Vitamin C supplementation had no side effect in non-cancer, but had anticancer properties in ovarian cancer cells.

Vitamin C (Vit C) has been widely used in the treatment and prevention of cancer. Nevertheless, the clinical results are still inconclusive. Using non-cancer (HOSEpiC) and cancer OVCAR-3 cells cultured in basal medium or in ovarian cancer-associated fibroblast (CAF)-supplemented medium, we estimated the dose-dependent effect of Vit C on sodium-ascorbate co-transporters (SVCT1, SVCT2) and glucose transporter (GLUT1) protein expression. Additionally, the action of Vit C on cell proliferation (alamarBlue), membrane permeability (LDH assay), caspase3 activity, the selected cell cycle and apoptosis pathway, poly(ADP-ribose) polymerase-1 (PARP) protein expression, and reactive oxygen species (ROS) activity was determined. We showed different effects of Vit C on the expression of the co-transporter in non-cancer and cancer cells. In non-cancer cells, Vit C, at a pharmacological concentration, increased SVCT2 and decreased GLUT1, while the opposite effect was noted in cancer cells. In cancer cells, Vit C, in a pharmacological dose, decreased cell proliferation through an inhibitory effect on cyclin-dependent kinase 2 (CDK2) (4.4-fold; p < 0.01), mainly due to the stimulatory effect on the expression of cyclin-dependent kinase (CDK) inhibitors, such as p21 and p53 (3.2- and 2.8-fold, respectively; p < 0.001), but not caspase pathway. The tumour microenvironment caused inefficiency of the lower doses of Vit C in ovarian cancer cells. At a pharmacological dose of 1 mM, Vit C decreased PARP expression (1.5-fold; p < 0.05). We suggest that it's nontoxic effects on non-cancer cells may be an indicator of its prophylactic use, while in a pharmacological dose Vit C should be considered a possible adjunctive drug in ovarian cancer. However, it is necessary to consider the effect of the CAF.

Vaspin in the pig ovarian follicles: expression and regulation by different hormones.

Vaspin, also known as visceral adipose tissue-derived serine protease inhibitor, is a member of the serine protease inhibitor family. Its expression is associated with obesity, insulin resistance and type 2 diabetes, and elevated concentration is observed in polycystic ovary syndrome. However, vaspin has never been studied in the ovary. Here, we identified vaspin in two prolific breeds of pigs: fat Meishan (MS) and lean Large White (LW). We then investigated the molecular mechanism involved in the regulation of its expression in response to gonadotropins, insulin, insulin-like growth factor type 1 (IGF-1) and steroids (progesterone, testosterone and oestradiol) in ovarian follicles cells. Using real-time PCR and Western blot, we found higher vaspin mRNA and protein expression in the ovarian follicles and adipose tissue at 10-12 days of the oestrous cycle in MS compared to LW. Moreover, vaspin expression, as well as its concentration in plasma and follicular fluid, decreased in ovarian follicles of LW during days of the oestrous cycle, while the opposite results were noted in MS. Immunohistochemistry showed vaspin in granulosa, theca, cumulus cells and oocytes as well as in adipocytes. Vaspin level in the ovary increased by gonadotropin, insulin, IGF-1 and steroids stimulation through kinases JAK/Stat, ERK1/2, PI3K and AMPK, as well as factor NF-κB. These findings all show vaspin expression and regulation in the pig ovary, indicating vaspin as a new regulator in female reproduction. Future studies will be necessary for understanding the role of vaspin on ovarian physiology providing new insights into the pathology of ovaries.

Maternal High-Fat Diet During Pregnancy and Lactation has Opposite Effects on Gonadal Expression of Leptin and Leptin Receptor in Rat Dams and Their Offspring.

This study investigated the effect of a high-fat (HF) diet on protein expression of leptin and its receptor in the gonads of dams and their offspring. Female Wistar rats were fed a HF diet (30% fat) or a standard breeding (BD) diet (5% fat) during pregnancy and lactation. At 21 days of lactation, mothers and both sexes of prepubertal offspring were killed by decapitation. The protein expression of leptin and its receptor was assayed by Western blot and immunohistochemistry in gonadal, periovarian, and epididymal white adipose tissue (WAT). We demonstrated that leptin protein expression in ovary, and both leptin and ObRb expression in periovarian WAT was decreased in HF dams compared with BD animals. Immunohistochemistry showed lower leptin expression in growing antral follicles and corpora lutea of HF dams. Conversely, in both gonads and epididymal WAT of HF offspring, leptin and its receptor were significantly higher expressed compared with BD. Immunolocalization of leptin system in HF offspring gonads showed higher expression in growing and antral follicles of the ovary, seminiferous tubules, and interstitial tissue of testes. In conclusion, high gonadal and gonadal-WAT expression of leptin system was observed in the offspring of dams fed a HF diet during pregnancy and lactation.

