cytochalasin-d and Carcinoma--Hepatocellular

Cytochalasin D targets actin and is ubiquitous in eukaryotic cells. When cytochalasin D is used as a cytotoxic agent in cancer therapy, it causes significant side effects. To prevent this, cytochalasin D can be encapsulated in polyethylene liposomes. In this study, high-performance liquid chromatography observation of the biodistribution of pegylated liposomal cytochalasin D in tumour-bearing mice showed that liposomal cytochalasin D could be conveniently dissolved in water for i.v. injection and that it specifically accumulated in tumour tissues, more than natural cytochalasin D did. The half-time of liposomal cytochalasin D in the plasma was also significantly longer than that of natural cytochalasin D (4h versus 10 min). MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that liposomal cytochalasin D treatment could cause significant inhibition of cell proliferation in vitro in a manner similar to that of natural cytochalasin D. The antitumour activities of liposomal cytochalasin D were investigated in B16 melanoma, CT26 colorectal carcinoma and H22 hepatoma models, and the results indicated that liposomal cytochalasin D could significantly inhibit tumour growth and prolong survival in a manner similar to that of cisplatin. TUNEL-based apoptosis assays showed that liposomal cytochalasin D induced significant tumour cell apoptosis. Significant inhibition of tumour angiogenesis was observed in mice treated with liposomal cytochalasin D. In addition, no significant side effects were observed in mice treated with liposomal cytochalasin D. Our results show that liposomal cytochalasin D increases solubility and bioavailability, a lower incidence of side effects and improves antitumour effects, indicating its potential as a chemical agent for cancer therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biological Availability; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chemistry, Pharmaceutical; Colorectal Neoplasms; Cytochalasin D; Dose-Response Relationship, Drug; Half-Life; Injections, Intravenous; Liposomes; Liver Neoplasms, Experimental; Male; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neovascularization, Pathologic; Polyethylene Glycols; Solubility; Tissue Distribution; Tumor Burden

Although Helicobacter pylori have been identified in the liver, the role of Helicobacter sp. in human liver diseases remains unclear. This study explored whether H. pylori were internalized and could persist in hepatocytes. The majority of an inoculum of H. pylori (1 x 10(7) colony forming units) adhered to hepatocytes. Using the gentamicin invasion assay we found that approximately 2% were internalized and persisted following passage for more than 2 months. Electron microscopy confirmed the presence of intracellular Helicobacter. The number of adherent or internalized H. pylori was significantly greater with hepatocytes than with gastric epithelial cells (P < 0.05) and was also dependent on cag pathogenicity island (PAI), VacA, OipA, or BabA status. Transmission electron microscopy was used to confirm adherence and invasion of H. pylori into hepatocytes. Internalization of H. pylori was inhibited by antibodies to beta1-integrin receptors, genistein, and cytochalasin D (P < 0.05) consistent with beta1-integrin acting as a surface receptor with additional requirements for tyrosine kinase phosphorylation and actin polymerization. In summary, H. pylori both adhered to and invaded into hepatocytes in vitro, depending on the virulent factors, and persisted within hepatocytes during subcultures. beta1-integrin is likely a receptor involved in internalization of H. pylori into hepatocytes.

Topics: Anti-Bacterial Agents; Bacterial Adhesion; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochalasin D; Endocytosis; Genistein; Gentamicins; Helicobacter Infections; Helicobacter pylori; Hepatocytes; Humans; Integrin beta1; Liver Neoplasms; Microscopy, Electron, Transmission; Nucleic Acid Synthesis Inhibitors; Protein Kinase Inhibitors

