Η ηπατική καρκινογένεση είναι μια πολυσταδιακή διαδικασία που οδηγεί προοδευτικά στον κακοήθη μετασχηματισμό του ηπατικού κυττάρου, μέσω ποικίλων μοριακών μηχανισμών. Πρόσφατα αναδεικνύεται ολοένα και σε μεγαλύτερο βαθμό, η τεράστια σημασία που έχει για την ανάπτυξη και εξέλιξη του καρκίνου το μικροπεριβάλλον του όγκου, η αλληλεπίδραση δηλαδή των καρκινικών κυττάρων με την εξωκυττάρια ουσία. Ιδιαίτερο ενδιαφέρον παρουσιάζει η αλληλεπίδραση των καρκινικών κυττάρων με την εξωκυττάρια ουσία μέσω των ιντεγκρινών, η οποία φαίνεται ότι εμπλέκεται σε όλα τα στάδια καρκινογένεσης. Σημαντικό μόριο στην προαναφερθείσα διαδικασία αποτελεί η ILK (Integrin-Linked Kinase), μία κινάση σερίνης-θρεονίνης που παρεμβάλλεται στα σηματοδοτικά μονοπάτια που ξεκινούν από ιντεγκρίνες, αυξητικούς παράγοντες και συμμετέχει στη ρύθμιση κομβικών για το καρκινικό κύτταρο λειτουργιών όπως έλεγχο του κυτταρικού κύκλου, απώλεια των δομών συνοχής του κυττάρου, αναστολή της απόπτωσης και ενεργοποίηση της αγγειογένεσης. Διαπιστώθηκε πως το σηματοδοτικό μονοπάτι ILK/p-Akt διαδραματίζει κομβικό ρόλο τόσο στη βιολογία της κίρρωσης όσο και του ηπατοκυτταρικού καρκίνου και πιθανόν αποτελεί μοριακό σύνδεσμο μεταξύ των δύο καταστάσεων, επιβεβαιώνοντας τη διατυπωμένη θεωρία περί άξονα χρόνιας φλεγμονής-ίνωσης (κίρρωσης)-καρκίνου. Επίσης στην παθογένεια της κίρρωσης και του καρκίνου ήπατος, φαίνεται πως ρόλο έχει το φαινόμενο της επιθηλιακής προς μεσεγχυματική μετατροπή (EMT-epithelial to mesenchymal transition). Αυτό αποδεικνύεται από την απώλεια έκφρασης της E-καντχερίνης και τη μεταφορά της β-κατενίνης στον πυρήνα που οδηγούν σε απώλεια των κυτταρικών συνδέσεων από τη μεμβράνη καθώς και από την υπερέκφραση των μορίων ILK και p-Akt. Τέλος η υπερέκφραση survivin και κυκλίνης-D1 στην κίρρωση και στο ηπακυτταρικό καρκίνωμα καταδεικνύουν τη σημασία της αντιαποπτωτικής δραστηριότητας και του αυξημένου κυτταρικού πολλαπλασιασμού στις συγκεκριμένες νοσολογικές οντότητες. / The hepatic carcinogenesis is a many phased process that leads progressively to the malignant transformation of hepatic cell via various molecular mechanisms. Recently, the enormous importance of the tumor microenvironment for the growth and development of cancer, i.e. the interaction between cancer cells and the extacellular matrix, has been proven. Particular interest is presented in the interaction of tumor cells with the extracellular matrix via integrins, which appears to be involved in all the stages of carcinogenesis. An important molecule in the aforementioned process is the ILK (Integrin-Linked Kinase), a serine-threonine kinase that is implicated in the signal transduction pathways that begin from integrines, growth factors and participate in the regulation of nodal for the tumor cell functions, such as control of cell cycle, loss of cell adhesion structures, suppresion of apoptosis and activation of angiogenesis. It has been realized that the signal transduction pathway ILK /p-Akt plays a nodal role so much in the biology of cirrhosis as well as in hepatocellular cancer and probably constitutes a molecular connection between the two conditions, confirming the formulated theory of axis of chronic inflamation-chirrosis-cancer. Also in the pathogenicity of cirrhosis and liver cancer, it appears that the epithelial to mesenchymal transition (EMT) phenomenon plays a role. This is proven by the loss of expression of E-cadherin and the transport of b-catenin in the nucleus that leads to loss of cellular connections from the membrane as well as from the overexpression of molecules ILK and p-Akt. Finally, the overexpression of survivin and cyclin-D1 in cirrhosis and hepatocellular carcinoma show the importance of antiapoptotic activity and increased cellular proliferation in the particular disease entities.

