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.

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.

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.

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.

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.

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.

"Real life" polycyclic aromatic hydrocarbon (PAH) mixtures modulate hCG, hPL and hPLGF levels and disrupt the physiological ratio of MMP-2 to MMP-9 and VEGF expression in human placenta cell lines.

Using JEG-3 and BeWo cells, we examined the effect of "real life" mixtures of polycyclic aromatic hydrocarbons (PAHs), at doses reported in maternal blood (Mix I) and in placental tissue (Mix II), on human chorionic gonadotropin (hCG), placental lactogen (hPL) and placental growth factor (hPLGF) secretion, protein expression and immunolocalization. Additionally, the action of PAH mixtures on basal and hormone-stimulated matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) protein expression was evaluated. Under basal conditions, the PAH mixtures increased hCG and decreased hPLGF levels in both cell lines, while hPL expression was stimulated in JEG-3 and inhibited in BeWo. There was no effect on the MMP-2/MMP-9 ratio or VEGF expression. In hormone-stimulated cells, PAH mixtures changed the MMP-2/MMP-9 ratio in JEG-3 cells in favor of MMP-9, while in BeWo MMP-2 was favored. The effect on VEGF expression was cell specific and dependent on the mixture. In hCG-treated cells, only Mix II inhibited VEGF expression in JEG-3 cells. Neither PAH mixtures affected this protein in BeWo cells. In hPL-treated cells, Mix I had a stimulatory effect in JEG-3 cells, while Mix II exerted an inhibitory effect in BeWo cells. In hPLGF-treated cells, Mix II decreased in JEG-3 cells, but in BeWo cells, both mixtures increased VEGF expression. Considering that the evaluated protein hormones play crucial roles in angiogenesis and neovascularization in the placenta, "real life" PAH mixtures by disrupting protein hormones levels, the MMP-2/MMP-9 ratio and VEGF expression can lead to insufficiency and many pregnancy-related disorders.

Local feedback loop of ghrelin-GH in the pig ovary: action on estradiol secretion, aromatase activity and cell apoptosis.

Ghrelin is recognized as an important regulator of growth hormone (GH) secretion, food intake and a factor which controls reproduction. In the present studies, the effect of GH and insulin-like growth factor (IGF-I) on ghrelin synthesis and secretion and the effects of ghrelin on GH synthesis and secretion in cultured whole porcine follicles were studied. Ghrelin and GH levels were measured in the follicular wall and in the culture medium. Moreover, the action of combined treatment with ghrelin and GH on estradiol secretion, aromatase activity and cell apoptosis were examined. We demonstrated that ghrelin increased GH secretion but not GH synthesis by ovarian follicles. GH stimulated both ghrelin synthesis and secretion in the ovarian follicles. The increase in estradiol secretion, aromatase activity and the decrease in caspase-3 activity were noted in ghrelin alone- and ghrelin in combination with GH-treated cells. In culture treated with combination of both these hormones, all investigated parameters were similar to those noted in ghrelin alone-treated cells. In conclusion, our study provides novel evidence for the gonadal feedback loop between GH and ghrelin secretion in the ovary. However, results of the presented research suggest independent action of GH and ghrelin in the ovary.

Endocrine disrupting compounds modulates adiponectin secretion, expression of its receptors and action on steroidogenesis in ovarian follicle.

We determined the effect of dioxin-like polychlorinated naphtalenes (PCN) (Halowax 1051) (100pg/ml), benzo(a)pyrene [B(a)P] (2.5ng/ml), hexachlorobenzene (HCBz) (0.2ng/ml) and non-dioxin like polybrominated diphenyl ether (BDE-47) (25ng/ml) and bisphenol A (BPA) (20ng/ml) on ovarian adiponectin secretion (ELISA) and its receptors expression (Western blot). Ovarian cells co-culture was used to examine action of endocrine disruptors (EDCs) on adiponectin (10µg/ml) stimulated steroidogenesis. B(a)P, HCBz, testosterone (T) decreased adiponectin secretion and receptors expression, BPA, BDE-47, estradiol (E2) had the opposite effect, while PCN had inhibitory effect only on adiponectin secretion. In adiponectin stimulated cells dioxin-like compounds decreased E2 and except of PCN had no effect on T, while non-dioxin like increased E2 and decreased T secretion. Results indicated to modulatory role of EDCs on adiponectin and its receptor and its action on ovarian steroidogenesis, suggest that dioxin- like compounds may contribute to the ovarian dysfunction in obesity-related disorders.

