Hepatocellular carcinoma (HCC) is one of deadliest cancers worldwide and ranking the third among all cancer-related mortalities. Current effective therapeutic approaches for HCC include surgical resection and trans-arterial embolization (TAE). Chemotherapy remains largely ineffective, and most popular used agents are epirubicin, doxorubicin, cisplatin and 5-FU. Besides, these chemotherapic drugs had potential serious side-effects such as low blood count, hair loss, vomiting, and they rarely present good anti-HCC effect in clinical practice. Our previous studies found that epirubicin injection attenuated the tumor burden of orthotopic Novikoff hepatoma, but caused serious side effects to hosts including reduction in spleen weight, white count, and body weight and high GOT level. Therefore, we aimed to evaluate possible alternative treatment such as COX-2 inhibitor for HCC. Celecoxib is a highly selective COX-2 inhibitor and less toxic than the traditional non-selective NSAIDs. Celecoxib showed relatively low cytotoxicity in Novikoff N1-S1 hepatoma cells and Clone 9 normal hepatocytes with an IC50 of up to 100 microM. Expression analysis revealed that COX-2 expression is very low in N1-S1 cells at protein and mRNA levels. Thus, N1-S1 is a kind of hepatoma cell line with low COX-II level. Interestingly, celecoxib upregulated PTEN expression and decreased AKT phosphorylation in vitro by COX-2 independent pathway, and then oral administration of celecoxib (30 mg/kg) for 7 days showed tendency of tumor suppression of Novikoff hepatoma in rats revealed by ultrasound and computed tomography (CT) scan. Histological analysis revealed that CD31-positive neo-vascularization¡BKi-67-positive cell-proliferation and FOXP3-positive regulatory T cells were found to reduce in celecoxib-treated rats, and then TUNEL-positive apoptotic cells were found to increase in celecoxib-treated rats. Besides, celecoxib-treated rats exhibited no significant side effect. Therefore, oral celecoxib may be a suitable chose of adjuvant therapy in combination with epirubicin or other chemotherapeutic agents for the treatment of HCC.

No description available.

ABSTRACT Vascular permeability and angiogenesis are crucial events in the rodent and human uterus in early pregnancy and are regulated by vascular endothelial growth factor (VEGF) and prostaglandins liberated from arachidonic acid by cyclooxygenase 2 (COX2). These events coincide with the typical morphological features of the receptive uterus and are regulated by synchronized release of ovarian hormones (oestrogen and progesterone). However, administration of follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG), commonly used in assisted reproduction, affect the synchrony of the hormonal milieu, particularly by increasing oestrogen levels. This causes detrimental changes to the uterine morphology and affects vascular permeability at the site of implantation. In the present study, the expression of COX2 and VEGF was compared between control and hyperstimulated rat uteri during the peri-implantation period using immunohistochemistry and Western blot analysis. While in control pregnant rats COX2 and VEGF immunolocalization occurred in the luminal epithelial cells and stroma on consecutive days, strong immunolocalization of COX2 and VEGF occurred in the luminal epithelial cells but was inhibited in the stroma of the hyperstimulated rats. This appears to have resulted in the suppression of stromal decidualization and vascular permeability. Western blot analysis did not show any results. This may be due to low concentrations of the protein in the sample. Since vascular permeability and angiogenesis are critical to the process of implantation and are influenced by VEGF and COX2, disturbance of the pattern of these two proteins by hyperstimulation may contribute to the low implantation rate in IVF programes. immunohistochemistry and Western blot analysis. While in control pregnant rats COX2 and VEGF immunolocalization occurred in the luminal epithelial cells and stroma on consecutive days, strong immunolocalization of COX2 and VEGF occurred in the luminal epithelial cells but was inhibited in the stroma of the hyperstimulated rats. This appears to have resulted in the suppression of stromal decidualization and vascular permeability. Western blot analysis did not show any results. This may be due to low concentrations of the protein in the sample. Since vascular permeability and angiogenesis are critical to the process of implantation and are influenced by VEGF and COX2, disturbance of the pattern of these two proteins by hyperstimulation may contribute to the low implantation rate in IVF programes.