To study the viscoelastic properties of human hepatocytes and hepatocellular carcinoma (HCC) cells under cytoskeletal perturbation, and to further to study the viscoelastic properties and the adhesive properties of mouse hepatoma cells (HTC) in different cell cycle.. Micropipette aspiration technique was adopted to measure viscoelastic coefficients and adhesion force to collagen coated surface of the cells. Three kinds of cytoskeleton perturbing agents, colchicines (Col), cytochalasin D (CD) and vinblastine (VBL), were used to treat HCC cells and hepatocytes and the effects of these treatment on cell viscoelastic coefficients were investigated. The experimental results were analyzed with a three-element standard linear solid. Further, the viscoelastic properties of HTC cells and the adhesion force of different cycle HTC cells were also investigated. The synchronous G(1) and S phase cells were achieved through thymine-2-desoryriboside and colchicines sequential blockage method and thymine-2-desoryriboside blockage method respectively.. The elastic coefficients, but not viscous coefficient of HCC cells (K(1)=103.6+/-12.6N.m(-2), K(2)=42.5 +/ 10.4N.m(-2), mu=4.5 +/- 1.9Pa.s), were significantly higher than the corresponding value for hepatocytes (K(1)=87.5 +/- 12.1N.m(-2), K(2)=33.3+/-10.3N.m(-2), mu=5.9+/-3.0Pa.s, P<0.01). Upon treatment with CD, the viscoelastic coefficients of both hepatocytes and HCC cells decreased consistently, with magnitudes for the decrease in elastic coefficients of HCC cells (K(1): 68.7 N.m(-2) to 81.7N.m(-2), 66.3% to 78.9%; K(2): 34.5N.m(-2) to 37.1N.m(-2), 81.2% to 87.3%, P<0.001) larger than those for normal hepatocytes (K(1): 42.6N.m(-2) to 49.8N.m(-2), 48.7% to 56.9%; K(2): 17.2N.m(-2) to 20.4N.m(-2), 51.7% to 61.3%, P<0.001). There was a little decrease in the viscous coefficient of HCC cells (2.0 to 3.4Pa.s, 44.4 to 75.6%, P<0.001) than that for hepatocytes (3.0 to 3.9Pa.s, 50.8 to 66.1% P<0.001). Upon treatment with Col and VBL, the elastic coefficients of hepatocytes generally increased or tended to increase while those of HCC cells decreased. HTC cells with 72.1% of G(1) phase and 98.9% of S phase were achieved and high K(1), K(2) value and low mu value were the general characteristics of HTC cells. G(1) phase cells had higher K(1) value and lower mu value than S phase cells had, and G(1) phase HTC cells had stronger adhesive forces ((275.9 +/- 232.8) x 10(-10)N) than S phase cells ((161.2 +/- 120.4) x 10(-10)N, P<0.001).. The difference in both the pattern and the magnitude of the effect of cytoskeletal perturbing agent on the viscoelastic properties between HCC cells and hepatocytes may reflect differences in the state of the cytoskeleton structure and function and in the sensitivity to perturbing agent treatment between these two types of cells. Change in the viscoelastic properties of cancer cells may affect significantly tumor cell invasion and metastasis as well as interactions between tumor cells and their micro-mechanical environments.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Adhesion; Cell Cycle; Colchicine; Cytochalasin D; Cytoskeleton; Elasticity; Hepatocytes; Humans; Liver Neoplasms; Mice; Nucleic Acid Synthesis Inhibitors; Tumor Cells, Cultured; Vinblastine

