Nitro-fatty acid inhibition of triple-negative breast cancer cell viability, migration, invasion, and tumor growth.

Triple-negative breast cancer (TNBC) comprises ∼20% of all breast cancers and is the most aggressive mammary cancer subtype. Devoid of the estrogen and progesterone receptors, along with the receptor tyrosine kinase ERB2 (HER2), that define most mammary cancers, there are no targeted therapies for patients with TNBC. This, combined with a high metastatic rate and a lower 5-year survival rate than for other breast cancer phenotypes, means there is significant unmet need for new therapeutic strategies. Herein, the anti-neoplastic effects of the electrophilic fatty acid nitroalkene derivative, 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA), were investigated in multiple preclinical models of TNBC. NO2-OA reduced TNBC cell growth and viability in vitro, attenuated TNFα-induced TNBC cell migration and invasion, and inhibited the tumor growth of MDA-MB-231 TNBC cell xenografts in the mammary fat pads of female nude mice. The up-regulation of these aggressive tumor cell growth, migration, and invasion phenotypes is mediated in part by the constitutive activation of pro-inflammatory nuclear factor κB (NF-κB) signaling in TNBC. NO2-OA inhibited TNFα-induced NF-κB transcriptional activity in human TNBC cells and suppressed downstream NF-κB target gene expression, including the metastasis-related proteins intercellular adhesion molecule-1 and urokinase-type plasminogen activator. The mechanisms accounting for NF-κB signaling inhibition by NO2-OA in TNBC cells were multifaceted, as NO2-OA (a) inhibited the inhibitor of NF-κB subunit kinase ß phosphorylation and downstream inhibitor of NF-κB degradation, (b) alkylated the NF-κB RelA protein to prevent DNA binding, and (c) promoted RelA polyubiquitination and proteasomal degradation. Comparisons with non-tumorigenic human breast epithelial MCF-10A and MCF7 cells revealed that NO2-OA more selectively inhibited TNBC function. This was attributed to more facile mechanisms for maintaining redox homeostasis in normal breast epithelium, including a more favorable thiol/disulfide balance, greater extents of multidrug resistance protein-1 (MRP1) expression, and greater MRP1-mediated efflux of NO2-OA-glutathione conjugates. These observations reveal that electrophilic fatty acid nitroalkenes react with more alkylation-sensitive targets in TNBC cells to inhibit growth and viability.

15-Hydroxyprostaglandin dehydrogenase generation of electrophilic lipid signaling mediators from hydroxy ω-3 fatty acids.

15-Hydroxyprostaglandin dehydrogenase (15PGDH) is the primary enzyme catalyzing the conversion of hydroxylated arachidonic acid species to their corresponding oxidized metabolites. The oxidation of hydroxylated fatty acids, such as the conversion of prostaglandin (PG) E2 to 15-ketoPGE2, by 15PGDH is viewed to inactivate signaling responses. In contrast, the typically electrophilic products can also induce anti-inflammatory and anti-proliferative responses. This study determined that hydroxylated docosahexaenoic acid metabolites (HDoHEs) are substrates for 15PGDH. Examination of 15PGDH substrate specificity was conducted in cell culture (A549 and primary human airway epithelia and alveolar macrophages) using chemical inhibition and shRNA knockdown of 15PGDH. Substrate specificity is broad and relies on the carbon position of the acyl chain hydroxyl group. 14-HDoHE was determined to be the optimal DHA substrate for 15PGDH, resulting in the formation of its electrophilic metabolite, 14-oxoDHA. Consistent with this, 14-HDoHE was detected in bronchoalveolar lavage cells of mild to moderate asthmatics, and the exogenous addition of 14-oxoDHA to primary alveolar macrophages inhibited LPS-induced proinflammatory cytokine mRNA expression. These data reveal that 15PGDH-derived DHA metabolites are biologically active and can contribute to the salutary signaling actions of Ω-3 fatty acids.

Conjugated linoleic acid is a preferential substrate for fatty acid nitration.

The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10(5) greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO(2)-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.

Covalent peroxisome proliferator-activated receptor gamma adduction by nitro-fatty acids: selective ligand activity and anti-diabetic signaling actions.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARgamma ligands are intermediates of lipid metabolism and oxidation that bind PPARgamma with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO(2)-FA) are endogenous products of nitric oxide ((*)NO) and nitrite (NO(2)(-))-mediated redox reactions that activate PPARgamma at nanomolar concentrations. We report that NO(2)-FA act as partial agonists of PPARgamma and covalently bind PPARgamma at Cys-285 via Michael addition. NO(2)-FA show selective PPARgamma modulator characteristics by inducing coregulator protein interactions, PPARgamma-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO(2)-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.

Nitro-fatty acid inhibition of neointima formation after endoluminal vessel injury.

