory action on E2 secretion. Co-cultures of porcine ovarian theca and granulosa cells were exposed for 48h to individual congeners of polychlorinated biphenyls (PCBs) alone or in combination, at environmentally relevant concentrations (PCB153 at 8microg/ml; PCB118 at 3microg/ml; PCB138 at 8microg/ml; PCB180 at 3microg/ml; PCB126 at 8ng/ml; DDT at 5microg/ml; p,p'-DDE at 10microg/ml). At the end of culture media samples were collected and used for cell viability and steroid determination, and cells were used for the measurement of caspase-3 activity. A threefold decrease in E2 secretion was noted when PCB126 was added in combination with PCB153. Only DDT and DDE could reverse the action of PCB126. Stimulatory secretion of E2 occurred in parallel with a decrease in testosterone (T) secretion. Among the single congeners used in the present study, DDE had the strongest stimulatory action on E2 secretion. In conclusion, the present findings indicate that the overall effect on steroid secretion by ovarian follicular cells could be dependent on the concentration of DDE in environmental POPs and that the predicted effect of a mixture based on a sum of independent effects of individual congeners is higher than that noted under the influence of defined mixture.

Bisphenol A and its derivatives decrease expression of chemerin, which reverses its stimulatory action in ovarian cancer cells.

Chemerin is an adipocyte-secreted protein that associates with obesity, inflammation, metabolic dysfunction, and carcinogenesis. Previous studies have shown human granulosa cells to produce bioactive chemerin and its receptor CMKLR1. In the present study, we demonstrated that the mRNA level of chemerin receptor is higher in a granulosa cell tumor cell line than in epithelial cancer cells, whereas chemerin expression and secretion were lower. Various exogenous factors, such as bisphenol A and its halogenated derivatives tetrabromobisphenol A and tetrachlorobisphenol A, can affect adipokine expression. For this reason, we investigated the effects of bisphenol A and its derivatives on the expression of chemerin and its receptor. At low nanomolar concentrations, BPA, TBBPA, and TCBPA decreased chemerin expression and secretion only in granulosa cell tumor COV434 cells by both peroxisome proliferator-activated receptor γ and estrogen receptor signaling pathways. Chemerin treatment had no effect on proliferation of ovarian non-cancer and cancer cell lines. However, we also found evidence to support the inhibition of BPA- and TBBPA-induced cell proliferation by chemerin. Taken together, our results indicate for the first time that BPA and its derivatives down-regulate chemerin expression, which can suppress the ability of BPA to induce proliferation. Moreover, both PPARγ and ERs were involved in the BPA-induced decrease in chemerin expression, and its ratio was crucial to exert these effects.