Although the virulence of coccoid H. pylori induced by water decrease, coccoid H. pylori still remains a considerable urease activity and the adhering ability to epithelial cells. Furthermore, the flagella, an important component responsible for bacterial movement and infection, were still observed as a cellular structure of coccoid H. pylori under electron microscope. The coccoid H. pylori induced by water is capable of colonizing in gastric mucosa and causing gastrititis in mice.

Vascular endothelial growth factor (VEGF)-C is an important lymphangiogenic factor involved in the lymphangiogenesis of gallbladder carcinoma (GBC) and the lymph node metastasis of the tumor. Tumor necrosis factor (TNF)-α, a key inflammatory cytokine responding to chronic inflammation of GBC, has been reported to stimulate the expression of VEGF-C in some nonneoplastic cells. But whether TNF-α promotes the expression of VEGF-C in GBC has yet to be determined. Therefore, in the present study, the concentration of TNF-α and VEGF-C and the lymphatic vessel density (LVD) in the clinical GBC specimens were analyzed, and a linear correlation was found between the concentration of TNF-α and that of VEGF-C, the lymphatic vessel density (LVD); The transcription and protein level of VEGF-C in NOZ cell line were detected by real-time polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA), and TNF-α enhanced the expression of VEGF-C in NOZ cell lines in a dose and time-dependent manner. Lymphatic tube formation in vitro was observed in a three-dimensional coculture system consisting of HDLECs and NOZ cell lines, and lymphatic vessels of GBC in nude mice model was detected by immunohistochemistry. TNF-α promoted the tube formation of lymphatic endothelial cells in vitro and the lymphangiogenesis of GBC in nude mice; The nuclear factor (NF)-κB binding site on the VEGF-C promoter was identified using Site-directed mutagenesis, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Taken together, TNF-α can upregulate the expression of VEGF-C and promote the lymphangiogenesis of GBC via NF-κB combining with the promoter of VEGF-C.

Background and Aim: Receptor interacting protein(RIP)-1 is thought to have a significant role in inflammation signaling pathways; however, the role of RIP-1 in malignant tumors is largely unknown. Methods: The present study examined the functions and underlying mechanisms of RIP-1 in gallbladder cancer in vitro and in vivo. In this study we determined the expression and role of RIP-1 in 60 clinical specimens from patients with gallbladder cancer and 3 gallbladder cancer cell lines. Using siRNA targeting RIP-1, plasmid vectors (phU6-EGFP-puro/siRIP-1) were constructed and transfected into the gallbladder cells to characterize the biological effect of RIP-1. Results: In vitro experiments indicated that silencing of RIP-1 in NOZ cells significantly suppressed growth and invasion. Furthermore, silencing of RIP-1 affected the RIP1-NF-κB/c-jun(AP-1)-VEGF-C pathways in NOZ cells. Silencing of RIP-1 in vivo inhibited tumor growth in a NOZ cell subcutaneous xenograft model. Immunohistochemstry analysis of the tumor in thesubcutaneous xenograft model also suggested that RIP-1 mediates the expression of VEGF-C. Conclusion: We have elucidated therelationship between RIP-1 overexpression and the growth and invasion of gallbladder cancer from clinical specimens using a xenograft model. We provide evidence that a reduction in the expression of RIP-1 in gallbladder cancer cells can exert inhibitory effects on the ability of cells to grow and invade in vitro. Thus, targeting RIP-1may be useful in the treatment of gallbladder cancer.

Tumor necrosis factor-α (TNF-α) has been suggested to be a putative tumor promoter gene, and autocrine of TNF-α expression has been found in colon cancer and ovarian cancer. As the role of autocrine TNF-α in human gallbladder cancer has not yet been elucidated, the present study examined the expression of TNF-α in gallbladder cancer-derived cell lines. Based on the data, TNF-α mRNA and TNF-α protein expression differed significantly different between the cell lines. In addition, using siRNA targeting TNF-α, the vector, pGPU-GFP-siTNF-α, was constructed and then transfected into the SGC-996 cells (gallbladder cancer cell line) which express high levels of endogenous TNF-α. In vitro experiments indicated that the silencing of TNF-α in the SGC-996 cells significantly suppressed proliferation and invasion. However, apoptosis was not induced by the silencing of TNF-α. Furthermore, we traced the mechanisms underlying these effects and found that the silencing of TNF-α affected the TNF-α-AKT-NF-κB-Bcl-2 pathway in the SGC-996 cells. Our data provide evidence that autocrine TNF-α plays a role as a tumor promoter gene in gallbladder cancer cells, possibly by promoting proliferation and invasion through autocrine mechanisms.

Vascular endothelial growth factor-C (VEGF-C) has a well-defined action on neoplastic lymphangiogenesis and angiogenesis through VEGF receptor-3 (VEGFR-3) and VEGFR-2, respectively, which are generally expressed in endothelial cells. The function of the VEGF-C/receptors pathway in tumor cell types is largely unknown. In this study, we examined the expression and role of VEGF-C/receptors in gallbladder cancer (GBC) cells. We examined the expression of VEGF-C in 50 surgical specimens from gallbladder cancer and three human gallbladder cancer cell lines. Both siRNA and neutralizing antibody to deplete the expression of VEGF-C were used to characterize the biological effect of VEGF-C in GBC NOZ cells. Furthermore, we examined the expression of its receptors, VEGFR-3 and VEGFR-2, in three human GBC cell lines. Our results are as follows: The expression of VEGF-C in the invasive marginal portion was significantly higher than the expression in the central portions. All the three GBC cell lines expressed VEGF-C. Treatment of NOZ cells with VEGF-C siRNA or a neutralizing antibody suppressed cell proliferation and invasion. Moreover, all the three GBC cell lines expressed VEGFR3, but only the NOZ cells expressed VEGFR-2 mRNA. Treatment of NOZ cells with a VEGFR-3 neutralizing antibody suppressed cell invasion, but treatment of NOZ cells with a VEGFR-2 neutralizing antibody suppressed cell proliferation and invasion. In conclusion, GBC cells express both VEGF-C and its receptors. VEGF-C may have a role in the progressive growth and invasion of human GBC through an autocrine mechanism.