Pamela A. Norton

Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis

BACKGROUND & AIMS The accelerated course of hepatic fibrosis that occurs in some patients after liver transplantation is a major clinical problem. This response may be caused by the antirejection therapeutics, and in an earlier report we showed that FK-506 enhanced the fibrogenic process in in vivo and in vitro models of liver fibrosis. In the present study, the aim was to determine whether a new immunosuppressive agent, rapamycin, enhances or inhibits liver fibrosis. METHODS Effects of rapamycin were investigated in a carbon tetrachloride model of hepatic fibrosis in rats and on hepatic stellate proliferation in vitro. RESULTS Rapamycin inhibited extracellular matrix deposition in the rat model of fibrogenesis as determined by histological analysis, collagen content, messenger RNA levels of procollagen and transforming growth factor beta1, and tissue transglutaminase activity. Moreover, rapamycin decreased platelet growth factor-induced proliferation of hepatic stellate cells. CONCLUSIONS These findings indicate that the new antirejection agent rapamycin inhibits hepatic fibrosis and thus may become a valuable addition to the immunosuppression armamentarium.

Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma

Background We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC). Methodology/Principal Findings Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (α-1,3) fucosylation. Increases in core (α-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific. Conclusions/Significance This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.

N-Linked Glycosylation of the Liver Cancer Biomarker GP73

The association between elevated circulating levels of GP73 (and fucosylated GP73 in particular) and hepatocellular carcinoma suggests that a thorough analysis of the extent of GP73 glycosylation is warranted. Detailed analysis of the glycosylation patterns of such low abundance proteins are hampered by technical difficulties. Using conventional lectin affinity chromatography, we have established that three quarters of the GP73 secreted from a cell line derived from HCC is fucosylated. Using mass spectrometry, we have established that at least two of three potential sites of N‐linked glycosylation are occupied on most molecules of GP73 secreted from cultured hepatoma cells. Furthermore, the oligosaccharides added to recombinant GP73 resemble those present in the bulk of secreted protein, mostly bi‐antennary with core fucose, with a smaller fraction of tri‐ and tetra‐antennary structures. The frequency of fucosylation observed on the recombinant protein agrees well with the pattern of lectin binding of the endogenous secreted protein. Finally, we have developed a method to interrogate the glycans added to either the near full length protein or at a particular sequon, providing proof of concept that a small peptide embedded in a heterologous context can preserve both fucosylation and a high level of branching of oligosaccharides added. J. Cell. Biochem. 104: 136–149, 2008.

A substituted tetrahydro-tetrazolo-pyrimidine is a specific and novel inhibitor of hepatitis B virus surface antigen secretion.

ABSTRACT The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected individuals are thought to play a role in suppressing the HBV-specific immune response. Current therapeutics are not directed at reducing this viral antigenemia; thus, our group has focused on identifying inhibitors of HBsAg secretion. By using the HBV-expressing cell line HepG2.2.15, high-throughput screening of an 80,288-compound synthetic small-molecule library identified HBF-0259, an aromatically substituted tetrahydro-tetrazolo-(1, 5-a)-pyrimidine. Following resynthesis, HBF-0259 had a 50% effective concentration of approximately 1.5 μM in a secondary, HBV-expressing cell line, with a concentration that exhibited 50% cytotoxicity of >50 μM. The equilibrium concentration of HBF-0259 in aqueous solution at physiological pH was 15 to 16 μM; the selective index was thus >9. As intended by our screening paradigm, HBF-0259 is a selective, potent inhibitor of secretion of both subviral and DNA-containing viral particles, while the secretion of α-1-acid glycoprotein and α-1-antitrypsin was unaffected. The HBV e antigen, which is not a constituent of HBV particles, was also unaffected, suggesting that the secretion of particles bearing HBV structural glycoproteins is targeted directly. Inhibitory activity was also confirmed by transfection of HBsAg, indicating that the action of the compound is independent of those of other viral proteins. HBF-0259 had no effect on HBV DNA synthesis, demonstrating that inhibition is independent of viral genomic replication. Finally, HBF-0259 had little or no effect on the cell-to-cell spread of two unrelated viruses, suggesting that it is a specific inhibitor of secretion of HBsAg. Possible mechanisms of action and the implications for its development are discussed.

