Introdução: O carcinoma hepatocelular (CHC) é o câncer primário de fígado mais comum e está associado com a segunda menor taxa de sobrevida em 5 anos de todos os tipos de tumores. A melatonina (Mel) é uma potente molécula antioxidante que se tem mostrado benéfica em diversas situações patológicas, incluindo o CHC. Objetivo: Avaliar o efeito da Mel sobre marcadores de estresse oxidativo e angiogênicos no tecido hepático de ratos Wistar no modelo experimental de carcinogênese hepática induzida por dietilnitrosamina (DEN) associado ao acetilaminofluoreno (2-AAF). Métodos: 32 ratos machos Wistar (150g) foram divididos em 4 grupos: Controle (CO); Controle+Mel (CO+Mel); DEN e DEN+Mel. O DEN (50mg/kg) foi administrado por via intraperitoneal duas/uma vez por semana, associado a uma única dose de 2-AAF (100mg/kg). A Mel foi administrada na água de beber dos animais na concentração final de 20 mg/L e o tratamento teve início na 12ª semana perdurando até o fim das 19 semanas de experimento. O sangue dos animais foi coletado para as análises de AST, ALT, FA, γ-GT e amostras de fígado utilizadas para avaliar a lipoperoxidação (LPO), a atividade das enzimas antioxidantes (CAT, GPx e GST), os níveis de GSH e de metabólitos do óxido nítrico, a análise histógica e as proteínas envolvidas na angiogênese tumoral (VEGF, PI3K, p-Akt e eNOS). Resultados: O dano tecidual e o processo fibrogênico presentes no parênquima hepático estavam diminuídos no grupo DEN+Mel, assim como o nível de TBARS e a atividade da enzima GST quando comparados ao grupo DEN. A atividade da CAT mostrou-se aumentada no grupo DEN+Mel quando comparada ao grupo DEN. No processo angiogênio, a expressão de VEGF, PI3K, p-Akt mostrou-se diminuída no grupo DEN+Mel enquanto a expressão da eNOS apresentou-se aumentada quando comparado ao grupo DEN. Conclusão: Constatamos que a Mel foi capaz de minimizar os danos no parênquima hepático, de diminuir a LPO, modular a atividade da CAT, além de mostrar-se eficaz na redução de VEGF e da via PI3K/Akt no modelo experimental de carcinogênese hepática. / Background: Hepatocellular carcinoma (CHC) is the most common primary liver cancer and is associated with the second lowest 5-year survival rate of all tumor types. Melatonin (Mel) is a powerful antioxidant molecule that has been demonstrated to be beneficial in various pathological conditions, including HCC. Objective: The aim of this study was to evaluate the effect of Mel on oxidative stress and angiogenic markers in liver tissue of Wistar rats in the experimental model of hepatic carcinogenesis induced by diethylnitrosamine (DEN) and acetylaminofluorene (2-AAF). Methods: 32 male Wistar rats (150g) were divided into 4 groups: Control (CO); Control+Mel (CO+Mel); DEN and DEN+Mel. DEN (50mg/kg) was administered intraperitoneally once or twice a week, associated with a single dose of 2-AAF (100mg/kg). Mel was given in drinking water at the final concentration of 20 mg/L and the treatment was started at 12th week and continued until the end of the 19 weeks of experiment. Blood samples were collected for AST, ALT, AP, γ-GT and liver samples were used to evaluate lipid peroxidation (LPO), activity of antioxidant enzymes (CAT, GPx and GST), levels of GSH and nitric oxide levels, histological analysis and expression of proteins involved in tumor angiogenesis (VEGF, PI3K, p-Akt and eNOS). Results: The tissue damage and the fibrogenic process present in the hepatic parenchyma were decreased as well as the levels of TBARS and the activity of GST in the group DEN+Mel when compared to DEN group. CAT activity was increased in DEN+Mel group when we compared with DEN group. The expression of VEGF, PI3K, p-Akt was decreased in DEN+Mel group while eNOS expression was increased when compared to DEN group. Conclusion: Mel was able to minimize damage in the hepatic parenchyma, reduce LPO, modulate the activity of CAT and reduce VEGF and the PI3K/Akt pathway in a experimental model of hepatic carcinogenesis.

