Tumor necrosis factor-α promotes the lymphangiogenesis of gallbladder carcinoma through nuclear factor-κB-mediated upregulation of vascular endothelial growth factor-C.
Bottom Line: 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).
Affiliation: Department of Hepatobiliary Surgery, The Affiliated Union Hospital of Fujian Medical University, Fuzhou, China; Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fuzhou, China.Show MeSH
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Mentions: To study the effect of TNF-α on lymphangiogenesis in vivo, we established three orhtotopic xenograft models of GBC in nude mice, which were respectively inoculated the abovementioned three NOZ cell lines: NOZ, NOZ/Ctrl and NOZ/VEGF-C siRNA in nude mice. Two weeks after inoculation, a daily dose of TNF-α (2 μg/kg) was injected into the abdominal cavity for 2 weeks. The lymphatic vessels of tumors were observed by immunohistochemistry. Since the D2-40 antibody against lymphatic vessel marker podoplanin does not recognize the murine antigen, mouse lymphatic vessels were detected using LYVE-1.31 The intratumoral lymphatic vessels of the tumors in the nude mice models were hardly to be observed, so all of the lymphatic vessels are from peritumor. As shown in Figure5, TNF-α increased the LVD (14.33 ± 1.35 vs 9.89 ± 0.80, P < 0.05) of orhtotopic xenograft tumors. In the VEGF-C siRNA group, TNF-α also increased the LVD (7.44 ± 0.44 vs 5.89 ± 0.29, P < 0.05) of the tumors, but the increase rate (26.37% ± 4.85%) was lower than that of control (44.19% ± 3.85%) (P < 0.05). Meanwhile, the lymph node metastasis rates of orhtotopic xenograft tumors were increased by TNF-α (Table2).
Affiliation: Department of Hepatobiliary Surgery, The Affiliated Union Hospital of Fujian Medical University, Fuzhou, China; Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fuzhou, China.