Detection of Mutated K-ras DNA in Urine, Plasma, and Serum of Patients with Colorectal Carcinoma or Adenomatous Polyps

Our previous studies demonstrated that urine contains DNA derived from the circulation and that this DNA originated, in part, from organ sites and tumors distal to the urinary tract. To explore the potential use of DNA from urine as compared to other body fluids as a source for circulating DNA for cancer detection, the DNA concentration and the frequency of detection of mutated Kristin‐ras (K‐ras) DNA in serum, plasma, and urine were examined. The concentration of DNA in the urine was similar to that in the serum, but the DNA concentration in plasma was significantly lower than in either urine or serum (P < 0.05). When DNA derived from 10 μL of body fluid was used in each mutation assay, the detection frequency of mutated K‐ras DNA was comparable among serum, plasma, and urine. However, when DNA derived from 200 μL of body fluid was used, the incidence of detecting mutated K‐ras DNA in urine was significant higher (95%) than in either serum (35%) or plasma (40%) (P < 0.0005), suggesting that inhibitory factors in serum/plasma may be more limiting than in urine. The use and practicality of urine as a source of circulating DNA for cancer detection are discussed.

Antiviral Profiles of Novel Iminocyclitol Compounds against Bovine Viral Diarrhea Virus, West Nile Virus, Dengue Virus and Hepatitis B Virus

The antiviral activity of iminocyclitol compounds with a deoxynojirimycin (DNJ) head group and either a straight chain alkyl or alkylcycloalkyl group attached to the nitrogen atom have been tested in vitro against multiple-enveloped viruses. Several of these analogues were superior to previously reported DNJ compounds. Iminocyclitols that inhibit the glycan-processing enzyme endoplasmic-reticular glucosidase have been shown to inhibit the morphogenesis of viruses that bud from the endoplasmic reticulum (ER) at non-cytotoxic concentrations. Bovine viral diarrhoea virus (BVDV) has been used as a surrogate system for study of the hepatitis C virus, which belong to the virus family (Flaviviridae) as West Nile virus (WNV) and dengue virus (DV). N-Nonyl-DNJ (NNDNJ) was previously reported to have micromolar antiviral activity against BVDV, but a limiting toxicity profile. N-Butylcyclohexyl-DNJ (SP169) was shown to be as potent as NNDNJ in assays against BVDV and less toxic. However, it was inactive against hepatitis B virus (HBV). The present study reports efforts to improve the performance profiles of these compounds. Introduction of an oxygen atom into the N-alkyl side chain of DNJ, either as an ether or a hydroxyl functionality, reduced toxicity but sacrificed potency. Introduction of a hydroxyl group at the tertiary carbon junction of the cycloalkyl and linear alkyl group, as in N-pentyl-(1-hydroxycyclohexyl)-DNJ (OSL-95II), led to a structure that was as well tolerated as DNJ (CC50>500 µM), but retained micromolar antiviral activity against all ER morphogenesis budding viruses tested: BVDV, WNV, DV and HBV. The implication of this modification to the development of broad-spectrum antiviral agents is discussed.

Transforming growth factor-β1 (TGF-β1) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression†

