The general public is increasingly interested in knowing whether antioxidant (vitamin E, vitamin C, coenzyme Q10 [Co-Q], and a litany of other anti-inflammatory and antioxidant agents sold over the counter) supplementation during cytotoxic cancer therapy or radiation therapy is beneficial or deleterious. There is great interest in knowing whether antioxidant supplementation can be used as a cancer prevention approach.
Results from clinical trials using beta-carotene (a molecule that inhibits the formation of oxidants derived from oxygen) and vitamin E revealed that the lung cancer patients treated with the antioxidants did worse than cancer patients who did not receive the antioxidant supplementation. While this finding is scientifically exciting and intriguing, it is confusing to lay public and raises the question: Do antioxidants enhance tumor formation and tumor metastasis?
To address this question, investigators from Sweden used vitamin E (a lipid-soluble antioxidant) and N-acetylcysteine (a water-soluble drug) in a mouse model of lung cancer. Results showed that lung cancer progression was elevated in mice treated with vitamin E or N-acetylcysteine. This intriguing and provocative study raises a fundamental question concerning the mechanism of action of these drugs. Are these structurally different drugs exerting pro-tumorigenic and pro-metastatic effects by decreasing the same oxidant formation in tissues or through different mechanisms? Similar paradoxical findings have also been reported in other areas of research including aging and cardiovascular diseases. The paradoxical roles of reactive oxygen species (ROS) and antioxidants, therefore, are not unique to the cancer biology field.
As discussed in the article, the dual nature of oxidants or ROS — the beneficial and the deleterious — in normal and cancer cells and tissues should be understood thoroughly before these drugs are prescribed for treating cancer and other diseases. This article focuses on the really fundamental aspects of the nature of oxidants that are produced in biological and pathophysiological systems as well as on the assays to detect and characterize the ROS and their molecular signaling pathways. The ultimate goal is to be able to develop an assay for detecting oxidant formation in cancer tissues isolated from patients that will assist clinicians in determining a suitable combination of chemotherapies.
ROS and their cellular sources
ROS do not relate to a single species; rather, they encompass many reactive species containing nitrogen, halogen, and sulfur in addition to oxygen. Peroxynitrite and nitrogen dioxide are examples of nitrogen-containing reactive species, hypochlorous acid (or bleach) is a chlorine-containing oxidant, and hydrogen sulfide or sulfur oxides represent a thiol-containing oxidant. ROS derived from oxygen generally are superoxide, hydrogen peroxide, and lipid-derived oxidants (lipid hydroperoxides).
Typically, oxidation of a fluorescent, chemiluminescent, or bioluminescent dye molecule is monitored, and the changes observed are related to increased oxidant formation. These dyes are generally a redox-active molecule that, in the reduced form, exhibits low fluorescence and, in the oxidized form, exhibits much higher fluorescence. The fluorescent quantum yield of oxidized dyes is much higher than that of the reduced form. The reduced and oxidized form of the bioluminescent dyes exhibits similar types of changes. Because many oxidants can oxidize a reduced fluorescent or bioluminescent dye molecule, one cannot infer the nature of oxidants by monitoring the changes in fluorescence or bioluminescence.
We characterized the chemical products derived from oxidation of redox-active dyes such as hydroethidine (HE) and related dyes. Superoxide selectively forms a highly characteristic diagnostic marker product, 2-hydroxyethidium, upon reaction with HE or an HE-derived radical. HE can also be used to monitor peroxidatic oxidation catalyzed by heme peroxidase and cytochrome c. Characteristic dimeric products formed from HE oxidation are detected. In addition, a nonspecific oxidation product, ethidium, that has previously been attributed to the superoxide reaction with HE is also detected. Thus, one can obtain semi-quantitative information about intracellular superoxide and derived oxidants by quantitating HE oxidation products.
Boronate-based fluorescent and bioluminescent probes are used to detect the other ROS such as hydrogen peroxide, hypochlorous acid, and peroxynitrite. These probes do not exhibit fluorescence; rather, they react with these species at different rates, forming a highly fluorescent product in cells. Thus, to decipher the actual species involved in the oxidation of boronates, one needs to use chemical and molecular biological tools, such as overexpression of catalase in mitochondria, that will catalytically detoxify hydrogen peroxide or L-NAME (a nonspecific inhibitor of nitric oxide synthase enzymes that will inhibit peroxynitrite formation). Interaction between hypochlorous acid or peroxynitrite and some boronate probes (ortho-Mito-boronate [Mito-B]) forms chlorinated or nitrated products of Mito-B as minor products that are diagnostic marker products. These products can be detected by liquid chromatography-mass spectrometry (LC-MS).
There are several intracellular sources of ROS, the most prominent of which are the mitochondria and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes. Inhibition of mitochondrial electron transport chain complexes induces the formation of superoxide and hydrogen peroxide. Generally, the probes used to detect the cytosolic ROS (hydroethidine and boronates) are modified through conjugation with the triphenylphosphonium cation (TPP+) and targeting to mitochondria. Mitochondria-targeted TPP+ probes may induce superoxide and hydrogen peroxide by inhibiting mitochondrial complexes (generally complex I). Thus, it is important to detect and verify mitochondrial ROS using several approaches as described in a recent manuscript (Redox Biol. 2018;15:347-62). As indicated earlier, the fluorescence techniques are not suitable for selectively detecting the various ROS in mitochondria and determining the characteristic marker products of fluorescent probes using LC-MS is necessary.
