Na etapa inicial do desenvolvimento de novos fármacos, a avaliação do metabolismo e da toxicidade é fundamental para definir seu potencial emprego como candidato a fármaco. Nestes estudos, diversos modelos in vitro são empregados, dentre eles linhagens de hepatoma humano. Entretanto, uma grande limitação ao uso deste modelo in vitro é a baixa expressão das enzimas do sistema citocromo P450. O carotenóide bixina, componente majoritário do anato (urucum), apresentou em estudos in vivo, a capacidade de induzir algumas isoformas do sistema citocromo P450, com a vantagem de apresentar baixa toxicidade. Neste trabalho, a fração lipossolúvel do anato (bixina) e hidrossolúvel (norbixina) foram avaliadas como indutores do sistema citocromo P450 em linhagens de hepatoma humano. Ensaios de MTT, empregando as linhagens HepG2, C3A e SK-HEP-1 indicaram que bixina e norbixina em concentrações abaixo de 0,22 mM são seguras quanto à citotoxicidade. A expressão dos genes CYP 1A1, 1A2, 2C9, 2B6, 2E1 e 3A4 foi avaliada, através de ensaios de RT-PCR em tempo real, em linhagens de hepatoma humano submetidas a tratamento com os compostos bixina e norbixina. Os resultados mostraram que células HepG2 e C3A tratadas com bixina nas concentrações de 0,05 e 0,1 mM, por períodos de 24 e 48 horas, apresentaram aumento de expressão da CYP 1A1 e CYP 1A2. Porém, a exposição de células HepG2 e C3A ao composto norbixina não resultou em aumento de expressão das isoformas avaliadas neste estudo. Os resultados deste trabalho indicaram o potencial emprego de bixina como agente indutor das CYPs 1A1 e 1A2, em linhagens de hepatoma humano utilizadas como modelo in vitro, para estudo de compostos cuja metabolização envolva uma destas vias, entretanto, estudos adicionais são fundamentais, a fim de avaliar a ação deste composto sobre outras isoformas do sistema citocromo P-450, bem como outros sistemas enzimáticos. / In the early development stage of the new drugs, the pharmacological and toxicological properties are critical to define the potential use of the candidate drug. During this stage, several in vitro models systems are employed, including human hepatoma cell lines. However, the main limitation of the use of cell lines as in vitro model is the low expression level of cytochrome P450 enzymes. A carotenoid knowed as bixin, the main pigment in the annatto (urucum), it has been reported to induce some isoforms of cytochrome P450 in rats, with the advantage of its low toxicity. In this work, the oil-soluble (bixin) and aqueous soluble extracts (norbixin) were evaluated as inducers of the cytochrome P450 system in human hepatoma cell lines (HepG2, C3A, SK-HEP-1). The results of MTT assays showed that bixin concentrations below 0.22 mM were not cytotoxic in HepG2, C3A and SK-HEP-1 cell lines. Expression changes in CYP 1A1, 1A2, 2C9, 2B6, 2E1 and 3A4 were evaluated, by real time RT-PCR and the results showed that the exposition to 0,05 mM and 0,1 mM bixin, for 24 and 48 hours of treatment, lead to an increase in CYP 1A1 and CYP 1A2 expression level. By contrast, the cytochrome P450 isoforms were not affected by the exposition to norbixin. In conclusion, this work indicated the potential use of bixin induced hepatoma cell lines as in vitro model for studies of biotransformation and toxicity of drugs involving CYP 1A, however, further studies are necessary to evaluate the effect of bixin on the other cytochrome P450 isoforms as well as other enzymatic systems.