Cell-specific and dose-dependent effects of PAHs on proliferation, cell cycle, and apoptosis protein expression and hormone secretion by placental cell lines.

In the preset study we measured the concentrations of 16 priority PAHs in maternal blood and placental tissue by using the GC-MS/MS system, and investigated the effects of selected PAHs (naphthalene, anthracene, phenanthrene, pyrene) and mixtures on BeWo and JEG-3 human placental cell line proliferation (Alamar Blue), cytotoxicity (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (XTT), lactate dehydrogenase (LDH), acid phosphatase (AP), endocrine activity (progesterone and estradiol secretion) and apoptosis (cyclin A1, cyclin D2, cdk 2, cdk 4, Bcl-xl, Bax, and caspase-3 protein expression). The concentrations of 16 PAHs in maternal blood were higher than in placental tissue. In JEG-3 cells except for naphthalene, all PAHs studied and their mixtures at maternal doses, and only naphthalene at placental doses, increased XTT, while in BeWo cells, placental doses increased XTT and AP activity. A cell-type dependent action: a proapoptotic effect (increased Bax and caspase-3) in BeWo cells and an antiapoptotic effect (decreased Bax and increased cdk2 and cyclin D1) in JEG-3 cells was observed. Naphthalene, pyrene, and phenanthrene exhibited an endocrine-disrupting effect in JEG3 cells but not in BeWo cells. Our results provide evidence of cell specific effects of selected low molecular weight PAHs on proliferation, the cell cycle, proapoptotic protein expression, and hormone secretion.

Halowax 1051 affects steroidogenesis by down-regulation of aryl hydrocarbon and estrogen receptors and up-regulation of androgen receptor in porcine ovarian follicles.

Polychlorinated naphthalenes (PCNs) are thought to interact with the aryl hydrocarbon receptor (AHR) and to have enzyme-inducing properties comparable to polychlorinated dibenzo-p-dioxins, therefore activation of steroid hormone receptors in endocrine tissues is also possible. The aim of the present study was to examine the effects of PCNs mixture, Halowax 1051 on gene and protein expression of receptors: estradiol (ERα/ß), androgen (AR) and AHRGene expression was evaluated by real-time PCR after 6 h of exposition and protein expression by Western blot after 24 h. Levels of sex steroids: androstenedione (A4), estradiol (E2) and testosterone (T) were measured by enzyme immunoassays. Results of the data show down-regulation of AHR gene expression after 6 h in parallel with an inhibition in AHR protein expression at doses 10 pg-10 ng/mL, down-regulation of ER at all doses used, and up-regulation of AR gene expression at doses 1 and 10 ng/mL without affecting their protein expression. To indicate the involvement of AHR, ERs and AR in the impact of PCNs on steroidogenesis, we used their specific blockers. Blocker of AHR reversed the inhibitory effect of Halowax 1051 on A4 secretion, and strengthened its effect on T secretion. Blockers of both ER and AR had no effect on Halowax 1051 action on steroids secretion. The results of this study suggest that AHR is involved in the effect of PCNs on steroidogenesis in the ovary. Additionally, we propose that cross-talk between AHR-ER and AHR-AR receptors mediates the effects of Halowax 1051 on ovarian follicles.

The molecular mechanism of action of superactive human leptin antagonist (SHLA) and quadruple leptin mutein Lan-2 on human ovarian epithelial cell lines.

