Tumor microenvironment-based feed-forward regulation of NOS2 in breast cancer progression.

Inflammation is widely recognized as an inducer of cancer progression. The inflammation-associated enzyme, inducible nitric oxide synthase (NOS2), has emerged as a candidate oncogene in estrogen receptor (ER)-negative breast cancer, and its increased expression is associated with disease aggressiveness and poor survival. Although these observations implicate NOS2 as an attractive therapeutic target, the mechanisms of both NOS2 induction in tumors and nitric oxide (NO)-driven cancer progression are not fully understood. To enhance our mechanistic understanding of NOS2 induction in tumors and its role in tumor biology, we used stimulants of NOS2 expression in ER(-) and ER(+) breast cancer cells and examined downstream NO-dependent effects. Herein, we show that up-regulation of NOS2 occurs in response to hypoxia, serum withdrawal, IFN-γ, and exogenous NO, consistent with a feed-forward regulation of NO production by the tumor microenvironment in breast cancer biology. Moreover, we found that key indicators of an aggressive cancer phenotype including increased S100 calcium binding protein A8, IL-6, IL-8, and tissue inhibitor matrix metalloproteinase-1 are up-regulated by these NOS2 stimulants, whereas inhibition of NOS2 in MDA-MB-231 breast cancer cells suppressed these markers. Moreover, NO altered cellular migration and chemoresistance of MDA-MB-231 cells to Taxol. Most notably, MDA-MB-231 tumor xenographs and cell metastases from the fat pad to the brain were significantly suppressed by NOS2 inhibition in nude mice. In summary, these results link elevated NOS2 to signals from the tumor microenvironment that arise with cancer progression and show that NO production regulates chemoresistance and metastasis of breast cancer cells.

Biological signaling by small inorganic molecules.

Small redox active molecules such as reactive nitrogen and oxygen species and hydrogen sulfide have emerged as important biological mediators that are involved in various physiological and pathophysiological processes. Advancement in understanding of cellular mechanisms that tightly regulate both generation and reactivity of these molecules is central to improved management of various disease states including cancer and cardiovascular dysfunction. Imbalance in the production of redox active molecules can lead to damage of critical cellular components such as cell membranes, proteins and DNA and thus may trigger the onset of disease. These small inorganic molecules react independently as well as in a concerted manner to mediate physiological responses. This review provides a general overview of the redox biology of these key molecules, their diverse chemistry relevant to physiological processes and their interrelated nature in cellular signaling.

Gene expression profiles of NO- and HNO-donor treated breast cancer cells: insights into tumor response and resistance pathways.

Nitric oxide (NO) synthase 2 (NOS2), a major inflammatory protein, modulates disease progression via NO in a number of pathologies, including cancer. The role of NOS2-derived NO is not only flux-dependent, which is higher in mouse vs human cells, but also varies based on spatial and temporal distribution both within tumor cells and in the tumor microenvironment. NO donors have been utilized to mimic NO flux conditions and to investigate the effects of varied NO concentrations. As a wide range of effects mediated by NO and other nitrogen oxides such as nitroxyl (HNO) have been elucidated, multiple NO- and HNO-releasing compounds have been developed as potential therapeutics, including as tumor modulators. One of the challenges is to determine differences in biomarker expression from extracellular vs intracellular generation of NO or HNO. Taking advantage of new NO and HNO releasing agents, we have characterized the gene expression profile of estrogen receptor-negative human breast cancer (MDA-MB-231) cells following exposure to aspirin, the NO donor DEA/NO, the HNO donor IPA/NO andtheir intracellularly-activated prodrug conjugates DEA/NO-aspirin and IPA/NO-aspirin. Comparison of the gene expression profiles demonstrated that several genes were uniquely expressed with respect to NO or HNO, such as miR-21, HSP70, cystathionine γ-lyase and IL24. These findings provide insight into targets and pathways that could be therapeutically exploited by the redox related species NO and HNO.

Nitrite reduction mediated by heme models. Routes to NO and HNO?

