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

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.

Fig. 1

2-DG induces ATP reduction, ER stress/UPR and autophagy. 1420 (a), MDA-MB-435 (b) and SKBR3 (c) cells were treated with 2-DG at doses as indicated. Upper panels, intracellular ATP levels were measured after 5 h of drug exposure. Lower panels, immunoblotting was performed to detect levels of Grp78, CHOP and LC3B II after 16 h (1420 and MDA-MB-435) or 24 h (SKBR3) of treatment. β-Actin was used as a loading control. *Bands of LC3B I were not detected by short-time film exposure in 1420 and SKBR3 cells. ^# P < 0.05 and ^##P < 0.01, compared to controls

Fig. 2

Mannose reverses 2-DG-induced ER stress and autophagy without affecting ATP depletion. a 1420 cells were treated either by 4 mM of 2-DG, 1 mM of mannose (Man) or both in the presence or absence of 4 μg/ml of EST for 16 h. Immunoblotting was performed to detect levels of Grp78, CHOP and LC3B II. b 1420 cells were treated either by 4 mM of 2-DG or 4 mM of 2-DG with 1 mM of Man for 16 h. Endogenous LC3B were detected by fluorescent microscopy using an anti-LC3B antibody. c Intracellular ATP levels were measured after 1420 cells were treated either by 4 mM of 2-DG, 1 mM of Man or both for 5 h. d 1420 cells were treated similarly as depicted in (a) without EST for 6 h. Immunoblotting was performed to detect phosphorylated (serine 79) and total ACC levels. Representative blots of one of two independent experiments are shown. e MDA-MB-435 cells were treated either by 10 mM of 2-DG, 1 mM of Man or both. Upper panel, intracellular levels of ATP were measured after 5 h of drug exposure. Lower panel, immunoblotting was performed to detect levels of Grp78, CHOP and LC3B II 16 h after treatment. *Bands of LC3B I were not detected by short-time film exposure in 1420 and SKBR3 cells. f MDA-MB-435 cells were treated and analyzed similarly as 1420 cells in (b) except that 2-DG was used at a dose of 10 mM. g SKBR3 cells were treated and analyzed similarly as MDA-MB-435 cells in (e) except that immunoblotting was performed 24 h after drug exposure. For immunoblotting analyses in (a, e, g), β-actin was used as a loading control. NS, not significant compared to 2-DG alone-treated samples

Fig. 3

2-DG induces autophagy in a similar kinetics as tunicamycin. 1420 cells were treated with 4 mM of 2-DG or 1 μg/ml of tunicamycin (TM) in the absence (a) or presence (b) of 4 μg/ml EST. At the indicated times, cells were harvested and immunoblotting was performed to detect levels of Grp78, CHOP and LC3B II. β-Actin was used as a loading control. Blots for TM treatment are from one of two independent experiments

Fig. 4

Oxamate induces ATP depletion but not autophagy. a Intracellular ATP levels were measured after 1420 cells were treated by 4 mM of 2-DG or different doses of oxamate (OX) as indicated for 5 h. b 1420 cells were treated by 4 mM of 2-DG or different doses of OX as indicated in the presence or absence of 4 μg/ml of EST for 16 h. Immunoblotting was performed to detect levels of Grp78, CHOP and LC3B II. Representative blots of one of two independent experiments are shown. c 1420 cells were treated with 5 mg/ml of OX in the presence or absence of 4 μg/ml EST. At the indicated times, cells were harvested and immunoblotting was performed to detect levels of LC3B II. d 1420 cells were treated by 5 mg/ml OX for 16 h. Endogenous LC3B was detected by fluorescent microscopy using an anti-LC3B antibody. e MDA-MB-435 cells were treated and analyzed similarly as 1420 cells in (a) except that 2-DG was used at a dose of 10 mM. f MDA-MB-435 cells were treated by 10 mM of 2-DG or different doses of OX as indicated for 16 h. Immunoblotting was performed to detect levels of Grp78, CHOP and LC3B II. *Bands of LC3B I were not detected by short-time film exposure in 1420 cells. Representative blots of one of two independent experiments are shown. g MDA-MB-435 cells were treated and analyzed similarly as 1420 cells in (d) except that 2-DG was used at a dose of 10 mM. For immunoblotting analyses in (b, c, f), β-actin was used as a loading control. ^# P < 0.05, ^## P < 0.01 and ^### P < 0.001, compared to controls

Fig. 5

Under anaerobic conditions, 2-DG severely depletes ATP and down-regulates autophagy. a Intracellular ATP levels were measured after 1420 cells were treated either by 4 mM of 2-DG, 0.1 μg/ml of oligomycin (OM) or both for 5 h. b 1420 cells were treated either by 4 mM of 2-DG, 0.1 μg/ml of OM or both in the presence or absence of 4 μg/ml of EST for 16 h. Immunoblotting was performed to detect levels of Grp78 and LC3B II. Representative blots of one of two independent experiments are shown. c 1420 cells were treated by 4 mM of 2-DG in combination with 0.1 μg/ml of OM in the presence or absence of 4 μg/ml EST. At the indicated times, cells were harvested and immunoblotting was performed to detect levels of LC3B II. d 1420 cells were treated by 4 mM of 2-DG in combination with 0.1 μg/ml of OM for 16 h. Endogenous LC3B were detected by fluorescent microscopy using an anti-LC3B antibody. e MDA-MB-435 cells were treated and analyzed similarly as 1420 cells in (a) except that 2-DG was used at a dose of 10 mM. f MDA-MB-435 cells were treated either by 4 mM of 2-DG, 0.1 μg/ml of OM or both for 16 h. Immunoblotting was performed to detect levels of Grp78 and LC3B II. *Bands of LC3B I were not detected by short-time film exposure in 1420 cells. For immunoblotting analyses in (b, c, f), β-actin was used as a loading control. ^##P < 0.01, compared to 2-DG alone-treated samples

Fig. 6

Autophagy protects cancer cells from 2-DG-induced cytotoxicity through relieving ER stress. a, b 1420 (a) and MDA-MB-435 (b) cells were treated with the indicated doses of 2-DG in the presence or absence of 10 mM of 3-methyladenine (3-MA) for 48 h. The percentages of dead cells were analyzed based on trypan blue exclusion. c, d 1420 (c) and SKBR3 (d) cells were treated with the indicated doses of 2-DG in the presence or absence of 0.1 μg/ml of rapamycin (Rap) for 48 h (SKBR3) or 72 h (1420). The percentages of dead cells were analyzed based on trypan blue exclusion. e 1420 cells were transfected with either a siRNA targeting luciferase (siLuc, negative control) or a pool of siRNAs against Atg7 (siAtg7) at 50 nM. Forty-eight hours post-transfection, cells were treated with different doses of 2-DG, and 16 h later, harvested and immunoblotted for expression levels of Atg7, LC3B II, Grp78, CHOP and cleaved caspase 3. β-Actin was used as a loading control. Representative blots of one of two independent experiments are shown. f 1420 cells were transfected as depicted above. Forty-eight hours post-transfection, cells were treated with different doses of 2-DG for another 48 h. The percentages of dead cells were then analyzed based on trypan blue exclusion. Representative results of one of two independent experiments are shown. ^# P < 0.05 and ^## P < 0.01, compared to the corresponding 2-DG alone-treated samples (a, b, c, d) or 2-DG-treated cells transfected with siLuc (f)