RATIONALE: Fatty acid nitroalkenes are endogenously generated electrophilic byproducts of nitric oxide and nitrite-dependent oxidative inflammatory reactions. Existing evidence indicates nitroalkenes support posttranslational protein modifications and transcriptional activation that promote the resolution of inflammation. OBJECTIVE: The aim of this study was to assess whether in vivo administration of a synthetic nitroalkene could elicit antiinflammatory actions in vivo using a murine model of vascular injury. METHODS AND RESULTS: The in vivo administration (21 days) of nitro-oleic acid (OA-NO(2)) inhibited neointimal hyperplasia after wire injury of the femoral artery in a murine model (OA-NO(2) treatment resulted in reduced intimal area and intima to media ratio versus vehicle- or oleic acid (OA)-treated animals,P<0.0001). Increased heme oxygenase (HO)-1 expression accounted for much of the vascular protection induced by OA-NO(2) in both cultured aortic smooth muscle cells and in vivo. Inhibition of HO by Sn(IV)-protoporphyrin or HO-1 small interfering RNA reversed OA-NO(2)-induced inhibition of platelet-derived growth factor-stimulated rat aortic smooth muscle cell migration. The upregulation of HO-1 expression also accounted for the antistenotic actions of OA-NO(2) in vivo, because inhibition of neointimal hyperplasia following femoral artery injury was abolished in HO-1(-/-) mice (OA-NO(2)-treated wild-type versus HO-1(-/-) mice, P=0.016). CONCLUSIONS: In summary, electrophilic nitro-fatty acids induce salutary gene expression and cell functional responses that are manifested by a clinically significant outcome, inhibition of neointimal hyperplasia induced by arterial injury.

15-Oxoeicosatetraenoic acid is a 15-hydroxyprostaglandin dehydrogenase-derived electrophilic mediator of inflammatory signaling pathways.

Bioactive lipids govern cellular homeostasis and pathogenic inflammatory processes. Current dogma holds that bioactive lipids, such as prostaglandins and lipoxins, are inactivated by 15-hydroxyprostaglandin dehydrogenase (15PGDH). In contrast, the present results reveal that catabolic "inactivation" of hydroxylated polyunsaturated fatty acids (PUFAs) yields electrophilic α,ß-unsaturated ketone derivatives. These endogenously produced species are chemically reactive signaling mediators that induce tissue protective events. Electrophilic fatty acids diversify the proteome through post-translational alkylation of nucleophilic cysteines in key transcriptional regulatory proteins and enzymes that govern cellular metabolic and inflammatory homeostasis. 15PGDH regulates these processes as it is responsible for the formation of numerous electrophilic fatty acids including the arachidonic acid metabolite, 15-oxoeicosatetraenoic acid (15-oxoETE). Herein, the role of 15-oxoETE in regulating signaling responses is reported. In cell cultures, 15-oxoETE activates Nrf2-regulated antioxidant responses (AR) and inhibits NF-κB-mediated pro-inflammatory responses via IKKß inhibition. Inhibition of glutathione S-transferases using ethacrynic acid incrementally increased the signaling capacity of 15-oxoETE by decreasing 15-oxoETE-GSH adduct formation. This work demonstrates that 15PGDH plays a role in the regulation of cell and tissue homeostasis via the production of electrophilic fatty acid signaling mediators.

Nitrite augments glucose uptake in adipocytes through the protein kinase A-dependent stimulation of mitochondrial fusion.

Though it is well accepted that adipose tissue is central in the regulation of glycemic homeostasis, the molecular mechanisms governing adipocyte glucose uptake remain unclear. Recent studies demonstrate that mitochondrial dynamics (fission and fusion) regulate lipid accumulation and differentiation in adipocytes. However, the role of mitochondrial dynamics in glucose homeostasis has not been explored. The nitric oxide oxidation products nitrite and nitrate are endogenous signaling molecules and dietary constituents that have recently been shown to modulate glucose metabolism, prevent weight gain, and reverse the development of metabolic syndrome in mice. Although the mechanism of this protection is unclear, the mitochondrion is a known subcellular target for nitrite signaling. Thus, we hypothesize that nitrite modulates mitochondrial dynamics and function to regulate glucose uptake in adipocytes. Herein, we demonstrate that nitrite significantly increases glucose uptake in differentiated murine adipocytes through a mechanism dependent on mitochondrial fusion. Specifically, nitrite promotes mitochondrial fusion by increasing the profusion protein mitofusin 1 while concomitantly activating protein kinase A (PKA), which phosphorylates and inhibits the profission protein dynamin-related protein 1 (Drp1). Functionally, this signaling augments cellular respiration, fatty acid oxidation, mitochondrial oxidant production, and glucose uptake. Importantly, inhibition of PKA or Drp1 significantly attenuates nitrite-induced mitochondrial respiration and glucose uptake. These findings demonstrate that mitochondria play an essential metabolic role in adipocytes, show a novel role for both nitrite and mitochondrial fusion in regulating adipocyte glucose homeostasis, and have implications for the potential therapeutic use of nitrite and mitochondrial modulators in glycemic regulation.

Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity.

AIMS: Obesity is a risk factor for diabetes and cardiovascular diseases, with the incidence of these disorders becoming epidemic. Pathogenic responses to obesity have been ascribed to adipose tissue (AT) dysfunction that promotes bioactive mediator secretion from visceral AT and the initiation of pro-inflammatory events that induce oxidative stress and tissue dysfunction. Current understanding supports that suppressing pro-inflammatory and oxidative events promotes improved metabolic and cardiovascular function. In this regard, electrophilic nitro-fatty acids display pleiotropic anti-inflammatory signalling actions. METHODS AND RESULTS: It was hypothesized that high-fat diet (HFD)-induced inflammatory and metabolic responses, manifested by loss of glucose tolerance and vascular dysfunction, would be attenuated by systemic administration of nitrooctadecenoic acid (OA-NO2). Male C57BL/6j mice subjected to a HFD for 20 weeks displayed increased adiposity, fasting glucose, and insulin levels, which led to glucose intolerance and pulmonary hypertension, characterized by increased right ventricular (RV) end-systolic pressure (RVESP) and pulmonary vascular resistance (PVR). This was associated with increased lung xanthine oxidoreductase (XO) activity, macrophage infiltration, and enhanced expression of pro-inflammatory cytokines. Left ventricular (LV) end-diastolic pressure remained unaltered, indicating that the HFD produces pulmonary vascular remodelling, rather than LV dysfunction and pulmonary venous hypertension. Administration of OA-NO2 for the final 6.5 weeks of HFD improved glucose tolerance and significantly attenuated HFD-induced RVESP, PVR, RV hypertrophy, lung XO activity, oxidative stress, and pro-inflammatory pulmonary cytokine levels. CONCLUSIONS: These observations support that the pleiotropic signalling actions of electrophilic fatty acids represent a therapeutic strategy for limiting the complex pathogenic responses instigated by obesity.

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids.

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO(2)) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO(2) via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO(2)-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO(2)-FAs stimulated the phosphorylation of eNOS at Ser(1179). These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO(2)-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.

Rab11b regulates the apical recycling of the cystic fibrosis transmembrane conductance regulator in polarized intestinal epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, DeltaF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

Nitro-fatty acid metabolome: saturation, desaturation, beta-oxidation, and protein adduction.

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via beta-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (*NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added beta-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and beta-oxidation reactions that terminate at the site of acyl-chain nitration.

Nitro-fatty acid reaction with glutathione and cysteine. Kinetic analysis of thiol alkylation by a Michael addition reaction.

Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M(-1)S(-1) and 355 M(-1)S(-1), respectively, at pH 7.4 and 37 degrees C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A2 and 15-deoxy-Delta(12,14)-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-beta-d-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.

STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G.

The cystic fibrosis transmembrane conductance regulator (CFTR) is critical to cAMP- and cGMP-activated intestinal anion secretion and the pathogenesis of secretory diarrhea. Enterotoxins released by Vibrio cholerae (cholera toxin) and Escherichia coli (heat stable enterotoxin, or STa) activate intracellular cAMP and cGMP and signal CFTR on the apical plasma membrane of small intestinal enterocytes to elicit chloride and fluid secretion. cAMP activates PKA, whereas cGMP signals a cGMP-dependent protein kinase (cGKII) to phosphorylate CFTR in the intestine. In the jejunum, cAMP also regulates CFTR and fluid secretion by insertion of CFTR from subapical vesicles to the surface of enterocytes. It is unknown whether cGMP signaling or phosphorylation regulates the insertion of CFTR associated vesicles from the cytoplasm to the surface of enterocytes. We used STa, cell-permeant cGMP, and cAMP agonists in conjunction with PKG and PKA inhibitors, respectively, in rat jejunum to examine whether 1) cGMP and cGK II regulate the translocation of CFTR to the apical membrane and its relevance to fluid secretion, and 2) PKA regulates cAMP-dependent translocation of CFTR because this intestinal segment is a primary target for toxigenic diarrhea. STa and cGMP induced a greater than fourfold increase in surface CFTR in enterocytes in association with fluid secretion that was inhibited by PKG inhibitors. cAMP agonists induced a translocation of CFTR to the cell surface of enterocytes that was prevented by PKA inhibitors. We conclude that cAMP and cGMP-dependent phosphorylation regulates fluid secretion and CFTR trafficking to the surface of enterocytes in rat jejunum.