INTRODUCTION: A number of leptin receptor antagonists have been synthesised for therapeutic use, with pre-clinical tests suggesting their future use in anticancer therapy. To our knowledge, there are no data concerning the possible application of leptin receptor blockers in ovarian cancer. METHODS: In this study, we evaluated two leptin receptor antagonists: superactive human leptin antagonist (SHLA) and quadruple leptin mutein, Lan-2 (L39A/D40A/F41A/I42A), on cell proliferation (Alamar Blue test, BrdU assay), cell cycle gene (qPCR) and protein expression (Western blot) and cell signalling pathways (Western blot) in three different types of cell lines: OVCAR-3, CaOV-3 and HOSEpiC. RESULTS: Both receptor blockers had no effect on non-cancerous HOSEpiC cell line proliferation; however, both reversed the stimulatory effect of leptin on CaOV-3 cell line proliferation to control levels and to below control levels in OVCAR-3 cells. In metastatic carcinoma CaOV-3, both ObR antagonists had an inhibitory effect on the cdk2/cyclin D1 complex, while in serous carcinoma, OVCAR-3, they only had an effect on cdk2 and cdk4 protein expression. SHLA had an inhibitory effect on all investigated signalling pathways in OVCAR-3, while only on Stat3 in CaOV-3. Lan-2 had an inhibitory effect on Stat3 and ERK1/2 in CaOV-3, while in OVCAR-3 it only affected Akt protein phosphorylation. CONCLUSION: Based on these results, we conclude that SHLA and Lan-2 are promising leptin receptor inhibitors which could be used to block leptin activity, eliminating its negative effects on activities related to carcinogenesis. However, the selection of a specific antagonist should be related to tumour type.

Lack of synergy between estrogen and progesterone on local IGF-I, IGFBP-3 and IGFBP-2 secretion by both hormone-dependent and hormone-independent breast cancer explants in vitro. Effect of tamoxifen and mifepristone (RU 486).

The aim of the present study was to investigate direct effects of estrogen (E2) or progesterone (P4) given separately vs. estrogen+progesterone on local IGF-I, IGFBP-3 and IGFBP-2 secretion. Explants obtained from estrogen receptor positive plus progesterone receptor positive (ER+/PR+) and hormone receptors negative (ER-/PR-) tumors were incubated with E2, P4 or both. Tamoxifen was added to E2-exposed cultures; mifepristone (RU 486) was added to P4, and both were given to E2+P4-supplemented cultures. In hormone-dependent and hormone-independent tissues, treatment with estrogen+progesterone increased IGF-I and IGFBP-2 secretion with concomitant decrease in IGFBP-3, in the same manner as E2 or P4 given alone. Tamoxifen decreased the E2- and E2+P4-stimulated IGF-I secretion by hormone-dependent breast cancer explants. RU 486 decreased the P4- and E2+P4-stimulated IGF-I secretion with parallel stimulation of IGFBP-3 secretion by ER+/PR+ explants. Estradiol and progesterone had a synergistic action on IGFBP-2 secretion by hormone-dependent breast cancer explants. In conclusion, the presented data suggest that there is no synergistic action of E2 and P4 in influencing IGF/IGFBPs ratio and, additionally, suggest a protective action of antiestrogen and antiprogestagen against excessive IGF-I secretion.

Action of methyl-, propyl- and butylparaben on GPR30 gene and protein expression, cAMP levels and activation of ERK1/2 and PI3K/Akt signaling pathways in MCF-7 breast cancer cells and MCF-10A non-transformed breast epithelial cells.

In the present study, we examined cAMP levels and activation of the MAPK/ERK1/2 and PI3K/Akt signaling pathways in response to the actions of parabens on GPR30 in MCF-7 and MCF-10A cells. Cells were exposed to methyl-, propyl- or butylparaben at a concentration of 20nM; 17-ß-estradiol (10nM) was used as a positive control. 17ß-estradiol and all tested parabens increased GPR30 gene and protein expression in MCF-7 and MCF-10A cells. No parabens affected cAMP levels in either cell line, with the exception of propylparaben in MCF-10A cells. 17ß-estradiol, propylparaben, and butylparaben increased phosphorylation of ERK1/2 in MCF-7 cells, whereas 17ß-estradiol, methyl- and butylparaben, but not propylparaben, increased phosphorylation of ERK1/2 in MCF-10A cells. Akt activation was noted only in MCF-7 cells and only with propylparaben treatment. Collectively, the data presented here point to a nongenomic mechanism of action of parabens in activation GPR30 in both cancer and non-cancer breast cell lines through ßγ dimer-mediated activation of the ERK1/2 pathway, but not the cAMP/PKA pathway. Moreover, among investigated parabens, propylparaben appears to inhibit apoptosis in cancer cells through activation of Akt kinases, confirming conclusions suggested by our previously published data. Nevertheless, continuing research on the carcinogenic action of parabens is warranted.

In vitro effect of leptin on steroids' secretion by FSH- and LH-treated porcine small, medium and large preovulatory follicles.

The role of exogenous leptin in the follicular steroidogenesis in pigs has not been fully elucidated and available data are controversial. In the current study porcine follicles were recovered from ovaries during early, middle, and preovulatory stages of the follicular phase of the estrous cycle. Follicles were cultured in the presence of the recombinant ovine leptin (oLEP) with or without LH (100 ng/ml) or FSH (100 ng/ml). Medium estradiol (E(2)), testosterone (T) and progesterone (P(4)) concentrations were determined after 48h of culture. Leptin at a dose of 2 ng/ml had no effect on basal E(2) and T secretion by small and medium follicles but decreased E(2) secretion by large follicles. Significant synergistic action of FSH and leptin resulting in a 2 - 5 fold stimulation of E(2) secretion by small and medium follicles was observed. The aromatase inhibitor, CGS 16949A augmented T secretion and inhibited E(2) secretion by control and FSH-treated medium follicles. In FSH and leptin-treated follicles, the inhibitory action of CGS 16949A on E(2) secretion was observed. However, there was no augmentation of T secretion. In leptin-treated follicles the stimulatory action on P(4) secretion was observed only during the preovulatory stage. In these follicles, significant synergistic action of leptin with LH on P(4) secretion was also noted. These results indicate that there is a maturation-dependent action of leptin on both E(2) and P(4) secretion. They also suggest a synergistic action of leptin and FSH on E(2) secretion by small and medium follicles as well as leptin and LH on P(4) secretion by large follicles in pigs.

Synergistic action of growth hormone and insulin-like growth factor I (IGF-I) on proliferation and estradiol secretion in porcine granulosa and theca cells cultured alone or in coculture.

The effect of growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion and the effects of GH and IGF-I on [(3)H] thymidine incorporation and estradiol (E2) secretion by theca interna (Tc) and granulosa cells (Gc) cultured alone and in coculture were studied in cultured porcine follicular cells, prepared from small (SF), medium (MF) and large (LF) preovulatory follicles. We demonstrated that both Tc and Gc secrete IGF-I and that GH had no effect on IGF-I secretion by Tc but, increased IGF-I secretion by Gc isolated from SF and cultured alone or in coculture. IGF-I stimulated secretion of E2 by all cells, except in Tc derived from SF in which the effect was not statistically significant. The only stimulatory effect of concurrent treatment with GH on E2 secretion was noted in Tc derived from MF. IGF-I increased the [(3)H] thymidine incorporation in all Tc cells but GH did not augment this effect. In Gc, IGF-I stimulated [(3)H] thymidine incorporation in cells derived from SF and MF but not from LF. GH had no stimulatory effect except on Gc derived from LF and grown alone. The highest stimulatory effect was observed in SF. This was smaller in MF and no effect was noted in LF. In conclusion, our work shows that both Gc and Tc are sites of IGF-I production and for the first time shows the stimulatory role of IGF-I in proliferation of Tc cells derived from all types of follicles and augmentation of E2 secretion in Tc derived from MF and LF. The promotion of the mitogenic activity in Tc by IGF-I during all stages of follicular development suggests an important role for theca cells in follicular growth.