Assessing Oxidative Stress in Tumors by Measuring the Rate of Hyperpolarized [1-13C]Dehydroascorbic Acid Reduction Using 13C Magnetic Resonance Spectroscopy

Abstract

Rapid cancer cell proliferation promotes the production of reducing equivalents, which counteract the effects of relatively high levels of reactive oxygen species. Reactive oxygen species levels increase in response to chemotherapy and cell death, whereas an increase in antioxidant capacity can confer resistance to chemotherapy and is associated with an aggressive tumor phenotype. The pentose phosphate pathway is a major site of NADPH production in the cell, which is used to maintain the main intracellular antioxidant, glutathione, in its reduced state. Previous studies have shown that the rate of hyperpolarized [1-¹³C]dehydroascorbic acid (DHA) reduction, which can be measured in vivo using non-invasive ¹³C magnetic resonance spectroscopic imaging, is increased in tumors and that this is correlated with the levels of reduced glutathione. We show here that the rate of hyperpolarized [1-¹³C]DHA reduction is increased in tumors that have been oxidatively prestressed by depleting the glutathione pool by buthionine sulfoximine treatment. This increase was associated with a corresponding increase in pentose phosphate pathway flux, assessed using ¹³C-labeled glucose, and an increase in glutaredoxin activity, which catalyzes the glutathione-dependent reduction of DHA. These results show that the rate of DHA reduction depends not only on the level of reduced glutathione, but also on the rate of NADPH production, contradicting the conclusions of some previous studies. Hyperpolarized [1-¹³C]DHA can be used, therefore, to assess the capacity of tumor cells to resist oxidative stress in vivo However, DHA administration resulted in transient respiratory arrest and cardiac depression, which may prevent translation to the clinic.

Keywords: 13C; dehydroascorbic acid; glutathione; glutathione peroxidase; hyperpolarization; in vivo imaging; oxidative stress; pentose phosphate pathway (PPP); tumor metabolism.

FIGURE 1.

PPP flux in oxidant-treated EL4 cells. a, DHA is reduced to AA by GSH, catalyzed by Grx, GST, and PDI and by NADPH-dependent reactions catalyzed by TrxR and 3α-hydroxysteroid dehydrogenase (3α-HSD). Reduction of GSSG is catalyzed by glutathione reductase (GR). 6PGL, 6-phosphogluconolactone; 6PGLase, 6-phosphogluconolactonase; LDH, lactate dehydrogenase. b–d, ratio of lactate singly labeled at C3 to the total labeled lactate concentration. Cells were incubated for 30 min with 11 mm [1,2-¹³C]glucose and 50 μm phenazine methosulfate (PMS) (b), 1 mm H₂O₂ (c), or 11 mm DHA (d). e–g, ratio of ¹³C to ¹²C-labeled 6PG in cells that were incubated for 30 s with 11 mm [U-¹³C]glucose and had been incubated previously for 30 min with 50 μm PMS (e), 1 mm H₂O₂ (f), or 11 mm DHA (g). Error bars, S.D. (n = 3). *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

FIGURE 2.

PPP flux in EL4 tumors treated with DHA. EL4 tumor-bearing mice were injected with 0.4 ml of 200 mm [1,2-¹³C₂]glucose (n = 4) or 0.4 ml of 200 mm [1,2-¹³C₂]glucose and 28 mm DHA (n = 3). Lactate labeling was measured in tissue extracts by ¹³C NMR. a, ratio of lactate singly labeled at C3 to total labeled lactate. b, ratio of ¹³C to ¹²C-labeled 6PG in control (Ctrl) EL4 tumors (n = 4) injected with 0.4 ml of 200 mm [U-¹³C]glucose and EL4 tumors injected simultaneously with 28 mm DHA and 0.4 ml of 200 mm [U-¹³C]glucose (n = 6). c, ¹³C spectrum of hyperpolarized [1-¹³C]lactate (185.1 ppm) and [1-¹³C]6PG (181.4 ppm) in a control EL4 tumor 15 s after the start of injection of 0.4 ml of 200 mm hyperpolarized [U-²H,U-¹³C]glucose. d, untreated mice (Ctrl, n = 5) or mice pretreated 3 min earlier with 28 mm DHA (n = 5) before injection of hyperpolarized [U-²H,U-¹³C]glucose (200 mm, 0.4 ml). The ratio of the sum of the intensities of the [1-¹³C]6PG resonance to the sum of the intensities of the [1-¹³C]lactate resonance in the first nine spectra is shown. Error bars, S.D. **, p < 0.01.

