Abstract 4335: Low dose 2 deoxy glucose induces ER stress and neuroblastoma cell death
Authors: Graham Regina M. , Barth Constantinos J. , Webster Keith A. , Vanni Steven 
Published: June 2012

Abstract
Introduction. The prognosis for relapsed neuroblastoma (NB) is extremely poor. While standard therapies can produce brief periods of remission, many NB patients will eventually die due to recurrent disease. Therefore novel approaches are urgently needed for the treatment of NB. Cancer cells are characterized by increased rates of aerobic glycolysis, which correlate with increased tumor aggressiveness and a poor prognosis. 2-deoxy-D-glucose (2-DG) is a glucose analogue which blocks glycolysis and has recently been investigated in a phase I trial for advanced solid tumors. Here we examine the potential of 2-DG for neuroblastoma treatment.
Methods. The effect of 2-DG was evaluated in a panel of neuroblastoma cell lines: N-Myc amplified: NB1691, SMS-KCNR, SK-N-BE2, and non N-Myc amplified: SK-N-SH, SH-SY5Y, and SVBM15, a newly developed cell line derived from a bone marrow aspirate of a stage IV relapsed NB patient. The effects of 2-DG were evaluated by MTS assay and LDH release. ATP levels were evaluated using an ATP colorimetric assay. Western blot analysis was used to determine protein levels of ER stress proteins and AKT.
Results. To determine the effective dose (ED50) of 2-DG, we exposed NB cells to 0.1-8mM 2-DG for 72 hours and measured cell viability. The ED50 was found to be close to 2mM for most cell lines examined; SH-SY5Y: 1.86mM, SK-N-SH: 2.23mM, SVBM15: 2.4mM, SK-N-BE2: 1.87mM, SMSKNR: 1.98mM, and NB1691: 5.44mM. LDH analysis of SH-SY5Y and SK-N-BE2 cells indicated that this effect was primarily due to cell death and not cell cycle inhibition. Inhibiting glycolysis with 2mM 2-DG reduced ATP levels by ∼30% at 24 hours (SK-N-BE2: 70±6.3% and SH-SY5Y: 64±4.4% of non-treated controls). Examination of cell signaling pathways induced by 2-DG in SH-SY5Y and SK-N-BE2 cells revealed a dose-dependent induction of AKT and ER stress associated proteins; GRP94, GRP78, and GADD153. To determine if inhibiting GRP94 or AKT would potentiate 2-DG induced cell death, cells were exposed to 2mM 2-DG with 17-AAG (500nM), an inhibitor of GRP94, or AKT inhibitor × (10uM). Inhibiting the activity of both GRP94 and AKT further reduced viability by an additional ∼50% over 2-DG alone in SH-SY5Y cells (2-DG: 45±1.4%; 17-AAG: 62±1.6%; 2-DG+17-AAG: 25±0.61%; AKT X: 49±1.8%; 2-DG+AKT X: 23±1.93%) and SK-N-BE2 cells (2-DG: 41±3.3%; 17-AAG: 85±2.0%; 2-DG+17-AAG: 24±0.43%; AKT X: 75±7.3%; 2-DG+AKT X: 22±1.2%).
Conclusions. These results demonstrate that clinically achievable levels of 2-DG reduce ATP concentrations and induce significant loss of NB cell viability. Furthermore, inhibiting cell survival signaling pathways activated by 2-DG exposure can increase the effectiveness of 2-DG treatment. Targeting NB cell metabolism is a novel approach to NB treatment, and when combined with additional chemotherapeutic agents, may be an effective treatment for NB.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4335. doi:10.1158/1538-7445.AM2011-4335