Feifei She

Vascular endothelial growth factor-C promotes the growth and invasion of gallbladder cancer via an autocrine mechanism

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.

Vascular endothelial growth factor-D promotes growth, lymphangiogenesis and lymphatic metastasis in gallbladder cancer.

Lymph node metastasis is a major prognostic factor for patients with gallbladder cancer (GBC), and greater understanding of the molecule mechanism of lymph node metastasis in GBC is needed to improve prognosis. VEGF-D has been implicated in the control of lymphangiogenesis in many carcinomas, but the biological function of VEGF-D in human GBC remains unclear. In this study, we analyzed the role of the VEGF-D in human GBC cells and addressed the functional role of VEGF-D using a xenograft mouse model. We examined the expression of VEGF-D in three human gallbladder cancer cell lines. A lentivirus-based effective VEGF-D siRNA vector was infected into GBC NOZ cells. The effect of VEGF-D siRNA on GBC NOZ cells was investigated by cell proliferation assay and invasion assay. Furthermore, we examined the role of VEGF-D-SiRNA on GBC NOZ cells in the mice of subcutaneous and orthotopic xenograft tumor. Our results are as follows: VEGF-D mRNA and protein were expressed in all three GBC cell lines (GBC-SD, NOZ, and SGC-996). We successfully selected D-3/siRNA as the most effective siRNA to silence VEGF-D expression after four VEGF-D siRNA plasmid transfection in NOZ cells. VEGF-D mRNA and protein expression were suppressed by lentivirus-mediated D-3/siRNA. D-3-RNAi-LV inhibited NOZ cells proliferation and invasion ability in vitro. D-3-RNAi-LV inhibited tumor growth and lymphangiogenesis in the NOZ cell subcutaneous xenograft model. D-3-RNAi-LV inhibited lymphangiogenesis and lymphatic metastasis in the NOZ cell orthotopic xenograft model. Furthermore, D-3-RNAi-LV inhibited tumor ascites and hepatic invasion in the NOZ cell orthotopic xenograft model. In conclusion, VEGF-D is involved and plays an important role in GBC progression, suggesting that VEGF-D may be a potential molecular target in the treatment of GBC.

Tumor necrosis factor-α promotes the lymphangiogenesis of gallbladder carcinoma through nuclear factor-κB-mediated upregulation of vascular endothelial growth factor-C.

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.

TNF-α promotes gallbladder cancer cell growth and invasion through autocrine mechanisms

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.

Expression of the RIP-1 Gene and its Role in Growth and Invasion of Human Gallbladder Carcinoma

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.

Establishment of and Comparison between Orthotopic Xenograft and Subcutaneous Xenograft Models of Gallbladder Carcinoma

BACKGROUND Gallbladder carcinoma (GBC) is the most common carcinoma of the biliary system. Among its research models, orthotopic xenograft models, important research tools, have been rarely reported in the literature however. AIM To explore establishment of an orthotopic xenograft model and to evaluate the advantage and disadvantage as compared with other models. MATERIALS AND METHODS Subcutaneous xenograft and orthotopic xenograft models of gallbladder carcinoma in nude mice were established and compared with human gallbladder carcinomas. RESULTS For the orthotopic xenograft model and clinical gallbladder carcinomas, the lymph node metastatic rates were 69.2% and 53.3% (p>0.05); ascites generation rates, 38.5% and 11.7%(p<0.05); liver invasive rates, 100% and 61.7%(p<0.05); and lymphatic vessel densities (LVD), 10.4 ± 3.02 and 8.77 ± 2.92 (p>0.05), respectively. In the subcutaneous xenograft model, no evidence of ascites generation, lymph node metastasis and liver metastasis were found, and its LVD was lower (4.56 ± 1.53, p<0.05). CONCLUSIONS Compared with the subcutaneous xenograft model, the orthotopic xenograft model better simulates clinical gallbladder carcinoma in terms of metastasis and invasion, which may be attributed to the difference in microenvironment and LVD.

Rescued expression of WIF-1 in gallbladder cancer inhibits tumor growth and induces tumor cell apoptosis with altered expression of proteins