Introdução: O carcinoma hepatocelular (CHC) é o câncer primário de fígado mais comum e está associado com a segunda menor taxa de sobrevida em 5 anos de todos os tipos de tumores. A melatonina (Mel) é uma potente molécula antioxidante que se tem mostrado benéfica em diversas situações patológicas, incluindo o CHC. Objetivo: Avaliar o efeito da Mel sobre marcadores de estresse oxidativo e angiogênicos no tecido hepático de ratos Wistar no modelo experimental de carcinogênese hepática induzida por dietilnitrosamina (DEN) associado ao acetilaminofluoreno (2-AAF). Métodos: 32 ratos machos Wistar (150g) foram divididos em 4 grupos: Controle (CO); Controle+Mel (CO+Mel); DEN e DEN+Mel. O DEN (50mg/kg) foi administrado por via intraperitoneal duas/uma vez por semana, associado a uma única dose de 2-AAF (100mg/kg). A Mel foi administrada na água de beber dos animais na concentração final de 20 mg/L e o tratamento teve início na 12ª semana perdurando até o fim das 19 semanas de experimento. O sangue dos animais foi coletado para as análises de AST, ALT, FA, γ-GT e amostras de fígado utilizadas para avaliar a lipoperoxidação (LPO), a atividade das enzimas antioxidantes (CAT, GPx e GST), os níveis de GSH e de metabólitos do óxido nítrico, a análise histógica e as proteínas envolvidas na angiogênese tumoral (VEGF, PI3K, p-Akt e eNOS). Resultados: O dano tecidual e o processo fibrogênico presentes no parênquima hepático estavam diminuídos no grupo DEN+Mel, assim como o nível de TBARS e a atividade da enzima GST quando comparados ao grupo DEN. A atividade da CAT mostrou-se aumentada no grupo DEN+Mel quando comparada ao grupo DEN. No processo angiogênio, a expressão de VEGF, PI3K, p-Akt mostrou-se diminuída no grupo DEN+Mel enquanto a expressão da eNOS apresentou-se aumentada quando comparado ao grupo DEN. Conclusão: Constatamos que a Mel foi capaz de minimizar os danos no parênquima hepático, de diminuir a LPO, modular a atividade da CAT, além de mostrar-se eficaz na redução de VEGF e da via PI3K/Akt no modelo experimental de carcinogênese hepática. / Background: Hepatocellular carcinoma (CHC) is the most common primary liver cancer and is associated with the second lowest 5-year survival rate of all tumor types. Melatonin (Mel) is a powerful antioxidant molecule that has been demonstrated to be beneficial in various pathological conditions, including HCC. Objective: The aim of this study was to evaluate the effect of Mel on oxidative stress and angiogenic markers in liver tissue of Wistar rats in the experimental model of hepatic carcinogenesis induced by diethylnitrosamine (DEN) and acetylaminofluorene (2-AAF). Methods: 32 male Wistar rats (150g) were divided into 4 groups: Control (CO); Control+Mel (CO+Mel); DEN and DEN+Mel. DEN (50mg/kg) was administered intraperitoneally once or twice a week, associated with a single dose of 2-AAF (100mg/kg). Mel was given in drinking water at the final concentration of 20 mg/L and the treatment was started at 12th week and continued until the end of the 19 weeks of experiment. Blood samples were collected for AST, ALT, AP, γ-GT and liver samples were used to evaluate lipid peroxidation (LPO), activity of antioxidant enzymes (CAT, GPx and GST), levels of GSH and nitric oxide levels, histological analysis and expression of proteins involved in tumor angiogenesis (VEGF, PI3K, p-Akt and eNOS). Results: The tissue damage and the fibrogenic process present in the hepatic parenchyma were decreased as well as the levels of TBARS and the activity of GST in the group DEN+Mel when compared to DEN group. CAT activity was increased in DEN+Mel group when we compared with DEN group. The expression of VEGF, PI3K, p-Akt was decreased in DEN+Mel group while eNOS expression was increased when compared to DEN group. Conclusion: Mel was able to minimize damage in the hepatic parenchyma, reduce LPO, modulate the activity of CAT and reduce VEGF and the PI3K/Akt pathway in a experimental model of hepatic carcinogenesis.

