El carcinoma hepatocelular (HCC) es una de las principales causas de mortalidad por cáncer en el mundo. Este estudio tiene por finalidad determinar las características Clínico-epidemiológicas de los pacientes conHCC Gigante y su Manejo Quirúrgico en el HNGAI durante el periodo 2009 – 2012. METODOLOGÍA: Estudio observacional, descriptivo, transversal y retrospectivo, que incluye a todos los pacientes hospitalizados en el HNGAI - Lima, que fueron sometidos a resecciones hepáticas, durante los años 2009-2012, con el diagnóstico de HCC gigante. RESULTADOS: Se incluyeron 21 pacientes con el diagnóstico de HCC Gigante, que se presentó con mayor frecuencia en pacientes de sexo femenino (57.1%). La edad promedio de presentación fue de 53.4 años. Asimismo, se presentó con mayor frecuencia en pacientes con infección por virus de Hepatitis B (81%), provenientes de zonas endémicas de este virus, sin cirrosis (76.2%), en estadío A de la clasificación de ChildPugh (90.5%), con niveles altos de Alfa-fetoproteína, con una media de 9,661.1 ng/ml. La forma de presentación fue un tumor único, grande, con una media de 15.1cm, con dolor abdominal como síntoma más frecuente (95.2%), en estadios tempranos de la enfermedad, Estadío I (71.4%), según la estadificación TNM – AJCC. CONCLUSIONES: Los resultados del presente estudio, nos muestran que el patrón del HCC Gigante en el Perú, es similar al HCC de áreas de alta incidencia. En nuestra serie el gran tamaño del HCC determinó que la hemihepatectomía y la hepatectomía extendida se efectuaran en la mayoría de los casos. PALABRAS CLAVE: Hepatocarcinoma Gigante, características clínico epidemiológicas, manejo quirúrgico, Perú

En los últimos años nuestro grupo ha estudiado las diferentes vías de señalización inducidas por TGF-β en hepatocitos fetales de rata. A dosis bajas, el TGF-β inhibe el crecimiento, pero a concentraciones más elevadas es capaz de inducir apoptosis (Sanchez et al. 1995; Sanchez et al. 1996). El proceso apoptótico está mediado por un incremento en el contenido intracelular de ROS, dependiente de la síntesis de novo de proteínas, y que correlaciona con una caída en los niveles intracelulares de glutatión (Sanchez et al. 1997). La muerte inducida por TGF-β en estas células se correlaciona con un descenso en los niveles de Bcl-xL, la despolarización de la membrana mitocondrial, la salida de citocromo c y posterior activación de caspasas (Herrera et al. 2001a; Herrera et al. 2001b). El incremento de ROS intracelulares se produce por activación de un sistema NADPH oxidasa y por disminución en la expresión de proteínas antioxidantes (Herrera et al. 2004). Recientemente hemos descrito que la NADPH oxidasa NOX4 se induce en condiciones pro-apoptóticas (Carmona-Cuenca et al. 2006), pero otras NADPH oxidasas podrían jugar un papel diferente en la señalización inducida por TGF-β (Murillo et al., 2007). La muerte inducida por TGF-β puede ser inhibida por EGF a través de la activación de PI3K (Fabregat et al. 2000), que contrarresta la expresión de Nox4 (Carmona-Cuenca et al. 2006). Sin embargo, el 40-50% de las células sobreviven a los efectos apoptóticos del TGF-β y adquieren una morfología fibroblastoide (Sanchez et al. 1999). Esto es debido a que el TGF-β también induce señales anti-apoptóticas en los hepatocitos fetales, proceso que requiere la activación del EGFR, producida por un aumento en los niveles de expresión de sus ligandos y activación de la metaloproteasa TACE/ADAM17 que los proteoliza y activa (Valdes et al. 2004; Murillo et al. 2005; Del Castillo et al. 2006; Murillo et al. 2007). Las células que sobreviven al TGF-β responden a esta citoquina en términos de migración e invasión, disminuyendo la expresión de marcadores hepáticos (Sanchez et al. 1999), e induciendo un proceso de EMT (Valdes et al. 2002). La población mesenquimática resultante es resistente a la muerte inducida por TGF-β, ha sufrido un proceso de desdiferenciación y expresa marcadores de célula madre (Del Castillo et al. 2006; del Castillo et al. 2008). Esta población puede rediferenciarse tanto a un linaje hepatocítico como hacia células biliares cuando se mantienen con los medios de diferenciación