By Western blotting and immunohistochemical analyses, we have demonstrated that Pin1 was overexpressed in 71.4% of hepatocellular carcinoma (HCC) and its levels correlated with the clinical survival rate. This conclusion was supported by the results from examining Pin1 protein in HCC cancer cell lines. RT-PCR was performed to examine the Pin1 transcription level in tumor part and was compared with that in non-tumor part. Our results indicated that pin1 overexpression was due to the upregulation of Pin1 transcription. Interestingly, most of the cases with upregulation of Pin1 have been shown to correlate with £]-catenin and Cyclin D1 accumulation in HCC specimens. These results were consistent with the previous studies that Pin1 caused £]-catenin and Cyclin D1 elevation in breast cancer. The concordance between hepatitis virus chronic infection and Pin1 overexpression of HCC patients was also analysis. Taken together, these data indicated that Pin1 overexpression leading to £]-catenin and Cyclin D1 accumulation might play a critical role in hepatocellular carcinogenesis and tumor progression. Pin1 levels therefore can be used as a prognostic marker for HCC, and our results suggested that Pin1 is a potential target for therapeutic intervention in hepatocellular carcinoma.

Erythropoietin (EPO) is produced in the kidney and in fetal liver in response to hypoxia as well as to CoCl2. The EPO protein and mRNA can be induced in response to both stimuli in the human hepatoma cell (HCC) lines Hep 3B and Hep G2. An oxygen sensing mechanism in which a ligand dependent conformational change in the heme protein produces H2O2 in respone to either hypoxia or Cobalt has been demonstrated. However, an intriguing question can be raised as to why some HCC sublines, such as Hep G2 and Hep 3B are capable of expressing EPO gene, whereas in other HCC sublines, such as J5 and SK-Hep-I are completely devoid of the ability to express EPO gene. Along this line, does ¡§differentiation status¡¨ of these HCC cells play a pivotal role in regulating the expression of EPO gene? Next in line, how a differentiation-associated upregulation of g-glutemylcysteine synthetase (g-GCS), which tightly regulating the biosynthesis of endogenous glutathione(GSH) can modulate the expression of EPO. The objective of this research project was designed to address all these questions. Reported herein are several lines of evidence to demonstrate that endogenous GSH contents do play a pivotal role in the control and regulation of the expression of EPO gene. Firstly, using a group of five HCC lines with varying degrees of differentiation as the experimental model, we demonstrated that the endogenous GSH contents of these HCC cells were differentially upregulated depending on the degree of differentiation with an order of abundance being Hep G2> Hep 3B> J5> Mahlavu> SK-Hep-I. Coincidently, we also found that g-GCS heavy subunit activities as well as its mRNA correlated precisely with this order. Among these HCC cell lines tested, only two well-differentiated sublines, Hep G2 and Hep 3B expressed EPO gene implying that the latter process was dependent upon GSH and suggested a notion that a threshold level might be required for its optimal reactivation. Secondly, to further obtain the evidence to substantiate this possible role of GSH, we then supplemented to the cell culture media with an excessive quantity of nonlethal N-acetylcysteine for the purpose of reinforcing the endogenous GSH biosynthesis. Interestingly, we found that this manipulation could revert the reactivation of EPO gene in cell lines, such as J5 and SK-Hep-I, in which their EPO gene expressions were ortherwise shut down under a normal circumstance. Finally, we were able to demonstrated using RT-PCR and western blotting that the expression of EPO gene was reverted in GCS30, a SK-Hep-I subline that was permanently transfected with g-GCSh and is capable of overly expressing endogenous GSH. Taken together, we demonstrated herein for the first time that, besides hypoxia and CoCl2, endogenous GSH contents can also act as a positive regulator for the expression of EPO gene. The underlying mechanism of how GSH exerts its action in the regulation of EPO expression awaits further clarification.

