On October 12, 1998, the Nobel Assembly awarded the Nobel Prize in Medicine and Physiology to scientists Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries concerning nitric oxide (NO) as a signaling molecule in the cardiovascular system. In contrast with the short research history of the enzymatic synthesis of NO, the introduction of nitrate-containing compounds for medicinal purposes marked its 150th anniversary in 1997. Glyceryl trinitrate (nitroglycerin; GTN) is the first compound of this category. Alfred Nobel (the founder of the Nobel Prize) himself had suffered from angina pectoris and was prescribed nitroglycerin for his chest pain while he refused to take due to the induction of headaches. Almost a century after its first chemical use, research in the nitric oxide and 3′,5′-cyclic guanosine monophosphate (NO/cGMP) pathway has dramatically expanded and the role of NO/cGMP in physiology and pathology has been extensively studied. Soluble guanylyl cyclase (sGC) is the receptor for NO. The α1β1 heterodimer is the predominant isoform of sGC that is obligatory for catalytic activity. NO binds to the ferrous (Fe2+) heme at histidine 105 of the β1 subunit and leads to an increase in sGC activity and cGMP production of at least 200-fold (1). On the other hand, the effects of NO can also be attributed to the cGMP-independent pathway, which is mediated mainly by reactive oxygen/nitrogen species such as highly reactive peroxynitrite (ONOO−) (1, 2). The direct NO effect (cGMP-independent) has been shown to interact with cancer biology through various signaling molecules (3), such as IκB-α (4), c-Jun NH2 terminal kinase (5), HIF-1α (6), Hdm2-p53 binding (7), Keap1/Nrf2 (8), and IRP-1(9). Thus, the steady-state concentration and the biological effects of NO/cGMP are critically determined not only by its rate of formation, but also by its rate of decomposition. Biotransformation of NO via different metabolic routes within the body presents another attractive field for our research as well as for drug discovery and development.
The role of NO and cGMP signaling in tumor biology has been extensively studied during the past three decades. The earliest studies with NO/cGMP were done in our lab in the 1970's and 1980s. Simple applications of NO or cGMP-regulating reagents to various cancer cell lines or animal models has generated controversial results. Whether the pathway is beneficial or detrimental in cancer is still open to question (10–13). We suggest several reasons for this ambiguity: first, although NO participates in normal signaling (e.g., vasodilation and neurotransmission), NO is also a cytotoxic or apoptotic molecule when produced at high concentrations by inducible nitric-oxide synthase (iNOS or NOS-2). In addition, the cGMP-dependent (NO/sGC/cGMP pathway) and cGMP-independent (NO oxidative pathway) components may vary among different tissues and cell types. Furthermore, solid tumors contain two compartments: the parenchyma (neoplastic cells) and the stroma (nonmalignant supporting tissues including connective tissue, blood vessels, and inflammatory cells) with different NO biology. Thus, the NO/sGC/cGMP signaling molecules in tumors as well as the surrounding tissue must be further characterized before targeting this signaling pathway for tumor therapy.
