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Physiological inflammation occurs in the female reproductive tract, but pathological inflammation is implicated in reproductive pathologies such as preterm labour and endometrial cancer. Preterm labour (PTL, before 37 weeks of gestation) is the leading cause of preterm birth, neonatal mortality and perinatal morbidities. Endometrial cancer is the commonest gynaecological cancer, and its pathogenesis is characterised by chronic inflammation. The overall aims of this thesis were (i) to develop an in vitro model of myometrial-monocyte interactions to replicate the events occurring in the myometrium in preterm labour (ii) to determine the effects of potential therapeutics such as lipoxins, IL-10 and progesterone, on inflammation, and (iii) to characterise the lipoxin pathway in endometrial adenocarcinoma. Macrophages infiltrate the pregnant myometrium during labour; however the role of these cells is unclear. A myometrial-monocyte coculture model was developed either using non-pregnant primary myometrial smooth muscle cells (UtSMCs), or immortalised pregnant human myometrial cells (PHM1-41), with primary monocytes from term (38-41 weeks of gestation), non-labouring pregnant women. Cultures were stimulated with the toll-like receptor 4 agonist lipopolysaccharide (LPS), in the presence or absence of each of lipoxins, IL-10 and progesterone. A significant and synergistic increase in IL-6 and IL-8 secretion was found in the UtSMC/monocyte coculture after stimulation with LPS for 24 hours, compared to LPS-treated UtSMCs, or monocytes alone, but the increase in IL-6 and IL-8 secretion was not inhibited by lipoxin, epi-lipoxin or benzo-lipoxin. The PHM1-41/monocyte coculture both alone and in response to LPS treatment generated significantly increased IL-6 and IL-8 secretion, compared to vehicle treatment in the coculture and compared to the culture of either cell type alone. IL-1β and TNFα secretion were only detected from the PHM1/monocyte coculture, and monocytes alone. Use of a TNFα blocking antibody partially suppressed LPS-induced IL-6 and IL-8 secretion in the coculture. Coculture of PHM1/monocytes resulted in increased secretion of multiple mediators including pro-inflammatory cytokines, chemokines and growth factors compared to culture of either PHM1 cells or primary monocytes separately, both with vehicle and with LPS. IL-10 inhibited LPS-induced IL-6 and IL-8 secretion from the coculture, as did progesterone, which also inhibited GM-CSF, MCP-1 and CXCL5 secretion. Myocyte contraction, measured by PHM1-41 cells embedded in collagen was increased by primary monocyte treatment. This suggests that not only do infiltrating monocytes increase myometrial inflammation but they can induce myometrial smooth muscle contraction. In endometrial adenocarcinoma, the lipoxin synthesis enzymes, ALOX-5 and -15 and FPR2 mRNA expression were upregulated compared to proliferative phase endometrium, with FPR2, a reported lipoxin receptor, immunolocalised in endometrial adenocarcinoma tissue. Additionally, TNFα treatment of Ishikawa endometrial adenocarcinoma cells increased FPR2 mRNA expression, and upregulation of FPR2 mRNA also occurred in xenograft tumours from CD1 nude mice, compared to the Ishikawa cells from which they originated. These findings highlight FPR2 expression in endometrial adenocarcinoma, and suggest this receptor could mediate inflammatory signals, and lipoxins could be produced by ALOX-5 and ALOX-15. Collectively, these data describe the novel effects of monocytes in the regulation of myometrial smooth muscle cell inflammation, and demonstrate a mechanism by which myometrial inflammation during both term and preterm labour is triggered by infiltrating macrophages. This myocyte/monocyte inflammation is regulated in part by TNFα, and can be suppressed by both IL-10 and progesterone co-treatment. Components of the lipoxin pathway are present in endometrial adenocarcinoma, but their role in regulation of inflammation is still to be elucidated. Future research to clarify the processes, by which leukocyte recruitment is regulated at labour and the role of monocyte/macrophages in altering myocyte properties, could help to elucidate the mechanisms coupling inflammation to labour and provide more appropriate targets for the treatment of PTL.

Le récepteur de haute affinité 1 (NTSR1) et son agoniste, la neurotensine (NTS), sont corrélés avec l’agressivité tumorale dans la plupart des tumeurs solides, y compris les cancers hormono-dépendants. Comme l’endomètre et l’ovaire sont également soumis à une régulation hormonale, nous avons évalué la contribution de NTS/NTSR1 à la carcinogenèse de l’endomètre et de l’ovaire. L’expression du récepteur de la neurotensine 1 (NTSR1) et la méthylation du promoteur NTSR1 (HM450) ont été analysées dans 385 cas de carcinome de l’endomètre du Cancer Genome Atlas (TCGA). De plus, à partir d’une série de 100 carcinomes de l’endomètre et de 66 échantillons d’endomètre bénin, l’expression de NTS et NTSR1 a été évalué par immunohistochimie. La série TCGA d’adénocarcinomes ovariens séreux de haut grade et une série de 46 tissus ovariens ont également été analysés. Dans la série TCGA de carcinomes de l’endomètre, le taux d’ARN messager de NTSR1 (ARNm) était négativement corrélé avec la survie globale (SG) et la survie sans progression (SSP) (p = 0,0012 et p = 0,0116, respectivement), et positivement corrélé avec le grade (p = 0,0008). En incluant seulement les carcinomes endométrioïdes, le niveau d’ARNm de NTSR1 était également négativement corrélé avec la SG (log-rank:p < 0.0001) et la SSP (log-rank: p = 0.002). Une forte expression d’ARNm de NTSR1 était significativement associée à une perte de méthylation du promoteur NTSR1. L’expression immunohistochimique de NTS et NTSR1 était significativement augmentée dans l’adénocarcinome (n = 100), par rapport à l’endomètre bénin (p <0,001). L’expression de NTSR1 était positivement corrélée avec le grade (p = 0,004). Une forte expression du NTSR1 cytoplasmique était significativement corrélée avec une