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. 2018 Oct;119(7):873-884.
doi: 10.1038/s41416-018-0263-y. Epub 2018 Oct 5.

Targeting Peroxiredoxin 1 Impairs Growth of Breast Cancer Cells and Potently Sensitises These Cells to Prooxidant Agents

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Free PMC article

Targeting Peroxiredoxin 1 Impairs Growth of Breast Cancer Cells and Potently Sensitises These Cells to Prooxidant Agents

Malgorzata Bajor et al. Br J Cancer. .
Free PMC article

Abstract

Background: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells.

Methods: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples.

Results: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate.

Conclusions: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Characterisation of PRDX1 and PRDX2 knockout in MCF-7 breast cancer cell line and in in vivo xenotransplantation model. a Analysis of the expression of PRDX1 (left panel) and PRDX2 (right panel) mRNA in normal and breast cancer tissues available in the analysed TCGA dataset (n = 108 pairs), regardless of the ethnicity of the patient. b Representative western blotting results showing the protein presence of PRDX1 and PRDX2 in HMEC, MCF-10A, MCF-7, ZR-75-1, T47D, SK-BR-3, MDA-MB-231, and HCC1806 cell lines. β-actin was used as a loading control. Bands were quantified by densitometry, RI was calculated as the quotient of the densitometry signal for PRDX1 (or PRDX2) band and that for β-actin and then normalised to that of the HMEC. Averaged RI value from two independent experiments was shown. c Western blotting shows efficient depletion of PRDX1 or PRDX2 proteins in the MCF-7 single-cell-derived clonal lines compared to parental and sgGFP controls. β-actin was used as a loading control. d Detection of DNA synthesis using EdU incorporation assay in PRDX1-knockout MCF-7 cells (red bars) compared to controls (black and grey bars) and PRDX2-knockout cells (green bars). *p < 0.05, ***p < 0.001. e Cell cycle analysis evaluated by the propidium iodide flow cytometry-based assay. Representative cell cycle profiles showing the distribution of cells in the different phases of the cell cycle for PRDX1-knockout MCF-7 cells compared to controls and PRDX2-knockout cells are presented (left panel). Summary of the percentage of cells in each phase of the cycle (right panel). Data shown are cumulative results from two independent experiments performed in triplicates, *p < 0.05, ***p < 0.001. f Representative images for the colony formation in MCF-7 cells (left panel) and a quantitative analysis of colony formation assay in PRDX1-knockout MCF-7 cells. Data shown are cumulative results from three independent experiments. *p < 0.05, ***p < 0.001. g Western blotting shows efficient depletion of PRDX1 protein in MCF-7-sgPRDX1-pool2 cells as compared to sgNTC-pool2 control. β-actin was used as a loading control. h Plots of mean tumour volumes in mice (initial n = 10 per group) inoculated with control (sgNTC-pool2) and sgPRDX1-pool2 of MCF-7 cells. Points are means and bars are SD. Statistical analysis was performed with two-way Anova test (****p < 0.0001)
Fig. 2
Fig. 2
Assessment of cellular responses to H2O2 using MCF-7 cells. a Western blotting shows efficient depletion of PRDX1 protein in the MCF-7-HyPer-3 cells. b Fluorescence of HyPer-3 (green) in live sgNTC-pool3 and sgPRDX1-pool3 MCF-7 cells after adding exogenous H2O2 at final concentration of 100 µM at the indicated time points. c Fluorescence intensities of HyPer-3 in the sgNTC-pool3 and sgPRDX1-pool3 MCF-7 cells untreated and incubated with 100 µM H2O2. Green arrow indicates adding of H2O2. Experiment was performed twice. d Concentration-dependent cytotoxicity of H2O2 produced by GOx in CRISPR/Cas9-engineered MCF-7 cells. Cells were treated with a range of GOx concentrations (0.25; 0.5; 1 mU/ml) for 24 h. Control cells were cultured without any reagent. At the end of treatment, the crystal violet staining (or MTT test, see Suppl. Fig. S3A) was performed and reported as percent growth relative to control. e Determination of cell death by propidium iodide staining followed by flow cytometry analysis. Cells were treated with a range of GOx concentrations (0.5; 1 mU/ml) for 24 h. Control cells were cultured without any reagent. Experiments were performed in duplicates and repeated three times, ***p < 0.001. f Relative fluorescence intensity (log2 values) of PY1 probe after reaction with H2O2 produced by GOx in sgPRDX1 and sgPRDX2 MCF-7 cells compared to controls. Cells treated with 1 mU/ml of GOx were preincubated with catalase (100 μg/ml) for 30 min. After 24 h of GOx treatment 10 μM PY1 dye was added to the medium for 30 min at 37 °C and the read was taken using EnVision reader at the excitation wavelength 514 nm and emission wavelength 550 nm. Each sample was at least triplicated, and data were obtained from three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test when significance was detected (**p < 0.01, ***p < 0.001)
Fig. 3
Fig. 3
Characterisation of PRDX1 and PRDX2-knockdown ZR-75-1 breast cancer cell line. a Western blotting shows downregulation of PRDX1 or PRDX2 proteins in ZR-75-1 cell line derivatives compared to parental and shNTC controls. β-actin was used as a loading control. b Representative images for the colony formation in ZR-75-1 cells (left panel) and a quantitative analysis of colony formation assay (right panel) shows a significant decrease of colony number in cells carrying shPRDX1, in contrast to controls and PRDX2-knockdown cells. Data shown are cumulative results from three independent experiments. **p < 0.01. c Concentration-dependent cytotoxicity of GOx in ZR-75-1 cells. Cells were treated with GOx (0.25; 0.5; 1 mU/ml) for 24 h. Control cells were cultured without any reagent. At the end of treatment, the crystal violet staining (or MTT test, see Suppl. Fig. S3D) was performed and reported as percent growth relative to control. d Relative fluorescence intensity (log2 values) of PY1 probe after reaction with H2O2 produced by GOx in shPRDX1 and shPRDX2 ZR-75-1 cells compared to controls was assessed as described in Fig. 2e