Differences in the mechanisms of action of BDE-47 and its metabolites on OVCAR-3 and MCF-7 cell apoptosis.

Data concerning possible carcinogenic action of polybrominated diphenyl ethers (PBDEs) in hormone-dependent tissues are limited. Our earlier studies showed that 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) stimulated OVCAR-3 and MCF-7 cell proliferation, while its hydroxylated metabolites (5-OH-BDE-47 and 6-OH-BDE-47) increased estrogen receptors protein expression and extracellular signal-regulated kinase 1/2 and protein kinase Cα phosphorylation in these cell lines. In addition to cell proliferative disorder, a failure in the regulation of apoptosis can also lead to the formation and development of tumors. Therefore, in the present study, we investigated the effect of BDE-47 and its metabolites (2.5-50 ng ml-1 ) on the expression of apoptosis regulatory genes and proteins, caspase-8 and -9 activity and DNA fragmentation induced by extracellular signal-regulated kinase inhibitor (PD098059) and protein kinase Cα inhibitor (GÓ§ 6976) in ovarian (OVCAR-3) and breast (MCF-7) cancer cells. In OVCAR-3 cells, BDE-47 upregulated expression of most of the investigated genes and increased protein expression of tumor necrosis factor (TNF)-α, TNF receptor 1, caspase-6, Bcl-xl and caspase-8 activity. Whereas in MCF-7 cells, BDE-47 resulted in the downregulation of most of the investigated genes, and decreased caspase-8 and -9 activity. In both OVCAR-3 and MCF-7 cells, the expression of most of the investigated genes were downregulated by metabolites. Exposure of OVCAR-3 cells to 5-OH-BDE-47 corresponded with a decrease in the protein expression of caspase-6, caspase-9 and Bcl-xl and treatment with 6-OH-BDE-47 decreased Bcl-xl and TNF receptor 1 expression in OVCAR-3 cells and caspase-9 expression in MCF-7 cells. Hydroxylated metabolites of BDE-47 have strong inhibitory effects on apoptosis in ovarian and breast tumor cells and thus should be considered potential carcinogens in hormone-dependent cancers. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Different mechanisms of action of 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47) and its metabolites (5-OH-BDE-47 and 6-OH-BDE-47) on cell proliferation in OVCAR-3 ovarian cancer cells and MCF-7 breast cancer cells.

Data concerning the possible action of polybrominated diphenyl ethers (PBDEs) in hormone-dependent cancer are scarce. Some data showed that PBDEs may directly affect breast cancer cells formation and only one research showed increased proliferation of the OVCAR-3 cells, but the results are ambiguous and the mechanisms are not clear. There is growing evidence that not only parent compounds but also its metabolites may be involved in cancer development. The present study was, therefore, designed to determine the effect of BDE-47 and its metabolites (2.5 to 50 ng ml-1 ) on proliferation (BrdU), cell-cycle genes (real-time PCR) and protein expression (Western blot), protein expression of oestrogen receptors (α ß), extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase Cα (PKCα) in OVCAR-3 ovarian and MCF-7 breast cancer cells. In OVCAR-3 cells, the parent compound stimulated cell proliferation by activating CDK1, CDK7, E2F1 and E2F2. Independent of time of exposure, BDE-47 had no effect on ERα and ERß protein expression and ERK1/2 and PKCα phosphorylation. Metabolites had no effect on cell proliferation but increased both ERs protein expression and ERK1/2 and PKCα phosphorylation. In MCF-7 cells, the parent compound displayed no effect on cell proliferation but decreased ERα and increased ERß protein expression with concomitant induction of PKCα phosphorylation. Both metabolites increased MCF-7 cell proliferation, ERK1/2 and PKCα phosphorylation and decreased ERα and ERß protein expression.We suggest that studies concerning PBDEs with fewer bromine atoms should be continued to understand environmental links to different hormone-dependent cancers. Copyright © 2016 John Wiley & Sons, Ltd.

Regulatory Role of Gonadotropins and Local Factors Produced by Ovarian Follicles on In Vitro Resistin Expression and Action on Porcine Follicular Steroidogenesis.

