herbimycin and Liver-Neoplasms

Chronic hepatitis B virus infection is strongly associated with the development of hepatocellular carcinoma (HCC). Epithelial tumors are frequently characterized by loss of cadherin expression or function. Cadherin-dependent adhesion prevents the acquisition of a migratory and invasive phenotype, and loss of its function is itself enough for the progression from adenoma to carcinoma. The HBx protein of hepatitis B virus is thought to contribute to the development of the carcinoma, however, its role in the oncogenic and metastatic processes is far from being fully understood. We report herein the ability of HBx to disrupt intercellular adhesion in three different cell lines stably transfected with an inducible HBx expression vector. The linkage between the actin cytoskeleton and cadherin complex, which is essential for its function, is disrupted in the presence of HBx, as indicated by detergent solubility and immunoprecipitation experiments. In addition, beta-catenin was tyrosine phosphorylated in HBx-expressing cells. Inhibition of the src family of tyrosine kinases resulted in the prevention of the disruption of adherens junctions. These results suggest that HBx is able to disrupt intercellular adhesion in a src-dependent manner, and provide a novel mechanism by which HBx may contribute to the development of HCC.

Topics: Adherens Junctions; Animals; Benzoquinones; beta Catenin; Cadherins; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cell Transformation, Viral; Cocarcinogenesis; Cytoskeletal Proteins; Enzyme Inhibitors; HeLa Cells; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; Lactams, Macrocyclic; Liver Neoplasms; Mice; Neoplasm Metastasis; Phosphorylation; Protein Processing, Post-Translational; Quinones; Recombinant Fusion Proteins; Rifabutin; src-Family Kinases; Trans-Activators; Transfection; Viral Regulatory and Accessory Proteins

Benzimidazole compounds, such as omeprazole and thiabendazole, are a different type of CYP1A1-inducer from Ah receptor-ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. In HepG2 cells, the commonly used tyrosine kinase-inhibitors, herbimycin-A and a series of tyrphostins, inhibited the induction of CYP1A1 produced by treatment with TCDD. Genistein, another type of tyrosine kinase inhibitor, inhibited the induction of CYP 1A1 whether it was produced by omeprazole or TCDD; however, this inhibition was caused by a dual effect of genistein, that is an anti-tyrosine kinase and an anti-topoisomerase I effect. An antagonist of Ah receptor, 3'-methoxy-4'-aminoflavone (1 microM), did not inhibit the induction of CYP1A1 produced in HepG2 cells by omeprazole or alpha-naphthoflavone (50 microM), but this antagonist did inhibit that produced by TCDD. Thus, omeprazole appears to induce CYP1A1 by initiating a protein tyrosine kinase-mediated signal transduction pathway, a different pathway from that initiated by TCDD.

Topics: Benzoflavones; Benzoquinones; Cytochrome P-450 CYP1A1; Enzyme Induction; Enzyme Inhibitors; Gene Expression; Genistein; Humans; Lactams, Macrocyclic; Liver Neoplasms; Omeprazole; Polychlorinated Dibenzodioxins; Protein-Tyrosine Kinases; Quinones; Receptors, Aryl Hydrocarbon; Rifabutin; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

Presentation of a wild-type secretory protein, apolipoprotein B100 (apoB), to the cytosol for ubiquitin-proteasome proteolysis has been observed in HepG2 cells. A currently accepted model for proteasomal degradation of secretory proteins is retrograde translocation of the substrate polypeptides from the lumen of endoplasmic reticulum (ER) back to the cytosol. In this report, we present evidence that newly synthesized apoB becomes exposed to the cytosol and targeted to the proteasomes in a co-translational manner. Thus, after protein translation was synchronized with puromycin, partially synthesized apoB polypeptides were found to be conjugated to ubiquitin. The magnitude of co-translational ubiquitination and subsequent degradation of apoB was increased when cells were pretreated with either herbimycin A to induce cytosolic Hsp70 or with an inhibitor of microsomal triglyceride transfer protein; both treatments impede translocation of nascent apoB across the ER membrane. These treatments also decreased secretion of apoB and increased its degradation via the ubiquitin-proteasome pathway. We suggest that translocation arrest with subsequent co-translational exposure to the cytosol provides an alternative model to explain how mammalian secretory proteins can overcome topological segregation by the ER membrane and undergo degradation by the ubiquitin-proteasome pathway.

Topics: Acetylcysteine; Animals; Apolipoprotein B-100; Apolipoproteins B; Benzoquinones; Carcinoma, Hepatocellular; CHO Cells; Cricetinae; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; Endoplasmic Reticulum; Enzyme Inhibitors; HSP70 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Liver Neoplasms; Models, Biological; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Biosynthesis; Protein Processing, Post-Translational; Puromycin; Quinones; Recombinant Proteins; Rifabutin; Transfection; Tumor Cells, Cultured; Ubiquitins

Cultured hepatic stellate cells were induced to elongate long, multipolar cellular processes by interstitial collagen gel used as a substratum, as compared to flattened or round cell shapes on polystyrene surface or on Matrigel containing the basement membrane components, respectively. The process induction was inhibited by several reagents as follows: (1) anti-integrin alpha2 antibody; (2) an oligopeptide, DGEA, an integrin-binding sequence in type I collagen molecule; (3) wortmannin, a phosphatidylinositol 3-kinase inhibitor. Protein tyrosine phosphorylation was enhanced throughout cells including cellular processes by culturing on type I collagen gel. Dual fluorescence staining showed that the core of the processes contained microtubules, whereas the periphery of the processes comprised fibrillar actin. Thus, the process extension was found to depend on integrin-binding to type I collagen fibres, followed by signal transduction and cytoskeleton assembly. The cellular processes included interstitial collagenase and vitamin A-containing lipid droplets. The lipid droplets and vitamin A-autofluorescence were increased by retinyl acetate addition to the culture medium, suggesting an important role of processes in hepatic stellate cell function.

Topics: Androstadienes; Animals; Antibodies, Monoclonal; Antigens, CD; Benzoquinones; Cells, Cultured; Collagen; Collagenases; Diterpenes; Drug Combinations; Enzyme Inhibitors; Gels; Humans; Integrin alpha2; Lactams, Macrocyclic; Laminin; Lipid Metabolism; Liver; Liver Neoplasms; Male; Matrix Metalloproteinase 1; Oligopeptides; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteoglycans; Quinones; Rats; Rats, Wistar; Retinyl Esters; Rifabutin; Signal Transduction; Staurosporine; Tumor Cells, Cultured; Vitamin A; Wortmannin