Skip to main content

Advertisement

Log in

2-Deoxy-d-glucose activates autophagy via endoplasmic reticulum stress rather than ATP depletion

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

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

References

  1. Wick AN, Drury DR, Nakada HI, Wolfe JB (1957) Localization of the primary metabolic block produced by 2-deoxyglucose. J Biol Chem 224:963–969

    PubMed  CAS  Google Scholar 

  2. Chen W, Gueron M (1992) The inhibition of bovine heart hexokinase by 2-deoxy-d-glucose-6-phosphate: characterization by 31P NMR and metabolic implications. Biochimie 74:867–873

    Article  PubMed  CAS  Google Scholar 

  3. Liu H, Hu YP, Savaraj N, Priebe W, Lampidis TJ (2001) Hypersensitization of tumor cells to glycolytic inhibitors. Biochemistry 40:5542–5547

    Article  PubMed  CAS  Google Scholar 

  4. Liu H, Savaraj N, Priebe W, Lampidis TJ (2002) Hypoxia increases tumor cell sensitivity to glycolytic inhibitors: a strategy for solid tumor therapy (model C). Biochem Pharmacol 64:1745–1751

    Article  PubMed  CAS  Google Scholar 

  5. Maher JC, Krishan A, Lampidis TJ (2004) Greater cell cycle inhibition and cytotoxicity induced by 2-deoxy-d-glucose in tumor cells treated under hypoxic vs aerobic conditions. Cancer Chemother Pharmacol 53:116–122

    Article  PubMed  CAS  Google Scholar 

  6. Raez LE, Rosenblatt J, Schlesselman J et al (2005) Combining glycolytic inhibitors with chemotherapy: phase I trial of 2-deoxyglucose and docetaxel in patients with solid tumors [abstract no: 3190]. In: American Society of Clinical Oncology Annual Meeting, May 13–17 Orlando, FL

  7. Kurtoglu M, Gao N, Shang J et al (2007) Under normoxia, 2-deoxy-d-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation. Mol Cancer Ther 6:3049–3058

    Article  PubMed  CAS  Google Scholar 

  8. Datema R, Schwarz RT (1978) Formation of 2-deoxyglucose-containing lipid-linked oligosaccharides Interference with glycosylation of glycoproteins. Eur J Biochem 90:505–516

    Article  PubMed  CAS  Google Scholar 

  9. Datema R, Schwarz RT (1979) Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo. Biochem J 184:113–123

    PubMed  CAS  Google Scholar 

  10. Kurtoglu M, Maher JC, Lampidis TJ (2007) Differential toxic mechanisms of 2-deoxy-d-glucose versus 2-fluorodeoxy-d-glucose in hypoxic and normoxic tumor cells. Antioxid Redox Signal 9:1383–1390

    Article  PubMed  CAS  Google Scholar 

  11. Szegezdi E, Fitzgerald U, Samali A (2003) Caspase-12 and ER-stress-mediated apoptosis: the story so far. Ann N Y Acad Sci 1010:186–194

    Article  PubMed  CAS  Google Scholar 

  12. Schroder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutat Res 569:29–63

    Article  PubMed  Google Scholar 

  13. Bernales S, McDonald KL, Walter P (2006) Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. PLoS Biol 4:e423

    Article  PubMed  Google Scholar 

  14. Ogata M, Hino S, Saito A et al (2006) Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 26:9220–9231

    Article  PubMed  CAS  Google Scholar 

  15. Yorimitsu T, Nair U, Yang Z, Klionsky DJ (2006) Endoplasmic reticulum stress triggers autophagy. J Biol Chem 281:30299–30304

    Article  PubMed  CAS  Google Scholar 

  16. Hoyer-Hansen M, Jaattela M (2007) Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium. Cell Death Differ 14:1576–1582

    Article  PubMed  CAS  Google Scholar 

  17. Ding WX, Ni HM, Gao W et al (2007) Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J Biol Chem 282:4702–4710

    Article  PubMed  CAS  Google Scholar 

  18. Kouroku Y, Fujita E, Tanida I et al (2007) ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell Death Differ 14:230–239

    Article  PubMed  CAS  Google Scholar 

  19. Sakaki K, Wu J, Kaufman RJ (2008) Protein kinase c theta is required for autophagy in response to stress in the endoplasmic reticulum. J Biol Chem 283:15370–15380

    Article  PubMed  CAS  Google Scholar 

  20. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6:463–477

    Article  PubMed  CAS  Google Scholar 

  21. He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93

    Article  PubMed  CAS  Google Scholar 

  22. Kuma A, Hatano M, Matsui M et al (2004) The role of autophagy during the early neonatal starvation period. Nature 432:1032–1036

    Article  PubMed  CAS  Google Scholar 

  23. Lum JJ, Bauer DE, Kong M, Harris MH, Li C, Lindsten T, Thompson CB (2005) Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120:237–248

    Article  PubMed  CAS  Google Scholar 

  24. Meley D, Bauvy C, Houben-Weerts JH, Dubbelhuis PF, Helmond MT, Codogno P, Meijer AJ (2006) AMP-activated protein kinase and the regulation of autophagic proteolysis. J Biol Chem 281:34870–34879

    Article  PubMed  CAS  Google Scholar 

  25. Xu ZX, Liang J, Haridas V, Gaikwad A, Connolly FP, Mills GB, Gutterman JU (2007) A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase. Cell Death Differ 14:1948–1957