<p>Selenium (Se) is an essential trace element and metalloid involved in several key metabolic activities: protection against oxidative damage, regulation of immune and thyroid function, and fertility. Several recent lines of evidence from epidemiology, genetic, and transgenic animal studies suggest that Se may play a protective role in Osteoarthritis (OA). However, the exact protective mechanism of Se is still unclear. </p><p>In this study, we hypothesized that Se exerts its chondroprotective benefit via an anti-oxidative and anti-inflammatory effect mediated by specific selenoproteins that neutralize cytokine-induced inflammatory responses in chondrocytes. We established an in vitro system for studying the effect of Se in the chondrosarcoma cell line SW-1353 and in human primary chondrocytes. Selenomethionine (SeMet) induced gene expression and enzyme activity of both antioxidative enzymes glutathione peroxidase (GPX) and thioredoxin reductase (TR) in SW-1353 cells. Our data suggest that Se may be protective against oxidative stress through regulation of the activity of these antioxidative enzymes.</p><p>As IL-1&beta; is one of the primary pro-inflammatory cytokines contributing to the progression in OA, we next investigated the effect of Se on the gene expression induced by physiological doses of IL-1&beta;. SeMet inhibited IL-1&beta; induced catabolic gene expression of matrix metalloproteinase 1 (MMP1) and MMP13 as well as total MMP activity in chondrocytes. Similarly, SeMet inhibited chondrocyte gene expression of IL-1&beta; induced nitric oxide synthase (iNOS) and cyclooxygenase (COX2) with corresponding reductions in nitric oxide (NO) and prostaglandin E2 (PGE2) production. In addition, SeMet pretreatment attenuated the IL-1&beta; induced activation of p38 MAPK but not the ERK, JNK or NFkB pathways. Taken together, our results suggest that Se inhibits IL-1&beta; induced expression of inflammatory and catabolic genes, partly through inhibition of IL-1&beta; cell signaling. </p><p>Since Se may function through selenoproteins, we evaluated the role of three specific major selenoproteins, GPX1, TR1 and DIO2, in modifying the inflammatory response stimulated by IL-1&beta; in chondrocytes by RNA interference. Based on RNA interference results, DIO2 and TR1 mediated the inhibitory effect of SeMet on IL-1&beta; induced COX2 gene expression, while GPX1 did not show a significant inhibitory effect on Se. Depletion of DIO2 increased the IL-1&beta; induced COX2 gene expression. This suggests that DIO2 may negatively modulate the IL-1&beta; response. Our data also suggest that part of this inhibitory effect of DIO2 could be through regulation of IL-1&beta; gene expression itself. These results highlight a potential new role of DIO2 in modulating the inflammatory response in chondrocytes </p><p>In summary, the result of this study suggests that Se may exert its chondroprotective effect through specific selenoproteins which neutralize oxidative stress and modify the inflammatory response in chondrocytes.</p> / Dissertation

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation the wound is repaired, however this process, and the mechanisms to maintain OSE homeostasis after the wound is repaired are poorly understood. We have shown the mouse OSE (mOSE) contains a stem cell population that is expanded by Transforming Growth Factor Beta 1 (TGFB1), a factor present in follicular fluid. These data suggest that components in the follicular fluid such as TGFB1 may promote wound repair and OSE homeostasis through maintenance of the OSE stem cell population. Additionally, TGFB1 may promote wound repair through induction of an epithelial-to-mesenchymal transition (EMT) and activation of pro-survival pathways, as seen in other tissues. To elucidate the mechanism for TGFB1-mediated ovulatory wound repair, mOSE cells were treated with TGFB1, which induced an EMT seen with increased Snai1 expression and cell migration. Snai1 overexpression also increased cell migration and sphere formation (a stem cell characteristic). RNA sequencing results suggest this is at least in part through elevated collagen deposition in SNAI1 overexpressing cells. A TGFB signalling targets array identified Cox2 induction following TGFB1 treatment. Constitutive Cox2 expression did not promote an EMT, but enhanced sphere formation and cell survival. Finally, TGFB1 treatment decreased Brca1 expression, which when deleted from mOSE cells also increased sphere formation. RNA sequencing results suggest that Brca1 deletion promotes stemness through activation of the stem cell genes Ly6a and Lgr5. RNA sequencing was also used to compare mOSE cells cultured as monolayers and as spheroids, with and without TGFB1. These results validate our findings that TGFB1 promotes an EMT partially through Snail induction and the upregulation of Cox2. mOSE cells cultured as spheroids acquire a mesenchymal transcriptional profile that is further enhanced with TGFB1 treatment. These data suggest that TGFB1 may promote ovulatory wound repair and maintain OSE homeostasis through the induction of an EMT, maintenance of the stem cell population and activation of a pro-survival pathway. Interestingly, mOSE spheroids also decrease Brca1 expression and upregulate cancer associated genes such as Pax8 and Greb1. The induction of survival pathways, while simultaneously increasing stemness and repressing Brca1 could render cells more susceptible to transformation. This work provides novel insights as to why ovulation is the primary non-hereditary risk factor for ovarian cancer.