SG et une SSP plus courtes (p < 0,001 et p = 0,001, respectivement). Cette corrélation restait significative en excluant les sous-types non-endométrioïdes (p = 0,04 et p = 0,02, respectivement). En analyse multivariée, l’expression de NTSR1 était un facteur de mauvais pronostic indépendant (p = 0,004). Nos résultats indiquent également la présence d’une corrélation positive entre l’expression du marqueur de prolifération MCM6 et le grade histologique dans le sous-type endométrioïde (grade I, 66,7 %, grade II, 75,3 %, grade III, 81,4 %, p <0,001) et une corrélation inverse à la SG et à la SSP (p = 0,02 pour les deux). Dans la cohorte TCGA, les analyses de Cox univariées et multivariées (p = 0,003 et p = 0,03, respectivement) ont révélé que les z-scores élevés de l’ARNm de MCM6 étaient associés à une SG réduite. Ces associations étaient absentes pour Ki-67. Il n’y avait pas de corrélation significative entre NTSR1 et MCM6 ou Ki-67. Dans l’adénocarcinome de l’ovaire, la NTS et NTSR1 ont été détectés dans 72 % et 74 % des cancers de l’ovaire, respectivement. En outre, dans la grande série d’adénocarcinomes ovariens séreux de haut grade, l’ARNm de NTSR1 s’est avéré être en corrélation avec des stades plus élevés et la résistance au platine (p = 0.02). Ceci est concordant avec les résultats expérimentaux montrant que l’antagoniste très spécifique de NTSR1, le SR48692, a augmenté la réponse au carboplatine dans les cellules cancéreuses de l’ovaire et les tumeurs expérimentales induites. Lorsque le SR48692 est combiné avec du carboplatine, une augmentation majeure des dommages à l’ADN induits par le platine et de la mort cellulaire, ainsi qu’une diminution de la croissance tumorale, a été notée. La surexpression de NTSR1 est un facteur de mauvais pronostic dans les cancers de l’endomètre et de l’ovaire, mettant en évidence la contribution de NTS dans la progression du cancer et ses utilisations en tant que marqueur pronostique, et en tant que cible thérapeutique potentielle. L’ajout d’un inhibiteur de NTSR1 en association avec une thérapie à base de sel de platine pourrait améliorer la réponse au traitement / The high affinity receptor 1 (NTSR1) and its agonist, neurotensin (NTS), are correlated with tumor cell aggressiveness in most solid tumors, including hormone-dependent cancers. As the endometrium and ovary are also subjected to hormonal regulation, we evaluated the contribution of NTS to endometrial and ovarian carcinogenesis. Neurotensin receptor 1 (NTSR1) expression and NTSR1 promoter methylation (HM450) were analyzed in 385 cases of endometrial carcinoma from The Cancer Genome Atlas (TCGA). Additionally, from a series of 100 endometrial carcinomas, and 66 benign endometrium samples, NTS and NTSR1 labeling was evaluated by immunohistochemistry. The TCGA series for ovarian high-grade serous adenocarcinoma and a series of 46 ovarian tissues were also analyzed. Using TCGA series, NTSR1 messenger RNA (mRNA) level was negatively correlated with overall survival (OS) and progression-free survival (PFS) (p = 0.0012 and p = 0.0116, respectively), and positively correlated with the grade (p = 0.0008). When including only endometrioid carcinomas, NTSR1 mRNA level continued to be negatively correlated with OS (log-rank: p < 0.0001) and PFS (log-rank: p = 0.002). A higher NTSR1 mRNA level was significantly associated with a loss of NTSR1 promoter methylation. Immunohistochemical expression of NTS and NTSR1 was significantly increased in adenocarcinoma (n = 100), as compared to benign endometrium (p < 0.001). NTSR1 expression was positively correlated with grade (p = 0.004). High immunohistochemical expression of cytoplasmic NTSR1 was significantly correlated with a shorter OS and PFS (p < 0.001 and p = 0.001, respectively). This correlation remained significant when excluding non-endometrioid subtypes (p = 0.04 and p = 0.02, respectively). In multivariate analysis, the expression of NTSR1 was an independent prognostic factor (p = 0.004). Our evidence indicated also the presence of a positive correlation between expression of proliferation marker, MCM6 and the histological grade of endometrioid endometrial adenocarcinoma (grade I, 66.7 %; grade II, 75.3 %; grade III, 81.4 %; p < 0.001) and an inverse correlation with OS ans PFS (p = 0.02 for both). For in silico analyses of the TCGA cohort, both univariate and multivariate Cox analyses (p = 0.003 and p = 0.03, respectively) revealed high MCM6 mRNA Z-scores associated with reduced OS. These associations were absent for Ki-67. No significant correlation between NTSR1 and MCM6 or Ki-67 was found. In ovarian adenocarcinoma, NTS and NTSR1 were detected in 72 % and 74 % of ovarian cancer, respectively. Furthermore, in a large series of high-grade ovarian cancer, NTSR1 mRNA was shown to correlate with higher stages and platinum resistance (p = 0.02). This correlates with experimental results showing the very specific NTSR1 antagonist, SR48692, enhanced the response to carboplatin in ovarian cancer cells and experimental tumors. When SR48692 is combined with carboplatin, a major improvement of platinum-induced DNA damage and cell death, as well as a decrease in tumor growth, was noted. NTSR1 overexpression is a poor prognostic factor in endometrial and ovarian cancer, highlighting the contribution of NTS in cancer progression and its uses as a prognostic marker, and as a potential therapeutic target. The addition of NTSR1 inhibitor in combination with platinum salt-based therapy could improve the response to the drug