    Article  PubMed  CAS  Google Scholar 

  26. Liang J, Shao SH, Xu ZX et al (2007) The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat Cell Biol 9:218–224

    Article  PubMed  CAS  Google Scholar 

  27. Papandreou I, Lim AL, Laderoute K, Denko NC (2008) Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L. Cell Death Differ 15:1572–1581

    Article  PubMed  CAS  Google Scholar 

  28. Wei S, Kulp SK, Chen CS (2010) Energy restriction as an antitumor target of thiazolidinediones. J Biol Chem 285:9780–9791

    Article  PubMed  CAS  Google Scholar 

  29. Christgen M, Lehmann U (2007) MDA-MB-435: the questionable use of a melanoma cell line as a model for human breast cancer is ongoing. Cancer Biol Ther 6:1355–1357

    Article  PubMed  CAS  Google Scholar 

  30. Klionsky DJ, Abeliovich H, Agostinis P et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175

    PubMed  CAS  Google Scholar 

  31. Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326

    Article  PubMed  CAS  Google Scholar 

  32. Duksin D, Mahoney WC (1982) Relationship of the structure and biological activity of the natural homologues of tunicamycin. J Biol Chem 257:3105–3109

    PubMed  CAS  Google Scholar 

  33. Maher JC, Wangpaichitr M, Savaraj N, Kurtoglu M, Lampidis TJ (2007) Hypoxia-inducible factor-1 confers resistance to the glycolytic inhibitor 2-deoxy-d-glucose. Mol Cancer Ther 6:732–741

    Article  PubMed  CAS  Google Scholar 

  34. Wouters BG, Koritzinsky M (2008) Hypoxia signalling through mTOR and the unfolded protein response in cancer. Nat Rev Cancer 8:851–864

    Article  PubMed  CAS  Google Scholar 

  35. Mazure NM, Pouyssegur J (2009) Hypoxia-induced autophagy: cell death or cell survival? Curr Opin Cell Biol 22:177–180

    Article  PubMed  Google Scholar 

  36. Maschek G, Savaraj N, Priebe W, Braunschweiger P, Hamilton K, Tidmarsh GF, De Young LR, Lampidis TJ (2004) 2-deoxy-d-glucose increases the efficacy of adriamycin and paclitaxel in human osteosarcoma and non-small cell lung cancers in vivo. Cancer Res 64:31–34

    Article  PubMed  CAS  Google Scholar 

  37. DiPaola RS, Dvorzhinski D, Thalasila A et al (2008) Therapeutic starvation and autophagy in prostate cancer: a new paradigm for targeting metabolism in cancer therapy. Prostate 68:1743–1752

    Article  PubMed  CAS  Google Scholar 

  38. Wu H, Zhu H, Liu DX, Niu TK, Ren X, Patel R, Hait WN, Yang JM (2009) Silencing of elongation factor-2 kinase potentiates the effect of 2-deoxy-d-glucose against human glioma cells through blunting of autophagy. Cancer Res 69:2453–2460

    Article  PubMed  CAS  Google Scholar 

  39. Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P (2000) Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 275:992–998

    Article  PubMed  CAS  Google Scholar 

  40. Ding WX, Ni HM, Gao W, Yoshimori T, Stolz DB, Ron D, Yin XM (2007) Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am J Pathol 171:513–524

    Article  PubMed  CAS  Google Scholar 

  41. Fujita E, Kouroku Y, Isoai A, Kumagai H, Misutani A, Matsuda C, Hayashi YK, Momoi T (2007) Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Hum Mol Genet 16:618–629

    Article  PubMed  CAS  Google Scholar 

  42. Boyce M, Py BF, Ryazanov AG, Minden JS, Long K, Ma D, Yuan J (2008) A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death. Cell Death Differ 15:589–599

    Article  PubMed  CAS  Google Scholar 

  43. Py BF, Boyce M, Yuan J (2009) A critical role of eEF-2K in mediating autophagy in response to multiple cellular stresses. Autophagy 5:393–396

    Article  PubMed  CAS  Google Scholar 

  44. Plomp PJ, Wolvetang EJ, Groen AK, Meijer AJ, Gordon PB, Seglen PO (1987) Energy dependence of autophagic protein degradation in isolated rat hepatocytes. Eur J Biochem 164:197–203

    Article  PubMed  CAS  Google Scholar 

  45. Plomp PJ, Gordon PB, Meijer AJ, Hoyvik H, Seglen PO (1989) Energy dependence of different steps in the autophagic-lysosomal pathway. J Biol Chem 264:6699–6704

    PubMed  CAS  Google Scholar 

  46. Schellens JP, Meijer AJ (1991) Energy depletion and autophagy. Cytochemical and biochemical studies in isolated rat hepatocytes. Histochem J 23:460–466

    Article  PubMed  CAS  Google Scholar 

  47. Meijer AJ (2009) Autophagy research: lessons from metabolism. Autophagy 5:3–5

    Article  PubMed  Google Scholar 

  48. Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ (2007) Potential therapeutic applications of autophagy. Nat Rev Drug Discov 6:304–312

    Article  PubMed  CAS  Google Scholar 

  49. Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132:27–42

    Article  PubMed  CAS  Google Scholar 

  50. Lee AS (2001) The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci 26:504–510

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Conflict of interest statement

None.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Theodore J. Lampidis.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00280-010-1391-0

Keywords

Navigation