In the present study we investigated in the reverse passive Arthus reaction elicited in the rat s... more In the present study we investigated in the reverse passive Arthus reaction elicited in the rat skin the anti-inflammatory effect of double-stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type-, but not mutant-decoy ODN, dose-dependently reduced both plasma leakage and neutrophil infiltration in rat skin. Molecular analysis performed on soft tissue obtained from rat skin demonstrated: (1) an inhibition of NF-kappaB/DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, as well as ammonium pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB activation, exhibited a similar profile of activity of decoy ODN. Our results indicate that decoy ODN, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate immune reactions.
We investigated the effect of PS ( 1) on nitrite and reactive oxygen species (ROS) production in ... more We investigated the effect of PS ( 1) on nitrite and reactive oxygen species (ROS) production in J774 macrophages stimulated with bacterial lipopolysaccharide (LPS) for 24 h. PS ( 1) inhibited in a concentration-dependent manner nitrite and ROS production as well as inducible nitric oxide synthase (iNOS) protein expression induced by LPS. Incubation of cells with PS ( 1) determined a significant decrease of nuclear factor-kappaB (NF-kappaB)/DNA binding activity which was correlated with a marked reduction of iNOS mRNA levels. These results show that PS ( 1) inhibits NF-kappaB activation and iNOS gene expression by preventing the reactive species production and suggest a role for this compound in controlling oxidative stress and/or inflammation.
Current Opinion in Molecular Therapeutics, Apr 1, 2010
Molecular therapy is emerging as a potential strategy for the treatment of inflammatory diseases.... more Molecular therapy is emerging as a potential strategy for the treatment of inflammatory diseases. Decoy oligonucleotides (ONs) against NFkappaB, an inducible transcription factor that plays a critical role in several inflammatory/immune diseases, can specifically block the transcriptional activity of this transcription factor. The therapeutic potential of such decoy ONs has been investigated in several chronic inflammatory-based diseases. However, the clinical use of decoy ONs is strongly hampered by several issues, including low bioavailability, a short half-life and limited intracellular uptake. Both chemical modifications to ONs and the use of delivery systems have been investigated in order to overcome these limitations. This review summarizes the most meaningful studies on the preclinical and clinical application of decoy ONs against NFkappaB in different diseases, and highlights successful strategies that have overcome the pharmacokinetic issues associated with ONs.
We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia ... more We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia linteiformis, on inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 microgram/ml) caused an increase of both iNOS and COX-2 protein expression, which was prevented in a concentration-dependent fashion by cyclolinteinone (12.5, 25 and 50 microM). Electrophoretic mobility-shift assay indicated that cyclolinteinone blocked the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor necessary for either iNOS or COX-2 induction. Cyclolinteinone also blocked disappearance of I(kappa)B-alpha from cytosolic fraction and nuclear translocation of NF-kappaB subunits p50 and p65. These results show that cyclolinteinone down-regulates iNOS and COX-2 protein expression by inhibiting NF-kappaB activation and suggest that it may represent a novel anti-inflammatory compound capable of controlling the excessive production of prostaglandins and nitric oxide occurring in several inflammatory diseases.
We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible ni... more We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible nitric oxide synthase expression in carrageenin-induced rat pleurisy. Injection of 0.2 ml of 1% lambda-carrageenin into the pleural cavity of male Wistar rats caused after 6 h: (a) exudate formation and leukocyte migration into the pleural cavity; (b) inducible NO synthase protein expression and accumulation of NO2- plus NO3- in pleural exudate; (c) increase in p50/p65 nuclear level as well as NF-kappaB/DNA binding activity. Treatment of rats with pyrrolidine dithiocarbamate (10, 30, and 100 mg/kg) and N-alpha-p-tosyl-L-lysine chloromethylketone (30 mg/kg), two inhibitors of NF-kappaB activation, given subcutaneously concomitantly with carrageenin, caused a significant inhibition of all the parameters assayed. These results suggest that in carrageenin-induced rat pleurisy the activation of NF-kappaB plays a key role in inducible NO synthase protein expression and in the development of inflammatory response.
