2-deoxyglucose-induced toxicity is regulated by Bcl-2 family members and is enhanced by antagonizing Bcl-2 in lymphoma cell lines

Abstract

Targeting altered cancer cell metabolism with the glycolysis inhibitor, 2-deoxyglucose (2DG), is a viable therapeutic strategy, but the effects of 2DG on lymphoma cells and the mechanism of action are unknown. Five T-cell lymphoma lines and two B-cell lymphoma lines were shown to be highly sensitive to 2DG. Examination of the cell death pathway demonstrated pro-apoptotic protein Bax 'activation' and caspase cleavage in 2DG-treated cells. However, Q-VD-OPh, a potent inhibitor of caspase activity provided minimal protection from death. In contrast, overexpressing the anti-apoptotic protein Bcl-2 dramatically enhanced the survival of 2DG-treated cells that was negated by a Bcl-2 antagonist. BH3-only members, Bim and Bmf, were upregulated by 2DG, and shRNAs targeting Bim protected from 2DG toxicity demonstrating that Bim is a critical mediator of 2DG toxicity. 2DG also induced GADD153/CHOP expression, a marker of endoplasmic reticulum (ER) stress and a known activator of Bim. Mannose, a reagent known to alleviate ER stress, transiently protected from 2DG-induced cell death. Examination of the effects of 2DG on energy metabolism showed a drop in ATP levels by 30 min that was not affected by either Bcl-2 or mannose. These results demonstrate that ER stress appears to be rate limiting in 2DG-induced cell death in lymphoma cells, and this cell killing is regulated by the Bcl-2 family of proteins. Bcl-2 inhibition combined with 2DG may be an effective therapeutic strategy for lymphoma.

Figure 1. TCLs are uniformly sensitive to 2DG treatment

(A) Mouse T cell lymphoma cells (TCLs) were plated at 200,000/mL in Adv.RPMI medium with 20 mM 2DG. Viability was assessed using Guava ViaCount dye exclusion assay (0-24 h). (B) Murine thymocytes (WT Thy) and TCLs were plated at 200,000/mL in Adv.RPMI medium with 20 mM 2DG for the time indicated. Viability was assessed by Guava ViaCount dye exclusion assay. Data represent average viabilities ± S.D. (C) TCL1 and TCL3 were treated with 20 mM 2DG and with 50 μM Q-VD-OPh (qVD) for the times indicated. caspase 3 and PARP cleavage were assessed by Western blot. U = uncleaved, C = cleaved. (D) Cells were treated with 20 mM 2DG and with 50 μM Q-VD-OPh (qVD) as indicated. Viability was assessed using Guava ViaCount dye exclusion assay at the times indicated.

Figure 2. Bax is activated following 2DG treatment

TCLs were treated with 2DG (20 mM) and with Q-VD-OPh (50 μM, qVD) as indicated. After 4 h (TCL3) or 6 h (TCL1) cells were labeled for the active conformation of Bax and analyzed by flow cytometry as described in methods. Histograms of the following samples are shown: 2DG treated cells stained with isotype control antibody (shaded gray), untreated cells stained with Anti-Bax (thin black line), 2DG treated cells stained with Anti-Bax (thick black line), 2DG+qVD treated cells stained with Anti-Bax (thick dotted gray line).

Figure 3. Bcl-2 expression protects from 2DG toxicity

(A) Western blot of TCL1 and TCL3 following lentiviral transfection with either Bcl-2 Luciferase plasmid or Luciferase control plasmid (as described in methods). Control (ctrl) and TCL-Bcl-2 cells (Bcl-2) were treated with 20 mM 2DG and viability was assessed by Guava ViaCount dye exclusion assay at the times indicated. Data represent average viabilities ± S.D. (B) Clonogenic survival of control (ctrl) and TCL-Bcl-2 (Bcl-2) lines was measured following 24 h 2DG (20 mM) treatment as described in methods. Data represent average calculations of CFU/well from three 96-well plates ± S.D. (C) TCLs were treated with 20 mM 2DG (TCL1, TCL1-Bcl-2 for 6 h; TCL3, TCL3-Bcl-2 for 4 h), harvested, fixed, and stained to examine Bax activation as described in Fig. 2 and the methods. Histograms of the following samples are shown: Untreated TCLs (thin black line), 20 mM 2DG treated TCLs (thick black line), 20 mM 2DG treated TCL-Bcl-2 cells (thick gray line). (D) TCLs and TCL-Bcl-2 cells were treated with 20 mM 2DG for the time indicated and ATP levels were measured using a bioluminescent cell assay kit as described in methods. Data represent average ATP levels ± S.D.

