Role And Mechanism Of RIP1 In TNF-α-mediated VEGF-C Expression And Lymphangiogenesis In Gallbladder Cancer

Background and purpose Previous studies has confirmed that cancer development and progression are closely associated with chronic inflammation.We previously reported that tumor necrosis factor alpha(TNF-α),a well-characterized pro-inflammatory factor,can activate NF-κB-dependent vascular endothelial growth factor-C(VEGF-C)to enhance lymphangiogenesis in gallbladder cancer(GBC).Receptor-interacting protein 1(RIP1)is a important molecule that involves in regulating both cell death and cell survival in TNF-α signaling pathway and highly expressed in GBC.However,whether RIP1 participates in the signaling pathway of TNF-α-induced VEGF-C expression that enhances lymphangiogenesis in GBC remains unclear.And our study will further explore the role and mechanism of RIP1 in TNF-α-NF-κB-VEGF-C pathway and lymphangiogenesis in GBC.Methods 1.We first examined the RIP1 protein levels in the GBC-SD and NOZ cells upon stimulation with increasing concentrations of TNF-α as indicated using Western blot,and determined the optimal concentration and time of TNF-α stimulation.2.We further constructed lentiviral RIP1 shRNA,siIκBα and transduced respectively them into NOZ and GBC-SD cells,and then PcDNA3.1-RIP1 vectors was transduced into LV-siRIP1 cell lines to reverse RIP1 expression.3.We explored the role of RIP1 in the TNF-α-NF-κB-VEGF-C pathway by detecting the protein expression of IκBα,p-IκBα,TAK1,NEMO,VEGF-C through immunoblotting or immunoprecipitation.Moreover,VEGF-C protein levels of the supernatant were measured by enzyme-linked immunosorbent assay(ELISA),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(ChIP).4.A three-dimensional coculture method and orthotopic transplantation nude mice model were used to evaluate lymphatic tube-forming and metastasis ability in GBC cells,and RIP1 can regulate TNF-α-mediated lymphatic tube-forming and metastasis in GBC cells in vitro and vivo.5.The expression of RIP1 protein,TNF-α protein and the lymphatic vessels in human GBC tissues was examined by immunohistochemistry,and we analyse the dependence between RIP1 protein with TNF-α protein and lymphatic vessels density(LVD).Results 1.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.2.RIP1 is indispensable for TNF-α-mediated activation of NF-κB in vitro.This enhancement by TNF-α-mediated activation of NF-κB was markedly abolished in RIP1-knockdown GBC-SD and NOZ cells.And the co-transfection of PcDNA3.1-RIP1 and siRIP1 reversed the abolished effect of RIP1 knockdown on the TNF-α-mediated activation of NF-κB.Importantly,when IκBα was co-knocked down with RIP1,the inhibitory effect of RIP1 knockdown on the TNF-α-mediated activation of NF-κB was again reversed.RIP1 was coprecipitated with TAK1 and NEMO that leads to IκBα(inhibitor of NF-κB alpha)phosphorylation and degradation and induction of NF-κB.3.RIP1 regulates TNF-α-mediated VEGF-C expression of GBC cells through the NF-κB pathway.We confirmed that VEGF-C protein expression and promoter-driven luciferase activity were markedly enhanced in LV-control(NC)(or control(NC))cell groups upon TNF-α stimulation.This enhancement by TNF-α was markedly abolished in LV-siRIP1(or siRIP1)cell groups or treatment with Bay-11-7082,an NF-κB(p65)inhibitor.And the PcDNA3.1-RIP1/LV-siRIP1 cell groups reversed the abolished effect of RIP1 knockdown on the TNF-α-mediated the expression of VEGF-C protein.Moreover,TNF-α enhanced the association of NF-κB with the VEGF-C promoter region in LV-control(NC)cell groups,and this enhancement by TNF-α was markedly impaired in LV-siRIP1 cell groups.4.RIP1 regulates TNF-α-mediated lymphangiogenesis in HDLECs in vitro.TNF-α markedly enhanced the tube-forming ability of HDLECs in the LV-control(NC)cell groups,and this enhancement by TNF-α was markedly impaired in LV-siRIP1 cell groups.PcDNA3.1-RIP1/LV-siRIP1 cell groups could balance the impaired effect of RIP1 knockdown on TNF-α-mediated lymphangiogenesis in HDLECs.Moreover,the supplementation of VEGF-C protein reversed the impairment of TNF-α-mediated tube-forming ability in the LV-siRIP1 cell groups.These results indicated that RIP1 has the ability to regulate TNF-α-mediated lymphangiogenesis,and this ability is related to VEGF-C levels.5.RIP1 regulates TNF-α-mediated lymphangiogenesis promoting lymphatic metastasis in GBC in vivo.we generated a murine orthotopic transplantation model of GBC using three established cell groups.The lymph node metastasis rate upon TNF-α stimulation was decreased in LV-siRIP1 cell group.The lymphatic vessel number(LVN)was markedly increased in the control and LV-control(NC)cell groups upon TNF-α stimulation,and this increase induced by TNF-α was markedly abolished in LV-siRIP1 cell group.6.We collected thirty gallbladder cancer samples and examined the expression of RIP1,TNF-α and LVD in clinical GBC specimens.The average optical density of RIP1 was linearly related to that of TNF-α in GBC tissues.In addition,linear dependence was also detected between the average optical density of RIP1 and LVD in GBC tissues.Conclusion 1.TNF-α dose-and time-dependently increased RIP1 protein expression in the GBC-SD and NOZ cells of GBC.2.RIP1 is indispensable for TNF-α-mediated activation of NF-κB in the GBC-SD and NOZ cells of GBC.3.RIP1 regulates TNF-α-mediated VEGF-C expression leading to the enhancement of lymphangiogenesis and lymphatic metastasis in GBC by modulating the NF-κB pathway.