Endometriumkrebs ist eine der häufigsten gynäkologischen Malignomen weltweit und wird in einen Typ I eingeteilt, welcher östrogenabhängig ist, und in eine Typ-II-Östrogen-unabhängige Form. Typ I ist der häufigste Fall und kommt in etwa für 75% bis 85% aller diagnostizierten Fälle in Frage. Erhöhte Östrogenspiegel haben gezeigt, das Risiko von Gebärmutterkrebsentwicklung zu erhöhen, genauso wie Östrogen die Proliferation von Endometriumzellen stimuliert und die Apoptose hemmt. Die Insulinresistenz scheint eine zentrale Rolle in der endometrialen Karzinogenese zu spielen und darüber hinaus werden Erkrankungen mit Insulinresistenz, wie zum Beispiel das polyzystische Ovarialsyndrom (PCOS) und Adipositas, sowie Typ II-Diabetes mellitus (DM) als signifikantes Risiko angesehen, Faktoren für die Entwicklung und Progression von Typ-I-Endometrium-Krebs zu sein. Zusätzlich können PCOS-Patienten durch eine Fettleibigkeit in einem normoglykämischen Status eine unabhängige Insulin-Resistenz haben. In diesem Fall scheint die Hyperinsulinämie der fördernde Faktor zu sein, nicht nur für die Entwicklung als auch für die Tumorprogression.Aber auch erhöhte Blutzuckerspiegel tragen zum Wachstum und die Karzinogenese in Endometriumkarzinom bei und dienen als wichtige Verbindung zwischen dem beobachteten erhöhten Krebsrisiko bei Patienten mit Typ-II-DM. Die Behandlung mit einem Anti-Diabetikum, welches den Insulinspiegel senken kann, könnte einen allgemeinen Ansatz bieten gegen die Entwicklung von Krebs und zur Verringerung der Metastasierung. Das Ziel dieser Studie war es, die Wirkung einer 0,1 mM Metformin-Dosis auf das proliferative und metastatische Potential von Endometriumkrebszellen bewerten zu können, sowie die Analyse der Auswirkungen von kurz- und langfristigen Behandlungen auf intrazelluläre Signalwege der Endometriumkrebszellen.(Fortsetzung) (Fortsetzung)Ebenso soll der Zusammenhang der Entwicklung und der Progression von Krebszellen untersucht werden, wenn sie einer Umgebung mit unterschiedlichen Glucosekonzentrationen und hohen Insulinspiegeln ausgesetzt werden. Darüber hinaus ist eine endometriale dreidimensionale (3D) Cokultur zu standardisieren, für eine viabele Kultur bei 20 Tagen Kultivierung. Das proliferative Potential wurde unter Verwendung des CellTitle-Glo-Tests durchgeführt, und das metastatische Potential wurde unter Verwendung von Transwell-Migration und Invasion untersucht. Die mRNAExpression von MKI67, mTOR, NOTCH1, NOTCH3 und JAG1 Gene wurden durch real-time PCR gemessen. Die kumulative Populationsverdopplungsrate wurde durch das Replikationsverhalten einer Endometriumkrebszelllinie durchgehend von 20 Tagen nach einer Behandlungsdauer bestimmt. In allen Assays wurden die Zellen durch Medien mit normaler (5,5 mM) oder hoher (17 mM) Glucosekonzentration, sowie in verschiedenen Gruppen behandelt: Kontrolle, Insulin, Metformin und Insulin+Metformin. Das 3D-Kokulturmodell wurde unter Verwendung von endometrialen Primärzellen und einer Endometriumkrebszelllinie hergestellt, wobei die Modellkonstruktion durch Matrigel® als extrazelluläre Matrix verwendet wurde. In dieser Studie hemmte die 0,1 mM Metformin-Dosis die Insulinwirkung stark und verringerte die Fähigkeit der endometrialen Krebszelllinie, in einer hohen und normalen Glukoseumgebung zu migrieren und einzudringen.(Fortsetzung) (Fortsetzung) Auf das proliferative Potential wurde dieser Effekt nicht beobachtet, allerdings reagierte die relative Zellproliferation empfindlich auf Metformin im Bereich zwischen 1 und 5 mM, unabhängig von der vorliegenden Glucosekonzentration. In den intrazellulären molekularen Mechanismen wurde beobachtet, dass die hohe Glukosekonzentration eine optimale Umgebung für endometriale Krebszellen schafft, um einen aggressiveren Genotyp und eine Resistenz gegenüber Metformin während einer Langzeitbehandlung zu zeigen. Darüber hinaus blieb das endometriale 3DKokulturmodell über 20 Kulturtage lebensfähig. Daher zeigte sich, trotz der Endometriumkrebszellen, eine Resistenz gegenüber dem Metformin-Effekt, wenn sie einer hohen Glucoseumgebung ausgesetzt waren. Die 0,1 mM Metformin-Dosis war in der Lage, die Insulinwirkung zu hemmen und das metastatische Potential der Zellen zu verringern, was darauf hindeutet, dass Metformin klinisch in Verbindung mit Insulin wirkt, sowie die indirekten und direkten Effekte als potentieller Wirkstoff in der Krebstherapie eingesetzt werden könnten. / O câncer de endométrio é uma das neoplasias ginecológicas com maior incidência, classificado como tipo I, estrógeno dependente, e tipo II, estrógeno nãodependente. O tipo I é a forma mais comum, ocorrendo em torno de 75 – 85 % dos casos de câncer de endométrio. Altos níveis de estrogênio têm sido relacionados ao aumento do risco de desenvolvimento do câncer de endométrio, pois estimula a proliferação celular e inibe a apoptose. A resistência à insulina parece desempenhar um papel central nesta neoplasia, e as doenças associadas à resistência à insulina como obesidade, Diabetes Mellitus (DM) tipo II e Síndrome dos Ovários Policísticos (PCOS) também são consideradas fatores de risco significantes para o desenvolvimento e progressão do câncer de endométrio tipo I. Adicionalmente, pacientes com PCOS podem apresentar um quadro de resistência à insulina independente de obesidade, permanecendo em um estado glicêmico normal. Neste caso, a hiperinsulinemia isolada seria um fator tanto para a promoção, como também para a progressão do câncer. Entretanto, o aumento de níveis séricos de glicose, a hiperglicemia, também é considerada um fator independente para o desenvolvimento e progressão do câncer de endométrio sendo um elo crítico entre o aumento do risco do desenvolvimento de câncer observado em pacientes com DM tipo II Dessa forma, tratamento utilizando agentes insulino-sensibilizantes, que atuam diminuindo a resistência à insulina e consequentemente reduzindo seus níveis pode ser uma estratégia interessante para prevenir o câncer e reduzir a disseminação metastática. Os objetivos deste trabalho foram avaliar o efeito da dose de 0,1 mM de metformina sobre o potencial proliferativo e metastático das células de câncer de endométrio, assim como, avaliar o efeito do tratamento a curto e a longo prazo sobre vias de sinalização intracelular relacionadas ao desenvolvimento e progressão do câncer de endométrio quando exposta a um ambiente com diferentes concentrações de glicose e níveis elevados de insulina. Por fim, a padronização de um modelo tridimensional (3D) de cocultura de células de endométrio que permanecesse viável ao longo de 20 dias de cultivo. O potencial proliferativo foi determinado pelo método luminescente CellTitle