NO^• is a free radical which was discovered in 1980 as a ubiquitous diffusible second messenger. While some authors use the term “nitric oxide” to refer to any of the nitric oxide reactive species (NO^•, NO⁻, and NO⁺), in biological settings, nitric oxide usually refers to the free radical. NO^• was determined to be a prominent constituent of what was then called endothelium-derived relaxing factor (EDRF) [1]. Later in 1985, Eschericia coli lipopolysacchride (LPS) was found to initiate production of NO^• by LPS-stimulated mouse macrophages [2]. We now know the molecule plays a significant role in both normal and abnormal physiology of human beings, as well as plants and invertebrates [3–5]. NO^• has a well characterized two-step synthetic metabolic pathway in which L-arginine is first converted to N_G-hydroxy-L-arginine and then to L-citrulline and NO^•. This reaction is catalyzed by the enzymatic family of nitric oxide synthases (NOSs). Three NOS isoforms exist: 1) nNOS/NOS1, a calcium-dependent enzyme discovered in neurons that is involved in neural transmission; 2) iNOS/NOS2, a calcium-independent enzyme that releases large amounts of NO^• in response to macrophage activation with endotoxin and cytokines, and is involved in cytotoxicity; and 3) eNOS/NOS3, also a calcium-dependent enzyme that is constitutively expressed, isolated from endothelial cells, and is found in normal vascular endothelium [6,7]. Once NO^• is produced, it can react with various molecules, resulting in more stable compounds such as S-nitrosothiols, metal adducts, peroxynitrites (while in the presence of oxygen), and tetrahydrobiopterin (THB) [6]. THB is recognized as a necessary prerequisite for the biosynthesis of key aromatic amino acid hydroxylase enzyme precursors necessary for synthesis of neurotransmitters such as serotonin, melatonin, epinephrine and dopamine.

It is believed NO^• regulates the physiology and pathophysiology of the body through one of three biomolecular mechanisms: 1) redox interactions with thiols, 2) coordinating interactions with metal functional centers, and 3) through protein kinase activity [8]. The role and impact of NO^• is believed to be widespread because of its ability to cross cell membranes in an unaltered chemical form, diffuse rapidly, interact with key generative and target cell-response proteins, and quickly interact with key transition metal containing proteins [6].

The various biomolecular mechanisms of NO^• result in numerous biological functions, including: 1) antitumor and microbial immunity, including against gram positive organisms [6], 2) immuno-modulation and allo-antigenicity [9,10], and 3) a signaling pathway [6]. Nearly every cell throughout the body has the ability to express calcium-independent iNOS [6]. Within the central nervous system, learning, sleep, feeding, male and female reproductive behavior are all impacted by NO^• [11]. It also influences the neurotransmitters in synapses between peripheral organs [11] and regulates angiogenesis and neurogenesis after stroke activity [12]. NO^• also delays the aging of oocytes [13], controls resting potential in skeletal muscle [14], regulates contraction-excitation coupling [14], and modulates chondrocyte development during endochondral ossification [15]. Significantly, NO^• depravation is a critical underlying cause for endothelial dysfunction, which in turn is a key common contributor to diabetes-related cardiovascular disease, myocardial infarction, and atherosclerosis [16,17].

NO^• in low concentrations is now known to have benign, modulating, and regulatory effects on normal mammalian and human biology and physiology. Higher concentration levels of NO^• are now shown to be both damaging and pathologic to physiologic processes [6]. What is defined as a high or low level can vary enormously depending upon which physiologic system the free radical is found and the apparent contradictory functional impact of NO^• presence on a particular molecular mechanism and biochemical microenvironment [6]. For instance, at NO^• concentrations of less than 100 nM, cyclic guanosine monophosphate (cGMP), cGMP-dependent protein kinase (PKG) and extracellular signal regulated kinase (ERK) activation can occur. As concentration levels increase Akt is phosphorylated, and in ranges between 300 to 800 nM hypoxia inducible factor-1 alpha and p53 are stabilized [18]. As concentrations increase further, nitrosation and oxidative processes become prominent and initiate stressful cellular events [19]. However, the role of NO^• can be either protective or toxic, depending upon the unique biochemical content of the microenvironment in which it exists [20].