No description available.

L’implantation retardée ou diapause embryonnaire décrit l'arrêt ou le retardement pendant l'embryogenèse. Chez le vison, la diapause est corrélée avec une sécrétion pituitaire insuffisante de la prolactine, ayant pour résultat la différentiation incomplète du corpus luteum et réduction de la progestérone. Des études antérieures suggèrent que le blastocyste de vison en diapause demeure dans un état de quiescence ou se développe lentement. Pour élucider ceci, la réplication de l'ADN a été étudiée. Les résultats démontrent synthèse de l’ADN et prolifération cellulaire dans les embryons au stade de morula, avant la diapause et dans les blastocystes après la réactivation. La réplication de l'ADN a été également détectée dans des blastocystes en diapause et en diapause prolongée. L'implantation est considérée comme une interaction bidirectionnelle entre le blastocyste et l'utérus. Il a été montré que les prostaglandines sont importantes pour la vascularisation de l’utérus au moment de l’implantation et peuvent réactiver l'utérus des visons après la diapause. La concentration protéinique et la localisation de la phospholipase citosolique A2 (CPLA2) et de la cyclooxygenase 2 (COX2) ont été étudiées dans l'utérus de vison. L’expression de la CPLA2 et COX2 étaient sur-régulées au moment de l'implantation. Il est connu que la prolactine active les corpus luteum des visons. L'idée de un lien entre la prolactine et la voie de signalisation des prostaglandines a été testée en mesurant les récepteurs de prolactine. Les résultats montrent une augmentation de l’expression des récepteurs de prolactine à l'implantation suggérant que la prolactine pourrait activer la voie de prostaglandine à l'utérus par son propre récepteur. La conclusion, les embryons pendant la diapause ne sont pas arrêtées complètement et les protéines liées à la voie de prostaglandine sont implique dans la réactivation de l'utérus. / Delayed implantation or diapause describes arrest or retardation during embryogenesis. In mink, diapause is related to insufficient pituitary prolactin secretion, resulting in incomplete differentiation of the corpus luteum with reduced progesterone concentration. The mink blastocyst at diapause was believed to be totally quiescent or expanding at a low rate. To explore this, DNA replication was studied. Results showed synthesis of DNA, and thus cell proliferation at the morulae stage before diapause and at the blastocyst following activation. DNA replication was detected not only at diapause but also at extended diapause. Furthermore, implantation is considered as a two-way interaction between the blastocyst and the uterus. It has been shown that prostaglandins are important for vascularization of the uterus and products of the prostaglandin pathway could reactivate the mink uterus following diapause. Protein concentration and localization was studied for cytosolic phospholipase A2 (CPLA2) and cyclooxygenase 2 (COX2) in mink uterus. Expression of CPLA2 and COX2 was up regulated at implantation. It is know that prolactin is the factor that activates the mink corpus luteum. The idea of a link between prolactin and prostaglandin pathway was investigated by quantifying the prolactin receptors in the uterus. Results showed an increase of prolactin receptors at implantation suggesting that prolactin could activate the prostaglandin pathway at the uterus through its own receptor. In conclusion, embryos during diapause are not completely arrested, and proteins related to the prostaglandin pathway are implicated in reactivation of the uterus.