ENZYMATIC SYNTHESIS OF NO AND NOS-2
The first nitric oxide synthase (NOS) isoform was purified from brain neuronal tissue and named neuronal NOS or type I NOS (nNOS or NOS-1; (14, 15). This was followed shortly thereafter by inducible NOS, also known as type II NOS (iNOS or NOS-2; (16), and then by endothelial NOS or type III NOS (eNOS or NOS-3; (17, 18). The genes for each NOS isoforms reside on three different chromosomes. All three isoforms of the enzyme function as a homodimer consisting of two identical monomers, which can be functionally and structurally divided into two major domains: a C-terminal reductase (carboxy) domain, and an N-terminal oxygenase (amino) domain (14, 16, 17). NOS enzymes produce ·NO by catalyzing a five electron oxidation of a guanidino nitrogen of L-arginine (L-Arg) via two successive mono-oxygenation reactions. NG-Hydroxy L-arginine is an intermediate, and two moles of O2 and 1.5 moles of NADPH are consumed per mole of ·NO formed. NOS enzymes are the only enzymes known to simultaneously require five bound cofactors/prosthetic groups: flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4), and Ca2+-calmodulin. The electron flow in the NO synthase reaction is: NADPH → FAD → FMN → heme → O2. The BH4 provides an additional electron during the catalytic cycle which is replaced during turnover. The catalytically active isoforms exist as homodimers with tetrahydrobiopterin and heme serving to facilitate dimer formation. The carboxy terminal domain has considerable homology among the isoforms, and is homologous to cytochrome P450. However, the amino terminal domain has less homology. The homology of the three isoforms is about 50–60% while the homology of a given isoform between species can be as great as 85 to 92%. All NOS isoforms have a calmodulin-binding site which is important for the transfer of electrons. NOS-1 and NOS-3 are expressed constitutively (cNOS), and the production of NO by cNOS is regulated mainly though signaling pathway which alters calmodulin binding and intracellular free calcium levels.
On the other hand, NOS-2 is unique because 1) it requires de novo synthesis in most cells; 2) upon exposure to stimuli such as lipopolysaccharide (LPS) and proinflammatory cytokines (IL-1, IFNγ, TNFα), it is rapidly expressed and results in the production of much larger quantities of NO relative to the two other isoforms; and 3) it is widely distributed in various cell types (19, 20, 21). NOS-2 transcript or protein does not appear to be present under normal conditions in most cells. The activation of NOS-2 and the subsequent production of large amounts of the free radical gas NO is an important anti-infectious and anti-tumor mechanism of innate immunity. However, overproduction of NO has been implicated in cancer or tumor cell proliferation (22, 23), and other pathological conditions that include (but are not limited to): (1) Tissue injury and various inflammatory disorders (24, 25); (2) Neuronal disease (26, 27); (3) Auto-immune diseases (28, 29); (4) Angiogenesis and related pathological changes (30, 31); and (5) Diabetes mellitus (32, 33).
TUMOR-ASSOCIATED MACROPHAGES (TAM) AND NOS-2
NOS-2 was first cloned and characterized from mouse macrophages (34). It has been shown that depletion of cytosolic L-Arg triggers generation from NOS-2. This NOS-2-mediated generation can be blocked by the NOS inhibitor N-nitro- L-arginine methyl ester or by L-Arg, but not by the noninhibitory enantiomer N-nitro-D-arginine methyl ester. In L-Arg-depleted macrophages NOS-2 generates both and NO that interact to form the potent oxidant peroxynitrite (ONOO−), whose formation was blocked by the presence of superoxide dismutase, urate salts, or L-Arg. Thus, macrophages NOS-2 generates both and NO that interact to form the potent oxidant peroxynitrite (ONOO−), and executes killing of microbes (35). However, excess amount of NO, , and ONOO− promote oxidative stress and tissue damage which may result in a number of chronic inflammatory pathological conditions. The involvement of macrophages in some of the chronic inflammatory diseases has been considered as one of the risk factors predisposing to cancer. Helicobacter pylori-driven gastric carcinoma (36), colitis-associated colon carcinoma (37), and hepatitis-mediated hepatocellular carcinoma (38) all have suggested a direct infectious origin for tumors and the involvement of macrophages in initiating and promoting tumorigenesis.