Το ηπατοκυτταρικό καρκίνωμα (ΗΚΚ) είναι η πιο σημαντική πρωτοπαθής νεοπλασία του ήπατος παγκοσμίως. Πολλοί αιτιολογικοί παράγοντες έχουν συσχετιστεί με την ανάπτυξη του ΗΚΚ, όπως η κίρρωση, οι ιοί της ηπατίτιδας και το αλκοόλ. Χρόνια λοίμωξη με ηπατίτιδα Β (HBV) και C (HCV) συχνά καταλήγει σε κίρρωση και ενισχύει την πιθανότητα ανάπτυξης ΗΚΚ. Ωστόσο οι υποκείμενοι μηχανισμοί που οδηγούν στην κακοήθη εξαλλαγή των κυττάρων παραμένουν αδιευκρίνιστοι. Ο HBV είναι ένας DNA ιός που ενσωματώνεται στο γονιδίωμα του ξενιστή και θεωρείται ότι με τον τρόπο αυτό προκαλεί καρκινογένεση. Επιπρόσθετα, ο ιός κωδικοποιεί μία πρωτεΐνη 17 kDa,την HBx, η οποία είναι γνωστό ότι αποτελεί αιτιολογικό παράγοντα ανάπτυξης ΗΚΚ. Η παρούσα ανασκόπηση αναλύει το ρόλο της HBx στους μοριακούς μηχανισμούς που σχετίζονται με την παθογένεση της επαγόμενης από τον HBV ηπατικής καρκινογένεσης. / Hepatocellular carcinoma (HCC) is the most important primary hepatic cancer, being a common cancer type worldwide. Many aetiological factors have been related with HCC development, such as cirrhosis, hepatitis viruses and alcohol. Chronic infection with hepatitis B (HBV) and C viruses (HCV) often results in cirrhosis and enhances the probability of developing HCC. The underlying mechanisms that lead to malignant transformation of infected cells, however, remain unclear. HBV is a DNA virus that integrates into the host genome, and this integration is believed, in part, to be carcinogenic. Besides, the virus encodes a 17 kDa protein, HBx, which is known to be a causative agent in the formation of HCC. This review examines the role of HBx in the molecular mechanisms involved in the pathogenesis of HBV-induced hepatocarcinogenesis.

Woodchucks are the preferred animal model to study chronic viral hepatitis and the development of hepatocellular carcinoma (HCC), which occurs as a result of infection with woodchuck hepatitis virus. Significant elevations in the phosphomonoester peak in 31P-MRS spectrum correlated to the presence of HCC. Ex vivo 31P-NMR determined that HCC tissue had significantly elevated concentrations of PC compared to uninfected control tissues, confirming that PME is specific to the tumour’s growth. Finally, a recombinant vaccinia virus was constructed to stimulate the immune systems of infected woodchucks against cells expressing core antigens. Despite reductions in surface antigen expression and viral load, elevations in serum GGT and the PME in 31P-MRS indicated that there was tumour growth in treated woodchucks. In conclusion, the PME peak represents a potential biomarker of cancerous growth when used in conjunction with serological tests to detect HCC in the liver due to chronic hepatitis virus infection.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine degradation pathway. The defective fumarylacetoacetate hydrolase enzyme causes the accumulation of upstream metabolites such as fumarylacetoacetate (FAA), maleylacetoacetate (MAA), succinylacetone (SA) and p-hydroxyphenylpyruvic acid (pHPPA). In vitro and in vivo studies showed that the accumulation of these metabolites are detrimental to cell homeostasis, by inducing cell cycle arrest, apoptosis, and endoplasmic reticulum stress, depleting GSH, inhibiting DNA ligase, causing chromosomal instability, etc. For in vivo studies different models of HT1 were developed. Most notably was the fah deficient mouse, whose neonatally lethal phenotype is rescued by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). Although, this model most closely resembles the human phenotype with elevated tyrosine levels and the development of hepatocellular carcinoma (HCC), the model is not human genome based. Both the in vitro and in vivo studies suggested that DNA repair is affected in HT1. However, it is not yet clear which DNA repair mechanisms are affected and if only protein functionality is affected, or if