adecuados. Por último, resultados preliminares al inicio de esta tesis doctoral proponían que la doble respuesta al TGF-β observada en hepatocitos fetales de rata en cultivo primario era exclusiva de este estadio del desarrollo hepático, ya que en hepatocitos adultos de rata el TGF-β sólo inducía apoptosis. La incapacidad del TGF-β de inducir señales de supervivencia parece deberse a la baja expresión de AKT y de TACE observada en hepatocitos adultos. Además, el TGF-β era incapaz de inducir un proceso de EMT en hepatocitos adultos de rata. A la vista de estos resultados se consideró de gran importancia analizar cuál podría ser la respuesta al TGF-β en células tumorales hepáticas. Así, nuestro principal objetivo en esta tesis ha sido analizar si las células de carcinoma hepatocelular responden a la muerte celular inducida por el TGF-β, y en el caso de que hayan adquirido resistencia, estudiar los mecanismos moleculares que la confieren. También queríamos saber si el TGF-β induce señales de supervivencia y un proceso de EMT en células tumorales hepáticas, y la relevancia de este proceso en la progresión del tumor hepático. Aunque quisimos iniciar el estudio con células de hepatoma de rata, debido a nuestra experiencia en este modelo de celular, consideramos muy importante también analizar la situación en células tumorales de hígado humano, ya que es conocido que los niveles de TGF-β son elevados en carcinoma hepatocelular (HCC) y diferentes evidencias han sugerido que la respuesta al TGF-β está alterada en células de HCC. / In the last years our research has focused on analyzing the signaling pathways induced by TGF-β in liver tumor cell lines, to understand the molecular mechanisms that confer resistance to its suppressor effects. TGF-β induces apoptosis in human fetal hepatocytes and in some liver tumor cells (FaO rat hepatoma, Hep3B and PLC/PRF/5 human hepatocarcinoma cells), which requires reactive oxygen species (ROS) production and up-regulation of the NADPH oxidase NOX4. This process is coincident with an increased expression of pro-apoptotic BCL-2 family members, such as BMF or BIM. However, in these same cells, TGF-β also induces anti-apoptotic signals, mediated by the activation of the epidermal growth factor receptor (EGFR) and coincident with up-regulation of the anti-apoptotic proteins BCL-XL, MCL1 or HIAP1. Inhibition of the EGFR, either by pharmacological inhibitors or through targeting knock-down with specific siRNA, significantly enhances the apoptotic response, which indicates that the EGFR plays a relevant role in conferring resistance to TGF-β-induced cell death. However, even when the EGFR is inhibited, some hepatocellular carcinoma cells, such as HepG2 or SK-Hep1, continue showing resistance to TGF-β-induced cell death. HepG2 cells are sensitized to TGF-β-induced apoptosis through the inhibition of the MEK pathway. MEK inhibition allows TGF-β to induce its pro-apoptotic program in these cells, which is coincident with NOX4 upregulation, modulation of the expression of BCL-2 family members and caspase-3 activation. It is worthy to note that activation of survival pathways, such as EGFR or MEK/ERK, in liver tumor cells confers resistance to TGF-β-induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis. Finally, our results have indicated that TGF-β is able to induce an epithelial to mesenchymal transition (EMT) process in human fetal hepatocytes, FaO rat hepatoma cells and Hep3B human hepatocarcinoma cells. TGF-β induces Snail expression, coincident with a decrease in E-cadherin mRNA and protein levels. Furthermore, cells show an increased expression of mesenchymal genes and reorganization of the actin cytoskeleton in stress fibers. Interestingly, these cells show loss of expression of specific hepatic markers and increased expression of stem cell markers. Indeed, chronic treatment with TGF-β selects a population of mesenchymal cells with a de-differentiated phenotype, reminiscent of progenitor-like cells. In summary, TGF-β induces different signals in liver tumor cells, some of them might contribute to tumor suppression (apoptosis), but others should mediate liver tumor progression and invasion.