FIGURE 3.

PPP flux in BSO-treated EL4 and Colo205 cells and tumors. The ratio of lactate singly labeled at C3 to total labeled lactate concentration in EL4 cells (10⁸) (a) and Colo205 cells (10⁷) (b) treated with 100 μm BSO and then incubated in RPMI medium containing 11 mm [1,2-¹³C₂]glucose with or without 11 mm DHA for 30 min. EL4 (e) and Colo205 (f) tumors were treated with 500 mg kg⁻¹ BSO and injected with 0.4 ml of 200 mm [1,2-¹³C₂]glucose and freeze-clamped 4 min later. Shown is the ratio of ¹³C- to ¹²C-labeled 6PG in extracts of EL4 (c) and Colo205 (d) cells treated with 100 μm BSO and incubated for 30 s with 11 mm [U-¹³C]glucose with or without 11 mm DHA. Shown are EL4 (g) and Colo205 (h) tumors treated with 500 mg kg⁻¹ BSO and then injected with 0.4 ml of 200 mm [U-¹³C]glucose and freeze-clamped 1 min later. Error bars, S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 4.

DHA reduction rate in EL4 and Colo205 tumors. a, chemical shift images of [1-¹³C]DHA and [1-¹³C]AA in an EL4 tumor-bearing mouse 27 s after injecting 0.2 ml of 28 mm hyperpolarized [1-¹³C]DHA. Color intensities were normalized to the respective maxima in the images. b–e, ¹³C spectroscopic measurements of DHA reduction in EL4 and Colo205 tumors treated with 500 mg kg⁻¹ BSO and then injected with 0.4 ml of 28 mm hyperpolarized [1-¹³C]DHA 6 or 24 h later. Spectra were acquired 15 s after the start of injection. Representative spectra, which are the sum of the 15 spectra acquired during the first 15 s, are shown for a control EL4 tumor (b) and an EL4 tumor 24 h after BSO treatment (c). The signal at 175.3 ppm is from [1-¹³C]DHA, and the signal at 179 ppm is from [1-¹³C]AA. Shown is the ratio of the sum of the hyperpolarized [1-¹³C]DHA and [1-¹³C]AA signals in the first 15 s of data acquisition (15 spectra) for EL4 tumors (d) and Colo205 tumors (e). Error bars, S.D. *, p < 0.05.

FIGURE 5.

Respiratory rate (a) and heart rate (b) of C57BL/6 mice (no injection, *) or injected with dissolution buffer (○) or 10 (▾), 25 (▴), 50 (♦), or 62.5 mg kg⁻¹ (■) DHA, produced by charcoal oxidation of ascorbic acid or 50 mg kg⁻¹ DHA from Sigma-Aldrich (●). Each condition was tested in a separate mouse. The black lines indicate time of injection.

FIGURE 6.

Oxidation of hyperpolarized [1-¹³C]AA in EL4 tumor cell suspensions. Shown is a representative time course of ¹³C signals from hyperpolarized [1-¹³C]AA (179.0 ppm) and [1-¹³C]DHA (175.3 ppm) in RPMI medium (a) and in a suspension of 10⁸ (2.5 × 10⁷ ml⁻¹) intact (b) or lysed (c) EL4 cells.