Teaching the basics of reactive oxygen species and their relevance to cancer biology: Mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies

Abstract

Reactive oxygen species (ROS) have been implicated in tumorigenesis (tumor initiation, tumor progression, and metastasis). Of the many cellular sources of ROS generation, the mitochondria and the NADPH oxidase family of enzymes are possibly the most prevalent intracellular sources. In this article, we discuss the methodologies to detect mitochondria-derived superoxide and hydrogen peroxide using conventional probes as well as newly developed assays and probes, and the necessity of characterizing the diagnostic marker products with HPLC and LC-MS in order to rigorously identify the oxidizing species. The redox signaling roles of mitochondrial ROS, mitochondrial thiol peroxidases, and transcription factors in response to mitochondria-targeted drugs are highlighted. ROS generation and ROS detoxification in drug-resistant cancer cells and the relationship to metabolic reprogramming are discussed. Understanding the subtle role of ROS in redox signaling and in tumor proliferation, progression, and metastasis as well as the molecular and cellular mechanisms (e.g., autophagy) could help in the development of combination therapies. The paradoxical aspects of antioxidants in cancer treatment are highlighted in relation to the ROS mechanisms in normal and cancer cells. Finally, the potential uses of newly synthesized exomarker probes for in vivo superoxide and hydrogen peroxide detection and the low-temperature electron paramagnetic resonance technique for monitoring oxidant production in tumor tissues are discussed.

Keywords: Mitochondrial inhibition; Oxidative phosphorylation; Oxidative stress; Reactive oxygen species; Triphenylphosphonium cation.

Fig. 1

HE-derived nonspecific oxidation and O₂^•–-specific hydroxylation products. The fluorescence spectra of the nonspecific oxidation product E⁺ (10 µM, right axis) and O₂^•–-dependent hydroxylation product 2-OH-E⁺ (10 µM, left axis) in the presence of DNA show a significant overlap. (Obtained from Ref. . Reprinted from H. Zhao, J. Joseph, H.M. Fales, E.A. Sokoloski, R.L. Levine, J. Vasquez-Vivar, B. Kalyanaraman, 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(16) (2005) 5727–5732, Copyright 2005 National Academy of Sciences.).

Fig. 2

H₂O₂and peroxynitrite-induced formation of major and minor products derived from o-MitoPhB(OH)₂. Reaction between H₂O₂ and o-MitoB stoichiometrically generates the only product, o-Mito-PhOH, whereas the reaction between ONOO^– and o-MitoB(OH)₂ produces a major product, o-Mito-PhOH, and a minor cyclized product that is highly characteristic for reaction with ONOO^–. (Obtained from Ref. . Reprinted from Interface Focus, 7, B. Kalyanaraman, G. Cheng, M. Hardy, O. Ouari, A. Sikora, J. Zielonka, M. Dwinell, Mitochondria-targeted metformins: anti-tumor and redox signaling mechanisms, 20160109, Copyright 2017.).

Fig. 3

Relationship between enhanced oxidation of redox dyes and redox-dependent processes. Enhanced oxidation of redox dyes leading to enhanced fluorescence intensity during tumorigenesis, metabolic reprogramming, and drug resistance.

Fig. 4

Lack of evidence for enhanced O₂^•–formation during tumorigenesis, metabolic reprogramming, and drug resistance. In order to categorically state that O₂^•– is formed during redox processes in tumor cells, it is essential to separate and detect the corresponding O₂^•–-specific hydroxylated products from HE or Mito-SOX.

Fig. 5

Mito-metformin induces increased O₂^•–and H₂O₂formation in pancreatic cancer cells. O₂^•–-dependent oxidation of the HE probe (A) and H₂O₂-dependent oxidation of the probe, o-MitoPhB(OH)₂ (B) in MiaPaCa-2 cells treated for 24 h with Mito-Met₁₀. (Obtained from Refs. , . Reprinted from Cancer Research, 76, G. Cheng, J. Zielonka, O. Ouari, M. Lopez, D. McAllister, K. Boyle, C.S. Barrios, J.J. Weber, B.D. Johnson, M. Hardy, M.B. Dwinell, B. Kalyanaraman, Mitochondria-targeted analogs of metformin exhibit enhanced antiproliferative and radiosensitizing effects in pancreatic cancer cells, 3904-15, Copyright 2016, and from Interface Focus, 7, B. Kalyanaraman, G. Cheng, M. Hardy, O. Ouari, A. Sikora, J. Zielonka, M. Dwinell, Mitochondria-targeted metformins: anti-tumor and redox signaling mechanisms, 20160109, Copyright 2017.).