Na etapa inicial do desenvolvimento de novos fármacos, a avaliação do metabolismo e da toxicidade é fundamental para definir seu potencial emprego como candidato a fármaco. Nestes estudos, diversos modelos in vitro são empregados, dentre eles linhagens de hepatoma humano. Entretanto, uma grande limitação ao uso deste modelo in vitro é a baixa expressão das enzimas do sistema citocromo P450. O carotenóide bixina, componente majoritário do anato (urucum), apresentou em estudos in vivo, a capacidade de induzir algumas isoformas do sistema citocromo P450, com a vantagem de apresentar baixa toxicidade. Neste trabalho, a fração lipossolúvel do anato (bixina) e hidrossolúvel (norbixina) foram avaliadas como indutores do sistema citocromo P450 em linhagens de hepatoma humano. Ensaios de MTT, empregando as linhagens HepG2, C3A e SK-HEP-1 indicaram que bixina e norbixina em concentrações abaixo de 0,22 mM são seguras quanto à citotoxicidade. A expressão dos genes CYP 1A1, 1A2, 2C9, 2B6, 2E1 e 3A4 foi avaliada, através de ensaios de RT-PCR em tempo real, em linhagens de hepatoma humano submetidas a tratamento com os compostos bixina e norbixina. Os resultados mostraram que células HepG2 e C3A tratadas com bixina nas concentrações de 0,05 e 0,1 mM, por períodos de 24 e 48 horas, apresentaram aumento de expressão da CYP 1A1 e CYP 1A2. Porém, a exposição de células HepG2 e C3A ao composto norbixina não resultou em aumento de expressão das isoformas avaliadas neste estudo. Os resultados deste trabalho indicaram o potencial emprego de bixina como agente indutor das CYPs 1A1 e 1A2, em linhagens de hepatoma humano utilizadas como modelo in vitro, para estudo de compostos cuja metabolização envolva uma destas vias, entretanto, estudos adicionais são fundamentais, a fim de avaliar a ação deste composto sobre outras isoformas do sistema citocromo P-450, bem como outros sistemas enzimáticos. / In the early development stage of the new drugs, the pharmacological and toxicological properties are critical to define the potential use of the candidate drug. During this stage, several in vitro models systems are employed, including human hepatoma cell lines. However, the main limitation of the use of cell lines as in vitro model is the low expression level of cytochrome P450 enzymes. A carotenoid knowed as bixin, the main pigment in the annatto (urucum), it has been reported to induce some isoforms of cytochrome P450 in rats, with the advantage of its low toxicity. In this work, the oil-soluble (bixin) and aqueous soluble extracts (norbixin) were evaluated as inducers of the cytochrome P450 system in human hepatoma cell lines (HepG2, C3A, SK-HEP-1). The results of MTT assays showed that bixin concentrations below 0.22 mM were not cytotoxic in HepG2, C3A and SK-HEP-1 cell lines. Expression changes in CYP 1A1, 1A2, 2C9, 2B6, 2E1 and 3A4 were evaluated, by real time RT-PCR and the results showed that the exposition to 0,05 mM and 0,1 mM bixin, for 24 and 48 hours of treatment, lead to an increase in CYP 1A1 and CYP 1A2 expression level. By contrast, the cytochrome P450 isoforms were not affected by the exposition to norbixin. In conclusion, this work indicated the potential use of bixin induced hepatoma cell lines as in vitro model for studies of biotransformation and toxicity of drugs involving CYP 1A, however, further studies are necessary to evaluate the effect of bixin on the other cytochrome P450 isoforms as well as other enzymatic systems.

