Targeting autophagic survival by inhibiting elongation factor-2 kinase: Effect of 2-deoxy-D-glucose in human glioma cell lines

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Elongation factor-2 kinase (eEF-2 kinase), also known as Ca⁺⁺/calmodulin-dependent protein kinase III, is a structurally and functionally unique enzyme whose activity is increased in most cancer cell lines and many human malignancies (Bagaglio et al, Cancer Res. 1993; Ryazanov et al, PNAS, 1997). eEF-2 kinase phosphorylates eEF-2, leading to loss of affinity of the elongation factor for the ribosome and termination of protein elongation, a major consumer of cellular energy. Autophagy is a highly conserved cellular process that is activated in times of metabolic stress and leads to large-scale degradation of proteins. Cells can use autophagy to recycle amino acids, fatty acids and nucleotides for macromolecular biosynthesis and ATP generation. eEF-2 kinase activity is markedly increased in glioblastoma, the most aggressive form of astrocytoma and a malignancy characterized by areas of decreased blood supply and necrosis. We previously reported that nutrient deprivation increased eEF-2 kinase activity in glioma and other cell lines, and that knockdown of eEF-2 kinase expression with siRNA blunted the autophagocytic response to nutrient deprivation and accelerated cell death (Wu H, et al., Cancer Res, 2006). To further analyze the role of eEF-2 kinase in response to metabolic stress, the human glioma cell line T98G was starved by exposure to varying concentrations of 2-Deoxy-D-glucose (2-DG), and eEF-2 kinase activity was determined by measuring the phosphorylation of eEF-2. We used shRNA to knockdown eEF-2 kinase to produce the T98G^{eEF2-K -/-} cell line. Glioma cells treated with 2-DG at 12.5, 25, and 50 mM increased eEF-2 kinase activity in parental T98G and T98G vector transfected controls. In contrast, eEF-2 kinase RNAi blunted eEF-2 phosphorylation induced by 2-DG. Treatment of the glioma cells with 2-DG also leads to decreases of ATP content. At same time, phospho-AMP-activated protein kinase (AMPK), a sensor of AMP/ATP ratios that can activate eEF-2 kinase, increased with the 2-DG treatment. Autophagy was determined by measuring cellular LC3-II, a component of autophagosome. We found that LC3-II increased after 2-DG treatment in T98G and T98G vector control cells but to a lesser extent in the T98G^{eEF2-K -/-} cell line. These data indicate that 2-DG treatment leads to depletion of ATP, which activates AMP kinase, and in turn increases the activity of eEF-2 kinase to limit protein synthesis for energy conservation.