RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway.

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.

Simultaneous recovery of dual pathways for ammonia metabolism do not improve further detoxification of ammonia in HepG2 cells.

BACKGROUND: Key enzyme deficiency in the dual-pathway of ammonia metabolism leads to low detoxification capacity of HepG2 cells. Previously, we established a HepG2/AFhGS cell line with overexpression of human glutamine synthetase (hGS) in pathway 1 and a HepG2/(hArgI+hOTC)4 cell line with overexpression of human arginase I (hArgI) and human ornithine transcarbamylase (hOTC) in pathway 2. The present study aimed to investigate whether simultaneous recovery of the two pathways contributes to the further improvement of ammonia detoxification in HepG2 cells. METHODS: We adopted a recombinant retrovirus carrying the hGS gene to infect HepG2/(hArgI+hOTC)4 cells and selected a new recombinant HepG2 cell line. The capacities of ammonia tolerance and detoxification in cells were detected by biochemical methods. Cell cycle PCR chip was used to assess the changes of gene expression. RESULTS: Introducing hGS into HepG2/(hArgI+hOTC)4 cells did not lead to hGS overexpression, but inhibited hArgI expression. The levels of synthetic glutamine and urea in HepG2/(hArgI+hOTC+AFhGS)1 cells were significantly lower than those in HepG2/(hArgI+hOTC)4 cells when cultured in the medium with 10 and 15 mmol/L glutamate (Glu) and with 60 and 180 mmol/L NH4Cl, respectively. In addition, the comparison of different cell growth showed that HepG2/AFhGS cells significantly lagged behind the other cells by the 5th and 7th day, indicating that introduction of hGS impedes HepG2 cell proliferation. Analysis of the mechanism suggested that the decreased expression of BCL2 played an important role. CONCLUSIONS: This study demonstrated that the recovery of two ammonia metabolic pathways in HepG2 cells is not helpful in increasing ammonia metabolism. The reinforcement of the pathway of urea metabolism is more important and valuable in improving the ammonia metabolism capacity in HepG2 cells.

Therapeutic evaluation of a microbioartificial liver with recombinant HepG2 cells for rats with hepatic failure.

BACKGROUND: HepG2/(ArgI+OTC)4 (previously constructed) is a recombinant human liver cell line with a strong ability to reduce ammonia in vitro. However, its application value ex vivo has not been investigated. OBJECTIVES: To examine the efficacy of HepG2/(ArgI+OTC)4 cells in a micro-bioartificial liver (micro-BAL) device for application ex vivo. METHODS: A simple micro-BAL device containing a microbioreactor and a small-type peristaltic pump was installed. The rats with hepatic failure were randomly divided into three groups (n = 10) and were treated with different micro-BAL loaded HepG2/(ArgI+OTC)4 cells, HepG2 cells and control (without cells), respectively. Changes in the liver and kidney function of the rats were determined before and after the treatment. The lifespan of the rats were observed and recorded. RESULTS: Despite the difference in survival time between experimental groups of rat model was not statistically significant, the capacity of HepG2/(ArgI+OTC)4 cells treatment group for tolerance and detoxifying ammonia was increased much more than that of HepG2 cells (p < 0.05), and other biochemical indicators of HepG2/(ArgI+OTC)4 cells treatment group were also better than that of HepG2 cells treatment group (p < 0.05). CONCLUSIONS: HepG2/(ArgI+OTC)4 cells can provide a better biological support for rats with hepatic failure in a short period of time, and they may be used as a convenient and useful choice for further cell material research of BAL.

N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells.

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 αvß3 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, ß3 and ß1 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.

[Expressions of VEGF-C and VEGF-D and their correlation with lymphangiogenesis and angiogenesis in gallbladder carcinoma].

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.

Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase.

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.

Hepatitis B virus X protein induces apoptosis and cell cycle deregulation through interfering with DNA repair and checkpoint responses.

AIM: To investigate the effects of hepatitis B virus X (HBx) gene on apoptosis and cell cycle in hepatocyte line HL-7702 and to discuss the possible mechanisms in the pathway. METHODS: The recombinant plasmid pcDNA3-X and vector pcDNA3 were transfected into HL-7702 cells and selected by G418 to construct two new cell lines, which were named HL-7702-HBx and HL-7702-con, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to confirm that HBx gene was expressed steadily in the HL-7702-HBx cells. Then apoptosis and cell cycle of the two cells were detected by DNA ladder, flow cytometric analysis, and electronic microscope observation. Apoptosis and cell cycle gene expressions in the two cells were subsequently evaluated by using gene arrays. Some of results were further confirmed by real-time PCR and western blot analysis. RESULTS: RT-PCR and the western blot analysis showed that HL-7702-HBx expressed the HBx gene steadily. Comparedwith the HL-7702-con cells, there was increased apoptosis and accumulation of the S phase in the HL-7702-HBx cells. The gene array analysis indicated that some DNA repair genes (XRCC1, DDB1, etc.) and DNA damage checkpoint-related genes (Cdc47, RAD17, etc.) played roles in the HBx-mediated imbalance of apoptosis and cell cycle. Both cDNA array analysis and real-time RT-PCR showed that mRNA of XRCC1, Cdc47 and RAD17 were upregulated by HBx. Unexpectedly, the western blot analysis revealed that HBx inhibited their protein expression. CONCLUSION: The expression of HBx in HL-7702 cells promoted apoptosis and accumulation of the S phase through the inhibition of DNA repair and checkpoints via post-transcriptional mechanisms.

