Electrophilic derivatives of omega-3 fatty acids counteract lung cancer cell growth.

PURPOSE: 17-oxo-DHA is an electrophilic keto-derivative of the omega-3 fatty acid docosahexaenoic acid (DHA) endogenously generated by cyclooxygenase-2 and a cellular dehydrogenase. 17-oxo-DHA displays anti-inflammatory and cytoprotective actions. DHA, alone or in combination with standard chemotherapy, displays antitumor activity. However, the effects of electrophilic keto-derivatives of DHA on cancer growth have never been evaluated. We investigated whether 17-oxo-DHA, alone or in combination with gemcitabine, displayed antitumor effects. Furthermore, we evaluated whether the enzyme 15-prostaglandin dehydrogenase (15-PGDH) was required for transducing the antitumor effects of DHA. METHODS: A panel of five histologically different human non-small cell lung cancer (NSCLC) cell lines was used. Cells were treated with 17-oxo-DHA and gemcitabine, alone or in combination, and apoptosis, proliferation, Fas and FasL expression (mRNA and protein) and active caspase-3/7 and -8 were assessed. Furthermore, an inhibitor of 15-PGDH was used to test the involvement of this enzyme in mediating the antitumor effects of DHA. RESULTS: 17-oxo-DHA (50 µM, 72 h) significantly reduced proliferation, increased cell apoptosis, Fas and FasL expression as well as active caspase-8 and -3/7. When 17-oxo-DHA was given in combination with gemcitabine, stronger effects were observed compared to gemcitabine alone. The enzyme 15-PGDH was required for DHA to promote its full anti-apoptotic effect suggesting that enzymatically generated keto-derivatives of DHA mediate its antitumor actions. CONCLUSIONS: Data herein provided, demonstrate that 17-oxo-DHA displays antitumor effects in NSCLC cell lines. Of note, the combination of 17-oxo-DHA plus gemcitabine, resulted in stronger anticancer effects compared to gemcitabine alone.

SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells.

Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke, by modulating the SIRT1/FoxO3 axis, affects airway epithelial pro-inflammatory responses. Human bronchial epithelial cells (16HBE) and primary bronchial epithelial cells (PBECs) from COPD patients and controls were treated with/without cigarette smoke extract (CSE), Sirtinol or FoxO3 siRNA. SIRT1, FoxO3 and NF-κB nuclear accumulation, SIRT1 deacetylase activity, IL-8 and CCL20 expression/release and the release of 12 cytokines, neutrophil and lymphocyte chemotaxis were assessed. In PBECs, the constitutive FoxO3 expression was lower in patients with COPD than in controls. Furthermore, CSE reduced FoxO3 expression only in PBECs from controls. In 16HBE, CSE decreased SIRT1 activity and nuclear expression, enhanced NF-κB binding to the IL-8 gene promoter thus increasing IL-8 expression, decreased CCL20 expression, increased the neutrophil chemotaxis and decreased lymphocyte chemotaxis. Similarly, SIRT1 inhibition reduced FoxO3 expression and increased nuclear NF-κB. FoxO3 siRNA treatment increased IL-8 and decreased CCL20 expression in 16HBE. In conclusion, CSE impairs the function of SIRT1/FoxO3 axis in bronchial epithelium, dysregulating NF-κB activity and inducing pro-inflammatory responses.

Cigarette smoke and non-neuronal cholinergic system in the airway epithelium of COPD patients.