Resistin, a hormone secreted by adipocytes, is thought to be important in reproduction. Our previous study demonstrated resistin expression in porcine ovarian follicles and its direct effect on steroidogenesis. The aim of the current study was to evaluate the effect of gonadotropins and the local ovarian factors, such as insulin-like growth factor type 1 (IGF1) and steroids (progesterone, testosterone, and 17 beta-estradiol), on the expression and secretion of resistin, as well as its steroidogenic action. Porcine ovarian follicles were exposed to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at 50-150 ng/ml, IGF1 (10-100 ng/ml), and steroids at 10(-8) to 10(-6) M for 24 h. Then, mRNA, protein expression, and medium concentration of resistin were determined using real-time PCR, Western blot analysis, and ELISA, respectively. In the subsequent experiments, ovarian follicles were exposed to resistin and/or FSH, LH, IGF1, and steroids, and ovarian steroidogenesis was analyzed. Additionally, we examined the direct effect of resistin on the protein expression of receptors for gonadotropins and investigated local factors. The results showed that gonadotropins and steroids have stimulatory effects but that IGF1 has an inhibitory effect on resistin expression and secretion. Resistin decreased gonadotropins and local hormone-induced steroid secretion and inhibited 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase protein expression. Additionally, we demonstrated that resistin increased the expression of receptors for progesterone and testosterone. These findings all show that the expression and function of resistin are regulated by gonadotropins and local factors produced by ovarian follicles.

Resistin is a survival factor for porcine ovarian follicular cells.

Previously, we demonstrated the expression of resistin in the porcine ovary, the regulation of its expression and its direct effect on ovarian steroidogenesis. The objective of this study was to examine the effect of resistin on cell proliferation and apoptosis in a co-culture model of porcine granulosa and theca cells. First, we analysed the effect of resistin at 1 and 10  ng/ml alone or in combination with FSH- and IGF1 on ovarian cell proliferation with an alamarBlue assay and protein expression of cyclins A and B using western blot. Next, the mRNA and protein expression of selected pro-apoptotic and pro-survival regulators of cell apoptosis, caspase-9, -8 and -3 activity and DNA fragmentation using real time PCR, western blot, fluorescent assay and an ELISA kit, respectively, were analysed after resistin treatment. Furthermore, we determined the effect of resistin on the protein expression of ERK1/2, Stat and Akt kinase. Using specific inhibitors of these kinases, we also checked caspase-3 activity and protein expression. We found that resistin, at both doses, has no effect on cell proliferation. The results showed that resistin decreased pro-apoptotic genes, which was confirmed on protein expression of selected factors. We demonstrate an inhibitory effect of resistin on caspase activity and DNA fragmentation. Finally, resistin stimulated phosphorylation of the ERK1/2, Stat and Akt and kinases inhibitors reversed resistin action on caspase-3 activity and protein expression to control. All of these results showed that resistin has an inhibitory effect on porcine ovarian cell apoptosis by activation of the MAPK/ERK, JAK/Stat and Akt/PI3 kinase signalling pathways.

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).

The toxicological effects of halogenated naphthalenes: a review of aryl hydrocarbon receptor-mediated (dioxin-like) relative potency factors.

