Serum apolipoprotein C3 levels are negatively associated with hepatitis B virus DNA in HBeAg-negative chronic hepatitis B patients.

BACKGROUND: Hepatitis B virus (HBV) infection remains a global health issue associated with substantial morbidity and mortality. Serum apolipoprotein C3 (ApoC3) and apolipoprotein A5 (ApoA5) levels were decreased in chronic hepatitis B (CHB) patients, however the relationship between ApoC3 or ApoA5 and HBV DNA load remains elusive. METHODS: A total of 384 CHB patients including 194 HBsAg(+) HBeAg(-) and 190 HBsAg(+) HBeAg(+) and 154 healthy individuals were recruited in our study. Serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), total cholesterol (Chol), triglycerides (TG), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), high-density lipoproteins cholesterol (HDL-C), low-density lipoproteins cholesterol (LDL-C) and lipoprotein a (Lpa) were examined in an automatic biochemical analyzer. Apolipoprotein A5 (ApoA5) and apolipoprotein C3 (ApoC3) were detected via ELISA. RESULTS: Serum ApoA1, ApoB, ApoC3 and ApoA5 levels were reduced in CHB patients. In HBeAg(-) CHB patients, plasma ApoC3 levels were negatively associated with HBV DNA load (r = 0.219, P < 0.001). But no correlation between ApoA5 and HBV DNA load was observed in CHB patients. CONCLUSIONS: These data showed that HBV infection inhibits lipid metabolism and ApoC3 is negatively associated with HBV DNA load in HBeAg (-) CHB patients. These findings provided new evidence about the link between ApoC3-related lipid metabolism and immune response.

Activation of mammalian target of rapamycin complex 1 (mTORC1) and Raf/Pyk2 by growth factor-mediated Eph receptor 2 (EphA2) is required for cholangiocarcinoma growth and metastasis.

Eph receptor 2 (EphA2) overexpression is frequently accompanied by the loss of its cognate ligand during tumor progression. However, the molecular mechanism of this ligand-independent promotion of tumor by EphA2 remains unclear in highly malignant and fatal cholangiocarcinoma (CC). We examined the biological role of EphA2 in tumor growth and metastasis in CC tissues and cells according to the degree of differentiation and we explored the downstream signaling pathways of EphA2. Growth factor-mediated EphA2 overexpression itself leads to the activation of the mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase (ERK) pathways through ligand-independent activation of EphA2 (phosphorylation of S897). An in vitro soft agar assay and in vivo orthotopic or subcutaneous tumor model showed that EphA2 enhanced colony formation and accelerated tumor growth, and which seemed to be mainly associated with Akt (T308)/mTORC1 activation. Aberrant expression and activation of EphA2 was also associated with poorer differentiation and higher metastatic ability. Enhanced metastatic ability was also observed in an orthotopic tumor model or lung metastasis model, correlating with Pyk2(Y402)/c-Src/ERK activation in addition to activation of the canonical Raf/MEK/ERK pathway. The mTORC1 and Raf/Pyk2 pathways also appeared to affect each other. These results suggest that growth factor-mediated EphA2 might be involved in tumor growth and metastasis through activation of the mTORC1 and Raf/Pyk2 pathways. Therapeutic strategies that target EphA2 and its downstream effectors may be useful to control CC. (HEPATOLOGY 2013;57:2248-2260).

Genome-wide expression patterns associated with oncogenesis and sarcomatous transdifferentation of cholangiocarcinoma.

BACKGROUND: The molecular mechanisms of CC (cholangiocarcinoma) oncogenesis and progression are poorly understood. This study aimed to determine the genome-wide expression of genes related to CC oncogenesis and sarcomatous transdifferentiation. METHODS: Genes that were differentially expressed between CC cell lines or tissues and cultured normal biliary epithelial (NBE) cells were identified using DNA microarray technology. Expressions were validated in human CC tissues and cells. RESULTS: Using unsupervised hierarchical clustering analysis of the cell line and tissue samples, we identified a set of 342 commonly regulated (>2-fold change) genes. Of these, 53, including tumor-related genes, were upregulated, and 289, including tumor suppressor genes, were downregulated (<0.5 fold change). Expression of SPP1, EFNB2, E2F2, IRX3, PTTG1, PPARγ, KRT17, UCHL1, IGFBP7 and SPARC proteins was immunohistochemically verified in human and hamster CC tissues. Additional unsupervised hierarchical clustering analysis of sarcomatoid CC cells compared to three adenocarcinomatous CC cell lines revealed 292 differentially upregulated genes (>4-fold change), and 267 differentially downregulated genes (<0.25 fold change). The expression of 12 proteins was validated in the CC cell lines by immunoblot analysis and immunohistochemical staining. Of the proteins analyzed, we found upregulation of the expression of the epithelial-mesenchymal transition (EMT)-related proteins VIM and TWIST1, and restoration of the methylation-silenced proteins LDHB, BNIP3, UCHL1, and NPTX2 during sarcomatoid transdifferentiation of CC. CONCLUSION: The deregulation of oncogenes, tumor suppressor genes, and methylation-related genes may be useful in identifying molecular targets for CC diagnosis and prognosis.

EFNA1 ligand and its receptor EphA2: potential biomarkers for hepatocellular carcinoma.

