New therapies for hepatocellular carcinoma

Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is often diagnosed at an advanced stage when most potentially curative therapies such as resection, transplantation or percutaneous and transarterial interventions are of limited efficacy. The fact that HCC is resistant to conventional chemotherapy, and is rarely amenable to radiotherapy, leaves this disease with no effective therapeutic options and a very poor prognosis. Therefore, the development of more effective therapeutic tools and strategies is much needed. HCCs are phenotypically and genetically heterogeneous tumors that commonly emerge on a background of chronic liver disease. However, in spite of this heterogeneity recent insights into the biology of HCC suggest that certain signaling pathways and molecular alterations are likely to play essential roles in HCC development by promoting cell growth and survival. The identification of such mechanisms may open new avenues for the prevention and treatment of HCC through the development of targeted therapies. In this review we will describe the new potential therapeutic targets and clinical developments that have emerged from progress in the knowledge of HCC biology, In addition, recent advances in gene therapy and combined cell and gene therapy, together with new radiotherapy techniques and immunotherapy in patients with HCC will be discussed

Papel de SLU7 en la diferenciación hepática y en la protección del hígado frente al daño: regulación de HNF4A

El hígado es un órgano con un papel central en el organismo puesto que realiza numerosas funciones esenciales para mantener la homeostasis sistémica. A nivel histológico, el hígado presenta una arquitectura compleja y muy organizada, formada por diferentes poblaciones celulares que desarrollan funciones concretas y, en conjunto, permiten el correcto funcionamiento del órgano. Así, el hígado está formado por las células parenquimales que incluyen a los hepatocitos y a los colangiocitos, y las células no parenquimales, que engloban a las células estrelladas hepáticas (HSC, hepatic stellate cells), las células de Kupffer o las células endoteliales sinusoidales. La población celular más abundante son los hepatocitos, que constituyen alrededor de un 80 % de la masa hepática total, y son los que más contribuyen al desarrollo de la función hepática. Se trata de unas células epiteliales polarizadas, muy diferenciadas y quiescentes, aunque presentan una gran capacidad de regeneración en respuesta a estímulos nocivos 1–4 . Las células hepáticas se organizan en el hígado alrededor de una estructura anatómica peculiar y compleja, determinada por el patrón del flujo sanguíneo, y que es esencial para que se puedan llevar a cabo las funciones hepáticas. Brevemente, el hígado recibe sangre de la vena porta rica en nutrientes y productos de deshecho, y sangre de la arteria hepática rica en oxígeno, que fluye a través de una red de capilares sinusoidales entre las células hepáticas, hasta drenar en las venas centrales. Junto a las venas porta y las arterias hepáticas se encuentran los conductos biliares que conducen la bilis hasta la vesícula biliar y el intestino, y los tres conductos juntos forman la triada portal. La unidad funcional del hígado son los lobulillos hepáticos, que consisten en fragmentos poligonales de células hepáticas dispuestas en torno a una vena central y rodeadas de triadas portales. Los capilares sinusoidales conectan la vena central con los espacios porta y, en paralelo a ellos, se organizan los hepatocitos formando cordones gruesos unicelulares (Figura 1A). Tanto la vena porta como la arteria hepática suministran la sangre a los hepatocitos conforme fluye a través de la red de sinusoides hasta drenar en las venas centrales. En función de si los hepatocitos se localizan más cerca de la región periportal o más cerca de la región pericentral, presentan diferencias fenotípicas en cuanto a la expresión de enzimas metabólicas, por lo que realizan funciones metabólicas diferentes. Esta distribución diferencial de las funciones metabólicas y secretoras del hígado a lo largo del eje porto-central se conoce como zonación, y constituye un marcador clave de la maduración hepática (Figura 1B) 1–5

Detection of anti-hepatitis C virus antibodies by ELISA using synthetic peptides

A novel ELISA assay for the detection of anti-hepatitis C virus antibodies in the sera of infected individuals is described. This assay is based on a mixture of three 15-amino acid synthetic peptides encompassing regions of core and NS4 proteins of hepatitis C virus. Comparison with other available ELISA assays based on recombinant polypeptides shows that, short synthetic peptides have the advantage over some larger recombinant peptides by giving higher specificity without loss of sensitivity

S-adenosylmethionine regulates MAT1A and MAT2A gene expression in cultured rat hepatocytes: a new role for S-adenosylmethionine in the maintenance of the differentiated status of the liver