There is no doubt that chloronaphthalenes (PCNs) and their brominated counterparts (PBNs) are dioxin-like compounds, but there is less evidence for mixed bromo/chloronaphthalenes (PXNs). In this article we review information relating to the dioxin-like potency of PCNs and PBNs obtained in vivo, in vitro, and in silico. The aim was to help and improve the quality of data when assessing the contribution of these compounds in the risk analysis of dioxin-like contaminants in foods and other sample types. In vivo and in vitro studies have demonstrated that PCN/PBN congeners are inducers of aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase, and luciferase enzymes that are features specifically indicative of planar diaromatic halogenated hydrocarbons such as dioxin and dioxin-like compounds. PCNs in the environment are of multisource origin. The limited data on PBNs in the environment suggest that these also appear to originate from different sources. The toxicological data on these compounds is even scarcer, most of it directed toward explaining the exposure risk from accidental contamination of feed with the commercial PBN containing product, Firemaster BP-6. The occurrence of PBNs and PXNs is possible as ultra-trace environmental and food-chain contaminants produced at least from combustion processes at unknown concentrations. Available toxicological and environmental data enable a focus on PCNs as dioxin analogues to an extent that specific local or regional environmental influences could result in a risk to human health. There is the possibility that they may act synergistically with the better-known classic dioxin and other dioxin-like compounds. PBNs and PXNs are much less studied than the dioxins, but are known to be products of anthropogenic processes that contaminate the environment. A continuously increasing use of bromine for manufacture of brominated flame retardants over the past three decades is anticipated as a stream of "brominated" wastes, that when degraded (combusted), will release PBNs and PXNs. This calls for advanced analytical methods and greater interest toxicologically to understand and control pollution and exposure by PBNs and PXNs. Particular congeners of bromonaphthalene in single studies were found to be much more toxic than their chlorinated counterparts. In addition, brominated/chlorinated naphthalenes also seem to be more potent toxicants than PCNs. About 20% of PCN congeners exhibit a dioxin-like toxicity with relative potencies varying between around 0.003 and 0.000001, but additional and more rigorous data are needed to confirm these figures. Recent food surveys have estimated a small but relevant human exposure to these compounds in foods, giving an additional source of dioxin-like toxicity to those compounds already covered by the World Health Organization-Toxic Equivalency Factors (TEFs) scheme. Given the additivity of response postulated for other dioxin-like compounds, it would seem unwise to ignore this additional contribution. Few data available showed that PBN congeners also exhibit a dioxin-like toxicity and are even more potent than PCN congeners, but the relative potency values were not derived for them until now. There are no toxicological data available for PXNs.

Differential effect of methyl-, butyl- and propylparaben and 17ß-estradiol on selected cell cycle and apoptosis gene and protein expression in MCF-7 breast cancer cells and MCF-10A non-malignant cells.

Parabens are alkyl esters of p-hydroxybenzoic acid used widely as antimicrobial preservatives in consumer products, including pharmaceuticals, foods and cosmetics. We showed previously that methyl-, butyl- and propylparaben parabens, even at low doses, stimulate the proliferation of MCF-7 breast cancer cells and non-transformed MCF-10A breast epithelial cells. The present study was undertaken to determine whether this represents a direct effect on cell cycle and apoptotic gene expression. MCF-7 and MCF-10A cells were exposed to methyl, butyl- and propylparaben (20 nm) or 17ß-estradiol (10 nm). Cell cycle and apoptotic gene expression were evaluated by real-time polymerase chain reaction and protein expression by Western blot. 17ß-estradiol upregulated G1 /S phase genes and downregulated cell cycle progression inhibitors in both MCF-7 and MCF-10A. Upregulation of Bcl-xL and downregulation of caspase 9 was observed in MCF-7, while upregulation of Bcl-xL, BCL2L2 and caspase 9 was noted in MCF-10A. Cyclins in MCF-7 cells were not affected by any of the parabens. Methyl- and butylparaben had no effect on the expression of selected apoptotic genes in MCF-7. In MCF-10A, all parabens tested increased the expression of G1 /S phase genes, and downregulated cell cycle inhibitors. Methylparaben increased pro-survival gene. Butylparaben increased BCL2L1 gene, as did 17ß-estradiol, while propylparaben upregulated both the extrinsic and intrinsic apoptotic pathways. There are differences in cell cycle and apoptosis gene expression between parabens and 17ß-estradiol in MCF-7 cells. In MCF-10A cells, most of the genes activated by parabens were comparable to those activated by 17ß-estradiol.

[Epilepsy, antiepileptic drugs and disorders of reproductive functions of women]. / Padaczka, stosowane leki a zaburzenia funkcji rozrodczych kobiet.

Epilepsy is one of the commonest chronic neurological disorders in developing countries. The disease itself and applied antiepileptic drugs cause fertility problems. As a result of seizures changes in hypothalamic and pituitary hormone secretion occur, resulting in hyperprolactinemia, menstrual disorders, premature ovarian failure as well as occurrence of polycystic ovary syndrome (PCOS). PCOS is the main problem in women with epilepsy caused by both the disease itself as well as treatment, mainly with valproic acid. Antiepileptic drugs exert an effect mainly on the level of active hormones, their synthesis in the ovaries and binding with sex hormone binding globulin. Due to impaired reproductive function, the probability of pregnancy in women with epilepsy is up to 2-fold lower than in healthy ones. It should be, however, consider that antiepileptic drugs cross the placenta, therefore it is very important to choose the appropriate treatment, not only to prevent epileptic seizures during pregnancy, but also not harmful to the developing fetus.

Supraphysiological leptin levels shift the profile of steroidogenesis in porcine ovarian follicles toward progesterone and testosterone secretion through increased expressions of CYP11A1 and 17b-HSD: a tissue culture approach.

