Differential functions of PKC-delta and PKC-zeta in cisplatin response of normal and transformed thyroid cells

We investigated the effects of cisplatin (cisPt) in normal PC Cl3 and in transformed and tumourigenic PC E1Araf cells. cisPt cytotoxicity was higher in PC Cl3 than in PC E1Araf cells. In both cell lines, cisPt provoked the ERK1/2 phosphorylation; this was unaltered by G?6976, a conventional PKC inhibitor, whilst it was blocked by low doses (0.1 microM) or high doses (10 microM) of GF109203X, an inhibitor of all PKC isozymes, in PC Cl3 and in PC E1Araf cells, respectively. In PC E1Araf, the cisPt-provoked ERK phosphorylation was also blocked by the use of a myristoylated PKC-zeta pseudosubstrate peptide. Conversely, in PC Cl3 the cisPt-provoked ERK phosphorylation was blocked by the use of rottlerin, a PKC-delta inhibitor. Results show that cisPt activates both PKC (the -delta and the -zeta isozymes in PC Cl3 and in PC E1Araf cells, respectively) and ERK in association with prolonged survival of thyroid cell lines.     

GUCY2C opposes systemic genotoxic tumorigenesis by regulating AKT-dependent intestinal barrier integrity

The barrier separating mucosal and systemic compartments comprises epithelial cells, annealed by tight junctions, limiting permeability. GUCY2C recently emerged as an intestinal tumor suppressor coordinating AKT1-dependent crypt-villus homeostasis. Here, the contribution of GUCY2C to barrier integrity opposing colitis and systemic tumorigenesis is defined. Mice deficient in GUCY2C (Gucy2c(-/-)) exhibited barrier hyperpermeability associated with reduced junctional proteins. Conversely, activation of GUCY2C in mice reduced barrier permeability associated with increased junctional proteins. Further, silencing GUCY2C exacerbated, while activation reduced, chemical barrier disruption and colitis. Moreover, eliminating GUCY2C amplified, while activation reduced, systemic oxidative DNA damage. This genotoxicity was associated with increased spontaneous and carcinogen-induced systemic tumorigenesis in Gucy2c(-/-) mice. GUCY2C regulated barrier integrity by repressing AKT1, associated with increased junction proteins occludin and claudin 4 in mice and Caco2 cells in vitro. Thus, GUCY2C defends the intestinal barrier, opposing colitis and systemic genotoxicity and tumorigenesis. The therapeutic potential of this observation is underscored by the emerging clinical development of oral GUCY2C ligands, which can be used for chemoprophylaxis in inflammatory bowel disease and cancer.     

Tacrine based human cholinesterase inhibitors: synthesis of peptidic-tethered derivatives and their effect on potency and selectivity

Tacrine based reversible inhibitors of cholinesterases (ChEIs) containing peptidic tethers were synthesized to interact with specific regions at the gorge level, and their potency was determined with human (h) acetylcholinesterase and butyrylcholinesterase. Analogues 3i,j and 3l,m were identified as promising hits and may pave the way for the development of a new series of tacrine based enzyme selective hChEIs.     

5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2) suppresses fMLP-mediated respiratory burst in human neutrophils by inhibiting phosphatidylinositol 3-kinase activity

Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4H-chromen-4-one (MSF-2), a lignan extracted from the fruit of Melicope Semecarprifolia, on fMLP-induced respiratory burst in human neutrophils and suggests a possible therapeutic approach to ameliorate disease associated with neutrophil hyperactivation. MSF-2 inhibited fMLP-induced neutrophil superoxide anion production, cathepsin G release and migration in human neutrophils isolated from healthy volunteers, reflecting inhibition of phosphatidylinositol 3-kinase (PI3K) activation. Specifically, PI3K/AKT activation results in migration, degranulation and superoxide anion production in neutrophils. MSF-2 suppresses PI3K activation and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production, and consequently inhibits downstream activation of PDK1 and AKT. Further, PI3K also stimulates respiratory burst via PLC-dependent elevation of intracellular calcium. MSF-2 reduces fMLP-mediated PLCγ2 activation and intracellular calcium accumulation notably through extracellular calcium influx in a PI3K and PLC-dependent manner. However, MSF-2 is not a competitive or allosteric antagonist of fMLP. Additionally, in an in vivo study, MSF-2 prevents fMLP-induced neutrophil infiltration and inflammation in mice. In conclusion, MSF-2 opposes fMLP-mediated neutrophil activation and inflammation by inhibiting PI3K activation and subsequent activation of AKT and PLCγ2.     