Methionine metabolism starts with the formation of S-adenosylmethionine (AdoMet), the most important biological methyl donor. This reaction is catalyzed by methionine adenosyltransferase (MAT). MAT is the product of two different genes: MAT1A, which is expressed only in the adult liver, and MAT2A, which is widely distributed, expressed in the fetal liver, and replaces MAT1A in hepatocarcinoma. In the liver, preservation of high expression of MAT1A and low expression of MAT2A is critical for the maintenance of a functional and differentiated organ. Here we describe that in cultured rat hepatocytes MAT1A expression progressively decreased, as described for other liver-specific genes, and MAT2A expression was induced. We find that this switch in gene expression was prevented by adding AdoMet to the culture medium. We also show that in cultured hepatocytes with decreased MAT1A expression AdoMet addition markedly increased MAT1A transcription in a dose-dependent fashion. This effect of AdoMet was mimicked by methionine, and blocked by 3-deazaadenosine and L-ethionine, but not D-ethionine, indicating that the effect was specific and mediated probably by a methylation reaction. These findings identify AdoMet as a key molecule that differentially regulates MAT1A and MAT2A expression and helps to maintain the differentiated status of the hepatocyte

Amphiregulin contributes to the transformed phenotype of human hepatocellular carcinoma cells

Hepatocellular carcinoma is a major cause of cancer-related deaths. Current treatments are not effective, and the identification of relevant pathways and novel therapeutic targets are much needed. Increasing evidences point to the activation of the epidermal growth factor receptor (EGFR) as an important mechanism in the development of hepatocarcinoma. We previously described that amphiregulin (AR), a ligand of the EGFR, is not expressed in healthy liver but is up-regulated during chronic liver injury, the background on which most liver tumors develop. Now, we have studied the expression and role of AR in human hepatocarcinoma. AR expression and function was studied in human liver tumors and cell lines. AR is expressed in human hepatocellular carcinoma tissues and cell lines and behaves as a mitogenic and antiapoptotic growth factor for hepatocarcinoma cells. We provide several lines of evidence, including AR silencing by small interfering RNAs and inhibition of amphiregulin by neutralizing antibodies, showing the existence of an AR-mediated autocrine loop that contributes to the transformed phenotype. Indeed, interference with endogenous AR production resulted in reduced constitutive EGFR signaling, inhibition of cell proliferation, anchorage-independent growth, and enhanced apoptosis. Moreover, knockdown of AR potentiated transforming growth factor-beta and doxorubicin-induced apoptosis. Conversely, overexpression of AR in SK-Hep1 cells enhanced their proliferation rate, anchorage-independent growth, drug resistance, and in vivo tumorigenic potential. These observations suggest that AR is involved in the acquisition of neoplastic traits in the liver and thus constitutes a novel therapeutic target in human hepatocarcinoma

Pathological and virological findings in patients with persistent hypertransaminasaemia of unknown aetiology

BACKGROUND: The histopathological spectrum and role of hepatitis viruses in cases of hypertransaminasaemia of unknown aetiology have not been correctly analysed in a sufficiently large number of patients. METHODS: We studied 1075 consecutive patients referred for liver biopsy because of elevation of alanine aminotransferase (ALT) levels for more than six months. From this population we selected those cases in whom the aetiology could not be defined from clinical, biochemical, and serological data obtained before biopsy. In these patients liver biopsies were reviewed, and hepatitis B virus (HBV)-DNA and hepatitis C virus (HCV)-RNA were assayed in serum by polymerase chain reaction (PCR). Serum hepatitis G virus (HGV)-RNA was determined by PCR in 74 patients. RESULTS: Of 1075 patients studied, the cause of the increased serum ALT levels remained elusive after appropriate testing in 109 patients (10.1%). Liver biopsies from these patients showed non-specific changes in 32.7% of cases, non-alcoholic steatohepatitis (NASH) in 15.8%, and chronic hepatitis or cirrhosis in 51.5%. HBV-DNA and/or HCV-RNA was detected more frequently in cryptogenic liver disease than in healthy blood donors (26.7% v 3.4%; p<0.001). HGV-RNA was found in only one patient. The proportion of cases with detectable HBV-DNA or HCV-RNA was 14.3% in patients with non-specific changes or NASH, 30.7% in patients with chronic hepatitis, and 61.5% in patients with cirrhosis. Cirrhosis was found more frequently in patients with positive HBV-DNA and/or HCV-RNA in serum than in those who tested negatively (p=0.005). CONCLUSIONS: In our series, patients in whom biochemical and serological data did not determine the aetiology of the disease represented 10% of all cases referred for liver biopsy for persistent elevation of serum transaminases. Approximately 50% of patients had chronic hepatitis or cirrhosis and the remainder had NASH or non-specific changes. Occult viral infections were found in a high proportion of cases in the first group and in a low percentage of patients in the second