Evidence from both clinical and animal studies suggests that exposure to excess androgens results in cyst formation. The present in vitro study assessed the effects of supraphysiological concentrations of leptin (20 and 40 ng/ml) on progesterone (P(4)), androstenedione androstendione (A(4)), testosterone and estradiol (E(2)) secretion by ELISA and the expression of CYP11A1, CYP17, 17b-hydroxysteroid dehydrogenase (17b-HSD) and CYP19 by western blot to answer the question of whether leptin could be independent risk factor for cystformation in pigs. Small- and medium-sized ovarian follicles were collected from prepubertal and cycling pigs. Increased P(4) and testosterone secretions were observed in both small- and medium-sized follicles in prepubertal and cycling animals whereas there was no change in E2 secretion. Leptin treatment resulted in an increase in CYP11A1 and 17b-HSD protein expression but had no effect on CYP17 and CYP19 expression in follicles of either size from prepubertal and cycling pigs. Results of presented data suggest that leptin in elevated doses, by stimulatory effect on CYP11A1 and 17b-HSD protein expression resulting in elevated P(4) and testosterone secretions could be an independent risk factor for cyst formation in both prepubertal and cycling pigs.

Effects of resistin on porcine ovarian follicle steroidogenesis in prepubertal animals: an in vitro study.

BACKGROUND: Resistin was first reported to be an adipocyte-specific hormone, but recent studies have indicated a connection between resistin and reproductive function. However, it is not yet known if resistin is expressed by the ovary and if it can affect steroidogenesis in ovarian follicles from prepubertal pigs. METHODS: In this study, using real time PCR, immunoblotting, and ELISA, we quantified resistin expression and concentration in maturing ovarian follicles (small, 3-4 mm; medium, 4-5 mm; large, 6-7 mm) collected from prepubertal pigs. In addition, the dose-responsive effects of recombinant human resistin (0.1, 1, 10, and 100 ng/ml) on steroid hormone (i.e., progesterone [P4], androstendione [A4], testosterone [T], and estradiol [E2]) secretion in culture medium and steroidogenic enzyme (i.e., CYP11A1, 3betaHSD, CYP17A1, 17betaHSD, and CYP19A1) expression in ovarian follicles were determined. RESULTS: We observed that resistin gene and protein expression increased significantly (P < 0.05) during follicular growth, with large follicles expressing the highest level of this adipokine. Recombinant resistin also increased P4, A4, and T secretion by up-regulating the steady state levels of CYP11A1, 3betaHSD, CYP17A1, and 17betaHSD. Recombinant resistin had no effects on E2 secretion and CYP19A1 expression in ovarian follicles. CONCLUSION: Our results show resistin expression in ovarian follicles from prepubertal pigs for the first time. We also show that recombinant resistin stimulates steroidogenesis in ovarian follicles by increasing the expression of CYP11A1, 3betaHSD, CYP17A1, and 17betaHSD. The presence of resistin in the porcine ovary and its direct effects on steroidogenesis suggest that resistin is a new regulator of ovary function in prepubertal animals.

Polybrominated diphenylethers (PBDEs) act as apoptotic factors in the corpus luteum in addition to having a short-term stimulatory effect on progesterone secretion by luteal cells.

To the best of our knowledge, there is a lack of data showing effect of polybrominated diphenyl ethers (PBDEs) on the corpus luteum (CL), a mini-endocrine gland responsible for a normal estrous cycle and the maintenance of pregnancy. Luteal cells obtained from corpora lutea (8-10 days after ovulation) were exposed to PBDE 47, 99, and 100 at doses of 50, 250, and 500 ng/ml for 24 and 48 hours. The progesterone (P4) level in the culture medium and caspase-3, -8, and -9 activities in the cells were estimated by ELISA. CYP11A1 and 3ß-HSD protein expression were evaluated by western blot. A 2-fold increase in P4 secretion after 24 hours and no effect after 48 hours of exposure were observed. We demonstrated that the increase in P4 secretion was the result of the stimulatory action of all PBDEs on 3ß-HSD protein expression and additionally, PBDE 99 alone on 3ß-HSD activity (measured by the conversion of P5 into P4). In contrast, the activation of caspase-8 and -9 but not caspase-3 during 24 hours of exposure, and activation of all investigated caspases during 48 hours was observed. In conclusion, the present findings provide evidence that despite the initial stimulatory effect of PBDEs on the secretion of progesterone (due to the fact that the biochemical apparatus responsible for the conversion of cholesterol into pregnenolone remains uninterrupted). PBDEs are also a key executor of apoptosis (by activating both the extrinsic and intrinsic pathways of apoptosis after longer exposure periods) which can lead to premature dysfunction of the corpus luteum.