The water-soluble ferriheme model Fe(III)(TPPS) mediates oxygen atom transfer from inorganic nitrite to a water-soluble phosphine (tppts), dimethyl sulfide, and the biological thiols cysteine (CysSH) and glutathione (GSH). The products with the latter reductant are the respective sulfenic acids CysS(O)H and GS(O)H, although these reactive intermediates are rapidly trapped by reaction with excess thiol. The nitrosyl complex Fe(II)(TPPS)(NO) is the dominant iron species while excess substrate is present. However, in slightly acidic media (pH ≈ 6), the system does not terminate at this very stable ferrous nitrosyl. Instead, it displays a matrix of redox transformations linking spontaneous regeneration of Fe(III)(TPPS) to the formation of both N2O and NO. Electrochemical sensor and trapping experiments demonstrate that HNO (nitroxyl) is formed, at least when tppts is the reductant. HNO is the likely predecessor of the N2O. A key pathway to NO formation is nitrite reduction by Fe(II)(TPPS), and the kinetics of this iron-mediated transformation are described. Given that inorganic nitrite has protective roles during ischemia/reperfusion (I/R) injury to organs, attributed in part to NO formation, and that HNO may also reduce net damage from I/R, the present studies are relevant to potential mechanisms of such nitrite protection.

Formation of cysteine sulfenic acid by oxygen atom transfer from nitrite.

Cysteine sulfenic acid CysS(O)H is shown to be formed for the reaction of cysteine (CysSH) with aqueous nitrite and the water-soluble ferriheme models Fe(III)(TPPS) (TPPS = meso-tetra(4-sulfonatophenyl)porphyrinato) or Fe(III)(TMPS) (TMPS = meso-tetra(sulfonatomesityl)porphyrinato) at pH 5.8 and 7.4. The other product is the respective ferrous nitrosyl complex Fe(II)(Por)(NO) (Por = TPPS or TMPS). Analogous oxygen atom transfers (OAT) were seen when glutathione (GSH) was used as the substrate. The sulfenic acids, CysS(O)H and GS(O)H, are transient species since they react rapidly with excess thiol to give the respective disulfides, so their presence as reactive intermediates was demonstrated by trapping with dimedone and detecting the resulting adduct using LC/MS. Preliminary kinetics studies are consistent with rate-limiting OAT from a ferric nitro complex Fe(III)(Por)(NO(2)(-)) to CysSH, although this reaction is complicated by a competing dead-end equilibrium to form the thiolate complex (Fe(III)(TPPS)(CysS(-)).

The distal pocket histidine residue in horse heart myoglobin directs the O-binding mode of nitrite to the heme iron.

It is now well-established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example, the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO(2)(-)) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMb(III) (i.e., ferric Mb(III)) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMb(III) H64V (2.0 A resolution) and its nitrite ion adduct metMb(III) H64V-nitrite (1.95 A resolution), and metMb(III) H64V/V67R (1.9 A resolution) and its nitrite ion adduct metMb(III) H64V/V67R-nitrite (2.0 A resolution). These are compared to the known structures of wild-type (wt) hh metMb(III) and its nitrite ion adduct hh metMb(III)-nitrite, which binds NO(2)(-) via an O-atom in a trans-FeONO configuration. Unlike wt metMb(III), no axial H(2)O is evident in either of the metMb(III) mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMb(III)s with cysteine as a reducing agent and of the (ferrous) Mb(II)s with nitrite ion under anaerobic conditions. The Mb(II)s were generated by reduction of the Mb(III) precursors in a second-order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the Mb(II)s by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.

Oxygen atom transfer from nitrite mediated by Fe(III) porphyrins in aqueous solution.

Aqueous solutions of the iron(III) porphyrin complex FeIII(TPPS) (1, TPPS = tetra(4-sulfonatophenyl)-porphyrinato) and nitrite ion react with various substrates S to generate the ferrous nitrosyl complex FeII(TPPS)(NO) (2) plus oxidized substrate. When S is a water-soluble sulfonated phosphine, the product is the resulting monoxide. When air is introduced to the product solutions, 2 is rapidly reoxidized to 1; however, even in the absence of air, there is a slow regeneration of the ferric species with concomitant production of nitrous oxide. Thus, in an anaerobic aqueous environment, FeIII(TPPS) catalyzes oxygen atom transfer from nitrite ion to substrates with the eventual formation of N2O.

Nitric Oxide Synthase-2-Derived Nitric Oxide Drives Multiple Pathways of Breast Cancer Progression.