Glo, e o potencial metastático pelo o ensaio transwell de migração e invasão. Análises de expressão do mRNA dos genes MKI67, mTOR, NOTCH1, NOTCH3 e JAG1 foram realizadas a partir da técnica de PCR em tempo real. O índice de duplicação populacional cumulativo das células determinou o comportamento de replicação da linhagem de câncer de endométrio ao longo do período de tratamento de 20 dias. Em todas os ensaios as células foram cultivadas em meio contendo concentrações normais (5,5 mM) ou altas (17 mM) de glicose, e divididas nos diferentes grupos de tratamento: controle, insulina, metformina e metformina associado a insulina. A padronização do modelo 3D de cocultura de células de endométrio foi realizado utilizando células primárias e células de linhagem de câncer de endométrio, a Matrigel® foi a matriz extracelular temporária utilizada para a construção do modelo. Neste estudo, a concentração de 0,1 mM de metformina inibiu a ação da insulina, diminuindo a habilidade de migração e invasão das células de câncer de endométrio independente da concentração de glicose presente no meio. Entretanto, este efeito não foi observado sobre o potencial proliferativo, sendo observada uma redução da proliferação das células de câncer de endométrio ao serem utilizadas concentrações maiores de metformina. Em relação aos mecanismos moleculares intracelulares, foi observado que na presença de altas concentrações de glicose as células de câncer de endométrio adquirem um genótipo mais agressivo e apresentam resistência ao efeito da metformina na dose de 0,1 mM durante o tratamento agudo. Além disso, foi possível a padronização de um modelo 3D de cocultura de células de câncer de endométrio que permanecesse viável ao longo dos 20 dias de cultivo. Contudo, apesar das células de câncer de endométrio apresentarem resistência ao efeito da metformina na presença de altas concentrações de glicose, a dose de 0,1 mM foi capaz de inibir o efeito da insulina e diminuir o potencial metastático dessas células, sugerindo que a metformina ao atuar clinicamente em combinação com seus efeitos indiretos e diretos pode ser um potencial agente adjuvante na terapia contra o câncer. / Endometrial cancer is one of the most common gynecological malignancies worldwide and is classified into a type I, which is estrogen-dependent, and a type II estrogen-independent form. The type I is the most common, accounting to 75%-85% of all cases of endometrial cancer. Elevated estrogen levels have been shown to increase the risk of endometrial cancer development, as estrogen stimulates endometrial cell proliferation and inhibits apoptosis. The insulin resistance seems to play a central role in endometrial carcinogenesis, furthermore, diseases associate with insulin resistance, as seen in polycystic ovary syndrome (PCOS), and obesity, as well as type II diabetes mellitus (DM) are considered as significant risk factors for the development and progression of type I endometrial cancer. Additionally, PCOS patients may have an insulin resistance independent of obesity remaining in a normoglycemic status. At this case, the hypeinsulinemia seems to be the promoter factor not only for the development but also for the cancer progression. However, also increased blood glucose levels are contributing to the growth and carcinogenesis in endometrial cancer and are acting as a critical link between the observed increased cancer risk in patients with type II DM. Therefore, the treatment with insulin-sensitizing agents that act through reducing insulin levels, could offer a general approach to prevent the development of cancer and reduce metastasis The aim of this study was to evaluate the effect of 0.1 mM metformin dose on the proliferative and metastatic potential of endometrial cancer cells, as well as, analyze the effects of short and long-term treatment on intracellular signaling pathways related to endometrial cancer development and progression when exposed to an environment with different glucose concentrations and high insulin levels. Additionally, the endometrial three-dimensional (3D) coculture standardization to remain viable over 20 culture days. The proliferative potential was performed by using CellTitle Glo assay, and the metastatic potential was performed by using transwell migration and invasion assay. The mRNA expression of MKI67, mTOR, NOTCH1, NOTCH3 and JAG1 genes were measured by real time PCR. The cumulative population doubling rate was evaluated to determine the replication behavior of an endometrial cancer cell line throughout 20 days of treatment period. In all assays the cells were cultured in medium containing normal (5.5 mM) or high (17 mM) glucose concentration, and treated in different groups: control, insulin,metformin or combined treatment The 3D coculture model was established by using endometrial primary cells and an endometrium cancer cell line, to the model construction Matrigel® was used as an extracellular matrix. In this study, the 0.1 mM metformin dose potently inhibited the insulin action, decreasing the ability of the endometrial cancer cell line to migrate and invade in a high and normal glucose environment. On the proliferative potential this effect was not observed, however, relative cell proliferation sensitivity to metformin was observed in the range between 1 and 5 mM regardless of the present glucose concentration. In the intracellular molecular mechanisms, it was observed that the high glucose concentration creates an optimal environment for endometrial cancer cells to exhibit a more aggressive genotype and resistance to metformin during a long-term treatment. Moreover, the endometrial 3D coculture model remained viable throughout 20 culture days. Therefore, despite of endometrial cancer cells show resistance to the metformin effect when exposed to high glucose environment, the 0.1 mM metformin dose was able to inhibit the insulin action and decrease the metastatic potential of the cells, suggesting that metformin is acting clinically in combination with indirectly and direct effects could emerge as a potential agent in cancer therapy.