O ácido 5-aminolevulínico (ALA) é o primeiro precursor do grupo heme acumulado, principalmente no fígado, em alguns tipos de porfirias hepáticas hereditárias (porfiria aguda intermitente-AIP e tirosinemia) ou adquiridas (intoxicação por chumbo) devido à diminuição da atividade da enzima porfobilinogênio deaminase. Amostras de biópsias de fígado de pacientes portadores de AIP revelaram alterações estruturais nas mitocôndrias e no retículo endoplasmático, acúmulo de lipofuscina, gordura e corpúsculos de ferritina. Têm sido demonstrado que mutações mitocondriais induzidas por pró-oxidantes também contribuem para o envelhecimento celular e para o desenvolvimento do câncer. Esses dados podem estar relacionados à maior incidência de carcinoma hepatocelular (HCC) em pacientes sintomáticos de AIP. In vitro, ALA produz espécies reativas de oxigênio (ROS), através da oxidação catalisada por metais, e pode ser visto como uma fonte endógena de ROS, iniciando danos oxidativos a estruturas celulares como o DNA, podendo estar envolvido na iniciação e promoção do câncer. Além disso, o produto final de oxidação do ALA, o ácido 4,5-dioxovalérico (DOVA) é capaz de induzir modificações nas bases do DNA como outros derivados carbonílicos reativos. Neste estudo, demonstramos que o ALA é capaz de produzir danos ao DNA como quebras de fita de DNA plasmidial, aumento do nível de 8-oxo-7,8- dihidro-2\'-desoxiguanosina e 5-hidroxi-2´-desoxicitidina em DNA de órgãos de ratos tratados com ALA e aumento da formação de diversas bases modificadas em DNA de timo de bezerro. O DOVA reagiu com 2´-desoxiguanosina e DNA de timo de bezerro isolado produzindo dois adutos diastereoisômeros. O ALA e o DOVA foram capazes de aumentar a mutagenicidade em S. typhimurium TA104 e induzir resposta SOS em E. coli PQ37. Danos ao DNA mitocondrial e nuclear também foram detectados através da técnica de reação quantitativa em cadeia da polimerase em fibroblastos humanos transformados tratados com ALA. Todos esses dados fornecem informações referentes ao potencial genotóxico do ALA e permitem estabelecer uma proposta de bases moleculares para conectar as lesões ao DNA promovidas pelo ALA com a maior incidência de carcinoma hepatocelular em pacientes sintomáticos de AIP. / 5-Aminolevulinic acid (ALA) is a heme precursor accumulated in some inborn (acute intermittent porphyria-AIP and tyrosinosis) or acquired (lead poisoning) types of hepatic porphyria. In AIP patients, ALA is overproduced and accumulated in the liver. Liver biopsy samples of AIP patients revealed mitochondrial and endoplasmic reticulum structural alterations and accumulation of lipofucsin, fat and ferritin bodies. Mitochondrial mutations induced by pro- oxidants were suggested to contribute to cellular aging and cancer. These findings may be connected to the higher frequency of hepatocellular carcinoma (HCC) associated to symptomatic AIP patients. In vitro, ALA produces reactive oxygen species (ROS) upon metal- catalyzed oxidation and can be viewed as a deleterious endogenous source of ROS, triggering oxidative damage to cell structures and organs and being involved in the initiation and promotion of cancer. Besides, the final oxidation product of ALA, the 4,5-dioxovaleric acid (DOVA) is expect to induce DNA base modifications as already shown for other reactive carbonyl derivatives. In this study we demonstrated that ALA is able to produce DNA lesions such as strand breaks in plasmid DNA, increased steady state level of 8-oxo-7,8- dihydro-2\'-deoxyguanosine and 5-hydroxy-2´-deoxycytidine in rat organs DNA of ALA-treated rats and increased formation of several modified DNA bases in calf thymus DNA. 4,5-Dioxovaleric acid was showed to react with 2\'-deoxyguanosine and isolated calf thymus DNA through Schiff?s base formation to produce two diastereisomeric adducts. Aminolevulinic acid and DOVA were able to increase mutagenicity of the S. typhimurim strain TA104 and induce SOS response in E. coli PQ37. The mitochondrial and nuclear DNA damage were also detected by quantitative polymerase chain reaction technique in transformed human fibroblasts treated with ALA. All these data provide additional information on the genotoxic potential of ALA and reinforce the hypothesis that ALA may be involved in the induction of HCC in symptomatic AIP patients.