SIGNIFICANCE: Breast cancer is the second leading cause of cancer-related deaths among women in the United States. Development and progression of malignancy are associated with diverse cell signaling pathways that control cell proliferation, survival, motility, invasion, and metastasis. Recent Advances: An increasing number of clinical studies have implicated a strong relationship between elevated tumor nitric oxide synthase-2 (NOS2) expression and poor patient survival. CRITICAL ISSUES: Herein, we review what we believe to be key mechanisms in the role(s) of NOS2-derived nitric oxide (NO) as a driver of breast cancer disease progression. High NO increases cyclooxygenase-2 activity, hypoxia inducible factor-1 alpha protein stabilization, and activation of important cell signaling pathways, including phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinase, epidermal growth factor receptor, and Ras, through post-translational protein modifications. Moreover, dysregulated NO flux within the tumor microenvironment has other important roles, including the promotion of angiogenesis and modulation of matrix metalloproteinase/tissue inhibitor matrix metalloproteinase associated with tumor progression. FUTURE DIRECTIONS: The elucidation of these and other NO-driven pathways implicates NOS2 as a key driver of breast cancer disease progression and provides a new perspective in the identification of novel targets that may be therapeutically beneficial in the treatment of estrogen receptor-negative disease. Antioxid. Redox Signal. 26, 1044-1058.

Variation of DNA photocleavage efficiency for [(TL)2Ru(dpp)]Cl2 complexes where TL=2,2'-bipyridine, 1,10-phenanthroline, or 4,7-diphenyl-1,10-phenanthroline.

The complexes [(bpy)(2)Ru(dpp)]Cl(2), [(phen)(2)Ru(dpp)]Cl(2), and [(Ph(2)phen)(2)Ru(dpp)]Cl(2) (where dpp=2,3-bis(2-pyridyl)pyrazine, bpy=2,2'-bipyridine, phen=1,10-phenanthroline, Ph(2)phen=4,7-diphenyl-1,10-phenanthroline) have been investigated and found to photocleave DNA via an oxygen-mediated pathway. These light absorbing complexes possess intense metal-to-ligand charge transfer (MLCT) transitions in the visible region of the spectrum. The [(TL)(2)Ru(dpp)](2+) systems populate (3)MLCT states after visible light excitation, giving rise to emissions in aqueous solution centered at 692, 690, and 698nm for TL=bpy, phen, and Ph(2)phen respectively. The (3)MLCT states and emissions are quenched by O(2), producing a reactive oxygen species. These complexes photocleave DNA with varying efficiencies, [(Ph(2)phen)(2)Ru(dpp)](2+)>[(phen)(2)Ru(dpp)](2+)>[(bpy)(2)Ru(dpp)](2+). The presence of the polyazine bridging ligand will allow these chromophores to be incorporated into larger supramolecular assemblies.

Signaling and stress: The redox landscape in NOS2 biology.

Nitric oxide (NO) has a highly diverse range of biological functions from physiological signaling and maintenance of homeostasis to serving as an effector molecule in the immune system. However, deleterious as well as beneficial roles of NO have been reported. Many of the dichotomous effects of NO and derivative reactive nitrogen species (RNS) can be explained by invoking precise interactions with different targets as a result of concentration and temporal constraints. Endogenous concentrations of NO span five orders of magnitude, with levels near the high picomolar range typically occurring in short bursts as compared to sustained production of low micromolar levels of NO during immune response. This article provides an overview of the redox landscape as it relates to increasing NO concentrations, which incrementally govern physiological signaling, nitrosative signaling and nitrosative stress-related signaling. Physiological signaling by NO primarily occurs upon interaction with the heme protein soluble guanylyl cyclase. As NO concentrations rise, interactions with nonheme iron complexes as well as indirect modification of thiols can stimulate additional signaling processes. At the highest levels of NO, production of a broader range of RNS, which subsequently interact with more diverse targets, can lead to chemical stress. However, even under such conditions, there is evidence that stress-related signaling mechanisms are triggered to protect cells or even resolve the stress. This review therefore also addresses the fundamental reactions and kinetics that initiate signaling through NO-dependent pathways, including processes that lead to interconversion of RNS and interactions with molecular targets.

NOS Inhibition Modulates Immune Polarization and Improves Radiation-Induced Tumor Growth Delay.