expression of DNA repair proteins are also affected. Characteristic of HT1 is the high prevalence of HCC and the presence of liver mosaicism. The liver mosaicism observed in HT1 patients are the result of reversion of the inherited mutation to wild-type. The general consensus is that the reversion is the result of a true back mutation. However, the mechanism underlying the back mutation is still unresolved. It was suggested that cancer develops either through a chromosomal instability mutator phenotype, a microsatellite instability mutator phenotype, or a point mutation instability mutator phenotype. In HT1 only chromosomal instability was reported. The aims of this study were to contribute to the understanding of the molecular basis of the genetic mosaicism in hereditary tyrosinemia type 1. More specifically, determine whether baseand nucleotide DNA repair mechanisms are affected and to what extent, and to determine if microsatellite instability is found in HT1. To achieve these aims, a parallel approach was followed: i.e. to develop a HT1 hepatic cell model and to use HT1 related models and HT1 patient material. To assess the molecular basis of the genetic mosaicism in HT1, the comet assay, gene expression assays, microsatellite instability assays, high resolution melting and dideoxy sequencing techniques were employed. Results from the comet assay showed that the HT1 accumulating metabolites, SA and pHPPA, decreased the capacity of cells for base- and nucleotide excision repair. Gene expression assays showed that short term exposure to SA and/or pHPPA do not affect expression of hOGG1 or ERCC1. The expression of these genes were, however, low in HT1 patient samples. Microsatellite instability assays showed allelic imbalance on chromosome 7 of the mouse genome, and microsatellite instability in the lymphocytes of HT1 patients. Although high resolution melt and sequencing results did not reveal any de novo mutations in fah or hprt1, the appearance of de novo mutations on other parts of the genome can not be ruled out. To conclude, results presented in this thesis, for the first time show that in HT1 the initiating proteins of the base- and nucleotide repair mechanisms are affected, the gene expression of DNA repair proteins are low, and microsatellite instability is found in HT1. By contributing to the elucidation of the mechanism underlying the development of HT1-associated HCC, and providing evidence for the development of a mutator phenotype, the results presented in this thesis contributes to the understanding of the molecular mechanisms underlying the genetic mosaicism in HT1. In addition to these contributions, a hypothesis is posited, which suggests that a point mutation instability (PIN) mutator phenotype is the mechanism underlying the mutation reversions seen in HT1. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Woodchucks are the preferred animal model to study chronic viral hepatitis and the development of hepatocellular carcinoma (HCC), which occurs as a result of infection with woodchuck hepatitis virus. Significant elevations in the phosphomonoester peak in 31P-MRS spectrum correlated to the presence of HCC. Ex vivo 31P-NMR determined that HCC tissue had significantly elevated concentrations of PC compared to uninfected control tissues, confirming that PME is specific to the tumour’s growth. Finally, a recombinant vaccinia virus was constructed to stimulate the immune systems of infected woodchucks against cells expressing core antigens. Despite reductions in surface antigen expression and viral load, elevations in serum GGT and the PME in 31P-MRS indicated that there was tumour growth in treated woodchucks. In conclusion, the PME peak represents a potential biomarker of cancerous growth when used in conjunction with serological tests to detect HCC in the liver due to chronic hepatitis virus infection.

