Malignant gliomas are highly invasive primary brain tumors resistant to conventional treatments primarily because of sustained angiogenesis and infiltrative nature. Despite multimodal therapeutic interventions high grade gliomas have a propensity for recurrence. We recently reported that T11-target structure (T11TS), a membrane glycopeptide isolated from sheep erythrocyte membrane inhibits VEGF signaling and prosurvival PI3K/Akt pathway in glioma associated brain endothelial cells. Angiopoietin signaling mediated through endothelial Tie2 receptors is a potent mediator of glioma angiogenesis. Apoptosis of tumor endothelial cells acts as a crucial regulator of tumor angiogenesis and some angiogenesis inhibitors mediate their anti-angiogenic effect through induction of endothelial cell apoptosis. The effect of T11TS on brain endothelial cell Angiopoietin-1/Tie2 signaling and endothelial cell apoptosis has not been investigated so far. The goal of this study is to determine the effects of T11TS administration on endothelial Angiopoietin-1/Tie 2 signaling and on apoptosis of brain endothelial cells in a chemically induced glioma model. Furthermore, this study investigated the mechanistic details of brain endothelial cell apoptosis induction caused by in vivo T11TS therapy. Expression of angiopoietin-1, Tie2, phospho-Tie2 proteins, apoptotic pathway regulator proteins and caspases were determined by flowcytometry, immunoblotting, immunofluorescence imaging and in situ immunofluorescent staining. Apoptosis in brain endothelial cells was directly evaluated by flowcytomeric Annexin V-PI assay and mitochondrial membrane JC-1 assay. T11TS administration in glioma induced rats significantly downregulate Angiopoietin-1 expression and Tie2 receptor activation in brain endothelial cells thereby blocking Angiopoietin-1/Tie2 signaling. T11TS therapy triggered dose-dependent externalization of membrane phosphatidylserine, reduction in mitochondrial membrane potential and cleavage of caspase 3 which strongly indicated induction of apoptosis in glioma associated brain endothelial cells. T11TS administration in glioma was found to activate both intrinsic and extrinsic pathways of apoptosis by modulating expression of apoptotic regulator proteins like p53, Bax, Bcl-2, Fas and FasL, triggering cytochorome c release into cytosol and activating caspases 8, 3 and 9 in brain endothelial cells. T11TS administration also stimulated cleavage of Bid in glioma associated brain endothelial cells. This preclinical study demonstrated that blockage of Angiopoietin-1/Tie2 signaling in glioma associated brain endothelial cells and induction of apoptosis in brain endothelial cells constitute the underlying mechanistic events that contribute to the anti-angiogenic efficacy of T11TS in malignant glioma.
Citation Format: Debanjan Bhattacharya, Suhnrita Chaudhuri, Swapna Chaudhuri. T11TS impedes glioma angiogenesis by attenuating brain endothelial Angiopoietin-1/Tie2 signaling and inducing apoptosis of glioma associated brain endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1024. doi:10.1158/1538-7445.AM2014-1024