Daniela De Stefano

The IKK complex contributes to the induction of autophagy

In response to stress, cells start transcriptional and transcription‐independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IκB kinase) complex that is best known for its essential role in the activation of the transcription factor NF‐κB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation‐induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF‐κB or ablation of the p65/RelA NF‐κB subunit failed to suppress IKK‐induced autophagy, indicating that IKK can promote the autophagic pathway in an NF‐κB‐independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK‐β suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

Cannabidiol inhibits inducible nitric oxide synthase protein expression and nitric oxide production in β-amyloid stimulated PC12 neurons through p38 MAP kinase and NF-κB involvement

In view of the pro-inflammatory scenario observed in Alzheimers disease, in the recent years anti-inflammatory drugs have been proposed as potential therapeutic agents. We have previously shown that cannabidiol, the main non-psychotropic component from Cannabis sativa, possess a variegate combination of anti-oxidant and anti-apoptotic effects that protect PC12 cells from Abeta toxicity. In parallel, cannabidiol has been described to have anti-inflammatory properties in acute models of inflammation but the possible inhibitory effect of cannabidiol on iNOS protein expression and nitrite production in the nitrosative stress induced by Abeta in neuronal cell-line is un-investigated. Stimulation of differentiated PC12 cells with Abeta (1-42) (1 microg/mL) for 36 h caused a significant increase of nitrite production, compared to un-stimulated cells, that was inhibited in a concentration-dependent manner by both the non-selective iNOS inhibitor, L-NAME (0.3-30 microM), and, at higher extent, by the selective iNOS inhibitor SMT (0.3-30 microM). CBD (10(-6) to 10(-4) M) inhibited both nitrite production and iNOS protein expression induced by Abeta (1-42). Cannabidiol effect was mediated through the inhibition of phosphorylated form of p38 MAP kinase and transcription factor nuclear factor-kappaB activation in a concentration-dependent manner. The here reported data increases our knowledge about the possible neuroprotective mechanism of cannabidiol, highlighting the importance of this compound to inhibit beta-amyloid induced neurodegeneration, in view of its low toxicity in humans.

Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation

We investigated the effect of hydroxytyrosol (HT), a phenolic compound from virgin olive oil, on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in J774 murine macrophages stimulated with lipopolysaccharide (LPS). Incubation of cells with LPS caused an increase in iNOS and COX-2 mRNA and protein level as well as ROS generation, which was prevented by HT. In addition, HT blocked the activation of nuclear factor-κB (NF-κB), signal transducer and activator of transcription-1α (STAT-1α) and interferon regulatory factor-1 (IRF-1). These results, showing that HT down-regulates iNOS and COX-2 gene expression by preventing NF-κB, STAT-1α and IRF-1 activation mediated through LPS-induced ROS generation, suggest that it may represent a non-toxic agent for the control of pro-inflammatory genes.

Nanocarriers for topical administration of resveratrol: A comparative study

The trans-resveratrol (t-res), a non-flavonoid polyphenol extracted from different plants, has recently earned interest for application on the skin for different applications. In this work, the potential of nanocarriers, namely transfersomes and ethanol-containing vesicles, to deliver t-res into/through the skin was investigated. Thus, transfersomes with different surfactants, namely polysorbate 80 (Tw80), sodium cholate (SC) and sodium deossicholate (SDC) and ethanol-containing vesicles with different lipid composition, namely soy phosphatidylcholine (SPC) and cholesterol (chol), encapsulating t-res were prepared and characterized. The nanocarriers had a mean diameter ranging between 83 and 116 nm with a high t-res encapsulation efficiency (≥ 70%). Moreover, cytotoxicity as well as the inhibition of production of reactive oxygen species (ROS) and lipid peroxidation, following incubation of H(2)O(2)-stimulated human keratinocyte (HaCaT) with t-res, as free or encapsulated into the nanocarriers, were investigated. Only blank nanocarriers containing Tw80 or ethanol were cytotoxic and led to increase of ROS, but this effect was not observed when using nanocarriers encapsulating t-res. Finally, permeation studies on porcine skin carried out on Franz diffusion cells, showed that only ethanol-containing vesicles based SPC were able to promote t-res permeation through the skin.

Nanomaterials Toxicity and Cell Death Modalities

In the last decade, the nanotechnology advancement has developed a plethora of novel and intriguing nanomaterial application in many sectors, including research and medicine. However, many risks have been highlighted in their use, particularly related to their unexpected toxicity in vitro and in vivo experimental models. This paper proposes an overview concerning the cell death modalities induced by the major nanomaterials.

