Abstract
The present study was directed to the development of EPR methodology for distinguishing cells with different proliferative activities, using “redox imaging.” Three nitroxide radicals were used as redox sensors: (a) mito-TEMPO—cell-penetrating and localized mainly in the mitochondria; (b) methoxy-TEMPO—cell-penetrating and randomly distributed between the cytoplasm and the intracellular organelles; and (c) carboxy-PROXYL—nonpenetrating in living cells and evenly distributed in the extracellular environment. The experiments were conducted on eleven cell lines with different proliferative activities and oxidative capacities, confirmed by conventional analytical tests. The data suggest that cancer cells and noncancer cells are characterized by a completely different redox status. This can be analyzed by EPR spectroscopy using mito-TEMPO and methoxy-TEMPO, but not carboxy-PROXYL. The correlation analysis shows that the EPR signal intensity of mito-TEMPO in cell suspensions is closely related to the superoxide level. The described methodology allows the detection of overproduction of superoxide in living cells and their identification based on the intracellular redox status. The experimental data provide evidences about the role of superoxide and hydroperoxides in cell proliferation and malignancy.
Highlights
Redox signaling is a key mechanism in maintaining cell homeostasis and normal functioning of the living organisms
We analyzed the dynamics of the electron paramagnetic resonance imaging (EPR) signal of mito-TEMPO in leukemic and normal lymphocytes at different incubation times and different concentration ratios of cells/nitroxide
Cell-penetrating nitroxide radicals are suitable contrast probes to distinguish between nonproliferative, slow proliferative, and fast proliferative cells, by using EPR spectroscopy
Summary
Redox signaling is a key mechanism in maintaining cell homeostasis and normal functioning of the living organisms. Violations of this mechanism play a crucial role in the pathogenesis of many diseases: cancer, neurodegeneration, atherosclerosis, inflammation, diabetes, etc., whose common characteristic is the development of oxidative stress and impairment of redox balance in cells, tissues, and body fluids [1]. Reactive oxygen species (ROS) are the main inducers of oxidative stress. Their production can be accelerated by exogenous and/or endogenous factors [2, 3]. Endogenous inducers of ROS are predominantly mitochondria and enzyme complexes [NAD(P)H-dependent oxidases (NOX), cytochrome P450dependent monooxygenases, xanthine oxidase, myeloperoxidase, and nitric oxide synthase (NOS)]
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