We previously observed that postnatal exposure of mice to the procarcinogen 4-aminobiphenyl (ABP) produced liver tumors only in wild-type males, while arylamine N-acetyltransferase deficient males and females of either strain were protected. Others have also observed a sex difference in liver tumors in mice using the procarcinogen diethylnitrosamine (DEN). Reasons for these sex and strain differences are unclear, but differences in acute hepatotoxicity and inflammation may be involved. In this thesis we found that neither ABP nor DEN produced overt hepatotoxicity in postnatally exposed mice, and only DEN caused an increase in levels of the pro-inflammatory cytokine interleukin-6 but was not sex-dependent. The lack of sex difference suggests that sex hormone modulation of inflammation following sexual maturation might favour growth of initiated cells in males. However, the lack of detectable inflammation following ABP exposure may be due to localized responses, or that inflammation may be a DEN-specific effect.

We previously observed that postnatal exposure of mice to the procarcinogen 4-aminobiphenyl (ABP) produced liver tumors only in wild-type males, while arylamine N-acetyltransferase deficient males and females of either strain were protected. Others have also observed a sex difference in liver tumors in mice using the procarcinogen diethylnitrosamine (DEN). Reasons for these sex and strain differences are unclear, but differences in acute hepatotoxicity and inflammation may be involved. In this thesis we found that neither ABP nor DEN produced overt hepatotoxicity in postnatally exposed mice, and only DEN caused an increase in levels of the pro-inflammatory cytokine interleukin-6 but was not sex-dependent. The lack of sex difference suggests that sex hormone modulation of inflammation following sexual maturation might favour growth of initiated cells in males. However, the lack of detectable inflammation following ABP exposure may be due to localized responses, or that inflammation may be a DEN-specific effect.

Introduction: Hepatic stellate cells (HSC) and myofibroblasts may be relevant stromal drivers of human hepatocellular carcinoma (HCC). It was hypothesised that targeted inhibition of αv integrin-mediated TGF-β activation, by HSC or hepatocytes, may result in reduced peri-tumoural and intra-tumoural extracellular matrix formation, and reduced hepatic carcinogenesis. The role of HSC in acute liver injury is less well characterised. It was anticipated that integrin signalling on HSC and hepatocytes might also be relevant in the acute setting. The emerging technique of intravital microscopy (IVM) allows detailed, real-time investigation of the cellular processes involved in hepatocyte injury, cell death and repair. It was hypothesised that this could be coupled with mouse models of HCC and acute liver injury, to perform sequential imaging under anaesthesia. Aims: (i) To determine the effect of targeted inhibition of αv integrins on HSC and hepatocytes, during hepatocarcinogenesis, in a mouse model of HCC. (ii) To investigate the effect of targeted inhibition of αv and other integrins on HSC, hepatocytes, and liver sinusoidal endothelial cells (LSEC), during acute liver injury, in the mouse model of paracetamol-induced liver injury. (iii) To develop IVM of the liver, via an abdominal imaging window, with optimisation of surgical and imaging techniques, to allow sequential imaging of the same animal. Methods: The diethylnitrosamine (DEN)-induced mouse model of hepatocarcinogenesis was used, and PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were employed to deplete αv integrins on HSC and hepatocytes respectively. Tumours were harvested at 40 weeks post-DEN. Tumour size and number was evaluated in all animals. PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were used in the paracetamol model, to investigate the role of αv integrins in acute liver injury. PDGFRβ-Cre;β8fl/fl and Alb-Cre;β 8fl/fl animals were also tested in this model. The role of integrins in liver sinusoidal endothelial cells (LSEC) during paracetamol-induced liver injury was evaluated using Cdh5-Cre mice. IVM of the liver was performed by surgical implantation of an abdominal imaging window, consisting of a titanium ring and coverslip, secured in place with a purse string suture. Fluorescent reporter mice were used to identify hepatic and vascular architecture, and other label-free microscope technologies were utilised to image collagen, lipid distribution, necrotic areas and blood flow within tissues. Results: In large cohorts of PDGFRβ-Cre;αvfl/fl, Alb-Cre;αvfl/fl, and control animals, there was no difference in mean tumour size or number, at 40 weeks. Targeted inhibition of α v integrins and β 8 integrin on hepatocytes, HSC or LSEC was not protective in paracetamol-induced liver injury. IVM of the liver can be performed on animals with HCC and throughout paracetamol-induced liver injury, to obtain high quality, real-time images of multiple cell lineages and the hepatic microenvironment. Conclusions: The role of TGF-β in HCC pathogenesis is complex and context-dependent. Targeted loss of αv integrin did not result in reduction in tumour burden in this non-cirrhotic model of HCC. IVM of the liver is a powerful tool to quantify inflammatory infiltrates and assessment of vascular remodelling throughout the course of acute liver injury and regeneration, providing insights into the biological processes determining recovery.

