Adenosine A2A Receptor Agonist, 2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine Hydrochloride Hydrate, Inhibits Inflammation and Increases Fibroblast Growth Factor-2 Tissue Expression in Carrageenan-Induced Rat Paw Edema.

Adenosine is the final product of ATP metabolism, mainly derived from the action of 5'-nucleotidase cleavage of AMP. Cellular production of adenosine is greatly enhanced in inflamed tissues, ischemic tissues, and under hypoxia, where ATP is released from damaged cells. Much evidence has been accumulated on adenosine anti-inflammatory effects mediated through A2A receptor activation; A2A adenosine receptor has also been shown to play a role in matrix deposition and wound healing in a damaged tissue, contributing to dermal tissue protection and repair. Fibroblast growth factor-2 (FGF-2) is a powerful mitogen for fibroblast; it is expressed by several inflammatory cell types and plays a pivotal role in angiogenesis, wound healing, gastric ulcer protection. Human recombinant FGF-2 has been shown to have anti-inflammatory effects. The purpose of the present work was to investigate on the anti-inflammatory effect of systemic administration of the adenosine A2A agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS21680) in the rat model of carrageenan-induced paw edema. We found that CGS21680 inhibits inflammation induced by carrageenan injection into the rat paw, and this effect is associated to the local reduction of cytokine levels and dermal increase of FGF-2 expression. Our results suggest that FGF-2 might be involved in the anti-inflammatory and tissue protective effect due to A2A receptor activation.

Design and characterization of a chitosan physical gel promoting wound healing in mice.

In this study, a sterile and biocompatible chitosan (CHI) gel for wound healing applications was formulated. CHI powder was treated in autoclave (ttCHI) to prepare sterile formulations. The heat treatment modified the CHI molecular weight, as evidenced by GPC analysis, and its physical-chemical features. Differential scanning calorimetry studies indicated that the macromolecules, before and after thermal treatment, differ in the strength of water-polymer interaction leading to different viscoelastic and flow properties. Thermally treated CHI exhibited the following effects: (i) increased the proliferation and migration of human foreskin foetal fibroblasts at 24 h; (ii) accelerated wound healing (measured as area of lesion) at 3 and 10 days in an in vivo model of pressure ulcers. These effects were linked to the increase of the hydroxyproline and haemoglobin content as well as Wnt protein expression. Moreover, we found a reduction of myeloperoxidase activity and TNF-α mRNA expression. These observations suggest the potential of this novel CHI gel in wound healing and other therapeutic applications.

Enhanced antioxidant effect of trans-resveratrol: potential of binary systems with polyethylene glycol and cyclodextrin.

Trans-resveratrol, a polyphenol extracted from Vitis vinifera, has different beneficial effects following its administration on the skin. Here the potential use of binary systems to enhance in vitro and in vivo activity of trans-resveratrol was investigated. Thus the aqueous solubility of trans-resveratrol was investigated in the presence of growing concentrations of polyethylene glycol (PEG) or ß-cyclodextrin (ßCD) as solubilizing excipients. Then, the solid dispersion of trans-resveratrol with PEG or inclusion complexes trans-resveratrol/ßCD were prepared and characterised by different methods. Cytotoxicity and inhibition of reactive oxygen species (ROS) following H2O2 challenge in the presence of trans-resveratrol, alone or associated to the excipients, was evaluated on human keratinocyte HaCaT cell line. Both the trans-resveratrol-containing binary systems induced significant reduction of H2O2-induced ROS production, especially in the case of ßCD that was selected for the following phase of the study. Thus, the effect of a cream containing trans-resveratrol, alone or associated to ßCD, on different skin parameters such as corneometry, colorimetry and elastometry, was evaluated on human volunteers. All patients showed a visible improvement of clinical conditions with a remarkable decrease of aging signs, but this effect was higher of the hemi face treated with the ßCD-containing formulation versus formulation containing trans-resveratrol alone.

Synthesis and pharmacological evaluation of modified adenosines joined to mono-functional platinum moieties.

The synthesis of four novel platinum complexes, bearing N6-(6-amino-hexyl)adenosine or a 1,6-di(adenosin-N6-yl)-hexane respectively, as ligands of mono-functional cisplatin or monochloro(ethylendiamine)platinum(II), is reported. The chemistry exploits the high affinity of the charged platinum centres towards the N7 position of the adenosine base system and a primary amine of an alkyl chain installed on the C6 position of the purine. The cytotoxic behaviour of the synthesized complexes has been studied in A549 adenocarcinomic human alveolar basal epithelial and MCF7 human breast adenocarcinomic cancer cell lines, in order to investigate their effects on cell viability and proliferation.

