RESUMEN
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.
Asunto(s)
Secuencia de Aminoácidos , Antineoplásicos , Farmacología , Adhesión Celular , Clonación Molecular , Escherichia coli , Genética , Metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos , Farmacología , Péptidos , Genética , Metabolismo , Farmacología , Proteínas Recombinantes , Genética , Células Tumorales CultivadasRESUMEN
<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Expression 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.</p><p><b>RESULTS</b>Expression 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.</p><p><b>CONCLUSION</b>p120tn 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.</p>
Asunto(s)
Humanos , Cadherinas , Metabolismo , Carcinoma Hepatocelular , Metabolismo , Patología , Cateninas , Adhesión Celular , Moléculas de Adhesión Celular , Metabolismo , Línea Celular Tumoral , Membrana Celular , Metabolismo , Movimiento Celular , Núcleo Celular , Metabolismo , Proliferación Celular , Proteínas del Citoesqueleto , Metabolismo , Citosol , Metabolismo , Factor de Crecimiento Epidérmico , Farmacología , Neoplasias Hepáticas , Metabolismo , Patología , Fosfoproteínas , Metabolismo , Fosforilación , Transporte de Proteínas , Transactivadores , Metabolismo , Tirosina , Metabolismo , beta CateninaRESUMEN
<p><b>BACKGROUND</b>PreS/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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>The 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.</p>