[Pt(O,O’-acac)(γ-acac)(DMS)] Alters SH-SY5Y Cell Migration and Invasion by the Inhibition of Na+/H+ Exchanger Isoform 1 Occurring through a PKC-ε/ERK/mTOR Pathway

We previously showed that [Pt(O,O’-acac)(γ-acac)(DMS)] ([Pt(acac)₂(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)₂(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)₂(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na⁺-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)₂(DMS)] decreases NHE1 activity, inhibites cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)₂(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)₂(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.     

Transcriptome-based identification of new anti-anti-inflammatory and vasodilating properties of the n-3 fatty acid docosahexaenoic acid in vascular endothelial cell under proinflammatory conditions

ScopeHigh intakes of n-3 fatty acids exert anti-inflammatory effects and cardiovascular protection, but the underlying molecular basis is incompletely defined. By genome-wide analysis we searched for novel effects of docosahexaenoic acid (DHA) on gene expression and pathways in human vascular endothelium under pro-inflammatory conditions.ConclusionsEndothelial exposure to DHA regulates novel genes and related pathways. Such unbiased identification should increase our understanding of mechanisms by which n-3 fatty acids affect human diseases.     

Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

Adiponectin, an adipocyte-derived insulin-sensitizing and anti-inflammatory hormone, is suppressed in obesity through mechanisms involving chronic inflammation and oxidative stress. Olive oil consumption is associated with beneficial cardiometabolic actions, with possible contributions from the antioxidant phenol hydroxytyrosol (HT) and the monounsaturated fatty acid oleic acid (OA, 18:1n-9 cis), both possessing anti-inflammatory and vasculo-protective properties. We determined the effects of HT and OA, alone and in combination, on adiponectin expression in human and murine adipocytes under pro-inflammatory conditions induced by the cytokine tumor necrosis factor(TNF)-α. We used human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes and murine 3T3-L1 adipocytes as cell model systems, and pretreated them with 1-100 μmol/L OA, 0.1-20 μmol/L HT or OA plus HT combination before stimulation with 10 ng/mL TNF-α. OA or HT significantly (P<0.05) prevented TNF-α-induced suppression of total adiponectin secretion (by 42% compared with TNF-α alone) as well as mRNA levels (by 30% compared with TNF-α alone). HT and OA also prevented—by 35%—TNF-α-induced downregulation of peroxisome proliferator-activated receptor PPARγ. Co-treatment with HT and OA restored adiponectin and PPARγ expression in an additive manner compared with single treatments. Exploring the activation of JNK, which is crucial for both adiponectin and PPARγ suppression by TNF-α, we found that HT and OA additively attenuated TNF-α-stimulated JNK phosphorylation (up to 55% inhibition). In conclusion, the virgin olive oil components OA and HT, at nutritionally relevant concentrations, have additive effects in preventing adiponectin downregulation in inflamed adipocytes through an attenuation of JNK-mediated PPARγ suppression.     

Antioxidant and anti-inflammatory properties of tomato fruits synthesizing different amounts of stilbenes

Resveratrol, a plant phenolic compound, is found in grapes and red wine, but is not widely distributed in other common food sources. The pathway for resveratrol biosynthesis is well characterized. Metabolic engineering of this compound has been achieved in tomato plants ( Lycopersicon esculentum Mill.) in order to improve their nutritional value. Tomato plants synthesizing resveratrol were obtained via the heterologous expression of a grape ( Vitis vinifera L.) cDNA encoding for the enzyme stilbene synthase (StSy), under the control of the fruit-specific promoter TomLoxB. The resulting LoxS transgenic plants accumulated trans -resveratrol and trans -piceid, in particular in the skin of the mature fruits. Quantitative analyses carried out on LoxS fruits were compared with those of a tomato line constitutively expressing the stsy gene (35SS). The LoxS fruits contained levels of trans -resveratrol that were 20-fold lower than those previously reported for the 35SS line. The total antioxidant capability and ascorbate content in transformed fruits were also evaluated, and a significant increase in both was found in the LoxS and 35SS lines. These results could explain the higher capability of transgenic fruits to counteract the pro-inflammatory effects of phorbol ester in monocyte–macrophages via the inhibition of induced cyclo-oxygenase-2 enzyme.     

Novel peptidomimetics as BACE-1 inhibitors: Synthesis,molecular modeling,and biological studies

Aiming at identifying new scaffolds for BACE-1 inhibition devoid of the pharmacokinetic drawbacks of peptide-like structures, we investigated a series of novel peptidomimetics based on a 1,4-benzodiazepine (BDZ) core 1a–h and their seco-analogues 2a–d. We herein discuss synthesis, molecular modeling and in vitro studies which, starting from 1a, led to the seco-analogues (R)-2c and (S)-2d endowed with BACE-1 inhibition properties in the micromolar range both on the isolated enzyme and in cellular studies. These data can encourage to pursue these analogues as hits for the development of a new series of BACE-1 inhibitors active on whole-cells.