Atomic force microscopy (AFM) can be used to acquire high-resolution topographical images of surfaces, but has the additional capability of detecting the local nanometer scale mechanical properties. For these reasons, it becomes a standard research tool in the surface science recently. In this paper, we used AFM to measure the several properties of two different human hepatocellular carcinoma cell lines, Hep G2 ( known as well differentiated and more highly carcinomatous hepatoma cell lines ) and SK-HEP-1 ( known as poorly differentiated and more lightly carcinomatous hepatoma cell lines ) cells fixed on the glass substrate, which including the surface morphology and the relationship between the cantilever deflections and loading forces ( force curve ). Considered the heterogeneous characteristics of the cell surface, the preferred experimental method is to make pixel-by-pixel force curves in a designated area ( force map ) , both adhesion forces and elasticity associated with different locations on the cell surfaces will be obtained. Finally, we use Hertzian model to calculate Young's modulus of Hep G2 and SK-HEP-1 respectively. Based on these results, we can understand the surface properties of two human hepatocarcinoma cell lines with different differentiated stage. The results showed the difference of the morphology, height, cell migration, degree of cell aggregation, roughness, elasticity, adhesive force of two cells. SK-HEP-1 cell has the wide distance of the folds, better cell migration, homogeneous properties of elasticity. It can be assumed that the SK-HEP-1 cells have a dense network structure of actin filaments under the cell membrane like branches (branched networks); Hep G2 cell has the narrow distance of the folds, poor cell migration, heterogeneous properties of elasticity. It can be assumed that the Hep G2 cells have the individual actin filaments and cross-linked network structure of actin filaments under the cell membrane. The above results can be speculated that the elastic properties of the membrane surface will be influenced of actin filaments.

Orientadora: Profa. Dra. Ana Carolina Santos de Souza Galvão / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2018. / O hepatorcarcinoma é um dos tipos tumorais mais comuns e está entre as três malignidades que mais causam óbito. Embora grandes avanços terapêuticos tenham sido realizados nos últimos anos, ainda se faz grande a necessidade de desenvolvimento de novas terapias mais efetivas na eliminação desta doença e menos severas quanto aos efeitos adversos resultantes. Diante deste cenário, neste trabalho a metformina foi avaliada quanto à sua capacidade citotóxica, antiproliferativa e antimetastática sobre células de carcinoma hepático humano - HepG2. Desta forma, objetivou-se no presente trabalho avaliar as alterações na viabilidade, proliferação e potencial metastático de HepG2 após o tratamento com metformina, A viabilidade celular foi verificada através do teste de redução do MTT e incorporação do vermelho neutro. A progressão do ciclo celular foi avaliada por citometria de fluxo e a análise de marcadores de morte celular foi realizada por meio de avaliação de expressão/atividade das moléculas sinalizadoras caspase 3, PARP e PARP clivada através de Western blotting. Variações no potencial metastático foram verificadas através de estudo das atividades de adesão, via teste de redução do MTT, migração celular, por Scratch Assay, migração e invasão por ensaios em placas do tipo transwell e determinação da atividade das metaloproteinases MMP2 e MMP9, por Zimografia em gelatina. Os resultados indicam que a metformina atua de forma citotóxica e citostática sobre HepG2 de modo concentração e tempo-dependente, e sugerem também a ocorrência de morte celular envolvendo ativação da caspase 3. Adicionalmente, o tratamento com metformina reduziu as atividades de migração e invasão celular, redução esta provavelmente associada à diminuição nas atividades de MMP2 e MMP9, ao mesmo tempo em que aumentou a atividade de adesão celular. Conclui-se, assim, que a ação antitumoral da metformina está associada com a indução de efeito citotóxico e citostático em HepG2, e que o euglicemiante parece reduzir o potencial metastático dessas células. Estes achados indicam que, futuramente, a metformina poderá embasar uma nova estratégia terapêutica e preventiva contra o hepatocarcinoma humano. / Hepatocellular carcinoma is one of the most common types of tumor and represents the third leading cause of cancer-related death. In despite to the major progresses made in recent years, there is still a great needed for the development of new therapies. More efficient treatments should eliminate the disease while triggers few smoother adverse effects as possible. Metformin is a traditional hypoglycemic prescribed for the treatment of type II diabetes. It has received considerable attention in the past years due to its potential anti-tumor activity. Indeed, previous studies involving this drug showed interesting results not only for the treatment itself, but also in preventing the cancer development. In this project, effects of metformin over human hepatic carcinoma cells (HepG2 lineage) were evaluated, assessing changes on cell proliferation, viability and metastatic potential. HepG2 cells were exposed to different metformin concentrations for 24 and 48 hours, and the cell viability/proliferation was determined by MTT assay. The metastatic potential analysis, adhesive and migratory abilities of HepG2 cells were evaluated by the determination of metalloproteinases (MMP-2 and -9) activities by zymography, cell motility by scratch assay and cell adhesion by MTT, respectively. Analysis of cell death markers was performed by the evaluation of caspase 3, PARP and cleaved-PARP expression by Western blotting. Results suggested that metformin promotes the reduction of cell viability in a concentration and time-dependent manner. HepG2 cells had their viability reduced through the cytotoxic and cystostatic action of metformin. Regulation over caspase 3 activation and cleavage of its substrate, PARP, indicated occurrence of programmed cell death by apoptosis. Metformin increased the adhesion simultaneously to the reduction of HepG2 migration and invasion abilities. Finally, it seems that the drug was able to modulate MMP-2 and -9 activities. Thus, confirming literature data, metformin seems able to decrease the metastatic potential of HepG2 cells. These findings indicate that, in the future, metformin may support a new therapeutic and preventive strategy against human hepatocarcinoma.