Acetylcholine (ACh), synthesized by Choline Acetyl-Transferase (ChAT), exerts its physiological effects via mAChRM3 in epithelial cells. We hypothesized that cigarette smoke affects ChAT, ACh, and mAChRM3 expression in the airways from COPD patients promoting airway disease. ChAT, ACh, and mAChRM3 were assessed: "ex vivo" in the epithelium from central and distal airways of COPD patients, Healthy Smoker (S) and Healthy Subjects (C), and "in vitro" in bronchial epithelial cells stimulated with cigarette smoke extract (CSE). In central airways, mAChRM3, ChAT, and ACh immunoreactivity was significantly higher in the epithelium from S and COPD than in C subjects. mAChRM3, ChAT, and ACh score of immunoreactivity was high in the metaplastia area of COPD patients. mAChRM3/ChAT and ACh/ChAT co-localization of immunoreactivity was observed in the bronchial epithelium from COPD. In vitro, CSE stimulation significantly increased mAChRM3, ChAT, and ACh expression and mAChRM3/ChAT and ACh/ChAT co-localization in 16HBE and NHBE, and increased 16HBE proliferation. Cigarette smoke modifies the levels of mAChMR3, ChAT expression, and ACh production in bronchial epithelial cells from COPD patients. Non-neuronal components of cholinergic system may have a role in the mechanism of bronchial epithelial cell proliferation, promoting alteration of normal tissue, and of related pulmonary functions.

Exposure to cigarette smoke extract and lipopolysaccharide modifies cytoskeleton organization in bronchial epithelial cells.

The integrity of the respiratory epithelium is crucial for airway homeostasis. Tobacco smoke exposure and recurrent infections of the airways play a crucial role in the progression and in the decline of the respiratory function in chronic obstructive pulmonary disease (COPD). The aim of this study was to detect differentially expressed proteins in a bronchial epithelial cell line (16-HBE) stimulated with cigarette smoke extract (CSE) and lipopolysaccharide (LPS), a constituent of gram-negative bacteria, alone and/or in combination, by using two-dimensional electrophoresis (2DE) analysis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot analysis was applied to confirm the expression of significantly modulated proteins. Flow cytometry and immunofluorescence were used to assess F-actin polimerization by phalloidin method. Fourteen proteins, with significant (p < 0.05) changes in intensity, were identified at various experimental points: 6 were up-regulated and 8 were down-regulated. As expected, bioinformatic analysis revealed that most of these proteins are involved in anti-oxidant and immune responses and in cytoskeleton stability. Western blot analysis confirmed that: Proteasome activator complex subunit 2 (PSME2), Peroxiredoxin-6 (PRDX6), Annexin A5 (ANXA5) and Heat shock protein beta-1 (HSPB1) were reduced and Coactosin-like protein (COTL-1) was increased by co-exposure of CSE and LPS. Furthermore, LPS and CSE increased actin polimerization. In conclusion, although further validation studies are needed, our findings suggest that, CSE and LPS could contribute to the progressive deterioration of lung function, altering the expression of proteins involved in metabolic processes and cytoskeleton rearrangement in bronchial epithelial cells.

Mucoadhesive solid lipid microparticles for controlled release of a corticosteroid in the chronic obstructive pulmonary disease treatment.

AIM: Therapeutic efficacy of pulmonary diseases is often limited and drug delivery systems offer new solutions to clinical problems. Solid lipid microparticles (SLMs) are suggested as systems for the delivery of therapeutics to the lung as, because of their size, they are able to deposit into secondary bronchi. MATERIALS & METHODS: Here, we describe two novel different SLMs using chitosan and alginate such as mucoadhesive polymers and we also studied their biocompatibility and their effectiveness compared with the free drug in controlling senescence and inflammatory processes in cigarette smoke extracts. RESULTS: Data reported show that fluticasone propionate (FP)-loaded SLMs are more effective than FP alone in controlling oxidative stress. CONCLUSION: The therapeutic approach using FP-loaded microparticles could be a promising strategy for the treatment of the chronic inflammatory pulmonary diseases.

Effects of Carbocysteine and Beclomethasone on Histone Acetylation/Deacetylation Processes in Cigarette Smoke Exposed Bronchial Epithelial Cells.

