title: 2-Deoxy-D-glucose activates autophagy via endoplasmic reticulum stress rather than ATP depletion.
abstract: OBJECTIVE
  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.
authors.affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, PAP Building, Room 115, 1550 NW 10th Ave, Miami, FL 33136, USA.
authors.forename: Haibin
authors.initials: H
authors.lastname: Xi
authors.forename: Metin
authors.initials: M
authors.lastname: Kurtoglu
authors.forename: Huaping
authors.initials: H
authors.lastname: Liu
authors.forename: Medhi
authors.initials: M
authors.lastname: Wangpaichitr
authors.forename: Min
authors.initials: M
authors.lastname: You
authors.forename: Xiongfei
authors.initials: X
authors.lastname: Liu
authors.forename: Niramol
authors.initials: N
authors.lastname: Savaraj
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Lampidis
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000963:Antimetabolites / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003847:Deoxyglucose / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008545:Melanoma / Q000188:drug therapy / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000473:pathology; D056811:Unfolded Protein Response / Q000187:drug effects
chemical_list: D000963:Antimetabolites; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D000255:Adenosine Triphosphate; D003847:Deoxyglucose