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Inhibiting the Activity of NADPH Oxidase in Cancer.

ABSTRACT: Significance: The primary function of NADPH oxidases (NOX1-5 and dual oxidases DUOX1/2) is to produce reactive oxygen species (ROS). If inadequately regulated, NOX-associated ROS can promote oxidative stress, aberrant signaling, and genomic instability. Correspondingly, NOX isoforms are known to be overexpressed in multiple malignancies, thus constituting potential therapeutic targets in cancer. Recent Advances: Multiple genetic studies aimed at suppressing the expression of NOX proteins in cellular and animal models of cancer have provided support for the notion that NOXs play a pro-tumorigenic role. Further, large drug screens and rational design efforts have yielded inhibitor compounds, such as the diphenylene iodonium (DPI) analog series developed by our group, with increased selectivity and potency over "first generation" NOX inhibitors such as apocynin and DPI. Critical Issues: The precise role of NOX enzymes in tumor biology remains poorly defined. The tumorigenic properties of NOXs vary with cancer type, and precise tools, such as selective inhibitors, are needed to deconvolute NOX contribution to cancer development. Most NOX inhibitors developed to date are unspecific, and/or their mechanistic and pharmacological characteristics are not well defined. A lack of high-resolution crystal structures for NOX functional domains has hindered the development of potent and selective inhibitors. Future Directions: In-depth studies of NOX interactions with the tumor microenvironment (e.g., cytokines, cell-surface antigens) will help identify new approaches for NOX inhibition in cancer.

SUBMITTER: Konate MM

PROVIDER: S-EPMC7370979 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed 15N-and deuterium-enriched spin probe 15N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are "primed" for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.

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Inhibiting the Activity of NADPH Oxidase in Cancer.

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