O ácido 5-aminolevulínico (ALA) é o primeiro precursor do grupo heme acumulado, principalmente no fígado, em alguns tipos de porfirias hepáticas hereditárias (porfiria aguda intermitente-AIP e tirosinemia) ou adquiridas (intoxicação por chumbo) devido à diminuição da atividade da enzima porfobilinogênio deaminase. Amostras de biópsias de fígado de pacientes portadores de AIP revelaram alterações estruturais nas mitocôndrias e no retículo endoplasmático, acúmulo de lipofuscina, gordura e corpúsculos de ferritina. Têm sido demonstrado que mutações mitocondriais induzidas por pró-oxidantes também contribuem para o envelhecimento celular e para o desenvolvimento do câncer. Esses dados podem estar relacionados à maior incidência de carcinoma hepatocelular (HCC) em pacientes sintomáticos de AIP. In vitro, ALA produz espécies reativas de oxigênio (ROS), através da oxidação catalisada por metais, e pode ser visto como uma fonte endógena de ROS, iniciando danos oxidativos a estruturas celulares como o DNA, podendo estar envolvido na iniciação e promoção do câncer. Além disso, o produto final de oxidação do ALA, o ácido 4,5-dioxovalérico (DOVA) é capaz de induzir modificações nas bases do DNA como outros derivados carbonílicos reativos. Neste estudo, demonstramos que o ALA é capaz de produzir danos ao DNA como quebras de fita de DNA plasmidial, aumento do nível de 8-oxo-7,8- dihidro-2\'-desoxiguanosina e 5-hidroxi-2´-desoxicitidina em DNA de órgãos de ratos tratados com ALA e aumento da formação de diversas bases modificadas em DNA de timo de bezerro. O DOVA reagiu com 2´-desoxiguanosina e DNA de timo de bezerro isolado produzindo dois adutos diastereoisômeros. O ALA e o DOVA foram capazes de aumentar a mutagenicidade em S. typhimurium TA104 e induzir resposta SOS em E. coli PQ37. Danos ao DNA mitocondrial e nuclear também foram detectados através da técnica de reação quantitativa em cadeia da polimerase em fibroblastos humanos transformados tratados com ALA. Todos esses dados fornecem informações referentes ao potencial genotóxico do ALA e permitem estabelecer uma proposta de bases moleculares para conectar as lesões ao DNA promovidas pelo ALA com a maior incidência de carcinoma hepatocelular em pacientes sintomáticos de AIP. / 5-Aminolevulinic acid (ALA) is a heme precursor accumulated in some inborn (acute intermittent porphyria-AIP and tyrosinosis) or acquired (lead poisoning) types of hepatic porphyria. In AIP patients, ALA is overproduced and accumulated in the liver. Liver biopsy samples of AIP patients revealed mitochondrial and endoplasmic reticulum structural alterations and accumulation of lipofucsin, fat and ferritin bodies. Mitochondrial mutations induced by pro- oxidants were suggested to contribute to cellular aging and cancer. These findings may be connected to the higher frequency of hepatocellular carcinoma (HCC) associated to symptomatic AIP patients. In vitro, ALA produces reactive oxygen species (ROS) upon metal- catalyzed oxidation and can be viewed as a deleterious endogenous source of ROS, triggering oxidative damage to cell structures and organs and being involved in the initiation and promotion of cancer. Besides, the final oxidation product of ALA, the 4,5-dioxovaleric acid (DOVA) is expect to induce DNA base modifications as already shown for other reactive carbonyl derivatives. In this study we demonstrated that ALA is able to produce DNA lesions such as strand breaks in plasmid DNA, increased steady state level of 8-oxo-7,8- dihydro-2\'-deoxyguanosine and 5-hydroxy-2´-deoxycytidine in rat organs DNA of ALA-treated rats and increased formation of several modified DNA bases in calf thymus DNA. 4,5-Dioxovaleric acid was showed to react with 2\'-deoxyguanosine and isolated calf thymus DNA through Schiff?s base formation to produce two diastereisomeric adducts. Aminolevulinic acid and DOVA were able to increase mutagenicity of the S. typhimurim strain TA104 and induce SOS response in E. coli PQ37. The mitochondrial and nuclear DNA damage were also detected by quantitative polymerase chain reaction technique in transformed human fibroblasts treated with ALA. All these data provide additional information on the genotoxic potential of ALA and reinforce the hypothesis that ALA may be involved in the induction of HCC in symptomatic AIP patients.

Untersuchung der Butyrateffekte auf die Genexpression von Zellkulturen während der Adenom-Karzinom-Sequenz mit Hilfe von Microarrays. Analyse des HDGF-Genclusters. Verwendete Zellkulturen Geki2, HT29 und SW620 / Examination of butyrate effects on genexpression of cellculteres during adenom-carcinom-sequence by use of microarrays. Analysis of hdgf-gencluster. USed cellcultures Geki2, HT29 und SW620.

