Inhibition of glycolysis and induction of autophagy in gliomas by 2-fluoro-deoxy-D-glucose.

B34

The metabolism of fast growing tumor cells requires increasing amounts of energy to support rapid proliferation, which provides an opportunity for targeting metabolism as therapeutic approach. In addition to apoptosis (extensively studied Type I programmed cell death), autophagy (Type II cell death), under normal physiological conditions serves as a natural way of disposing of unwanted cellular material, regeneration of both cellular building blocs and energy resources. However, when the cells undergo metabolic stress, such as nutrient deprivation, the process of autophagy dramatically increases and ultimately leads to cell death. In this study we investigated the potential of inhibiting the glycolytic pathway in glioma U87 cell lines using 2-fluoro-deoxy-D-glucose (2-FG) and determined whether or not 2-FG induced autophagic cell death. U87 cells were treated with increasing concentrations of 2-FG and allowed to grow under normoxic (21% O₂) or hypoxic (0.1% O₂) conditions. The cytotoxicity of 2-FG was assessed in treated cultures at various time points (24, 48 and 72 h). Our results show that 2-FG induces Type II cell death in U87 glioma cells in dose dependent manner and that it is initiated even in the absence of nutritional stress. The cytotoxicity studies show IC50 (2-FG) to be 3.4 mM and that U87 sensitivity towards 2-FG increases 40% in cells grown under hypoxic conditions. Additionally, to test the cytotoxicity of 2-FG in U87 cells during glucose deprivation, this cytotoxicity analysis was repeated with cells grown in low glucose (5.6 mM) media. Under these conditions U87 cells were increasingly sensitive to 2-FG effects; growth was inhibited by 78% under normoxic conditions and by 86% when grown under hypoxia conditions. Confirmation of autophagy in 2-FG treated cells was demonstrated using transmission electron microscopy (TEM). U87 cells treated with 5 mM 2-FG X 72 hrs showed the presence of autophagic vacuoles in the cytoplasm - a hallmark of autophagy. Cell cycle analysis of U87 cells treated with 5 mM 2-FG X 72 hrs showed marked accumulation in G2/M phase with no increase in Sub G0/G1, demonstrating that cell death following treatment is not attributable to apoptosis. These studies show 2-FG to be a potent inhibitor of cell proliferation and an inducer of autophagic cell death in the U87 cell line and that targeting autophagic survival response using inhibitors of glycolysis should be a reasonable therapeutic approach to the treatment of cancers that are heavily dependent on glycolysis for survival.