Inhibition of glycolysis modulates prednisolone resistance in acute lymphoblastic leukemia cells - PubMed (original) (raw)

Inhibition of glycolysis modulates prednisolone resistance in acute lymphoblastic leukemia cells

Esther Hulleman et al. Blood. 2009.

Abstract

Treatment failure in pediatric acute lymphoblastic leukemia (ALL) is related to cellular resistance to glucocorticoids (eg, prednisolone). Recently, we demonstrated that genes associated with glucose metabolism are differentially expressed between prednisolone-sensitive and prednisolone-resistant precursor B-lineage leukemic patients. Here, we show that prednisolone resistance is associated with increased glucose consumption and that inhibition of glycolysis sensitizes prednisolone-resistant ALL cell lines to glucocorticoids. Treatment of prednisolone-resistant Jurkat and Molt4 cells with 2-deoxy-D-glucose (2-DG), lonidamine (LND), or 3-bromopyruvate (3-BrPA) increased the in vitro sensitivity to glucocorticoids, while treatment of the prednisolone-sensitive cell lines Tom-1 and RS4; 11 did not influence drug cytotoxicity. This sensitizing effect of the glycolysis inhibitors in glucocorticoid-resistant ALL cells was not found for other classes of antileukemic drugs (ie, vincristine and daunorubicin). Moreover, down-regulation of the expression of GAPDH by RNA interference also sensitized to prednisolone, comparable with treatment with glycolytic inhibitors. Importantly, the ability of 2-DG to reverse glucocorticoid resistance was not limited to cell lines, but was also observed in isolated primary ALL cells from patients. Together, these findings indicate the importance of the glycolytic pathway in glucocorticoid resistance in ALL and suggest that targeting glycolysis is a viable strategy for modulating prednisolone resistance in ALL.

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Figures

Figure 1

Figure 1

Glycolysis is up-regulated in prednisolone-resistant human leukemia cells. Graphic representation of in vitro prednisolone responsiveness (left panel) and glucose consumption (right panel) of 2 prednisolone-resistant and 2 prednisolone-sensitive human ALL cell lines. Response to prednisolone was measured by the MTT assay; glucose consumption was calculated per cell by measuring the conversion of glucose to 6-phosphogluconate. The glucose consumption in Jurkat cells after 4 days of incubation was set to be 100%, corresponding to approximately 75% of the total glucose present in the medium (1.5 g/L). A representative experiment is shown; data are presented as means plus or minus SD (n = 3).

Figure 2

Figure 2

Schematic representation of glucose metabolism in mammalian cells. Glycolytic inhibitors used in this study are indicated as 2-DG, 3-BrPA, and LND.

Figure 3

Figure 3

Effect of 2-DG treatment on glucose consumption and prednisolone- induced cytotoxicity in human ALL cell lines. Graphic representation of relative glucose consumption (A) or in vitro prednisolone responsiveness (B) in 2 prednisolone-resistant and 2 prednisolone-sensitive ALL cell lines after 2-DG treatment. Glucose consumption was calculated by measuring the conversion of glucose to 6-phosphogluconate, and glucose consumption in Jurkat cells was set at 100%. Response to prednisolone was measured by the MTT assay; cell survival in nontreated cells was set at 100%. Used concentrations of prednisolone and 2-DG varied depending on cellular toxicity (550 μg/mL and 0.078 μg/mL prednisolone, and 2 mM and 0.5 mM for -resistant and -sensitive cell lines, respectively). Representative experiments are shown; data are presented as means plus or minus SD (n = 3).

Figure 4

Figure 4

Modulation of drug-resistance by 2-DG. Graphic representation of in vitro responsiveness to cytotoxic drugs in 2 prednisolone-resistant ALL cell lines after 2-DG treatment, as assessed by the MTT assay. Concentrations used were 1 mM (Molt4) or 2 mM 2-DG (Jurkat), 550 μg/mL prednisolone, 100 μg/mL dexamethasone, 0.5 ng/mL vincristine, and 0.0098 U/mL L-asparaginase. Cell survival in cells treated only with 2-DG was set at 100% to visualize the effect of synergy. Representative experiments are shown; data are presented as means plus or minus SD (n = 3).

Figure 5

Figure 5

Effect of glycolytic inhibitors on prednisolone-induced cytotoxicity in prednisolone–resistant and prednisolone-sensitive cell lines. Cell survival curves representing in vitro prednisolone responsiveness, as assessed by the MTT assay in 2 prednisolone-resistant (top panel) and 2 prednisolone-sensitive ALL cell lines (bottom panel) after treatment with glycolytic inhibitors. Cells were treated with 1 mM (Jurkat, Molt4) or 0.5 mM 2-DG (▲), 62.5 μM LND (formula image), 30 μM 3-BrPA (●) in combination with prednisolone, as indicated in the figure. Cells treated only with prednisolone served as controls (■). To visualize synergy, the survival rate of prednisolone in combination with an inhibitor was corrected for the toxicity caused by the inhibitor itself (set to 100%). A representative experiment is shown; data are presented as means plus or minus SD (n = 3).

Figure 6

Figure 6

Inhibition of GAPDH expression by RNA interference increases prednisolone-induced cytotoxicity. (A) mRNA levels of GAPDH in Jurkat cells as measured by qPCR. Two different shRNA sequences targeting GAPDH were used (shGAPDH-1 and -2). Expression of cells infected with nonsilencing shRNA sequences was set at 100% and relative expression levels were calculated. (B) Cell survival curves representing in vitro prednisolone responsiveness after RNA interference in cells interfered for GAPDH (formula image, ▲) or in cells infected with a nonsilencing shRNA sequence (■). (C) mRNA levels of HIF-1α in Jurkat cells as measured by qPCR. Two different shRNA sequences targeting HIF-1α were used (sh HIF-1α-1 and -2). Expression of cells infected with nonsilencing shRNA sequences was set at 100%, and relative expression levels were calculated. (D) Cell survival curves representing in vitro prednisolone responsiveness after RNA interference in cells interfered for HIF-1α (formula image, ▲) or in cells infected with a nonsilencing shRNA sequence (■). A representative experiment is shown; data are presented as means plus or minus SD (n = 3).

Figure 7

Figure 7

Effect of 2-DG treatment on prednisolone-induced cytotoxicity in primary ALL cells. Cell survival curves representing in vitro prednisolone responsiveness, in prednisolone-resistant ALL patients (top panel), -intermediate patients (middle panel), and -sensitive patients (bottom panel) as assessed by the MTT assay. Concentrations 2-DG varied from 0.2 mM to 2 mM (▼), depending on the patient sample sensitivity. To visualize the synergy, the survival of cells treated with prednisolone and 2-DG was corrected for the toxic effect of 2-DG given as single drug (set at 100%). Cells treated only with prednisolone served as controls (■). A representative experiment is shown; data are presented as means plus or minus SD (n = 3).

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