Im Rahmen dieser Arbeit wurde die Expression des putativen Onkogens HDGF (Hepatoma Derived Growth Factor) in der humanen Kolonkarzinomzelllinie HT29 mittels RNA Interferenz herunterreguliert. Zwei unterschiedliche, für das Zielgen HDGF spezifische siRNAs wurden hierzu jeweils in einen Plasmidvektor kloniert. Zellen der Kolonkarzinomzelllinie HT29 wurden zunächst transient und stabil mit den beiden resultierenden Plasmidvektoren transfiziert, die Expression von HDGF in den transfizierten Zellen mittels Realtime-PCR quantifiziert und mit der Expression in mit Lipofektamin behandelten Kontrollzellen verglichen. Durch die stabile Transfektion beider Plasmidvektoren konnte die HDGF-Expression fast komplett supprimiert werden. Im Rahmen eines cDNA-Array konnten außer einer Expressionsverminderung von HDGF noch multiple Expressionsveränderungen anderer Gene identifiziert werden. Dies ist zum einen durch unspezifische, durch den Plasmidvektor bedingte Effekte erklärbar. Zum anderen aber ist die Deregulation vieler dieser Gene ein Effekt der Inaktivierung von HDGF. / In this thesis the expression of the putative oncogene HDGF (Hepatoma Derived Growth Factor) in a colorectal cancer cell-line was down-regulated by means of RNA interference. The expression of HDGF was almost completely suppressed. The expression of various other genes was also influenced. This is on the one hand due to unspecific effects caused by the plasmidvector. On the other hand it is also an effect of the gene silencing of HDGF.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Current therapeutic approaches for HCC including surgical resection and trans-arterial embolization (TAE) remain largely ineffective, underscoring the need for development of novel therapeutic strategies. Because HCC is high vascularized, continuous administration angiogenesis inhibitor using gene therapy approach may facilitate long-term blockade tumor vasculature, thereby perturbing the growth of HCC. Vasostatin 112 (VS112) encodes an alternatively spliced fragment of angiogenesis inhibitor vasostatin, which encompasses residues 1-64 and 133-180 of calreticulin. In this study, recombinant adenovirus encoding VS112 (Ad-VS112) was generated to evaluate its potential for suppression of orthotopic Novikoff hepatoma in syngenic Sprague-Dawley (SD) rats. Adenovirus-mediated VS112 overexpression significantly inhibited the migration and tube formation of endothelial cells, indicating the anti-angiogenic potency of VS112 gene delivery. However, VS112 overexpression had no influence on the viability of N1-S1 Novikoff hepatoma cells. To investigate the prophylactic effect of VS112 expression on hepatoma growth, N1-S1 cells were infected with Ad-VS112 or adenovirus encoding green fluorescent protein (Ad-GFP) then implanted into the liver of SD rats. After 14 days, rats implanted with VS112-expressing showed significantly reduced incidence and size of hepatoma compared with those implanted with Ad-GFP-infected cells. To investigate the therapeutic efficacy of VS112 gene delivery, the SD rats were implanted with N1-S1 cells on day 0, treated with adenovirus vectors (2 x 1010 plaques forming units) via intravenous route on day 1, then sacrificed on day 14 to monitor hepatoma growth. By measuring tumor weight, it was found that Ad-VS112-treated rats exhibited significantly decreased tumor burden compared with control groups, which was in accordance with their lower serum GOT level. Histological analysis revealed a significant reduction of vWF-positive blood vessels in Ad-VS112-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, the expression of pro-inflammatory nuclear factor kappa B (NF£eB) and cyclooxgenase II (COXII) was also effectively attenuated in Ad-VS112-treated hepatoma. In conclusion, prior or post VS112 gene delivery potently suppresses the growth of orthotopic hepatoma,thereby holding promises for future treatment of HCC.