A decoy oligonucleotide to NF-κB delivered through inhalable particles prevents LPS-induced rat airway inflammation.

The inflammatory process plays a crucial role in the onset and progression of several lung pathologies, including cystic fibrosis (CF), and the involvement of NF-κB is widely recognized. The specific inhibition of NF-κB by decoy oligonucleotides delivered within the lung may be beneficial, although rationally designed systems are needed to optimize their pharmacological response. Prompted by this need, we have developed and tested in vivo an inhalable dry powder for the prolonged delivery of a decoy oligodeoxynucleotide to NF-κB (dec-ODN), consisting of large porous particles (LPPs) based on poly(lactic-co-glycolic) acid. First, LPPs containing dec-ODN (dec-ODN LPPs) were engineered to meet the aerodynamic criteria crucial for pulmonary delivery, to gain an effective loading of dec-ODN, to sustain its release, and to preserve its structural integrity in lung lining fluids. We then investigated the effects of dec-ODN LPPs in a rat model of lung inflammation induced by the intratracheal aerosolization of LPS from Pseudomonas aeruginosa. The results show that a single intratracheal insufflation of dec-ODN LPPs reduced the bronchoalveolar neutrophil infiltration induced by LPS for up to 72 hours, whereas naked dec-ODN was able to inhibit it only at 6 hours. The persistent inhibition of neutrophil infiltrate was associated with reduced NF-κB/DNA binding activity, as well as reduced IL-6, IL-8, and mucin-2 mRNA expression in lung homogenates. We consider it noteworthy that the developed LPPs, preventing the accumulation of neutrophils and NF-κB-related gene expression, may provide a new therapeutic option for the local treatment of inflammation associated with lung disease.

Macrophage autophagy in atherosclerosis.

Macrophages play crucial roles in atherosclerotic immune responses. Recent investigation into macrophage autophagy (AP) in atherosclerosis has demonstrated a novel pathway through which these cells contribute to vascular inflammation. AP is a cellular catabolic process involving the delivery of cytoplasmic contents to the lysosomal machinery for ultimate degradation and recycling. Basal levels of macrophage AP play an essential role in atheroprotection during early atherosclerosis. However, AP becomes dysfunctional in the more advanced stages of the pathology and its deficiency promotes vascular inflammation, oxidative stress, and plaque necrosis. In this paper, we will discuss the role of macrophages and AP in atherosclerosis and the emerging evidence demonstrating the contribution of macrophage AP to vascular pathology. Finally, we will discuss how AP could be targeted for therapeutic utility.

Palmitoylethanolamide counteracts reactive astrogliosis induced by ß-amyloid peptide.

Emerging evidence indicates that astrogliosis is involved in the pathogenesis of neurodegenerative disorders. Our previous findings suggested cannabinoids and Autacoid Local Injury Antagonism Amides (ALIAmides) attenuate glial response in models of neurodegeneration. The present study was aimed at exploring palmitoylethanolamide (PEA) ability to mitigate ß-amyloid (Aß)-induced astrogliosis. Experiments were carried out to investigate PEA's (10(-7) M) effects upon the expression and release of pro-inflammatory molecules in rat primary astrocytes activated by soluble Aß(1-42) (1 µg/ml) as well as to identify mechanisms responsible for such actions. The effects of Aß and exogenous PEA on the astrocyte levels of the endocannabinoidsand of endogenous ALIAmides were also studied. The peroxisome proliferator-activated receptor (PPAR)-α (MK886, 3 µM) or PPAR-γ (GW9662, 9 nM) antagonists were co-administered with PEA. Aß elevated endogenous PEA and d5-2-arachidonoylglycerol (2-AG) levels. Exogenous PEA blunted the Aß-induced expression of pro-inflammatory molecules. This effect was reduced by PPAR-α antagonist. Moreover, this ALIAmide, like Aß, increased 2-AG levels. These results indicate that PEA exhibits anti-inflammatory properties able to counteract Aß-induced astrogliosis, and suggest novel treatment for neuroinflammatory/ neurodegenerative processes.

Sustained inhibition of IL-6 and IL-8 expression by decoy ODN to NF-κB delivered through respirable large porous particles in LPS-stimulated cystic fibrosis bronchial cells.

BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Neutrophil-dominated inflammation and chronic bacterial infection are still considered the primary cause of bronchioectasis, respiratory failure and consequent death in CF patients. Activation of nuclear factor (NF)-κB is responsible for overproduction of cytokines, such as interleukin (IL)-6 and IL-8, in airways of CF patients. Thus, decoy oligodeoxynucleotides against NF-κB (dec-ODN) may limit lung inflammation in CF. In the present study, we studied the effects of dec-ODN delivered through biodegradable and respirable poly(D,L-lactide-co-glycolide) large porous particles (LPP) on IL-6 and IL-8 mRNA expression as well as NF-κB/DNA binding activity in cystic fibrosis cells stimulated with lipopolysaccharide (LPS) from Pseudomonas aeruginosa. METHODS: dec-ODN LPP were prepared by a modified double emulsion technique and characterized in terms of size, morphology, tapped density and dec-ODN loading. Human epithelial bronchial IB3-1 (CFTR-mutated) as well as S9 (CFTR-corrected) were stimulated with LPS from P. aeruginosa for 24 and 72 h in the absence or presence of naked dec-ODN or dec-ODN LPP. RESULTS: Stimulation of cells with LPS from P. aeruginosa caused an increase of IL-6 and IL-8 mRNA levels, which were significantly inhibited by dec-ODN LPP at 24 and 72 h, whereas naked dec-ODN inhibited those only at 24 h. Similar effects were exhibited by dec-ODN LPP or naked dec-ODN on NF-κB/DNA binding activity. CONCLUSIONS: Our observations indicate that respirable biodegradable dec-ODN LPP may represent a promising strategy for inhibiting NF-κB transcriptional activity and related gene expression and, thus, reduce lung chronic inflammation in CF patients.

The I{kappa}B kinase inhibitor nuclear factor-{kappa}B essential modulator-binding domain peptide for inhibition of injury-induced neointimal formation.

OBJECTIVE: The activation of nuclear factor-κB (NF-κB) is a crucial step in the arterial wall's response to injury. The identification and characterization of the NF-κB essential modulator-binding domain (NBD) peptide, which can block the activation of the IκB kinase complex, have provided an opportunity to selectively abrogate the inflammation-induced activation of NF-κB. The aim of the present study was to evaluate the effect of the NBD peptide on neointimal formation. METHODS AND RESULTS: In the rat carotid artery balloon angioplasty model, local treatment with the NBD peptide (300 µg/site) significantly reduced the number of proliferating cells at day 7 (by 40%; P<0.01) and reduced injury-induced neointimal formation (by 50%; P<0.01) at day 14. These effects were associated with a significant reduction of NF-κB activation and monocyte chemotactic protein-1 expression in the carotid arteries of rats treated with the peptide. In addition, the NBD peptide (0.01 to 1 µmol/L) reduced rat smooth muscle cell proliferation, migration, and invasion in vitro. Similar results were observed in apolipoprotein E(-/-) mice in which the NBD peptide (150 µg/site) reduced wire-induced neointimal formation at day 28 (by 47%; P<0.01). CONCLUSIONS: The NBD peptide reduces neointimal formation and smooth muscle cell proliferation/migration, both effects associated with the inhibition of NF-κB activation.

Oligonucleotide decoy to NF-kappaB slowly released from PLGA microspheres reduces chronic inflammation in rat.

Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of several genes involved in the immune and inflammatory process. Previously, we demonstrated that NF-kappaB activation can be significantly inhibited by a double stranded oligodeoxynucleotide (ODN). Nevertheless, the therapeutic use of ODN requires a delivery system able to improve poor crossing of cell membranes and rapid in vivo enzymatic degradation. Poly(D,L-lactide-co-glycolide) (PLGA) microspheres can increase ODN stability in biological environment and release the encapsulated drug in long time frames. Here, we used a decoy ODN against NF-kappaB and we investigated its effect, when administered in naked form or when delivered by PLGA microspheres, in a rat model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges caused leukocyte infiltration and formation of granulation tissue which were inhibited up to 15 days by co-injection of microspheres releasing decoy ODN whereas naked decoy ODN showed this effect only up to 5 days. Molecular analysis performed on granulation tissue demonstrated an inhibition of NF-kappaB activation correlated to a decrease of tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) expression. Our results suggest that microspheres could be an useful tool to improve pharmacokinetics of decoy ODN and may represent a strategy to inhibit NF-kappaB activation in chronic inflammation.

