Article Figures & Data
Figures
- Figure 1.
Effects of 2-DG and 2-FDG on ATP levels and viability in tumor cells under aerobic and anaerobic conditions: reversal by mannose. A, cells were treated with 4 mmol/L of 2-DG for 72 h and the percentage of dead cells was assayed by trypan bue exclusion. B, 2-DG–sensitive cells were treated with 4 mmol/L of 2-DG or 2-FDG under aerobic or anaerobic (oligomycin 0.05 μg/mL) conditions and the percentage of dead cells was assayed by trypan bue exclusion. C, in cells treated with either 4 mmol/L of 2-DG or 2-FDG under normoxic conditions, the percent reduction of ATP was calculated by dividing the amount found in treated samples by the amount in untreated controls. ATP concentrations were normalized to total protein as measured by bicinchoninic acid protein assay. To minimize complications in data interpretation arising from differential toxicity induced by 2-DG versus 2-FDG, treatment with each sugar analogue was applied for 24 h. D, 1420 and SKBR3 cells were treated with increasing concentrations of 2-DG in conjunction with 1 mmol/L of various sugars followed by analysis of cytotoxicity with trypan blue exclusion assays. Note that only mannose reverses 2-DG toxicity. Columns, average of triplicates; bars, SD.
- Figure 2.
2-DG interference with N-linked glycosylation in tumor cells growing under normoxia. A FACE technique was used to investigate the effects of 2-DG on LLO assembly (A) as well as on N-glycans (B). Cells were treated with either 0.5 or 4 mmol/L of 2-DG for 24 h, followed by extraction and FACE of LLOs. The standard oligosaccharides used in these studies are as follows: G4 to G7, glucose oligomers; G3M9, mature oligosaccharide (G3M9Gn2); M5, oligosaccharide intermediate (M5Gn2). C, the levels of mature LLO were quantified by measuring the density of the G3M9 bands in all cell types treated with either 0.5 or 4 mmol/L of 2-DG. The amount of LLO is shown as arbitrary units, which is calculated by the percentage of reduction in band intensity in treated as compared with control samples. Columns, average of two samples; bars, SD.
- Figure 3.
Reversal of the effects of 2-DG on LLO synthesis by exogenous mannose. A, using the FACE technique, LLO levels were investigated in two sensitive cell lines, SKBR3 and 1420, following treatment with 0.5 mmol/L 2-DG in the presence or absence of 1 mmol/L mannose for 24 h. B, the G3M9 band was quantified by measuring the density of the band, and the amount of LLO is shown as arbitrary units, which are calculated by percent reduction in the band intensity of treated as compared with control samples. Note that mannose is without effect on LLO synthesis in untreated control cultures. Standards are as in Fig. 2.
- Figure 4.
UPR induction by 2-DG in sensitive and resistant cells. A, GRP78/Bip expression was assayed by Western blot in cells treated with 0.5 mmol/L 2-DG for 24 h. Note the marked increase in GRP78/Bip expression in 1420 and SKBR3 but not in 1469 and MDA 231 cells. B, using a gel reader, the amount of GRP78/Bip was quantified. Induction of GRP78/Bip was calculated by dividing its amount in 2-DG–treated samples by the amount in untreated control samples. Each amount was normalized to the expression of β-actin. Western blot analysis of GRP78/Bip and GRP94 in SKBR3 and 1420 cells (C) or 1469 and MDA 231 cells (D) treated with the indicated sugar analogues or tunicamycin (TUN) for 24 h. β-Actin was assayed to compare the loading of samples.
- Figure 5.
Assay of UPR-specific apoptotic factor CHOP/GADD153 and apoptosis in 2-DG–sensitive cells. A, CHOP/GADD153 expression was assayed by Western blot after 48 h of treatment with 4 mmol/L of either 2-DG or 2-FDG or 1 μg/mL of tunicamycin. Due to high toxicity, treatment with 1 μmol/L staurosporine (STA) was restricted to 12 h. B, parallel experiments were done to quantify the apoptotic cell percentage by Annexin V/propidium iodide staining and analysis via flow cytometer. The quadrants that divide cell populations into four groups (control, early apoptotic, late apoptotic, and necrotic) were set by treating these same cells with a known inducer of apoptosis, staurosporine, or an inducer of necrosis, 70% ethanol.
- Figure 6.
Overall scheme of the effects of 2-DG and 2-FDG on glycolysis and N-linked glycosylation. 2-FDG inhibits glycolysis better than does 2-DG. In contrast, 2-DG is more portent than 2-FDG in interfering with N-linked glycosylation. Due to the resemblance of 2-DG with mannose, it not only competes with mannose metabolism in this pathway but also incorporates fraudulently into dolichol-pyrophosphate (lipid)–linked oligosaccharides, which are the precursors for N-linked glycosylation. Although 2-FDG has been shown to decrease mannose metabolites, the position of the fluorine group in 2-FDG restricts it from resembling mannose and, therefore, does not have a direct inhibitory effect on mannose incorporation into LLO as does 2-DG. The greater activity of 2-DG versus 2-FDG on N-linked glycosylation correlates with its toxic activity in selected tumor types growing under normal oxygen tension, which can be reversed by addition of exogenous mannose (▴, glucose;
, mannose; and ▪, N-acetyl-glucosamine).
Tables
- Table 1.
The comparison of oxygen consumption in 2-DG–sensitive versus 2-DG–resistant cell lines
Cell line Tissue type Average O2 consumption (nmol/106 cells/min) 143B + oligomycin Osteosarcoma with oxidative phosphorylation inhibitor 0.97 ± 0.11 ρ0 Osteosarcoma 0.09 ± 0.004 1469 Pancreatic cancer 3.38 ± 0.32 SKBR3 Breast adenocarcinoma 2.01 ± 0.29 1420 Pancreatic adenocarcinoma 3.50 ± 0.03 MDA 231 Breast cancer 2.76 ± 0.04
Volume 6, Issue 11