Introdução: O carcinoma hepatocelular (CHC) é o câncer primário de fígado mais comum e está associado com a segunda menor taxa de sobrevida em 5 anos de todos os tipos de tumores. A melatonina (Mel) é uma potente molécula antioxidante que se tem mostrado benéfica em diversas situações patológicas, incluindo o CHC. Objetivo: Avaliar o efeito da Mel sobre marcadores de estresse oxidativo e angiogênicos no tecido hepático de ratos Wistar no modelo experimental de carcinogênese hepática induzida por dietilnitrosamina (DEN) associado ao acetilaminofluoreno (2-AAF). Métodos: 32 ratos machos Wistar (150g) foram divididos em 4 grupos: Controle (CO); Controle+Mel (CO+Mel); DEN e DEN+Mel. O DEN (50mg/kg) foi administrado por via intraperitoneal duas/uma vez por semana, associado a uma única dose de 2-AAF (100mg/kg). A Mel foi administrada na água de beber dos animais na concentração final de 20 mg/L e o tratamento teve início na 12ª semana perdurando até o fim das 19 semanas de experimento. O sangue dos animais foi coletado para as análises de AST, ALT, FA, γ-GT e amostras de fígado utilizadas para avaliar a lipoperoxidação (LPO), a atividade das enzimas antioxidantes (CAT, GPx e GST), os níveis de GSH e de metabólitos do óxido nítrico, a análise histógica e as proteínas envolvidas na angiogênese tumoral (VEGF, PI3K, p-Akt e eNOS). Resultados: O dano tecidual e o processo fibrogênico presentes no parênquima hepático estavam diminuídos no grupo DEN+Mel, assim como o nível de TBARS e a atividade da enzima GST quando comparados ao grupo DEN. A atividade da CAT mostrou-se aumentada no grupo DEN+Mel quando comparada ao grupo DEN. No processo angiogênio, a expressão de VEGF, PI3K, p-Akt mostrou-se diminuída no grupo DEN+Mel enquanto a expressão da eNOS apresentou-se aumentada quando comparado ao grupo DEN. Conclusão: Constatamos que a Mel foi capaz de minimizar os danos no parênquima hepático, de diminuir a LPO, modular a atividade da CAT, além de mostrar-se eficaz na redução de VEGF e da via PI3K/Akt no modelo experimental de carcinogênese hepática. / Background: Hepatocellular carcinoma (CHC) is the most common primary liver cancer and is associated with the second lowest 5-year survival rate of all tumor types. Melatonin (Mel) is a powerful antioxidant molecule that has been demonstrated to be beneficial in various pathological conditions, including HCC. Objective: The aim of this study was to evaluate the effect of Mel on oxidative stress and angiogenic markers in liver tissue of Wistar rats in the experimental model of hepatic carcinogenesis induced by diethylnitrosamine (DEN) and acetylaminofluorene (2-AAF). Methods: 32 male Wistar rats (150g) were divided into 4 groups: Control (CO); Control+Mel (CO+Mel); DEN and DEN+Mel. DEN (50mg/kg) was administered intraperitoneally once or twice a week, associated with a single dose of 2-AAF (100mg/kg). Mel was given in drinking water at the final concentration of 20 mg/L and the treatment was started at 12th week and continued until the end of the 19 weeks of experiment. Blood samples were collected for AST, ALT, AP, γ-GT and liver samples were used to evaluate lipid peroxidation (LPO), activity of antioxidant enzymes (CAT, GPx and GST), levels of GSH and nitric oxide levels, histological analysis and expression of proteins involved in tumor angiogenesis (VEGF, PI3K, p-Akt and eNOS). Results: The tissue damage and the fibrogenic process present in the hepatic parenchyma were decreased as well as the levels of TBARS and the activity of GST in the group DEN+Mel when compared to DEN group. CAT activity was increased in DEN+Mel group when we compared with DEN group. The expression of VEGF, PI3K, p-Akt was decreased in DEN+Mel group while eNOS expression was increased when compared to DEN group. Conclusion: Mel was able to minimize damage in the hepatic parenchyma, reduce LPO, modulate the activity of CAT and reduce VEGF and the PI3K/Akt pathway in a experimental model of hepatic carcinogenesis.