It has long been recognized that solid tumors, both primary lesions and metastases, are infiltrated by large numbers of tumor-associated inflammatory cells consisting of subsets of T cells (helper, suppressor and cytotoxic), B cells, natural killer cells, and macrophages (39). Tumor-associated macrophages (TAM) represent a population of tissue macrophages which can be divided into two phenotypes, M1 and M2. Differential cytokine production is a key feature of polarized macrophages. The M1 phenotype encounter with interferon-γ (IFN-γ) and microbial stimuli such as LPS and are characterized by interleukin-12 (IL-12) and tumor necrosis factor (TNF) production and consequent activation of polarized type I T cell response. M2 macrophages can further divide into at least 3 groups based on different stimuli: M2a are stimulated with IL-4 and IL-13; M2b are induced by immune complexes and agonists of Toll-like receptors (TLRs) or IL-1 receptors; and M2c are sensitive to IL-10, TGF-β, and glucocorticoid hormones (40). In general, M1 macrophages exert host defensive function against viral and microbial infections through producing large amounts of inflammatory cytokines and NO. The cytotoxic activity at high NO level and pro-inflammatory cytokines serve as a major mechanism against phagocytized microorganisms and neoplastic cells (41). On the other hand, M2 cells participate by scavenging of debris, angiogenesis, remodeling, and repair of wounded/damaged tissues. It has been well documented that NOS-2 dominates arginine metabolism in M1 and produces high levels of NO, while the arginase pathway plays a key role in the M2 cells with generation of ornithine and polyamines (42, 43).
Inflammation has been known to contribute to tumor growth, progression, and immunosuppression suggesting that cancer is an inflammatory disease (44). The tumor microenvironment containing cytokine and chemokine networks functions as a driving force in TAMs recruitment. Clinical studies have shown a correlation between the infiltration of TAMs and a poor prognosis in the breast, cervix, and bladder cancers, while there is conflicting evidence for their role in prostate, lung and brain tumors (for review see 45). As we have reviewed above different components of inflammatory cytokines may result in mixed populations of TAMs. For example, approximately 70% of TAMs are M2 macrophages (CD68/CD163) and the remaining 30% are M1 macrophages (CD68/HLA-DR) in human non-small cell lung cancer. The researchers have found that M1 densities in the tumor islets, stroma, or islets and stroma are positively associated with patient's survival time. Multivariate Cox proportional hazards analysis also showed that M1 macrophage density in the tumor islets is an independent predictor of patient's survival time (46). A further molecular mechanism has been proposed through the study with STA6–/– tumor-bearing mice which display an M1 phenotype with a low level of arginase and a high level of NO. As a result, these mice immunologically rejected spontaneous mammary carcinoma (47). The aforementioned experimental results promoted TAM-targeted therapeutic strategy aimed at either switching infiltrating macrophages from M2 to M1; and/or restoring/enhancing M1 phenotype. For instance, TAMs from both mouse and human tumors show defective production of IL-12 and lack of p50/p65 NF-κB activation. Sica et al. has reported that (48) an activation of IL-10-dependent pathway in tumor microenvironment promoted the IL-10 positive/IL-12 negative phenotype of TAMs. Blocking IL-10 results in the restoration of NF-jB-dependent inflammatory functions (e.g., expression of cytotoxic mediators, NO) and cytokines (TNFα, IL-1, IL-12), therefore recovering M1 inflammation and intra-tumoral cytotoxicity (48). Alternatively, TAMs can also be redirected from M2 to the M1 phenotype. Toll-like receptor 9 ligand CpG plus anti-interleukin-10 receptor antibody promptly switched infiltrating macrophages infiltrate from M2 to M1 and triggered pre-established colon carcinoma rejection (49). It has been demonstrated that homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) plays a critical role in programming of the macrophages. Mice deficient for SHIP1 display a propensity of development of M2 (which are high arginase levels and produce ornithine to promote host-cell growth and collagen formation) instead of M1 (which have high inducible nitric oxide synthase levels and produce NO to kill microorganisms and tumor cells) (50). Of interest, M2, but not M1, can produce legumain, a robust acidic cysteine endopeptidase (asparaginyl endopeptidase), which has been found to be highly up-regulated in many murine and human tumor tissues. Luo et al. (51) created a legumain-based DNA vaccine that successfully induced a CD8+ T cell response against TAMs. The vaccine treatment dramatically reduced legumain density in tumor tissues and resulted in a marked decrease in proangiogenic factors released by TAMs such as TGF-beta, TNF-alpha, matrix metalloproteinase (MMP)-9, and vascular endothelial growth factor (VEGF). This, in turn, led to a suppression of both tumor angiogenesis and tumor growth and metastasis. Importantly, the success of this strategy was demonstrated in murine models of metastatic breast, colon, and non-small cell lung cancers, where 75% of vaccinated mice survived lethal tumor cell challenges and 62% were completely free of metastases.