Endometriumkrebs ist eine der häufigsten gynäkologischen Malignomen weltweit und wird in einen Typ I eingeteilt, welcher östrogenabhängig ist, und in eine Typ-II-Östrogen-unabhängige Form. Typ I ist der häufigste Fall und kommt in etwa für 75% bis 85% aller diagnostizierten Fälle in Frage. Erhöhte Östrogenspiegel haben gezeigt, das Risiko von Gebärmutterkrebsentwicklung zu erhöhen, genauso wie Östrogen die Proliferation von Endometriumzellen stimuliert und die Apoptose hemmt. Die Insulinresistenz scheint eine zentrale Rolle in der endometrialen Karzinogenese zu spielen und darüber hinaus werden Erkrankungen mit Insulinresistenz, wie zum Beispiel das polyzystische Ovarialsyndrom (PCOS) und Adipositas, sowie Typ II-Diabetes mellitus (DM) als signifikantes Risiko angesehen, Faktoren für die Entwicklung und Progression von Typ-I-Endometrium-Krebs zu sein. Zusätzlich können PCOS-Patienten durch eine Fettleibigkeit in einem normoglykämischen Status eine unabhängige Insulin-Resistenz haben. In diesem Fall scheint die Hyperinsulinämie der fördernde Faktor zu sein, nicht nur für die Entwicklung als auch für die Tumorprogression.Aber auch erhöhte Blutzuckerspiegel tragen zum Wachstum und die Karzinogenese in Endometriumkarzinom bei und dienen als wichtige Verbindung zwischen dem beobachteten erhöhten Krebsrisiko bei Patienten mit Typ-II-DM. Die Behandlung mit einem Anti-Diabetikum, welches den Insulinspiegel senken kann, könnte einen allgemeinen Ansatz bieten gegen die Entwicklung von Krebs und zur Verringerung der Metastasierung. Das Ziel dieser Studie war es, die Wirkung einer 0,1 mM Metformin-Dosis auf das proliferative und metastatische Potential von Endometriumkrebszellen bewerten zu können, sowie die Analyse der Auswirkungen von kurz- und langfristigen Behandlungen auf intrazelluläre Signalwege der Endometriumkrebszellen.(Fortsetzung) (Fortsetzung)Ebenso soll der Zusammenhang der Entwicklung und der Progression von Krebszellen untersucht werden, wenn sie einer Umgebung mit unterschiedlichen Glucosekonzentrationen und hohen Insulinspiegeln ausgesetzt werden. Darüber hinaus ist eine endometriale dreidimensionale (3D) Cokultur zu standardisieren, für eine viabele Kultur bei 20 Tagen Kultivierung. Das proliferative Potential wurde unter Verwendung des CellTitle-Glo-Tests durchgeführt, und das metastatische Potential wurde unter Verwendung von Transwell-Migration und Invasion untersucht. Die mRNAExpression von MKI67, mTOR, NOTCH1, NOTCH3 und JAG1 Gene wurden durch real-time PCR gemessen. Die kumulative Populationsverdopplungsrate wurde durch das Replikationsverhalten einer Endometriumkrebszelllinie durchgehend von 20 Tagen nach einer Behandlungsdauer bestimmt. In allen Assays wurden die Zellen durch Medien mit normaler (5,5 mM) oder hoher (17 mM) Glucosekonzentration, sowie in verschiedenen Gruppen behandelt: Kontrolle, Insulin, Metformin und Insulin+Metformin. Das 3D-Kokulturmodell wurde unter Verwendung von endometrialen Primärzellen und einer Endometriumkrebszelllinie hergestellt, wobei die Modellkonstruktion durch Matrigel® als extrazelluläre Matrix verwendet wurde. In dieser Studie hemmte die 0,1 mM Metformin-Dosis die Insulinwirkung stark und verringerte die Fähigkeit der endometrialen Krebszelllinie, in einer hohen und normalen Glukoseumgebung zu migrieren und einzudringen.(Fortsetzung) (Fortsetzung) Auf das proliferative Potential wurde dieser Effekt nicht beobachtet, allerdings reagierte die relative Zellproliferation empfindlich auf Metformin im Bereich zwischen 1 und 5 mM, unabhängig von der vorliegenden Glucosekonzentration. In den intrazellulären molekularen Mechanismen wurde beobachtet, dass die hohe Glukosekonzentration eine optimale Umgebung für endometriale Krebszellen schafft, um einen aggressiveren Genotyp und eine Resistenz gegenüber Metformin während einer Langzeitbehandlung zu zeigen. Darüber hinaus blieb das endometriale 3DKokulturmodell über 20 Kulturtage lebensfähig. Daher zeigte sich, trotz der Endometriumkrebszellen, eine Resistenz gegenüber dem Metformin-Effekt, wenn sie einer hohen Glucoseumgebung ausgesetzt waren. Die 0,1 mM Metformin-Dosis war in der Lage, die Insulinwirkung zu hemmen und das metastatische Potential der Zellen zu verringern, was darauf hindeutet, dass Metformin klinisch in Verbindung mit Insulin wirkt, sowie die indirekten und direkten Effekte als potentieller Wirkstoff in der Krebstherapie eingesetzt werden könnten. / O câncer de endométrio é uma das neoplasias ginecológicas com maior incidência, classificado como tipo I, estrógeno dependente, e tipo II, estrógeno nãodependente. O tipo I é a forma mais comum, ocorrendo em torno de 75 – 85 % dos casos de câncer de endométrio. Altos níveis de estrogênio têm sido relacionados ao aumento do risco de desenvolvimento do câncer de endométrio, pois estimula a proliferação celular e inibe a apoptose. A resistência à insulina parece desempenhar um papel central nesta neoplasia, e as doenças associadas à resistência à insulina como obesidade, Diabetes Mellitus (DM) tipo II e Síndrome dos Ovários Policísticos (PCOS) também são consideradas fatores de risco significantes para o desenvolvimento e progressão do câncer de endométrio tipo I. Adicionalmente, pacientes com PCOS podem apresentar um quadro de resistência à insulina independente de obesidade, permanecendo em um estado glicêmico normal. Neste caso, a hiperinsulinemia isolada seria um fator tanto para a promoção, como também para a progressão do câncer. Entretanto, o aumento de níveis séricos de glicose, a hiperglicemia, também é considerada um fator independente para o desenvolvimento e progressão do câncer de endométrio sendo um elo crítico entre o aumento do risco do desenvolvimento de câncer observado em pacientes com DM tipo II Dessa forma, tratamento utilizando agentes insulino-sensibilizantes, que atuam diminuindo a resistência à insulina e consequentemente reduzindo seus níveis pode ser uma estratégia interessante para prevenir o câncer e reduzir a disseminação metastática. Os objetivos deste trabalho foram avaliar o efeito da dose de 0,1 mM de metformina sobre o potencial proliferativo e metastático das células de câncer de endométrio, assim como, avaliar o efeito do tratamento a curto e a longo prazo sobre vias de sinalização intracelular relacionadas ao desenvolvimento e progressão do câncer de endométrio quando exposta a um ambiente com diferentes