Identification of plasma microRNAs as new potential biomarkers with high diagnostic power in human cutaneous melanoma.

Melanoma is a devastating disease with few therapeutic options in the advanced stage and with the urgent need of reliable biomarkers for early detection. In this context, circulating microRNAs are raising great interest as diagnostic biomarkers. We analyzed the expression profiles of 21 selected microRNAs in plasma samples from melanoma patients and healthy donors to identify potential diagnostic biomarkers. Data analysis was performed using global mean normalization and NormFinder algorithm. Linear regression followed by receiver operating characteristic analyses was carried out to evaluate whether selected plasma miRNAs were able to discriminate between cases and controls. We found five microRNAs that were differently expressed among cases and controls after Bonferroni correction for multiple testing. Specifically, miR-15b-5p, miR-149-3p, and miR-150-5p were up-regulated in plasma of melanoma patients compared with healthy controls, while miR-193a-3p and miR-524-5p were down-regulated. Receiver operating characteristic analyses of these selected microRNAs provided area under the receiver operating characteristic curve values ranging from 0.80 to 0.95. Diagnostic value of microRNAs is improved when considering the combination of miR-149-3p, miR-150-5p, and miR-193a-3p. The triple classifier had a high capacity to discriminate between melanoma patients and healthy controls, making it suitable to be used in early melanoma diagnosis.

Cytotoxic Activity of Oleocanthal Isolated from Virgin Olive Oil on Human Melanoma Cells.

Oleocanthal is one of the phenolic compounds of extra virgin olive oil with important anti-inflammatory properties. Although its potential anticancer activity has been reported, only limited evidence has been provided in cutaneous malignant melanoma. The present study is aimed at investigating the selective in vitro antiproliferative activity of oleocanthal against human malignant melanoma cells. Since oleocanthal is not commercially available, it was obtained as a pure standard by direct extraction and purification from extra virgin olive oil. Cell viability experiments carried out by WST-1 assay demonstrated that oleocanthal had a remarkable and selective activity for human melanoma cells versus normal dermal fibroblasts with IC50s in the low micromolar range of concentrations. Such an effect was paralleled by a significant inhibition of ERK1/2 and AKT phosphorylation and downregulation of Bcl-2 expression. These findings may suggest that extra virgin olive oil phenolic extract enriched in oleocanthal deserves further investigation in skin cancer.

AM251 induces apoptosis and G2/M cell cycle arrest in A375 human melanoma cells.

Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.

Montelukast prevents microparticle-induced inflammatory and functional alterations in human bronchial smooth muscle cells.

Microparticles (MPs) are membrane fragments that may play a role in the pathogenesis of chronic respiratory diseases. We aimed to investigate whether human monocytes/macrophage-derived MPs could induce a pro-inflammatory phenotype in human bronchial smooth muscle cells (BSMC) and the effect of montelukast in this setting. Experimental methods included isolation of human monocytes/macrophages and generation of monocyte-derived MPs, RT-PCR analysis of gene expression, immunoenzymatic determination of pro-inflammatory factor release, bioluminescent assay of intracellular cAMP levels and electromobility shift assay analysis of NF-κB nuclear translocation. Stimulation of human BSMC with monocyte-derived MPs induced a pro-inflammatory switch in human BSMC by inducing gene expression (COX-2 and IL-8), protein release in the supernatant (PGE2 and IL-8), and heterologous ß2-adrenoceptor desensitization. The latter effect was most likely related to autocrine PGE2 since pre-treatment with COX inhibitors restored the ability of salbutamol to induce cAMP synthesis in desensitized cells. Challenge with MPs induced nuclear translocation of NF-κB and selective NF-κB inhibition decreased MP-induced cytokine release in the supernatant. Montelukast treatment prevented IL-8 release and heterologous ß2-adrenoceptor desensitization in human BSMC exposed to monocyte-derived MPs by blocking NF-κB nuclear translocation. These findings provide evidence on the role of human monocyte-derived MPs in the airway smooth muscle phenotype switch as a novel potential mechanism in the progression of chronic respiratory diseases and on the protective effects by montelukast in this setting.

Protective effect of high-dose montelukast on salbutamol-induced homologous desensitisation in airway smooth muscle.