NOS-2 EXPRESSING HUMAN TUMOR TISSUES, DEAD OR ALIVE?
The research data from our studies and others have clearly indicated that NOS-2 expression is associated with several human malignant tumors including those of the brain, head and neck, thyroid, breast, lung, esophagus, stomach, pancreas, liver, gallbladder, colon, bladder, prostate, and melanoma (for details see 52, 53). However, localized NOS-2 expression patterns vary as a consequence of complexity of tumor tissue components. In brain tumor samples, NOS-2 can be detected in both tumor and stromal cells while little to no expression is observed in vascular endothelial cells (54–58). Although NOS-2 expression has been reported in Kaposi's sarcoma, it was clarified that the tumor body mainly contains NOS-3, and the surrounding stromal and immune cells are the major source of NOS-2 (59).
In contrast to NOS-2-expressing M1 macrophages, high concentrations of NO produced by NOS-2 in tumor tissues are thought to promote neoplastic transformation and tumor growth in most cases. NO-derived reactive nitrogen species (RNS) include •NO (nitrogen monoxide) that can undergo interconversion to form NO+ (nitrosonium), and NO– (nitroxyl anion) in certain cellular conditions (20, 60–62). Excessive •NO reacts with O2•– to form peroxynitrite (ONOO–) that can further form peroxynitrous acid (ONOOH), a very unstable and reactive oxidizing species. Involvement of ONOO– in inflammatory conditions has been determined by detection of nitrotyrosine (NO2-Tyr) formation in various forms of human cancer (63–67). Nitrite (NO2–) is another major oxidation product derived from NO, and can be oxidized by myeloperoxidase (MPO), lactoperoxidase (LPO), or horseradish peroxidase to form an reactive nitrogen intermediate(s) such as nitrogen dioxide that is capable of nitrating tyrosine (68). MPO and other peroxidases are also able to use halides and pseudohalides as co-substrates to generate another reactive intermediate, hypochlorous acid, which further forms nitryl chloride and induce the formation of NO2-Tyr (69, 70). Thus, multiple pathways participate in RNS and tyrosine nitration which may be associated with subsequent consequences, including genotoxic and cytotoxic effects: genotoxic effects include deamination of nucleic acid bases, transition and/or transversion of nucleic acids, alkylation and DNA strand breakage, and inhibition of DNA repair enzymes, alkyl transferase and DNA ligase. Cytotoxic effects include inhibition of mitochondrial respiration, suppression of protein and related signaling and serious damage by direct or indirect mechanisms.
Initiation and/or promotion of tumorigenesis by NOS-2 has been well documented in animal models also. For example, NOS-2 function deletion suppressed intestinal polyposis in ApcMin/+ mice (71). Genetic ablation of NOS-2 showed decreased incidence of Helicobacter pylori associated gastric carcinogenesis (72); urethane-induced lung tumorigenesis (73); as well as PyV-mT-induced mammary hyperplasia (74). The nature of NOS-2 expression in certain tumors may offer therapeutic target for controlling tumor initiation, growth, and metastasis by selectively inhibiting NOS-2 function. The selective NOS-2 inhibitors L-N6-(1-iminoethyl)lysine 5-tetrazole-amide (SC-51) and aminoguanidine (AG) suppressed incidence of azoxymethane (AOM)-induced colonic aberrant crypt foci which demonstrates a chemopreventive potential of NOS-2 inhibitors (75). Another NOS-2 blocker N-(3-(aminomethyl)benzyl)acetamidine (1400W), has also showed significant inhibitory action on the human colon adenocarcinoma DLD-1 xenograft (76). Pharmacological inhibition of NOS-2 also markedly attenuated the growth of human colon cancer, KB epidermoid carcinoma and SK-N-MC neuroblastoma (77–79).