It is also now well established that NO^• plays a multifaceted and contradictory role in the biology and growth of tumors [21]. Over-expression of NOS has been shown to be responsible for tumor angiogenesis and maintaining vascular tone within tumor blood vessel systems [22–25], as well as the facilitation of neoplastic transformation [22,26,27]. Studies have shown that in cancer patients, NO^• regulates blood flow to tumors, and by down-regulating NO^• synthesis, a distinct vasoconstricting event results [22]. This has been demonstrated through the use of N-nitro-L-arginine (L-NNA) to reduce blood flow to tumors in BD9 rats with P22 carcinosarcoma [22,28]. In humans cancer patients reducing NOS results in an increase in blood pressure [22,29,30]. At higher concentrations in the proper microenvironment, for an extended period of time, NO^• exposure initiates inflammation, can stimulate tumor growth and/or metastatic behavior [6,7], and can lead to mutations and the clinical presentation of cancer [9,31,32]. Exogenous sources of NO^•, such as cigarette smoke, contribute to subcellular damage through the formation of N-nitrosoamines and N-nitrosamides, contributing to elevated expression of head and neck cancers [7,33–36].

It is also well recognized that reactive oxygen species (ROSs) play an important function in either the protective or pathologic expression of NO^• reactions with oxygen [6,7]. ROSs provide beneficial impacts through killing of microorganisms and malignant cells, or a pathologic effect, again depending upon the microenvironment. Higher concentrations of ROS generate oxidative and nitrosative environmental stressors leading to: 1) DNA damage, 2) down-regulated antioxidants, and 3) an impact on transcription/translation activities, thereby generally impairing normal cellular function [7,37,38]. Should DNA become damaged from either endogenous or exogenous sources of NO^•, a number of defensive apoptotic systems are initiated to protect against unwanted cellular transformation [6,7]. Amongst the most important defensive apoptotic systems is the up regulation of DNA damage sensing proteins, such as p53. Damage not repaired typically results in cellular death through apoptosis. NO^• is also known to inhibit caspase activation, which in turn is known to induce normal apoptosis. Studies have also shown NO^• prohibits apoptosis in a variety of cell types, as well as in some tumor cells [39,40].

Thiols are a group of biological molecules which act as intracellular antioxidants. Among the most studied forms is glutathione (GSH), known to react with and neutralize electrophilic centers of a number of environmental and oxidative cellular stressors. The enzyme which catalyzes these reactions is glutathione S-transferase-pi (GST-pi), one of a family of Phase II detoxifying glutathione S-transferases (GSTs) responsible not only for detoxification, but also activation of significant biochemical pathways essential to normal physiology [41]. These oxidative stressors include elevated levels of NO^•. The GST isoenzymes and their behavior are essential to providing yet another tool toward protection of DNA from a variety of endogenous and exogenous pathogenic sources [42–44]. Equally important, the GSTs catalyze the conjugation of GSH to an array of xenobiotic or toxic compounds rendering them non-toxic [45]. Due to these behaviors GSTs are an important area of research in molecular biology.

While GST isoenzymes relieve the source of toxicity, the catalytic group is also strongly implicated in the development of cellular resistance to anti-cancer drug therapy [42,45]. It provides a mechanism to explain cancer patients’ observed resistance to anticancer drug therapies [45]. Tumor cell lines which over-express GST-pi have heightened detoxification responses and acquire increased resistance to compounds perceived by the body as being toxic, including chemotherapeutic drugs [42,45,46]. GST isoenzymes are categorized into three primary groups: 1) cytosolic, 2) membrane-bound microsomal, and 3) mitochondrial [45]. The cytosolic type is further divided into seven classes: 1) Alpha, 2) Mu, 3) Omega, 4) Pi, 5) Sigma, 6) Theta, and 7) Zeta [45]. GST-pi is now recognized to be the predominant isoform subclass [42,44].

