To Study The Mechanism Of Tumor Necrosis Factor-α Induces Vascular Endothelial Growth Factor In MCF-7 Cells

The cytokine TNF-αhas paradoxical role in the evolution and treatment of malignant disease.High dose local administration of TNF-αselectively destroys tumor blood vessels and has powerful anti-cancer action.However,TNF-αappears to act as an endogenous tumor promoter when chronically produced in microenvironment of tumor.It has been proved that TNF-αis a mediator of angiogenesis.The expression of vascular endothelial growth factor(VEGF)has been implicated in breast cancer angiogenesis.And the promoter region of the VEGF gene contains AP-1 binding motifs.Although some associations have been implicated between TNF-αand VEGF,how TNF-αregulates VEGF in breast cancer is not clear.AP-1 is known to be involved in TNF-αreceptor signaling,enabling TNF-αto alter the expression of many genes,some of which may be important in tumor development.Analysis of the VEGF promoter region reveals several potential binding sites for the transcription factors AP-1. The mitogen-activated protein kinase(MAPK)cascade is AP-1 upstream. AP-1 can be activated by any or all three of the MAPK pathways, increasing its sensitivity to a variety of external signals.The MAPK family of protein kinases includes the extracellar signal-regulated kinases (ERK),the c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK),and p38.Using a luciferase reporter assay,we demonstrated that AP-1 activity was significantly upregulated by TNF-α.Using western-blot,we proved that the expression levels of c-Jun,c-Fos,JunB were induced by TNF-α, and the phosphorylation levels of c-Jun were also increased by treatment of TNF-α,but other AP-1 family members such as Fra-1,Fra-2 and JunD were not changed significantly.Using immunoprecipitation assay,we found that the activation of AP-1 family member appeared as c-jun-c-jun and c-jun-JunB homodimers.After incubation of the breast cancer cell line MCF-7 with TNF-αfor different periods of time,using RT-PCR and western-blot,we detected the increases of VEGF mRNA and protein levels.Blocking of JNK pathway with specific inhibitors significantly reduced TNF-αinduced VEGF expression,indicating that JNK pathway may be involved in TNF-αinduced AP-1 activation and VEGF expression in MCF-7 cells.Finally,we demonstrated that phosphated c-jun(p-c-jun)binded to the VEGF promoter and regulated VEGF directly using the chromatin immunoprecipitation(CHIP)assay.In conclusion,in the present study,we found that TNF-α.induced VEGF expression in breast cancer cell line MCF-7 via p-c-Jun,in a JNK and AP- 1 pathway dependent manner.