Li Lok Yee Mandy. / "June 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 155-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A doença hepática associada a sobrecarga de ferro pode ocorrer devido a causas genéticas ou secundárias. Esse estudo avaliou pacientes com hepatopatia crônica com sobrecarga de ferro submetidos à pesquisa das mutações HFE no período de 2007-2009 e classificou como portadores de hemocromatose hereditária HFE (HH-HFE) aqueles que apresentavam as mutações C282Y/C282Y ou C282Y/H63D e como sobrecarga de ferro não HFE aqueles que apresentavam outras mutações no gene HFE como C282Y/-, H63D/- e H63D/H63D ou pacientes sem qualquer uma dessas mutações mencionadas. Os objetivos do estudo foram 1) analisar e correlacionar os aspectos fenotípicos e genotípicos de grupo de indivíduos com doença hepática crônica e sobrecarga de ferro; 2) caracterizar o quadro clínico, laboratorial e anatomopatológico, em busca de achados compatíveis com o fenótipo de hemocromatose; 3) Correlacionar o quadro clínico com as mutações no gene HFE. Foram analisados 108 indivíduos portadores de hepatopatia crônica selecionados a partir de saturação de transferrina (ST) > 45% e ferritina sérica > 350 ng/mL. Foram estudados e comparados 16 pacientes no grupo HH-HFE com 92 no grupo sobrecarga de ferro não HFE. Da casuística geral, a idade média ao diagnóstico da doença foi de 46,69 anos (16-77), com 70,73% constituída por indivíduos de cor branca, 77,57% do sexo masculino e 64,8% tinham cirrose hepática. A frequência de cirrose hepática não diferiu entre os grupos, entretanto, artropatia, carcinoma hepatocelular, diabetes e osteoporose foram mais frequentes no grupo HH- HFE (53,8% x 15,9%, 31,2% x 7,06%, 56,2% x 30%, 72,7% x 32,1%, respectivamente, p < 0,05). Os pacientes com mutações HFE diagnósticas de HH apresentaram maior chance de ter carcinoma hepatocelular (OR= 5,0, p= 0,032) quando comparados com os portadores de outros genótipos HFE e aqueles sem mutação. Os níveis de ST, ferro e ferritina também foram maiores naquele grupo, bem como os graus de siderose 3 e 4 (p= 0,026). A ST foi a variável que se correlacionou independentemente com o diagnóstico das mutações C282Y/C282Y e C282Y/H63D. A frequência de fatores de risco para sobrecarga de ferro não diferiu entre os grupos. Observou-se, entretanto, que no grupo HH-HFE havia maior número de pacientes sem qualquer fator de risco detectado (p= 0,019). Níveis de ST > 82% apresentaram maior valor preditivo negativo para o diagnóstico de HH-HFE do que os de ferritina, ferro, capacidade total de ligação de ferro e de transferrina. Concluímos que os portadores de HH-HFE têm maiores graus de sobrecarga de ferro quando comparados ao grupo de sobrecarga de ferro não-HFE; em indivíduos com doença hepática crônica. ST > 82% tem maior acurácia para diagnóstico de HH-HFE; portadores de mutações C282Y em homozigose ou em heterozigose composta com H63D têm maior chance de apresentar carcinoma hepatocelular do que os portadores de outras mutações no gene HFE e pacientes sem mutação / Chronic liver disease related to iron overload may occur due to genetic or secondary causes. This study analyzed patients with chronic liver diseases and iron overload who were tested for HFE mutations from 2007 to 2009. Patients with C282Y/C282Y or C282Y/H63D mutations were diagnosed with HFE hereditary hemochromatosis (HFE-HH) and those with other HFE genotypes (C282Y/-, H63D/- or H63D/H63D) or individuals without HFE mutations (wild type) were designed as non-HFE iron overload. The aims of this study were: 1) to analyze and to establish correlations between phenotypic and genotypic aspects of individuals with chronic liver disease and with iron overload; 2) to charachterize the clinical manifestations, laboratory and histological findings consistent with the phenotype of hemochromatosis; 3) to verify associations between clinical manifestations and HFE mutations. One hundred and eight patients