Figure 1.
2-DG inhibits endothelial cell growth, migration, and induces endothelial cytotoxicity in vitro.
HUVECs were stimulated with bFGF (10 ng/ml), treated with different concentrations of 2-DG for 72 hours, and its effects on cell growth (A) and cytotoxicity (B) were assessed as in materials and methods. A. 2-DG significantly inhibited bFGF induced HUVEC cell growth, with 72% inhibition by a concentration of 0.6 mM of 2-DG (* p<0.01, vs. control (0 mM)). Results (percent of control) are presented as the average of triplicate experiments and 95% confidence intervals. B. 2-DG induced significant cytotoxic effects on HUVECs in a dose dependent manner. ** p = 0.02; 0.6 mM 2-DG vs. control. C. HMVEC-L (lane 7) and HUVECs (lane 8) were significantly more sensitive to the cytotoxic effects of low doses (0.6 mM) of 2-DG than cancer (lanes 1–5) and non-cancer epithelial cells (lane 6). Lane 1: HT-29; 2: CAKI-1; 3: MDA-MB231; 4: 786-0; 5: HT-1080; 6: HREC. *** p<0.001 HUVEC and HMVEC vs. all other cell lines. Results (percent cell death) are presented as the average of triplicate experiments and 95% confidence intervals. D. HUVEC migration was assessed by the scratch assay. Significant inhibition of migration at 24 hours was observed, in a dose dependent manner. **** p = 0.009; 0.6 mM 2-DG vs. control. Results (percent of control) are presented as the average and 95% confidence intervals of triplicate experiments. All experiments were repeated at least twice.
Figure 2.
2-DG inhibits HUVEC capillary formation, but does not disrupt already established tubes.
HUVECs plated on matrigel were exposed to different concentrations of 2-DG before (upper panel) and after (lower panel) they organized into capillaries. A. Significant inhibition of HUVEC tube formation was observed in a dose dependent manner. * p = 0.005, 0.6 mM 2-DG (C) compared with control (B). In the lower panel, HUVEC capillaries were allowed to form overnight, before they were exposed to 2-DG. Changes in total tube length were assessed 24 hours after 2-DG exposure. D. 2-DG did not disrupt already established HUVEC capillaries. E, F: Representative pictures of control capillary tubes (E) and tubes treated with 0.6 mM 2-DG (F). Scale bar = 100 µm.
Figure 3.
Differential effects of 2-DG and other glycolytic inhibitors on in vitro angiogenesis and reversal of 2-DG's antiangiogenic effects by mannose.
HUVECs were exposed to 2-DG and the glycolytic inhibitors, 2-FDG and oxamate, and cell growth and cytotoxicity were measured at 72 hours. A. 2-DG had significantly more potent cytotoxic effects than 2-FDG and oxamate at equimolar concentrations. * p<0.05, 2-DG vs. 2-FDG and oxamate. B. 2-DG and 2-FDG inhibited HUVEC growth more potently than oxamate (p<0.001). The differences between the growth inhibitory effects of 2-DG and 2-FDG were not statistically significant (p>0.5). C. HUVECs were exposed to 0.6 mM of 2-DG, 2-FDG and oxamate, and tube formation assay was performed. Quantitative analysis of total tube length was performed as in materials and methods. 2-DG inhibited tube formation more potently than 2-FDG and oxamate. Histogram bars represent the average (and 95% confidence intervals) total tube length (percent of control) of triplicate experiments. ** P<0.0001, 2-DG vs. 2-FDG and oxamate. D. Co-treatment of HUVECs with 2-DG and mannose reverted the cytotoxic effects of 2-DG. 1: Control. 2: 2-DG at 0.6 mM. 3: 2-DG (0.6 mM) and mannose (1 mM). E. Mannose rescued 2-DG's inhibitory effects on HUVEC tube formation. I = control, II = 2-DG (0.6 mM), III = 2-DG (0.6 mM) + mannose (1 mM), IV = mannose (1 mM). Scale bar: 100 µm.
Figure 4.
2-DG interferes with N-linked glycosylation by inhibiting lipid-linked oligosaccharide (LLO) assembly.