The NADPH oxidase family of enzymes (Nox1 through Nox5) induces superoxide and hydrogen peroxide in the cytosolic compartments. Many probes are currently available that can react with Nox-derived ROS and form specific products. Under inflammatory conditions, both Nox and inducible nitric oxide synthase are induced, thus enabling the formation of peroxynitrite. Many boronate-based probes rapidly react with peroxynitrite, forming diagnostic marker products. The boronate probes effectively compete with cellular antioxidants such as glutathione for reaction with peroxynitrite. Most other tyrosine-containing probes react with hemolysis-derived radicals (hydroxyl radicals and nitrogen-dioxide radicals), forming nitrated products.
Redox signaling, cancer drug resistance, metabolic reprogramming, and mitochondrial ROS
Why is it important to understand more about the ROS mechanism and reaction?Â
Although cytotoxic antitumor drugs initially exert a positive response in many cancer patients, the patients develop resistance to cytotoxic antitumor drug therapy with continued use. Most chemotherapeutic agents are faced with the drug resistance phenomenon that frustrates and impedes the cancer treatment modality. Studies indicate that drug-resistant cancer cells undergo metabolic reprogramming from glycolysis to enhanced oxidative phosphorylation to sustain their growth. Using ROS-sensitive redox probes, it was shown that increased mitochondrial superoxide formation (associated with enhanced oxidative phosphorylation) occurs in metabolically reprogrammed drug-resistant cancer cells. However, the investigators followed the increase in fluorescence intensity of oxidized redox probes and attributed the chemical identity of ROS. Clearly, based on this article, it is necessary to use rigorous methods to determine the chemical identity of ROS formed during drug resistance. In order to develop strategies to overcome drug resistance, it is necessary to fully understand the mechanisms of ROS formation and their chemical identity.
OXPHOS-inhibiting drugs, mitophagy, ROS, and cancer cell proliferation
The oxidative phosphorylation (OXPHOS) inhibiting (anti-OXPHOS) drugs refer to mitochondria-targeted drugs that decrease mitochondrial respiration via inhibition of electron transport chain proteins such as complex I. Delocalized cations (e.g., rhodamine dye) accumulate more selectively in cancer cell mitochondria (due to enhanced negative mitochondrial membrane potential) than in normal cell mitochondria and inhibit mitochondrial respiration in cancer cells. Although it is well known that cationic compounds are taken up into mitochondria of cancer cells and induce antiproliferative effects, only recently was it shown that several bioactive agents can be selectively targeted to mitochondria via fine-tuning of the linker side chain attached to a TPP+ group.
Mito-Q, Mito-Vit-E, Mito-metformin, and Mito-honokiol — molecules that are conjugated to TPP+ — preferentially target the mitochondria of cancer cells as compared with normal, non-transformed cells. This class of mitochondria-targeted agents selectively and potently inhibits complex I more so than other mitochondrial complexes. We believe that it is necessary to use the extracellular flux analyzer in real time in order to simultaneously assess the activity of different complexes of the electron transport chain. Results indicate that inhibition of complex I is a primary mechanism leading to its antiproliferative effects.
Mitochondria-targeted OXPHOS-inhibiting therapeutics (mito compounds) activate AMP-activated protein kinase (AMPK), a master energy sensor within the cell. AMPK activation results in the upregulation of adenosine triphosphate (ATP)-generating pathways and decreased ATP-consuming pathways. The relationship between AMPK activation and cancer cell proliferation became evident in light of the protective effects of AMPK inhibitors. Inhibiting AMPK signaling reversed the antiproliferative effects of OXPHOS inhibitors such as Mito-metformin. The causative role of ROS in AMPK activation still needs to be understood.
Mitophagy, a highly regulated process of removing modified or damaged mitochondrial proteins, has been linked to tumorigenesis, although its functional role (cytoprotective or cytotoxic) in normal and cancer cells is different and context dependent. Whereas starvation or nutrient deprivation induces autophagy, OXPHOS inhibitors promote mitophagy that plays a critical role in tumorigenesis, tumor progression, and metastasis. Understanding the role of autophagy-inducing pathways in detail will play a major role in developing innovative cancer therapeutics.
Tumor microenvironment and ROS formation
ROS generated from NADPH oxidases have been attributed to inactivation of T cells in the tumor microenvironment. One of the consequences is the decreased immune response to tumor cells (e.g., pancreatic cancer microenvironment). Determining the identity and reactivity of ROS in the tumor microenvironment will help prevent inactivation of T cells and also in the suppression of the Nox activity that is responsible for ROS generation. Both mechanisms will result in an increase in tumor-specific immune responses.
Future perspectives
This review has set the tone for the rigorous identification of ROS in cells. The ex vivo electron paramagnetic resonance technique discussed in the review can be used to assess oxidant formation in tissues. Monitoring dynamic changes in specific ROS following drug resistance and metabolic reprogramming in tumors could help in the development of precise antitumor drug therapy. Going forward, it is crucial to unraveling the paradoxical roles of ROS and autophagy/mitophagy in tumor growth and tumor regression.
These findings are described in the article entitled Teaching the basics of reactive oxygen species and their relevance to cancer biology: Mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies, recently published in the journal Redox Biology. This work was conducted by Balaraman Kalyanaraman, Gang Cheng, and Jacek Zielonka from the Medical College of Wisconsin, Micael Hardy and Olivier Ouari from Aix Marseille Univ, and Brian Bennett from Marquette University.