Pharmacological Research the Official Journal of the Italian Pharmacological Society, 1992
The anti-inflammatory effect of glucocorticoids depends, at least in part, on the induction of tw... more The anti-inflammatory effect of glucocorticoids depends, at least in part, on the induction of two regulatory proteins, lipocortin and vasocortin, both preventing the release of inflammatory mediators. Lipocortin inhibits phospholipase A2 (PLA2) and therefore reduces arachidonic acid metabolites formation. Vasocortin inhibits histamine release from mast cells. Lipocortin and vasocortin may be regarded as the first two identified members of the (perhaps greater) family of glucocorticoid-induced proteins.
We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible ni... more We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible nitric oxide synthase expression in carrageenin-induced rat pleurisy. Injection of 0.2 ml of 1% lambda-carrageenin into the pleural cavity of male Wistar rats caused after 6 h: (a) exudate formation and leukocyte migration into the pleural cavity; (b) inducible NO synthase protein expression and accumulation of NO2- plus NO3- in pleural exudate; (c) increase in p50/p65 nuclear level as well as NF-kappaB/DNA binding activity. Treatment of rats with pyrrolidine dithiocarbamate (10, 30, and 100 mg/kg) and N-alpha-p-tosyl-L-lysine chloromethylketone (30 mg/kg), two inhibitors of NF-kappaB activation, given subcutaneously concomitantly with carrageenin, caused a significant inhibition of all the parameters assayed. These results suggest that in carrageenin-induced rat pleurisy the activation of NF-kappaB plays a key role in inducible NO synthase protein expression and in the development of inflammatory response.
Naunyn Schmiedeberg S Archives of Pharmacology, Aug 1, 2001
Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amoun... more Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amounts of prostaglandins (PGs) generated by the inducible isoform of cyclooxygenase (COX-2). Nitric oxide (NO), a pleiotropic free radical, has been demonstrated to modulate the release of a broad range of inflammatory mediators, amongst these PGs. In the present study we investigated the molecular mechanism by which NO affects cyclooxygenase pathway. Incubation of J774 cells with LPS caused an increase of prostaglandin E2 production and COX-2 protein expression which was prevented in a concentration-dependent fashion by pre-incubating cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating agents. Electrophoretic mobility shift assay indicated that both NO-generating agents blocked LPS-induced activation of nuclear factor-kappaB (NF-kappaB) by increasing IkappaB-alpha protein expression and blocking nuclear translocation of NF-kappaB subunits p50 and p65. SNP and GSNO also inhibited nuclear factor-interleukin-6 (NF-IL6) activation. These results show for the first time that SNP and GSNO down-regulate LPS-induced COX-2 expression by inhibiting NF-kappaB and NF-IL6 activation and suggest a negative feed-back mechanism that may be important for limiting excessive or prolonged PGs production in pathological events.
Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amoun... more Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amounts of prostaglandins (PGs) generated by the inducible isoform of cyclooxygenase (COX-2). Nitric oxide (NO), a pleiotropic free radical, has been demonstrated to modulate the release of a broad range of inflammatory mediators, amongst these PGs. In the present study we investigated the molecular mechanism by which NO affects cyclooxygenase pathway. Incubation of J774 cells with LPS caused an increase of prostaglandin E2 production and COX-2 protein expression which was prevented in a concentration-dependent fashion by pre-incubating cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating agents. Electrophoretic mobility shift assay indicated that both NO-generating agents blocked LPS-induced activation of nuclear factor-kappaB (NF-kappaB) by increasing IkappaB-alpha protein expression and blocking nuclear translocation of NF-kappaB subunits p50 and p65. SNP and GSNO also inhibited nuclear factor-interleukin-6 (NF-IL6) activation. These results show for the first time that SNP and GSNO down-regulate LPS-induced COX-2 expression by inhibiting NF-kappaB and NF-IL6 activation and suggest a negative feed-back mechanism that may be important for limiting excessive or prolonged PGs production in pathological events.
