Article | Published:

Cellular and Molecular Biology

Targeting peroxiredoxin 1 impairs growth of breast cancer cells and potently sensitises these cells to prooxidant agents

British Journal of Cancervolume 119pages873884 (2018) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

Note: This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).

References

  1. 1.

    Barrera, G. Oxidative stress and lipid peroxidation products in cancer progression and therapy. ISRN Oncol. 2012, 137289 (2012).

  2. 2.

    Mahalingaiah, P. K., Ponnusamy, L. & Singh, K. P. 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. 230, 1916–1928 (2015).

  3. 3.

    Gorrini, C., Harris, I. S. & Mak, T. W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 12, 931–947 (2013).

  4. 4.

    Graczyk-Jarzynka, A. et al. New insights into redox homeostasis as a therapeutic target in B-cell malignancies. Curr. Opin. Hematol. 24, 393–401 (2017).

  5. 5.

    Moses, C., Garcia-Bloj, B., Harvey, A. R. & Blancafort, P. Hallmarks of cancer: The CRISPR generation. Eur. J. Cancer 93, 10–18 (2018).

  6. 6.

    Rhee, S. G. & Kil, I. S. Multiple functions and regulation of mammalian peroxiredoxins. Annu Rev. Biochem 86, 749–775 (2017).

  7. 7.

    Muchowicz, A. et al. SK053 triggers tumor cells apoptosis by oxidative stress-mediated endoplasmic reticulum stress. Biochem Pharmacol. 93, 418–427 (2015).

  8. 8.

    Trzeciecka, A. et al. Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma. Oncotarget 7, 1717–1731 (2016).

  9. 9.

    O’Leary, P. C. et al. Peroxiredoxin-1 protects estrogen receptor alpha from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer. Breast Cancer Res 16, R79 (2014).

  10. 10.

    The Cancer Genome Atlas - Cancer Genome - TCGA http://cancergenome.nih.gov/ Accessed 2017

  11. 11.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  12. 12.

    McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res 40, 4288–4297 (2012).

  13. 13.

    Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

  14. 14.

    Bilan, D. S. et al. HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging. ACS Chem. Biol. 8, 535–542 (2013).

  15. 15.

    Chou, T. C. Drug combination studies and their synergy quantification using the Chou–Talalay method. Cancer Res 70, 440–446 (2010).

  16. 16.

    Noh, D. Y. et al. Overexpression of peroxiredoxin in human breast cancer. Anticancer Res 21, 2085–2090 (2001).

  17. 17.

    Zhang, D. et al. Reactive oxygen species formation and bystander effects in gradient irradiation on human breast cancer cells. Oncotarget 7, 41622–41636 (2016).

  18. 18.

    Chen, W. C. et al. Induction of radioprotective peroxiredoxin-I by ionizing irradiation. J. Neurosci. Res 70, 794–798 (2002).

  19. 19.

    Jezierska-Drutel, A., Rosenzweig, S. A. & Neumann, C. A. Role of oxidative stress and the microenvironment in breast cancer development and progression. Adv. Cancer Res 119, 107–125 (2013).

  20. 20.

    Dickinson, B. C., Huynh, C. & Chang, C. J. A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells. J. Am. Chem. Soc. 132, 5906–5915 (2010).

  21. 21.

    Lippert, A. R., Van de Bittner, G. C. & Chang, C. J. Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems. Acc. Chem. Res 44, 793–804 (2011).

  22. 22.

    Veal, E. A., Underwood, Z. E., Tomalin, L. E., Morgan, B. A. & Pillay, C. S. Hyperoxidation of peroxiredoxins: gain or loss of function? Antioxid. Redox Signal 28, 574–590 (2018).

  23. 23.

    Carvalho, L. A. C. et al. Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2. J. Biol. Chem. 292, 8705–8715 (2017).

  24. 24.

    He, T., Hatem, E., Vernis, L., Lei, M. & Huang, M. E. PRX1 knockdown potentiates vitamin K3 toxicity in cancer cells: a potential new therapeutic perspective for an old drug. J. Exp. Clin. Cancer Res 34, 152 (2015).

  25. 25.

    Kim, J. H. et al. Peroxiredoxin 2 mediates insulin sensitivity of skeletal muscles through regulation of protein tyrosine phosphatase oxidation. Int J. Biochem Cell Biol. 99, 80–90 (2018).

  26. 26.

    Chen, Q., Polireddy, K., Chen, P. & Dong, R. The unpaved journey of vitamin C in cancer treatment. Can. J. Physiol. Pharmacol. 93, 1055–1063 (2015).

  27. 27.

