Alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV

The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Galα1-3(Fucα1-2)Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies.     

Gene expression of ABC proteins in hepatocellular carcinoma,perineoplastic tissue,and liver diseases

BACKGROUND: The development of hepatocellular carcinoma (HCC) is a frequent event during the natural history of cirrhosis. Effective treatment is, however, hampered by drug resistance related to the expression of multidrug resistance (MDR) proteins belonging to the ABC family transporters. Studying expression of genes coding for these proteins may help to explain the potential sensitivity of HCC to chemotherapy. MATERIAL AND METHODS: The expression of MRP1, MRP2, MRP3, MDR1, and MDR3 was investigated by quantitative RT-PCR analyses in paraffin-embedded tissues obtained from 9 cases of HCC, 16 cases of cirrhosis, 10 cases of chronic extrahepatic cholestasis, and 16 cases of normal liver. In HCC cases, gene expression was assessed both in neoplastic and perineoplastic tissue after microscopically assisted microdissection. RESULTS: MRP1 was significantly and similarly overexpressed in HCC and perineoplastic tissue. MRP2 and MDR1 were also increased in HCC, but the level of expression did not correlate with that of perineoplastic tissue. The level of expression was either reduced or normal in cirrhotic liver and during chronic cholestasis. Expression of MDR3 was unchanged in all conditions investigated. CONCLUSIONS: The genetic expression of multi-drug resistance proteins, in particular MRP1, MRP2, and MDR1, is increased during HCC. In the case of MRP1, the extent of expression is similar in neoplastic and perineoplastic tissue, but this is not the case for MRP2 and MDR1. The assessment of ABC protein expression pattern may provide important information for the diagnosis and treatment of HCC.     

Immunohistochemical expression of pi class glutathione S-transferase and alpha-fetoprotein in hepatocellular carcinoma and chronic liver disease

OBJECTIVE: To determine whether tumor marker pi glutathione transferase (GST-pi) is expressed in hepatocellular carcinoma (HCC) and other chronic liver diseases and to compare its expression with that of alpha-fetoprotein (AFP). STUDY DESIGN: Samples used were formalin-fixed, paraffin-embedded liver tissues: normal (n = 3), chronic hepatitis B (n = 15), cirrhosis (n = 15) and HCC (n = 30). The expression of AFP and GST-pi was detected by using immunohistochemistry with the peroxidase-antiperoxidase method. AFP immunoreactivity was based on the cytoplasm of the hepatocytes, while GST-pi immunoreactivity was based on the nuclei of hepatocytes. RESULTS: In normal liver tissues, AFP was not expressed. However, there was strong staining of GST-pi in bile duct epithelium cells and weak staining in hepatocytes. Our results showed higher AFP immunoreactivity in cases of HCC (36.7%) as compared to cirrhosis (6.7%) and hepatitis B (0%), whereas GST-pi immunoreactivity was lower in cases of HCC (53.3%) as compared to cases of cirrhosis (100.0%) and hepatitis B (93.3%). Percent sensitivity of AFP determination for HCC was 36.7% as compared to 53.3% for GST-pi, thus making GST-pi a more sensitive marker for detection of HCC. This study showed a significant relationship between the intensity and percentage of cells stained in hepatitis B, cirrhosis and HCC for GST-pi immunoreactivity (P < .001, .001 and .05, respectively) but not for AFP (P > .05). Statistical analysis showed that there was no significant relationship between expression of AFP and GST-pi in cirrhosis and HCC cases. Hepatitis B virus infection in HCC cases showed a positive rate of 46.7%, with AFP staining positively in 42.9% of tissues and GST-pi staining positively in 57.1% of tissues. CONCLUSION: AFP is a diagnostic but rather insensitive tissue marker for HCC. However, the absence of AFP in benign chronic liver disease makes this marker useful in differentiating between HCC and other chronic liver diseases, whereas GST-pi can be used as a diagnostic marker for HCC as well as in detecting other chronic liver diseases.     

Expression of cyclooxygenase-2 and transforming growth factor-beta1 in HCV-induced chronic liver disease and hepatocellular carcinoma

Cyclooxygenase-2 (COX-2) and transforming growth factor-beta1 (TGF-beta1) were modulated in a variety of viral infections, but there is a paucity of data about their role in the pathologic process of cirrhosis and/or hepatocellular carcinoma (HCC) following chronic hepatitis C virus (HCV) infection. The material of the current study included 50 cases of chronic hepatitis C (CHC) without cirrhosis, 30 cases of CHC with cirrhosis, and 30 cases of HCC with HCV admitted to the Gastroenterology and Hepatology Department of Theodor Bilharz Research Institute, Giza, Egypt. Fifteen wedge liver biopsies, taken during laparoscopic cholecystectomy, were included in the study as normal controls. Laboratory investigations, serologic markers for viral hepatitis, and serum alpha fetoprotein levels (alpha-FP) were done for all cases of the study. Immunohistochemistry using primary antibodies against both factors revealed weak to faint immunoreactivity to COX-2 and TGF-beta1 in normal hepatic tissue (< 30% and < 50% of the cells, respectively). COX-2 expression was upregulated in patients with CHC with and without cirrhosis, yet 80% of positively stained cirrhotic cases showed marked staining intensity. Higher COX-2 expression was observed in well-differentiated HCC cases (80%) with marked staining intensity (75%) compared with advanced HCC tumors (P < .001). TGF-beta1 was expressed in the hepatocytes of all cases of CHC with and without cirrhosis as well as in 67% of HCC cases. Extensive cytoplasmic expression was detected in 52%, 93.3%, and 46.6% of CHC patients without cirrhosis, patients with cirrhosis, and patients with HCC, respectively. A positive correlation was observed between hepatic expression of COX-2 and TGF-beta1 (r = 0.67, P < .05); however, no correlation was detected between the latter and grade of HCC differentiation (r = 0.33, P > .05). CONCLUSION: These findings may suggest that TGF-beta1 plays a role in hepatic cell damage following HCV infection thus stressing the usefulness of this cytokine as a prognostic marker for liver cell injury. However, COX-2 is a predictive marker for malignant transformation and has a role in the early stages of hepatocarcinogenesis, but not in the advanced stages. The combined expression of both factors in HCV-related HCC suggests their synergistic action in the pathophysiology of hepatocarcinogenesis.     