Introduction: Most literatures researched radiofrequency ablation (RFA) for early hepatocellular carcinoma (HCC) defined the early tumor size as 3cm or less. However, detection rate of HCC smaller than 2 cm became increasing since high risk patients had received regular screening and the imaging techniques has been much improved. Whether RFA or percutaneous ethanol injection (PEI) is better for a patient with such a small HCC is still controversial. Methods: We retrospectively obtained patients with single HCC 2 cm in diameter or smaller from the computerized medical records database in a local teaching hospital located at southern Taiwan, diagnosed during January 1, 2002 to April 30, 2008. Those patients received RFA (RFA group) or PEI (PEI group) as the first-line nonsurgical treatments were enrolled for further analysis. We compared baseline characteristics of RFA and PEI groups, including gender, age, possible risk factors of recurrence, and prognostic factors. Then, we analyzed recurrent rate, time to recurrence, survival rate, complication rate, mean cost of each treatment, and hospital stay of RFA and PEI groups. Results: There were 32 patients qualified for the study design, including 22 in PEI group¡G13 males and 9 females with mean age was 63.73 years; and 10 in RFA group¡G7 males and 3 females with mea age was 58.30 years¡CNo statistically significant differences between RFA and PEI groups were observed with respect to baseline characteristics. Nevertheless, there was significant difference between these two groups with respect to mean hospital stay (p=0.007) and mean cost (p¡Õ0.001): mean cost of PEI was NTD $16934.7; mean cost of RFA was NTD $51677.6, the difference was NTD $34732.9. There was no difference respect to complication rate, recurrent rate, time to recurrence and overall survival rate between RFA and PEI groups. Conclusion: For patients with single HCC 2 cm in diameter or smaller (i.e. very early HCC), we concluded that: if under similar basic background, the cost of RFA was much higher than that of PEI, but no difference in the complication rate, recurrent rate, time to recurrence and overall survival rate between these two treatment.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide, accounting for over 600,000 deaths per year. The most common treatment strategy for intermediate and advanced stage unresectable HCC is transarterial chemoembolization (TACE), which involves the local administration of a chemotherapeutic drug combined with arterial occlusion resulting in ischemic tumor necrosis. However, TACE suffers from inadvertent exposure of noncancerous liver parenchyma to embolic agents resulting in liver injury. In some cases, over-embolization has lead to infection, necrosis of unaffected liver tissue, and even liver failure which suggests the need for a biocompatible, multifunctional embolic material which can deliver anticancer drugs with high target specificity. Our laboratory has recently developed a method to fabricate porous silicon (pSi) microparticles with defined physicochemical properties based on photolithography and anodic etching. These microparticles function as multistage drug delivery systems that can circumvent the biobarriers present in the systemic circulation enabling site-specific localization and release of chemotherapy and imaging agents. The versatility of the fabrication process enables the realization of microparticles ranging in size from 600nm to 116[mu]m in diameter with varying shapes, including discoidal, cylindrical and hemispherical, and varying porosity with pore sizes ranging from 6nm to greater than 50nm in diameter. Nanoparticles, such as quantum dots, siRNA-loaded nanoliposomes, gadolinium-based contrast agents, gold and iron oxide nanoparticles, are loaded in pSi microparticles by tailoring their pore sizes and surface chemistries. This thesis presents preliminary results on the applicability of biocompatible, engineered pSi microparticles as an embolic agent for HCC chemoembolization therapy. Hemispherical microparticles with 116[mu]m diameter were successfully fabricated and suspended in phosphate buffered saline (PBS). A microvascular construct was rapid prototyped in polydimethylsiloxane (PDMS) as an in vitro experimental platform to study the embolization behavior of pSi microparticles. Oxidized pSi microparticles were introduced into the microfluidic device at an appropriate flow rate and time-lapse images were taken showing the formation of occlusions at the bifurcation within minutes of administration. Furthermore, penetration through the bifurcation was completely hindered suggesting that pSi microparticles can potentially be used as a biocompatible, multifunctional chemoembolization agent. Although these results are promising, further investigations are warranted.

No description available.

Hepatitis B virus (HBV) causes an estimated 600,000 deaths annually, largely due to hepatocellular carcinoma (HCC). HBx, a promiscuous transactivator, is a viral oncoprotein, but its exact functions are poorly understood. Many studies have suggested that NF-κB signaling mediates HBx functions, but the underlying molecular mechanisms remain yet to be elucidated. Here, we provide evidence that HBx-mediated NF-κB activation depends on the physical interaction between HBx and a transcription factor, p65. In the cytoplasm, HBx-p65 interaction may promote IκBα phosphorylation and subsequent p65 nuclear localization. A cytokine assay using qPCR and RT-PCR indicates that HBx is associated with a unique profile of cytokine mRNA expression. As shown by chromatin immunoprecipitation (ChIP), HBx in the nuclues can be recruited to the gene promoter by p65. These findings support the importance of HBx-p65 interaction and suggest that it is potentially a promising target of novel therapeutics for HBV-associated liver diseases, including HCC.

