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

Abstract

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 (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.

Quantification of different iron forms in the aceruloplasminemia brain to explore iron-related neurodegeneration

“…Our work has some limitations, the first being the long-term formalin fixation of the tissue. Since formalin fixation is known to reduce total tissue iron content and may have a detrimental effect on protein conformations ( Schrag et al, 2010 , Gellein et al, 2008 , Ghosh et al, 2010 , Kalyanaraman et al, 2019 ), it is likely that the presented absolute concentrations of Fe 3+ ions, ferrihydrite-iron and maghemite-iron in the aceruloplasminemia brain are underestimated compared to in vivo . In addition, formalin fixation has likely increased the R 2 * values, compared to in vivo conditions ( Birkl et al, 2016 ).…”

Quantification of different iron forms in the aceruloplasminemia brain to explore iron-related neurodegeneration

“…Our work has some limitations, the first being the long-term formalin fixation of the tissue. Since formalin fixation is known to reduce total tissue iron content and may have a detrimental effect on protein conformations (44)(45)(46)(47), it is likely that the presented absolute concentrations of Fe 3+ ions, ferrihydrite-iron and maghemite-iron in the aceruloplasminemia brain are underestimated compared to in vivo. Therefore, future studies should aim to minimize the duration of formalin fixation (34), and ideally include both aceruloplasminemia and control brain tissue to enable more in-depth analyses of the exact relationship between the different molecular forms of iron that are present in healthy and severely diseased states.…”

Cryogenic electron paramagnetic resonance spectroscopy of flash-frozen tissue for characterization of mitochondrial disease

“…The low-temperature EPR signals from tissues including liver, muscle, heart, and brain, have largely been assigned from comparison of signals from tissues with those from fractionated extracts, purified proteins or protein complexes, modified or truncated proteins or protein complexes, and isolated mitochondria [80][81][82] . The predominant signals are those from the MRC complexes I-IV, with additional signals from semiquinones, aconitase, catalase, and from ferriheme and transferrin in residual blood.…”

“…A second challenge is that many of the signals overlap, sometimes extensively. From the 24 [79,82,[108][109][110][111] . Additional well-characterized tissue-specific signals include Fe(III) from transferrin (Tf), Cu(II) from ceruloplasmin (Cp), high-and low-spin hemes from catalase, and Mn(II).…”

“…EPR as a stand-alone technique provides three pieces of information relevant to MD and other diseases and conditions with metabolic components including cancer, neurological diseases, and cardiac dysfuncti on [79,82,106,[108][109][110][111]115] . The first is a measure of the redox potential across the MRC by quantitation of signals due to oxidized and reduced redox centers.…”