Novel biomarkers are needed for early detection and progression evaluation of hepatocellular carcinoma (HCC). The purpose of this study was to identify useful biomolecular markers for HCC. The 26 genes that encode membrane or secretory proteins were identified from cDNA microarray data. We further examined the expression of EFNA1 and its receptor EphA2 and determined their biological implications during the development and progression of HCC. The EFNA1 mRNA was overexpressed in most HCCs as compared with its expression in corresponding nontumor tissues (36 out of 40 cases, 90%), but EphA2 expression was noted in only half of the HCC tissues (20 of 40 cases, 50%). In most of the hepatoma cell lines, the EFNA1 protein expression was positively associated with alpha-fetoprotien (AFP) expression but inversely associated with EphA2 expression. Furthermore, EFNA1 levels were detectable in the supernatant of the cultured hepatoma cells and in the serum of patients with HCC. In contrast, EphA2 expression was prominent in highly invasive hepatoma cells, and its overexpression was significantly correlated with decreased differentiation (r = 0.0248, p < 0.010) and poor survival (p = 0.0453) for HCC patients. EFNA1 and EphA2 may be useful serum markers for the detection of HCC development and progression, respectively.

Inhibition of PSMD4 blocks the tumorigenesis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver with high mortality and frequent recurrence. Although various therapies provide potential cure for HCC patients, unfortunately the five-year survival rate of advanced HCC remains dismal. It is critical to explore the pathogenesis of HCC and identify novel biomarkers for early HCC diagnosis. PSMD4 is a major receptor of the 26S proteasome involved in ubiquitindependent and proteasome-mediated protein degradation. In our study, PSMD4 was overexpressed in HCC tissues and cell lines determined by Northern blot, western blot and immunohistochemistry. The silencing of PSMD4 blocked cell proliferation and tumor growth, induced cell apoptosis and inhibited the proteasome activity. Western blot results showed that the knockdown of PSMD4 blocked the expression of cyclooxygenase 2 (COX2), phosphorylated Sarcoma tyrosine kinase (P-SRC) and Bcl-2, but improved the levels of p53 and Bax in HCC, lung cancer, colorectal cancer, breast cancer and endometrial cancer cell lines. Taken together, these findings indicated that the subunit of 26S proteasome PSMD4 exerts as an oncogene in HCC and other cancers via regulating the expression p53, Bcl-2 and Bax. These findings enriched the pathogenesis of HCC, and provided a new biomarker for cancers diagnosis and a new target for cancers therapy.

Identification of molecular markers for the oncogenic differentiation of hepatocellular carcinoma.

The aim of this study was to identify molecular markers associated with oncogenic differentiation in hepatocellular carcinoma (HCC). Using an unsupervised clustering method with a cDNA microarray, HCC (T) gene expression profiles and corresponding non-tumor tissues (NT) from 40 patients were analyzed. Of total 217 genes, 72 were expressed preferentially in HCC tissues. Among 186 differentially regulated genes, there were molecular chaperone and tumor suppressor gene clusters in the Edmondson grades I and II (GI/II) subclass compared with the liver cirrhosis (LC) subclass. The Edmondson grades III and IV (GIII/IV) subclass with a poor survival (P=0.0133) contained 122 differentially regulated genes with a cluster containing various metastasis- and invasion-related genes compared with the GI/II subclass. Immunohistochemical analysis revealed that ANXA2, one of the 72 genes preferentially expressed in HCC, was over-expressed in the sinusoidal endothelium and in malignant hepatocytes in HCC. The genes identified in the HCC subclasses will be useful molecular markers for the genesis and progression of HCC. In addition, ANXA2 might be a novel marker for tumor angiogenesis in HCC.

[Relationship among hepatocyte apoptosis, P450 2E1 and oxidative stress in alcoholic liver disease of rats].

OBJECTIVE: To observe the pathological changes and investigate the correlation of hepatocyte apoptosis with cytochrome P450 2E1(CYP2E1) expression and oxygen free radical in alcoholic liver disease (ALD) in rat. METHODS: 40% ethanol in the dose of 8 g/kg body weight was given to rats by gavage twice daily for 8 weeks in model group (n=37), and rats in control group (n=33) received same volume of saline by gavage. At the end of the 8th week, the contents of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. The pathological changes in the liver was observed under light microscope with hematoxylin and eosin (HE) staining, and hepatocyte apoptosis was detected by the terminal deoxynucleotidyl transferase mediated dUTP biotin nick and labeling (TUNEL) method. Expression of serum CYP2E1 was determined by polymerase chain reaction (PCR), the contents of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) in serum were detected by thibabituric acid (TBA) quantifying method. RESULTS: The TUNEL positive cells were located around the central vein, and spotty and focal necrosis was found in the ALD group. The apoptotic index (AI) in the ALD group was significantly higher than in the control group (P<0.05). To express in CYP2E1, the allelic frequency of c1 and c2 were 91.65%, and 8.35% respectively, in control group, while the allelic frequency of c1 and c2 were 53.35% and 46.65%, respectively, in ALD group, and there were significantly differences between two groups (all P<0.05). The content of MDA in serum had positive correlation with hepatocyte apoptosis index (MDA vs. AI, r( MDA ) =0.644), and the activity of SOD in serum had negative correlation with AI (SOD vs. AI, r( SOD ) =-0.511, all P<0.05) in the ALD group, and there was negative correlation between MDA and SOD (r=-0.582, P<0.05). CONCLUSION: Chronic alcohol administration induced alcoholic liver disease and liver dysfunction, and hepatocyte apoptosis is enhanced. Rsa I and Pst I RFLPs are related with ALD in model rats, and c2 gene might be related with the development of ALD. The content of MDA and activity of SOD play an important role in the process of hepatocyte apoptosis and lipid peroxidation process.