cytochalasin-b and Liver-Neoplasms

Topics: Animals; Biological Transport; Biological Transport, Active; Blood Glucose; Carcinoma, Hepatocellular; Cytochalasin B; Cytochalasins; Deoxyglucose; Erythrocytes; Hexoses; Humans; Kinetics; Liver Neoplasms; Methylglucosides; Neoplasms, Experimental; Rats; Thermodynamics

Topics: Animals; Carcinoma, Hepatocellular; Cell Fractionation; Cell Fusion; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cells; Cells, Cultured; Chickens; Cytochalasin B; Erythrocytes; Hybrid Cells; L Cells; Liver Neoplasms; Macrophages; Methods; Parainfluenza Virus 1, Human; RNA, Messenger; Ultracentrifugation; Virus Replication

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that accumulates in the early endosomal membrane, and acts as a scaffold to recruit proteins that contain a PI3P-binding domain, such as the FYVE domain. In this study, we examined the effect of PI3P depletion on the insulin response in rat hepatoma-derived H4IIEC3 cells. We found that insulin treatment induced the transient formation of an actin domain structure, a mesh-like tangled network of actin filaments where phosphorylated Akt, endosomal proteins, and PI3P accumulated. Actin domain formation was repressed by the depletion of PI3P by SAR405, an inhibitor of the class III PI3 kinase, Vps34, by the inhibition of PI3P function by the competitive binding of an excess amount of GST-fused 2xFYVE protein to intracellular PI3P, and by the use of diabetic model cells, in which PI3P was depleted. SAR405 did not affect the phosphorylation level of Akt, and the transcriptional regulation of gluconeogenic and cholesterol synthetic genes after insulin treatment. Interestingly, insulin-induced DNA synthesis was specifically inhibited by SAR405, cytochalasin B, and also in diabetic model cells. These results suggest that PI3P is required for the formation of actin domains, which affected a signaling pathway downstream of Akt associated with DNA synthesis in H4IIEC3 cells.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Class III Phosphatidylinositol 3-Kinases; Cytochalasin B; DNA, Neoplasm; Insulin; Liver Neoplasms; Phosphatidylinositol Phosphates; Protein Domains; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidinones; Rats

Phloretin (Ph), a natural product found in apples and pears with glucose transporter (GLUT) inhibitory activity, exerts antitumor effects. However, little is known about its effects on human liver cancer. The purpose of this study is to test the cytotoxic effects of Ph on HepG2 cells and to identify the underlying molecular pathways. Human hepatocellular carcinoma specimens and HepG2 show a high level of GLUT2 transporter activity in the cell membrane. Real-time PCR and MTT assays demonstrate that Ph-induced cytotoxicity correlates with the expression of GLUT2. Flow cytometry and DNA fragmentation studies show that 200 microM Ph induces apoptosis in HepG2, which was reversed by glucose pretreatment. GLUT2 siRNA knockdown induced HepG2 apoptosis, which was not reversed by glucose. Western blot analysis demonstrates that both intrinsic and extrinsic apoptotic pathways in addition to Akt and Bcl-2 family signaling pathways are involved in Ph-induced cell death in HepG2 cells. Furthermore, using flow cytometry analysis, a mitochondrial membrane potential assay and Western blot analysis, we show that cytochalasin B, a glucose transport inhibitor, enhances the Ph-induced apoptotic effect on HepG2 cells, which was reversed by pretreatment with glucose. Furthermore, we found significant antitumor effects in vivo by administering Ph at 10 mg/kg intraperitoneally to severe combined immune deficiency mice carrying a HepG2 xenograft. A microPET study in the HepG2 tumor-bearing mice showed a 10-fold decrease in (18)F-FDG uptake in Ph-treated tumors compared to controls. Taken together, these results suggest that Ph-induced apoptosis in HepG2 cells involves inhibition of GLUT2 glucose transport mechanisms.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspases; Cell Proliferation; Cells, Cultured; Cytochalasin B; Flow Cytometry; Fluorescent Antibody Technique; Glucose; Glucose Transporter Type 2; Hepatocytes; Humans; Immunoenzyme Techniques; In Vitro Techniques; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Mice, SCID; Phloretin; Positron-Emission Tomography; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Transplantation, Heterologous

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.