In the present study we investigated in the reverse passive Arthus reaction elicited in the rat s... more In the present study we investigated in the reverse passive Arthus reaction elicited in the rat skin the anti-inflammatory effect of double-stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type-, but not mutant-decoy ODN, dose-dependently reduced both plasma leakage and neutrophil infiltration in rat skin. Molecular analysis performed on soft tissue obtained from rat skin demonstrated: (1) an inhibition of NF-kappaB/DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, as well as ammonium pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB activation, exhibited a similar profile of activity of decoy ODN. Our results indicate that decoy ODN, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate immune reactions.
We investigated the effect of PS ( 1) on nitrite and reactive oxygen species (ROS) production in ... more We investigated the effect of PS ( 1) on nitrite and reactive oxygen species (ROS) production in J774 macrophages stimulated with bacterial lipopolysaccharide (LPS) for 24 h. PS ( 1) inhibited in a concentration-dependent manner nitrite and ROS production as well as inducible nitric oxide synthase (iNOS) protein expression induced by LPS. Incubation of cells with PS ( 1) determined a significant decrease of nuclear factor-kappaB (NF-kappaB)/DNA binding activity which was correlated with a marked reduction of iNOS mRNA levels. These results show that PS ( 1) inhibits NF-kappaB activation and iNOS gene expression by preventing the reactive species production and suggest a role for this compound in controlling oxidative stress and/or inflammation.
Current Opinion in Molecular Therapeutics, Apr 1, 2010
Molecular therapy is emerging as a potential strategy for the treatment of inflammatory diseases.... more Molecular therapy is emerging as a potential strategy for the treatment of inflammatory diseases. Decoy oligonucleotides (ONs) against NFkappaB, an inducible transcription factor that plays a critical role in several inflammatory/immune diseases, can specifically block the transcriptional activity of this transcription factor. The therapeutic potential of such decoy ONs has been investigated in several chronic inflammatory-based diseases. However, the clinical use of decoy ONs is strongly hampered by several issues, including low bioavailability, a short half-life and limited intracellular uptake. Both chemical modifications to ONs and the use of delivery systems have been investigated in order to overcome these limitations. This review summarizes the most meaningful studies on the preclinical and clinical application of decoy ONs against NFkappaB in different diseases, and highlights successful strategies that have overcome the pharmacokinetic issues associated with ONs.
We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia ... more We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia linteiformis, on inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 microgram/ml) caused an increase of both iNOS and COX-2 protein expression, which was prevented in a concentration-dependent fashion by cyclolinteinone (12.5, 25 and 50 microM). Electrophoretic mobility-shift assay indicated that cyclolinteinone blocked the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor necessary for either iNOS or COX-2 induction. Cyclolinteinone also blocked disappearance of I(kappa)B-alpha from cytosolic fraction and nuclear translocation of NF-kappaB subunits p50 and p65. These results show that cyclolinteinone down-regulates iNOS and COX-2 protein expression by inhibiting NF-kappaB activation and suggest that it may represent a novel anti-inflammatory compound capable of controlling the excessive production of prostaglandins and nitric oxide occurring in several inflammatory diseases.
We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible ni... more We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible nitric oxide synthase expression in carrageenin-induced rat pleurisy. Injection of 0.2 ml of 1% lambda-carrageenin into the pleural cavity of male Wistar rats caused after 6 h: (a) exudate formation and leukocyte migration into the pleural cavity; (b) inducible NO synthase protein expression and accumulation of NO2- plus NO3- in pleural exudate; (c) increase in p50/p65 nuclear level as well as NF-kappaB/DNA binding activity. Treatment of rats with pyrrolidine dithiocarbamate (10, 30, and 100 mg/kg) and N-alpha-p-tosyl-L-lysine chloromethylketone (30 mg/kg), two inhibitors of NF-kappaB activation, given subcutaneously concomitantly with carrageenin, caused a significant inhibition of all the parameters assayed. These results suggest that in carrageenin-induced rat pleurisy the activation of NF-kappaB plays a key role in inducible NO synthase protein expression and in the development of inflammatory response.
