Elisabetta Panza

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic‐regulated current (IKM), a sub‐threshold voltage‐dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of IKM in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the IKM activator retigabine (0.01–30 μmol/L; Emax = 54.80 ± 3.85%; IC50 = 0.50 ± 0.36 μmol/L). The IKM blockers tetraethylammonium (0.1–3 mmol/L) and XE‐991 (0.1–30 μmol/L) enhanced K+‐evoked [3H]DA release and prevented retigabine‐induced inhibition of depolarization‐evoked [3H]DA release. Retigabine‐induced inhibition of K+‐evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti‐KCNQ2 polyclonal antibodies, an effect prevented by antibody pre‐absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1–300 μmol/L) potentiated 9 mmol/L [K+]e‐evoked [3H]DA release (Emax = 155 ± 9.50%; EC50 = 25 ± 1.80 μmol/L). OXO (100 μmol/L)‐induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1–10 nmol/L) and abolished by the M3‐preferring antagonist 4‐diphenylacetoxy N‐methylpiperidine methiodide (1 μmol/L), but was unaffected by the M1‐selective antagonist MT‐7 (10–100 nmol/L) or by Pertussis toxin (1.5–3 μg/mL), which uncouples M2‐ and M4‐mediated responses. Finally, OXO‐induced potentiation of depolarization‐induced [3H]DA release was not additive to that produced by XE‐991 (10 μmol/L), was unaffected by retigabine (10 μmol/L), and was abolished by synaptosomal entrapment of anti‐KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, IKM channels containing KCNQ2 subunits regulate depolarization‐induced DA release and that IKM suppression is involved in the reinforcement of depolarization‐induced DA release triggered by the activation of pre‐synaptic muscarinic heteroreceptors.

Nanocapsules Based on Linear and Y-Shaped 3-Miktoarm Star-Block PEO-PCL Copolymers as Sustained Delivery System for Hydrophilic Molecules

Well-defined amphiphilic Y-shaped miktoarm star-block copolymers of PEO and PCL were synthesized by ring-opening polymerization of ε-caprolactone initiated by a PEO-bound lysine macroinitiator. The copolymers were characterized by (1)H NMR, SEC, DSC, and WAXD techniques. Separate PCL and PEO crystalline phases occur in melt-crystallized copolymers when their segmental lengths were comparable and the PCL content was ≤80 wt %. Self-assembling of these copolymers in aqueous medium led to nanoaggregates with low critical aggregation concentration values (0.35 to 1.6 mg·L(-1)) and size depending on composition. Despite the fact that copolymers were not prone to self-organize in vesicles, once processed by a novel w/o emulsion-melting-sonication technique, they gave nanocapsules with a water core and a hydrophilic surface. A macromolecular fluorescent dye was effectively loaded and released at sustained rate by optimizing nanocapsule formulation. The results demonstrate that amphiphilic block copolymers can be assembled in different kinds of nanomorphologies independently of their hydrophilic/hydrophobic balance and architecture through specifically designed preparation techniques.

EXPRESSION, LOCALIZATION, AND PHARMACOLOGICAL ROLE OF Kv7 POTASSIUM CHANNELS IN SKELETAL MUSCLE PROLIFERATION, DIFFERENTIATION AND SURVIVAL AFTER MYOTOXIC INSULTS

Changes in the expression of potassium channels regulate skeletal muscle development. The purpose of this study was to investigate the expression profile and pharmacological role of Kv7 voltage-gated potassium channels in skeletal muscle differentiation, proliferation, and survival after myotoxic insults. Transcripts for all Kv7 genes (Kv7.1–Kv7.5) were detected by polymerase chain reaction (PCR) and/or real-time PCR in murine C2C12 myoblasts; Kv7.1, Kv7.3, and Kv7.4 transcripts were up-regulated after myotube formation. Western blot experiments confirmed Kv7.2, Kv7.3, and Kv7.4 subunit expression, and the up-regulation of Kv7.3 and Kv7.4 subunits during in vitro differentiation. In adult skeletal muscles from mice and humans, Kv7.2 and Kv7.3 immunoreactivity was mainly localized at the level of intracellular striations positioned between ankyrinG-positive triads, whereas that of Kv7.4 subunits was largely restricted to the sarcolemmal membrane. In C2C12 cells, retigabine (10 μM), a specific activator of neuronally expressed Kv7.2 to Kv7.5 subunits, reduced proliferation, accelerated myogenin expression, and inhibited the myotoxic effect of mevastatin (IC50 ≈ 7 μM); all these effects of retigabine were prevented by the Kv7 channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 μM). These data collectively highlight neural Kv7 channels as significant pharmacological targets to regulate skeletal muscle proliferation, differentiation, and myotoxic effects of drugs.

