Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are two major liver malignancies. Although some phenotypic overlap is known, HCC and CCA are usually different with regard to etiology, histology, and prognosis. Gene expression and deacetylase activity of the class III histone deacetylase SIRT1 are up-regulated in cancer cells due to oncogene overexpression or loss of function of tumor suppressor genes. SIRT1 may play a critical role in tumor initiation, progression, and drug resistance by blocking senescence and apoptosis, and promoting cell growth and angiogenesis, but pleiotropic effects (synchronous or metachronous anti-proliferation and anti-apoptotic mechanisms) have been suggested in some cancers. Our aim was to investigate the expression of SIRT1 in liver epithelial malignancies. Thirty carcinomas of the liver, including 16 HCC and 14 CCA cases, were investigated by immunohistochemistry using monoclonal antibodies against SIRT1 and p53. Western blot analysis (WBA) was carried out for expression of SIRT1 in three CCA cell lines, one HCC cell line, and one cell line of Papova-immortalized normal hepatocytes. An expression of SIRT1 was found in 11 of 16 (68.75%) HCC and in 5 of 14 (35.71%) CCA. Moreover, we found an expression of p53 in 8 out of 16 (50%) HCC and 13 out of 14 (92.86%) CCA. WBA showed expression of SIRT1 in all cell lines studied, although a stronger signal was seen in the HCC cell line. Immunohistochemical data did not correlate to clinical stage or other clinical or histopathological parameters. Sirtuin 1 is a phylogenetically-conserved family of deacetylases and our data seem to indicate that (1) pleiotropic effects may be present in hepatic epithelial malignancies, and (2) there is no specificity of SIRT1 for either HCC or CCA.

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) are the two most common primary liver malignancies in adult patients. The molecular mechanisms underlying the pathogenesis of HCC and CCA are still poorly understood. Sonic hedgehog (SHH) signaling plays an essential role during mammalian development, i.e., promoting organ growth, tissue differentiation, and cell polarity. The upregulation of SHH has been observed during carcinogenesis, including colorectal carcinoma. Our aim was to investigate the expression pattern of SHH in HCC and CCA. We investigated 40 malignant tumors of the liver, including 21 HCC and 19 of intrahepatic CCA cases by immunohistochemistry (IHC) using a polyclonal antibody against SHH and Avidin-Biotin Complex method. We also investigated the co-localization of SHH and Bone morphogenetic protein 4 (BMP4) in CCA using indirect double IHC. Moreover, we examined whether SHH is expressed in two HCC cell lines HepG2 and HuH-7 and three CCA cell lines OZ, HuCCT1 and HuH28. We found that SHH was expressed in 15 out of 21 cases (71.4 %) of HCC and 100 % of CCA cases by immunohistochemistry. SHH expression showed a positive trend in liver tumors (HCC, CCA) with high grade (G2-G3). SHH localized to the epithelial cells, while BMP4 was expressed in the stromal cells in CCA by double IHC. However, both HCC and CCA cell lines showed SHH expression by Western blot analysis. In conclusion, SHH seems to be an interesting marker of de-differentiation in liver tumors and the simultaneous epithelial-mesenchymal expression may be an intriguing prompt to investigate cross-talks between SHH and BMP4.

Cholangiocarcinoma (CCA) is one of the most frequent malignant epithelial liver tumors after hepatocellular carcinoma (HCC). Its incidence seems to be increasing worldwide, although risk factors are heterogeneous and differ globally. Although diagnostic and therapeutic medicine have advanced in several countries, tackling this tumor remains a challenge. The causes of CCA's increasing incidence are likely a differential increment of some factors according to the geographical area, which will be considered in this review. Environment-linked risk factors may play a critical role in the carcinogenesis. Liver flukes may play a major role in East Asia, while exposure to chemical compounds, such as naphthenic acids, has been postulated as a source of the rate increase in Western countries. Carcinogenesis is variable and confounding factors also need to be taken into account. Carcinogenesis depends on a sequential process and most probably involves both cholestasis and chronic inflammation as promoting steps after induction. The release and interaction of interleukin-6 (IL-6), transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), and platelet-derived growth factor (PDGF) are at the basis of the proliferation of biliary epithelial cells or cholangiocytes. Additional steps for the final development of CCA may also involve an increase of the mutation rate of tumor suppressor genes, such as TP53, and the evasion of apoptosis.