Pharmacological Research the Official Journal of the Italian Pharmacological Society, 1992
The anti-inflammatory effect of glucocorticoids depends, at least in part, on the induction of tw... more The anti-inflammatory effect of glucocorticoids depends, at least in part, on the induction of two regulatory proteins, lipocortin and vasocortin, both preventing the release of inflammatory mediators. Lipocortin inhibits phospholipase A2 (PLA2) and therefore reduces arachidonic acid metabolites formation. Vasocortin inhibits histamine release from mast cells. Lipocortin and vasocortin may be regarded as the first two identified members of the (perhaps greater) family of glucocorticoid-induced proteins.
We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible ni... more We studied the involvement of nuclear factor-kappaB (NF-kappaB) in the regulation of inducible nitric oxide synthase expression in carrageenin-induced rat pleurisy. Injection of 0.2 ml of 1% lambda-carrageenin into the pleural cavity of male Wistar rats caused after 6 h: (a) exudate formation and leukocyte migration into the pleural cavity; (b) inducible NO synthase protein expression and accumulation of NO2- plus NO3- in pleural exudate; (c) increase in p50/p65 nuclear level as well as NF-kappaB/DNA binding activity. Treatment of rats with pyrrolidine dithiocarbamate (10, 30, and 100 mg/kg) and N-alpha-p-tosyl-L-lysine chloromethylketone (30 mg/kg), two inhibitors of NF-kappaB activation, given subcutaneously concomitantly with carrageenin, caused a significant inhibition of all the parameters assayed. These results suggest that in carrageenin-induced rat pleurisy the activation of NF-kappaB plays a key role in inducible NO synthase protein expression and in the development of inflammatory response.
Naunyn Schmiedeberg S Archives of Pharmacology, Aug 1, 2001
Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amoun... more Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amounts of prostaglandins (PGs) generated by the inducible isoform of cyclooxygenase (COX-2). Nitric oxide (NO), a pleiotropic free radical, has been demonstrated to modulate the release of a broad range of inflammatory mediators, amongst these PGs. In the present study we investigated the molecular mechanism by which NO affects cyclooxygenase pathway. Incubation of J774 cells with LPS caused an increase of prostaglandin E2 production and COX-2 protein expression which was prevented in a concentration-dependent fashion by pre-incubating cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating agents. Electrophoretic mobility shift assay indicated that both NO-generating agents blocked LPS-induced activation of nuclear factor-kappaB (NF-kappaB) by increasing IkappaB-alpha protein expression and blocking nuclear translocation of NF-kappaB subunits p50 and p65. SNP and GSNO also inhibited nuclear factor-interleukin-6 (NF-IL6) activation. These results show for the first time that SNP and GSNO down-regulate LPS-induced COX-2 expression by inhibiting NF-kappaB and NF-IL6 activation and suggest a negative feed-back mechanism that may be important for limiting excessive or prolonged PGs production in pathological events.
Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amoun... more Stimulation of J774 macrophages with lipopolysaccharide (LPS) leads to the release of large amounts of prostaglandins (PGs) generated by the inducible isoform of cyclooxygenase (COX-2). Nitric oxide (NO), a pleiotropic free radical, has been demonstrated to modulate the release of a broad range of inflammatory mediators, amongst these PGs. In the present study we investigated the molecular mechanism by which NO affects cyclooxygenase pathway. Incubation of J774 cells with LPS caused an increase of prostaglandin E2 production and COX-2 protein expression which was prevented in a concentration-dependent fashion by pre-incubating cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating agents. Electrophoretic mobility shift assay indicated that both NO-generating agents blocked LPS-induced activation of nuclear factor-kappaB (NF-kappaB) by increasing IkappaB-alpha protein expression and blocking nuclear translocation of NF-kappaB subunits p50 and p65. SNP and GSNO also inhibited nuclear factor-interleukin-6 (NF-IL6) activation. These results show for the first time that SNP and GSNO down-regulate LPS-induced COX-2 expression by inhibiting NF-kappaB and NF-IL6 activation and suggest a negative feed-back mechanism that may be important for limiting excessive or prolonged PGs production in pathological events.
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