Histone deacetylase expression/activity may control inflammation, cell senescence, and responses to corticosteroids. Cigarette smoke exposure, increasing oxidative stress, may negatively affect deacetylase expression/activity. The effects of cigarette smoke extracts (CSE), carbocysteine, and beclomethasone dipropionate on chromatin remodeling processes in human bronchial epithelial cells are largely unknown. The present study was aimed to assess the effects of cigarette smoke, carbocysteine, and beclomethasone dipropionate on histone deacetylase 3 (HDAC3) expression/activity, N-CoR (nuclear receptor corepressor) expression, histone acetyltransferases (HAT) (p300/CBP) expression, p-CREB and IL-1 m-RNA expression, neutrophil chemotaxis. Increased p-CREB expression was observed in the bronchial epithelium of smokers. CSE increased p-CREB expression and decreased HDAC3 expression and activity and N-CoR m-RNA and protein expression. At the same time, CSE increased the expression of the HAT, p300/CBP. All these events increased acetylation processes within the cells and were associated to increased IL-1 m-RNA expression and neutrophil chemotaxis. The incubation of CSE exposed cells with carbocysteine and beclomethasone counteracted the effects of cigarette smoke on HDAC3 and N-CoR but not on p300/CBP. The increased deacetylation processes due to carbocysteine and beclomethasone dipropionate incubation is associated to reduced p-CREB, IL-1 m-RNA expression, neutrophil chemotaxis. These findings suggest a new role of combination therapy with carbocysteine and beclomethasone dipropionate in restoring deacetylation processes compromised by cigarette smoke exposure. J. Cell. Physiol. 232: 2851-2859, 2017. © 2016 Wiley Periodicals, Inc.

17-oxo-DHA displays additive anti-inflammatory effects with fluticasone propionate and inhibits the NLRP3 inflammasome.

Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function associated with increased local and systemic inflammatory markers, such as TNFα and IL-1ß. Glucocorticoids are used to treat this chronic disease, however their efficacy is low and new drugs are very much required. 17-oxo-DHA is a cyclooxygenase-2-dependent, electrophilic, α,ß-unsaturated keto-derivative of docosahexaenoic acid with anti-inflammatory properties. We evaluated the action of 17-oxo-DHA alone or in combination with the steroid fluticasone propionate (FP) in peripheral blood mononuclear cells (PBMCs) from COPD patients and healthy individuals exposed to lipopolysaccharide. We show that PBMCs from COPD patients released higher levels of TNFα and IL-1ß compared to controls. 17-oxo-DHA displayed strong anti-inflammatory effects. The addition of 17-oxo-DHA in combination with FP showed enhanced anti-inflammatory effects through the modulation of transcriptional and post-transcriptional mechanisms. 17-oxo-DHA, but not FP, was able to suppress the release of mature IL-1ß through inhibition of the NLRP3 inflammasome. Furthermore, 17-oxo-DHA inhibited inflammasome-dependent degradation of the glucocorticoid receptor (GR). Our findings suggest that 17-oxo-DHA in combination with FP or other steroids might achieve higher therapeutic efficacy than steroids alone. Combined treatment might be particularly relevant in those conditions where increased inflammasome activation may lead to GR degradation and steroid-unresponsive inflammation.

Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells.

IL-17A is overexpressed in the lung during acute neutrophilic inflammation. Acetylcholine (ACh) increases IL-8 and Muc5AC production in airway epithelial cells. We aimed to characterize the involvement of nonneuronal components of cholinergic system on IL-8 and Muc5AC production in bronchial epithelial cells stimulated with IL-17A. Bronchial epithelial cells were stimulated with recombinant human IL-17A (rhIL-17A) to evaluate the ChAT expression, the ACh binding and production, the IL-8 release, and the Muc5AC production. Furthermore, the effectiveness of PD098,059 (inhibitor of MAPKK activation), Bay11-7082 (inhibitor of IkBα phosphorylation), Hemicholinium-3 (HCh-3) (choline uptake blocker), and Tiotropium bromide (Spiriva®) (anticholinergic drug) was tested in our in vitro model. We showed that rhIL-17A increased the expression of ChAT, the levels of ACh binding and production, and the IL-8 and Muc5AC production in stimulated bronchial epithelial cells compared with untreated cells. The pretreatment of the cells with PD098,059 and Bay11-7082 decreased the ChAT expression and the ACh production/binding, while HCh-3 and Tiotropium decreased the IL-8 and Muc5AC synthesis in bronchial epithelial cells stimulated with rhIL-17A. IL-17A is involved in the IL-8 and Muc5AC production promoting, via NFκB and ERK1/2 pathway activation, the synthesis of ChAT, and the related activity of autocrine ACh in bronchial epithelial cells.