The Ecto-5'-Nucleotidase/CD73 Inhibitor, α,ß-Methylene Adenosine 5'-Diphosphate, Exacerbates Carrageenan-Induced Pleurisy in Rat.

The ecto-5'-nucleotidase (ecto-5'NT/CD73) represents a crucial enzyme for endogenous adenosine generation. Several findings have shown that CD73 plays an important role in regulating vascular permeability and immune cell function. Adenosine 5'-(α,ß-methylene)diphosphate (APCP) is a CD73 inhibitor, widely used as pharmacological tool to investigate the role of CD73/adenosine pathway in several in vitro and in vivo models, although it has been also shown to inhibit other ectoenzymes involved in adenosinergic pathway. Here, we evaluated the effect of APCP in the development of inflammation in carrageenan-induced pleurisy model. We found that treatment with APCP (400 µg/rat) significantly increased cell accumulation, exudate formation, and pro-inflammatory cytokine content into the pleural cavity in the acute phase (4 h) of inflammation, with no differences in the sub-acute phase (72 h) except for the regulation of monocyte chemotactic protein-1 levels. In addition, cells collected by pleural lavage fluids of APCP-treated rats, 4 h following carrageenan injection, showed increased ability to migrate in vitro, both in presence and in absence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine as chemotactic stimulus, compared to cells obtained by control rats. Our results demonstrate that APCP exacerbates the early phase of carrageenan-induced pleurisy by controlling pleural effusion and polymorphonuclear migration in vivo and ex vivo. This effect is likely dependent upon CD73 inhibition, although an inhibitory effect of other ectoenzymes cannot be ruled out.

Novel cationic liposome formulation for the delivery of an oligonucleotide decoy to NF-kappaB into activated macrophages.

Nuclear factor-kappaB (NF-kappaB) is involved in several pathological processes, such as inflammation. Pro-inflammatory genes expression can be down-regulated by using an oligonucleotide (ODN) decoy to NF-kappaB. Cationic liposomes are largely used to improve ODN uptake into cells, although a higher transfection efficiency and a lower toxicity are required to use them in therapy. In this work, we investigated the potential of a novel liposome formulation, based on the recently synthesised cationic lipid (2,3-didodecyloxypropyl) (2-hydroxyethyl) dimethylammonium bromide (DE), as the delivery system for a double stranded ODN decoy to NF-kappaB. Liposomes composed of DE or DE mixed with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine or cholesterol as helper lipids were complexed with ODN at different +/- charge ratios. In vitro uptake and the effect of ODN, naked or complexed with DE-containing liposomes, were evaluated in lipopolysaccharide-stimulated RAW 264.7 macrophages. The use of helper lipids increased liposome physical stability up to 1 year at 4 degrees C. ODN complexed with DE/cholesterol liposomes, at the +/- charge ratio of 8, showed a limited cytotoxicity and the highest inhibition of nitrite production, inducible nitric oxide synthase protein expression and NF-kappaB/DNA binding activity. Confocal microscopy confirmed a high ODN cell uptake obtained with DE/cholesterol liposomes at the highest +/- charge ratio.

Local administration of WIN 55,212-2 reduces chronic granuloma-associated angiogenesis in rat by inhibiting NF-kappaB activation.

Chronic inflammation is often associated with granuloma formation that is a hallmark of many human diseases. The transcription factor nuclear factor-kappa B (NF-kappaB) plays a central role in this process by regulating the expression of several pro-inflammatory genes. Cannabinoids (CBs) from Cannabis sativa L. exert a large number of biological effects including anti-inflammatory and anti-angiogenic effects. In this study, we investigated the role of CBs on granuloma formation induced by lambda-carrageenin-soaked sponge implant in rat. Our results show that local administration of WIN 55,212-2, a CB(1)/CB(2) agonist, given daily or at time of implantation significantly decreased weight and neo-angiogenesis in granuloma tissue and inhibited nuclear factor-kappa B (NF-kappaB)/DNA binding that was associated with a reduced inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor alpha (TNF-alpha), and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and protein expression. Also, arachidonyl-2-chloroethylamide (ACEA), a CB(1) selective agonist, and JWH-015, a CB(2) selective agonist, exhibited the same effects that were reversed by SR141716-A and SR144528, respectively, CB(1) and CB(2) selective antagonists. These results indicate that CBs given locally may represent a potential therapeutic tool in controlling chronic inflammation avoiding psychotropic effects.

Neutralization of interleukin-18 inhibits neointimal formation in a rat model of vascular injury.