The GST-pi gene has four functional polymorphisms (GSTP1*A, GSTP1*B, GSTP1*C, and GSTP1*D), with each allelic genotype having different treatment response outcomes among individual cancer patients [45,47]. Examples include: 1) GSTP1*A is responsible for acquired resistance to cisplatin treatment due to creation of platinum-GSH conjugates [48], 2) GSTP1*B, which in certain circumstances, is associated with an impaired ability to detoxify platinum based therapeutic treatment [49], and 3) patients testing positive for GSTP1*C appear to experience breast cancer with less frequency [50]. All polymorphisms have been shown to impact, to varying degrees: 1) anticancer therapy treatment, 2) chemotherapeutic response, and 3) susceptibility to cancer. Most importantly, GSTP1 has been reported to be over-expressed in a number of different tumor types including: colon, lymphoma, pancreas, breast, NSCLC and ovarian [42,51]. One effort analyzed GST enzymes, GST composition, and GSH concentration levels in normal and squamous cell carcinoma tissues among 25 patients (14 with oropharyngeal or oral tumors, 11 with laryngeal tumors) [44]. GST-pi levels increased in 11 of the 14 oral cavity tumors, and elevated expression of GST-pi was found in all laryngeal tumors [44]. Another report provides evidence for heighten risk of relapse for laryngeal cancer associated with GST-pi over-expression [52]. Others propose the possibility that up-regulated GST-pi, GST-mu, and GST-alpha can be predictors of a second primary tumor in head and neck cancers [53]. Additional studies demonstrate over-expression of GST-pi within normal mucosa adjacent to tumors, dysplastic mucosal lesions, and head & neck squamous cell carcinoma (HNSCC) [54,55]. GST-pi expression increases through a step-wise progression, correlating with up-regulated NOS and molecular markers of oxidative injury [54,55]. It has been hypothesized that GST-pi is over-expressed in mucosal cells in response to oxidative injury by toxins such as NO^• and known nitrosative carcinogens resulting from smoking cigarettes [54,55].

Presented herein is a study in which we investigated GST-pi expression in laryngeal tumors (Table 1). Patient charts were reviewed for TNM stage and course of treatment. Tissue sections were reviewed, and the intensity of tumor staining was graded on an immunohistochemical scale (0–4).

In ten patients, seven had previously undergone radiation therapy; five of the seven patients were concurrently treated with chemotherapy and radiation therapy. All patients failed treatment or had recurrence or persistent disease. The seven patients who received radiation exhibited higher levels of GST-pi expression than the three patients who were not treated with radiation. Figure 1 shows examples of GST-pi immunostaining observed in human this study.

To investigate the commonality among squamous cell carcinomas arising in different sites, we also investigated the NOS and GST-pi expression of cervical squamous cell carcinomas (CSCC). Presented herein are results showing expression of eNOS, iNOS, and GST-pi in a series of patients with CSCC. Patient charts were reviewed for TNM stage, tumor grade, and course of treatment. All samples were obtained prior to treatment. Tissue sections were reviewed; the intensity of tumor staining was graded on an immunohistochemical scale (0–3). Table 2 summarizes the results.

Both iNOS and GST-pi were highly expressed in CSCC, whereas eNOS showed only limited expression. Examples of the observed staining are shown in Figure 2. The eNOS expression in CSCC was in contrast to previously reported HNSCC work which showed highly expressed eNOS [56,57].

Another reported study confirms our findings that NO^• is a significant contributor to cervical cancer, and suggests a link between NO^• and a number of prominent risk factors associated with the onset of cervical cancer. These factors include: 1) chronic inflammation, 2) HPV infections, 3) extended use of oral contraceptives, 4) sexually transmitted diseases, and 5) smoking tobacco [58]. All of these factors cause increases in NO^• levels [58–61] and markers of NO^•-mediated mutagenesis in patients with cervical intraepithelial neoplasia [58,62,63].