Le carcinome hépatocellulaire (HCC) est le cancer du foie le plus répandu et représente la seconde cause de décès par cancer dans le monde. Un mauvais pronostic et l'absence de traitement efficace en font un problème majeur de santé publique dans les pays en voie de développement, notamment en Asie du Sud-Est, justifiant pleinement la recherche de molécules ou d'approches thérapeutiques nouvelles contre l'HCC. Ce travail porte sur la recherche de molécules isolées de plantes vietnamiennes actives contre l'HCC. La première approche a consisté en un criblage pharmacologique de 33 substances naturelles qui a conduit à l'identification de 7 ent-kaurane diterpénoïdes isolés de Croton kongensis Gagnep. présentant des propriétés antiprolifératives originales. La seconde approche, par criblage in silico d'une banque de 354 substances naturelles, a permis d'identifier la solasonine comme inhibiteur de l'interaction mortalin - p53 induisant l'apoptose dans la lignée cellulaire humaine HepG2. / Human hepatocellular carcinoma (HCC) is the most common type of liver cancer, the second most common cause of death from cancer worldwide. A very poor prognosis and a lack of effective treatments make liver cancer a major public health problem, notably in less developed regions, particularly in Eastern Asia. This fully justifies the search of new molecules and therapeutic strategies against HCC. The present work focused on finding bioactive compounds from Vietnamese plants against HCC. The first approach used classical screening of 33 natural compounds which resulted in the identification of 7 ent-kaurane diterpenoids isolated from Croton kongensis Gagnep. as potential agents. The second approach aimed at identifying molecules that could abrogate the interaction between Mortalin and p53 by in silico screening of a database of 354 natural compounds, which allowed the identification of Solasonine as a potent inhibitor of p53 - mortalin interactions.

Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hep- atocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKa activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 signifi- cantly activated AMPKa and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a po- tential treatment option for HCC.

La inflamación hepática crónica es un fenómeno crucial en la patogénesis del carcinoma hepatocelular (HCC). En los últimos años, el proceso inflamatorio desencadenado por la activación del complejo del inflamasoma se ha asociado con diferentes enfermedades hepáticas. Sin embargo, su papel en el desarrollo de HCC está prácticamente sin explorar. En esta tesis, se estudia y analiza el impacto de diferentes componentes del inflamasoma, incluyendo la proteína caspasa 1 y los receptores AIM2 (del inglés absent in melanoma 2) y NLRP3 (del inglés NOD-like receptor family pyrin domain containing 3), en el desarrollo de HCC inducido por dietilnitrosamina (DEN) en ratones. La inactivación genética de AIM2, pero no de NLRP3, reduce el daño hepático y el desarrollo de HCC en este modelo. La deficiencia de AIM2 está asociada a una disminución en la activación del inflamasoma, la inflamación hepática y la respuesta proliferativa durante la fase de iniciación del HCC. También se identificó que AIM2 está altamente expresado en células de Kupffer, y que la producción de interleucina (IL)-1β, mediada por AIM2, en estas células se ve aumentada tras el daño hepático inducido por DEN. Además, la inhibición de IL-1β resultó en menor daño hepático, inflamación y proliferación durante la fase inicial de hepatocarcinogénesis; sin embargo, esta inhibición no fue suficiente para frenar la progresión de tumores hepáticos ya establecidos. Así, los datos de esta tesis indican que AIM2 promueve el proceso inflamatorio durante el daño hepático carcinogénico, contribuyendo al desarrollo genotóxico de HCC en ratones y, por tanto, reconociendo AIM2 como una posible diana terapéutica en esta enfermedad.

