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.

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.

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.

Adiponectin Reverses the Proliferative Effects of Estradiol and IGF-1 in Human Epithelial Ovarian Cancer Cells by Downregulating the Expression of Their Receptors.

The expression of adiponectin receptors AdipoR1 and AdipoR2 has been reported in the human ovary and ovarian cancer tissues. Moreover, adiponectin has been reported to act as an anti-tumor factor by inhibiting cancer cell proliferation. Thus, we investigate whether adiponectin and its receptors influence ovarian cancer development. In the present study, we found that adiponectin was not expressed in the granulosa cell line (COV434), and epithelial ovarian cancer cell lines (OVCAR-3, SKOV-3, and Caov-3). Additionally, we found that AdipoR1 and AdipoR2 expression is lower in epithelial ovarian cancer cells than in granulosa tumor cells. Endogenous 17ß-estradiol as well as exogenous estrogens, such as bisphenol A and its chlorinated and brominated analogs do not affect adiponectin receptor expression. We found that adiponectin inhibited the growth of OVCAR-3 and SKOV-3 cells, and that this effect was independent of apoptosis. Moreover, adiponectin reverses the stimulatory effects of 17ß-estradiol and insulin-like growth factor 1 on cell proliferation by downregulating the expression of their receptors, whereas progesterone increased the sensitivity of cancer cells to adiponectin by upregulating AdipoR1 and AdipoR2 expression. These results suggest interactions between adiponectin and various ovarian steroid hormone and growth factor pathways in ovarian cancer cells.

Stimulation of ovarian cell proliferation by tetrabromobisphenol A but not tetrachlorobisphenol A through G protein-coupled receptor 30.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) are bisphenol A (BPA) analogs, where the phenolic moieties are substituted with halogens (Br or Cl). Previous studies indicate that BPA has significant proliferative effects on in vitro cultured epithelial ovarian cancer (EOC) cells. Considering this, we analyzed the effects of both TBBPA and TCBPA at 1, 10, and 50nM on ovarian cancer cell proliferation. The majority of malignant ovarian tumors are epithelial in origin, but approximately 10% are classified as ovarian sex cord tumors, with the most common type being granulosa cell tumors (GCTs). OVCAR-3 and KGN cells were used as in vitro models to represent EOCs and GCTs, respectively. Here, we found that TBBPA, but not TCBPA, stimulated OVCAR-3 and KGN cell proliferation, with lower potency than BPA. The stimulatory effects of TBBPA and BPA on cell proliferation were reversed by pre-treatment with a G protein-coupled receptor 30 (GPR30) antagonist in both cell lines, which possess similar basal GPR30 expression levels. Taken together, our results show for the first time that TBBPA, which has lower potency than BPA, stimulates ovarian cancer cell proliferation through the GPR30 pathway.