L’implantation retardée ou diapause embryonnaire décrit l'arrêt ou le retardement pendant l'embryogenèse. Chez le vison, la diapause est corrélée avec une sécrétion pituitaire insuffisante de la prolactine, ayant pour résultat la différentiation incomplète du corpus luteum et réduction de la progestérone. Des études antérieures suggèrent que le blastocyste de vison en diapause demeure dans un état de quiescence ou se développe lentement. Pour élucider ceci, la réplication de l'ADN a été étudiée. Les résultats démontrent synthèse de l’ADN et prolifération cellulaire dans les embryons au stade de morula, avant la diapause et dans les blastocystes après la réactivation. La réplication de l'ADN a été également détectée dans des blastocystes en diapause et en diapause prolongée. L'implantation est considérée comme une interaction bidirectionnelle entre le blastocyste et l'utérus. Il a été montré que les prostaglandines sont importantes pour la vascularisation de l’utérus au moment de l’implantation et peuvent réactiver l'utérus des visons après la diapause. La concentration protéinique et la localisation de la phospholipase citosolique A2 (CPLA2) et de la cyclooxygenase 2 (COX2) ont été étudiées dans l'utérus de vison. L’expression de la CPLA2 et COX2 étaient sur-régulées au moment de l'implantation. Il est connu que la prolactine active les corpus luteum des visons. L'idée de un lien entre la prolactine et la voie de signalisation des prostaglandines a été testée en mesurant les récepteurs de prolactine. Les résultats montrent une augmentation de l’expression des récepteurs de prolactine à l'implantation suggérant que la prolactine pourrait activer la voie de prostaglandine à l'utérus par son propre récepteur. La conclusion, les embryons pendant la diapause ne sont pas arrêtées complètement et les protéines liées à la voie de prostaglandine sont implique dans la réactivation de l'utérus. / Delayed implantation or diapause describes arrest or retardation during embryogenesis. In mink, diapause is related to insufficient pituitary prolactin secretion, resulting in incomplete differentiation of the corpus luteum with reduced progesterone concentration. The mink blastocyst at diapause was believed to be totally quiescent or expanding at a low rate. To explore this, DNA replication was studied. Results showed synthesis of DNA, and thus cell proliferation at the morulae stage before diapause and at the blastocyst following activation. DNA replication was detected not only at diapause but also at extended diapause. Furthermore, implantation is considered as a two-way interaction between the blastocyst and the uterus. It has been shown that prostaglandins are important for vascularization of the uterus and products of the prostaglandin pathway could reactivate the mink uterus following diapause. Protein concentration and localization was studied for cytosolic phospholipase A2 (CPLA2) and cyclooxygenase 2 (COX2) in mink uterus. Expression of CPLA2 and COX2 was up regulated at implantation. It is know that prolactin is the factor that activates the mink corpus luteum. The idea of a link between prolactin and prostaglandin pathway was investigated by quantifying the prolactin receptors in the uterus. Results showed an increase of prolactin receptors at implantation suggesting that prolactin could activate the prostaglandin pathway at the uterus through its own receptor. In conclusion, embryos during diapause are not completely arrested, and proteins related to the prostaglandin pathway are implicated in reactivation of the uterus.