Montelukast (MK) is a potent cysteinyl-leukotriene receptor antagonist that causes dose-related improvements in chronic asthma. We sought to determine whether MK was able to prevent salbutamol-induced tolerance in airway smooth muscle. Homologous ß2-adrenoceptor desensitisation models were established in guinea-pigs and in human bronchial smooth muscle cells (BSMC) by chronic salbutamol administration. Characterisation tools included measurement of the response of tracheal smooth muscle tissues to salbutamol, analysis of gene expression and receptor trafficking, evaluation of intracellular cAMP levels and phosphodiesterase (PDE) activity in human bronchial smooth muscle cells. Salbutamol-induced ß2-adrenoceptor desensitisation was characterised by ß2-agonist hyporesponsiveness (-30%, p < 0.001) in desensitised tracheal smooth muscle, as compared to controls. MK, given intraperitoneally at 5 mg/kg/day for 6 consecutive days, completely restored tissue responsiveness to salbutamol. Prolonged salbutamol treatment significantly decreased cAMP synthesis, induced a complete removal of the ß2-adrenoceptor from plasma membrane with a parallel increase in the cytosol and increased PDE4D5 gene transcription and PDE activity in human bronchial smooth muscle cells. In homologously desensitised BSMC, MK 30 µM for 24 h was able to prevent salbutamol subsensitivity and such an effect was associated with inhibition of salbutamol-induced PDE4 activity and restoration of membrane ß2-adrenoceptor expression and function. These findings suggest the presence of a favourable interaction between MK and ß2-adrenoceptor agonists that might improve the therapeutic index of bronchodilators in patients with chronic respiratory diseases.

Hydrogen sulphide: novel opportunity for drug discovery.

Hydrogen sulphide (H(2)S) is emerging as an important endogenous modulator, which exhibits the beneficial effects of nitric oxide (NO) on the cardiovascular (CV) system, without producing toxic metabolites. H(2)S is biosynthesized in mammalian tissues by cystathionine-ß-synthase and cystathionine-γ-lyase. H(2)S exhibits the antioxidant properties of inorganic and organic sulphites, behaving as a scavenger of reactive oxygen species. There is also clear evidence that H(2)S triggers other important effects, mainly mediated by the activation of ATP-sensitive potassium channels (K(ATP)). This mechanism accounts for the vasorelaxing and cardioprotective effects of H(2)S. Furthermore, H(2)S inhibits smooth muscle proliferation and platelet aggregation. In non-CV systems, H(2)S regulates the functions of the central nervous system, as well as respiratory, gastroenteric, and endocrine systems. Conversely, H(2)S deficiency contributes to the pathogenesis of hypertension. Likewise, impairment of H(2)S biosynthesis is involved in CV complications associated with diabetes mellitus. There is also evidence of a cross-talk between the H(2)S and the endothelial NO pathways. In particular, recent observations indicate a possible pathogenic link between deficiencies of H(2 S activity and the progress of endothelial dysfunction. These biological aspects of endogenous H(2)S have led several authors to look at this mediator as "the new NO" that has given attractive opportunities to develop innovative classes of drugs. In this review, the main biological actions of H(2)S are discussed. Moreover, some examples of H(2)S-donors are shown, as well as some hybrids, in which H(2)S-releasing moieties are added to well-known drugs, for improving their pharmacodynamic profile or reducing the potential for adverse effects, are reported.

Enhancer and competitive allosteric modulation model for G-protein-coupled receptors.

A new mathematical model, referred to as Enhancer and Competitive Allosteric Modulator (ECAM) model, developed with the aim of quantitatively describing the interaction of an allosteric modulator with both enhancer and competitive properties towards G-protein-coupled receptors is described here. Model simulations for equilibrium (displacement-like and saturation-like), and kinetic (association and dissociation) binding experiments were performed. The results showed the ability of the model to interpret a number of possible ligand-receptor binding behaviors. In particular, the binding properties of PD81723, an enhancer and competitive allosteric modulator for the adenosine A(1) receptor, were experimentally evaluated by radioligand binding assays and interpreted by the ECAM model. The results also offer a theoretical background enabling the design and optimization of compounds endowed with allosteric enhancer, competitive, agonist, antagonist, and inverse agonist properties.

Erucin exhibits vasorelaxing effects and antihypertensive activity by H2 S-releasing properties.