IL-17A induces chromatin remodeling promoting IL-8 release in bronchial epithelial cells: Effect of Tiotropium.

AIMS: IL-17A plays a key role in the persistence of airway inflammation, oxidative stress, and reduction of steroid-sensitivity in COPD. We studied the effect of IL-17A on chromatin remodeling and IL-8 production. MAIN METHODS: We measured the levels of IL-8 and IL-17A in induced sputum supernatants (ISS) from healthy controls (HCs), healthy smokers (HSs), and COPD patients by enzyme-linked immunosorbent assay (ELISA). A human bronchial epithelial cell line (16HBE) was stimulated with ISS from HCs, HSs, or COPD subjects. IL-8 was evaluated in 16HBE by Western blot and real-time polymerase chain reaction (PCR). Histone deacetylase 2 (HDAC2), acetyl histone H3 (Ac-His H3) (k9) and inhibitor kappa kinase alpha (IKKα) levels were evaluated in the nuclear extract by Western blot. Finally, we evaluated the effect of IL-17A depletion in ISS, the silencing of IKKα, and the anti-inflammatory effects of Tiotropium Spiriva® (100nM) on 16HBE. KEY FINDINGS: IL-8 and IL-17A levels were higher in ISS from COPD patients and HSs than from HCs. IL-8 protein and messenger RNA (mRNA) levels were increased in 16HBE stimulated with ISS from COPD patients compared with untreated cells. Furthermore, ISS from COPD patients reduced the nuclear levels of HDAC2 while increasing the activity of both Ac-His H3 (k9) and IKKα in stimulated 16HBE. IL-17A depletion in ISS and the IKKα silencing in 16HBE significantly increased the nuclear levels of HDAC2, reduced Ac-His H3 (k9), and promoted IL-8 synthesis in stimulated 16HBE. Tiotropium controls the proinflammatory activity generated by ISS from COPD patients in 16HBE. SIGNIFICANCE: IL-17A present in the airway of COPD patients, which induces chromatin remodeling, promotes the release of IL-8 in the bronchial epithelium. Tiotropium is able to control this proinflammatory activity.

MD2 expression is reduced in large airways of smokers and COPD smokers.

Toll-like receptor 4 (TLR4) signaling requires a number of accessory proteins to initiate a signal. MD-2 is one of the accessory proteins with a relevant role in lipopolysaccharide responses. Although cigarette smoke increases TLR4 expression, TLR4 signaling is altered in smokers and in smokers COPD patients. The main aims of this study were to explore whether MD2 is altered in large and small airways of COPD and of smokers without COPD. The expression of MD2 ex vivo was assessed by immunohistochemistry in surgical specimens from current smokers COPD (s-COPD; n = 14), smokers without COPD (S; n = 7), and from non-smoker non-COPD subjects (C; n = 11. The in vitro effects of cigarette smoke extracts on the MD2 expression in a human bronchial epithelial cell line (16-HBE) were also assessed by flow cytometry. MD2 is reduced in the epithelium and in the submucosa in large airways but not in the epithelium and in the submucosa in small airways of smokers and of s-COPD. The expression of MD2 in the submucosa of the large airways is significantly higher in comparison to the submucosa of the small airways in all the studied groups. In vitro, cigarette smoke is able to increase TLR4 but it reduces MD2 in a dose-dependent manner in bronchial epithelial cells. Cigarette smoke may alter innate immune responses reducing the expression of the MD2, a molecule with an important role in TLR4 signaling.

Cigarette smoke alters the proteomic profile of lung fibroblasts.