Fig. 4
Fig. 4
Knockout of PRDX1, but not PRDX2, sensitises breast cancer cells to prooxidant agents. MCF-7 (a) and ZR-75-1 (b) cells were treated with increasing concentration of sodium ascorbate (0.2, 0.4 mM) for 24 h. Cells treated with 0.4 mM of L-ASC were preincubated with catalase (100 μg/ml) for 30 min. b, c Effects of 0.4 mM L-ASC treatment for 24 h on MCF-7 control, sgPRDX1 and 2 cells viability (c) and cytotoxicity (d). Fluorescence was measured at 400Ex/505Em (viability) and 485Ex/520Em (cytotoxicity) using EnVision reader. Each sample was at least triplicated, and data were obtained from three independent experiments. e Determination of cell death by propidium iodide staining followed by flow cytometry analysis. Cells were treated with a range of L-ASC concentrations (0.2–0.8 mM) for 24 h. Control cells were cultured without any reagent. Experiments were performed in duplicates and repeated three times, **p < 0.01, ***p < 0.001. f Expression of the senescence marker SA–β-Gal in parental and CRISPR/Cas9-engineered MCF-7 cells was detected by X-Gal staining at pH 6. Microphotographs were taken at 10 magnification (inverted microscope, Nikon). g Representative flow cytometry histograms of SA-β-Gal activity and bar graphs showing flow cytometry analysis of SA-β-Gal activity for MCF-7 parental, sgPRDX1-A and sgPRDX1-B cells control (untreated) and after treatment with 0.2 mM L-ASC for 24 h. Experiments were performed in triplicates and repeated twice.**p < 0.01
Fig. 5
Fig. 5
Catalytically inactive PRDX1 variants, in contrast to C83A mutant and wtPRDX1 variant, do not rescue survival of PRDX1-knockout MCF-7 cells. a anti-PRDX1 and anti-V5-tag western blotting analysis shows overexpression of PRDX1 mutated protein variants with the following mutations: C83A, C173A, and C52/C173A (lanes 3–5, respectively) or overexpressing wtPRDX1 protein (lane 6) in MCF-7 sgPRDX1-A cells compared to parental (lane 1) and knockout sgPRDX1-A (lane 2) cells. β-actin was used as a loading control. b, c Cells were treated with increasing concentrations of glucose oxidase (0.125–2 mU) (b) or sodium L-ascorbate (0.1–1.6 mM) (c) for 24 h. Catalytically inactive PRDX1 variants, in contrast to C83A mutant and wtPRDX1 variant, do not rescue survival of PRDX1-knockout MCF-7 cells. For all cytotoxicity assays, control cells were cultured without any reagent. At the end of treatment, the crystal violet staining was performed and reported as percent growth relative to control. Experiments were performed in triplicates and repeated at least twice. Statistical analysis was performed with one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test when significance was detected (*p < 0.05, **p < 0.01, ***p < 0.001)
Fig. 6
Fig. 6
Cytotoxic effects of combinations of adenanthin and GOx or L-ASC in MCF-7 cell lines. Cells were treated with increasing concentrations of ADNT in the absence or presence of either a GOx (0.25; 0.5; 1 mU/ml) or b L-ASC (0.4, 0.8 mM) for 48 h. At the end of treatment, cell proliferation was determined by crystal violet staining and reported as percent growth relative to controls. The combination index (CI) calculated by the Chou–Talalay method was used to determine drug interaction. The CI is reported at different doses of prooxidants and ADNT as indicated in tables. CI values < 0.9 suggest synergism. c Representative results and the quantitative analysis of colony formation assay in MCF-7 cells incubated with 0.3 µM ADNT in combination with 50 µM L-ASC shows a significant decrease in colony area as compared to cells treated with single drugs. Mean ± SEM of the three independent experiments is shown. Statistical analysis was performed with one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test when significance was detected (***p < 0.001). d Non-malignant HMEC (left panel) or MCF-10A (right panel) cells were treated with increasing doses of ADNT in the absence or presence of increasing concentration of L-ASC for 48 h. e Scheme representing a dual role for PRDX1 in human breast cancer (please see the text for details)

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References

    1. Barrera G. Oxidative stress and lipid peroxidation products in cancer progression and therapy. ISRN Oncol. 2012;2012:137289. - PMC - PubMed
    1. Mahalingaiah PK, Ponnusamy L, Singh KP. Chronic oxidative stress leads to malignant transformation along with acquisition of stem cell characteristics, and epithelial to mesenchymal transition in human renal epithelial cells. J. Cell Physiol. 2015;230:1916–1928. doi: 10.1002/jcp.24922. - DOI - PubMed
    1. Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 2013;12:931–947. doi: 10.1038/nrd4002. - DOI - PubMed
    1. Graczyk-Jarzynka A, et al. New insights into redox homeostasis as a therapeutic target in B-cell malignancies. Curr. Opin. Hematol. 2017;24:393–401. doi: 10.1097/MOH.0000000000000351. - DOI - PMC - PubMed
    1. Moses C, Garcia-Bloj B, Harvey AR, Blancafort P. Hallmarks of cancer: The CRISPR generation. Eur. J. Cancer. 2018;93:10–18. doi: 10.1016/j.ejca.2018.01.002. - DOI - PubMed

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