phalloidine and Liver-Neoplasms

To establish a method for optical sections of HepG2 human hepatoblastoma cells with confocal laser scanning microscope (CLSM) and to study the spatial structure of filamentous actin (F-actin) in HepG2 cells.. HepG2 cells were stained with FITC-phalloidin that specifically binds F-actin, with propidium iodide (PI) to the nucleus, and scanned with a CLSM to generate optically sectioned images. A series of optical sections taken successively at different focal levels in steps of 0.7 microm were reconstructed with the CLSM reconstruction program.. CLSM images showed that the FITC-stained F-actin was abundant microfilament bundles parallel or netted through the whole cell and its processes. Most F-actin microfilaments extended through the cell from one part toward the other or run through the process. Some microfilaments were attached to the plasma membrane, or formed a structural bridge connecting to the neighboring cells.. A method for double labeling HepG2 human hepatoblastoma cells and CLSM imaging F-actin microfilaments and nuclei by image thin optical sections and spatial structure was developed. It provides a very useful way to study the spatial structure of F-actin.

Topics: Actin Cytoskeleton; Actins; Carcinoma, Hepatocellular; Cell Line, Tumor; Fluorescent Dyes; Humans; Imaging, Three-Dimensional; Liver Neoplasms; Microscopy, Confocal; Phalloidine

Low-power laser irradiation (LPLI) has come into a wide range of use in medical field. Considering basic research, LPLI can enhance DNA synthesis and increases proliferation rate of human cells. But only a few data about the effects of LPLI on human liver or hepatoma cells are available. The cytoskeleton plays important roles in cell function and therefore is implicated in the pathogenesis of many human liver diseases, including malignant tumors. In our previous study, we found the stability of cytokeratin molecules in human hepatocytes was related to the intact microtubule network that was influenced by colchicine. In this study, we are going to search the effect of LPLI on proliferation of human hepatoma cell line HepG2 and J-5 cells. In addition, the stability of cytokeratin and synemin (one of the intermediate filament-associated proteins) were analyzed under the action of LPLI to evaluate the possible mechanism of LPLI effects on proliferation of human hepatoma cells. In experiment, HepG2 and J-5 cells were cultured in 24-well plate for 24 hours. After irradiation by 130 mW diode 808 nm GaAlAs continue wave laser in different time intervals, the cell numbers were counted. Western blot and immunofluorescent staining examined the expression and distribution of PCNA, cytokeratin and synemin. The cell number counting and PCNA expression were evaluated to determine the proliferation. The organization and expression of cytokeratin and synemin were studied to identify the stability of cytoskeleton affected by LPLI. The results revealed that proliferation of HepG2 and J-5 cells was inhibited by LPLI since the cell number and PCNA expression was reduced. Maximal effect was achieved with 90 and 120 seconds of exposure time (of energy density 5.85 J/cm2 and 7.8 J/cm2, respectively) for HepG2 and J-5, respectively. The decreased ratio of cell number by this dose of irradiation was 72% and 66% in HepG2 and J-5 cells, respectively. Besides that, the architecture of intermediate filaments in these cells was disorganized by laser irradiation. The expression of intermediate filament-associated protein, synemin, was also reduced. Two significant findings are raised in this study: (1) Diode 808 nm GaAlAs continuous wave laser has an inhibitory effect on the proliferation of human hepatoma cells line HepG2 and J-5. (2) The mechanism of inhibition might be due to down-regulation of synemin expression and alteration of cytokeratin organization that was caused by laser irr

Topics: Analysis of Variance; Blotting, Western; Carcinoma, Hepatocellular; Cell Count; Cell Line, Tumor; Cell Proliferation; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Humans; In Vitro Techniques; Intermediate Filament Proteins; Keratins; Liver Neoplasms; Low-Level Light Therapy; Phalloidine; Proliferating Cell Nuclear Antigen; Radiation Dosage; Time Factors

Male inbred F344 rats weighing about 150 g were fed continuously a diet containing 0.02% N-2-fluorenylacetamide for 14-15 weeks. The presumptively preneoplastic hepatocytes were transferred to an in vitro system after dispersion by a collagenase-perfusion technique. Sensitivity to phalloidin in terms of formation of cytoplasmic blebs on the cell surface was less in the presumptively preneoplastic hepatocytes than in normal hepatocytes. Among the presumptively preneoplastic hepatocytes, the gamma-glutamyltransferase (GGT)-positive cells were less sensitive to phalloidin than GGT-negative cells, indicating a greater contribution to the decrease in the sensitivity to phalloidin of the presumptively preneoplastic cells.

Topics: 2-Acetylaminofluorene; Animals; Drug Resistance; gamma-Glutamyltransferase; Histocytochemistry; In Vitro Techniques; Liver; Liver Neoplasms; Male; Neoplasms, Experimental; Oligopeptides; Phalloidine; Precancerous Conditions; Rats; Rats, Inbred F344

In vitro treatment of isolated rat hepatocytes with brominated taurodehydrocholic acid (BTC) reduced their sensitivity against phalloidin and inhibited the uptake of phalloidin as well as of cholate in an irreversible and concentration dependent manner. BTC was taken up itself by liver cells; this process was inhibited by 4,4'-diisothiocyano 2,2'-stilbene disulfonate (DIDS). When hepatocytes were incubated with 35S-BTC their plasma membranes contained five labeled protein species with molecular weights of 67,000, 49,000, 38,000, 32,000 and 24,000 as shown by SDS-electrophoresis. No marked difference was observed when isolated plasma membranes from livers were directly treated with the affinity label. DIDS suppressed covalent binding of 35S-BTC to membrane components drastically. Incubation of phalloidin insensitive AS-30D ascites hepatoma cells with 35S-BTC did not result in a chemical modification of the above five proteins. This agrees with an earlier observation that hepatoma cells are unable to take up phalloidin and bile acids (Petzinger et al. 1979; Rufeger and Grundmann 1977; Kroker et al. 1978).

Topics: Affinity Labels; Animals; Carcinoma, Hepatocellular; Cholic Acids; Deoxycholic Acid; In Vitro Techniques; Liver; Liver Neoplasms; Membrane Proteins; Oligopeptides; Phalloidine; Rats; Steroids, Brominated; Sulfur Radioisotopes; Taurodeoxycholic Acid

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Liver Neoplasms; Neoplasm Transplantation; Oligopeptides; Phalloidine; Rats

Topics: Animals; Carcinoma, Hepatocellular; Diethylnitrosamine; Drug Tolerance; Lethal Dose 50; Liver Neoplasms; Neoplasm Transplantation; Oligopeptides; Phalloidine; Rats; Skin