The Study On Curcumin And TNF-a Regulating The Expression Of Notch1 In Raji Cell And Modulating Angiogenesis

Notch signaling plays an important role in diverse cellular and developmental processes, including differentiation, proliferation, survival, and apoptosis. Notch signaling plays also a critical role in vasculogenesis and angiogenesis. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. VEGF can induce gene expression of Notch1 and its ligand in human arterial endothelial cells.Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. Angiogenesis is essential for cancer development and growth. Inhibiting angiogenesis would therefore seem to be a reasonable approach to prevent or treat cancer. Vascular endothelial growth factor (VEGF) is the key mediator of angiogenesis in cancer, in which it is up-regulated by oncogene expression, a variety of growth factors and also hypoxia. The transcription factor nuclear factor kappa B (NF-κB) can intervene in oncogenesis by virtue of its capacity to regulate the expression of a plethora of genes that modulate apoptosis, and cell survival as well as proliferation, inflammation, tumor metastasis and angiogenesis. Activation or inactivation of transcription factors promote cancer development, cell survival and proliferation and induce tumor angiogenesis.Several lines of evidence implicate NF-κB activity in regulating endothelial cell phenotype during inflammation and angiogenesis. NF-κB is involved in the upregulation of VEGF and its activity is associated with the high expression of VEGF mRNA .TNF-αhas previously been shown to exert its angiogenic effect on human microvascular cells by modulating VEGF and IL-8 expression. TNF-α-induced angiogenesis is also mediated through up-regulation of various angiogenic factors such as VEGF.Human B cells Burkitt's lymphoma Raji cell are resistant to nuclear apoptosis induced by various stimuli. Curcumin, the yellow pigment in the spice turmeric, has been shown to exhibit chemopreventive and growth inhibitory activities against multiple tumor cell lines. In this study, we investigated the expressions of Notch1 in Raji and a human umbilical vein endothelial cell (HUVECs)-derived cell line (ECV304), IκB-αand P65/ NF-κB in Raji cells treated by curcumin and TNF-α,and network formation of endothelial cells on Matrigel . In order to elucidate the mechanisms controlling the process of lymphoma neovascularization. .PART I The Study on curcumin and TNF-αregulating the expression of VEGF in Raji cells and Modulating Angiogenesis in ECV304 cellsBackground and objective Curcumin, the yellow pigment in the spice turmeric, has been shown to exhibit chemopreventive and growth inhibitory activities against multiple tumor cell lines,and inhibit tumor angiogenesis.Vascular endothelial growth factor (VEGF) is the key mediator of angiogenesis in cancer. Tumour necrosis factor-alpha (TNF-α) has previously been shown to exert its angiogenic effect on human microvascular cells by modulating VEGF expression. But the effects of curcumin and TNF-αon the protein VEGF of Raji cells and network formation of endothelial cells on Matrigel are unknown. To better understand the possibilities of antiangiogenic tumor therapy and to assess possible side effects, we investigated the effect of TNF-αand curcumin on the expression of vascular endothelial growth factor (VEGF) in Raji cell line and their effect on angiogenesis in a human umbilical vein endothelial cell (HUVECs)-derived cell line (ECV304).The aim of this study was to elucidate potential mechanisms controlling tumor neovascularization.Methods Raji cell line was treated with curcumin at different dose for virous times ,and MTT method was performed to assay the proliferation inhibition of Raji cells.VEGF secreted by Raji cell line was determined by ELISA. Angiogenesis was tested by network formation of endothelial cells on Matrigel. Levels of VEGF mRNA in Raji cell was determined by RT-PCR.Results The proliferation of Raji cell was inhibited by curcumin at a dose and time dependent manner, and the volume of inhinbition IC50 was 25μmol/L at 24h in Raji cell. Secretion of VEGF by Raji cell was increased by TNF-alpha treatment and suppressed by curcumin (P < 0.01). The mRNA expression of VEGF165 and VEGF121 (containing 165 and 121 amino acid residues, respectively) were detected in any fractions. TNF-αaugmented the expression of VEGF165 and VEGF121 mRNA and curcumin reduced the expression (P < 0.01). No networks or cords formed in control and curcumin groups. There was tube formation on matrigel in the supernatants of the Raji culture group and the supernatants groups treated by VEGF group and TNF-αin Raji cell.Conclusion Expressions of VEGF mRNA in Raji cell was increased by TNF-αand suppressed by curcumin. VEGF and TNF-αcan induce angiogenesis, and curcumin can inhibit angiogenesis in ECV304 cells.PART II The study on curcumin and TNF-αregulating the expression of Notch1 in Raji and ECV304 cell linesBackground and objective Growing evidence also suggests involvement of Notch signaling in the regulation of vascular formation. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. Notch signaling plays a critical role in tumor angiogenesis and metastasis. Notch activation can be oncogenic VEGF can induce gene expression of Notch1 and its ligand in human arterial endothelial cells. To better understand a novel mechanism for the oncogenic capacity of Notch1 in Raji cell line, and a role in tumor angiogenesis and metastasis, we investigate the effect of curcumin and TNF-αon the expression of Notch1 in Raji and ECV304 cell lines,In order to elucidate the effect of Notch1 controlling the process of lymphoma neovascularization.Methods Levels of Notch1 mRNA in Raji and ECV304 cell lines was determined by RT-PCR.The changes of expressions of Notch1 protein was tested by flowcytometer.Results The mRNA expression of Notch1 was detected in Raji and ECV304 cell lines. TNF-αaugmented the expression of Notch1 mRNA and curcumin reduced the expression (P < 0.01). Flowcytometer showed the level of Notch1 protein was increased induced by TNF-αand decreased by curcumin.Conclusion Notch1 protein was expressed in Raji and ECV304 cell lines. Curcumin and TNF-αcan play a role through affecting Notch1 signaling in Raji and ECV304 cell lines .Notch1 signaling might be a potential new target for treatment.PART III The study on curcumin and TNF-αregulating the expression of IκB-αand NF-κB/p65 in Raji cellBackground and objective The transcription factor nuclear factor kappa B (NF-κB) can modulate tumor metastasis and angiogenesis.NF-κB is involved in the upregulation of VEGF and its activity is associated with the high expression of VEGF mRNA. Curcumin inhibits constitutive active NF-κB, leading to suppression of cell growth in leukemia cells.In this study we investigate the effect of curcumin and TNF-αon the expressions of IkappaBalpha(IκB-α) and NF-κB/p65 to determine whether curcumin can regulate the expression of nuclear factor kappa B (NF-kappaB) binding protein IκB-αin Raji human B lymphoma cells.Methods The expression of IκB-αand NF-κB/p65 proteins in Raji cell were investigated by using Western blotting and flowcytometer.Results The expression of IκB-αwas downregulated by curcumin in Raji cells. After treatment with TNF-αor curcumin plus TNF-αfor 15 min, there was a substantial reduction in the amount of IκB-αprotein.There was p65 protein expression in the nucleus after treatment with TNF-a for 15 min, but no p65 protein was found in control and curcumin-treated groups Conclusion Curcumin inhibited the expression of IκB-αprotein in Raji cell.Curcumin can play a role through affecting NF-κB signaling.