Gamma secretase inhibitor impairs epithelial-to-mesenchymal transition induced by TGF-ß in ovarian tumor cell lines.

Ovarian cancer is characterized by being highly metastatic, a feature that represents the main cause of failure of the treatment. This study investigated the effects of γ-secretase inhibition on the TGF-ß-induced epithelial-mesenchymal transition (EMT) process in ovarian cancer cell lines. SKOV3 cells incubated in the presence of TGF-ß showed morphological and biochemical changes related to EMT, which were blocked by co-stimulation with TGF-ß and the γ-secretase inhibitor DAPT. In SKOV3 and IGROV1 cells, the co-stimulation blocked the cadherin switch and the increase in the transcription factors Snail, Slug, Twist and Zeb1 induced by TGF-ß. DAPT impaired the translocation of phospho-ß-catenin to the inner cell compartment observed in TGF-ß-treated cells, but was not able to block the induction at protein level induced by TGF-ß. Moreover, the inhibitor blocked the increased cell migration and invasiveness ability of both cell lines induced by TGF-ß. Notch target genes (Hes1 and Hey1) were induced by TGF-ß, decreased by DAPT treatment and remained low in the presence of both stimuli. However, DAPT alone caused no effects on most of the parameters analyzed. These results demonstrate that the γ-secretase inhibitor used in this study exerted a blockade on TGF-ß-induced EMT in ovarian cancer cells.

Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome.

STUDY QUESTION: Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ovarian hyperstimulation syndrome (OHSS) altered and in part responsible for the high vascular permeability observed in these patients. STUDY ANSWER: FF S1P levels are lower in FF from patients at risk of OHSS and treatment with S1P may reduce vascular permeability in these patients. WHAT IS KNOWN ALREADY: Although advances have been made in the diagnosis, and management of OHSS and in basic knowledge of its development, complete prevention has proven difficult. STUDY DESIGN, SIZE, DURATION: A total of 40 FF aspirates were collected from patients undergoing ART. The women (aged 25-39 years old) were classified into a control group (n = 20) or a group at risk of OHSS (n = 20). The EA.hy926 endothelial cell line was used to assess the efffects of FF from patients at risk of OHSS with or without the addition of S1P. An animal model that develops OHSS in immature Sprague-Dawley rats were also used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Migration assays, confocal microscopy analysis of actin filaments, immunoblotting and quail chorioallantoic membrane (CAM) assays of in-vivo angiogenesis were performed and statistical comparisons between groups were made. MAIN RESULTS AND THE ROLE OF CHANCE: The S1P concentration was significantly lower in FF from patients at risk of OHSS (P = 0.03). The addition of S1P to this FF decreased cell migration (P < 0.05) and prevented VE-cadherin phosphorylation in endothelial cells (P < 0.05). S1P in the FF from patients at risk of OHSS increased the levels of VE-cadherin (P < 0.05), N-cadherin (P < 0.05) and ß-catenin (P < 0.05), and partially reversed actin redistribution in endothelial cells. The addition of S1P in FF from patients at risk of OHSS also decreased the levels of vascular endothelial growth factor (VEGF121; P < 0.01) and S1P lyase (SPL; P < 0.05) and increased the levels of S1PR1 (P < 0.05) in endothelial cells. In CAMs incubated with FF from patients at risk of OHSS with S1P, the number of vessel branch points decreased while the periendothelial cell coverage increased. Additionally, in a rat OHSS model, we demonstrated that vascular permeability and VEGF121 and its receptor KDR expression were increased in the OHSS group compared to the control group and that S1P administration decreased these parameters. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The results of this study were generated from an in-vitro system. This model reflects the microvasculature in vivo. Even though the ideal model would be the use of human endothelial cells from the ovary, it is obviously not possible to carry out this kind of approach in ovaries of patients from ART. More studies will be necessary to delineate the effects of S1P in the pathogenesis of OHSS. Hence, clinical studies are needed in order to choose the most appropriate method of prevention and management. WIDER IMPLICATIONS OF THE FINDINGS: The use of bioactive sphingolipid metabolites may contribute to finding better and safer therapeutic strategies for the treatment of OHSS and other human diseases that display aberrant vascular leakage. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants ANPCyT (PICT 2012-897), CONICET (PIP 5471), Roemmers and Baron Foundation, Argentina. The authors declare no conflict of interest.

Extracellular histones reduce survival and angiogenic responses of late outgrowth progenitor and mature endothelial cells.

