Three Arachidonoylamide Derivatives Inhibit Pro-Inflammatory Genes Expression by Modulating NF-kB and AP1 Activities.

BACKGROUND: Since the anti-inflammatory activity of arachidonic acid derivatives was previously reported, we synthesized three new amide derivatives of arachidonic acid (AA-Ds) and tested their anti-inflammatory effects on an in vitro skin inflammation model. Aim of our study was to find derivatives of natural compounds able to down regulate inflammatory signal transduction pathway. METHODS: Human keratinocytes cell line (HaCaT) was cultured and induced by cytokines in the presence of AA-Ds. Cytokines administration elicited an inflammatory response mediated by NF-kB and STAT-1 activation that induced proinflammatory genes expression. RESULTS: By real time PCR we found that 24 hours after induction all AA-Ds significantly inhibit inducible Nitric Oxide Synthase (iNOS), TNFα, Inhibitor α of NF-kB, chemokine (C-X-C motif) ligand 9 and 10 genes expression. We analyzed their molecular effects in particular on the iNOS gene expression. Since iNOS transcript half-life did not change with AA-Ds treatment, we excluded a prominent role of post-transcriptional regulation for this gene and focused our attention on its transcriptional regulation. Starting three-five hours after cytokines induction, HaCaT cells, pretreated with each compound, showed inhibition of both NF-kB DNA-binding and NF-kB p65-Ser536 phosphorylation. STAT1 activation was inhibited only by AA-D4 derivative. To explain why the inhibition of iNOS expression began late after induction we analyzed activities of others key transcription factors. AA-Ds treatment elicited early increases of AP1 DNA binding as well as c-Jun, c-Fos and Fra-1 mRNA levels. Our data agree with the repressing effects of AP1 on human iNOS promoter previously described in others cell systems (Kleinert et al.). CONCLUSION: AA-Ds shown to be good candidates as inhibitors of several pro-inflammatory genes induction and our study provides indications for their possible use as new antiinflammatory drugs.

St. John's wort extract and hyperforin protect rat and human pancreatic islets against cytokine toxicity.

The extract of Hypericum perforatum (St. John's wort, SJW) and its component hyperforin (HPF) were previously shown to inhibit cytokine-induced activation of signal transducer and activator of transcription-1 and nuclear factor κB and prevent apoptosis in a cultured ß-cell line. Objective of this study was to assess the protection exerted by SJW and HPF on isolated rat and human islets exposed to cytokines in vitro. Functional, ultrastructural, biomolecular and cell death evaluation studies were performed. In both rat and human islets, SJW and HPF counteracted cytokine-induced functional impairment and down-regulated mRNA expression of pro-inflammatory target genes, such as iNOS, CXCL9, CXCL10, COX2. Cytokine-induced NO production from cultured islets, evaluated by nitrites measurement in the medium, was significantly reduced in the presence of the vegetal compounds. Noteworthy, the increase in apoptosis and necrosis following 48-h exposure to cytokines was fully prevented by SJW and partially by HPF. Ultrastructural morphometric analysis in human islets exposed to cytokines for 20 h showed that SJW or HPF avoided early ß-cell damage (e.g., mitochondrial alterations and loss of insulin granules). In conclusion, SJW compounds protect rat and human islets against cytokine effects by counteracting key mechanisms of cytokine-mediated ß-cell injury and represent promising pharmacological tools for prevention or limitation of ß-cell dysfunction and loss in type 1 diabetes.

Phenylpropanoid glycosides from plant cell cultures induce heme oxygenase 1 gene expression in a human keratinocyte cell line by affecting the balance of NRF2 and BACH1 transcription factors.

Phenylpropanoids have several highly significant biological properties in both plants and animals. Four phenylpropanoid glycosides (PPGs), verbascoside (VB), forsythoside B (FB), echinacoside (EC) and campneoside I (CP), were purified and tested for their capability to activate NRF2 and induce phase II cytoprotective enzymes in a human keratinocyte cell line (HaCaT). All four substances showed similar strong antioxidant and radical-scavenging activities as determined by diphenylpicrylhydrazyl assay. Furthermore, in HaCaT cells, FB and EC are strong activators of NRF2, the nuclear transcription factor regulating many phase II detoxifying and cytoprotective enzymes, such as heme oxygenase 1 (HMOX1). In HaCaT cells, FB and EC (200 µM) induced nuclear translocation of NRF2 protein after 24 h and reduced nuclear protein levels of BACH1, a repressor of the antioxidant response element. FB and EC greatly HMOX1 mRNA levels by more than 40-fold in 72 h. Cytoplasmic HMOX1 protein levels were also increased at 48 h after treatment. VB was less active compared to FB and EC, and CP was slightly active only at later times of treatment. We suggest that hydroxytyrosol (HYD) could be a potential bioactive metabolite of PPGs since HYD, in equimolar amounts to PGGs, is able to both activate HO-1 transcription and modify Nrf2/Bach1 nuclear protein levels. This is in agreement with the poor activity of CP, which contains a HYD moiety modified by an O-methyl group. In conclusion, FB and EC from plant cell cultures may provide long-lasting skin protection by induction of phase II cytoprotective capabilities.

Role of mitochondrial uncoupling protein 2 in cancer cell resistance to gemcitabine.

Cancer cells exhibit an endogenous constitutive oxidative stress higher than that of normal cells, which renders tumours vulnerable to further reactive oxygen species (ROS) production. Mitochondrial uncoupling protein 2 (UCP2) can mitigate oxidative stress by increasing the influx of protons into the mitochondrial matrix and reducing electron leakage and mitochondrial superoxide generation. Here, we demonstrate that chemical uncouplers or UCP2 over-expression strongly decrease mitochondrial superoxide induction by the anticancer drug gemcitabine (GEM) and protect cancer cells from GEM-induced apoptosis. Moreover, we show that GEM IC(50) values well correlate with the endogenous level of UCP2 mRNA, suggesting a critical role for mitochondrial uncoupling in GEM resistance. Interestingly, GEM treatment stimulates UCP2 mRNA expression suggesting that mitochondrial uncoupling could have a role also in the acquired resistance to GEM. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing strongly enhances GEM-induced mitochondrial superoxide generation and apoptosis, synergistically inhibiting cancer cell proliferation. These events are significantly reduced by the addition of the radical scavenger N-acetyl-l-cysteine or MnSOD over-expression, demonstrating a critical role of the oxidative stress. Normal primary fibroblasts are much less sensitive to GEM/genipin combination. Our results demonstrate for the first time that UCP2 has a role in cancer cell resistance to GEM supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to GEM treatment.