O colágeno XVIII é um componente das membranas basais com diversos domínios funcionais, como a endostatina e o domínio frizzled, que têm importante papel em processos celulares como proliferação e diferenciação. COL18A1 possui dois promotores alternativos: o promotor 1, que regula a síntese da variante NC11-303, e o promotor 2 responsável pelas variantes NC11- 728 e NC11-493, expressas por hepatócitos. Existe uma variação interindividual da endostatina circulante e da expressão do colágeno XVIII no fígado. A expressão do colágeno XVIII/endostatina foi correlacionada com a progressão tanto do hepatocarcinoma (HCC), quanto da fibrose/cirrose hepática. Elucidar a regulação da expressão de COL18A1 pode auxiliar na compreensão dessa variação interindividual e da progressão dessas doenças. Neste trabalho demos início a caracterização do promotor 2 do COL18A1. Identificamos na seqüência predita como promotora cinco regiões conservadas entre humanos e camundongos. A análise in silico e funcional dessas regiões revelou que os fatores de transcrição, Sp1, Sp3, YY1, Oct-1, C/EBPα e C/EBPβ, interagem com as mesmas. Demonstramos que C/EBPβaumenta a taxa de transcrição do promotor 2 em hepatócitos, e que existe uma correlação positiva da expressão de NC11-493 com C/EBPαe C/EBPβem tecido hepático cirrótico e tumoral. As expressões de C/EBPαem tecido hepático cirrótico e tumoral estão diretamente correlacionadas, enquanto que os níveis de NC11-493 nos tumores estão inversamente correlacionados com o tamanho dos mesmos. Mostramos a existência de diversos SNPs no promotor 2. O SNP-700T/G, funcional in vitro, afeta a interação de Sp3 e YY1 com essa região regulatória. A deleção da região do SNP indicou que ela possui elementos importantes para a transcrição em hepatócitos, apesar deste SNP não estar relacionado com o nível de expressão do colágeno XVIII em fígado fibrótico ou com susceptibilidade a HCC. O SNP- 700T/G está em desequilíbrio de ligação com o SNPc.1135C/T, no domínio frizzled do colágeno XVIII. Não foi possível elucidar a funcionalidade do SNPs c.1135C/T in vitro, mas os haplótipos formados por esses dois SNPs têm diferentes frequências entre descendentes de europeus e de africanos. Nosso trabalho traz importantes contribuições e abre novas perspectivas para a compreensão da regulação do colágeno XVIII em fígado humano, tanto em situações fisiológicas, quanto em processos fibrogênicos e tumorigênicos¶ / Collagen XVIII is a basal membrane component with several funcional domains, such as endostatin and frizzled domains, which have important roles in cellular processes such as proliferation and differentiation. COL18A1 has two promoter regions: promoter 1, that regulates the synthesis of NC11-303 isoform, and promoter 2, localized in intron 2, responsible for NC11-728 and NC11-493 isoforms expressed by hepatocytes. There is a large interindividual variation in circulating endostatin and in collagen XVIII liver expression. Collagen XVIII/endostatin levels were correlated with hepatocellular carcinoma (HCC) progression, as well as liver fibrosis/cirrhosis, conditions that precede HCC. Elucidating the mechanisms that regulate COL18A1 expression in hepatocytes may help understanding its variation among individuals and liver disease stages, as well as contribute to new treatment strategies. In this work we began to characterize COL18A1 promoter region 2. We identified in the predicted promoter sequence five conserved regions between human and mouse. The in silico and functional analysis of these regions revealed that transcription factors Sp1, Sp3, YY1, Oct-1, C/EBPα and C/EBPβ interact with them. We have demonstrated that C/EBPβ increases promoter 2 transcription rate in hepatocytes, and that there is a positive correlation of NC11-493 expression with that of C/EBPα and C/EBPβ in cirrhotic and tumor liver samples. Non-tumor and tumor C/EBPα expressions positively correlate between themselves, while NC11-493 tumor expression inversely correlates with tumor size. We also showed that there are several SNPs in COL18A1 promoter 2 region. SNP-700T/G, functional in vitro, affects Sp3 and YY1 interaction with the promoter 2 region and deletion of the SNP region indicated that this sequence has important hepatocyte regulatory elements. Our results suggest that this SNP does not significantly affects COL18A1 expression in fibrotic/cirrhotic liver and is not associated with HCC susceptibility. SNP-700T/G is in linkage disequilibrium with SNPc.1135C/T, at collagen XVIII frizzled domain. We could not elucidate SNPc.1135C/T functionality in vitro, but the haplotypes formed by these two SNPs have different frequencies in European and African descendants. In conclusion, our work brings important contributions and opens new perspectives for the comprehension of collagen XVIII regulation in human liver in physiological situations, as well as in fibrotic/cirrhotic and tumorigenic process.