Due to the fact that higher concentration of NO exhibits a tumoricidal effect, several attempts have been made to increase NO in tumor tissue through either delivery of the NOS-2 gene or induction of NOS-2 expression. Xie et al. showed that NOS-2 gene transfection suppresses tumorigenicity and abrogates metastasis of K-1735 murine melanoma cells (80). Delivery of human cell lines that are capable of expressing high levels of NOS-2 to nude mouse implanted with human ovarian cancer SKOV-3 cells or human colon cancer DLD-1 cells resulted in significant tumor cell death (81).
NEW STRATEGY OF NOS-2/NO SIGNALING BASED CANCER THERAPY
Despite considerable progress in NOS-2/NO signaling research, the NOS-2-targeted cancer therapy is complicated by the interactions between the NO signaling network and the tumor tissue complex. Here, we would like to introduce two representative studies by our group and others to propose a new hypothesis of NOS-2/NO signaling in cancer biology and related therapeutic strategy.
Sikora et al. (82) has performed experiments of inhibiting NOS-2 expressed in human melanoma cells, however, the effects on tumor cell proliferation differed between in vitro and in vivo settings. The NOS-2-selective antagonist N6-(1-iminoethyl)- L-lysine dihydrochloride (L-NIL) strongly suppressed NOS-2-derived NO production in human melanoma lines mel624, A375, and mel526. However, the NOS-2 blocker failed to inhibit melanoma cell proliferation measured with XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)-assay. On the other hand, L-NIL significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. In addition, L-NIL treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. These discrepant data suggest that a proper cell proliferation measurement should be selected when NO/cGMP signaling is involved. The MTT assay is a colorimetric assay for measuring the activity of reductase enzymes that reduce MTT or structurally similar dyes (XTT, MTS, WSTs) to the corresponding formazan yelling a purple color (83). MTT quantifies cell survival, proliferation, and mitochondrial function. However, it should be noted that NO, a molecule with oxidative properties, may interfere with the outcome of the MTT (XTT) assays which riling on the redox reaction. Furthermore, it has been reported that cGMP increases mitochondrial function, which may result in increased MTT reading (84). Another important consideration derived from this study is that smaller effect of the NOS-2 inhibitor in the cell culture MTT assay versus the tumor xenografts assay may imply an inhibition of cell-cell communication by the NOS-2 inhibitor. One of the most important cell to cell interactions in cancer is the tumor-derived angiogenesis. The NOS-2–/– mice showed markedly lower VEGF expression and slower growth of B16-F1 melanoma xenograft indicating an role of NOS-2 in tumor stroma-mediated tumor growth and VEGF expression/angiogenesis (85). In contrast, NOS-2-overexpression in DLD-1 colon carcinoma cells increases tumor growth as well as the degree of functionally perfused vasculature and angiogenesis (86). Pharmacological inhibition of NOS-2 by either 1400W or L-nil also reduced subcutaneous tumor growth and vessel development in human melanoma (82) and colon adenocarcinoma (76). Gauthier et al. (87) reported an interesting finding that NOS-2 expression in tumor cells reduces, whereas NOS-2 expression in host stromal cells increases, the development of metastatic nodules of murine breast carcinomas. The authors found that in vivo, low NO-producing tumor cells (EMT-6H cells) developed more tumor nodules in the lungs than the high NO-producing tumor cells (EMT-6 J cells). However, in NOS-2-expressing mice, NO was protective against metastasis. In NOS-2-deficient mice, the development of tumor nodules in the lungs was suppressed and survival rate was increased with both low and high NO-producing tumor cell clones. While the investigators may interpret the data by emphasizing the importance of the cellular origin of NO which affects tumor growth differently, we propose that higher intracellular NO exhibits a cytotoxic effect and thus limits tumor growth and/or release of pro-inflammatory mediators. However, the NO concentration in the tumor microenvironment plays a key role in the promotion of tumor growth, angiogenesis, and metastasis.
