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3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs - PubMed

3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs

Linda Strandberg Ihrlund et al. Mol Oncol. 2008 Jun.

Abstract

Tumour cells depend on aerobic glycolysis for adenosine triphosphate (ATP) production, making energy metabolism an interesting therapeutic target. 3-Bromopyruvate (BP) has been shown by others to inhibit hexokinase and eradicate mouse hepatocarcinomas. We report that similar to the glycolysis inhibitor 2-deoxyglucose (DG), BP rapidly decreased cellular ATP within hours, but unlike DG, BP concomitantly induced mitochondrial depolarization without affecting levels of reducing equivalents. Over 24h, and at equitoxic doses, DG reduced glucose consumption more than did BP. The observed BP-induced loss of ATP is therefore largely due to mitochondrial effects. Cell death induced over 24h by BP, but not DG, was blocked by N-acetylcysteine, indicating involvement of reactive oxygen species. BP-induced cytotoxicity was independent of p53. When combined with cisplatin or oxaliplatin, BP led to massive cell death. The anti-proliferative effects of low-dose platinum were strikingly potentiated also in resistant p53-deficient cells. Together with the reported lack of toxicity, this indicates the potential of BP as a clinical chemopotentiating agent.

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Figures

Figure 1
Figure 1

Early effects of 2‐deoxyglucose and 3‐bromopyruvate on ATP and reducing equivalents. (A) HCT116 cells were treated with the indicated concentrations of 2‐deoxyglucose (DG) and 3‐bromopyruvate (BP) for 5h. Cellular ATP levels were then determined using the Aposensor ATP Assay kit (Alexis Biochemicals) and are expressed as fold signal in untreated control samples. Data represent the averages of three independent experiments. Bars indicate standard error of the mean (SEM). (B) HCT116 cells were treated with the indicated concentrations of DG and BP for 3h, and were then subjected to the MTT reduction assay (Promega) to assess levels of reducing equivalents (NAD(P)H) to reflect glycolytic activity. Results are shown as fold signal in untreated control samples. Data represent the averages of three independent experiments, except with 2.4mM BP, which was done twice. Bars indicate standard error of the mean (SEM). (C) HCT116 cells were treated with the indicated concentrations of DG and BP for 3 and 6h, and the mitochondrial inner membrane potential was then examined using the fluorescent TMRE dye which is taken up only by mitochondria with intact membrane potential. The experiment was performed three times and assessment of the M1 populations yielded a statistically significant difference (p=0.04; Student's t‐test) between 40mM DG and 50μM BP treatments at 3h.

Figure 2
Figure 2

Effects of 2‐deoxyglucose and 3‐bromopyruvate on 24‐h survival and glucose consumption. (A) HCT116 cells were treated in 96‐well plates with the indicated drugs and concentrations for 24h, after which the amount of surviving cells in each well was quantitated as total protein using the sulphorhodamine B spectrophotometric assay (Sigma–Aldrich). Data are represented by black bars showing percent survival compared to untreated controls. Glucose concentrations in the supernatants were assessed using the QuantiChrome Glucose Assay (BioVision), and the amount of glucose consumed over 24h was calculated for each well. Data are represented by grey bars showing glucose consumption relative to the amount consumed in untreated controls. Data are the averages of two independent experiments with duplicate samples in each. (B) Cells were treated with DG or BP at indicated concentrations in the absence (black bars) or presence (grey bars) of N‐acetylcysteine (NAC, 5mM), and the resulting survival levels were assessed after 24h using the sulphorhodamine B assay. Data are the averages of two independent experiments with duplicate samples in each.

Figure 3
Figure 3

Anti‐proliferative effects of 2‐deoxyglucose and 3‐bromopyruvate. (A) HCT116 cells were treated with 20mM DG or 50μM BP for 18h, after which time samples were prepared for cell cycle analysis. Data represent the averages of two separate experiments. (B) Four different human tumour cell lines were treated for 24h with 20mM DG or 50μM BP. Accumulated apoptosis was then quantitated using the Apoptosense assay (PEVIVA, Sweden). Data represent averages±SEM of at least three experiments per cell line. (C) HCT116 cells were treated with 20mM DG for 48h, and low‐level cell death (upper right quadrant) was confirmed using the propidium iodide/annexin‐V assay by flow cytometry, while 50μM BP for 48h led to near complete loss of viable cells (lower left quadrant). (D) HCT116 wild‐type‐p53 and HCT116 p53−/− cells were treated with the indicated concentrations of DG and BP for 48h, after which time survival was assessed by cell counting. Data represent averages±SEM of three separate experiments.

Figure 4
Figure 4

Potentiation of platinum drugs by BP. (A) HCT116 cells were treated with 5μM cisplatin (cDDP) or 5μM oxaliplatin (OXP) in the presence or absence of 25μM BP. To assess induction of necrosis, released and accumulated LDH, and LDH remaining in the surviving cells were both quantified separately, using the LDH release assay (Promega). Results are expressed as fold release compared to untreated controls (filled bars; left‐side y‐axis). Cumulative levels of apoptosis in total lysates (supernatant plus cells) after similar treatment were assessed using the Apoptosense assay (PEVIVA) and expressed as fold increase compared to untreated controls (empty bars; right‐side y‐axis). The scale on the right‐side y‐axis was chosen in order to let 10‐fold induction represent a “maximum” in the sense that this is apoptosis induction by cisplatin at 20μM, i.e., a near‐maximal clinically attainable concentration. Data represent the average of two experiments with triplicate or quadruplicate samples in each. (B, C) BP‐induced potentiation of overall anti‐proliferative effects of 3μM cisplatin and 1μM oxaliplatin, respectively, was examined after 48h in HCT116 p53‐wild‐type (B) and in HCT116 p53‐deficient (C) cells. The numbers of remaining cells were counted using a Burker chamber, and the results are expressed as percent of numbers in untreated controls. Data represent averages±SEM of three separate experiments.

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