2-Deoxyglucose, an Inhibitor of Glycolysis, Enhances the Oncolytic Effect of Coxsackievirus

Abstract

Glioblastoma multiforme (GBM) is one of the most common types of brain tumor. Despite intensive research, patients with GBM have a poor prognosis due to a very high rate of relapse and significant side effects of the treatment, with a median survival of 14.6 months. Oncolytic viruses are considered a promising strategy to eliminate GBM and other types of cancer, and several viruses have already been introduced into clinical practice. However, identification of the factors that underly the sensitivity of tumor species to oncolytic viruses or that modulate their clinical efficacy remains an important target. Here, we show that Coxsackievirus B5 (CVB5) demonstrates high oncolytic potential towards GBM primary cell species and cell lines. Moreover, 2-deoxyglucose (2DG), an inhibitor of glycolysis, potentiates the cytopathic effects of CVB5 in most of the cancer cell lines tested. The cells in which the inhibition of glycolysis enhanced oncolysis are characterized by high mitochondrial respiratory activity and glycolytic capacity, as determined by Seahorse analysis. Thus, 2-deoxyglucose and other analogs should be considered as adjuvants for oncolytic therapy of glioblastoma multiforme.

Keywords: 2-deoxyglucose; coxsackievirus; metabolism; oncolytic virus; seahorse.

Figure 1

CVB5 exhibits the highest oncolytic activity towards glioblastoma multiforme (high grade) cell lines, stable glioblastoma cell lines: DBTRG-05MG (a), U251-MG (e), primary gliomas: GBM3821 (f), GBM5522 (b), GBM6067 (c), GBM6138 (d), and Hela cell line (h), as cancerous non-glioma cell line, and HEF (g) as non-cancerous cell line. The cells were seeded 24 h prior to infection and inoculated with MOIs from 0.001–1000 with polioviruses type 1–3 (PV1–3), Coxsackievirus A (CVA), or B (CVB). Cell viability was assessed 48 h later by MTT test. The depicted values are means ± S.D.

Figure 2

Cytotoxicity of metabolic inhibitors towards glioblastoma cell lines assessed using CellTracker reagent. Cells were seeded 24 h before exposure to increasing concentrations of 2DG (a), metformin (b), or phenformin (c). Cell viability was assessed 48 h after drug addition using CellTracker reagent.

Figure 3

Oncolytic activity of CVB5 towards glioblastoma cells in the presence of 2DG increased in DBTRG-05MG (a), GBM5522 (b), GBM6067 (c), GBM6138 (d) and HeLa (h), decreased in GBM3821 (f) cell line, and did not change in U251-MG (e) and HEF (g) cell line. Cells were seeded 24 h before treatment with 2, 4, and 10 mM 2DG and simultaneous infection with CVB5 at MOIs of 0.001–1000. Viability was determined using CellTracker method 48 h later, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

Oncolytic activity of CVB5 towards glioblastoma cells in the presence of metformin decreased in U251-MG (e) and HeLa (h) but not in DBTRG-05MG (a), GBM5522 (b), GBM6067 (c), GBM6138 (d), GBM3821 (f) and HEF (g) cell lines. Cells were seeded 24 h before treatment with 2, 4, and 10 mM metformin and simultaneous infection with CVB5 at MOIs of 0.001-1000. Viability was determined using CellTracker 48 h later, * p ≤ 0.05, ** p ≤ 0.01.

Figure 5

Oncolytic activity of CVB5 towards glioblastoma cells in the presence of phenformin decreased in DBTRG-05MG (a), GBM6067 (c), and HeLa (h), and increased in GBM3821 (f). In GBM5522 (b), GBM6138 (d), U251-MG (e), and HEF (g) cells it did not affect oncolytic activity of CVB5. Cells were seeded 24 h before treatment with 10, 20, and 50 µM phenformin and simultaneous infection with CVB5 at MOIs of 0.001–1000. Viability was determined using CellTracker method 48 h later, * p ≤ 0.05, ** p ≤ 0.01.

Figure 6

2DG does not affect CVB5 replication. HEK293T cells were infected with the conditioned medium from DBTRG-05MG (a), GBM5522 (b), GBM6138 (c), U251-MG (d) or GBM3821 (e) glioblastoma cells treated with 4 mM 2DG simultaneously with CVB5, harvested 48 h post-infection. Cell viability was assessed 72 h later using MTT assay. Values are means ± S.D.

Figure 7

CVB5 does not affect the glycolytic activity of glioblastoma cells. DBTRG-05MG cells were infected with CBV5 at MOI 1, and the cells were subjected to GlycoStress assay according to manufacturer’s instructions at 7 h post-infection. In GlycoStress, glucose at final concentrations of 11 and 30 mM was followed by oligomycin (1 µM) and 2DG (50 µM) addition. Depicted values are means ± S.D.

Figure 8

Enhanced oncolytic activity of CVB5 towards glioblastoma cell lines correlates with their basal mitochondrial respiration and spare respiratory capacity. Non-mitochondrial oxygen consumption (a), ATP production (b), basal (c) and maximum (e) respiration and spare respiratory capacity (d) as well as coupling efficiency (f) and proton leak (g) were assessed by the Seahorse technology in MitoStress assay. Oligomycin, (1 µM), FCCP (0.75 and 1.5 µM), and a mixture of antimycin and rotenone (1 µM each) were added. Values are means ± S.D. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 9

Enhanced oncolytic activity of CVB5 towards glioblastoma cell lines correlates with their glycolytic capacity. Basal glycolysis (a), glycolytic reserve (b) and maximum capacity (c) as well as non-glycolytic acidification (d) were assessed by the Seahorse technology in GlycoStress assay. Glucose was added to the final concentrations of 11 and 30 mM followed by addition of oligomycin (Oligo, 1 µM), and 2DG (50 µM). Values are means ± S.D, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 10

Effect of antioxidant N-acetylcysteine (NAC) on cytopathic effect of CVB5 in the presence of 2DG. DBTRG-05MG (a), GBM5522 (b), GBM6067 (c), U251-MG (d) or GBM3821 (e) cells were seeded day before treatment with 4 mM 2DG, NAC (1 or 5 mM), and simultaneous infection with CVB5 at MOIs of 0.001-1000. Viability was assessed 48 h post-infection. Values are means ± S.D. * p < 0.05.

Figure 11

Influence of 2DG on the cytopathic effects of polioviruses and Coxsackieviruses depends on the virus as well as the cell line. DBTRG (a), GBM5522 (b), GBM6067 (c), GBM6138 (d), U251-MG (e), GBM3821 (f) or HeLa (g) cells were seeded day before treatment with 4 mM 2DG and simultaneous infection with viruses at MOIs of 0.001–1000. Viability was assessed after 48 h using MTT assay. Values are means ± S.D.

Figure 12

Levels of transcription of genes that are the markers of cancer stem cells. The cells were cultivated in standard condition, and CD44 (a), CD133 (b), cMyc (c) and VEGF (d) mRNA levels were assessed by RT-qPCR, GUS mRNA levels were used for normalization. The data were analyzed using ∆∆Ct methods, normalized to HeLa cells line. Values are means ± S.D. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.