Racional: O carcinoma hepatocelular é uma neoplasia maligna agressiva com elevada morbidade e mortalidade. Objetivo: Revisão da literatura sobre o diagnóstico e o manejo do carcinoma hepatocelular nos vários estágios da doença. Método: Revisão da literatura utilizando a base Medline/PubMed e literatura adicional. Resultados: O carcinoma hepatocelular é geralmente complicação da cirrose hepática. As hepatites virais crônicas B e C também são fatores de risco para o surgimento do carcinoma hepatocelular. Quando associado à cirrose hepática, o carcinoma hepatocelular geralmente surge a partir da evolução de um nódulo regenerativo hepatocitário que sofre degeneração maligna. O diagnóstico é efetuado através de tomografia computadorizada de abdome com contraste endovenoso (efeito wash in e wash out), e a ressonância magnética pode auxiliar nos casos que não possam ser definidos pela tomografia computadorizada. O único tratamento potencialmente curativo para o carcinoma hepatocelular é a ressecção do tumor, seja ela realizada através de hepatectomia parcial ou de transplante. Infelizmente, apenas cerca de 15% dos carcinomas hepatocelulares são passíveis de tratamento cirúrgico. Pacientes portadores de cirrose hepática estágio Child B e C não devem ser submetidos à ressecção hepática parcial. Para esses pacientes, as opções terapêuticas curativas restringem-se ao transplante de fígado, desde que selecionáveis para esse procedimento, o que na maioria dos países dá-se através dos Critérios de Milão (lesão única com até 5 cm de diâmetro ou até três lesões de até 3 cm de diâmetro). A sobrevida em 5 anos para pacientes transplantados para o carcinoma hepatocelular pode alcançar 70% Conclusão: Quando diagnosticado em seus estágios iniciais, o carcinoma hepatocelular é potencialmente curável. O conhecimento das estratégias de 17 diagnóstico e tratamento do carcinoma hepatocelular a fim propiciam sua identificação precoce e a indicação de tratamento apropriado. / Introduction: Hepatocellular carcinoma is an aggressive malignant tumor with high lethality. Aim: A literature review on diagnosis and management of hepatocellular carcinoma was performed. Methods: Literature review utilizing databases Medline/PubMed. Results: Hepatocellular carcinoma is a common complication of hepatic cirrhosis. Chronic viral hepatitis B and C also constitute as risk factors for development of hepatocellular carcinoma. In patients with cirrhosis, hepatocelular carcinoma usually develops from a malignant transformation of a dysplastic regenerative nodule. Diagnosis is confirmed through computed tomography scan with intravenous contrast (wash in and wash out effect), and magnetic resonance may be helpful in some instances. Curative treatment for hepatocellular carcinoma may be performed through partial liver resection or liver transplantation. Only 15% of all hepatocellular carcinomas are localized and amenable to operative treatment. Patients with Child C liver cirrhosis are not amenable to partial liver resections. The only curative treatment for hepatocellular carcinomas in patients with Child B or C cirrhosis is liver transplantation. In most countries, only patients with hepatocellular carcinoma under Milan Criteria (single tumor with up to 5 cm diameter or up to three nodules with a maximum diameter of 3 cm) are considered candidates for liver transplant. Five-year survival following liver transplantation for hepatocellular carcinoma may reach 70%. Conclusion: Hepatocellular carcinoma is a potentially curable neoplasm if discovered in its initial stages. Clinicians and surgeons should be familiar with strategies for early diagnosis and treatment of hepatocellular carcinoma as a way to decrease mortality associated with this malignant neoplasm.

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.