Figure 4. ABT-737, a Bcl-2 antagonist, restores and induces sensitivity to 2DG

(A) TCLs with Bcl-2 overexpression were treated as indicated with 2DG and ABT-737 (0.5 μM). Viability was assessed by Guava ViaCount at the times indicated. Data represent average viabilities ± S.D. (B) Non-transduced TCLs were treated as indicated with 2DG and ABT-737 (0.5 μM) for 24 hours. Viability was assessed by Guava ViaCount. Data represent average viabilities ± S.D.

Figure 5. ABT-737 inhibits 2DG-caused binding of Bim and Bmf BH3-only proteins to Bcl-2

(A) TCL cells were treated with 2DG (20 mM) for 6 hours as indicated. Protein levels were assessed by Western blot. (B) TCL-Bcl-2 cells were treated with 2DG (20 mM) for 24 hours as indicated. Protein levels were verified by Western blot. (C) TCL-Bcl-2 cells were treated with 2DG (20 mM) for 24 hours and with 0.5 μM ABT-737 for the final 3h as indicated. Immunoprecipitation was done with Bcl-2 antibody as described in methods. Protein levels were verified by Western blot. B = beads, L = lysates, S = supernatant.

Figure 6. Downregulation of Bim protein inhibits 2DG toxicity

(A) Cells were transduced with shBIM retroviral plasmids. Bim protein levels were assessed by Western blot and actin was used as a loading control. The ratios of Bim to actin levels were determined by UVP (methods) and the untreated vector control cells were assigned a normalized ratio of 1.0. (B) Cells were treated with various concentrations of 2DG and viability was monitored by Guava ViaCount at the time indicated. The duration of 2DG treatment was individually selected for each line such that control cells would be expected to be between 30 and 70% viable at that time point. Data represent average viabilities ± S.D.

Figure 7. Bim upregulation and ER stress following 2DG treatment

(A) Cells were treated with 20 mM 2DG and 20 mM mannose as indicated. Viability was monitored by Guava ViaCount. Data represent average viabilities ± S.D. (B) Cells were treated with 20 mM 2DG and/or 20 mM mannose for 6 h as indicated. mRNA levels of Gadd153 and Gadd34 are expressed relative to the levels in untreated samples. Data represent average mRNA levels ± S.D. from three independent samples. (C) Cells were treated with 20 mM 2DG, 20 mM mannose or 5 μM tunicamycin (TM; a positive control for ER stress) for 6 h as indicated. Splicing of Xbp1 mRNA was assessed by conventional RT-PCR, with spliced (S) and unspliced (Us) forms indicated. Replicate samples are shown. (D) Cells were treated with 20 mM 2DG and 20 mM mannose for 6 h as indicated. Protein levels were determined by Western blot as described in the methods section. ATP levels were measured as described in methods. Data represent average ATP levels ± S.D. (E) Cells were treated with 20 mM 2DG and 20 mM mannose for 6 h and 24 h as indicated. Protein levels were assessed by Western blot and ATP levels were measured as in (D).

Figure 8. B cell lymphoma cell lines are sensitive to 2DG treatment

Mouse B cell lymphoma cell lines were treated with various concentrations of 2DG either alone (Ctrl) or in combination with 2 μM ABT-737 (ABT) or 2 mM mannose (Mann) as indicated. Viability was assessed by PI exclusion 18 h after the treatment.