AimsTo assess the expression of Twisted gastrulation (TWSG1) protein, which regulates the activity of bone morphogenetic proteins (BMPs) in the extracellular space in malignant epithelial tumours of the liver.MethodsThirteen hepatocellular carcinoma (HCC) samples and 12 intrahepatic cholangiocellular carcinoma (CCA) samples were compiled into diagnosis-specific tissue microarrays. Sections were immunostained with a monoclonal antibody against TWSG1 and a polyclonal antibody against BMP4. Human cell lines were also used, including one HCC cell line (HepG2), three CCA cell lines (OZ, Huh-28, HuCCT-1) and a Papova-immortalised normal hepatocyte cell line (THLE-3) for western blot analysis (WBA).ResultsImmunostaining and WBA showed a stronger TWSG1 expression in CCA than in HCC. The difference in expression was significant (p<0.05), and the immunohistochemical signal was particularly evident in the malignant epithelial areas close to desmoplastic stroma in CCA and in the areas of glandular differentiation in HCC. No expression was seen in normal hepatocytes. Interestingly, BMP4 was fully expressed in CCA and only partly in HCC. WBA showed a band for BMP4 in both CCA and HCC cell lines.ConclusionsTWSG1 is expressed in both malignant epithelial carcinomas, although the level of expression is higher in CCA than in HCC and seems to correlate at least partially with BMP4 expression.

Approach to an infant with jaundice and persistent conjugated hyperbilirubinemia includes several pediatric investigations with a different spectrum of invasiveness ranging from clinical biochemistry to liver biopsy. The broad and intense level of investigation needs to be set up soon to exclude surgical conditions that would prompt the child to a beneficial at least temporary solution. Paucity of the interlobular bile ducts (PIBD) is defined as a low ratio of interlobular bile ducts to portal tract ratios. However, obtaining adequate tissue for a definitive diagnosis can be a problem in young children. The interlobular bile duct to portal tract ratio is a value that has been considered differently from several authors, but major consensus and discussion platforms among pathologists and hepatologists seem indicate a cut-off value of 0.6 as highly likely for PIBD. PIBD may occur in a non-syndromic setting with various conditions or in genetic syndromes with a peculiar association with simple or complex congenital heart disease. Two wellestablished syndromes have been identified as genetic syndromes with a PIBD, although the list of the syndromes may be growing in the future. The first syndrome described in the literature is Alagille syndrome (AGS) or arteriohepatic dysplasia with pulmonary stenosis as the most common cardiac single finding and tetralogy of Fallot as the most common complex cardiac defect. Alagille syndrome can be caused by either mutation in the Jagged-1 gene (JAG1) mutation or in the NOTCH2 gene (Bauer et al. 2010). The second syndrome is Williams-Beuren syndrome (WBS), which is a neurodevelopmental disorder with supravalvular aortic or pulmonary stenosis. The WBS is associated with a microdeletion within the 7q11.23 chromosomal band, which encompasses 28 genes and specific low copy repeats serve as substrate for non-allelic homologous recombination leading to the deletion. The most common deletion, which occurs in about 95% of cases, involves a 1.5 megabase DNA segment (Henrichsen et al. 2011). Interestingly, in both genetic syndromes an abnormality of the outlet tract of the ventricular pump of the heart represents one of the most salient feature.

BACKGROUND:Cholangiocarcinoma (CC) is the most frequent malignant epithelial tumor of the biliary system. CC has received increasing interest due to its different etiologic factors, invasiveness, and the difficulty of diagnosis at an early stage. The pathogenesis of CC has not been clearly defined, but cohesiveness of tumor cells seems to be a critical factor. Calcium-dependent adherence proteins or cadherins are a family of proteins essential for connecting the plasma membrane of adjacent cells. Linkage of cadherins with the cytoskeleton occurs through another class of proteins, called catenins. E-cadherin forms a mutually exclusive complex or unit with β-catenin. Loss of E-cadherin -β-catenin adhesion represents an important step in the progression of many epithelial malignancies. Cell lines arising from CC are not often investigated and may show a differential expression of cell adhesion molecules, particularly E-cadherin - β-catenin. We hypothesized that a moderately invasive cell line of CC may co-localize both molecules in cytoplasm and cytoplasmic membrane, indicating a greater "tightness" of the tumor cells, while a metastasizing cell line may show isolated cytoplasmic membrane localization, indicating tumor cells probably more keen to reach the blood stream and give metastases. Thus, our aim was to investigate the expression and localization of E-cadherin and β-catenin in two CC cell lines, including a rapidly metastasizing cell line and a moderately invasive cell line, correlating to a different degree of invasiveness of the primary tumor.MATERIALS AND METHODS:OZ and HuCCT1 cells represent homogeneous, functional human biliary epithelial tumor cell lines that were originally isolated in Japan. Following cell line growth we extracted total proteins. Western blot analysis, immunofluorescence and confocal laser microscopy were used to identify the protein expression and their cyto-localization and co-localization.RESULTS:Both CC cell lines expressed E-cadherin and β-catenin, but they showed remarkably different localization patterns. In HuCCT1, both E-cadherin and β-catenin were localized in the cytoplasm, while in OZ these proteins were localized in the cytoplasmic membrane only. This was attributed to a different degree of invasiveness of the primitive CC from which the cell lines were characterized, OZ being a metastasizing cell line, HuCCT1 being a moderately invasive cell line.CONCLUSION:To the best of our knowledge, this is the first time that E-cadherin and β-catenin have been studied in detail in these two cell lines. These data seem to be very promising in terms of adding insight into the cell biology of CC and initiating investigations that aim to identify cytoskeletal dynamics and ultimately provide guidelines for developing new therapeutic strategies.