Abstract
Purpose
The glucose analog and glycolytic inhibitor 2-deoxy-d-glucose (2-DG), which is currently under clinical evaluation for targeting cancer cells, not only blocks glycolysis thereby reducing cellular ATP, but also interferes with N-linked glycosylation, which leads to endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). Both bioenergetic challenge and ER stress have been shown to activate autophagy, a bulk cellular degradation process that plays either a pro- or anti-death role. Here, we investigate which pathway 2-DG interferes with that activates autophagy and the role of this process in modulating 2-DG-induced toxicity.
Methods
Pancreatic cancer cell line 1420, melanoma cell line MDA-MB-435 and breast cancer cell line SKBR3 were used to investigate the relationship between induction by 2-DG treatment of ER stress/UPR, ATP reduction and activation of autophagy. ER stress/UPR (Grp78 and CHOP) and autophagy (LC3B II) markers were assayed by immunoblotting, while ATP levels were measured using the CellTiter-Glo Luminescent Cell Viability Assay. Autophagy was also measured by immunofluorescence utilizing LC3B antibody. Cell death was detected with a Vi-Cell cell viability analyzer using trypan blue exclusion.
Results
In the three different cancer cell lines described earlier, we find that 2-DG upregulates autophagy, increases ER stress and lowers ATP levels. Addition of exogenous mannose reverses 2-DG-induced autophagy and ER stress but does not recover the lowered levels of ATP. Moreover, under anaerobic conditions where 2-DG severely depletes ATP, autophagy is diminished rather than activated, which correlates with lowered levels of the ER stress marker Grp78. Additionally, when autophagy is blocked by siRNA, cell sensitivity to 2-DG is increased corresponding with upregulation of ER stress-mediated apoptosis. Similar increased toxicity is observed with 3-methyladenine, a known autophagy inhibitor. In contrast, rapamycin which enhances autophagy reduces 2-DG-induced toxicity.
Conclusions
Overall, these results indicate that the major mechanism by which 2-DG stimulates autophagy is through ER stress/UPR and not by lowering ATP levels. Furthermore, autophagy plays a protective role against 2-DG-elicited cell death apparently by relieving ER stress. These data suggest that combining autophagy inhibitors with 2-DG may be useful clinically.
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Abbreviations
- 2-DG:
-
2-Deoxy-d-glucose
- 3-MA:
-
3-Methyladenine
- Atg7:
-
Autophagy-related gene 7
- CHOP:
-
C/EBP homologous protein
- eEF-2K:
-
Eukaryotic elongation factor-2 kinase
- ER:
-
Endoplasmic reticulum
- Grp78:
-
Glucose-regulated protein 78 kD
- LC3B:
-
Microtubule-associated protein-1 light chain 3B
- UPR:
-
Unfolded protein response
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Acknowledgments
This work is supported by the National Cancer Institute grant# CA37109 to T.J.L. and V.A. Research Merit Award to N.S.
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Xi, H., Kurtoglu, M., Liu, H. et al. 2-Deoxy-d-glucose activates autophagy via endoplasmic reticulum stress rather than ATP depletion. Cancer Chemother Pharmacol 67, 899–910 (2011). https://doi.org/10.1007/s00280-010-1391-0
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DOI: https://doi.org/10.1007/s00280-010-1391-0