Topics: Actin Cytoskeleton; Antibodies, Monoclonal; Carbocyanines; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Colchicine; Cytochalasin B; Fluorescein-5-isothiocyanate; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Hepacivirus; Humans; Indoles; Kidney; Liver Neoplasms; Microtubules; Replicon; RNA Helicases; RNA, Viral; Serine Endopeptidases; Tubulin; Tubulin Modulators; Viral Nonstructural Proteins; Virus Replication

Although cisplatin is a frequently used cancer chemotherapeutic drug, its effectiveness is hindered by the development of resistance in cancer cells. In order to understand the reason(s) for this resistance, the mechanism of uptake of cisplatin into cells must be characterized. While several previous studies showed structural differences between cisplatin-sensitive and resistant cells, the influence of microfilaments, known to affect transport of molecules into cells, and the influence of certain biophysical characteristics of the plasma membrane needed clarification.. We show that resistant human epidermal carcinoma (KB-CP20) and liver carcinoma (BEL-7404-CP20) cells become relatively more resistant if their already weak microfilaments are degraded by cytochalasin B treatment (.5-2 microM). The sensitive counterparts of these cells with intact microfilaments are not significantly affected by this treatment. We also show that the "fluidity" of the plasma membrane and the membrane potential of the sensitive and resistant cells studied do not appear to influence the uptake of cisplatin into the cells.. Our results suggest that the status of the microfilament system influences the mechanism of uptake of cisplatin into cells.

Topics: Actin Cytoskeleton; Biological Transport; Carcinoma; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Membrane Permeability; Cisplatin; Cytochalasin B; Drug Resistance, Neoplasm; Humans; KB Cells; Liver Neoplasms; Membrane Fluidity; Membrane Potentials; Skin Neoplasms

We have previously shown that microtubules in nonneuronal cells form long, stable bundles after transfection with the embryonic neuronal microtubule-associated protein MAP2c. In this study, we found that treating MAP2c-transfected cells with the actin depolymerising drug cytochalasin B led to the outgrowth of microtubule-containing processes from the cell surface. This effect was specific to MAP2c and did not occur in untransfected cells whose microtubules had been stabilised by treatment with taxol. The outgrowth and retraction of these processes during repeated cycles of cytochalasin addition and removal was followed by video time-lapse microscopy and was suggestive of a physical interaction between compressive forces exerted by the MAP2c-stabilised microtubule bundles and tensile forces originating in the cortical actin network. We suggest that MAP2c confers three properties on cellular microtubules that are essential for process outgrowth: stability, bundling and stiffness. The latter probably arises from the linking together of neighbouring tubulin subunits by three closely spaced tubulin-binding motifs in the MAP2 molecule that limits their motion relative to one another and thus reduces the flexibility of the polymer. Similar multimeric tubulin-binding domains in other proteins of the MAP2 class, including tau in axons and MAP4 in glial cells, may play the same role in the development and support of asymmetric cell morphology. Axial bundles of microtubules are found in growing neurites but not in growth cones, suggesting that the regulated expression of these MAP-induced properties makes an important contribution to the establishment of a stable process behind the advancing growth cone.

Topics: Actins; Carcinoma, Hepatocellular; Cytochalasin B; Humans; Liver Neoplasms; Microscopy, Electron; Microscopy, Fluorescence; Microtubule-Associated Proteins; Microtubules; Polymers; Transfection; Tumor Cells, Cultured; Video Recording

The subcellular distribution of glucose transporters in rat hepatocytes and HepG2 cells was studied in the absence and in the presence of insulin. Glucose transporters were quantitated by measuring glucose-sensitive cytochalasin B binding and by protein immunoblotting using isoform-specific antibodies. Plasma membrane contamination into subcellular fractions was assessed by measuring distribution of 5'-nucleotidase and cell surface carbohydrate label. In hepatocytes, GLUT-2 occurred in a low-density microsomal (LDM) fraction at a significant concentration, and as much as 15% of cellular GLUT-2 was found intracellularly that cannot be accounted for by plasma membrane contamination. In HepG2 cells which express GLUT-1 and GLUT-2, the two isoforms showed distinct subcellular distribution patterns: GLUT-2 was highly concentrated in LDM while very little GLUT-1 was found in this fraction, indicating that a large portion of GLUT-2 occurs in intracellular organelles. Insulin treatment did not change the subcellular distribution patterns of glucose transporters in both cell types. Our results suggest that rat hepatocytes and HepG2 cells possess an intracellular storage pool for GLUT-2, but lack the insulin-responsive glucose transporter translocation mechanism.