To investigate the relevance of the rheological properties, i.e., viscoelasticities and adhesion to basement membrane components coated surface, of both hepatocytes and hepatocellular carcinoma (HCC) cells to the cytoskeleton structure.. Micropipette aspiration technique was adopted to measure viscoelastic coefficients and adhesion forces to 2 microg/ml collagen IV/1.25 microg/ml laminin coated surface of the cells. Two kinds of cytoskeleton perturbing agents, colchicine and cytochalasin D, were used to treat both HCC cells and hepatocytes and the effects of these treatments on cell viscoelastic coefficients and cell adhesion forces were investigated.. Upon treatment of cells with colchicine in a concentration range of 1 to 60 mg/L, the elastic coefficients, especially the first elastic coefficient K1, and adhesion forces of hepatocytes generally tended to increase or increased significantly while, in contrast, viscoelastic coefficients and adhesion forces of HCC cells decreased obviously. Upon treatment of cells with cytochalasin D in a concentration range of 0.25-5.00 mg/L, viscoelastic coefficients of both hepatocytes and HCC cells decreased uniformly, with a larger magnitude for the decrease in elastic coefficients and adhesion forces of HCC cells than for those of hepatocytes. Adhesion forces of hepatocytes and HCC cells onto collagen IV/laminin coated surface varied similarly as viscoelastic coefficients under the action of the cytoskeleton perturbing agents. A significant positive correlation existed between changes of HCC cell adhesion forces on collagen IV/laminin coated surfaces and those of cell elastic coefficients (P<0.01).. The effects of cochicine and cytochalasin D on rheological properties of HCC cells differed significantly either in ways or extents from those on rheological properties of hepatocytes. These results might reflect the difference in the state of cytoskeleton structure and function among these two kinds of cells.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Colchicine; Cytochalasin D; Cytoskeleton; Elasticity; Hepatocytes; Humans; Liver Neoplasms; Viscosity

Tumor necrosis factor-alpha (TNF-alpha) is an important component of the early signaling pathways leading to liver regeneration and proliferation, but it is also responsible for several hepatotoxic effects. We have investigated the effect of TNF-alpha on thapsigargin (TG)-induced store-mediated Ca2+ entry (SMCE) in the human hepatocellular carcinoma cell line HepG2. In these cells, short-term (10 min) exposure to TNF-alpha slightly increased SMCE. In contrast, long-term (12 h) exposure to TNF-alpha significantly reduced SMCE. This effect was reversed by coincubation with atrial natriuretic peptide (ANP), which itself had no effect on SMCE. Cytochalasin D and latrunculin A, inhibitors of actin polymerization, abolished SMCE. Long-term exposure of HepG2 cells to TNF-alpha abolished TG-induced actin polymerization and membrane association of Ras proteins. When TNF-alpha was added in combination with ANP, these effects were reduced. These findings suggest that in HepG2 cells, TNF-alpha inhibits SMCE by affecting reorganization of the actin cytoskeleton, probably by interfering with the activation of Ras proteins, and that ANP protects against these inhibitory effects of TNF-alpha.

Topics: Actins; Atrial Natriuretic Factor; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Carcinogens; Carcinoma, Hepatocellular; Cell Compartmentation; Cytochalasin D; Cytoskeleton; Humans; Liver Neoplasms; Nucleic Acid Synthesis Inhibitors; Polymers; ras Proteins; Thapsigargin; Thiazoles; Thiazolidines; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Following hypoosmotic stress-induced cell volume change, the actin cytoskeleton reorganizes itself. The role of this reorganization in the activation of the phosphatidylinositol 3-OH-kinase/protein kinase B/activator protein 1 (PI-3-K/PKB/AP-1) proliferative signaling cascade is unknown. Focal adhesion kinase (FAK) participates in the cytoskeleton-based activation of PI-3-K. We hypothesized that hypoosmotic stress-induced activation of PKB and AP-1 in HepG2 cells is dependent on an intact actin cytoskeleton and subsequent FAK phosphorylation.. HepG2 cells were incubated for 1 h with or without 20 microM cytochalasin D, an actin disrupter, and were then exposed for up to 30 min to hypoosmotic medium (200 mOsm/L) to induce swelling. Tumor necrosis factor alpha (1.4 nM) and medium alone served as positive and negative controls, respectively. Western blots measured cytoplasmic phosphorylated or total FAK and PKB. EMSAs measured nuclear AP-1. All experiments were performed in triplicate.. Exposure to hypoosmotic stress resulted in activation of the following signaling messengers in a sequential fashion: (1) phosphorylation of FAK occurred by 2 min, (2) phosphorylation of PKB occurred by 10 min, (3) nuclear translocation of AP-1 occurred by 30 min. All three signaling events were abolished when these cells were pretreated with cytochalasin D.. Actin reorganization following hypoosmotic stress is essential for the FAK-mediated activation of the PI-3-K/PKB/AP-1 proliferative cascade. These data delineate a possible mechanism by which the cell swelling-induced cytoskeletal changes can initiate proliferative signal transduction in human liver cancer.

