Enhancement of Radiation Response in Breast Cancer Stem Cells by Inhibition of Thioredoxin- and Glutathione-Dependent Metabolism - PubMed (original) (raw)

. 2016 Oct;186(4):385-395.

doi: 10.1667/RR14463.1. Epub 2016 Sep 19.

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Enhancement of Radiation Response in Breast Cancer Stem Cells by Inhibition of Thioredoxin- and Glutathione-Dependent Metabolism

Samuel N Rodman et al. Radiat Res. 2016 Oct.

Abstract

The goal of this study was to determine if depletion of glutathione (GSH) and inhibition of thioredoxin (Trx) reductase (TrxR) activity could enhance radiation responses in human breast cancer stem cells by a mechanism involving thiol-dependent oxidative stress. The following were used to inhibit GSH and Trx metabolism: buthionine sulfoximine (BSO), a GSH synthesis inhibitor; sulfasalazine (SSZ), an inhibitor of xc- cysteine/glutamate antiporter; auranofin (Au), a thioredoxin reductase inhibitor; or 2-AAPA, a GSH-reductase inhibitor. Clonogenic survival, Matrigel assays, flow cytometry cancer stem cell assays (CD44+CD24-ESA+ or ALDH1) and human tumor xenograft models were used to determine the antitumor activity of drug and radiation combinations. Combined inhibition of GSH and Trx metabolism enhanced cancer cell clonogenic killing and radiation responses in human breast and pancreatic cancer cells via a mechanism that could be inhibited by N-acetylcysteine (NAC). Au, BSO and radiation also significantly decreased breast cancer cell migration and invasion in a thiol-dependent manner that could be inhibited by NAC. In addition, pretreating cells with Au sensitized breast cancer stem cell populations to radiation in vitro as determined by CD44+CD24-ESA+ or ALDH1. Combined administration of Au and BSO, given prior to irradiation, significantly increased the survival of mice with human breast cancer xenografts, and decreased the number of ALDH1+ cancer stem cells. These results indicate that combined inhibition of GSH- and Trx-dependent thiol metabolism using pharmacologically relevant agents can enhance responses of human breast cancer stem cells to radiation both in vitro and in vivo.

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Figures

Figure 1

Figure 1. Au, BSO, and SSZ are effective at depleting GSH and inhibiting TR in pancreatic (PANC-1; MIA PaCa-2) and breast cancer cells (MDA-MB-231)

Cells were treated with BSO (0.1 mM), SSZ (0.1–0.5 mM), or NAC (15 mM) for 24 hours and/or with Au (250 nM for MIA PaCa-2 and SUM159 or 500 nM PANC-1 and MDA-MB-231) for 3 hours followed by analysis for TR activity * p<0.05 vs. no drug (MDA-MB-231 and SUM159) or vs. 500 nM (PANC-1 and MIA PaCa-2) (A), total GSH * p<0.05 vs. control (B) or plated for clonogenic survival assay (C). Error bars ±1 SEM of at least 3 separate experiments. * p<0.05 vs. control, α p<0.05 compared to treatments without NAC.

Figure 2

Figure 2. 2-AAPA induces cytotoxicity that is reversible with NAC as well as inhibiting both GR and TR activity

MDA-MB-231 (black bars) and SUM159 cells (gray bars) were treated with 15 mM NAC for 24 hours and/or 20 µM – 100 µM 2-AAPA for 3 hours. Cells were harvested and then analyzed for GR activity (A), TR activity (B), or clonogenic survival (C). Errors ± 1 SEM of at least 3 separate experiments. * p<0.05 vs. control. α p<0.001 vs. treatment without NAC.