Members of the membrane-spanning four-domains, subfamily A (MS4A) family are small polypeptides that share the structural features of four-transmembrane domains and unevenly sized extracellular loops. The family includes CD20, FcεRIβ and HtM4, plus a number of relatively uncharacterised proteins / predicted proteins. MS4A proteins are discussed in relation to other protein families, such as the tetraspanins, that are also characterised by four-transmembrane domains. The aim of this study was to identify the cell and tissue distribution, subcellular localisation, and function of a newly discovered member of the MS4A family, hepatocellular carcinoma-associated antigen 112 (HCA112). At a subcellular level, HCA112 was found on the plasma membrane of transfected COS-7 cells, and also within the Golgi complex, trans-Golgi-network, and early endosomes. The molecule is orientated such that the large loop is extracellular and the Nand C-terminal domains are cytoplasmic. The presence of HCA112 associated with components of the endocytic pathway raised the question of whether some originated from the surface membrane. Antibody was used to label a HA epitope tag engineered into the large extracellular loop of HCA112, and the bound antibody was tracked through early endosomes to the recycling compartment. Here it co-localised with internalised transferrin, indicating strongly that HCA112 is internalised via clathrin-dependent mechanisms. Several endocytic sorting motifs within the intracellular domains of HCA112 were investigated for their ability to direct internalisation of HCA112. Deletion of a di-leucine motif was found to slow but not prevent endocytosis, suggesting that it is involved in endocytosis of HCA112, although not essential for the process. When HCA112 expression constructs featuring N- and C-terminal domain truncations were examined, it was found that the N-terminal tail does not affect the subcellular localisation or trafficking of HCA112, while deletion of the C-terminal intracellular domain resulted in retention of the mutant protein in the ER. HCA112 has a wide tissue distribution and is highly expressed in the lining/covering and parenchymal epithelium of some tissues, proximal renal tubules, ductal epithelium in a number of organs, endothelial cells, some steroidogenic endocrine cells, adipocytes, smooth muscle cells, follicular dendritic cells and macrophages. The expression of HCA112 by a wide range of cell types suggests that its function(s) has general importance and is not limited to any specific cell type(s). After reflection on the functions of the HCA112-expressing cells, a common theme that emerged was one of endocytic activity. This lead to speculate that one function of HCA112 might be related to uptake of macromolecules, for instance, in antigen processing and presentation. This might be a general function, such as facilitating uptake of other cell membrane proteins, or directing the traffic of endocytic vesicles. It was noted that HCA112 has a similar cell and tissue distribution to the scavenger receptor and fatty acid translocase FAT/CD36 (Zhang et al., 2003). Furthermore, in cells co-transfected with HCA112 and FAT/CD36, the two molecules co-localise in early endosomes and co-immunoprecipitate, suggesting that the molecules physically and spatially associate. Thus, HCA112 could be involved with (or complement) FAT/CD36 in its functions as a long chain fatty acid transporter and scavenger receptor. A proteomics study of proteins that co-immunoprecipitated with HCA112 detected putative interactions with a number of proteins. These included LR8, transferrin receptor, interferon induced transmembrane proteins 2 and 3, Calpain-6, stomatin, PDGF α receptor, and heat shock 70 kDa protein 8 (HSPA8, formerly known as clathrin un-coating ATPase). Of these, LR8 and the transferrin receptor were investigated in more detail. The results provide strong evidence that HCA112 forms a novel complex with LR8, and that this may be involved in macromolecule internalisation or trafficking of membrane proteins, such as FAT/CD36 or the transferrin receptor. In the case of the transferrin receptor, this traffic appears to involve the clathrin-dependent pathway, but it is possible that when HCA112 is associated with FAT/CD36, it functions within lipid raft domains. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375454 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 64-83).