As a highly conserved metabolic pathway, the Wnt signaling pathway is involved in cell differentiation, proliferation and several other processes. In normal cells, this pathway is suppressed, and abnormal activation is often associated with tumor occurrence and development. In certain types of tumor, Wnt inhibitory factor 1 (WIF-1), an inhibitor of the Wnt pathway, inhibits tumor growth. However, the effect of the expression of WIF-1 on gallbladder cancer remains to be fully elucidated. In the current study, reverse transcription-quantitative polymerase chain reaction and western blotting were conducted. The present study demonstrated that, in gallbladder cancer, WIF-1 generally exhibited low levels of expression as a result of gene promoter methylation. Treatment with the drug, 5-aza-2-deoxycytidine, increased the expression of WIF-1 in the GBC-SD gallbladder cell line. In addition, a WIF-1-expression plasmid was transfected into GBC-SD cells, and it was found that cell proliferation, invasion and metastasis declined significantly, whereas the apoptotic rate increased. A nude mouse tumor transplantation experiment showed that the oncogenicity of the GBC-SD cells expressing WIF-1 was substantially lower, compared with that of the untransfected GBC-SD cells and of GBD-SD cells expressing the control plasmid. A fluorescent protein chip experiment showed that the restored expression of WIF-1 affected the expression of several cellular proteins. These alterations may explain the different biological behavior of the tumor cells expressing WIF-1. As an effective inhibitory factor of the Wnt signaling pathway, WIF-1 modulated the expression of proteins controlling the proliferation, apoptosis and metastasis of gallbladder tumor cells, thus suppressing the tumor. Therefore, WIF-1 may be an effective treatment target for gallbladder cancer.

CCR7 mediates the TNF-α-induced lymphatic metastasis of gallbladder cancer through the “ERK1/2 - AP-1” and “JNK - AP-1” pathways

BackgroundCC-chemokine receptor 7 (CCR7), which plays an important role in cell directional movement, is highly expressed in various cancers and positively related to lymph node metastasis. The inflammatory cytokine tumour necrosis factor (TNF)-α promotes tumour progression and lymph node metastasis in gallbladder cancer (GBC). However, the expression of CCR7 in GBC is unclear, and its role in the TNF-α-induced lymphatic metastasis of GBC requires further research.MethodsThe expression of CCR7 in clinical samples was detected by immunohistochemistry, and the relationship between CCR7 and clinicopathological factors or the TNF-α level of the bile was analyzed. After treatment with various concentrations of TNF-α, CCR7 expression in GBC cell lines was measured by Western blotting. The relative luciferase reporter assay, site-directed mutagenesis and chromatin immunoprecipitation were used to analyze the promoter activity and transcriptional regulation of CCR7. MAPKs inhibitors were used to explore the upstream signalling molecules of AP-1. We established a NOZ cell line stably expressing lentiviral CCR7 shRNA that effectively silenced the expression of CCR7, and to determine the role of TNF-α - CCR7 axis in the migration of GBC cells to the lymphatic system by transwell assays and animal experiments.ResultsCCR7 was highly expressed in GBC samples. Higher expression of CCR7 was associated with American Joint Committee on Cancer (AJCC) staging and lymph node metastasis. Moreover, we found that CCR7 expression in GBC tissue was positively correlated with the levels of TNF-α in the bile, and that TNF-α enhanced the promoter activity and protein expression of CCR7 through the “ERK1/2-AP-1” and “JNK-AP-1” pathways. Finally, we revealed that TNF-α could promote GBC cell migration to lymphatic endothelial cells or lymph nodes through upregulation of CCR7 in vitro and in vivo.ConclusionsOur study suggests that CCR7 is highly expressed in GBC, and mediates the TNF-α-induced lymphatic metastasis of GBC through the “TNF-α - ERK1/2 - AP-1 - CCR7” and “TNF-α - JNK - AP-1 - CCR7” pathways.

TNF-alpha promotes lymphangiogenesis and lymphatic metastasis of gallbladder cancer through the ERK1/2/AP-1/VEGF-D pathway

BackgroundTumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined.MethodsThe expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment.ResultsTNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo.ConclusionTaken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.

WNT inhibitory factor 1 promoter hypermethylation is an early event during gallbladder cancer tumorigenesis that predicts poor survival

Gallbladder cancer (GBC) is the most common malignant tumor in the human biliary tract, but the lack of a marker for timely diagnosis leads to an extremely poor prognosis. In this study, we assessed CpG sites in the WIF-1 promoter using bisulfite sequencing PCR and methylation-specific PCR to detect methylation in gallbladder cancer and cholecystitis tissues. WIF-1 promoter methylation was present in 36 of 50 (72.0%) gallbladder cancers but only 5 of 20 (25.0%) cholecystitis tissues (P=0.000<0.05), suggesting that WIF-1 promoter methylation might participate in the malignant transformation of cholecystitis into gallbladder cancer. WIF-1 methylation was negatively correlated with WIF-1 protein expression by immunohistochemistry, demonstrating that WIF-1 expression is downregulated by promoter hypermethylation. We analyzed the prognosis of 50 GBC patients with 5years of follow-up. Univariate analysis revealed that patients with hypermethylated WIF-1 exhibited worse overall survival than those with hypomethylated WIF-1 (χ2=8.137, P=0.004<0.05). Furthermore, multivariate analysis revealed that WIF-1 methylation was an independent prognostic factor for 5-year overall survival (P=0.011). Therefore, WIF-1 methylation is a candidate as a marker for early gallbladder cancer diagnosis and prognosis.