Made available in DSpace on 2015-04-01T14:16:01Z (GMT). No. of bitstreams: 1 ArquivoTotal.pdf: 1987821 bytes, checksum: 0557f7c6083cf4200e6fc6542c383ea7 (MD5) Previous issue date: 2013-05-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cancer is a disease with a high mortality rate in Brazil, including, stomach cancer is currently the fourth most common type of cancer worldwide, responsible for countless deaths. Its etiology is multifactorial, because studies suggest associations to various factors such as dietary habits, environmental factors, genetic and epigenetic factors and gastric infection by Helicobacter pylori. Epigenetic changes such as methylation of the promoter regions of genes involved in cellular homeostasis may contribute to gastric carcinogenesis. To verify the methylation status of THBS1, GPX3 and COX2 genes and to evaluate their association with H. pylori in gastric adenocarcinomas, Methylation-Sensitive Restriction Enzyme PCR (MSRE-PCR) assay was performed in 39 gastric carcinomas (intestinal and diffuse types) and 15 normal stomach tissue samples. The presence of H. pylori was performed by amplification of the fragment of the 16S rRNA. Statistical analysies were performed using Fisher s exact test. The hypermethylation of GPX3, THBS1 and COX2 occurred in 18% (n = 7), 5% (n = 2) 36% (n = 14) of gastric cancer samples, respectively, whereas in normal samples was found in 13%, 7% and 67%. The presence of H. pylori was detected in 67% of gastric cancer samples and 67% in normal gastric samples. No correlation was found between the methylation profile of the studied samples and clinicopathological variables and the presence of H. pylori (P ≥ 0,05). The presence of H. pylori in gastric cancer samples and normal was not associated with clinicopathologic variables analyzed (P> 0.05). / O câncer é uma doença com alta taxa de mortalidade no Brasil, dentre eles, o câncer de estômago constitui atualmente, o quarto tipo de câncer mais comum a nível mundial. No ano de 2012, estimam-se, para o Brasil, 12.670 casos novos de câncer do estômago em homens e 7.420 em mulheres. A sua etiologia é multifatorial, pois estudos sugerem associações a diversos fatores como: hábitos alimentares, fatores ambientais, fatores genéticos e epigenéticos e a infecção gástrica por Helicobacter pylori. Alterações epigenéticas tais como a metilação das regiões promotoras de genes envolvidos na homeostase celular podem contribuir para carcinogênese gástrica. Para verificar o estado de metilação de genes THBS1, GPX3 e COX2 e avaliar a sua associação com a Helicobacter pylori (H. pylori) em adenocarcinomas gástricos, Methylation-Sensitive Restriction Enzyme PCR (MSRE-PCR) foi realizada em 39 carcinomas gástricos (intestinal e tipo difusa) e 15 amostras de tecido normal do estômago. A presença de H. pylori foi realizada por amplificação de um fragmento de rRNA 16S. Analysies estatísticas foram realizadas utilizando o teste exato de Fisher. A hipermetilação de GPX3, THBS1 e COX2 ocorreu em 18% (n = 7), 5% (n = 2) 36% (n = 14) das amostras de câncer gástrico, respectivamente, ao passo que em amostras normais foi encontrada em 13%, 7 % e 67%. A presença de H. pylorifoi detectada em 67% das amostras de câncer gástrico e 67% em amostras gástricas normais. Não foi encontrada correlação entre o perfil de metilação das amostras estudadas com variáveis clínico-patológicas e com presença de H. pylori (P > 0,05). A presença de H. pylori nas amostras de câncer gástrico e normais não foi associada com as variáveis clínico-patológicas analisadas (P > 0.05).

Nous montrons pour la première fois qu’en plus de la MAPK ERK1/2, l’endothéline-1 (ET-1) via les récepteurs ETA et ETB active une autre MAP kinase : la p38 uniquement dans les cellules de léiomyomes utérins de rate (ELT3) mais pas dans les cellules myométriales saines. Dans les cellules ELT3, l’analyse des voies de signalisation montre que malgré les similitudes observées entre les modes d’activation des voies p38 et ERK1/2 par ET-1, celles-ci sont activées de façon indépendante l’une de l’autre. En plus, la forskoline active p38 (mais pas ERK1/2), par contre l’activation de p38 par ET-1 n’implique pas une production d’AMPc. Par ailleurs ERK1/2 et p38 coactivées par ET-1 coopèrent pour augmenter l’expression de COX2 et la production des prostaglandines E2 (PGE2) pour favoriser l’effet antiapoptotique de ET-1. De plus p38 activée par ET-1 contribue à la prolifération des léiomyomes. Nos résultats élucident les mécanismes par lesquels ET-1 contribue à la croissance des léiomyomes. / We demonstrated for the first time, that in addition to the MAPK ERK1/2, Endothelin-1 (ET-1) through ETA and ETB receptors activated another MAP kinase: p38 only in uterine leiomyoma cells (ELT3) but not in normal myometrial cells. In ELT3 cells, analysis of signaling pathways showed that, despite the similarities between the mechanisms involved in the activation of p38 and ERK1/2 pathways by ET-1, these kinases are activated independently one of another. In addition, forskolin (a cAMP inducer), activated p38 (but not ERK1/2), whereas the activation of p38 by ET-1 did not involve production cAMP. Moreover the coactivated ERK1/2 and p38 pathways by ET-1 cooperated to increase expression of COX2 and prostaglandin E2 (PGE2) production. This PGE2 like ET-1 exerted an antiapoptotic effect in ELT3 cells. Furthermore, p38 activated by ET-1 contributes to the proliferation of ELT3 leiomyoma cells. Our data highlight the mechanisms by which ET-1 could promote uterine leiomyoma growth.