    Chen, Q. et al. Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo. Proc. Natl Acad. Sci. USA 104, 8749–8754 (2007).

  28. 28.

    Niles, A. L., Moravec, R. A. & Riss, T. L. In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening. Curr. Chem. Genom. 3, 33–41 (2009).

  29. 29.

    Park, Y. H. et al. Peroxiredoxin I participates in the protection of reactive oxygen species-mediated cellular senescence. BMB Rep. 50, 528–533 (2017).

  30. 30.

    Tait, L., Soule, H. D. & Russo, J. Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10. Cancer Res. 50, 6087–6094 (1990).

  31. 31.

    Lee, W. et al. Human peroxiredoxin 1 and 2 are not duplicate proteins: the unique presence of CYS83 in Prx1 underscores the structural and functional differences between Prx1 and Prx2. J. Biol. Chem. 282, 22011–22022 (2007).

  32. 32.

    Jiang, B. et al. Diterpenoids from Isodon adenantha. J. Nat. Prod. 65, 1111–1116 (2002).

  33. 33.

    Muchowicz, A. et al. Adenanthin targets proteins involved in the regulation of disulphide bonds. Biochem Pharmacol. 89, 210–216 (2014).

  34. 34.

    Siernicka, M. et al. Adenanthin, a new inhibitor of thiol-dependent antioxidant enzymes, impairs the effector functions of human natural killer cells. Immunology 146, 173–183 (2015).

  35. 35.

    Mahalingaiah, P. K. & Singh, K. P. Chronic oxidative stress increases growth and tumorigenic potential of MCF-7 breast cancer cells. PLoS ONE 9, e87371 (2014).

  36. 36.

    Hampton, M. B., Vick, K. A., Skoko, J. & Neumann, C. A. Peroxiredoxin involvement in the initiation and progression of human cancer. Antioxid. Redox Signal 28, 591–608 (2018).

  37. 37.

    Neumann, C. A. et al. Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression. Nature 424, 561–565 (2003).

  38. 38.

    Niu, W. et al. Peroxiredoxin 1 promotes invasion and migration by regulating epithelial-to-mesenchymal transition during oral carcinogenesis. Oncotarget 7, 47042–47051 (2016).

  39. 39.

    He, T., Hatem, E., Vernis, L. & Huang, M. E. Peroxiredoxin 1 knockdown sensitizes cancer cells to reactive oxygen species-generating drugs - an alternative approach for chemotherapy. Free Radic. Biol. Med 75(Suppl 1), S13 (2014).

  40. 40.

    Karim, M. E., Tha, K. K., Othman, I., Borhan Uddin, M. & Chowdhury, E. H. Therapeutic potency of nanoformulations of siRNAs and shRNAs in animal models of cancers. Pharmaceutics 10, 65 (2018).

  41. 41.

    Cinci, M., Mamidi, S., Li, W., Fehring, V. & Kirschfink, M. Targeted delivery of siRNA using transferrin-coupled lipoplexes specifically sensitizes CD71 high expressing malignant cells to antibody-mediated complement attack. Target Oncol. 10, 405–413 (2015).

  42. 42.

    Schoenfeld, J. D. et al. O2- and H2O2-mediated disruption of Fe metabolism causes the differential susceptibility of NSCLC and GBM cancer cells to pharmacological ascorbate. Cancer Cell 31, 487–500 e8 (2017).

  43. 43.

    Levine, M., Espey, M. G. & Chen, Q. Losing and finding a way at C: new promise for pharmacologic ascorbate in cancer treatment. Free Radic. Biol. Med 47, 27–29 (2009).

  44. 44.

    Jacobs, C., Hutton, B., Ng, T., Shorr, R. & Clemons, M. Is there a role for oral or intravenous ascorbate (vitamin C) in treating patients with cancer? A systematic review. Oncologist 20, 210–223 (2015).

Download references

Acknowledgements

The authors wish to thank Prof. Rafal Ploski and Dr. Malgorzata Rydzanicz (both of Medical University of Warsaw) for their invaluable contribution to RNASeq studies. We also thank Dr. Beata Pyrzynska (Medical University of Warsaw) for her help in obtaining HEK-293FT cells. We would like to thank Dr. Rut Klinger (University College Dublin) for her help in generating the HyPer3-encoding lentiviral vector. We want to thank Mr. Tomasz Jarzynka (International Institute of Molecular and Cell Biology in Warsaw) for help with script writing for ImageJ automated analysis. We also want to thank Prof. Pawel Wlodarski and Dr. Ilona Kalaszczynska (both of Medical University of Warsaw) for providing an access to Nikon microscope.