Topics: Adipose Tissue; Animals; Biological Transport; Blotting, Western; Borohydrides; Cell Fractionation; Cell Membrane; Cells, Cultured; Cytochalasin B; Glucose; Humans; Insulin; Insulin Resistance; Isomerism; Liver; Liver Neoplasms; Microsomes, Liver; Monosaccharide Transport Proteins; Organelles; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

The liver is frequently colonized by metastatic tumor cells despite its dense population of macrophages (Kupffer cells). We have studied the interactions between metastatic colon carcinoma cells (DHD) and syngeneic Kupffer cells under different experimental conditions in vitro. In an adhesion assay the binding of DHD cells to Kupffer cell monolayers was shown to be time and temperature dependent, reaching a maximum level after about 90 min of incubation at 37 degrees C. In contrast, only a low level of binding could be observed at 4 degrees C. The level of binding could be increased by pretreatment of the Kupffer cells with phorbol 12-myristate 13-acetate. A firm interaction between the two cell types was shown to be dependent on the presence of calcium- and trypsin-sensitive structures on the surface of the Kupffer cells. Pretreatment of the macrophages with the cytoskeletal inhibitors colchicine and cytochalasin B was also found to reduce significantly the binding of tumor cells. This binding was also inhibited to a large extent by D-mannose and N-acetyl-D-galactosamine. The Kupffer cells were not cytotoxic against the colon carcinoma cells.

Topics: Animals; Carcinoma; Cell Adhesion; Cell Line; Colchicine; Colonic Neoplasms; Cytochalasin B; Cytotoxicity, Immunologic; Egtazic Acid; Kupffer Cells; Liver Neoplasms; Macrophages; Male; Monosaccharides; Peritoneal Cavity; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Trypsin

The gene encoding the human erythrocyte glucose transporter, cloned from HepG2 hepatoma cells, was expressed in Escherichia coli by introducing a prokaryote-type ribosome binding site, subcloning the gene into the T7 promoter/T7 polymerase expression system, and transforming a strain that is defective in glucose transport. Cells bearing plasmids with the transporter gene take up 2-deoxy-D-glucose and D-glucose, unlike cells bearing plasmids without the transporter gene. Moreover, 2-deoxy-D-glucose uptake is inhibited by unlabeled D-glucose, cytochalasin B, or mercuric chloride but not by L-glucose. The glucose transport protein is inserted into the membrane of E. coli, as evidenced by immunoblotting experiments with two site-directed polyclonal antibodies, one directed against the COOH terminus of the glucose transporter and the other directed against a synthetic peptide containing amino acid residues 225-238. As detected with both antibodies, the protein migrates with apparent molecular mass of 34 kDa in sodium dodecyl sulfate/12% polyacrylamide, a size similar to that of the unglycosylated glucose-transport protein synthesized in vitro.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cloning, Molecular; Cytochalasin B; Deoxyglucose; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Escherichia coli; Gene Expression Regulation; Glucose; Humans; Immunoassay; Liver Neoplasms; Mercuric Chloride; Molecular Sequence Data; Monosaccharide Transport Proteins; Plasmids; Tumor Cells, Cultured

Topics: Animals; Bucladesine; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cells, Cultured; Cytochalasin B; DNA, Neoplasm; Liver Neoplasms; Neoplasm Proteins; Pyruvates; Rats; RNA, Neoplasm

The increase in susceptibility to killing by rabbit antibody and guinea pig complement of guinea pig hepatoma cells (line-10), after treatment with certain metabolic inhibitors, did not correlate with the mobility of antigen on the cell surface as measured by indirect immunofluorescence.

Topics: Animals; Antibodies; Antibodies, Neoplasm; Antigens; Azides; Carcinoma, Hepatocellular; Cell Membrane; Colchicine; Complement System Proteins; Cyanides; Cycloheximide; Cytochalasin B; Cytotoxicity Tests, Immunologic; Dactinomycin; Fluorescent Antibody Technique; Fluorides; Forssman Antigen; Guinea Pigs; Immunity, Cellular; Iodoacetates; Liver Neoplasms; Puromycin; Staining and Labeling

Nutrient transport rates and cyclic AMP levels have been implicated in the regulation of cell proliferation. In the present study, however, changes in intracellular cyclic AMP level in several lines of cultured cells (normal 3T3 and SV40 and polyomavirus-transformed 3T3 cells; 3T6, C6 GLIOMA, MOUSE L, and Novikoff rat hepatoma cells) by treatment with papaverine, prostaglandine E1 or isoproterenol did not correlate with the inhibition of the uridine, hypoxanthine or deoxyglucose transport rates by these chemicals. Transport inhibitions by above chemicals or Persantin or Cytochalasin B occurred in most cell lines in the absence of any measurable change in intracellular cyclic AMP concentration. Furthermore, treatment of several cell lines with 1 mM dibutyryl cyclic AMP had no immediate effect on the transport of uridine, thymidine or deoxyglucose, although the transport capacity of the cells for uridine and thymidine, but not that for deoxyglucose, decreased progressively with time of treatment. We also observed that the uridine transport system of all cell lines derived from 3T3 cells and the hypoxanthine transport system of L cells exhibited high degrees of resistance to inhibition by the various chemicals. On the other hand, deoxyglucose transport was inhibited to about the same extent by these chemicals in all the cell lines investigated.