Topics: Actins; Carcinoma, Hepatocellular; Cell Nucleus; Cytochalasin D; Cytoskeleton; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Liver Neoplasms; Nucleic Acid Synthesis Inhibitors; Osmotic Pressure; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factor AP-1; Tumor Cells, Cultured

Helicobacter pylori vacuolating toxin (VacA) causes vacuolation in a variety of cultured cell lines, sensitivity to VacA differing greatly, however, among the different cell types. We found that the high sensitivity of HEp-2 cells to VacA was impaired by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function of sphingolipid-cholesterol-rich membrane microdomains ("lipid rafts"), also impaired VacA-induced cell vacuolation. Overexpression into HEp-2 cells of proteins inhibiting clathrin-dependent endocytosis (i.e., a dominant-negative mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant of dynamin II) did not affect vacuolation induced by VacA. Nevertheless, F-actin depolymerization, known to block the different types of endocytic mechanisms, strongly impaired VacA vacuolating activity. Taken together, our data suggest that the high cell sensitivity to VacA depends on the presence of one or several GPI-anchored protein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin-independent endocytic pathway.

Topics: Actin Cytoskeleton; Actins; Animals; Bacterial Proteins; Bacterial Toxins; Carcinoma, Hepatocellular; Cell Line; CHO Cells; Clathrin; Cricetinae; Cytochalasin D; Dogs; Dose-Response Relationship, Drug; Endocytosis; Humans; Iodine Radioisotopes; Nystatin; Phosphatidylinositols; Proteins; Type C Phospholipases; Vacuoles

The viscoelastic properties of both hepatocytes and hepatocellular carcinoma (HCC) cells were measured by means of a micropipette aspiration technique. Experimental results were analyzed with a three-element standard linear solid model, in which an elastic element, K1, is in parallel with a Maxwell element composed of another elastic element, K2, in series with a viscous element, mu. Further, we investigated the relevance of viscoelastic properties of these two types of cells to the cytoskeleton structures by treating cells with three cytoskeletal perturbing agents, namely cytochalasin D (CD), colchicine (Col) and vinblastine (VBL). The results showed that the elastic coefficients, but not viscous coefficient of HCC cells (K1 = 103.6 +/- 12.6 N m-2, K2 = 42.5 +/- 10.4 N m-2, mu = 4.5 +/- 1.9 Pa s, n = 30), were significantly higher than the corresponding values for hepatocytes (K1 = 87.5 +/- 12.1 N m-2, K2 = 33.3 +/- 10.3 N m-2, mu = 5.9 +/- 3.0 Pa s, n = 24). Upon treatment with CD, the viscoelastic coefficients of both hepatocytes and HCC cells decreased uniformly, with magnitudes for the decrease in elastic coefficients of HCC cells (K1: 68.7 to 81.7 N m-2, 66.3 to 78.9%; K2: 34.5 to 37.1 N m-2, 81.2 to 87.3%) larger than those for normal hepatocytes (K1: 42.6 to 49.8 N m-2, 48.7 to 56.9%; K2: 17.2 to 20.4 N m-2, 51.7 to 61.3%). There was a smaller decrease in the viscous coefficient of HCC cells (2.0 to 3.4 Pa s, 44.4 to 75.6%) than that for hepatocytes (3.0 to 3.9 Pa s, 50.8 to 66.1%). Upon treatment with Col and VBL, the elastic coefficients of hepatocytes generally increased or tended to increase while those of HCC cells decreased. The differences in either the pattern or the magnitude of the effect of cytoskeletal perturbing agent on the viscoelastic properties between HCC cells and hepatocytes might possibly reflect differences in the state of the cytoskeleton structure and function, or in the cells' sensitivity to perturbing agent treatment between these two types of cells. Changes in the viscoelastic properties of cancer cells might well affect tumor cell invasion and metastasis as well as interactions between tumor cells and their micro-mechanical environments.