Authors contributions

M.B., A.O.Z. and A.M. cultured the cells; A.O.Z. and M.B. carried out the cell growth/survival assays and western blotting experiments; M.B. performed flow cytometry-based study; A.G.-J. performed the experiments with PY1 probe; A.D. and A.G.-J. carried out digital analysis of the colony formation images; A.O.Z., M.B. and A.D. carried out selection of sgRNA clones for the in vitro study; A.M. generated the MCF-7 cells for the in vivo study; R.Z., A.M., M.F., A.O.Z., M.B., A.G.-J. and M.S. carried out the lentiviral transduction procedures; A.M. and A.Z. carried out the in vivo study; M.F. generated the MCF-7sgRNA derivatives for the in vivo study and oversaw the in vivo study; P.S. carried out the in silico analysis of ADNT:PRDX interactions; M.B. and A.O.Z. carried out life imaging studies; P.G. analysed the clinical data; L.T. carried out the RNAseq study; P.C.O'.L. generated the sgRNA sequences and consulted the CRISPR/Cas9-mediated generation of MCF-7 cell line derivatives; M.K. and M.B. carried out the Cs-137-irradiation study; M.B. carried out the statistical analysis; M.B. and R.Z. designed the experiments and drafted the manuscript; J.G. provided critical insights into the study design, provided research materials for oxidative stress assessment, and contributed to supervision of the study; and R.Z. conceived the study, supervised the project, and coordinated all experiments in this work. All authors critically revised the initial draft of the manuscript and subsequent revisions. All authors approved the manuscript in its current form.

Author information

Author notes

  1. These authors contributed equally: Malgorzata Bajor and Agata O. Zych

Affiliations

  1. Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Warsaw, Poland

    • Malgorzata Bajor
    • , Agata O. Zych
    • , Agnieszka Graczyk-Jarzynka
    • , Angelika Muchowicz
    • , Malgorzata Firczuk
    • , Antoni Domagala
    • , Marta Siernicka
    • , Agnieszka Zagozdzon
    • , Jakub Golab
    •  & Radoslaw Zagozdzon
  2. Department of Clinical Immunology, Transplantation Institute, Medical University of Warsaw, Warsaw, Poland

    • Malgorzata Bajor
    • , Monika Kniotek
    •  & Radoslaw Zagozdzon
  3. Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland

    • Agata O. Zych
    •  & Marta Siernicka
  4. Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland

    • Lech Trzeciak
  5. Center for Advanced Materials and Technologies, Warsaw University of Technology, Warsaw, Poland

    • Lech Trzeciak
  6. Laboratory of Human Cancer Genetics, Centre of New Technologies, University of Warsaw, Warsaw, Poland

    • Pawel Gaj
  7. Department of Bioinformatics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

    • Pawel Siedlecki
    •  & Radoslaw Zagozdzon
  8. Department of Systems Biology, Institute of Experimental Plant Biology and Biotechnology, University of Warsaw, Warsaw, Poland

    • Pawel Siedlecki
  9. Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA

    • Patrick C. O’Leary
  10. Centre for Preclinical Research and Technology, Medical University of Warsaw, Warsaw, Poland

    • Jakub Golab

Authors

  1. Search for Malgorzata Bajor in:

  2. Search for Agata O. Zych in:

  3. Search for Agnieszka Graczyk-Jarzynka in:

  4. Search for Angelika Muchowicz in:

  5. Search for Malgorzata Firczuk in:

  6. Search for Lech Trzeciak in:

  7. Search for Pawel Gaj in:

  8. Search for Antoni Domagala in:

  9. Search for Marta Siernicka in:

  10. Search for Agnieszka Zagozdzon in:

  11. Search for Pawel Siedlecki in:

  12. Search for Monika Kniotek in:

  13. Search for Patrick C. O’Leary in:

  14. Search for Jakub Golab in:

  15. Search for Radoslaw Zagozdzon in:

Ethics approval and consent to participate

All animal experiments were performed in accordance with the guidelines approved by the Ethics Committee of the Medical University of Warsaw (approval No. 40/2015).

Funding

The work was supported by National Science Centre, Poland (grant No. 2014/13/B/NZ5/01354; RZ), European Commission Horizon 2020 Programme (692180-STREAM-H2020-TWINN-2015; J.G.), European Commission 7th Framework Programme (FP7-REGPOT-2012-CT2012-316254-BASTION; J.G.), and Medical University of Warsaw (1M19/PM14/14; M.B.).

Competing interests

The authors declare no competing interests.

Note

This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 4.0 Unported License).

Corresponding author

Correspondence to Radoslaw Zagozdzon.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41416-018-0263-y