concentrações de glicose e níveis elevados de insulina. Por fim, a padronização de um modelo tridimensional (3D) de cocultura de células de endométrio que permanecesse viável ao longo de 20 dias de cultivo. O potencial proliferativo foi determinado pelo método luminescente CellTitle Glo, e o potencial metastático pelo o ensaio transwell de migração e invasão. Análises de expressão do mRNA dos genes MKI67, mTOR, NOTCH1, NOTCH3 e JAG1 foram realizadas a partir da técnica de PCR em tempo real. O índice de duplicação populacional cumulativo das células determinou o comportamento de replicação da linhagem de câncer de endométrio ao longo do período de tratamento de 20 dias. Em todas os ensaios as células foram cultivadas em meio contendo concentrações normais (5,5 mM) ou altas (17 mM) de glicose, e divididas nos diferentes grupos de tratamento: controle, insulina, metformina e metformina associado a insulina. A padronização do modelo 3D de cocultura de células de endométrio foi realizado utilizando células primárias e células de linhagem de câncer de endométrio, a Matrigel® foi a matriz extracelular temporária utilizada para a construção do modelo. Neste estudo, a concentração de 0,1 mM de metformina inibiu a ação da insulina, diminuindo a habilidade de migração e invasão das células de câncer de endométrio independente da concentração de glicose presente no meio. Entretanto, este efeito não foi observado sobre o potencial proliferativo, sendo observada uma redução da proliferação das células de câncer de endométrio ao serem utilizadas concentrações maiores de metformina. Em relação aos mecanismos moleculares intracelulares, foi observado que na presença de altas concentrações de glicose as células de câncer de endométrio adquirem um genótipo mais agressivo e apresentam resistência ao efeito da metformina na dose de 0,1 mM durante o tratamento agudo. Além disso, foi possível a padronização de um modelo 3D de cocultura de células de câncer de endométrio que permanecesse viável ao longo dos 20 dias de cultivo. Contudo, apesar das células de câncer de endométrio apresentarem resistência ao efeito da metformina na presença de altas concentrações de glicose, a dose de 0,1 mM foi capaz de inibir o efeito da insulina e diminuir o potencial metastático dessas células, sugerindo que a metformina ao atuar clinicamente em combinação com seus efeitos indiretos e diretos pode ser um potencial agente adjuvante na terapia contra o câncer. / Endometrial cancer is one of the most common gynecological malignancies worldwide and is classified into a type I, which is estrogen-dependent, and a type II estrogen-independent form. The type I is the most common, accounting to 75%-85% of all cases of endometrial cancer. Elevated estrogen levels have been shown to increase the risk of endometrial cancer development, as estrogen stimulates endometrial cell proliferation and inhibits apoptosis. The insulin resistance seems to play a central role in endometrial carcinogenesis, furthermore, diseases associate with insulin resistance, as seen in polycystic ovary syndrome (PCOS), and obesity, as well as type II diabetes mellitus (DM) are considered as significant risk factors for the development and progression of type I endometrial cancer. Additionally, PCOS patients may have an insulin resistance independent of obesity remaining in a normoglycemic status. At this case, the hypeinsulinemia seems to be the promoter factor not only for the development but also for the cancer progression. However, also increased blood glucose levels are contributing to the growth and carcinogenesis in endometrial cancer and are acting as a critical link between the observed increased cancer risk in patients with type II DM. Therefore, the treatment with insulin-sensitizing agents that act through reducing insulin levels, could offer a general approach to prevent the development of cancer and reduce metastasis The aim of this study was to evaluate the effect of 0.1 mM metformin dose on the proliferative and metastatic potential of endometrial cancer cells, as well as, analyze the effects of short and long-term treatment on intracellular signaling pathways related to endometrial cancer development and progression when exposed to an environment with different glucose concentrations and high insulin levels. Additionally, the endometrial three-dimensional (3D) coculture standardization to remain viable over 20 culture days. The proliferative potential was performed by using CellTitle Glo assay, and the metastatic potential was performed by using transwell migration and invasion assay. The mRNA expression of MKI67, mTOR, NOTCH1, NOTCH3 and JAG1 genes were measured by real time PCR. The cumulative population doubling rate was evaluated to determine the replication behavior of an endometrial cancer cell line throughout 20 days of treatment period. In all assays the cells were cultured in medium containing normal (5.5 mM) or high (17 mM) glucose concentration, and treated in different groups: control, insulin,metformin or combined treatment The 3D coculture model was established by using endometrial primary cells and an endometrium cancer cell line, to the model construction Matrigel® was used as an extracellular matrix. In this study, the 0.1 mM metformin dose potently inhibited the insulin action, decreasing the ability of the endometrial cancer cell line to migrate and invade in a high and normal glucose environment. On the proliferative potential this effect was not observed, however, relative cell proliferation sensitivity to metformin was observed in the range between 1 and 5 mM regardless of the present glucose concentration. In the intracellular molecular mechanisms, it was observed that the high glucose concentration creates an optimal environment for endometrial cancer cells to exhibit a more aggressive genotype and resistance to metformin during a long-term treatment. Moreover, the endometrial 3D coculture model remained viable throughout 20 culture days. Therefore, despite of endometrial cancer cells show resistance to the metformin effect when exposed to high glucose environment, the 0.1 mM metformin dose was able to inhibit the insulin action and decrease the metastatic potential of the cells, suggesting that metformin is acting clinically in combination with indirectly and direct effects could emerge as a potential agent in cancer therapy.