IKK connects autophagy to major stress pathways

Cells respond to stress by activating cytoplasmic mechanisms as well as transcriptional programs that can lead to adaptation or death. Autophagy represents an important cytoprotective response that is regulated by both transcriptional and transcription-independent pathways. NFκB is perhaps the transcription factor most frequently activated by stress, and has been ascribed with either pro- or anti-autophagic functions, depending on the cellular context. Our results demonstrate that activation of the IKK (IκB kinase) complex, which is critical for the stress-elicited activation of NF-κB, is sufficient to promote autophagy independent of NFκB, and that IKK is required for the optimal induction of autophagy by both physiological and pharmacological autophagic triggers.

Nuclear Factor-κB Regulates Inflammatory Cell Apoptosis and Phagocytosis in Rat Carrageenin-Sponge Implant Model

In the present study we investigated whether apoptosis and phagocytosis are regulated by nuclear factor (NF)-kappaB in a model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges elicited an inflammatory response, characterized by a time-related increase of leukocyte infiltration into the sponge and tissue formation, which was inhibited by simultaneous injection of wild-type oligodeoxynucleotide decoy to NF-kappaB. Molecular and morphological analysis performed on infiltrated cells demonstrated: 1) an inhibition of NF-kappaB/DNA binding activity; 2) an increase of polymorphonuclear leukocyte apoptosis correlated either to an increase of p53 or Bax and decrease of Bcl-2 protein expression; and 3) an increase of phagocytosis of apoptotic polymorphonuclear leukocytes by macrophages associated with an increase of transforming growth factor-beta1 and decrease of tumor necrosis factor-alpha as well as nitrite/nitrate production. Our results, showing that blockade of NF-kappaB by oligodeoxynucleotide decoy increases inflammatory cell apoptosis and phagocytosis, may contribute to lead to new insights into the mechanisms governing the inflammatory process.

Local administration of WIN 55,212-2 reduces chronic granuloma-associated angiogenesis in rat by inhibiting NF-κB 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-κB) 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 λ-carrageenin-soaked sponge implant in rat. Our results show that local administration of WIN 55,212-2, a CB1/CB2 agonist, given daily or at time of implantation significantly decreased weight and neo-angiogenesis in granuloma tissue and inhibited nuclear factor-kappa B (NF-κB)/DNA binding that was associated with a reduced inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and protein expression. Also, arachidonyl-2-chloroethylamide (ACEA), a CB1 selective agonist, and JWH-015, a CB2 selective agonist, exhibited the same effects that were reversed by SR141716-A and SR144528, respectively, CB1 and CB2 selective antagonists. These results indicate that CBs given locally may represent a potential therapeutic tool in controlling chronic inflammation avoiding psychotropic effects.

Enhanced intracellular uptake and inhibition of NF‐κB activation by decoy oligonucleotide released from PLGA microspheres

Nuclear factor‐κB (NF‐κB) transcription factor regulates the expression of genes involved in immune response and inflammation. NF‐κB activity can be efficiently inhibited by double‐stranded oligodeoxynucleotides (ODNs). In the present study, we investigated the potential of poly(DL‐lactic‐co‐glycolic acid) (PLGA) microspheres as delivery system for an ODN against NF‐κB in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS).

Gliadin increases iNOS gene expression in interferon-γ-stimulated RAW 264.7 cells through a mechanism involving NF-κB

Nitric oxide (NO) plays an important role in the pathogenesis of the histological changes seen in coeliac disease. We have investigated the effect of peptic-tryptic digest of gliadin (Pt-G) and gliadin (G) on inducible nitric oxide synthase (iNOS) protein expression in RAW 264.7 macrophages stimulated with interferon-γ (IFN-γ). Pt-G and G enhanced in a concentration and time-dependent manner NO production by IFN-γ-stimulated RAW 264.7 cells. The increase of iNOS protein expression was correlated with NF-κB/DNA binding activity and occurred at transcriptional level. Pyrrolidine dithiocarbamate and N-α-para-tosyl-L-lysine chloromethyl ketone, two known inhibitors of NF-κB activation, decreased significantly NO production and iNOS protein expression as well as NF-κB/DNA binding activity. Our results show that the effect of Pt-G and G on enhancement of iNOS protein expression in IFN-γ-treated RAW 264.7 cells is mainly mediated through NF-κB and suggest that blockage of NF-κB activation reduces enhancing effect of gluten on NO production in inflamed mucosa of coeliac patients.