Skip to main content

Advertisement

SpringerLink
Log in

An EPR Study Using Cyclic Hydroxylamines To Assess The Level of Mitochondrial ROS in Superinvasive Cancer Cells

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

It has been proposed that a mitochondrial switch involving a high mitochondrial superoxide production is associated with cancer metastasis. We here report an EPR analysis of ROS production using cyclic hydroxylamines in superinvasive SiHa-F3 compared with less invasive SiHa wild-type human cervix cancer cells. Using the CMH probe, no significant difference was observed in the overall level of ROS between SiHa and SiHa-F3 cells. However, using mitochondria-targeted cyclic hydroxylamine probe mitoTEMPO-H, we detected a significantly higher mitochondrial ROS content in SiHa-F3 compared with the wild-type SiHa cells. To investigate the nature of mitochondrial ROS, we overexpressed superoxide dismutase 2, a SOD isoform exclusively localized in mitochondria, in SiHa-F3 superinvasive cells. A significantly lower signal was detected in SiHa-F3 cells overexpressing SOD2 compared with SiHa-F3. Despite some limitations discussed in the paper, our EPR results suggest that mitochondrial ROS (at least partly superoxide) are produced to a larger extent in superinvasive cancer cells compared with less invasive wild-type cancer cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Porporato, P. E., Payen, V. L., Perez-Escuredo, J., De Saedeleer, C. J., Danhier, P., Copetti, T., Dhup, S., Tardy, M., Vazeille, T., & Bouzin, C., et al. (2014). A mitochondrial switch promotes tumor metastasis. Cell Reports, 8, 754–766.

    Article  CAS  Google Scholar 

  2. Zhao, H., Joseph, J., Fales, H. M., Sokoloski, E. A., Levine, R. L., Vasquez-Vivar, J., & Kalyanaraman, B. (2005). Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proceedings of the National Academy of Sciences of the United States of America, 102, 5727–5732.

    Article  CAS  Google Scholar 

  3. Zielonka, J., & Kalyanaraman, B. (2010). Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth. Free Radical Biology and Medicine, 48, 983–1001.

    Article  CAS  Google Scholar 

  4. Kalyanaraman, B., Darley-Usmar, V., Davies, K. J., Dennery, P. A., Forman, H. J., Grisham, M. B., Mann, G. E., Moore, K., Roberts, 2nd, L. J., & Ischiropoulos, H. (2012). Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radical Biology and Medicine, 52, 1–6.

    Article  CAS  Google Scholar 

  5. Kalyanaraman, B., Dranka, B. P., Hardy, M., Michalski, R., & Zielonka, J. (2014). HPLC-based monitoring of products formed from hydroethidine-based fluorogenic probes–the ultimate approach for intra- and extracellular superoxide detection. Biochimica et Biophysica Acta, 1840, 739–744.

    Article  CAS  Google Scholar 

  6. Zielonka, J., Hardy, M., Michalski, R., Sikora, A., Zielonka, M., Cheng, G., Ouari, O., Podsiadly, R., & Kalyanaraman, B. (2017). Recent developments in the probes and assays for measurement of the activity of NADPH oxidases. Cell Biochemistry and Biophysics, 75, 335–349.

    Article  CAS  Google Scholar 

  7. Kalyanaraman, B., Cheng, G., Hardy, M., Ouari, O., Bennett, B., & Zielonka, J. (2018). Teaching the basics of reactive oxygen species and their relevance to cancer biology: Mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies. Redox Biology, 15, 347–362.

    Article  CAS  Google Scholar 

  8. Scheinok, S., Leveque, P., & Gallez, B. (2018). Comparison of different methods for measuring the superoxide radical by EPR spectroscopy in buffer, cell lysates and cells. Free Radical Research, 52, 1182–1196.

    Article  CAS  Google Scholar 

  9. Dikalov, S. I., Kirilyuk, I. A., Voinov, M., & Grigor’ev, I. A. (2011). EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines. Free Radical Research, 45, 417–430.

    Article  CAS  Google Scholar 

  10. Scheinok, S., Driesschaert, B., d’Hose, D., Sonveaux, P., Robiette, R., & Gallez, B. (2019). Synthesis and characterization of a 5-membered ring cyclic hydroxylamine coupled to triphenylphosphonium to detect mitochondrial superoxide by EPR spectrometry. Free Radical Research, 53, 1135–1143.

    Article  CAS  Google Scholar 

  11. Van Hee, V. F., Perez-Escuredo, J., Cacace, A., Copetti, T., & Sonveaux, P. (2015). Lactate does not activate NF-κB in oxidative tumor cells. Frontiers in Pharmacology, 6, 228.