Overexpression of P53 protein and local hGH, IGF-I, IGFBP-3, IGFBP-2 and PRL secretion by human breast cancer explants.

BACKGROUND: Insulin-like growth factor-I (IGF-I) in concert with insulin-like binding protein 3 (IGFBP-3), insulin-like binding protein 2 (IGFBP-2), human growth hormone (GH) and P53 protein is involved in autocrine/paracrine growth signaling pathways as an adaptive response to environmental stimuli. OBJECTIVE: The study evaluated the local secretion of PRL, hGH, IGF-I, IGFBP-2 and IGFBP-3 by breast cancer tissue explants in relation to the overexpression of P53 protein in breast cancer tissue. MATERIALS AND METHODS: Breast cancer explants were obtained during radical mastectomies. The overexpression of P53 protein was assessed immunohistochemically using monoclonal antibody (DAKO, Anti-Human P53 protein, clone DO-7); the results of the reaction were stratified into 5 groups. The lack of P53 protein overexpression was defined as 0% of cells that overexpressed P53 protein. IGF-I, IGFBP-3, IGFBP-2, and hGH levels were measured with RIA kits, and prolactin was measured with the MEIA kit. RESULTS: The local secretion of hGH by tumour explants - presenting a positive immunohistochemical reaction (IHCR) to the product of P53 gene - was twice as high as those with no IHCR to product of P53 gene; the opposite was noted in the case of IGF-I, IGFBP-2 and IGFBP-3 secretion. In both cases, the level of hGH, IGF-I and IGFBP-3 secretion did not correlate with the ratio of cells overexpressing P53 protein. There was a significant decrease in local, basic IGFBP-2 secretion along with an increased ratio of cells with positive IHCR to product of P53 gene. Furthermore, local PRL secretion was not correlated with the ratio of cells overexpressing P53 protein in breast cancer tissue. Prolactin also exerts no influence on IGF-I secretion. CONCLUSION: Our results may suggest the presence of local hGH/IGF-I feedback in breast tissue as well as the possibility of P53/hGH/IGF-I/IGFBP-3 but not P53/PRL/IGF-I axis.

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.

Effect of ghrelin on proliferation, apoptosis and secretion of progesterone and hCG in the placental JEG-3 cell line.

To determine the effect of ghrelin on placental cell proliferation, apoptosis and hormone secretion we cultured human JEG-3 cells with 100, 250, 500 or 1000 pg/ml of ghrelin for 48 hours. Ghrelin stimulated cell proliferation and decreased caspase-3 activity. All of the investigated ghrelin concentrations decreased progesterone (P(3)) but had no effect on human chorionic gonadotrophin (hCG) secretion. Stimulatory effects on cell proliferation paralleled inhibitory effects on cell apoptosis suggesting a possible role for ghrelin in placental formation or remodeling.

Effect of pituitary growth hormone and insulin-like growth factor type-I on proliferation, apoptosis and hormone secretion of the placental cell line JEG-3.

OBJECTIVE: Placenta human choriocarcinoma JEG-3 cells were used to study the possibility that pituitary growth hormone (GH) and insulin-like growth factor type I (IGF-I) act on first trimester of pregnancy progesterone (P4) and human chorionic gonadotropin (hCG) secretion, cell proliferation and apoptosis. MATERIAL AND METHODS: The JEG-3 cell line was cultured in Dulbecco modified eagle medium without phenol red containing 10% FBS. The cells were plated in 96-well plates at the density of 3 x 10(3) for 24 h and treated with 10, 50, 100, or 200 ng/ml of GH or 10, 30, 100, or 250 ng/ml of IGF-I for 24 h. At the end of the culture period, cell proliferation was measured using the BrdU colorimetric assay and caspase-3 activity, as a marker of apoptosis, was determined by the colorimetric method. Media were frozen for hormone analysis by enzyme immunoassay. Results. We found that the stimulatory activities of GH and IGF-I on both P4 and hCG secretion paralleled the stimulation of cell proliferation and inhibition of apoptosis. CONCLUSION: Our findings suggest an important role for pituitary GH and IGF-I in normal placental function during the first trimester of pregnancy.