Transfection and expression of hepatitis B virus x gene and its effect on apoptosis in HL-7702 cells.

AIM: To investigate the effects of hepatitis B virus x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702. METHODS: The reconstituted plasmid pcDNA3-x was established through recombination DNA technique; pcDNA3-X was transfected into HL-7702 cells by lipid-mediated trasfection. Positive clones were screened by G418, and HL-7702/HBx cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells. The apoptosis rate in HL-7702 cells was determined by flow cytometry, TUNEL technology, electronic microscope. At the mean time, pcDNA3-X was transfected transiently into HL-7702 cells, and total RNA from HL-7702 cells was extracted 24, 48, 72, 96 and 120 h after the transient transfection, and semi-quantitative analysis was performed by RT-PCR to detect the expression of HBV X gene. Furthermore, apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times. RESULTS: RT-PCR analysis showed that HBV X gene could be expressed stably in HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HBx cells were much higher than those of HL-7702/pcDNA3 and HL-7702 cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/HBx cells under electronic microscope, but not in HL-7702/pcDNA3 and HL-7702 cells. In the experiment of transient transfection, RT-PCR reveals that X gene was expressed most at 72 h after transfection; and the apoptosis rate reached the highest at the same time. After that, the apoptosis rate was reduced with the decrease of the X gene expression. CONCLUSION: HBV X gene and X protein can promote the apoptosis in hepatocyte. And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.

Cooperative inhibitory effects of antisense oligonucleotide of cell adhesion molecules and cimetidine on cancer cell adhesion.

AIM: To explore the cooperative effects of antisense oligonucleotide (ASON) of cell adhesion molecules and cimetidine on the expression of E-selectin and ICAM-1 in endothelial cells and their adhesion to tumor cells. METHODS: After treatment of endothelial cells with ASON and/or cimetidine and induction with TNF-alpha, the protein and mRNA changes of E-selectin and ICAM-1 in endothelial cells were examined by flow cytometry and RT-PCR, respectively. The adhesion rates of endothelial cells to tumor cells were measured by cell adhesion experiment. RESULTS: In comparison with TNF-alpha inducing group, lipo-ASON and lipo-ASON/cimetidine could significantly decrease the protein and mRNA levels of E-selectin and ICAM-1 in endothelial cells, and lipo-ASON/cimetidine had most significant inhibitory effect on E-selectin expression (from 36.37+/-1.56% to 14.23+/-1.07%, P<0.001). Meanwhile, cimetidine alone could inhibit the expression of E-selectin(36.37+/-1.56% vs 27.2+/-1.31%, P<0.001), but not ICAM-1 (69.34+/-2.50% vs 68.07+/-2.10%,P>0.05) and the two kinds of mRNA, either. Compared with TNF-alpha inducing group, the rate of adhesion was markedly decreased in lipo-E-selectin ASON and lipo-E-selectin ASON/cimetidine treated groups(P<0.05), and lipo-E-selectin ASON/cimetidine worked better than lipo-E-selectin ASON alone except for HepG2/ECV304 group (P<0.05). However, the decrease of adhesion was not significant in lipo-ICAM-1 ASON and lipo-ICAM-1 ASON/cimetidine treated groups except for HepG2/ECV304 group (P>0.05). CONCLUSION: These data demonstrate that ASON in combination with cimetidine in vitro can significantly reduce the adhesion between endothelial cells and hepatic or colorectal cancer cells, which is stronger than ASON or cimetidine alone. This study provides some useful proofs for gene therapy of antiadhesion.

Construction of IL-2 gene-modified human hepatocyte and its cultivation with microcarrier.

AIM: To construct interleukin-2 gene-modified human hepatocyte line (L-02/IL-2) and investigate the changes of the function of liver cells and IL-2 secretion in culture with microcarrier,laying the foundation for further experimentation on hepatocyte transplantation. METHODS: hIL-2 gene was transduced into L-02 hepatocytes by recombinant retroviral vector pLNCIL-2, and the changes of morphology and clonogenicity rate of the transduced cells were observed, the secretion levels of hIL-2 in cultural supernatant were detected by ELISA and NeoR gene was amplified by PCR. The growth of L-02/IL-2, the special biochemistry items and the levels of IL-2 were detected after cultivation with microcarrier. RESULTS: The clonogenicity rate of the L-02/IL-2 cells was lower than that of L-02/Neo cells and L-02 cells. The levels of hIL-2 could reach 32,000 pg/10(6) cells per day and kept secreting for more than ten weeks. NeoR gene segment was respectively obtained by PCR from both L-02/IL-2 and L-02/Neo cell's genomic DNA. At the 6(th) day in culture with microcarrier, the matrix-induced liver cell aggregates were formed, the number of alive L-02/IL-2 cell were 16.8+/-0.53 x 10(6)/flask and the levels of ALB and UREA were 52.54+/-1.28 mg/L and 5.29+/-0.17 mmol/L, respectively. These data had not significantly changed as compared with those of L-02 cells (P>0.05); However, the levels of IL-2 in IL-2/L-02 cells remarkably exceeded that in L-02 cells in the whole culture process (P<0.001). CONCLUSION: The IL-2 gene-modified hepatocyte line has been successfully constructed. The L-02/IL-2 cellular aggregates cultured with microcarrier have a high capacity of IL-2 production as well as protein synthesis and amino acid metabolism.