Topics: Animals; Biological Transport, Active; Bucladesine; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Cyclic AMP; Cytochalasin B; Deoxy Sugars; Deoxyglucose; Depression, Chemical; Dipyridamole; Glioma; Hypoxanthines; Isoproterenol; L Cells; Liver Neoplasms; Mice; Papaverine; Prostaglandins; Rats; Thymidine; Uridine

Adenine, guanine, and hypoxanthine were rapidly incorporated into the acid-soluble nucleotide pool and nucleic acids by wild type Novikoff cells. Incorporation followed normal Michaelis-Menten kinetics, but the following evidence indicates that specific transport processes precede the phosphoribosyltransferase reactions and are the rate-limiting step in purine incorporation by whole cells. Cells of an azaguanine-resistant subline of Novikoff cells which lacked hypoxanthine-guanine phosphoribosyltransferase activity and failed to incorporate guanine or hypoxanthine into the nucleotide pool, exhibited uptake of guanine and hypoxanthine by a saturable process. Similarly, wild type cells which had been preincubated in a glucose-free basal medium containing KCN and iodoacetate transported guanine and hypoxanthine normally, although a conversion of these purines to nucleotides did not occur in these cells. The mutant and KCN-iodoacetate treated wild type cells also exhibited countertransport of guanine and hypoxanthine when preloaded with various purines, uracil, and pyrimidine nucleosides. The cells also possess a saturable transport system for uracil although they lack phosphoribosyltransferase activity for uracil. In the absence of phosphoribosylation, none of the substrates was accumulated against a concentration gradient. Thus transport is by facilitated diffusion (nonconcentrative transport). Furthermore, the apparent Km values for purine uptake by untreated wild type and azaguanine-resistant cells were higher and the apparent Vmax values were lower than those for the corresponding phosphoribosyltransferases...

Topics: Adenine; Animals; Biological Transport, Active; Calorimetry; Carcinoma, Hepatocellular; Cell Line; Centrifugation, Density Gradient; Chloromercuribenzoates; Cytochalasin B; Cytosine; Dipyridamole; Dithiothreitol; Guanine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Kinetics; Liver Neoplasms; Neoplasms, Experimental; Pentosyltransferases; Purines; Pyrimidines; Rats; Thermodynamics; Time Factors; Uracil

Anucleate HTC cells have been used to determine the importance of the nucleus in the regulation of the intracellular levels of tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells. In the absence of the nucleus, neither the induction of the enzyme by dexamethasone nor its deinduction upon removal of the hormone occurs. Degradation of the enzyme takes place when protein synthesis is inhibited in anucleates by cycloheximide. Therefore, the maintenance of induced levels of enzyme activity after dexamethasone withdrawal from pre-induced anucleates suggest that the nucleus is required for the inactivation of the TAT mRNA.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Cell Nucleus; Cycloheximide; Cytochalasin B; Dexamethasone; Enzyme Induction; Liver Neoplasms; Neoplasm Proteins; Rats; Tyrosine Transaminase

Topics: Animals; Biological Transport; Carcinoma, Hepatocellular; Cell Line; Cell Membrane Permeability; Cells, Cultured; Cytochalasin B; Deoxyribonucleosides; Deoxyuridine; Depression, Chemical; Dipyridamole; Fluorouracil; Kinetics; Liver Neoplasms; Neoplasms, Experimental; Rats; Thymidine; Time Factors; Tritium

Topics: Adenine Nucleotides; Adenosine; Animals; Biological Transport; Carbon Dioxide; Carbon Isotopes; Carcinoma, Hepatocellular; Cells, Cultured; Chromatography, Paper; Cycloheximide; Cytochalasin B; Diffusion; Dipyridamole; Glucosamine; Glucose; Lactates; Liver Neoplasms; Neoplasms, Experimental; Puromycin; Rats; Ribonucleotides; Tritium; Uracil Nucleotides; Uridine; Uridine Diphosphate Sugars

Topics: Agglutination; Animals; Azides; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cell Membrane; Cells; Concanavalin A; Cycloheximide; Cytochalasin B; Dimethyl Sulfoxide; Dinitrophenols; Glycosides; Lectins; Liver Neoplasms; Rats; Sarcoma, Yoshida; Temperature; Tritium

Topics: Butyrates; Carcinoma, Hepatocellular; Cell Line; Cyclic AMP; Cytochalasin B; Dimethyl Sulfoxide; Enzyme Induction; Hydrocortisone; Indoles; Insulin; Liver Neoplasms; Tyrosine Transaminase