Nitric oxide synthases (NOS) are important mediators of progrowth signaling in tumor cells, as they regulate angiogenesis, immune response, and immune-mediated wound healing. Ionizing radiation (IR) is also an immune modulator and inducer of wound response. We hypothesized that radiation therapeutic efficacy could be improved by targeting NOS following tumor irradiation. Herein, we show enhanced radiation-induced (10 Gy) tumor growth delay in a syngeneic model (C3H) but not immunosuppressed (Nu/Nu) squamous cell carcinoma tumor-bearing mice treated post-IR with the constitutive NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME). These results suggest a requirement of T cells for improved radiation tumor response. In support of this observation, tumor irradiation induced a rapid increase in the immunosuppressive Th2 cytokine IL10, which was abated by post-IR administration of L-NAME. In vivo suppression of IL10 using an antisense IL10 morpholino also extended the tumor growth delay induced by radiation in a manner similar to L-NAME. Further examination of this mechanism in cultured Jurkat T cells revealed L-NAME suppression of IR-induced IL10 expression, which reaccumulated in the presence of exogenous NO donor. In addition to L-NAME, the guanylyl cyclase inhibitors ODQ and thrombospondin-1 also abated IR-induced IL10 expression in Jurkat T cells and ANA-1 macrophages, which further suggests that the immunosuppressive effects involve eNOS. Moreover, cytotoxic Th1 cytokines, including IL2, IL12p40, and IFNγ, as well as activated CD8(+) T cells were elevated in tumors receiving post-IR L-NAME. Together, these results suggest that post-IR NOS inhibition improves radiation tumor response via Th1 immune polarization within the tumor microenvironment.

Molecular pathways: toll-like receptors in the tumor microenvironment--poor prognosis or new therapeutic opportunity.

Numerous reports have described Toll-like receptor (TLR) expression in the tumor microenvironment as it relates to cancer progression, as well as their involvement in inflammation. While TLRs mediate immune surveillance, clinical studies have associated TLR expression in the tumor with poor patient survival, indicating that TLR expression may affect cancer treatment and survival. This review will examine mechanisms in which TLR activation upregulates protumorigenic pathways, including the induction of inducible nitric oxide synthase (iNOS2) and COX2, which in turn increase TLR expression and promote a feed-forward loop leading to tumor progression and the development of more aggressive tumor phenotypes. These propagating loops involve cancer cell, stroma, and/or immune cell TLR expression. Because of abundant TLR expression in many human tumors, several TLR agonists are now in clinical and preclinical trials and some have shown enhanced efficacy when used as adjuvant with radiation, chemotherapy, or cancer vaccines. These findings suggest that TLR expression influences cancer biology and therapeutic response, which may involve specific interactions within the tumor microenvironment, including mediators of inflammation such as nitric oxide and the arachidonic acid signaling pathways.

Synthesis and chemical and biological comparison of nitroxyl- and nitric oxide-releasing diazeniumdiolate-based aspirin derivatives.

Structural modifications of nonsteroidal anti-inflammatory drugs (NSAIDs) have successfully reduced the side effect of gastrointestinal ulceration without affecting anti-inflammatory activity, but they may increase the risk of myocardial infarction with chronic use. The fact that nitroxyl (HNO) reduces platelet aggregation, preconditions against myocardial infarction, and enhances contractility led us to synthesize a diazeniumdiolate-based HNO-releasing aspirin and to compare it to an NO-releasing analogue. Here, the decomposition mechanisms are described for these compounds. In addition to protection against stomach ulceration, these prodrugs exhibited significantly enhanced cytotoxcity compared to either aspirin or the parent diazeniumdiolate toward nonsmall cell lung carcinoma cells (A549), but they were not appreciably toxic toward endothelial cells (HUVECs). The HNO-NSAID prodrug inhibited cylcooxgenase-2 and glyceraldehyde 3-phosphate dehydrogenase activity and triggered significant sarcomere shortening on murine ventricular myocytes compared to control. Together, these anti-inflammatory, antineoplasic, and contractile properties suggest the potential of HNO-NSAIDs in the treatment of inflammation, cancer, or heart failure.

Nitrite reduction by Co(II) and Mn(II) substituted myoglobins: towards understanding necessary components of Mb nitrite reductase activity.