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. An extensive array of growth factors and their receptors have been identified and may act as positive and negative modulators in different stages of liver carcinogenesis. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from conditioned medium of Huh-7 hepatoma cell line. HDGF has growth stimulating activity for various types of cells. Recent evidence indicates that HDGF upregulation is associated with poor survival outcome and tumor progression in HCC, non-small cell lung carcinoma and melanoma. However, the exact function and molecular mechanism of HDGF overexpression during HCC progression remain largely unknown. In the first project (Chapter 2) of this thesis study, we started with characterizing in HDGF release and response to exogenous HDGF between benign HepG2 and malignant SK-Hep-1 hepatoma cells. It was found that serum deprivation significantly stimulated the HDGF secretion in SK-Hep-1 cells but not HepG2 cells. Interestingly, SK-Hep-1 cells did not increase the secretion of vascular endothelial growth factor (VEGF), a potent angiogenic factor, during serum deprivation. Besides, SK-Hep-1 cells were more responsive to the growth- and migration-promoting effect of exogenous HDGF. We also validated the angiogenic functions of recombinant HDGF protein in vitro and in vivo. In the second project (Chapter 3), we investigated the influence of cellular HDGF level on the neoplastic potential of hepatoma cells. Adenovirus vectors encoding HDGF, Ad-HDGF, and antisense HDGF, Ad-HDGF (-), were generated to modulate the cellular HDGF levels in SK-Hep-1 cells. Adenovirus-mediated HDGF gene delivery increased the HDGF expression and release, and stimulated the proliferation, migration and anchorage-independent growth of SK-Hep-1 cells. In contrast, infection with Ad-HDGF (-) reduced the HDGF expression and secretion, and attenuated the oncogenic behaviors of SK-Hep-1 cells. Implanting HDGF-overexpressing SK-Hep-1 cells led to the accelerated growth of xenografted hepatoma in SCID mice while implantation of HDGF-downregulated SK-Hep-1 cells caused retarded tumor growth. Histological analysis revealed the increased proliferation and neovascularization in HDGF-overexpressing tumors. This could be attributed to elevated VEGF expression and activation of the nuclear factor kappa B (NF£eB) activities by HDGF upregulation in SK-Hep-1 cells. In the third project (Chapter 4), we delineated the mechanism underlying HDGF-induced VEGF secretion and activation of NFB pathway in SK-Hep-1 cells. Adding recombinant HDGF protein enhanced the VEGF release by SK-Hep-1 cells particularly during serum starvation. This was associated with a concomitant increment in VEGF protein and mRNA levels in SK-Hep-1 cells. Like many mitogens, HDGF increased the production of reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide in a dose-dependent manner. Pretreatment with antioxidants abolished the HDGF-induced VEGF secretion. NF£eB is a pivotal transcription factor for regulation of pro-inflammatory cytokines and genes such as VEGF and cycloxygenase¡V2 (COX-2). Application of HDGF stimulated NF£eB-driven luciferase activities. This was correlated with a dose- and time-depedent increment of NF£eB (p65) by HDGF. HDGF treatment also elevated the COX-2 protein levels and activities in SK-Hep-1 cells. In addition, blockade of COX-2 by NS-398 attenuated the HDGF-induced VEGF secretion, suggesting the involvement of COX-2. Finally, it was found that HDGF stimulated the phosphorylation of Akt, Erk1/2, and p38 MAPK. Inhibition of Akt by LY294002 also diminished the HDGF-induced VEGF secretion. These studies suggest that HDGF induces oxidative stress to activate NF£eB/COX-2/Akt pathway, thereby stimulating VEGF expression and release. In summary, this thesis study brings functional and mechanistic insights on how aberrant HDGF expression contributes to angiogenesis and tumorigenesis during liver carcinogenesis.

