2-Deoxy-D-glucose kills select tumor cell types under normoxia: reversal by mannose indicates interference with glycosylation

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Cells growing under hypoxia are solely dependent on glucose metabolism via glycolysis for energy production. Consequently, when this pathway is blocked, with 2-deoxy-D-glucose (2DG), they die. In contrast, under normoxia when glycolysis is blocked most cells survive since fats and proteins can substitute as energy sources to fuel mitochondrial oxidative phosphorylation. Here, utilizing trypan blue exclusion assays, as well as flow cytometric analysis of DNA content, we find that 500 μg/ml of 2-DG kills a variant of the human breast carcinoma cell line, SKBR3, even when growing under normal oxygen tension. Previous studies demonstrated that 2-DG, under normal oxygen conditions, interferes with the process of N-linked glycosylation in viral coat glycoprotein synthesis, which can be reversed by addition of exogenous mannose. Thus, our results which show that 2-DG toxicity in SKBR3 variants growing under normoxia, can be completely reversed by low dose mannose (180 μg/ml) implicates glycosylation and not glycolysis as the mechanism responsible for this phenomenon. Moreover, mannose is not able to reverse the toxicity of 2-DG in tumor cells that are sensitive to it under hypoxic conditions. Additionally, 2-fluro-deoxy-D-glucose (2-FG) containing a fluoro group in the gluco configuration, which prevents it from resembling mannose, is not toxic to SKBR3 cells under normoxia at equivalent 2-DG doses. 2-FG, however, is as good or better than 2-DG in killing hypoxic tumor cells via blockage of glycolysis. To investigate the effect of 2-DG on glycoprotein synthesis, we measured an over-expressed glycoprotein in the SKBR3 variant, ErbB2, by western blot analysis and found it to be inhibited. Furthermore, the inhibition of ErbB2 synthesis by 2-DG could be reversed by mannose. However, SKOV3, an ovarian cancer line that also over-expresses Erb2 is found to be resistant to 2-DG under normoxic conditions suggesting that ErbB2 is not the sole target of -2DG in SKBR3. Overall, our results raise the possibility that in the appropriate tumor cell types, 2-DG could be used clinically as a single agent to selectively kill both the aerobic (via interference with glycosylation) as well as the hypoxic (via inhibition of glycolysis) cells of a solid tumor.