Topics: Carcinoma, Hepatocellular; Cells, Cultured; Colchicine; Cytochalasin D; Cytoskeleton; Elasticity; Hepatocytes; Humans; Linear Models; Liver; Micromanipulation; Suction; Time Factors; Tumor Cells, Cultured; Vinblastine; Viscosity

The Na+-taurocholate cotransport polypeptide (ntcp) is the primary transporter for the uptake of bile acids in the liver. The second messenger adenosine 3':5'-cyclic monophosphate (cAMP) rapidly increases ntcp protein concentration in the plasma membrane, yet the mechanism is unknown. To investigate this, HepG2 cells were transiently transfected with a carboxy-terminal-tagged green fluorescence protein (GFP) conjugate of ntcp, and then examined by confocal video microscopy. Transporter activity was directly assayed with 3H-taurocholic acid (TC) scintigraphy. ntcp-GFP targeted to the plasma membrane in transfected cells, and the conjugate protein transported 3H-TC as effectively as unmodified rat ntcp. Stimulation of ntcp-GFP cells with cAMP increased GFP fluorescence in the plasma membrane by 40% (P <.0001) within 2.5 minutes and by 55% within 10 minutes. Similarly, cAMP increased transport of bile acids by 30%. Cytochalasin D, an inhibitor of microfilaments, did not prevent ntcp-GFP from targeting to the plasma membrane, but completely abolished the increase in GFP fluorescence seen in response to cAMP. In contrast, the microtubule inhibitor, nocodazole, prevented development of membrane fluorescence in 48 (96%) of 50 cells. Cells regained plasma membrane fluorescence within 2 hours after nocodazole removal. These findings suggest that targeting of ntcp to the plasma membrane consists of 2 steps: 1) delivery of ntcp to the region of the plasma membrane via microtubules; and 2) insertion of ntcp into the plasma membrane, in a microfilament- and cAMP-sensitive fashion.

Topics: Actin Cytoskeleton; Animals; Base Sequence; Bile Acids and Salts; Biological Transport; Carcinoma, Hepatocellular; Carrier Proteins; Cell Membrane; Cyclic AMP; Cytochalasin D; Green Fluorescent Proteins; Humans; Kinetics; Liver Neoplasms; Luminescent Proteins; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Open Reading Frames; Organic Anion Transporters, Sodium-Dependent; Protein Structure, Secondary; Rats; Recombinant Fusion Proteins; Symporters; Taurocholic Acid; Transfection; Tritium; Tumor Cells, Cultured

Using micropipette aspiration technique, the authors investigated the viscoelastic properties of human fetal hepatocytes (HFH) and hepatocellular carcinoma (HCC) cells. And further, the effects of two cytoskeleton interferents, cytochalasin D and colchicine on the viscoelastic properties of HFHs were investigated. The results showed: the elastic coefficients K1 and K2 of HCC cells were significantly higher than those of HFHs; the action of cytochalasin D led to an obvious decrease in elastic coefficients K1, K2 as well as the viscous coefficient mu of HFHs; under the action of colchicine, the K1 value of HFHs increased while the K2 and mu values decreased obviously or tended to decrease. A brief discussion was presented about these results.

Topics: Carcinoma, Hepatocellular; Cells, Cultured; Colchicine; Cytochalasin D; Cytoskeleton; Elasticity; Fetus; Humans; Liver; Liver Neoplasms; Tumor Cells, Cultured; Viscosity