Fig. 6

Dimeric product formed from one-electron oxidation of HE and analogs. (Modified from Ref. . Reprinted from Cell Biochemistry and Biophysics, Modified Metformin as a More Potent Anticancer Drug: Mitochondrial Inhibition, Redox Signaling, Antiproliferative Effects and Future EPR Studies, 75, 2017, 311-7, B. Kalyanaraman, G. Cheng, M. Hardy, O. Ouari, A. Sikora, J. Zielonka, M.B. Dwinell with permission of Springer.).

Fig. 7

O₂^•–-specific and nonspecific oxidation products formed from the mitochondrial superoxide probe, MitoNeoD. (Modified from Ref. . Reprinted from Cell Chemical Biology, 24, M.M. Shchepinova, A.G. Cairns, T.A. Prime, A. Logan, A.M. James, A.R. Hall, S. Vidoni, S. Arndt, S.T. Caldwell, H.A. Prag, V.R. Pell, T. Krieg, J.F. Mulvey, P. Yadav, J.N. Cobley, T.P. Bright, H.M. Senn, R.F. Anderson, M.P. Murphy, R.C. Hartley, MitoNeoD: A Mitochondria-Targeted Superoxide Probe, 1285-98.e12, https://doi.org/10.1016/j.chembiol.2017.08.003, https://creativecommons.org/licenses/by/4.0/, Copyright 2017.).

Fig. 8

Some examples of TPP⁺-based positively charged anticancer agents. Note that it is critical to clearly define the complete structure indicating the side chain length of mitochondria-targeted TPP⁺-based compounds. (Modified from Ref. . Adapted with permission from J. Zielonka, J. Joseph, A. Sikora, M. Hardy, O. Ouari, J. Vasquez-Vivar, G. Cheng, M. Lopez, B. Kalyanaraman, Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications, Chemical Reviews 117(15) (2017) 10043-120. Copyright 2017 American Chemical Society.).

Fig. 9

Chemical structures of Mito-CP and its redox inactive analog, Mito-CP-acetamide.

Fig. 10

Chemical structures of Mito-Tempo and Mito-Tempo acetamide.

Fig. 11

Peroxiredoxin pathway of H₂O₂metabolism. (Modified from Ref. . Reprinted by permission from Macmillan Publishers Ltd: Nature Chemical Biology, M.C. Sobotta, W. Liou, S. Stöcker, D. Talwar, M. Oehler, T. Ruppert, A.N.D. Scharf, T.P. Dick, Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling, Nature Chemical Biology 11(1) (2015) 64–70, copyright 2015.).

Fig. 12

PGC1α regulates mitochondrial biogenesis and oxidative stress in melanoma cells. (Modified from Ref. . Reprinted from Cancer Cell, 23, F. Vazquez, J.H. Lim, H. Chim, K. Bhalla, G. Girnun, K. Pierce, C.B. Clish, S.R. Granter, H.R. Widlund, B.M. Spiegelman, P. Puigserver, PGC1alpha expression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress, 287–301, Copyright 2013, with permission from Elsevier.).

Fig. 13

Mito-metformin and activation of AMPK: Potential signaling role of H₂O₂. (Obtained from Ref. . Reprinted from Cancer Research, 76, G. Cheng, J. Zielonka, O. Ouari, M. Lopez, D. McAllister, K. Boyle, C.S. Barrios, J.J. Weber, B.D. Johnson, M. Hardy, M.B. Dwinell, B. Kalyanaraman, Mitochondria-targeted analogs of metformin exhibit enhanced antiproliferative and radiosensitizing effects in pancreatic cancer cells, 3904-15, Copyright 2016.).

Fig. 14

Redox signaling of H₂O₂via peroxiredoxin/Trx redox relay including STAT3 transcription factor. (Obtained from Ref. . Reprinted by permission from Macmillan Publishers Ltd: Nature Chemical Biology, M.C. Sobotta, W. Liou, S. Stöcker, D. Talwar, M. Oehler, T. Ruppert, A.N.D. Scharf, T.P. Dick, Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling, Nature Chemical Biology 11(1) (2015) 64–70, copyright 2015.).

Fig. 15

Schematic of the Keap1–Nrf2–ARE pathway. (Modified from Ref. . Reprinted from Oncotarget, 8, X. Gao, B. Schottker, Reduction-oxidation pathways involved in cancer development: a systematic review of literature reviews, 51888-906, copyright 2017.).

Fig. 16

O₂^•–-mediated inactivation of aconitase: A possible source of hydroxyl radical generation in mitochondria. (Modified from Ref. . This research was originally published in Journal of Biological Chemistry. J. Vasquez-Vivar, B. Kalyanaraman, M.C. Kennedy. Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation. 2000; 275:14064-9. © the American Society for Biochemistry and Molecular Biology.).