Cell Biochemistry and Biophysics

, Volume 77, Issue 1, pp 89–98 | Cite as

Low-Temperature EPR Spectroscopy as a Probe-Free Technique for Monitoring Oxidants Formed in Tumor Cells and Tissues: Implications in Drug Resistance and OXPHOS-Targeted Therapies

  • Balaraman KalyanaramanEmail author
  • Gang Cheng
  • Jacek Zielonka
  • Brian BennettEmail author
Original Paper


Oxidants formed from oxidative and nitrative metabolism include reactive oxygen species (ROS) such as superoxide, hydrogen peroxide/lipid hydroperoxides and reactive nitrogen species (RNS) (e.g., peroxynitrite [ONOO] and nitrogen dioxide), and reactive halogenated species (e.g., hypochlorous acid [HOCl]). Increasingly, ROS and RNS are implicated in tumorigenesis as well as tumor growth, progression, and metastasis. Recently, ROS were implicated in drug resistance, metabolic reprogramming, and T-cell metabolism in immunotherapy. Mostly, fluorescent probes have been used in cell culture systems. The identity of species is obtained by LC–MS analyses of diagnostic marker products. However, extrapolation of these assays to cancer xenografts is difficult if not impossible. Thus, development of a probe-free assay for monitoring and assessing oxidant formation in tumor cells and tumor xenografts is critical and timely. Here, we describe the use of ex vivo electron paramagnetic resonance (EPR) spectroscopy at cryogenic temperatures as a uniquely useful probe-free technique for assessing intracellular oxidation and oxidants via EPR signals from redox centers, particularly iron-sulfur clusters, in mitochondrial and cytosolic redox proteins. Examples of cancer cells subjected to inhibition of mitochondrial oxidative phosphorylation are presented. This ex vivo methodology can be readily extended to monitor oxidant formation in tumor tissues isolated from mice and humans.


Electron paramagnetic resonance Reactive oxygen species Oxidative phosphorylation Mitochondrial targeting 



This research was supported by NIH NCI U01 CA178960 to B.K., NIH NCI R01 CA208648 to B.K., the Quadracci Endowment to B.K., and an MCW Cancer Center award to B.K. and B.B. EPR was supported by an NSF Major Research Instrumentation award (CHE-1532168 to B.B.) and by Bruker BioSpin.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiophysicsMedical College of WisconsinMilwaukeeUSA
  2. 2.Free Radical Research CenterMedical College of WisconsinMilwaukeeUSA
  3. 3.Cancer CenterMedical College of WisconsinMilwaukeeUSA
  4. 4.Department of PhysicsMarquette UniversityMilwaukeeUSA

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