Indicaxanthin from Cactus Pear Fruit Exerts Anti-Inflammatory Effects in Carrageenin-Induced Rat Pleurisy

Nutritional research has shifted recently from alleviating nutrient deficiencies to chronic disease prevention. We investigated the activity of indicaxanthin, a bioavailable phytochemical of the betalain class from the edible fruit of Opuntia ficus-indica (L. Miller) in a rat model of acute inflammation. Rat pleurisy was achieved by injection of 0.2 mL of λ-carrageenin in the pleural cavity, and rats were killed 4, 24, and 48 h later; exudates were collected to analyze inflammatory parameters, such as nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α); cells recruited in pleura were analyzed for cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) expression, and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation. Indicaxanthin (0.5, 1, or 2 μmol/kg), given orally before carrageenin, time- and dose-dependently, reduced the exudate volume (up to 70%) and the number of leukocytes recruited in the pleural cavity (up to 95%) at 24 h. Pretreatment with indicaxanthin at 2 μmol/kg inhibited the carrageenin-induced release of PGE(2) (91.4%), NO (67.7%), IL-1β (53.6%), and TNF-α (71.1%), and caused a decrease of IL-1β (34.5%), TNF-α (81.6%), iNOS (75.2%), and COX2 (87.7%) mRNA, as well as iNOS (71.9%) and COX-2 (65.9%) protein expression, in the recruited leukocytes. Indicaxanthin inhibited time- and dose- dependently the activation of NF-κB, a key transcription factor in the whole inflammatory cascade. A pharmacokinetic study with a single 2 μmol/kg oral administration showed a maximum 0.22 ± 0.02 μmol/L (n = 15) plasma concentration of indicaxanthin, with a half-life of 1.15 ± 0.11 h. When considering the high bioavailability of indicaxanthin in humans, our findings suggest that this dietary pigment has the potential to improve health and prevent inflammation-based disorders.

Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression.

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H2S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST), synthesizes H2S in the presence of the substrate 3‐mercaptopyruvate (3‐MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non‐lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H2S donors, the most active of which was diallyl trisulfide (DATS). The main pro‐apoptotic mechanisms involved were suppression of nuclear factor‐κB activity and inhibition of AKT and extracellular signal‐regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l‐cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l‐cysteine/CSE/H2S pathway is involved in melanoma progression.

Activation of pre-synaptic M-type K+ channels inhibits [ 3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (IKM) on cytoplasmic Ca2+ intracellular Ca2+concentration ([Ca2+]i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. Kv7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K+]e, the IKM activator retigabine (RT, 10 μM) inhibited [3H]d‐aspartate ([3H]d‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the IKM blocker XE‐991 (20 μM). The IKM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K+]e‐induced synaptosomal [Ca2+]i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca2+]i transients. The P/Q‐type voltage‐sensitive Ca2+channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca2+]i increase and [3H]d‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca2+]i and the resulting enhancement of [3H]d‐Asp release induced by [Ca2+]i mobilization from intracellular stores, or by store‐operated Ca2+channel activation. Collectively, the present data reveal that the pharmacological activation of IKM regulates depolarization‐induced [3H]d‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca2+]i occurring through P/Q‐type VSCCs.

Tedarenes A and B: Structural and Stereochemical Analysis of Two New Strained Cyclic Diarylheptanoids from the Marine Sponge Tedania ignis

Ring strain causes planar chirality in tedarenes A and B, two cyclic diarylheptanoids isolated from the marine sponge Tedania ignis. In both molecules, the chiral plane is an olefinic system, which is very rare among natural products. In tedarene A (1), interconversion is too fast to allow isolation of the enantiomeric atropisomers but still slow enough to cause coalescence of some (1)H and (13)C NMR signals at room temperature. In tedarene B (2), which also shows stable central and axial chirality, the two planar diastereomers are in slow equilibrium. Tedarene B is the smallest natural product with central, axial, and planar chirality in the same simple molecule. The identification of planar chirality as the difference between its conformational isomers allowed the use of theoretical prediction of the CD spectrum to determine the absolute configuration of the stereogenic carbon C-9 as well as of the biphenyl chiral axis.

