Inhibition of Glutamate Cysteine Ligase Activity Sensitizes Human Breast Cancer Cells to the Toxicity of 2-Deoxy-d-Glucose

Abstract

It has been hypothesized that cancer cells increase glucose metabolism to protect against metabolic fluxes of hydroperoxides via glutathione-dependent peroxidases. 2-Deoxy-D-glucose, inhibits glucose metabolism and has been shown to cause cytotoxicity in cancer cells that is partially mediated by disruptions in thiol metabolism. In the current study, human breast cancer cells were continuously treated (24 hours) with 2-deoxy-D-glucose, and total glutathione content as well as the expression of the first enzyme in the glutathione synthetic pathway [glutamate cysteine ligase (GCL)] were found to be induced 2.0-fold. Inhibiting GCL activity during 2-deoxy-Dglucose exposure using L-buthionine-[S ,R]-sulfoximine (BSO) significantly enhanced the cytotoxic effects of 2-deoxy-Dglucose and caused increases in endpoints indicative of oxidative stress, including % oxidized glutathione and steadystate levels of pro-oxidants as assayed using an oxidationsensitive fluorescent probe. These results show that treatment of human breast cancer cells with 2-deoxy-D-glucose causes metabolic oxidative stress that is accompanied by increases in steady-state levels of GCL mRNA, GCL activity, and glutathione content. Furthermore, inhibition of 2-deoxy-D-glucosemediated induction of GCL activity with BSO increases endpoints indicative of oxidative stress and sensitizes cancer cells to 2-deoxy-D-glucose-induced cytotoxicity. These results support the hypothesis that drug combinations capable of inhibiting both glucose and hydroperoxide metabolism may provide an effective biochemical strategy for sensitizing human cancer cells to metabolic oxidative stress. (Cancer Res 2006; 66(3): 1605-10)

2-Deoxyglucose combined with wild-type p53 overexpression enhances cytotoxicity in human prostate cancer cells via oxidative stress

“…Samples were thawed and whole homogenates were prepared as described [3,4,15,22,23]. Total glutathione (GSH+GSSG), and glutathione disulfide (GSSG) were determined using a spectrophotometric recycling assay [3,4,15,22,23].…”

“…2-Deoxy-d-glucose competitively inhibits metabolism of glucose and has been suggested to be selectively cytotoxic to fully transformed cells [3], via a mechanism that involves hydroperoxide-mediated oxidative stress [4]. Since glucose is a major source of electrons for hydroperoxide metabolism [15][16][17][18] and tumor cells are believed to produce relatively high steady-state levels of hydroperoxides [19], the mechanism by which 2DG enhances oxidative stress in cancer cells was suggested to involve limitation of hydroperoxide detoxification [4].…”

“…Since glucose is a major source of electrons for hydroperoxide metabolism [15][16][17][18] and tumor cells are believed to produce relatively high steady-state levels of hydroperoxides [19], the mechanism by which 2DG enhances oxidative stress in cancer cells was suggested to involve limitation of hydroperoxide detoxification [4]. Recently p53-induced apoptosis in cancer cells has been shown to occur through induction of p53-induced genes (PIGs) and the tumor suppressive effects of some PIGs are thought to be the result of increased formation of reactive oxygen species and mitochondrial respiration (ROS) (i.e.…”

“…O 2 •-and H 2 O 2 ), which in turn lead to oxidative stress [1,2,9,10,20]. Since 2DG would be expected to inhibit the formation of pyruvate and NADPH, which function in the detoxification pathways of H 2 O 2 [3,4,15], we hypothesized that the combination of 2DG and wt p53 over expression would lead to increased steady-state levels of ROS and enhanced cell killing by oxidative stress. In the current report, the interaction between 2DG and p53 gene therapy in two androgen-independent prostate cancer cell lines (DU-145 and PC-3) was evaluated in vitro.…”

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AbstractOver expression of the tumor suppressor gene, wild type p53 (wtp53), using adenoviral vectors (Adp53) has been suggested to kill cancer cells by hydroperoxide-mediated oxidative stress [1,2] and nutrient distress induced by the glucose analog, 2-deoxyglucose (2DG), has been suggested to enhance tumor cell killing by agents that induce oxidative stress via disrupting hydroperoxide metabolism [3,4]. In the current study clonogenic cell killing of PC-3 and DU-145 human prostate cancer cells (lacking functional p53) mediated by 4 h exposure to 50 plaque forming units (pfus)/ cell of Adp53 (that caused the enforced over expression of wtp53) was significantly enhanced by treatment with 2DG.

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