Regulated splicing of fibronectin (FN) occurs during the mesenchymal to chondrocyte transition and ultimately results in the relative enrichment of an extra domain B (EDB) exon‐containing FN isoform with the suggestion that FN isoforms may play a functional role in chondrogenesis. Promotion of chondrogenesis can also be achieved by treatment with transforming growth factor‐β (TGF‐β), which also regulates FN isoform expression. We have examined the effects of TGF‐β treatment on the assumption of the chondrogenic phenotype in the teratoma‐derived cell line ATDC5 and tested whether these effects on chondrogenesis are paralleled by appropriate changes in FN isoform expression. ATDC5 cells were maintained in a pre‐chondrogenic state and, in this state, treated with 10 ng/ml TGF‐β. The cells started to elaborate a matrix rich in sulfated proteoglycans, such that within the first 12 days of culture, TGF‐β1 treatment appeared to slightly accelerate early acquisition of an Alcian blue‐stained matrix, and caused a dose‐ and time‐dependent decrease in collagen type I expression; changes in collagen type II expression were variable. At later times, cells treated with TGF‐β became indistinguishable from those of the controls. Interestingly, TGF‐β treatment caused a significant dose‐ and time‐dependent decrease in the proportion of FN containing the extra domain A (EDA) and the EDB exons. These data suggest that TGF‐β induces the early stages of chondrogenic maturation in this pre‐chondrogenic line and that TGF‐β treatment increases expression of FN isoforms that lack the EDA and EDB exons. Published 2005 Wiley‐Liss, Inc.

Increased levels of tetra-antennary N-linked glycan but not core fucosylation are associated with hepatocellular carcinoma tissue.

Background: Alterations in glycosylation have long been associated with the development of cancer. In the case of primary hepatocellular carcinoma (HCC), one alteration that has often been associated is increased amounts of fucose attached to the N-glycans of serum proteins secreted by the liver. Methods: In an effort to determine the origin of this increased fucosylation, we have conducted N-linked glycan analysis of HCC tissue, the surrounding nontumor tissue, and compared this to tissue from a nondiseased adult liver. Results: Surprisingly, no difference in the level of fucosylation was observed from the three donor groups, suggesting that the increased levels of fucosylation observed in serum of those with HCC is not the result of increased synthesis of fucosylated proteins in the cancer tissue. On the other hand, increased levels of a tetra-antennary glycan were observed in the HCC tissue as compared with the surrounding tissue or to the nondiseased livers. Conclusions: This represents, to our knowledge, one of the first reports associating increased levels of branching with the development of HCC. Impact: The identification of increased levels of tetra-antennary glycan on liver tumor tissue, as opposed to adjacent or nondiseased tissue may lead to improved detection of HCC. Cancer Epidemiol Biomarkers Prev; 21(6); 925–33. ©2012 AACR.

Activation of fibronectin gene expression by hepatitis B virus x antigen.

Summary.  The development of fibrosis and cirrhosis during chronic hepatitis B virus (HBV) infection correlates with the persistent expression of HBV x antigen (HBxAg), which acts in part, by stimulating selected signal transduction pathways, including nuclear factor κB (NF‐κB). To identify NF‐κB responsive genes that are differentially expressed in HBxAg‐positive cells, HepG2 cells were stably transfected with HBxAg, and then with pZeoSV2 or pZeoSV2‐IκBα. When RNAs from each culture were compared by PCR‐select cDNA subtraction, fibronectin (FN) mRNA was shown to be strongly down‐regulated by IκBα. Up‐regulated expression of FN and co‐expression between FN and HBxAg were observed in liver sections from HBV carriers that were stained for HBxAg and analysed for FN mRNA by in situ hybridization (ISH). In liver cell cultures, HBxAg increased the levels of FN mRNA and protein. This was because of the HBxAg‐mediated trans‐activation of the FN promoter, which was NF‐κB‐dependent. HBxAg also antagonized the repression of the FN promoter by the tumour suppressor, p53. Hence, the FN gene may be a natural target for HBxAg trans‐activation, perhaps through activation of NF‐κB and inactivation of p53, thereby contributing to the accumulation of FN in the liver over the course of chronic HBV infection.

Hepatitis B Virus-Mediated Changes of Apolipoprotein mRNA Abundance in Cultured Hepatoma Cells

ABSTRACT An inverse correlation between hepatitis B virus (HBV) and steady-state levels of apolipoprotein AI and CIII mRNAs was observed in two hepatoma cell lines. Analysis of a third line containing an inducible viral genome implicated viral pregenomic RNA in apolipoprotein mRNA reduction. We conclude that HBV alters infected cells despite the absence of overt cytopathogenicity.