ATB-346, a novel hydrogen sulfide-releasing anti-inflammatory drug, induces apoptosis of human melanoma cells and inhibits melanoma development in vivo

Inflammation plays a key role in tumor promotion and development. Indeed, cyclooxygenase-2 (COX-2) expression is strongly associated with different types of cancer. An emerging class of compounds with significant anti-inflammatory properties is the hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (H2S-NSAIDs). They consist of a traditional NSAID to which an H2S-releasing moiety is covalently attached. We have recently demonstrated that H2S donors inhibit melanoma cell proliferation. In the current study, we evaluated the potential beneficial effects of a new H2S-releasing derivative of naproxen, ATB-346 [2-(6-methoxynapthalen-2-yl)-propionic acid 4-thiocarbamoyl phenyl ester] which inhibits COX activity but also releases H2S. We used cell culture and a mouse melanoma model to evaluate the effect of ATB-346 on: i) in vitro growth of human melanoma cells; ii) in vivo melanoma development in mice. Cell culture studies demonstrated that ATB-346 reduced the in vitro proliferation of human melanoma cells and this effect was associated to induction of apoptosis and inhibition of NF-κB activation. Moreover, ATB-346 had novel Akt signaling inhibitory properties. Daily oral dosing of ATB-346 (43μmol/kg) significantly reduced melanoma development in vivo. This study shows that ATB-346, a novel H2S-NSAID, inhibits human melanoma cell proliferation by inhibiting pro-survival pathways associated with NF-κB and Akt activation. Furthermore, oral treatment with ATB-346 inhibits melanoma growth in mice. In conclusion, the combination of inhibition of cyclooxygenase and delivery of H2S by ATB-346 may offer a promising alternative to existing therapies for melanoma.

Sinularioside, a triacetylated glycolipid from the Indonesian soft coral Sinularia sp., is an inhibitor of NO release

Chemical analysis of the Indonesian soft coral Sinularia sp. (order Alcyonacea, family Alcyoniidae) afforded a known glucosylcerebroside of the sarcoehrenoside-type and sinularioside (2), a new naturally triacetylated glycolipid containing two α-D-arabinopyranosyl residues and a myristyl alcohol unit. Their complete stereostructures were solved by interpretation of MS and NMR data along with CD analysis of degradation products. Sinularioside proved to moderately inhibit LPS-induced NO release, providing interesting clues into the poorly understood structure-activity relationships for anti-inflammatory glycolipids.

Differential expression of cyclooxygenase-2 in metastatic melanoma affects progression free survival

The possible correlation between cyclooxygenase-2 (COX-2) expression and disease progression in melanoma is still a matter of debate. Analysis of COX-2 expression in 45 lymph node melanoma metastases demonstrates a significant correlation between the percent of expression and progression free survival (PFS). A positive COX-2 expression ≥10% (COX-2high), as opposite to a positive expression ≤9% (COX-2low), translated into a striking significant reduction of PFS of about 3 years. The reduction in PFS correlated neither with BRAFV600E nor with NRASQ61 expression in the analyzed samples. This concept was reinforced by the finding that tumour development in COX-2−/− mice was almost blunted. Similarly, inhibition of COX-2 protein expression in human melanoma cell lines, by using siRNAs technology as well as selective inhibition of COX-2 activity by celecoxib, reduced cellular proliferation and invasiveness. In conclusion we show that COX-2high is a negative prognostic factor in metastatic melanoma. Our study also clarifies that the uncertainty about the role of COX-2 in metastatic malignant melanoma, found in the current relevant literature, is probably due to the fact that a threshold in COX-2 expression has to be reached in order to impact on cancer malignancy. Our findings suggest that COX-2 expression may become an useful diagnostic tool in defining melanoma malignancy as well as argue for a possible therapeutic use of NSAID as add on therapy in selected cases.