    PubMed  PubMed Central  Google Scholar 

  12. Dikalov, S. I., Li, W., Mehranpour, P., Wang, S. S., & Zafari, A. M. (2007). Production of extracellular superoxide by human lymphoblast cell lines: comparison of electron spin resonance techniques and cytochrome C reduction assay. Biochemical Pharmacology, 73, 972–980.

    Article  CAS  Google Scholar 

  13. Yang, W., Zou, L., Huang, C., & Lei, Y. (2014). Redox regulation of cancer metastasis: molecular signaling and therapeutic opportunities. Drug Development Research, 75, 331–341.

    Article  CAS  Google Scholar 

  14. Ishikawa, K., Takenaga, K., Akimoto, M., Koshikawa, N., Yamaguchi, A., Imanishi, H., Nakada, K., Honma, Y., & Hayashi, J. (2008). ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science, 320, 661–664.

    Article  CAS  Google Scholar 

  15. Dikalov, S. I., Polienko, Y. F., & Kirilyuk, I. (2018). Electron paramagnetic resonance measurements of reactive oxygen species by cyclic hydroxylamine spin probes. Antioxidants & Redox Signaling, 28, 1433–1443.

    Article  CAS  Google Scholar 

  16. Chen, K., & Swartz, H. M. (1988). Oxidation of hydroxylamines to nitroxide spin labels in living cells. Biochimica et Biophysica Acta, 970, 270–277.

    Article  CAS  Google Scholar 

  17. Palazzolo-Balance, A. M., Suquet, C., & Hurst, J. K. (2007). Pathways for intracellular generation of oxidants and tyrosine nitration by a macrophage cell line. Biochemistry, 46, 7536–7548.

    Article  Google Scholar 

  18. Dikalova, A. E., Bikineyeva, A. T., Budzyn, K., Nazarewicz, R. R., McCann, L., Lewis, W., Harrison, D. G., & Dikalov, S. I. (2010). Therapeutic targeting of mitochondrial superoxide in hypertension. Circulation Research, 107, 106–116.

    Article  CAS  Google Scholar 

  19. Keana, J. F. W., Pou, S., & Rosen, G. M. (1987). Nitroxides as potential contrast enhancing agents for MRI application: influence of structure on the rate of reduction by rat hepatocytes, whole liver homogenate, subcellular fractions, and ascorbate. Magnetic Resonance in Medicine, 5, 525–536.

    Article  CAS  Google Scholar 

  20. Cheng, G., Zielonka, J., McAllister, D., Hardy, M., Ouari, O., Joseph, J., Dwinell, M. B., & Kalyanaraman, B. (2015). Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: role of mitochondrial bioenergetics and energy-sensing mechanism. Cancer Letters, 365, 96–106.

    Article  CAS  Google Scholar 

  21. Reily, C., Mitchell, T., Chacko, B. K., Benavides, G., Murphy, M. P., & Darley-Usmar, V. (2013). Mitochondrially targeted compounds and their impact on cellular bioenergetics. Redox Biology, 1, 86–93.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by grants from French Community of Belgium (ARC 14/19-058), the Belgian Fonds National de la Recherche Scientifique (F.R.S.-FNRS, J0.209.16), the Belgian Télévie (7.4557.15) and the Fondation Louvain. PS is a F.R.S.-FNRS Senior Research Associate.

Author information

Authors and Affiliations

  1. Biomedical Magnetic Resonance, Louvain Drug Research Institute (LDRI), Université catholique de Louvain (UCLouvain), Brussels, Belgium

    Samantha Scheinok & Bernard Gallez

  2. Pole of Pharmacology, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain (UCLouvain), Brussels, Belgium

    Tania Capeloa & Pierre Sonveaux

  3. Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Center, University of Torino, Torino, Italy

    Paolo E. Porporato

Authors
  1. Samantha Scheinok
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tania Capeloa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paolo E. Porporato
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pierre Sonveaux
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bernard Gallez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bernard Gallez.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Special Issue: XIth International Workshop on EPR in Biology and Medicine. This paper was presented as a poster during the meeting.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Scheinok, S., Capeloa, T., Porporato, P.E. et al. An EPR Study Using Cyclic Hydroxylamines To Assess The Level of Mitochondrial ROS in Superinvasive Cancer Cells. Cell Biochem Biophys 78, 249–254 (2020). https://doi.org/10.1007/s12013-020-00921-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-020-00921-6

Search

Navigation