Smoking is strongly associated with diseases such as lung cancer and chronic obstructive pulmonary disease (COPD). Lung fibroblasts are crucial for the integrity of alveolar structure by producing extracellular matrix proteins which are required for attachment, structure, and function of alveolar epithelial cells. Despite the well-known association between cigarette smoke exposure and pulmonary and cardiovascular diseases, many questions remain regarding the mechanisms by which smoking induces diseases. The aim of this study is to detect differentially expressed proteins in human foetal lung cells (HFL-1) after 5 and 10% doses of cigarette smoke extract (CSE) exposure, combining two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In order to evaluate cellular ability to recover as well as lasting damage, we analysed the proteomic pattern 24 hours after the CSE removal (release). Eleven proteins had significant changes at various experimental points. Among these, 7 were up-regulated after CSE-treatments and 4 were down-regulated. Some spots seemed to be modified permanently or in a transient manner, in fact they returned to baseline levels after CSE-removal (normalisation after CSE release) and others were modified by selective CSE concentrations or only after release. MS identified, differentially expressed proteins are involved in stress response, mitochondrial activity, and aging. These findings may improve our understanding about molecular mechanisms underlying CSE caused damage and they may also integrate the comprehension of cigarette smoke effects on human health.

Advances in asthma pathophysiology: stepping forward from the Maurizio Vignola experience.

Maurizio Vignola was a superb and innovative researcher, who wrote seminal papers on the biology of airway epithelium in asthma. Inflammation and remodelling were the main topics of his research, mostly conducted in biopsy specimens from patients with asthma of variable severity, encompassing the entire spectrum of the disease from mild to severe asthma. His observations contributed to define the biology of asthma as we know it today, and opened the way to the personalised treatment of asthma. His group has successfully continued to investigate the biology and clinical aspects of bronchial asthma, with major interest in the clinical use of biomarkers to monitor disease activity, and in the development of new therapeutic perspectives. This review summarises the latest work on these topics proudly conducted by Maurizio's closest collaborators. The results indicate significant progress in our understanding of asthma in the last 10 years, in particular increased knowledge of the complex interaction between inflammatory and remodelling pathways, improved recognition of biological and clinical asthma phenotypes, and development of new treatment strategies, especially for patients with severe corticosteroid-resistant asthma.

Effects in cigarette smoke stimulated bronchial epithelial cells of a corticosteroid entrapped into nanostructured lipid carriers.

BACKGROUND: Nanomedicine studies have showed a great potential for drug delivery into the lung. In this manuscript nanostructured lipid carriers (NLC) containing Fluticasone propionate (FP) were prepared and their biocompatibility and effects in a human bronchial epithelial cell line (16-HBE) stimulated with cigarette smoke extracts (CSE) were tested. RESULTS: Biocompatibility studies showed that the NLC did not induce cell necrosis or apoptosis. Moreover, it was confirmed that CSE increased intracellular ROS production and TLR4 expression in bronchial epithelial cells and that FP-loaded NLC were more effective than free drug in modulating these processes. Finally, the nanoparticles increased GSH levels improving cell protection against oxidative stress. CONCLUSIONS: The present study shows that NLC may be considered a promising strategy to improve corticosteroid mediated effects in cellular models associated to corticosteroid resistance. The NLC containing FP can be considered good systems for dosage forms useful for increasing the effectiveness of fluticasone decreasing its side effects.

Altered expression of p21, activated caspase-3, and PCNA in bronchiolar epithelium of smokers with and without chronic obstructive pulmonary disease.