Recently, we (52) have shown that despite a high NOS-2 expression levels in malignant brain tumor, the malignant cells did not respond to the treatment with an NOS-2 inhibitor. We compared the NO/sGC/cGMP pathway in human glioma tissues and cell lines with normal controls, and demonstrated that the expression of soluble guanylyl cyclase (sGC), the only endogenous NO receptor, is significantly lower in glioma preparations. Our analysis of GEO databases (National Cancer Institute) further revealed a statistically significant reduction in sGC transcript levels in human glioma specimens. On the other hand, the expression levels of particulate (membrane) guanylyl cyclases (pGC) and cGMP specific phosphodiesterase (PDE) were intact in the glioma cells that we have tested. Pharmacologically manipulating endogenous cGMP generation in glioma cells through either stimulating pGC by atrial natriuretic peptide (ANP) and/or brain natriuretic peptide (BNP), or blocking PDE by 3-isobutyl-1-methylxanthine (IBMX) or zaprinast caused significant inhibition of proliferation and colony formation of glioma cells. Genetically restoring sGC expression also inversely correlated with glioma cell growth. Orthotopic implantation of glioma cells transfected with a constitutively active mutant form of sGC (sGCα1β1cys-105) in athymic mice increased the survival time by 4-fold over the control. Histological analysis of xenografts overexpressing α1β1cys-105 sGC revealed changes in cellular architecture which resemble the morphology of normal cells. In addition, a decrease in angiogenesis contributed to glioma inhibition by sGC/cGMP therapy.
To explain these controversial studies that we reviewed here, we proposed that there are two possible roles of NO/cGMP signaling in malignant tumors (Scheme 1). First, NOS-2 expression and NO overproduction may contribute to the formation of an inflammatory cancer micro-environment. Second, sGC/cGMP signaling may influence proliferation and/or differentiation of the tumor cells. Progress of genetic and proteomic research is expected to identify additional molecular alterations in the NO/cGMP pathway in tumors, these will yield more therapeutic targets. For example, cGMP-dependent protein kinase (cGK), also called protein kinase G (PKG), is a serine/threonine-specific protein kinase which exists in two isoforms, type-I and type II. PGK-1 has been shown to have tumor suppressor properties in colon carcinoma (88). However, PKG-1α has been recently reported to promote DNA synthesis and cell proliferation in human ovarian cancer cells (89). PKG-2 has been known to be widely expressed in normal brain tissue. We, thus, screened human glioma cell lines for expression of PKG isoforms and found four different phenotypes. While some cell lines express PKG-1 (e.g., U87), PKG-2 (e.g., A172 or Ln18), or both (e.g., D54), other cells are PKG-negative (e.g., U373). The U87 glioma cells have been characterized as highly tumorigenic with low-invasiveness, and PKG-2 deficient. Westermark's group (90) reported that over-expression of PKG-2 in U87 cells inhibited cell proliferation and xenograft growth.
Our current finding and the data of other studies support our hypothesis that a normal function of the sGC-cGMP signaling axis may be important for the prevention and/or treatment of malignant tumors. Inhibiting NOS-2 overexpression and the tumor inflammatory microenvironment, combined with normalization of the sGC/cGMP signaling may be a favorable alternative to chemotherapy and radiotherapy for malignant tumors. For example, restoring cGMP levels in tumors with low sGC expression can be achieved by application of cGMP analogs, gene therapy to restore sGC formation, activation of the particulate isoforms of GC, inhibition of PDE isoforms, and/or NOS-2 inhibition. Genomic and proteomic status of tumors as well as biochemical characterization of the GC/cGMP signaling pathway could provide reliable clues to develop these therapeutic approaches.
The authors express their appreciation for the grant supports from the Society of Neurological Surgeons (A. Siu); The Undergraduate Research Fellowship from the Office of the Vice President of Research (OVPR) of GWU (A. Sotolongo); Federal Work Student Study (L. S), and the George Washington University. The authors also want to acknowledge Dr Rakesh Kumar for his helps and support towards our research.