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.

Tumor 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. The 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. TNF-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. Taken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.