Endometriumkrebs ist eine der häufigsten gynäkologischen Malignomen weltweit und wird in einen Typ I eingeteilt, welcher östrogenabhängig ist, und in eine Typ-II-Östrogen-unabhängige Form. Typ I ist der häufigste Fall und kommt in etwa für 75% bis 85% aller diagnostizierten Fälle in Frage. Erhöhte Östrogenspiegel haben gezeigt, das Risiko von Gebärmutterkrebsentwicklung zu erhöhen, genauso wie Östrogen die Proliferation von Endometriumzellen stimuliert und die Apoptose hemmt. Die Insulinresistenz scheint eine zentrale Rolle in der endometrialen Karzinogenese zu spielen und darüber hinaus werden Erkrankungen mit Insulinresistenz, wie zum Beispiel das polyzystische Ovarialsyndrom (PCOS) und Adipositas, sowie Typ II-Diabetes mellitus (DM) als signifikantes Risiko angesehen, Faktoren für die Entwicklung und Progression von Typ-I-Endometrium-Krebs zu sein. Zusätzlich können PCOS-Patienten durch eine Fettleibigkeit in einem normoglykämischen Status eine unabhängige Insulin-Resistenz haben. In diesem Fall scheint die Hyperinsulinämie der fördernde Faktor zu sein, nicht nur für die Entwicklung als auch für die Tumorprogression.Aber auch erhöhte Blutzuckerspiegel tragen zum Wachstum und die Karzinogenese in Endometriumkarzinom bei und dienen als wichtige Verbindung zwischen dem beobachteten erhöhten Krebsrisiko bei Patienten mit Typ-II-DM. Die Behandlung mit einem Anti-Diabetikum, welches den Insulinspiegel senken kann, könnte einen allgemeinen Ansatz bieten gegen die Entwicklung von Krebs und zur Verringerung der Metastasierung. Das Ziel dieser Studie war es, die Wirkung einer 0,1 mM Metformin-Dosis auf das proliferative und metastatische Potential von Endometriumkrebszellen bewerten zu können, sowie die Analyse der Auswirkungen von kurz- und langfristigen Behandlungen auf intrazelluläre Signalwege der Endometriumkrebszellen.(Fortsetzung) (Fortsetzung)Ebenso soll der Zusammenhang der Entwicklung und der Progression von Krebszellen untersucht werden, wenn sie einer Umgebung mit unterschiedlichen Glucosekonzentrationen und hohen Insulinspiegeln ausgesetzt werden. Darüber hinaus ist eine endometriale dreidimensionale (3D) Cokultur zu standardisieren, für eine viabele Kultur bei 20 Tagen Kultivierung. Das proliferative Potential wurde unter Verwendung des CellTitle-Glo-Tests durchgeführt, und das metastatische Potential wurde unter Verwendung von Transwell-Migration und Invasion untersucht. Die mRNAExpression von MKI67, mTOR, NOTCH1, NOTCH3 und JAG1 Gene wurden durch real-time PCR gemessen. Die kumulative Populationsverdopplungsrate wurde durch das Replikationsverhalten einer Endometriumkrebszelllinie durchgehend von 20 Tagen nach einer Behandlungsdauer bestimmt. In allen Assays wurden die Zellen durch Medien mit normaler (5,5 mM) oder hoher (17 mM) Glucosekonzentration, sowie in verschiedenen Gruppen behandelt: Kontrolle, Insulin, Metformin und Insulin+Metformin. Das 3D-Kokulturmodell wurde unter Verwendung von endometrialen Primärzellen und einer Endometriumkrebszelllinie hergestellt, wobei die Modellkonstruktion durch Matrigel® als extrazelluläre Matrix verwendet wurde. In dieser Studie hemmte die 0,1 mM Metformin-Dosis die Insulinwirkung stark und verringerte die Fähigkeit der endometrialen Krebszelllinie, in einer hohen und normalen Glukoseumgebung zu migrieren und einzudringen.(Fortsetzung) (Fortsetzung) Auf das proliferative Potential wurde dieser Effekt nicht beobachtet, allerdings reagierte die relative Zellproliferation empfindlich auf Metformin im Bereich zwischen 1 und 5 mM, unabhängig von der vorliegenden Glucosekonzentration. In den intrazellulären molekularen Mechanismen wurde beobachtet, dass die hohe Glukosekonzentration eine optimale Umgebung für endometriale Krebszellen schafft, um einen aggressiveren Genotyp und eine Resistenz gegenüber Metformin während einer Langzeitbehandlung zu zeigen. Darüber hinaus blieb das endometriale 3DKokulturmodell über 20 Kulturtage lebensfähig. Daher zeigte sich, trotz der Endometriumkrebszellen, eine Resistenz gegenüber dem Metformin-Effekt, wenn sie einer hohen Glucoseumgebung ausgesetzt waren. Die 0,1 mM Metformin-Dosis war in der Lage, die Insulinwirkung zu hemmen und das metastatische Potential der Zellen zu verringern, was darauf hindeutet, dass Metformin klinisch in Verbindung mit Insulin wirkt, sowie die indirekten und direkten Effekte als potentieller Wirkstoff in der Krebstherapie eingesetzt werden könnten. / O câncer de endométrio é uma das neoplasias ginecológicas com maior incidência, classificado como tipo I, estrógeno dependente, e tipo II, estrógeno nãodependente. O tipo I é a forma mais comum, ocorrendo em torno de 75 – 85 % dos casos de câncer de endométrio. Altos níveis de estrogênio têm sido relacionados ao aumento do risco de desenvolvimento do câncer de endométrio, pois estimula a proliferação celular e inibe a apoptose. A resistência à insulina parece desempenhar um papel central nesta neoplasia, e as doenças associadas à resistência à insulina como obesidade, Diabetes Mellitus (DM) tipo II e Síndrome dos Ovários Policísticos (PCOS) também são consideradas fatores de risco significantes para o desenvolvimento e progressão do câncer de endométrio tipo I. Adicionalmente, pacientes com PCOS podem apresentar um quadro de resistência à insulina independente de obesidade, permanecendo em um estado glicêmico normal. Neste caso, a hiperinsulinemia isolada seria um fator tanto para a promoção, como também para a progressão do câncer. Entretanto, o aumento de níveis séricos de glicose, a hiperglicemia, também é considerada um fator independente para o desenvolvimento e progressão do câncer de endométrio sendo um elo crítico entre o aumento do risco do desenvolvimento de câncer observado em pacientes com DM tipo II Dessa forma, tratamento utilizando agentes insulino-sensibilizantes, que atuam diminuindo a resistência à insulina e consequentemente reduzindo seus níveis pode ser uma estratégia interessante para prevenir o câncer e reduzir a disseminação metastática. Os objetivos deste trabalho foram avaliar o efeito da dose de 0,1 mM de metformina sobre o potencial proliferativo e metastático das células de câncer de endométrio, assim como, avaliar o efeito do tratamento a curto e a longo prazo sobre vias de sinalização intracelular relacionadas ao desenvolvimento e progressão do câncer de endométrio quando exposta a um ambiente com diferentes