<p>Mammalian development is orchestrated by global transcriptional changes, which drive cellular differentiation, giving rise to diverse cell types. The mechanisms that mediate the temporal control of early differentiation can be studied using embryonic stem cell (ESCs) and embryonal carcinoma cells (ECCs) as model systems. In these stem cells, differentiation signals induce transcriptional repression of genes that maintain pluripotency (PpG) and activation of genes required for lineage specification. Expression of PpGs is controlled by these genes’ proximal and distal regulatory elements, promoters and enhancers, respectively. Previously published work from our laboratory showed that during differentiation of ESCs, the repression of PpGs is accompanied by enhancer silencing mediated by the Lsd1/Mi2-NuRD-Dnmt3a complex. The enzymes in this complex catalyze histone H3K27Ac deacetylation and H3K4me1/2 demethylation followed by a gain of DNA methylation mediated by the DNA methyltransferase, Dnmt3a. The absence of these chromatin changes at PpG enhancers during ESC differentiation leads to their incomplete repression. In cancer, abnormal expression of PpG is commonly observed. Our studies show that in differentiating F9 embryonal carcinoma cells (F9 ECCs), PpG maintain substantial expression concomitant with an absence of Lsd1-mediated H3K4me1 demethylation at their respective enhancers. The continued presence of H3K4me1 blocks the downstream activity of Dnmt3a, leading to the absence of DNA methylation at these sites. The absence of Lsd1 activity at PpG enhancers establishes a “primed” chromatin state distinguished by the absence of DNA methylation and the presence of H3K4me1. We further established that the activity of Lsd1 in these cells was inhibited by Oct3/4, which was partially repressed post-differentiation. Our data reveal that sustained expression of the pioneer pluripotency factor Oct3/4 disrupts the enhancer silencing mechanism. This generates an aberrant “primed” enhancer state, which is susceptible to activation and supports tumorigenicity. </p> <p>As differentiation proceeds and multiple layers of cells are produced in the early embryo, the inner cells are depleted of O₂, which triggers endothelial cell differentiation. These cells form vascular structures that allow transport of O₂ and nutrients to cells. Using ESC differentiation to endothelial cells as a model system, studies covered in this thesis work elucidated a mechanism by which the transcription factor Vascular endothelial zinc finger 1 (Vezf1) regulates endothelial differentiation and formation of vascular structures. Our data show that Vezf1-deficient ESCs fail to upregulate the expression of pro-angiogenic genes in response to endothelial differentiation induction. This defect was shown to be the result of the elevated expression of the stemness factor Cbp/p300-interacting transactivator 2 (Cited2) at the onset of differentiation. The improper expression of Cited2 sequesters histone acetyltransferase p300 from depositing active histone modifications at the regulatory elements of angiogenesis-specific genes that, in turn, impedes their activation. </p> <p>Besides the discovery of epigenetic mechanisms that regulate gene expression during differentiation, our studies also include development of a sensitive method to identify activities of a specific DNA methyltransferase at genomic regions. In mammals, DNA methylation occurs at the C5 position of cytosine bases. The addition of this chemical modification is catalyzed by a family of enzymes called DNA methyltransferases (Dnmts). Current methodologies, which determine the distribution of Dnmts or DNA methylation levels in genomes, show the combined activity of multiple Dnmts at their target sites. To determine the activity of a particular Dnmt in response to an external stimulus, we developed a method, Transition State Covalent Crosslinking DNA Immunoprecipitation (TSCC-DIP), which traps catalytically active Dnmts at their transition state with the DNA substrate. Our goal is to produce a strategy that would enable the determination of the direct genomic targets of specific Dnmts, creating a valuable tool for studying the dynamic changes in DNA methylation in any biological process.</p>