Nitrite reduction to nitric oxide by heme proteins is drawing increasing attention as a protective mechanism to hypoxic injury in mammalian physiology. Here we probe the nitrite reductase (NiR) activities of manganese(II)- and cobalt(II)-substituted myoglobins, and compare with data obtained previously for the iron(II) analog wt Mb(II). Both Mn(II)Mb and Co(II)Mb displayed NiR activity, and it was shown that the kinetics are first order each in [protein], [nitrite], and [H(+)], as previously determined for the Fe(II) analog wt Mb(II). The second order rate constants (k(2)) at pH 7.4 and T=25 °C, were 0.0066 and 0.015 M(-1)s(-1) for Co(II)Mb and Mn(II)Mb, respectively, both orders of magnitude slower than the k(2) (6M(-1)s(-1)) for wt Mb(II). The final reaction products for Mn(II)Mb consisted of a mixture of the nitrosyl Mn(II)Mb(NO) and Mn(III)Mb, similar to the products from the analogous NiR reaction by wt Mb. In contrast, the products of NiR by Co(II)Mb were found to be the nitrito complex Co(III)Mb(ONO(-)) plus roughly an equivalent of free NO. The differences can be attributed in part to the stronger coordination of inorganic nitrite to Co(III)Mb as reflected in the respective M(III)Mb(ONO(-)) formation constants K(nitrite): 2100 M(-1) (Co(III)) and <~0.4M(-1) (Mn(III)). We also report the formation constants (3.7 and 30 M(-1), respectively) for the nitrite complexes of the mutant metmyoglobins H64V Mb(III)(NO(2)(-)) and H64V/V67R Mb(III)(ONO(-)) and a K(nitrite) revised value (120 M(-1)) for the nitrite complex of wt metMb. The respective K(nitrite) values for the three ferric proteins emphasize the importance of a H-bonding residue, such as His64 in the Mb(III) distal pocket or the Arg67 in H64V/V67R Mb(III), in stabilizing nitrite coordination. Notably, the NiR activities of the corresponding ferrous Mbs follow a similar sequence suggesting that nitrite binding to these centers are analogously affected by the H-bonding residues.

S-Nitrosation Mediates Multiple Pathways That Lead to Tumor Progression in Estrogen Receptor-Negative Breast Cancer.

Chronic inflammation within the tumor microenvironment is a major driver of tumor progression and poor prognosis. Inducible nitric oxide synthase (NOS2) is present in numerous solid tumors. Estrogen receptor-negative (ER-) patients with high expression of tumor NOS2 have a poorer outcome than patients with low expression of NOS2. Furthermore, expression of NOS2 is associated with the basal-like breast cancer phenotype. Using an in vitro model, we have found that nitrosation of critical thiols and nitration of tyrosines lead to the activation of membrane receptors such as epithelial growth factor receptor, Src, Ras, and CD63. These nitric oxide-mediated events in itiate oncogenic signaling pathways such as PI3K/Akt, Ras/ERK, ß-catenin, nuclear factor-κB, and AP-1. These data suggest that NOS2 can serve as a major "nonmutatational driver" of ER- breast cancer.

Nitric oxide synthase and breast cancer: role of TIMP-1 in NO-mediated Akt activation.

Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation.

Luminescently tagged 2,2'-bipyridine complex of FeII: synthesis and photophysical studies of 4-[N-(2-anthryl)carbamoyl]-4'-methyl-2,2'-bipyridine.

The anthracene lumiphore was linked to the chelating ligand 2,2'-bipyridine, forming 4-[N-(2-anthryl)carbamoyl]-4'-methyl-2,2'-bipyridine (bpyAnth). Coupling through an amide linkage provides some electronic isolation of the anthracene lumiphore. Electrochemistry suggested little change of the anthracene oxidation whether free (1.35 V) linked to 2,2'-bipyridine as bpyAnth (1.30 V) or appended to Fe(II) (1.29 V). The bpyAnth ligand retained the structured luminescence characteristic of anthracene at 375, 400, 419, and 441 nm. This anthracene emission persists even when bpyAnth is complexed to an Fe(II) center. The complex [Fe(bpyAnth)3]2+ is emissive, in marked contrast to typical polyazine iron(II) complexes. This bpyAnth ligand serves as a luminescently tagged analogue of 2,2'-bipyridine, useful for coordination to a variety of metals.