Glutathione and GSH-dependent enzymes in patients with liver cirrhosis and hepatocellular carcinoma

We investigated glutathione level, activities of selenium independent GSH peroxidase, selenium dependent GSH peroxidase, GSH S-transferase, GSH reductase and the rate of lipid peroxidation expressed as the level of malondialdehyde in liver tissues obtained from patients diagnosed with cirrhosis or hepatocellular carcinoma. GSH level was found to be lower in malignant tissues compared to adjacent normal tissues and it was higher in cancer than in cirrhotic tissue. Non-Se-GSH-Px activity was lower in cancer tissue compared with adjacent normal liver or cirrhotic tissue, while Se-GSH-Px activity in cancer was found to be similar to its activity in cirrhotic tissue and lower compared to control tissue. An increase in GST activity was observed in cirrhotic tissue compared with cancer tissue, whereas the GST activity in cancer was lower than in adjacent normal tissue. The activity of GSH-R was similar in cirrhotic and cancer tissues, but higher in cancer tissue compared to control liver tissue. An increased level of MDA was found in cancer tissue in comparison with control tissue, besides its level was higher in cancer tissue than in cirrhotic tissue. Our results show that the antioxidant system of cirrhosis and hepatocellular carcinoma is severely impaired. This is associated with changes of glutathione level and activities of GSH-dependent enzymes in liver tissue. GSH and enzymes cooperating with it are important factors in the process of liver diseases development.     

Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker

Background

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.

Methodology and Principal Findings

Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.

Conclusion

HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.     

Molecular profiling of hepatocellular carcinoma in mice with a chronic deficiency of hepatic s-adenosylmethionine: relevance in human liver diseases

S-adenosylmethionine arises as a central molecule in the preservation of liver homeostasis as a chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. In the present work, we have attempted a comprehensive analysis of proteins associated with hepatocarcinogenesis in MAT1A knock out mice using a combination of two-dimensional electrophoresis and mass spectrometry, to then apply the resulting information to identify hallmarks of human HCC. Our results suggest the existence of individual-specific factors that might condition the development of preneoplastic lesions. Proteomic analysis allowed the identification of 151 differential proteins in MAT1A-/- mice tumors. Among all differential proteins, 27 changed in at least 50% of the analyzed tumors, and some of these alterations were already detected months before the development of HCC in the KO liver. The expression level of genes coding for 13 of these proteins was markedly decreased in human HCC. Interestingly, seven of these genes were also found to be down-regulated in a pretumoral condition such as cirrhosis, while depletion of only one marker was assessed in less severe liver disorders.     

Differential expression of nuclear lamin proteins during chicken development

By immunocytochemistry, quantitative immunoblotting, and two-dimensional gel electrophoresis, we have analyzed the distribution of nuclear lamin proteins during chicken embryonic development. Whereas no qualitative differences in the patterns of expression of lamins A, B1, and B2 were observed during gametogenesis in either the female or the male germ line, profound changes in the composition of the nuclear lamina occurred during the development of somatic tissues. Most unexpectedly, early chicken embryos were found to contain little if any lamin A, although they contained substantial amounts of lamins B1 and B2. During embryonic development, lamin A became increasingly prominent, whereas the amounts of lamin B1 decreased in many tissues. Interestingly, the extent and the developmental timing of these changes displayed pronounced tissue-specific variations. Lamin B2 was expressed in fairly constant amounts in all cell types investigated (except for pachytene-stage germ cells). These results have implications for the purported functional specializations of individual lamin proteins. In addition, they suggest that alterations in the composition of the nuclear lamina may be important for the establishment of cell- or tissue-specific differences in nuclear architecture.     

Monitoring liver alterations during hepatic tumorigenesis by NMR profiling and pattern recognition

Human hepatocellular carcinoma (HCC) is the most recurrent malignancy of the liver and represents one of the main causes of cancer death worldwide. Furthermore, the liver is the most frequent site of metastatic colonization, and hepatic metastases are far more common than primary cancers in Western countries. A possible way of investigating liver diseases is to study the tissue metabolic profiles. High-resolution nuclear magnetic resonance (NMR) spectroscopy of hepatic tissue extracts was combined with pattern-recognition and visualization techniques to uncover metabolic differences among analyzed tissue types. Extracts were from primary HCC, chronic hepatitis-C-virus related cirrhotic tissues, hepatic metastases from colorectal carcinomas, and non-cirrhotic normal liver tissues adjacent to metastases as controls. We identified all metabolites present in the NMR spectra, and after statistical evaluation of all spectral classes, we were able to define the metabolic changes underlying the different liver conditions and diseases. In particular, the lactate and the glucose tissue signals were found to primarily discriminate the different histological samples. We followed the biochemical changes of human hepatic lesions through primary (HCC) and secondary (metastases from colorectal carcinoma) liver tumors, cirrhotic tissues, and non-cirrhotic histologically-confirmed normal liver tissues adjacent to metastases, achieving a metabolic differentiation of the various pathological states based upon the variation of the intracellular lactate/glucose ratio. It is suggested that such a signal pattern may act as a potential marker for assessing pathological hepatic lesions.