O carcinoma hepatocelular (CHC) é a quarta causa mais frequente de morte por câncer em todo o mundo. Este estudo teve dois grandes objetivos, o primeiro foi estabelecer o carcinoma hpatocelular experimental por indução química e o segundo estudar os efeitos da melatonina sobre o estresse oxidativo, estresse de retículo endoplasmático e apoptose durante a hepatocarcinogênese. Foram realizados dois experimentos, ambos utilizaram dietilnitrosamina (DEN) mais acetilaminofluoreno (2-AAF) em ratos Wistar machos pesando 145-150 g. O primeiro estudo testou 3 protocolos de indução do câncer hepático. Os animais foram divididos em três grupos testes: DEN50: que recebeu DEN 50 mg duas vezes por semana até a 6ª semana e uma vez por semana nas semanas 11 a 13. DEN75: recebeu DEN 75mg uma vez por semana nas semanas até a 6ª semana e um reforço nas semanas de 11 a 13. DEN100: recebeu 5 doses de DEN 100mg uma a cada seis semanas por 28 semanas. Todos receberam 2- AAF dose única de 100 mg na 4ª semana. Após a indução foi comprovado por testes bioquímicos, macroscópico e histológico que o protocolo DEN50 desenvolve CHC avançado em 19 semanas, apresentou a fase inflamatória na 5ª semana e cirrose na 12ª semana. O protocolo DEN100 exibe padrão de lesões pré-cancerosas com cirrose em 28 semanas. O protocolo DEN75 foi o mais heterogêneo dos três, pois desenvolveu lesões pré-cancerosas, cancer inicial e CHC avançado. O segundo estudo, repetiu o protocolo DEN50 e administrou melatonina 20mg/L. Os tratamentos começaram nas semanas 5 e 12. Ao final de 19 semanas foi observado que animais do grupo que só recebeu DEN+2-AAF (DEN-CHC) desenvolveram carcinoma avançado, exibiram mais expressão de proteinas pró-inflamatórias (iNOS, COX-2 e NFkB). E animais tratados com Melatonina (DEN+MEL5 e DEN+MEL 12) apresentaram padrão histológico de cirrose e reduzida expressão destas proteinas. Quanto ao comportamento oxidativo foi observado que grupo DEN-CHC apresentou menor lipoperoxidação (LPO) por redução de ácidos graxos poliinsaturados, maior oxidação proteica, menor atividade da SOD e maior índice de danos ao DNA. O tratamento com melatonina ao longo da hepatocarcinogênese se mostrou efetivo para proteger a membrana lipidica, reduziu a oxidação proteica, aumentou a atividade da SOD e atenuou o dano ao DNA. Por fim, referente ao estresse de retículo endoplasmático e apoptose, animais com DEN-CHC não apresentaram ativação das proteinas de estresse de retículo (BiP, ATF6 e CHOP) nem acionaram as rotas apoptóticas. Entretanto, animais tratados com Melatonina tiveram aumento significativo na expressão de proteinas como BiP, ATF6 e CHOP, assim como proteinas pró-apoptóticas. Nossos resultados apontam que a melatonin, durante o processo de hepatocarcinogênese experimental, atuou como anti-inflamatório, antioxidante e pró-apoptótico. E estas ações contribuiram para evitar a progressão do carcinoma hepatocelular. / Hepatocellular carcinoma (HCC) is the fourth most frequent cause of cancer death worldwide. This study had two main objectives, the first was to establish the experimental hpatocelular carcinoma by chemical induction and the second study the effects of melatonin on oxidative stress, endoplasmic reticulum stress and apoptosis during hepatocarcinogenesis. Two experiments were conducted, both used Diethylnitrosamine (DEN) + acetylaminofluorene (2-AAF) in male Wistar rats weighing 145-150 g. The first study tested three induction protocols of liver cancer. The animals were divided into three test groups: DEN50: DEN that received 50 mg twice a week until 6 weeks and once a week during the weeks 11 and 13. DEN75: DEN received 75mg once a week during the weeks to 6 weeks and another reinforcement in weeks 11 to 