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Abstract Background Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved. Methods The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively. Results Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity. Conclusions Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

Background: Tumor necrosis factor alpha (TNF-α) enhances lymphangiogenesis in gallbladder carcinoma (GBC) via activation of nuclear factor (NF-κB)-dependent vascular endothelial growth factor-C (VEGF-C). Receptor-interacting protein 1 (RIP1) is a multifunctional protein in the TNF-α signaling pathway and is highly expressed in GBC. However, whether RIP1 participates in the signaling pathway of TNF-α-mediated VEGF-C expression that enhances lymphangiogenesis in GBC remains unclear. Methods: The RIP1 protein levels in the GBC-SD and NOZ cells upon stimulation with increasing concentrations of TNF-α as indicated was examined using Western blot. Lentiviral RIP1 shRNA and siIκBα were constructed and transduced respectively them into NOZ and GBC-SD cells, and then PcDNA3.1-RIP1 vectors was transduced into siRIP1 cell lines to reverse RIP1 expression. The protein expression of RIP1, inhibitor of NF-κB alpha (IκBα), p-IκBα, TAK1, NF-κB essential modulator were examined through immunoblotting or immunoprecipitation. Moreover, VEGF-C mRNA levels were measured by quantitative real-time polymerase chain reaction, VEGF-C protein levels were measured by immunoblotting and enzyme-linked immunosorbent assay, and VEGF-C promoter and NF-κB activities were quantified using a dual luciferase reporter assay. The association of NF-κB with the VEGF-C promoter was analysed by chromatin immunoprecipitation assay. A three-dimensional coculture method and orthotopic transplantation nude mice model were used to evaluate lymphatic tube-forming and metastasis ability in GBC cells. The expression of RIP1 protein, TNF-α protein and lymphatic vessels in human GBC tissues was examined by immunohistochemistry, and the dependence between RIP1 protein with TNF-α protein and lymphatic vessel density was analysed. Results: TNF-α dose- and time-dependently increased RIP1 protein expression in the GBC-SD and NOZ cells of GBC, and the strongest effect was observed with a concentration of 50 ng/ml. RIP1 is fundamental for TNF-α-mediated NF-κB activation in GBC cells and can regulate TNF-α-mediated VEGF-C expression at the protein and transcriptional levels through the NF-κB pathway. RIP1 can regulate TNF-α-mediated lymphatic tube formation and metastasis in GBC cells both in vitro and vivo. The average optical density of RIP1 was linearly related to that of TNF-α protein and the lymphatic vessel density in GBC tissues. Conclusion: We conclude that RIP1 regulates TNF-α-mediated lymphangiogenesis and lymph node metastasis in GBC by modulating the NF-κB-VEGF-C pathway. [ABSTRACT FROM AUTHOR]

Objective: To investigate the expression of VEGF-C and VEGF-D and their correlations with lymphangiogenesis and angiogenesis in gallbladder carcinoma., Methods: Fifty cases of gallbladder carcinoma with complete clinical and pathological data were analyzed. The expression of VEGF-C and -D, D2-40, CD31 was assayed by immunohistochemical staining, with 10 samples of normal gallbladder tissues away from cancer and 19 samples of chronic cholecystitis as controls, and their correlation with clinicopathological findings were analyzed retrospectively., Results: Thirty-two (64.0%) of the 50 gallbladder cancers were positive for VEGF-C protein expression by immunohistochemistry and the positive rate of VEGF-D protein expression was 62.0% (31/50). The protein expression of VEGF-C and VEGF-D in tumor tissues was significantly higher than that in normal gallbladder tissues away from the tumor (P < 0.05), but no correlation with that in chronic cholecystitis (P < 0.05). The VEGF-C expression correlated with the patient age and lymph node metastasis (both P < 0.05). The VEGF-D expression only correlated with lymph node metastasis (P < 0.05). In the 50 gallbladder cancers, the MLVD was 6.9 + or - 3.6 and the MVD was 36.1 + or - 12.8. The MLVD in both VEGF-C and -D positive groups was significantly higher than that in the negative groups (P = 0.000), and the lymph node metastasis also increased. MVD in both VEGF-C and -D positive groups was higher than that in the negative groups (P < 0.05), and it was also correlated with tumor differentiation (P < 0.05). A significant positive correlation was also found between VEGF-C and VEGF-D expression (r = 0.498, P < 0.01)., Conclusion: VEGF-C and VEGF-D are involved in the lymphangiogenesis and angiogenesis in gallbladder carcinoma, promote lymph node metastasis of the tumor, and both are important in the regulation of lymphangiogenesis and angiogenesis in this cancer. VEGF-C and VEGF-D are of clinical significance in evaluating lymph node metastatic potency and estimation of prognosis in gallbladder carcinoma.

Background: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxification. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated., Methods: hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and amplified. Then hGS mRNA was measured by semi-quantitative RT-PCR; hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH4+ were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry., Results: The expression of hGS mRNA in HepG2/pLNChGS cells (8.306+/-0.336) and HepG2/pLNAFhGS cells (21.358+/-1.716) was much stronger than in control cells (P<0.05), and that in HepG2/pLNAFhGS cells was markedly stronger than in HepG2/pLNChGS cells (P<0.05). The hGS enzymatic activities of HepG2/pLNChGS cells (3.279+/-0.328 U/mg prot) and HepG2/pLNAFhGS cells (4.557+/-0.253 U/mg prot) were higher than those of control cells (P<0.05), and those of HepG2/pLNAFhGS cells were also higher than the activities of HepG2/pLNChGS cells (P<0.05). In addition, the effect of hGS introduction on HepG2 cell proliferation was not significant. The amount of glutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH4+ was higher than in the two control cells (P<0.05). The amount of glutamine synthesis and cell proliferation in the higher concentrations of NH4+ (5 or 10 mmol/L) in HepG2/pLNAFhGS cells increased more than those in HepG2/pLNChGS cells (P<0.05). NH4+ at a high concentration (10 mmol/L) was toxic to HepG2 and HepG2/pLNCX cells, but less toxic to HepG2/pLNChGS and HepG2/pLNAFhGS cells., Conclusion: The constructed hepatocytes (HepG2 cells) with specific high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.