Introdução: O carcinoma hepatocelular (CHC) é o câncer primário de fígado mais comum. A cirrose hepática é o principal fator de risco para esse tumor. O rastreamento para o CHC em pacientes com cirrose tem sido recomendado há anos. Acredita-se que a detecção e o tratamento precoce do CHC melhorem a sobrevida dos pacientes. O objetivo deste estudo foi analisar a sobrevida dos pacientes cirróticos com CHC pequeno e identificar fatores preditivos de sobrevida no Brasil. Casuística e Métodos: Entre janeiro de 1998 e dezembro de 2003, 74 pacientes cirróticos com CHC foram avaliados. Eles preenchiam os seguintes critérios: CHC com até três nódulos e no máximo 30 mm de diâmetro cada. Os fatores preditores de sobrevida foram identificados através do método de Kaplan-Meier e o modelo de Cox. Resultados: A média de idade foi de 58 anos (32-77); 71% dos pacientes eram do sexo masculino; 64% tinham hepatite C; 60% eram Child-Pugh A, o valor mediano da pontuação de MELD foi de 11; 79% tinham hipertensão portal. No momento do diagnóstico, 71% tinham uma única lesão; o tamanho do principal tumor era menor que 20 mm em 47%; o valor médio de AFP foi de 131 ng/ml. Três pacientes tinham trombose de veia porta, sugestiva de invasão vascular. Cinqüenta pacientes (67,5%) foram incluídos na lista de transplante hepático, que foi realizado só em quatro pacientes. A ressecção cirúrgica do tumor foi possível em quatro pacientes. Quarenta e oito (64,8%) pacientes receberam tratamento ablativo percutâneo (ablação por radiofreqüência ou injeção percutânea de etanol). Nove pacientes não receberam nenhum tratamento específico para o tumor. A taxa de sobrevida geral foi de 80%; 62%; 41% e 17% em 12, 24, 36 e sessenta meses, respectivamente. O tempo médio de seguimento após o diagnóstico do CHC foi de 23 meses (mediana de 22 meses, variando de um a 86 meses) para todo o grupo. Durante o seguimento, ocorreram 39 óbitos ocorreram relacionados com insuficiência hepática ou progressão do CHC. A análise univariada dos 74 pacientes mostrou que escore MELD maior que 11 (p = 0,016), classificação de Child-Pugh B e C (p = 0,007), AFP > 100 ng/ml (p = 0,006), mais de uma lesão (p = 0,041), diâmetro do tumor > 20 mm (p = 0,009) e presença de invasão vascular (p < 0,0001) foram preditores independentes de sobrevida. A análise de regressão de Cox identificou invasão vascular (RR = 14,60 - IC 95% = 3,3 - 64,56 - p < 0,001) e tamanho do tumor > 20mm (RR = 2,14 - IC 95% = 1,07 - 4,2 - p = 0,030) como preditores independentes de pior sobrevida. O tratamento do CHC esteve relacionado com melhor sobrevida. Conclusão: A identificação de CHC pequeno com até 20 mm de diâmetro está relacionada com melhores taxas de sobrevida. Por outro lado, a presença de invasão vascular, apesar do tamanho pequeno das lesões, é um fator associado a péssimo prognóstico. / Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Liver cirrhosis is the major risk factor for this tumor. Screening for HCC in patients with cirrhosis has been recommended, in the belief that detection and treatment of early HCC improves patient survival. The aims of this study were to analyze the overall survival of small HCC in cirrhotic patients and identify independent predictors of survival, in Brazil. Methods: Between January 1998 and December 2003, seventy-four cirrhotic patients with hepatocellular carcinoma were evaluated satisfying the following criteria: HCC of 30 mm or smaller and a maximum of three lesions. Predictors of survival were identified using the Kaplan-Meier and the Cox model. Results: Mean age was 58 years-old (32-77), 71% of patients was male, 64% had hepatitis C, 60% were Child-Pugh A, mean MELD score was 11 and 79% had portal hypertension. At the time of diagnosis, 71% had one tumor, the size of the main tumor was smaller than 20 mm in 47%, mean AFP level was 131 ng/ml. Three patients had portal vein thrombosis, suggesting vascular invasion. Fifty patients (67.5%) were included in the liver transplant list, but it was performed in only four patients. Tumor resection was possible in four patients. Forty-eight (64.8%) patients received percutaneous treatment (radiofrequency ablation or percutaneous ethanol injection). Nine patients did not receive any cancer treatment. The overall survival rates were 80%, 62%, 41% and 17% at 12, 24, 36 and 60 months, respectively. The mean length of follow-up after HCC diagnosis was 23 months (median 22 months, range 1-86 months) for the entire group. During follow-up a total of 39 deaths related to liver failure or HCC progression occurred. Univariate analysis of the 74 patients showed that MELD score greater than 11 (p = 0.016), Child-Pugh classification (p = 0.007), AFP > 100 ng/ml (p = 0.006), more than one lesion (p = 0.041), tumor diameter > 20 mm (p = 0.009) and presence of vascular invasion (p < 0.0001) were significant predictors of survival. Cox regression analysis identified vascular invasion (RR = 14.60 - IC 95% = 3.3 - 64.56 - p < 0.001) and tumor size > 20mm (RR = 2.14 - IC 95% = 1.07 - 4.2 - p = 0.030) as independent predictors of decreased survival. Treatment of HCC was related to increased overall survival. Conclusion: Identification of small tumors of up to 20 mm diameter is related to increase survival. Nevertheless, vascular invasion, in spite of the small diameter of the lesions, is a factor associated with dismal prognosis.

Intra-arterial or percutaneous locoregional therapies (LRT) are often employed to maintain potential liver transplant (LT) recipients with hepatocellular carcinoma (HCC) within T2/Milan criteria. Predictors of survival when LRT is used as destination therapy in those who are either ineligible or unwilling for LT remain poorly defined. We evaluated predictors of 3-year survival with destination LRT in a population of cirrhotic patients diagnosed with HCC, presenting within T2 criteria, and either ineligible or unwilling for LT. The cohort surviving 3 years had a significantly lower model for end-stage liver disease (MELD) score at HCC diagnosis (9.7 vs. 11.4, P= 0.037) and MELD following initial locoregional therapy (10.7 vs. 13.3, P= 0.008) compared to those not surviving three years despite similar demographic, tumor, and treatment variables. LRT as destination therapy results in modest intermediate term survival, with liver function at presentation and immediately following initiation of LRT predicting intermediate survival with this approach.