with chronic liver diseases and with iron overload, defined as transferrin saturation (TS) > 45% and serum ferritin levels > 350 ng/mL were included. Sixteen patients had HH-HFE and were compared with 92 patients with non-HFE iron overload group. The average of age at diagnosis was 46.69 years (16-77), 70.73% were Caucasians, 77.57% were male and 64.8% had hepatic cirrhosis. The proportion of hepatic cirrhosis was similar in both groups, nevertheless arthropathy, hepatocellular carcinoma, diabetes and osteoporosis were more frequent in the HFE-HH group (53,8% x 15,9%, 31,2% x 7,06%, 56,2% x 30%, 72,7% x 32,1%, respectively, p < 0,05). The HFE C282Y/C282Y or C282Y/ H63D genotypes had a higher chance to develop hepatocellular carcinoma (OR= 5.0, p= 0.032) when compared with the other HFE genotypes and with those wild type. The levels of TS, serum iron and ferritin were greater in HFE-HH group, as well as hepatic siderosis grade 3 and 4 (p= 0.026). TS was the biochemical marker of iron overload with the higher independent correlation with the presence of C282Y/C282Y and C282Y/H63D mutations. The frequency of risk factors for iron overload was not different between the groups, however, in HFE-HH group a greater number of patients without any risk factor was detected (p= 0.019). TS > 82% had a higher predictive negative value for diagnosing HFE-HH when compared to the levels of ferritin, serum iron, total iron binding capacity and transferrin. We concluded that the HFE-HH patients had a greater iron overload than patients with chronic liver diseases with non-HFE iron overload. TS > 82% had more accuracy to diagnose HFE-HH. The carriers of C282Y/C282Y or C282Y/H63D mutations had a higher probability to develop hepatocellular carcinoma, when compared to the patients with HFE genotypes and patients wild type

INTRODUÇÃO E OBJETIVOS: A doença hepática gordurosa não alcoólica (DHGNA) tem sido associada ao carcinoma hepatocelular (CHC), muitas vezes em paciente com hepatopatia avançada. Este estudo objetivou avaliar o efeito do sorafenibe no modelo experimental de CHC avançado secundário à DHGNA, padronizar o PET com 18F-FDG para avaliar o CHC neste modelo e avaliar se há relação entre o grau de avidez pelo 18F-FDG e o grau de diferenciação tumoral do CHC. METODOLOGIA: Estudo foi aprovado pela Comissão de Ética no Uso de Animais. Foram utilizados trinta ratos Sprague-Dawley, machos, com 3 meses de vida, pesando entre 300-400g. O CHC secundário à DHGNA foi induzido pela combinação de dieta hiperlipídica deficiente em colina e dietilnitrosamina na dose de 100 mg/ L na água de beber ad libitum por 16 semanas. Após este período foram suspensos os estímulos carcinogênicos, realizou-se ultrassonografia abdominal para caracterização dos nódulos hepáticos maiores que 2 mm, e foi feita a divisão dos dois grupos segundo randomização e iniciada a administração diária do fármaco por gavagem durante 3 semanas: Controle (n=10) - 1 mL salina, Sorafenibe (n=20): 5mg/ kg/ dia. Ao término do tratamento, os animais realizaram PET (Gamma Medica-Ideas, USA) com 18F-FDG (média de 18F-FDG injetada de 1,02 ± 0,17 mCi ou 37,7 ± 6,29 MBq). Três dias após o PET, os animais foram anestesiados e foi feita a eutanásia, quando foi coletado material hepático. As lâminas foram avaliadas, por patologista veterinário experiente, na coloração de hematoxilina-eosina e imunohistoquímica para glutamina sintetase, antígeno específico de hepatócitos 1 e citoqueratina-19. RESULTADOS: A mortalidade nos dois grupos foi de 60% (p=0,07). Os achados ultrassonográficos mostraram grupos homogêneos com média de nódulos por animal: 4,88 ± 2,75 no controle e 4,95 ± 3,11 no sorafenibe (p=0,48). Na 19ª semana, viu-se que a média de lesões hipercaptantes por animal no PET foi de 4,37 ± 1,59 no grupo sorafenibe e 8,5 ± 3,7 no controle (p=0,006). A avidez máxima do 18F-FDG (SUVmáx) foi diferente entre os grupos estudados: 2,4 ± 1,98 no sorafenibe e 3,8 ± 1,74 no controle (p=0,01). Houve correlação direta entre o CHC pouco diferenciado/indiferenciado e os maiores valores de SUVmed (R2 = 0,34, p=0,04), SUVmax (R2 = 0,44, p=0,01), relação Tumor SUVmax/Fígado SUVmax (R2 = 0,42, p=0,02) e relação Tumor SUVmax/ Músculo SUVmax (R2 = 0,54, p=0,006). A média por animal de CHC confirmado pela histologia foi menor no grupo sorafenibe que no controle (5,5 ± 1,5 vs 3,3 ± 0,48, p=0,01). E o grupo tratado com sorafenibe apresentou mais CHC bem diferenciado que o controle (39% vs 5%, respectivamente, p=0,01), bem como, menor presença de CHC pouco diferenciado que o grupo controle (52% vs 81%, p-0,003). CONCLUSÃO: O sorafenibe reduziu o número médio de CHC, a agressividade dos CHC e menor SUVmax dos tumores. A metodologia do PET foi padronizada para este modelo animal específico. O PET 18F-FDG pode ser utilizado para avaliar não invasivamente o grau de diferenciação histológica do CHC, pois valores maiores de SUVmed, SUVmax, Tumor SUVmax/Fígado SUVmax e Tumor SUVmax/ Músculo SUVmax foram correlacionados com CHC pouco diferenciado / BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) has been linked to hepatocellular carcinoma (HCC), often in patients with advanced liver disease. This study aimed to: assess the effect of sorafenib in the experimental model of NAFLD related HCC, standardize PET 18F-FDG to be an assessment tool of HCC in this model and to assess if there is a correlation between the degree of avidity for 18F-FDG and the degree of tumor differentiation. METHODS: The ethics committee on animal use approved this study. Thirty male sprague-dawley rats were used, weighing between 300-400g. NAFLD related HCC was induced by the combination of fat and choline deficient diet with diethylnitrosamine (100 mg/L) in drinking water for 16 weeks. After this period these carcinogenic stimuli were suspended, and liver nodules were identified by abdominal ultrasound. Two groups were randomized: control (n=10) and treatment (n=20). Rats received daily gavage administration of 1 mL saline in the control group and sorafenib (5mg/kg/day) in the treatment group. After treatment, animals performed PET (Gamma Medica-Ideas, USA) with 18F-FDG (average of 18F-FDG injected 1.02 ± 0.17 mCi or 37.7 ± 6.29 MBq). Three days after the PET, the animals were anesthetized and euthanized. Histological aspect was evaluated by experienced veterinary pathologist. RESULTS: The mortality in both groups was 60% (p = 0.07). The sonographic findings showed homogeneous groups with average nodules per animal of: 4.88 ± 2.75 in control and 4.95 ± 3.11 in sorafenib (p = 0.48). On the 19th week, it was observed that the average hypercaptant lesion per animal in PET was 4.37 ± 1.59 in the sorafenib group and 8.5 ± 3.7 in control group (p = 0.006). Average SUVmax was different between groups: 2.4 ± 1.98 in the sorafenib group and 3.8 ± 1.74 in the control group (p = 0.01). A direct correlation was found between the poorly differentiated HCC and larger values of: SUVmed (R2 = 0.34, p = 0.04), SUVmax (R2 = 0.44, p = 0.01) tumorSUVmax / LiverSUVmax ratio (R2 = 0.42, p = 0.02) and tumorSUVmax / MuscleSUVmax ratio (R2 = 0.54, p = 0.006). HCC average per animal was lower in the sorafenib group than in the control group (5.5 ± 1.5 vs. 3.3 ± 0.48; p = 0.01). And sorafenib group had more well differentiated HCC (39% vs 5%, respectively, p = 0.01) and lower presence of poorly differentiated HCC (52% vs 81%, p -0.003) than the control group. CONCLUSION: Sorafenib reduced the average number of HCC, the aggressiveness of HCC and lowered values of tumors SUVmax. The methodology of PET was standardized for this particular animal model. PET 18F-FDG can be used noninvasively to assess the degree of histological differentiation of HCC, as higher values of SUVmed, SUVmax, tumorSUVmax/ LiverSUVmax ratio and tumorSUVmax / MuscleSUVmax ratio were correlated with poorly differentiated HCC

Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.