BACKGROUND: Studies in humans and animal models suggest that interleukin-18 (IL-18) plays a crucial role in vascular pathologies. IL-18 is a predictor of cardiovascular death in angina and is involved in atherotic plaque destabilization. Higher IL-18 plasma levels also are associated with restenosis after coronary artery angioplasty performed in patients with acute myocardial infarction. We investigated the effective role of IL-18 in neointimal formation in a balloon-induced rat model of vascular injury. METHODS AND RESULTS: Endothelial denudation of the left carotid artery was performed by use of a balloon embolectomy catheter. Increased expression of IL-18 and IL-18Ralpha/beta mRNA was detectable in carotid arteries from days 2 to 14 after angioplasty. The active form of IL-18 was highly expressed in injured arteries. Strong immunoreactivity for IL-18 was detected in the medial smooth muscle cells at days 2 and 7 after balloon injury and in proliferating/migrating smooth muscle cells in neointima at day 14. Moreover, serum concentrations of IL-18 were significantly higher among rats subjected to vascular injury. Treatment with neutralizing rabbit anti-rat IL-18 immunoglobulin G significantly reduced neointimal formation (by 27%; P < 0.01), reduced the number of proliferating cells, and inhibited interferon-gamma, IL-6, and IL-8 mRNA expression and nuclear factor-kappaB activation in injured arteries. In addition, in vitro data show that IL-18 affects smooth muscle cell proliferation. CONCLUSIONS: These results identify a critical role for IL-18 in neointimal formation in a rat model of vascular injury and suggest a potential role for IL-18 neutralization in the reduction of neointimal development.

Lycopene, quercetin and tyrosol prevent macrophage activation induced by gliadin and IFN-gamma.

Oxidative stress plays an important role in inflammatory process of celiac disease. We have studied the effect of the lycopene, quercetin and tyrosol natural antioxidants on the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression in RAW 264.7 macrophages stimulated by gliadin in association with IFN-gamma. The IFN-gamma plus gliadin combination treatment was capable of enhancing iNOS and COX-2 gene expression and nuclear factor-kappaB (NF-kappaB), interferon regulatory factor-1 (IRF-1) and signal transducer and activator of transcription-1alpha (STAT-1alpha) activation induced by reactive oxygen species generation at 24 h. Lycopene, quercetin and tyrosol inhibited all these effects. The results here reported suggest that these compounds may represent non toxic agents for the control of pro-inflammatory genes involved in celiac disease.

Oxalomalate affects the inducible nitric oxide synthase expression and activity.

Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.

The marijuana component cannabidiol inhibits beta-amyloid-induced tau protein hyperphosphorylation through Wnt/beta-catenin pathway rescue in PC12 cells.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. A massive accumulation of beta-amyloid (Abeta) peptide aggregates has been proposed as pivotal event in AD. Abeta-induced toxicity is accompanied by a variegated combination of events including oxidative stress. The Wnt pathway has multiple actions in the cascade of events triggered by Abeta, and drugs that rescue Wnt activity may be considered as novel therapeutics for AD treatment. Cannabidiol, a non-psychoactive marijuana component, has been recently proposed as an antioxidant neuroprotective agent in neurodegenerative diseases. Moreover, it has been shown to rescue PC12 cells from toxicity induced by Abeta peptide. However, the molecular mechanism of cannabidiol-induced neuroprotective effect is still unknown. Here, we report that cannabidiol inhibits hyperphosphorylation of tau protein in Abeta-stimulated PC12 neuronal cells, which is one of the most representative hallmarks in AD. The effect of cannabidiol is mediated through the Wnt/beta-catenin pathway rescue in Abeta-stimulated PC12 cells. These results provide new molecular insight regarding the neuroprotective effect of cannabidiol and suggest its possible role in the pharmacological management of AD, especially in view of its low toxicity in humans.

The role of NF-kappaB, IRF-1, and STAT-1alpha transcription factors in the iNOS gene induction by gliadin and IFN-gamma in RAW 264.7 macrophages.

