Wilmarie Flores-Santana

The chemical biology of nitric oxide: implications in cellular signaling.

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO]<1-30 nM), Akt phosphorylation ([NO] = 30-100 nM), stabilization of HIF-1alpha ([NO] = 100-300 nM), phosphorylation of p53 ([NO]>400 nM), and nitrosative stress (1 microM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.

Nitric oxide and redox mechanisms in the immune response

The role of redox molecules, such as NO and ROS, as key mediators of immunity has recently garnered renewed interest and appreciation. To regulate immune responses, these species trigger the eradication of pathogens on the one hand and modulate immunosuppression during tissue‐restoration and wound‐healing processes on the other. In the acidic environment of the phagosome, a variety of RNS and ROS is produced, thereby providing a cauldron of redox chemistry, which is the first line in fighting infection. Interestingly, fluctuations in the levels of these same reactive intermediates orchestrate other phases of the immune response. NO activates specific signal transduction pathways in tumor cells, endothelial cells, and monocytes in a concentration‐dependent manner. As ROS can react directly with NO‐forming RNS, NO bioavailability and therefore, NO response(s) are changed. The NO/ROS balance is also important during Th1 to Th2 transition. In this review, we discuss the chemistry of NO and ROS in the context of antipathogen activity and immune regulation and also discuss similarities and differences between murine and human production of these intermediates.

Molecular mechanisms for discrete nitric oxide levels in cancer

Nitric oxide (NO) has been invoked in nearly every normal and pathological condition associated with human physiology. In tumor biology, nitrogen oxides have both positive and negative affects as they have been implicated in both promoting and preventing cancer. Our work has focused on NO chemistry and how it correlates with cytotoxicity and cancer. Toward this end, we have studied both concentration- and time-dependent NO regulation of specific signaling pathways in response to defined nitrosative stress levels that may occur within the tumor microenvironment. Threshold levels of NO required for activation and stabilization of key proteins involved in carcinogenesis including p53, ERK, Akt and HIF have been identified. Importantly, threshold NO levels are further influenced by reactive oxygen species (ROS) including superoxide, which can shift or attenuate NO-mediated signaling as observed in both tumor and endothelial cells. Our studies have been extended to determine levels of NO that are critical during angiogenic response through regulation of the anti-angiogenic agent thrombospondin-1 (TSP-1) and pro-angiogenic agent matrix metalloproteinase-9 (MMP-9). The quantification of redox events at the cellular level has revealed potential mechanisms that may either limit or potentiate tumor growth, and helped define the positive and negative function of nitric oxide in cancer.

The Emergence of Nitroxyl (HNO) as a Pharmacological Agent

Once a virtually unknown nitrogen oxide, nitroxyl (HNO) has emerged as a potential pharmacological agent. Recent advances in the understanding of the chemistry of HNO has led to the an understanding of HNO biochemistry which is vastly different from the known chemistry and biochemistry of nitric oxide (NO), the one-electron oxidation product of HNO. The cardiovascular roles of NO have been extensively studied, as NO is a key modulator of vascular tone and is involved in a number of vascular related pathologies. HNO displays unique cardiovascular properties and has been shown to have positive lusitropic and ionotropic effects in failing hearts without a chronotropic effect. Additionally, HNO causes a release of CGRP and modulates calcium channels such as ryanodine receptors. HNO has shown beneficial effects in ischemia reperfusion injury, as HNO treatment before ischemia-reperfusion reduces infarct size. In addition to the cardiovascular effects observed, HNO has shown initial promise in the realm of cancer therapy. HNO has been demonstrated to inhibit GAPDH, a key glycolytic enzyme. Due to the Warburg effect, inhibiting glycolysis is an attractive target for inhibiting tumor proliferation. Indeed, HNO has recently been shown to inhibit tumor proliferation in mouse xenografts. Additionally, HNO inhibits tumor angiogenesis and induces cancer cell apoptosis. The effects seen with HNO donors are quite different from NO donors and in some cases are opposite. The chemical nature of HNO explains how HNO and NO, although closely chemically related, act so differently in biochemical systems. This also gives insight into the potential molecular motifs that may be reactive towards HNO and opens up a novel field of pharmacological development.

The specificity of nitroxyl chemistry is unique among nitrogen oxides in biological systems

The importance of nitric oxide in mammalian physiology has been known for nearly 30 years. Similar attention for other nitrogen oxides such as nitroxyl (HNO) has been more recent. While there has been speculation as to the biosynthesis of HNO, its pharmacological benefits have been demonstrated in several pathophysiological settings such as cardiovascular disorders, cancer, and alcoholism. The chemical biology of HNO has been identified as related to, but unique from, that of its redox congener nitric oxide. A summary of these findings as well as a discussion of possible endogenous sources of HNO is presented in this review.

Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine.