In this study, we investigated the mechanisms of HDGF on cell migration in non-transformed NIH/3T3 cells. HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression, Akt phosphorylation and PTEN phosphorylation as well as stimulated the RhoA, Rac1, and Cdc42 activities. Furthermore, Adenoviral gene transfer of PTEN attenuated migration and PI3K/Akt/Rho GTPases signaling in HDGF-overexpressing transfectants. Pharmaceutical intervention using the PI3K inhibitor, LY294002, potently reversed HDGF-stimulated cell migration, dorsal ruffles formation and podosome formation as well as the RhoA, Rac1, and Cdc42 activities. Thus, HDGF elicits the activation of PI3K/Akt /Rho GTPases signaling cascade and promotes cytoskeleton remodeling to stimulate cellular migration. Moreover, we investigate the expression profile of HDGF during breast carcinogenesis. Immunohistochemical studies revealed elevated HDGF expression in human breast cancer. Nuclear HDGF labelling index was positively correlated with tumour grade, stage and proliferation index, but negatively correlated with survival rate in breast cancer patients. Our data also showed that HDGF over-expression was associated with lymph node metastasis and represented an independent prognostic factor for tumor recurrence. Furthermore, Immunoblot study revealed that elevated HDGF expression significantly higher in breast cancer cells (MDA-MB-231 cells) than that in non-transformed breast cells (MCF-7 cells). Consistently, higher invasive potency and colony formation also observed in MDA-MB-231 cells than in MCF-7 cells. Adenovirus-mediated HDGF over-expression and exogenous HDGF treatment stimulated the invasiveness and colony formation as well as E-cadherin down-regulation and Vimentin up-regulation. Conversely, either HDGF knockdown by RNA interference, HDGF antibody neutralization or BITC-induced EMT suppression in MDA-MB-231 cells attenuated the malignant behavior and elicited EMT reversal by enhancing E-cadherin expression while depleting Vimentin expression. In summary, HDGF elicits the activation of PI3K/Akt/Rho GTPases signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration. Moreover, the formation of podosome rosettes is correlated with cell invasion, the podosome-stimulating capability of HDGF is consistent with HDGF regulates the metastasis of breast cancer through modulating of epithelial-mesenchymal transition. Therefore, our results provide not only novel insights into the role of the HDGF in cell migration and tumor metastasis, but also validate a novel prognostic indicator for breast cancer. Key words: Hepatoma-derived growth factor, PI3K, Akt, epithelal-mesenchemal transition, E-cadherin, Vimentin

Liver fibrosis, a major medical problem with significant morbidity and mortality, is considered as a wound-healing response to a variety of chronic stimuli. It is characterized by an excessive deposition of extracellular matrix (ECM) proteins, which disrupts the normal architecture of liver and ultimately leads to pathophysiological damage to liver. Hepatoma-derived growth factor (HDGF), a growth factor originally purified from hepatoma cells, is highly expressed in fetal hepatocytes and hepatoma. It is known to play multifunctional roles in mitogenesis, organogenesis, embryogenesis, and tumorigenesis. Its expression correlates with the proliferating state of hepatocellular carcinoma (HCC) and serves as a prognostic factor. Since liver fibrosis frequently occurs prior to HCC development, the specific aim of this study is to investigate the role of HDGF in the progression of liver fibrosis by using animal models of mice receiving either bile duct ligation surgery or carbon tetrachloride administration. Quantitative real-time PCR and Western blotting analysis showed a significant elevation of HDGF expression in both models. HDGF levels correlated with progression of liver fibrosis in a time-dependent manner as well as paralleled with the expression of other two fibrotic markers, transforming growth factor-b1 (TGF-b1) and pro-collagen type I, in fibrotic livers. Intriguingly, the over-expressed HDGF protein was localized mainly in perivenous hepatocytes of fibrotic livers. Besides, adenovirus-mediated HDGF gene delivery potentiated the production of TGF-b1 and pro-collagen type I, thereby enhancing the intrahepatic collagen matrix deposits as evidenced by Sirius red stain and morphometrical analysis. In cultured hepatocytes, TGF-b1 and HDGF mutually up-regulated their de novo synthesis only when grown on collagen-coated matrix, strongly suggesting that the TGF-b1- and/or HDGF-driven pro-fibrogenic signaling is collagen-dependent and a vicious circle may exist at the initial stage of hepatic fibrogenesis. Moreover, administration with recombinant HDGF stimulated BrdU uptake and synthesis of both a-smooth muscle actin and pro-collagen type I in cultured hepatic stellate cells, implicating that a mode of paracrinal action lies between these two cell types. In conclusion, HDGF plays a pro-fibrogenic role during liver fibrosis and blockade of HDGF pathway may potentially constitute the preventive or therapeutic strategies for chronic liver diseases.