Novel role for amphiregulin in protection from liver injury

Clinically, the Fas and Fas ligand system plays a central role in the development of hepatocyte apoptosis, a process contributing to a broad spectrum of liver diseases. Therefore, the development of therapies aimed at the inhibition of hepatocyte apoptosis is a major issue. Activation of the epidermal growth factor receptor has been shown to convey survival signals to the hepatocyte. To learn about the endogenous response of epidermal growth factor receptor ligands during Fas-mediated liver injury we investigated the expression of epidermal growth factor, transforming growth factor alpha, heparin-binding epidermal growth factor-like growth factor, betacellulin, epiregulin, and amphiregulin in the liver of mice challenged with Fas-agonist antibody. Amphiregulin expression, barely detectable in healthy liver, was significantly up-regulated. Amphiregulin administration abrogated Fas-mediated liver injury in mice and showed direct anti-apoptotic effects in primary hepatocytes. Amphiregulin activated the Akt and signal transducer and activator of transcription-3 survival pathways, and up-regulated Bcl-xL expression. Amphiregulin knock-out mice showed signs of chronic liver damage in the absence of any noxious treatment, and died faster than wild type mice in response to lethal doses of Fas-agonist antibody. In contrast, these mice were more resistant against sublethal liver damage, supporting the hypothesis that chronic liver injury can precondition hepatocytes inducing resistance to subsequent cell death. These results show that amphiregulin is a protective factor induced in response to liver damage and that it may be therapeutic in liver diseases

Immunogenicity of variable regions of hepatitis C virus proteins: selection and modification of peptide epitopes to assess hepatitis C virus genotypes by ELISA

The immunogenicity of variable regions of hepatitis C virus (HCV) proteins was studied by ELISA by using 543 synthetic peptides from 120 variable regions and 90 sera from HCV-infected patients. Some regions from certain genotypes were less immunogenic, or even non-immunogenic, compared with their equivalents in other genotypes. However, the mean recognition of all peptides from genotypes 1a, 1b and 3 by sera infected with genotypes 1a, 1b and 3, respectively, showed no significant differences, suggesting a similar overall immunogenicity of variable regions from these genotypes. Proteins NS4a, NS4b and NS5a were found to be the most immunogenic. Recognition of individual peptides by the sera of infected patients showed that the humoral response against HCV is patient-dependent. The work shows that 15-mer peptides may encompass several B-cell epitopes. These epitopes may lie in slightly different positions in different genotypes. Thirty-one percent of the 543 peptides were recognized by some of the 35 healthy donors. This may be a reflection of the large number of antigens to which they had been exposed, but it may also reflect a strategy of HCV to respond to immune pressure. After selection and modification, a set of 40 peptides was used to assess genotypes 1a, 1b, 1, 2 and 3 in the sera of HCV-infected patients, with sensitivities of 34.1, 48.5, 68.8, 58.3 and 48.9% and specificities of 100, 99.1, 97.1, 99.5 and 99%, respectively. The overall sensitivity and specificity for the assessment of genotypes 1, 2 and 3 were 64 and 98%, respectively

Hepatitis C virus infection of primary tupaia hepatocytes leads to selection of quasispecies variants, induction of interferon-stimulated genes and NF-kappaB nuclear translocation

Systems for in vitro culture of Hepatitis C virus (HCV) are essential tools to analyse virus-cell interactions and to investigate relevant pathophysiological aspects of HCV infection. Although the HCV replicon methodology has increased our understanding of HCV biology, this system does not reproduce the natural infection. Recently, tupaia (Tupaia belangeri chinensis) hepatocytes have been utilized for in vitro culture of HCV. In the present work, primary tupaia hepatocytes infected in vitro with HCV were used to analyse the evolution of HCV quasispecies in infected cells and the ability of the virus to influence antiviral and proinflammatory responses in cells sustaining virus replication. The results confirmed the potential of tupaia hepatocytes as a model for HCV infection, although this system is limited by rapid loss of differentiated cell phenotype in culture. These findings revealed an extraordinary plasticity of HCV quasispecies, which underwent rapid evolution to tupaia-tropic variants as early as 24 h after infection. It was also shown that HCV could activate interferon-sensitive genes, albeit modestly in comparison with other viruses such as Semliki Forest virus. Importantly, HCV activated NF-kappaB in primary hepatocytes and upregulated NF-kappaB-responsive genes including the chemokines MCP-1 and CXCL2 (MIP-2). This effect may play a role in induction of the hepatic inflammatory reaction in vivo. In summary, HCV quasispecies adapt rapidly to the specific biology of the host and HCV stimulates a blunted interferon response while inducing a proinflammatory phenotype in the infected cell