Figure 3

Figure 3. Simultaneous inhibition of Trx- and GSH-metabolism enhances radiation responses

(A) Normalized clonogenic survival is shown for MDA-MB-231 (black bars) and SUM159 cells (gray bars). 2-AAPA treatment for 3 hours followed by 2 Gy IR. * p<0.05 compared to control, δ p<0.05 compared to treatment groups alone. (B) PANC-1 cells were treated 24 hours with 100 units/ml PEG-CAT or PEG alone, 200 µM SSZ and 500 nM Au for the last 3 hours of incubation followed by 2 Gy IR. Errors ± 1 SEM of at least 3 separate experiments. * p<0.05 vs control, ** p<0.05 vs treatments alone and α - p<0.05 vs treatment without PEG-CAT. (C, D, E) MDA-MB-231 cells were treated with 100 µM BSO for 24 hours and/or 500 nM Au for 1 hour followed by 1,2,4,6 and 8 Gy IR immediately followed by the clonogenic survival assay. Results were normalized to treatment alone and the resulting graph analyzed using IGOR Pro 6.36. (F) Normalized clonogenic survival is shown for MDA-MB-231 (black bars) and SUM159 cells (gray bars). 100 µM BSO for 24 hours +Au (250 nM SUM159 or 500 nM MDA-MB-231) for the last hour, followed by 2 Gy IR. * p<0.05 compared to control, δ p<0.05 compared to treatment groups alone.

Figure 4

Figure 4. Addition of Au+BSO to radiation increases DNA damage in breast cancer cells

MDA-MB-231 cells were treated with 100 µM BSO for 24 h, 500 nM Au for 1 hour followed by 2 Gy IR. Cells were harvested and analyzed using Western blot analysis with a γ H2AX antibody and an α tubulin loading control. The gels from 4 independent experiments were quantified using Image J. * p<0.03

Figure 5

Figure 5. Disruption of redox balance inhibits cancer cell migration and invasion and sensitizes cancer stem cells to IR

MDA-MB-231 were treated with BSO, Au and IR as in Figure 3B. Immediately following IR the media was changed to fresh media, incubated 2.5 hours, trypsinized and assessed for viability. 2.5 × 104 viable cells were reseeded in BD BioCoat 8 micron migration (A) or BioCoat Matrigel coated invasion (B) chambers. After 16 hours chambers were stained and invading cells counted. Error bars represent ± 1 SEM of at least four separate experiments.* p<0.05 compared to control, ** p<0.05 compared to IR, α p<0.05 compared to Au+BSO. Exponentially growing (C) MDA-MB-231 or (D) SUM159 cells were treated with 500 nM Au followed in one hour by 2 Gy IR and an additional 2 Gy fraction 24 hours later. One hour after the second dose of radiation cells were trypsinized and then stained with CD44-BV high, CD24-PE low, ESA-APC high (MDA-MB-231 cells) or ALDFLUOR (SUM159 cells) and analyzed with flow cytometry. * p<0.05 compared to control.

Figure 6

Figure 6. Pre-treatment with Au+BSO before radiation decreases tumor growth rate and breast cancer stem cells in vivo

MDA-MB-231 breast cancer xenografts were treated with BSO 450 mg/kg i.p. followed in 2 hours with Au 1.7 mg/kg i.p. followed in 3 hours with 2 × 6 Gy IR separated by 48 hours. (A) Tumor growth curves. Error bars are ± 1 SEM. (B) Kaplan-Meier survival plots. Criteria for euthanasia was reached when xenograft tumors measured 15 mm in one direction. (C) Three separate SUM159 xenograft tumors were grown in nude mice. Tumors were harvested, digested and plated in a tissue culture flask at 4% O2 for 48 hours. Cells were then treated with 1 mM BSO for 24 hours then harvested and subjected to flow cytometry with CM-SNARF and ALDFLUOR. At least 5,000 ALDH1+ cells were analyzed from each sample to determine the MFI of CM-SNARF (D) SUM159 xenograft tumors were grown on nude mice, and treated with 675 mg/kg BSO followed in two hours with Au 2.7 mg/kg and in four hours with 6 Gy IR. Tumors were harvested 20 hour after IR and subjected to the ALDEFLUOR flow cytometry assay. Error bars represents ± 1 SEM of at least three tumors assayed on at least two different occasions. * p<0.05 vs. control, δ p<0.05 vs. treatment groups alone.

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