Hepatocellular carcinoma (HCC) is the third commonest cancer worldwide. However HCC is considered to be highly resistant to chemotherapy. Gene therapies aimed to regulate Bd-2 proteins may sensitize HCC cells to chemotherapy. Studies have demonstrated that Bid/tBid are crucial in hepatocyte apoptosis. Bid also plays important roles in the development and chemotherapeutic sensitivity of HCC. The objective of this study is to test effect of Ad/AFPtBid and chemotherapeutic agents in combination on an orthotopic HCC model. / In conclusion, (1) Ad/AFPtBid can specifically target and effectively suppress the AFP-producing HCC. (2) Ad/AFPtBid can significantly sensitize HCC to 5-FU, their combination can significantly increase the anti-tumor effectiveness. (3) Ad/AFPtBid shows little toxicity in vivo. (4) The complementary effect of tBid and 5-FU on different phases of the cell cycle may explain the better therapeutic result if both are used to treat HCC. (5) The elucidation of phase specific effect of tBid points to a possible therapeutic option that combines tBid with different phase specific agents to treat HCC. / It is well established that many apoptosis inducers act in a cell cycle-specific fashion. This leads us to hypothesize that tBid might have phase specific effect. So, we tested the susceptibility of Hep3B cells at 00/01, S or G2/M phases to tBid. The results revealed that tBid significantly reduced Hep3B cells in G0/G1 phase, increased cells in G2/M phase. On the contrary, 5-FU arrested Hep3B cells in G0/G1 phase, and significantly reduced cells in G2/M phase. The levels of cell cycle-related proteins were altered in line with the result of the cell cycle. This suggests Hep3B cells in G0/G1 phase may be more susceptible to tBid. The complementary effects tBid and 5-FU on different phases of the cell cycle may explain the better therapeutic result if both are used to treat HCC. / The mice bearing orthotopic HCC tumors were treated with Ad/AFPtBid alone or in combination with 5-FU/Dox. Serum AFP levels were measured to mornitor tumor progression. The mice were killed four weeks after treatment. Liver tissues were subjected to immunohistochemical staining of proliferation cell nuclear antigen (PCNA) and TUNEL staining. Another batch of mice was observed for survival rate over a six month period. In addition, possible side effects of Ad/AFPtBid were tested in BALB/c mice. Results demonstrated that Ad/AFPtBid significantly inhibited Hep3B tumor growth. The combination of Ad/AFPtBid with 5-FU was more effective in tumor regression than either agent alone. However, the combination of Ad/AFPtBid with Dox treatment failed to demonstrated better effect than Dox treatment alone because the mice that received Dox exhibited serious weight loss. Tumor tissues from Ad/AFPtBid alone or combination treatment groups showed a decrease in cells positive for PCNA, and an increase in apoptosis by TUNEL staining, indicating that Ad/AFPtBid induced tumor regression through its pro-apoptotic effect. Inflammatory cell infiltration was also increased. Furthermore, Ad/AFPtBid did not suppress the hepatic tumor formed by non-AFP producing SK-HEP-1 or DLD-1. Finally, Ad/AFPtBid and 5-FU in combination results in better survival rate. No acute toxic effect of Ad/AFPtBid was observed. / Ma, Shihong. / "December 2009." / Adviser: CHEN Gong George. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 114-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.