Nitric oxide (NO) plays an important role in the pathogenesis of celiac disease. We have examined the involvement of nuclear factor-kappaB (NF-kappaB), interferon regulatory factor-1 (IRF-1), and signal transducer and activator of transcription-1alpha (STAT-1alpha) on the synergistic induction of inducible nitric oxide synthase (iNOS) gene expression by gliadin (G) in association with interferon-gamma (IFN-gamma) in RAW 264.7 macrophages. We found that IFN-gamma was efficient in enhancing the basal transcription of the iNOS promoter at 1, 6, and 24 h, whereas G had no effect. The G plus IFN-gamma association caused an increase in iNOS promoter activity which was inhibited by pyrrolidine dithiocarbammate (PDTC) at 6 and 24 h as well as by genistein (Gen) and tyrphostine B42 (TB42) at 1 h, inhibitors of NF-kappaB, IRF-1, and STAT-1alpha activation, respectively. Similarly, the IFN-gamma and G combination treatment led to a higher increase in iNOS mRNA levels at 1, 6, and 24 h compared with IFN-gamma alone. Gen and TB42 inhibited iNOS mRNA levels at 1 h, whereas PDTC inhibited iNOS mRNA levels at 6 and 24 h. In addition, the synergistic induction of iNOS gene expression by G plus IFN-gamma correlated with the induction of NF-kappaB, IRF-1, and STAT-1alpha/DNA binding activity and mRNA expression. In conclusion, our study, which provides evidence that the effect of G on iNOS gene transcription in IFN-gamma-stimulated RAW 264.7 cells can be ascribed to all three transcription factors, may contribute to lead to new insights into the molecular mechanisms governing the inflammatory process in celiac disease.

Simvastatin inhibits the expression of stemness­related genes and the metastatic invasion of human cancer cells via destruction of the cytoskeleton.

Statins are a class of drugs that inhibit the rate-limiting steps in the cholesterol biosynthesis pathway. They act by inhibiting 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate. Blocking of mevalonate synthesis leads to inhibition of the farnesylation and geranylgeranylation of several functional proteins, such as RhoA and other small guanosine triphosphate-binding proteins, that are important in maintaining the undifferentiated status of the cells. In the present study, we hypothesized that simvastatin, likely through the inhibition of farnesylation and geranylgeranylation of Rac1, Cd42 and RhoA, induces a destruction/restructuration of the cytoskeleton that decreases mechanical strain transfer to the nuclei, inducing the loss of transmission of regulatory signals from the cytoskeleton to the nucleoskeleton. Although this remains at present a hypothesis and is not easy to define if the de-structuration of the cytoskeleton is a secondary effect of simvastatin treatment or the inhibition of post-translational protein modification have a precise role in the structuration of actin cytoskeleton, we speculate that these signal variations could inhibit the expression of certain stemness genes, which could therefore be considered nucleoskeleton-associated and mechanically regulated genes. On the other hand, the restructuration of the cytoskeleton inhibits the formation of lamellipodia and filopodia, which likely decreases the capability of cancer cells to invade the extracellular matrix, thereby modulating the equilibrium between proliferation, differentiation and metastatic invasion in human cancer cells. On the basis of our results we think that simvastatin, alone or in combination with conventional drugs, may have a possible role in cancer therapy.

Nitric oxide-induced endoplasmic reticulum stress activates the expression of cargo receptor proteins and alters the glycoprotein transport to the Golgi complex.

The endoplasmic reticulum Golgi intermediate compartment 53 protein recycles continuously between the endoplasmic reticulum and the Golgi complex and ensures the anterograde transport of specific glycoproteins with the assistance of the Multiple Clotting Factor Deficiency adaptor protein. Therefore, to analyze the effect of the endoplasmic reticulum stress on the secretory pathway beyond the endoplasmic reticulum, we analyzed the expression of both proteins in J774 macrophages incubated with the nitric oxide donor DETA NONOate or with thapsigargin. Both proteins accumulated progressively, by a transcriptional mechanism, in response to these inducers. Nitric oxide also induced a higher level of calreticulin and glucose regulated 78 protein, two endoplasmic reticulum proteins controlled by the unfolded protein response. Interestingly, nitric oxide induced the processing of the activating transcription factor 6alpha of the unfolded protein response, while thapsigargin also induced the activation of the transcription factor X-box Binding Protein 1. In addition, we showed that the accumulation of both transporters occurred simultaneously with the activation of endoplasmic reticulum-stress-dependent apoptosis, suggesting that these proteins may participate in the events that will eventually decide the fate of the cell. Induction of endoplasmic reticulum stress affected the rate of anterograde transport of a reporter glycoprotein, indicating that the endoplasmic reticulum to Golgi transport is remarkably impaired. Our results indicate that increased levels of cargo receptor proteins might have a function either in the quality control of protein folding in the endoplasmic reticulum or in the homeostasis of the intermediate compartment and Golgi complex during cell stress.