Blockade Of Notch Signaling With γ-Secretase Inhibitor In Glioma Antiangiogenesis Therapy

Part 1:Expression and significance of Delta-like 4 in gliomaObjectives:As one of Notch legands, Delta-like 4 (DLL4) was mainly expressed on endothelial cells. The objective of our research was to study the expression of Delta-like 4 (DLL4), as well as the correlation with microvessel density (MVD) in glioma.Methods:40 cases of glioma (classⅠ,12 cases; classⅡ,13 cases; classⅢ～Ⅳ,15 cases) were immunohistochemistically analyzed with monoclone anti-DLL4 antibody and polyclone anti-CD31 antibody, expression level of DLL4 was measured with Image-Pro Plus 6.0 via optical density analysis. MVD was calculated.Results:OD of classⅠ,Ⅱ,Ⅲ～Ⅳwere 514.71±179.56,961.97±88.53, 1125.31±225.35, respectly (p<0.01). MVD of classⅠ,Ⅱ,Ⅲ～Ⅳwere 24.75±10.33, 57.62±12.45,114.60±25.28, respectly (p<0.01). Correlation coefficient between MVD and OD of DLL4 was 0.68 (p<0.01).Conclusions:The level of DLL4 closely correlates with glioma histological grade and MVD. It is significant to detect the expression level of DLL4 to evaluate the biological behavior and blood supply of glioma. Considering the difficulty of DLL4 inhibition in clinical practice and the specific blockade of y-secretase inhibitor on DLL4 receptor Notch, we will investigate the effects and mechanisms ofγ-secretase inhibitor in glioma antiangiogenic therapy.Part 2:Effects ofγ-secretase inhibitor on endothelial cellsObjective:Previous researches had documented that Notch signaling had important role in angiogenesis and vascular function development. The objective of this study was to investigate the regulation of Vascular Endothelial Growth Factor Receptor (VEGFR) and endothelial cell proliferation byγ-secretase inhibitor. Materials and Methods:1×106 H5V cells were cultured with DAPT (final concentration was 5μmol/L) or equivalent amount of DMSO as a negative control. Total protein or RNA was harvested 6 h later. Protein and mRNA expression of VEGFR1, VEGFR2 and VEGFR3 were measured by western blot and realtime-PCR, respectly. For cell proliferation assays, H5V cells were seeded in 24-well plates at a seeding density of 1×104/cm2. Media was supplemented with DMSO, DMSO+ VEGF-A164, DAPT, or DAPT+VEGF-A164. The final concentration of DAPT and VEGF-A164 was 5μmol/L and lOng/ml, respectly. Media was changed every 24 h and cells were counted 72 h later with a coulter counter.Results:The protein levels of NICD and Hes-1 declined significantly under 5μmol/L DAPT treatment, which meant Notch signaling was effectually blocked; VEGFR1 expression level was decreased by DAPT, and the mRNA level of VEGFR1 was down-regulated to 29% compared with DMSO group; protein of VEGDR-2 were significantly elevated by DAPT, and mRNA level of VEGFR2 in DAPT-treated groups was increased to 3.16 fold compared with control groups; the expression of VEGFR3 between DAPT treated cell and DMSO treated had no significant difference. DAPT enhanced endothelial cell proliferation in presence of VEGF compared with control groups or VEGF groups, but exerted no effect if used alone.Conclusions:γ-secretase inhibitor could effectually block Notch signaling; y-secretase inhibitor regulates VEGF pathway via altering VEGF receptors expression; y-secretase inhibitor enhances the effect of endothelial cell proliferation induced by VEGF.Part 3:y-secretase inhibitor impresses vessel development and suppresses glioma growthPurpose:Glioma is a common intracranial malignant tumor, the objective of this study was to investigate the role of y-secretase inhibitor in antiangiogenesis therapy for glioma.Experimental Design:U87MG cells (10×106) were suspended in 200μ1 serum-free DMEM and injected subcutaneously into the flank of SCID mice. One week later, mice were treated with daily subcutaneously injections of DAPT (100mg/kg/day), or equivalent amount of DMSO as a negative control. Tumor growth was monitored every 5 days. All mice were sacrificed 25 days later, and the tumors were excised. Sections of tissues were immunohistochemistry analysis for the expression level of VEGF,VEGFR1,VEGFR2; immunostained with anti-CD31 antibody to measure vessel density,vessel size; 15mg/kg Hoechst 33342 (Sigma Aldrich) in PBS was injected via a lateral tail vein to tumor-bearing mice. One minute later, the mice were sacrificed and Hoechst 33342 fluorescence signals were recorded to measure tumor perfusion ratio; To measure tumor hypoxia, Hypoxyprobe-1 (HPI, Inc; 60mg/kg) was injected intraperitoneally one hour before sacrificed. Monoclonal anti-Hypoxyprobe antibody was used to analyze tumor hypoxia ratio. The fluorescence signals were also recorded and analyzed with Image-Pro Plus 6.0 software.Results:Compared with control tumors, VEGFR1 expression was reduced, but VEGFR2 expression was increased in DAPT treated tumors. On the contrary, VEGF levels had no significant difference between DAPT and vehicle groups in vivo. DAPT treated tumors had a 2.45-fold increase in vessel density compared with control tumors, but such increased vessels were small in lumen. Perfusion area stained with Hoechst 33342 was significantly decreased while the area of hypoxia greatly increased. The tumor volumes of DAPT treated groups were quite smaller than that of vehicle treated (P< 0.01). Microscopic analysis showed that many vessels in DAPT tumors were stuffed with endothelial-like abnormal cells within vascular lumens, but no stuffed vessel could be found in control tumor vessels.Conclusions:y-secretase inhibitor increases tumor vessel density, but depresses vessel function development. Such uncoupling of tumor vessel density from vessel function suppresses glioma growth. So,γ-secretase inhibitor might be used as a novel therapeutic agent for glioma antiangiogenesis therapy.