UNLABELLED: ABSTRACT Background: The cyclin-dependent kinase inhibitor p21CIP1/WAF1 is involved in cell-cycle growth arrest due to cell stressors, such as cigarette smoke. The role of p21 in cell apoptosis is controversial as it exerts pro- or antiapoptotic effects in different cells. In the present study, we investigated whether, in the epithelium of small airways of smokers with and without COPD, altered p21 expression is associated with an imbalance between proliferation and apoptosis. OBJECTIVES AND METHODS: The expression of specific molecules involved in the regulation of apoptosis, such as activated caspase-3 and cytoplasmic p21, cell quiescence (G0) or proliferation markers such as Ki67 and PCNA, and cell-cycle markers such as the nuclear p21, was assessed in the small airway (bronchiolar) epithelium of smokers with and without COPD and in nonsmoker non-COPD subjects. RESULTS: In smokers with and without COPD, we found an increase of cytoplasmic nuclear p21 and activated caspase-3 expression. By contrast, we verified in all the studied groups a similar low expression of the proliferation marker Ki67 and a reduced expression of PCNA in smokers and smokers with COPD. CONCLUSIONS: In the small airway epithelium, cytoplasmic p21 correlating with increased activated caspase-3 expression might play a proapoptotic role. Furthermore, p21 alteration may be associated with the inhibition of tissue repair in smokers and smokers with COPD as confirmed by the low expression of proliferation markers such as PCNA. All these events may play a role in the permanent cellular damage leading to the destruction of bronchiolar tissue.

Cigarette smoke alters IL-33 expression and release in airway epithelial cells.

Airway epithelium is a regulator of innate immune responses to a variety of insults including cigarette smoke. Cigarette smoke alters the expression and the activation of Toll Like Receptor 4 (TLR4), an innate immunity receptor. IL-33, an alarmin, increases innate immunity Th2 responses. The aims of this study were to explore whether mini-bronchoalveolar lavage (mini-BAL) or sera from smokers have altered concentrations of IL-33 and whether cigarette smoke extracts (CSE) alter both intracellular expression (mRNA and protein) and release of IL-33 in bronchial epithelial cells. The role of TLR4 in the expression of IL-33 was also explored. Mini-BALs, but not sera, from smokers show reduced concentrations of IL-33. The expression of IL-33 was increased also in bronchial epithelium from smokers. 20% CSE reduced IL-33 release but increased the mRNA for IL-33 by real time PCR and the intracellular expression of IL-33 in bronchial epithelial cells as confirmed by flow cytometry, immunocytochemistry and western blot analysis. The effect of CSE on IL-33 expression was also observed in primary bronchial epithelial cells. IL-33 expression was mainly concentrated within the cytoplasm of the cells. LPS, an agonist of TLR4, reduced IL-33 expression, and an inhibitor of TLR4 increased the intracellular expression of IL-33. In conclusion, the release of IL-33 is tightly controlled and, in smokers, an altered activation of TLR4 may lead to an increased intracellular expression of IL-33 with a limited IL-33 release.

Cigarette smoke alters non-neuronal cholinergic system components inducing MUC5AC production in the H292 cell line.

Cigarette smoke extract (CSE) affects the expression of Choline Acetyl-Transferase (ChAT), muscarinic acetylcholine receptors, and mucin production in bronchial epithelial cells. Mucin 5AC (MUC5AC), muscarinic acetylcholine receptor M3, ChAT expression, acetylcholine levels and acetylcholine binding were measured in a human pulmonary mucoepidermoid carcinoma cell line (H292) stimulated with CSE. We performed ChAT/RNA interference experiments in H292 cells stimulated with CSE to study the role of ChAT/acetylcholine in MUC5AC production. The effects of Hemicholinium-3 (HCh-3) (50 µM) (a potent and selective choline uptake blocker) and Tiotropium bromide (Spiriva(®)) (100 nM), alone or in combination with Salmeterol (SL) and Fluticasone propionate (FP), were tested in this model. MUC5AC, muscarinic acetylcholine receptor M3, ChAT, acetylcholine expression and acetylcholine binding significantly increased in H292 cells stimulated with CSE (5%) compared to untreated cells. HCh-3 reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. ChAT/RNA interference eliminated the effect of CSE on MUC5AC production. FP reduced ChAT and acetylcholine binding in unstimulated cells, while showing a partial effect in CSE stimulated cells. SL increased the ChAT expression and acetylcholine binding in H292 cells stimulated with or without CSE. Tiotropium, alone or together with FP and SL, reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. CSE affects the ChAT/acetylcholine expression, increasing MUC5AC production in H292 cells. Pharmacological treatment with anticholinergic drugs reduces the secretion of MUC5AC generated by autocrine acetylcholine activity in airway epithelial cells.