concentrações de glicose e níveis elevados de insulina. Por fim, a padronização de um modelo tridimensional (3D) de cocultura de células de endométrio que permanecesse viável ao longo de 20 dias de cultivo. O potencial proliferativo foi determinado pelo método luminescente CellTitle Glo, e o potencial metastático pelo o ensaio transwell de migração e invasão. Análises de expressão do mRNA dos genes MKI67, mTOR, NOTCH1, NOTCH3 e JAG1 foram realizadas a partir da técnica de PCR em tempo real. O índice de duplicação populacional cumulativo das células determinou o comportamento de replicação da linhagem de câncer de endométrio ao longo do período de tratamento de 20 dias. Em todas os ensaios as células foram cultivadas em meio contendo concentrações normais (5,5 mM) ou altas (17 mM) de glicose, e divididas nos diferentes grupos de tratamento: controle, insulina, metformina e metformina associado a insulina. A padronização do modelo 3D de cocultura de células de endométrio foi realizado utilizando células primárias e células de linhagem de câncer de endométrio, a Matrigel® foi a matriz extracelular temporária utilizada para a construção do modelo. Neste estudo, a concentração de 0,1 mM de metformina inibiu a ação da insulina, diminuindo a habilidade de migração e invasão das células de câncer de endométrio independente da concentração de glicose presente no meio. Entretanto, este efeito não foi observado sobre o potencial proliferativo, sendo observada uma redução da proliferação das células de câncer de endométrio ao serem utilizadas concentrações maiores de metformina. Em relação aos mecanismos moleculares intracelulares, foi observado que na presença de altas concentrações de glicose as células de câncer de endométrio adquirem um genótipo mais agressivo e apresentam resistência ao efeito da metformina na dose de 0,1 mM durante o tratamento agudo. Além disso, foi possível a padronização de um modelo 3D de cocultura de células de câncer de endométrio que permanecesse viável ao longo dos 20 dias de cultivo. Contudo, apesar das células de câncer de endométrio apresentarem resistência ao efeito da metformina na presença de altas concentrações de glicose, a dose de 0,1 mM foi capaz de inibir o efeito da insulina e diminuir o potencial metastático dessas células, sugerindo que a metformina ao atuar clinicamente em combinação com seus efeitos indiretos e diretos pode ser um potencial agente adjuvante na terapia contra o câncer. / Endometrial cancer is one of the most common gynecological malignancies worldwide and is classified into a type I, which is estrogen-dependent, and a type II estrogen-independent form. The type I is the most common, accounting to 75%-85% of all cases of endometrial cancer. Elevated estrogen levels have been shown to increase the risk of endometrial cancer development, as estrogen stimulates endometrial cell proliferation and inhibits apoptosis. The insulin resistance seems to play a central role in endometrial carcinogenesis, furthermore, diseases associate with insulin resistance, as seen in polycystic ovary syndrome (PCOS), and obesity, as well as type II diabetes mellitus (DM) are considered as significant risk factors for the development and progression of type I endometrial cancer. Additionally, PCOS patients may have an insulin resistance independent of obesity remaining in a normoglycemic status. At this case, the hypeinsulinemia seems to be the promoter factor not only for the development but also for the cancer progression. However, also increased blood glucose levels are contributing to the growth and carcinogenesis in endometrial cancer and are acting as a critical link between the observed increased cancer risk in patients with type II DM. Therefore, the treatment with insulin-sensitizing agents that act through reducing insulin levels, could offer a general approach to prevent the development of cancer and reduce metastasis The aim of this study was to evaluate the effect of 0.1 mM metformin dose on the proliferative and metastatic potential of endometrial cancer cells, as well as, analyze the effects of short and long-term treatment on intracellular signaling pathways related to endometrial cancer development and progression when exposed to an environment with different glucose concentrations and high insulin levels. Additionally, the endometrial three-dimensional (3D) coculture standardization to remain viable over 20 culture days. The proliferative potential was performed by using CellTitle Glo assay, and the metastatic potential was performed by using transwell migration and invasion assay. The mRNA expression of MKI67, mTOR, NOTCH1, NOTCH3 and JAG1 genes were measured by real time PCR. The cumulative population doubling rate was evaluated to determine the replication behavior of an endometrial cancer cell line throughout 20 days of treatment period. In all assays the cells were cultured in medium containing normal (5.5 mM) or high (17 mM) glucose concentration, and treated in different groups: control, insulin,metformin or combined treatment The 3D coculture model was established by using endometrial primary cells and an endometrium cancer cell line, to the model construction Matrigel® was used as an extracellular matrix. In this study, the 0.1 mM metformin dose potently inhibited the insulin action, decreasing the ability of the endometrial cancer cell line to migrate and invade in a high and normal glucose environment. On the proliferative potential this effect was not observed, however, relative cell proliferation sensitivity to metformin was observed in the range between 1 and 5 mM regardless of the present glucose concentration. In the intracellular molecular mechanisms, it was observed that the high glucose concentration creates an optimal environment for endometrial cancer cells to exhibit a more aggressive genotype and resistance to metformin during a long-term treatment. Moreover, the endometrial 3D coculture model remained viable throughout 20 culture days. Therefore, despite of endometrial cancer cells show resistance to the metformin effect when exposed to high glucose environment, the 0.1 mM metformin dose was able to inhibit the insulin action and decrease the metastatic potential of the cells, suggesting that metformin is acting clinically in combination with indirectly and direct effects could emerge as a potential agent in cancer therapy.