The chemical reactivity, toxicology, and pharmacological responses to nitroxyl (HNO) are often distinctly different from those of nitric oxide (NO). The discovery that HNO donors may have pharmacological utility for treatment of cardiovascular disorders such as heart failure and ischemia reperfusion has led to increased speculation of potential endogenous pathways for HNO biosynthesis. Here, the ability of heme proteins to utilize H2O2 to oxidize hydroxylamine (NH2OH) or N-hydroxy-L-arginine (NOHA) to HNO was examined. Formation of HNO was evaluated with a recently developed selective assay in which the reaction products in the presence of reduced glutathione (GSH) were quantified by HPLC. Release of HNO from the heme pocket was indicated by formation of sulfinamide (GS(O)NH2), while the yields of nitrite and nitrate signified the degree of intramolecular recombination of HNO with the heme. Formation of GS(O)NH2 was observed upon oxidation of NH2OH, whereas NOHA, the primary intermediate in oxidation of L-arginine by NO synthase, was apparently resistant to oxidation by the heme proteins utilized. In the presence of NH2OH, the highest yields of GS(O)NH2 were observed with proteins in which the heme was coordinated to a histidine (horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin, and hemoglobin) in contrast to a tyrosine (catalase) or cysteine (cytochrome P450). That peroxidation of NH2OH by horseradish peroxidase produced free HNO, which was able to affect intracellular targets, was verified by conversion of 4,5-diaminofluorescein to the corresponding fluorophore within intact cells.

Comparing the chemical biology of NO and HNO.

For the past couple of decades nitric oxide (NO) and nitroxyl (HNO) have been extensively studied due to the important role they play in many physiological and/or pharmacological processes. Many researchers have reported important signaling pathways as well as mechanisms of action of these species, showing direct and indirect effects depending on the environment. Both NO and HNO can react with, among others, metals, proteins, thiols and heme proteins via unique and distinct chemistry leading to improvement of some clinical conditions. Understanding the basic chemistry of NO and HNO and distinguishing their mechanisms of action as well as methods of detection are crucial for understanding the current and potential clinical applications. In this review, we summarize some of the most important findings regarding NO and HNO chemistry, revealing some of the possible mechanisms of their beneficial actions.

The inhibitors of histone deacetylase suberoylanilide hydroxamate and trichostatin A release nitric oxide upon oxidation

Suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) is the lead compound of a new class of histone deacetylase (HDAC) inhibitors used as anticancer drugs that have been shown to affect multiple proteins associated with gene expression, cell proliferation, and migration. Studies have also demonstrated the essential role of the hydroxamate moiety of SAHA in HDAC inhibition. The ability of SAHA and its structural analog trichostatin A (TSA) to generate NO upon oxidation was tested directly, by spin trapping of NO using electron paramagnetic resonance spectroscopy, and also indirectly, via the determination of nitrite using the Griess assay. H2O2/metmyoglobin was used to oxidize SAHA and TSA. These studies demonstrate, for the first time, the release of NO from SAHA and its structural analog TSA. We tested the protective effects of SAHA, TSA, and valproic acid (VPA) in mammalian Chinese hamster V79 cells exposed to a bolus of H2O2 for 1 h and monitored the clonogenic cell survival. Both SAHA and TSA afforded significant cytoprotection when co-incubated with H2O2, whereas VPA was ineffective. These studies provide evidence for the release of NO by hydroxamate-containing HDAC inhibitors and their antioxidant effects. Such roles may be an added advantage of this class of HDAC agents used for epigenetic therapies in cancer.

Nitroxide derivatives of non-steroidal anti-inflammatory drugs exert anti-inflammatory and superoxide dismutase scavenging properties in A459 cells

Inflammation and reactive oxygen species are associated with the promotion of various cancers. The use of non‐steroidal anti‐inflammatory drugs (NSAIDs) in cancer prevention treatments has been promising in numerous cancers. We report the evaluation of NSAIDs chemically modified by the addition of a redox‐active nitroxide group. TEMPO‐aspirin (TEMPO‐ASA) and TEMPO‐indomethacin (TEMPO‐IND) were synthesized and evaluated in the lung cancer cell line A549.

Nitric Oxide and Cancer: An Overview

An involvement of nitric oxide, a diatomic radical, has been described for numerous areas from environmental pollution to cardiovascular disease, carcinogenesis, tumor progression, genotoxicity, and angiogenesis. Previously, it has been demonstrated that NO may perform different functions dependent on NO levels achieved in a particular microenvironment. Furthermore, researchers also have discovered and identified the various sources of NO, which can elicit different biological responses of NO. In order to better understand the biological consequences of NO responses, one must first understand the chemical biology of NO. Since the first discussions during the early 1990s, it became widely accepted that NO chemical biology can be classified into two classes: direct interaction and indirect interaction. These two classes provided us with the means to understand the basic chemical toxicological effects of NO and its resulting reactive nitrogen species (RNS). NO has been reported to be involved in several steps of carcinogenesis, including interactions with p53 at both the genetic and the protein level and through regulation of the apoptotic pathways and DNA repair mechanisms. Recently, NO has also been linked to various immune and inflammation responses, especially in cancer development and wound healing process. Tumors are known to alter the immune response and tissue vascularization which involves NO. Therefore, a better understanding of the roles of NO in immune response modulation and wound healing would allow us to design a better treatment plan and improve NO drug efficacy.