Dual anti-oxidant and anti-inflammatory actions of the electrophilic cyclooxygenase-2-derived 17-oxo-DHA in lipopolysaccharide- and cigarette smoke-induced inflammation.

BACKGROUND: 17-Oxo-DHA is an endogenous electrophilic derivative of the omega-3 fatty acid docosahexaenoic acid (DHA) which is generated in activated macrophages by the action of cyclooxygenase-2. METHODS: The ability of 17-oxo-DHA to control inflammation and oxidative stress was tested in human macrophages (THP-1) and bronchial epithelial cell line (16HBE) stimulated with cigarette smoke extract (CSE) and lipopolysaccharide (LPS). All data were further confirmed using primary bronchial epithelial cells, alveolar macrophages and peripheral blood mononuclear cells. RESULTS: 17-Oxo-DHA was a strong inducer of the anti-oxidant response promoting Nrf2 nuclear accumulation, leading to the expression of heme oxygenase 1 and more than doubling glutathione levels. This resulted in suppression of CSE-induced ROS generation in macrophages. In macrophages, 17-oxo-DHA potently suppressed TNFα release in response to LPS, CSE and IL-1ß acting at transcriptional level via a mechanism independent of Nrf2. Externally supplemented 17-oxo-DHA displayed the same effects in the presence of the Cox-inhibitor indomethacin. The non-electrophilic 17-oxo-DHA precursor DHA did not show any biological actions, indicating that the electrophilic moiety was required for this compound to become bioactive. CONCLUSIONS: 17-Oxo-DHA promotes cytoprotective actions both in immune and structural cells. In immune cells, 17-oxo-DHA is effective in contrasting CSE- and LPS-induced oxidative damage and inflammation acting via multiple independent pathways. GENERAL SIGNIFICANCE: Herein we provide insights on how the novel endogenous electrophilic DHA-derivative 17-oxo-DHA promotes anti-oxidant and anti-inflammatory actions. Data herein reported indicate that 17-oxo-DHA is an attractive lead compound for the development of new treatments for cigarette smoke-related airway inflammatory disorders.

Oxidative stress and innate immunity responses in cigarette smoke stimulated nasal epithelial cells.

Cigarette smoke extracts (CSE) may play a significant role in diseases of the upper airway including chronic rhinosinusitis. Even short term exposure of cigarette smoke has adverse effects on mitochondrial functions and redox homeostasis in tissues which may progress to further complications associated with chronic smoking. Cigarette smoke alters toll-like receptor 4 (TLR4) expression and activation in bronchial epithelial cells. Carbocysteine is an anti-oxidant and mucolytic agent. The effects of carbocysteine on CSE induced oxidative stress and on associated innate immune and inflammatory responses in nasal epithelial cells are largely unknown. The present study was aimed to assess in CSE stimulated nasal epithelial cells (RPMI 2650) the effects of carbocysteine (10(-4)M) on: cell survival, intracellular reactive oxygen species (ROS) production, TLR4 expression, LPS binding and neutrophil chemotaxis (actin reorganization). We found that CSE increased ROS production, TLR4 expression, LPS binding and neutrophil chemotaxis and all these events were counteracted by pre-incubating CSE stimulated RPMI 2650 cells with carbocysteine. In conclusion, the present study provides compelling evidence that carbocysteine may be considered a promising therapeutic strategy in chronic inflammatory nasal diseases.

Gemcitabine sensitizes lung cancer cells to Fas/FasL system-mediated killing.

Gemcitabine is a chemotherapy agent commonly used in the treatment of non-small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine-induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real-time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL-expressing cells was evaluated by flow cytometry, and caspase-8 and caspase-3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine-activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti-Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane-bound FasL (mFasL) and of mFasL-positive apoptotic H292 cells, as well as caspase-8 and caspase-3 cleavage. Moreover, gemcitabine increased CH11-induced caspase-8 and caspase-3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine-treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up-regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas-dependent apoptosis mediated by caspase-8 and caspase-3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing.