Carcinoma, Hepatocellular ; pathology ; Humans ; Liver Neoplasms ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Peptide Fragments ; pharmacology ; Peptides ; pharmacology ; Tumor Cells, Cultured

BACKGROUNDPreS/S gene was derived from hepatitis B virus (HBV) integration fragment of human hepatocellular carcinoma genome, containing the promoter of preS/S and the C terminal truncated preS/S open reading frame. PreS/S protein may have important roles in processing hepatoma in some HBV-infected patients. The aim of the study was to study the activity of HBV preS/S protein on proliferating cell nuclear antigen (PCNA) promoter and to localize compartment of the preS/S protein in the liver cell line L02.

METHODSThe authors studied the effect of the 3 -truncated preS/S on human PCNA promoter by co-transfecting the expression plasmids of luciferase reporter gene, used the immunohistochemical method to localize the preS/S protein.

RESULTSThe expression product of the plasmid, pKSH7C-HpaI which contained the 3 -truncated preS/S and the flanking cellular sequences, stimulated the expression of PCNA promoter dose dependently,and its effect was 0.5 folds higher than control. Immunohistochemistry showed that the preS/S protein located in the cytosolic region of the liver cell line L02.

CONCLUSIONSThe HBV preS/S protein could stimulate the PCNA promoter of the liver cell, its effect was not direct, which suggests that the effect of preS/S protein on PCNA promoter was probably through the cell signal transduction pathway.

Carcinoma, Hepatocellular ; genetics ; virology ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; genetics ; virology ; Proliferating Cell Nuclear Antigen ; genetics ; Promoter Regions, Genetic ; Protein Precursors ; genetics ; Tumor Cells, Cultured ; Virus Integration

To block tumor cell adhesion, inhibit tumor metastasis and recurrence, the anti-adhesion peptide-trimeric beta peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK, beta3) was designed. The DNA fragment of beta3 was cloned into expression vector pET-His and the fusion protein His-beta3 was expressed in E. coli. BL21(DE3)plysS. After 1.5 hours' induction with IPTG, His-beta3 peptide was expressed significantly amounting to 10% of the insoluble proteins and 4% of the total proteins. 20mg of beta3 peptide was obtained from one litter culture medium after purification by using metal-chelating sepharose 6B FF. The purity of beta3 is 92.2% according to Gel-Pro analysis. The anti-adhesion effects of beta3 peptide, beta1 peptide (DLYYLMDLSYSMK) and GRGDS on the hepatocellular carcinoma cell line SMMC-7721 and the high metastasis hepatocellular carcinoma cell line HCCLM6 were studied. The result showed the beta3 blocked the adhesion of HCCLM6 cells and SMMC-7721 cells to fibronectin (FN) specifically. The inhibition effect was dose-dependent and time-dependent and the inhibition rate of beta3 was higher than three times concentration of beta1 and GRGDS. This suggested that pET-His-beta3/BL21(DE3)plysS was a suitable expression system for beta3, and the expressed beta3 specially inhibited the adhesion of cancer cells.
Amino Acid Sequence ; Antineoplastic Agents ; pharmacology ; Cell Adhesion ; drug effects ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Peptide Fragments ; pharmacology ; Peptides ; genetics ; metabolism ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect of catenin p120 (p120ctn) translocation on the malignant features of hepatocellular carcinoma and its interrelation with beta-catenin in E-cadherin-mediated cell signaling.

METHODSExpression and translocation of p120ctn, tyrosine phosphorylation, and its binding capacity to E-cadherin were detected by DNA transfection, immunoblotting and immunoprecipitation. Cellular localization of p120ctn and beta-catenin was detected by immunofluorescent microscopy. Cell adhesion, cell migration and cell proliferation were also studied.

RESULTSExpression of p120ctn increased after cells transfected with p120ctn isoform 3A, and it was located mainly at cell-cell contact region. Its binding to E-cadherin was enhanced. After EGF stimulation, tyrosine phosphorylation of p120ctn was increased, membrane expression of p120ctn and beta-catenin was decreased while cytosol expression was increased. It was translocated into the nucleus, cell adhesiveness was increased but mobility decreased. With over-expression of p120ctn, beta-catenin was recruited by nucleus export. Cell proliferation was reduced but it was increased after EGF treatment.

CONCLUSIONp120tn plays an important role in cell adhesion, migration and proliferation of hepatocellular carcinoma, and its tyrosine phosphorylation might contribute to this mechanism. There might be a competitive relationship between p120ctn and beta-catenin.

Cadherins ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Catenins ; Cell Adhesion ; Cell Adhesion Molecules ; metabolism ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Cell Movement ; Cell Nucleus ; metabolism ; Cell Proliferation ; Cytoskeletal Proteins ; metabolism ; Cytosol ; metabolism ; Epidermal Growth Factor ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Phosphoproteins ; metabolism ; Phosphorylation ; Protein Transport ; Trans-Activators ; metabolism ; Tyrosine ; metabolism ; beta Catenin