UNLABELLED: ESSENTIALS: Extracellular histones are highly augmented in sites of neovessel formation, such as regeneration tissues. We studied histone effect on survival and angiogenic activity of mature and progenitor endothelial cells. Extracellular histones trigger apoptosis and pyroptosis and reduce angiogenesis in vivo and in vitro. Histone blockade can be useful as a therapeutic strategy to improve angiogenesis and tissue regeneration. BACKGROUND: Extracellular histones are highly augmented in sites of neovessel formation, like regeneration tissues. Their cytotoxic effect has been studied in endothelial cells, although the mechanism involved and their action on endothelial colony-forming cells (ECFCs) remain unknown. OBJECTIVE: To study the effect of histones on ECFC survival and angiogenic functions and compare it with mature endothelial cells. METHODS AND RESULTS: Nuclear morphology analysis showed that each human recombinant histone triggered both apoptotic-like and necrotic-like cell deaths in both mature and progenitor endothelial cells. While H1 and H2A exerted a weak toxicity, H2B, H3 and H4 were the most powerful. The percentage of apoptosis correlated with the percentage of ECFCs exhibiting caspase-3 activation and was zeroed by the pan-caspase inhibitor Z-VAD-FMK. Necrotic-like cell death was also suppressed by this compound and the caspase-1 inhibitor Ac-YVAD-CMK, indicating that histones triggered ECFC pyroptosis. All histones, at non-cytotoxic concentrations, reduced migration and H2B, H3 and H4 induced cell cycle arrest and impaired tubulogenesis via p38 activation. Neutrophil-derived histones exerted similar effects. In vivo blood vessel formation in the quail chorioallantoic membrane was also reduced by H2B, H3 and H4. Their cytotoxic and antiangiogenic effects were suppressed by unfractioned and low-molecular-weight heparins and the combination of TLR2 and TLR4 blocking antibodies. CONCLUSIONS: Histones trigger both apoptosis and pyroptosis of ECFCs and inhibit their angiogenic functions. Their cytotoxic and antiangiogenic effects are similar in mature endothelial cells and disappear after heparin addition or TLR2/TLR4 blockade, suggesting both as therapeutic strategies to improve tissue regeneration.

Gonadotropin-releasing hormone agonist affects rat ovarian follicle development by interfering with FSH and growth factors on the prevention of apoptosis.

Apoptosis is the biological process by which follicular cells are eliminated in atretic follicles. The aim of the present study was to examine the in vitro effect of a GnRH-a (leuprolide acetate, LA) and its interactions with FSH, dibutyryl cAMP, and growth factors (IGF-I, EGF, and FGF) on follicular apoptosis in early antral ovarian follicles obtained from prepubertal DES- treated rats. Follicles cultured 24 hr in the absence of hormones showed spontaneous onset of apoptotic DNA fragmentation. The presence of FSH suppressed the spontaneous onset of apoptotic DNA fragmentation (75-85%). Quantitative estimation of DNA cleavage from ovarian follicles revealed no significant changes in DNA fragmentation after in vitro LA treatment (1-100 ng/ml). However, coincubation with LA interfered partially with the effects of FSH on apoptosis suppression. This apoptosis suppression was also obtained by treatment with dibutyryl cAMP (80%), and was partially prevented by the presence of LA in the cultures. Follicles were cultured 24 hr with FGF, EGF, or IGF-I, and these factors suppressed DNA fragmentation (70, 60, and 70% respectively), while the presence of LA (100 ng/ml) in the culture medium prevented this effect. In conclusion, we show that the rescue from apoptotic DNA fragmentation produced in early antral follicles by FSH, cAMP, and growth factors, is prevented by coincubation with LA. This GnRH analog would thus interfere in the pathway of FSH, cAMP and/or growth factors by an as yet unknown mechanism.

Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: relationship between steroidogenesis and apoptosis.

The purpose of this study was to evaluate the effects of GnRH-analog (Leuprolide acetate, LA) administration on follicular luteinization in equine chorionic gonadotropin plus human chorionic gonadotropin (eCG + hCG)-superovulated prepubertal treated rats. Results indicate that LA treatment decreases circulating levels of progesterone (P) and P accumulation in collagenase-dispersed ovarian cell cultures, though estradiol (E2) production is increased. These data suggest that cells from the LA group may be less luteinized following gonadotropin treatment. Studies performed on histological ovarian sections after different times of eCG administration showed that LA injections produce lower amounts of corpora lutea and antral follicles, and a greater number of atretic and preantral follicles. The basal and LH-stimulated P and progestagen accumulations are decreased in incubations of corpora lutea isolated from the LA group. In addition, the mitochondrial cholesterol side-chain cleavage (P450SCC) levels in corpora lutea from LA-treated rats are reduced, indicating that the decrease in P production observed is due in part to an alteration in the steroidogenic luteal capability. Immunocytochemical localization of nuclei exhibiting DNA fragmentation by the technique of terminal deoxynucleotidyl transferase end-labeling showed that LA treatment causes an increase in the number of apoptotic cells in preantral and antral follicles at all times studied (1, 2, 4, or 7 days of LA administration). A similar effect, though less pronounced, was observed in corpora lutea. It is concluded that LA treatment produces a failure in the steroidogenic luteal capability and an increase of apoptotic mechanisms in the ovary, producing as a consequence an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins.

Regulation of ovarian follicle differentiation in gonadotrophin-stimulated rats.

The aim of the present study was to investigate the regulation of the in vitro DNA synthesis of ovarian cells recovered from prepubertal rats 48 h after administration of pregnant mare's serum gonadotrophin alone (granulosa cells) or followed by human chorionic gonadotrophin (luteal cells). Isolated granulosa cells were cultured in serum-free medium, different stimuli added for periods of 48 h, and 3H-thymidine incorporation was measured. Both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) inhibited 3H-thymidine incorporation by cultured granulosa cells in a dose-dependent manner (FSH: 10, 100, 200 ng/mL = 26, 41, 49% inhibition, respectively; LH: 0.1, 1, 10 ng/mL = 11, 37, 75% inhibition, respectively). On the other hand, estradiol was found to stimulate 3H-thymidine incorporation in granulosa cells (Estradiol: 5, 50, 500 ng/mL = 17, 37, 76% stimulation, respectively). In luteal cells, the rate of basal 3H-thymidine incorporation was very low (granulosa cells: 2560 +/- 310; luteal cells: 661 +/- 92 cpm/100,000 cells) and not modified by any stimulus. To determine the possible production of an inhibitory growth factor by the early corpus luteum, 3H-thymidine incorporation by granulosa cells was assessed in the presence of 10% conditioned media (CM) recovered from luteal cell cultures. A marked inhibition both in basal and estradiol-stimulated 3H-thymidine incorporation was observed (74 and 76% of inhibition, respectively). Results suggest that an inhibitory growth factor produced by luteal cells after luteinizing gonadotrophin stimulus could be involved in the differentiation of growing follicles to corpus luteum.

[Regulation of follicular luteinization by an agonist of gonadotropin-releasing hormone (GnRH)]. / Regulación de la luteinización folicular por un análogo de la hormona liberadora de gonadotrofinas (GnRH).

Long treatments with GnRH agonist are used in patients to suppress the endogenous secretion of gonadotropins, however; these analogs have a direct effect on the ovary. The aim of this work was to study the in vivo effect of the GnRH analogue, leuprolide acetate (LA) on ovarian steroidogenesis and apoptosis mechanisms. LA (1 microgram/rat/day) was injected to PMSG/hCG superovulated rats. Corpora lutea were isolated by microdissection and incubated (4/0.5 ml) during 3 h with LH (10 ng/ml) or dibutyryl cAMP (dcAMP 1 mM). Progesterone production was measured observing in LA treated rats a decrease in basal and LH stimulated values (Basal = Control C: 96.6 +/- 9.6; LA: 22.9 +/- 2.8; LH = C: 145.7 +/- 4.9; LA: 23.6 +/- 2.0 ng/ml, p < 0.001). In contrast, dcAMP stimulated significantly both groups (C: 153.9 +/- 11.8; LA: 83.15 +/- 8.2). cAMP production was lower in LA corpora lutea and LH was not able to stimulate them (Basal = C: 7.29 +/- 1.6; LA: 1.17 +/- 0.6; LH = C: 13.2 +/- 0.4; LA: 2.5 +/- 0.4 ng/ml, p < 0.01). Corpus luteum of both groups showed similar protein content. On the other hand, taking into account that in ovarian histological slides of LA treated rats we observed more atresic follicles and less corpora lutea, we determined the amount of apoptotic cells. The criterion used was the presence of apoptotic bodies and nuclear chromatin aggregated in dense masses beneath the nuclear envelope. An increase of apoptotic cells in the LA group ovaries was detected. This result was confirmed by immunohistochemical technics (TUNEL). It was concluded that LA treatment produces in ovarian cells a failure in the LH receptor-adenilate cyclase system and an increase in cellular apoptosis.