Abstract 2332: The glucose analog 2-DG selectively inhibits AKT and ERK in endothelial cells via interference of N-linked glycosylation and induces endothelial cell apoptosis in vitro and in vivo.

Abstract

Abstract BACKGROUND: As every step in tumor angiogenesis is an energy consuming process, targeting endothelial (EC) glucose metabolism is a rational strategy for angiogenesis inhibition. We recently showed that interference with endothelial glucose metabolism by the glucose analog 2-deoxy-D-Glucose (2-DG) inhibits angiogenesis in vitro and in vivo, at concentrations lower than those required to induce tumor cell cytotoxicity. The objectives of the current study are to further understand the basis for EC sensitivity to 2-DG in vitro and in vivo. METHODS: Time dependent effects of 2-DG were studied on EC tube formation at 4, 8 and 18 hrs. Apoptosis was assessed by the TUNEL assay at 24, 48 and 72 hours. The effects of low antiangiogenic concentrations (0.6 mM) of 2-DG on ERK, AKT and mTOR pathways were assessed in endothelial (HUVEC) and cancer (MDA-MB-231, HT-29, 786-0) cells by western blot. The in vivo effects of intraocular 2-DG administration on newly forming (CD-105) and total (lectin) tumor vessels were evaluated in the LHBETATAG retinoblastoma model. In vivo EC apoptosis after 2-DG treatment was assessed by TUNEL/CD31 staining of treated tumors. RESULTS: Inhibition of HUVEC capillary formation by 2-DG occurred as early as 8 hours after treatment, and the effects were clear at 18 hours compared to controls. EC apoptosis was non-significantly increased at 24 hours, and was prominent at 48 and 72 hours. Antiangiogenic 2-DG concentrations (0.6 mM) significantly inhibited phosphorylation of Akt473, S6 and Erk at 24 hours. On the other hand, 2-DG did not affect these pathways in MDA-MB-231 or HT-29 cells, while they occurred at a lower level in 786-0 cells. Mannose successfully reversed AKT, S6 and ERK inhibition only in HUVECs, but not in 786-0 cells, suggesting that the EC selective inhibition of ERK and AKT are mediated by inhibition of N-linked glycosylation, while other mechanisms mediate the effects in cancer cells. Importantly, these effects were 2-DG specific, as other glucose analogs (FDG) and the glycolytic inhibitor oxamate did not significantly affect ERK or AKT. Intraocular 2-DG administration decreased tumor microvasculature, and importantly, induced EC apoptosis, as demonstrated by TUNEL+/CD 31 co-localization. CONCLUSIONS: Our data indicate that in angiogenic ECs, 2-DG's inhibition of N-linked glycosylation targets critical proliferation and survival pathways. 2-DG mediated Inhibition of AKT and ERK pathways may explain the significant endothelial sensitivity cytotoxic and pro-apoptotic effects of 2-DG in vitro and in vivo, and further support targeting EC glucose metabolism as a viable strategy for angiogenesis inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2332. doi:1538-7445.AM2012-2332