The role of reactive nitrogen species (RNSs) has been well documented for many decades. The impact of RNSs originates in inflammatory tissues and can result in mutations in tumor suppressor genes, leading to subsequent tumor neoplastic growth [64]. RNSs also cause post-translational modifications of proteins involved in fundamental cellular functions such as apoptosis, cell cycle check point, and DNA repair [65]. RNSs are known to cause both oxidation and nitration reactions resulting in DNA strand breaks, mutations in DNA base pairs, and helix modifications [65]. What has also become increasingly evident over time is how important the molecular composition of the microenvironment is relative to the degree of DNA alterations. The molecular composition of the microenvironment is influenced by a number of RNS factors, including: 1) biomolecular profile, 2) type, 3) concentration level, 4) accessibility, 5) bioavailability, and 6) half life [65]. RNSs can evolve further into a variety of related molecules including 4-hydroxynoneal (4-HNE) and reactive aldehydes-malondialdehydes (MDA), both of which are associated with increased cancer risk in chronic inflammatory diseases [65–67]. Both 4-HNE and MDA are also known to cause point mutations within tumor suppressor genes [65–67]. Further, RNSs play a critical role as a facilitator between signal transduction receptors such as the MAPK signaling cascade. This can lead to the expression of proto-oncogenes such as c-Jun, c-Fos, and AP-1. These proto-oncogenes impact differentiation, proliferation, cellular death, and transformation [65,68,69]. Free radical exposure is also well recognized to cause post-translational modifications which affect the functionality of key cellular proteins. For example, exposure to NO^•, an abundant RNS, leads to post-translational modifications of both p53 and Rb tumor suppressor genes at critical concentration levels [65,70,71]. Exposure to NO^• also activates DNA repair and signal transduction species including DNA protein kinases [65,72,73].

In the early 1940’s C.H. Waddington first used “epigenetics” to describe the mechanisms responsible for the developmental pathway from fertilized egg to an adult [74–76]. Epigenetics is known to regulate primary biological functions, including, but not limited to: 1) memory function, 2) development and aging, 3) mobile elements activity, 4) genomic imprinting, 5) viral infections, 6) somatic gene therapy, 7) cloning, 8) X-inactivation, and 9) the biology of cancer [77–81]. The list of diseases associated with epigenetic dysregulation continues to grow as research efforts progress [77–81]. Over time and with the expanded knowledge base created by the efforts of many, the term has evolved to reference the heritable modifications to chromatin, which regulate gene expression, but do not change the underlying DNA sequence [75,78,82]. The impact on chromatin composition can be rapid and reversible, originating from endogenous and exogenous sources and which may well modulate gene expression behavior [47,74,75,82].

Gene expression and silencing can be carried out via a number of interrelated epigenetic mechanisms that may be modulated by NO^•. These mechanisms include, but are not limited to: 1) DNA methylation [75,82–84], 2) microRNA (miRNA) [82,85,86], and 3) histone modifications [75,78,82,83,85,87–89]. The three mechanisms combine synergistically to regulate and affect epigenetic programming and reprogramming behavior [82,85,90–97]. Herein we discuss these three mechanisms and the emerging research to date as to how these may be affected by NO^•. Interestingly, a recent study involving Duchenne muscular dystrophy indicated that a diminution of NO^• results in global epigenetic changes, thereby implicating NO^• as an “epigenetic molecule” [5].

As indicated earlier in this review, our prior work has focused on the role of NO^• in both squamous cell carcinomas (head & neck, cervix) and adenocarcinomas (lung, breast). We and others have reported a spectrum of NOS expression in patient populations of these tumors, as well as other tumor types. It has also been observed that patients who present with and/or progress to high levels of NOS expression portend to have poorer clinical outcomes than those with low level expression. It has been hypothesized that immune system cells are being killed by the comparatively high free radical NO^• environment encountered in the tumor bed [185]. Since there is no practical way to study this in human patients, we sought to produce a unique, in vitro tissue culture model system of free radical stressed tumor cells to determine if in fact they could adapt to increasing levels of NO^•. The resulting model system would mimic the spectrum of NO^• expression found clinically [186].