A. Cells were treated with 2-DG with or without 1 mM mannose, and 2-FDG for 24 h, followed by extraction and fluorophore assisted carbohydrate electrophoresis (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). 2-DG inhibited assembly of mature LLOs. Lane 1 = untreated control. Lane 2 = 0.6 mM 2-DG; lane 3 = 3 mM 2-DG; Mannose reverted 2-DG inhibitory effects on LLO synthesis (lane 4, 2-DG at 0.6 mM + mannose, 1 mM; lane 5, 2-DG 3 mM + mannose, 1 mM). 2-FDG (lane 6: 0.6 mM; lane 7: 3 mM) treatment also decreased LLO synthesis, albeit at a lesser degree than 2-DG. Lane 8: glucose oligomer standards. B. The levels of mature LLOs were quantitated by measuring the fluorescence of G3M9Gn2-ANDS bands in each lane (arbitrary units), which is calculated by the percentage of band intensity in treated as compared with control samples. Bars represent the averages of single determinations in two separate experiments. C. To show minor LLO intermediates as well as the effects of mannose rescue more clearly (from figure 4A), electronically-generated traces of M5Gn2 through G3M9Gn2 in lanes 1–5 (from panel A) are displayed.
Figure 5.
2-DG induces HUVEC unfolded protein response (UPR) and UPR mediated apoptosis.
HUVECs were treated with 2-DG, with or without mannose, and 2-FDG for 24 hours, and immunoblotting was performed of cell lysates. 2-DG induced upregulation of Grp 94 (first panel) and Grp 78 (second panel) chaperone proteins and markers of the unfolded protein response. These effects were reversed by mannose (0.5 mM). CHOP/GADD 153, a transcription factor involved in ER stress mediated apoptosis, was potently upregulated by 2-DG (0.06, 0.6, and 3 mM) and partially reversed by mannose (third panel). CHOP/GADD 153 induction was associated with increased levels of cleaved caspase 3 in HUVECs treated by 2-DG (fourth panel). 2-FDG (0.06, 0.6, and 3 mM) induced a mild to moderate UPR response, especially at higher concentrations. However, CHOP/GADD 153 was not significantly induced, and levels of cleaved caspase 3 were not increased. Tunicamycin was used as positive control of the induction of UPR.
Figure 6.
Induction of endothelial cell apoptosis by 2-DG and reversal by mannose.
HUVEC cells in chamber slides were treated with 0.6 or 6 mM 2DG with or without 1 mM mannose and incubated for 24 (A), 48 (B), and 72 (C) hrs. Apoptosis (determined by TUNEL assay) was significantly induced upon 2DG treatment in a dose and time dependent manner at all time points tested. The pro-apoptotic effects of 2-DG were reversed by mannose treatment of HUVECs. Results are presented as percentage of TUNEL positive cells over total cells, normalized to untreated controls (+/− 95% CI). Experiments were performed in triplicate and repeated twice. * = p<0.05.
Figure 7.
2-DG inhibits in vivo angiogenesis.
A. Mice were injected with matrigel alone ((negative)control) or mixed with bFGF/VEGF and glucose (6 mM) or bFGF/VEGF and 6 mM 2-DG. Twelve days later mice were injected with FITC/dextran, plugs were removed and perfusion was determined by fluorescence. I. Matrigels mixed with bFGF/VEGF and glucose were associated with significant neovascularization (lane 2) compared to negative controls (lane-1). 2-DG, on the other hand, significantly inhibited in vivo angiogenesis, (lane 3 vs. 2). Bars represent means and 95% CIs of 5–6 mice per group. Matrigel plugs from additional mice were extracted for IHC analysis. Representative pictures of matrigel plugs stained with H&E (II, III, IV) and CD31 (V, VI, VII) are presented. II, V: Negative control. III, VI: positive control; IV, VII: 2-DG. Arrows indicate microvessels. Scale bar: 20 µm. B. The effects of 2-DG on tumor angiogenesis in vivo were evaluated in the HBETATAG model of retinoblastoma. 2-DG was administered intraperitoneally (3 times per week, for 5 weeks) to tumor bearing mice as described in materials and methods. At the end of the treatment period, mice were euthanized and retinal tumors were extracted for analysis of tumor vasculature (lectin staining). I: Tumors in mice treated with 2-DG had a significant reduction of tumor microvessels (measured by quantification of lectin fluorescence) compared to saline treated mice (p<0.001). MFI = mean fluorescence intensity. Bars represent means and 95% CIs of at least 4 independent samples per group. II–VII: Representative fluorescent pictures of retinal tumors from mice treated with saline (II, III, IV), and with 2-DG (V, VI, VII). II, V: DAPI staining. III, VI: Lectin staining III, VII: Overlay. Arrows indicate microvessels. Scale bar: 10 µm.