13 DEN100: DEN received 5 doses of 100mg one every six weeks for 28 weeks. All received 2 AAF single dose of 100 mg at week 4. After induction was confirmed by biochemical, macroscopic and histological tests that DEN50 protocol develops advanced HCC in 19 weeks, presents inflammatory phase in the 5th week and cirrhosis at 12 weeks. The default display protocol DEN100 of precancerous lesions and cirrhosis in 28 weeks. DEN75 The protocol was the most heterogeneous of the three, as developed precancerous lesions, early cancer and advanced HCC. The second study, repeated the DEN50 protocol and administered melatonin 20mg / L. The treatments began on week 5 and 12. At the end of 19 weeks was observed that animals in the group that received only DEN (DEN-CHC) developed advanced carcinoma exhibited over expression of proinflammatory proteins (iNOS, COX-2 and NFkB). And animals treated with melatonin (DEN+ MEL5W and DEN+MEL 12W) showed histological pattern of cirrhosis and reduced expression of these proteins. As for the oxidative behavior was observed that DEN-HCC group had lower lipid peroxidation (LPO) by reducing polyunsaturated fatty acids, higher protein oxidation, lower activity of SOD and higher rate of DNA damage. Treatment with melatonin throughout hepatocarcinogenesis was effective to protect the lipid membrane, protein oxidation decreased, increased SOD activity and attenuated DNA damage. Finally, referring to the endoplasmic reticulum stress and apoptosis, animal DEN-CHC did not show activation of reticulum stress protein (BiP, ATF6 and CHOP) or triggered apoptotic routes. However, melatonin treated animals had a significant increase in the expression of proteins and BiP, CHOP and ATF6, as well as pro-apoptotic proteins. Our results indicate that melatonin, during the process of experimental hepatocarcinogenesis, acted as anti-inflammatory, antioxidant and pro-apoptotic. And these actions contributed to prevent progression of hepatocellular carcinoma.

La chronothérapie des cancers administre les médicaments anticancéreux à des moments précis de la journée afin d’en optimiser la tolérance et l’efficacité. Cependant le système circadien qui règle sur 24 h la prolifération et le métabolisme cellulaires peut être altéré par un décalage horaire chronique, la mutation d’un gène de l’horloge, ou un traitement, favorisant ainsi la survenue de pathologies métaboliques, comportementales ou malignes. La disparité des profils circadiens de température corporelle des patients cancéreux ainsi que leurs modifications sous chimiothérapie fournit les bases d’une personnalisation de la chronothérapeutique. La capacité d’un cycle thermique à entraîner sur 24 h l’horloge circadienne de cellules d’hépatocarcinome en culture indique que ce biomarqueur est aussi un effecteur de la synchronisation des cellules cancéreuses, et constitue un repère circadien pour la chronothérapeutique in vitro et in vivo. / Chronotherapy delivers anticancer drugs at specific times of the day to optimize tolerability and efficacy. However, the circadian system that controls cell proliferation and metabolism over 24 h, can be altered by a chronic jet lag, a clock gene mutation, or a xeniobiotic treatment, thus favoring the occurrence of metabolic, behavioral or malignancies. The disparity of circadian body temperature patterns of cancer patients as well as its disruption during the treatment provides a clincher for chronotherapy personalization. The ability of a thermal cycle to drive the circadian clock in cultured hepatocarcinoma cells of 24 h indicates that this biomarker is also an effector of the synchronization of cancer cells, as well as a marker for the circadian in vitro and in vivo chronotherapeutic.