Combined inhibition of glycolysis, the pentose cycle, and thioredoxin metabolism selectively increases cytotoxicity and oxidative stress in human breast and prostate cancer - PubMed (original) (raw)

Combined inhibition of glycolysis, the pentose cycle, and thioredoxin metabolism selectively increases cytotoxicity and oxidative stress in human breast and prostate cancer

Ling Li et al. Redox Biol. 2015.

Abstract

Inhibition of glycolysis using 2-deoxy-d-glucose (2DG, 20mM, 24-48h) combined with inhibition of the pentose cycle using dehydroepiandrosterone (DHEA, 300µM, 24-48h) increased clonogenic cell killing in both human prostate (PC-3 and DU145) and human breast (MDA-MB231) cancer cells via a mechanism involving thiol-mediated oxidative stress. Surprisingly, when 2DG+DHEA treatment was combined with an inhibitor of glutathione (GSH) synthesis (l-buthionine sulfoximine; BSO, 1mM) that depleted GSH>90% of control, no further increase in cell killing was observed during 48h exposures. In contrast, when an inhibitor of thioredoxin reductase (TrxR) activity (Auranofin; Au, 1µM), was combined with 2DG+DHEA or DHEA-alone for 24h, clonogenic cell killing was significantly increased in all three human cancer cell lines. Furthermore, enhanced clonogenic cell killing seen with the combination of DHEA+Au was nearly completely inhibited using the thiol antioxidant, N-acetylcysteine (NAC, 20mM). Redox Western blot analysis of PC-3 cells also supported the conclusion that thioredoxin-1 (Trx-1) oxidation was enhanced by treatment DHEA+Au and inhibited by NAC. Importantly, normal human mammary epithelial cells (HMEC) were not as sensitive to 2DG, DHEA, and Au combinations as their cancer cell counterparts (MDA-MB-231). Overall, these results support the hypothesis that inhibition of glycolysis and pentose cycle activity, combined with inhibition of Trx metabolism, may provide a promising strategy for selectively sensitizing human cancer cells to oxidative stress-induced cell killing.

Keywords: 2-Deoxy-d-glucose; Auranofin; Buthionine sulfoximine; Cancer; Dehydroepiandrosterone; Glutathione; Oxidative stress; Pentose phosphate pathway; Thioredoxin.

Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

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Figures

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Graphical abstract

Fig. 1

Fig. 1

The pathways involving glucose and hydroperoxide metabolism believed to be involved with protection of cancer cells from metabolic oxidative stress (inhibitors of Trx and GSH metabolism are shown in italics). 2DG competes with glucose for uptake into the cells competitively inhibiting pyruvate production and the pentose cycle after glucose-6-phosphate-dehydrogenase (G6PD). DHEA inhibits G6PD. The GSH and Trx dependent systems participate in the detoxification of H2O2 and organic hydroperoxides. NADPH is a source of reducing equivalents for the Trx/GSH-dependent systems. BSO inhibits glutamate cysteine ligase (γ-GCL) preventing glutathione synthesis. Auranofin is the inhibitor of thioredoxin reductase (TrxR), which reduces the oxidized Trx to the reduced form. These inhibitors were used alone and in combination to increase the cancer cell oxidative stress, resulting in cancer cell cytotoxicity.

Fig. 2

Fig. 2

Clonogenic cell survival curves for PC-3 cells (A), DU145 cells (B), and MDA-MB-231 cells (C) treated with 2DG, DHEA, and BSO. 500,000–1,000,000 Cells were plated in 60 mm dishes. After 24 h cells were treated with 20 mM 2DG, 300 µM DHEA, and 1 mM BSO. Cells were collected for clonogenic survival assay at the 24, and 48 h after the treatment started. Each measurement represents mean±1 SD from two experiments. *p<0.001 difference versus control treatment, #p<0.001 difference versus 2DG or DHEA treatment alone. One-way ANOVA was used with Tukey's post-hoc analysis was used to test for statistical significance.

Fig. 3

Fig. 3

Effect of 2DG, DHEA and BSO treatment on glutathione levels in PC-3, DU145, and MDA-MB-231 cells. 1,000,000 Cells were plated into 100 mm dishes and after 24 h treated with 20 mM 2DG, 300 µM DHEA, and 1 mM BSO. At the end of 24 and 48 h total glutathione (A) and glutathione disulfide (GSSG) (B) were measured using spectrophotometric recycling assay. One way ANOVA with least significant difference (LSD) post-hoc analysis was performed resulting in *P<0.05, compared to control at the same time point.

Fig. 4

Fig. 4

Au treatment enhances cancer cell killing from 2DG and DHEA treatment in PC-3 (A), DU145 (B), and MDA-MB-231 (C) cells. Cells were plated in 60 mm dishes and treated with 2DG and DHEA as before with the addition of 1 µM Au for 24 h after which clonogenic cell survivals assay was performed. One-way ANOVA was used with Tukey's post-hoc analysis resulted in *p<0.001 differences versus control, #p<0.01 versus all other experimental conditions without Au.

Fig. 5

Fig. 5

NAC rescues cancer cells from the cytotoxicity induced by combined DHEA and Au. Cells were plated and treated as above with the addition of 20 mM NAC for 24 h to select dishes followed by the clonogenic survival assay for PC-3 (A) and MB231 (B). Results were normalized to control and error bars represent mean±1SD from two different experiment treatment dishes. Each treatment dish was then plated into at least three cloning dishes each. One-way ANOVA with Tukey's post-hoc analysis resulted in *p<0.001 versus control, #p<0.01 versus treatment without NAC. After 24 h treatment, PC-3 cells were also harvested for redox western blotting for oxidized and reduced Trx-1 as described in methods (_N_=4) (C). DTT and H2O2 were added during the final 10 min of incubation as positive and negative controls but were not included in the quantitative analysis (C). Quantification of the treatment groups included in the immuno-blot was performed using ImageJ (_N_=4) (D). One-way ANOVA followed by the LSD post-hoc analysis was used to test for statistical significance (*p<0.05).

Fig. 6

Fig. 6

Effect of 2DG, DHEA and Au on normal breast HMEC cells versus breast cancer MDA-MB-231 cells. Cells were plated and 24 h later treated with 20 mM 2DG and 300 µM DHEA for 17–18 h. 1 µM Au was added 15 min before cells were collected for clonogenic assay. One-way ANOVA was used with Tukey's post-hoc analysis resulted in *p<0.001 when MD-MB231 cells are compared with the same treatment groups in HMEC cells.

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