Our model system was developed by “adapting” low NO^• expressing cell lines to increasing levels of NO^• donor. These “parent” cells were gradually exposed to high NO^• (HNO) levels, resulting in a new set of HNO cell lines. DETA-NONOate was selected as the NO^• donor for the adaptation process due to: a) its high level of free radical donation (two moles of NO^• per mole of DETA-NONOate), and relatively long half-life (approximately 24 h. at 37 °C and pH 7.4). During the adaptation process, the cell lines successfully withstood incremental increases of 25 μM DETA-NONOate. For each cell line, the adaptation endpoint was selected as the concentration in which the exogenous NO^• introduced to the cells was lethal to the parent cell lines. At this endpoint concentration, the HNO cells still grow robustly and are not morphologically altered from the original (untreated) parent cells. Six different parent/HNO cell line pairs have already been developed: one human lung adenocarcinoma cell line (A549) [186], one mouse lung adenocarcinoma (LP07) [186], and four human breast adenocarcinomas (T-47D, Hs578t, BT-20, and MCF-7) [187]. Ongoing work is focusing on extending this model to human head & neck, colon, prostate, and liver tumor cell lines.

While the A549 cells were adapted to DETA-NONOate (see Figure 3 below), the A549-HNO cell lines were also found to be resistant to other nitrogen-based free radical donors [186,187]. This suggests the A549-HNO cell line could have been generated by using any appropriate NO^• donor, and that the cells were adapted to the NO^• free radical, and not the donor per se [186,187].

Additionally, the lung and breast tumor HNO cell lines were exposed to various concentrations of hydrogen peroxide (H₂O₂), an oxygen-based free radical donor [186,187]. The HNO cell lines were more resistant to exposure than the corresponding parent cell lines (see Figure 4 as an example). These results show that the HNO cells are similarly resistant to oxygen-based free radicals.

The reported adaptation process resulted in major biological changes, between the parent and HNO cells despite the identical morphology between the two. HNO cancer cell lines exhibited more aggressive growth than did their corresponding parent cell lines under both normal and low-nutrient growth conditions [186,187].

The HNO adapted cell lines are comparable to aggressive, fast growing tumors growing in high NO^• environments, while the parent cell lines represent less aggressive, slower growing tumors existing in relatively lower NO^• environments. Furthermore, our adaptation process demonstrated that long-term NO^• exposure can alter slow growing, less resistant tumors, into faster growing and more resistant cancer cells [186,187]. The molecular mechanism for this parent-to-HNO transformation remains to be elucidated; however, high concentration levels of NO^• (above 1 μM) are known to increase nitrosative cellular stress, which interferes with DNA repair and inhibits zinc finger complexes [187–189]. Our model system has proven that tumor cells are able to adapt to comparatively high NO^• concentrations, regardless of tumor origin or their histological type. Understanding the role of NO^• in tumor cells may in part lie with NO^•-mediated epigenetics.

As was discussed above, epigenetic alterations that involve aberrant DNA methylation of CpG sequences in genes is increasingly being recognized as a key mechanism involved in transcriptional silencing of genes in both disease states and, healthy ageing populations [65,82,190]. Our HNO-adapted cell line system provides a robust, in vitro model for the identification of novel genetic targets that are associated with antioxidant stress. We also have evidence that, relative to the MCF-7 breast cancer parent cells, the HNO adapted MCF-7 cells demonstrate a significant increase in hypermethylation of both HPP1 (70-fold increase) and APC (22-fold increase) tumor suppressor genes (see Figure 5 below).

Research of NO^• has evolved greatly over time. The protective/cytotoxic duality of NO^•, once in question, is now generally accepted. As such, current studies are now more intently focused on understanding the role of NO^• in cellular toxicity, particularly as it relates to tumor development and progression. The association between NOS and GST may be a key component of this story, given that over-expression of NOS (regardless of isotype) is often observed in parallel with the over-expression of GST (particularly GST-pi). As discussed above, these results are consistent across a number of different tumor types, suggesting that NO^• behavior may be more consistent across different tumor types than originally thought. Whether this commonality in NO^• behavior exists among different tumor types will become more apparent as research is further pursued in the field. The HNO cell line model system described above may prove to be a valuable tool for such studies. Similarly, work will continue in the area of NO^• and epigenetics. While epigenetic research is still in its infancy, it is already clear that NO^• may play an important role in a number of epigenetic functions, including DNA methylation, microRNAs, and histone modifications. Thus, while much is already known about the biological role of NO^•, even more has yet to be discovered.