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2 S)-releasing agents are viewed as potential antihypertensive drugs. Recently, natural isothiocyanates emerged as original H2 S-donor agents. Among them, erucin, present in some edible cruciferous plants, shows suitable H2 S-releasing properties and features of "druggability." The aim of this work was to investigate the erucin-mediated release of H2 S inside vascular cells, its vasorelaxing effects, and activity on BP of normo and hypertensive animals. EXPERIMENTAL APPROACH: Intracellular H2 S-release and the hyperpolarizing effect of erucin were tested using fluorescent dye, in human aortic smooth muscle cells (HASMCs). Its direct vasorelaxing effect and ability to inhibit noradrenaline-induced vasoconstriction were evaluated on endothelium-intact or -denuded rat aortic rings. Its vasodilator properties were tested in coronary arteries using Langendorff-perfused rat hearts. Finally, erucin's antihypertensive activity was evaluated in vivo in normotensive and spontaneously hypertensive rats (SHRs) by recording systolic BP using the tail-cuff method. KEY RESULTS: Erucin induced the release of H2 S inside HASMCs. Moreover, erucin hyperpolarized the membrane of HASMCs membrane in a concentration-dependent manner. It induced vasodilatation of rat aortic rings, in endothelium-denuded vessels. This effect was further improved by the presence of endothelial NO. When pre-incubated with rat aortic rings, erucin induced concentration-dependent inhibition of noradrenaline-induced vasoconstriction. Erucin did not affect basal coronary flow but restored the flow to normal in pre-contracted coronary vessels. Finally, in vivo, erucin decreased systolic BP in SHRs by about 25%, and restored the BP to values observed in normotensive rats. CONCLUSIONS AND IMPLICATIONS: Erucin is an H2 S donor endowed with vasorelaxing and antihypertensive effects. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Synthesis and in-vitro antitumour activity of new naphthyridine derivatives on human pancreatic cancer cells.

OBJECTIVES: The aim of the study was to evaluate the antitumour effect in vitro of newly synthesized 7-substituted 2,3-dihydro-1,8-naphthyridines. METHODS: Characterization tools included cell viability assay, caspase 3/7 induction, DNA fragmentation, fibroblast growth factor type 1 receptor kinase inhibition, and in-vitro antiangiogenic analysis. KEY FINDINGS: Treatment of MIA PaCa-2 human pancreatic cancer cells with test compounds showed time- and concentration-dependent cytotoxicity with IC50 values in the micromolar range. Compounds with an aminoalkyl or a diaminoalkyl side chain at the 7-position exhibited remarkable cytotoxicity, whereas the presence of a methyl group or a cyclic amine in the same position led to a significant decrease in their biological activity. Cytotoxicity screening demonstrated that the most active was compound 11 (mean 50% inhibition of cell proliferation (IC50) 11 mum). This compound had an in-vitro antitumour efficacy superior to 5-fluorouracil (the lowest cell viability value after treatment (E(max)) 0.2% and 19%, respectively) and proved to be less toxic than 5-fluorouracil against non-cancerous human oral epithelial cells. In addition, compound 11 induced apoptosis in MIA PaCa-2 cells and it was able to promote antiangiogenic effects in vitro. Finally, its cytotoxicity was enhanced in pancreatic cancer cells stimulated with fibroblast growth factor, while no substantial effect was observed on human bronchial smooth muscle cells stimulated with the same growth factor. CONCLUSIONS: These findings suggest that 1,8-naphthyridine derivatives are a promising class of compounds in cancer research. In particular, the antitumour activity of compound 11 is worth further investigation.

Circulating cell-free microRNAs in cutaneous melanoma staging and recurrence or survival prognosis.

Cutaneous melanoma is a skin cancer with increasing incidence. Identification of novel clinical biomarkers able to detect the stage of disease and suggest prognosis could improve treatment and outcome for melanoma patients. Cell-free microRNAs (cf-miRNAs) are the circulating copies of short non-coding RNAs involved in gene expression regulation. They are released into the interstitial fluid, are detectable in blood and other body fluids and have interesting features of ideal biomarker candidates. They are stable outside the cell, tissue specific, vary along with cancer development and are sensitive to change in the disease course such as progression or therapeutic response. Moreover, they are accessible by non-invasive methods or venipuncture. Some articles have reported different cf-miRNAs with the potential of diagnostic tools for melanoma staging, recurrence and survival prediction. Although some concordance of results is already emerging, differences in analytical methods, normalization strategies and tumour staging still will require further research and standardization prior to clinical usage of cf-miRNA analysis. This article reviews this literature with the aim of contributing to a shared focusing on these new promising tools for melanoma treatment and care.

The Role of Hydrogen Sulfide and H2S-donors in Myocardial Protection Against Ischemia/Reperfusion Injury.

Hydrogen sulfide (H2S), previously known only as a toxic agent, in the last decades has been recognized as an important endogenous gasotransmitter, playing a key role in the homeostasis of the cardiovascular system. In the last years, the growing evidence about a protective role exhibited by H2S against myocardial ischemia/reperfusion (I/R), led to an increasing interest for the possible mechanism of action accounting for the H2S cardioprotective effect, and to the discovery of the involvement of several targets. Currently, many mechanisms of action have been proposed and verified through in vitro and in vivo models of I/R injury, such as the anti-inflammatory or the anti-oxidant ones, or mechanisms of Ssufhydration able to modify proteins such as ion channels. Particular attention was focused on the mitochondrial preservation and on anti-apoptotic mechanisms, and finally even a pro-angiogenesis effect has been described. At the same time, the design, the development and the pharmacological characterization of moieties able to release H2S, employed alone as H2S-donor, or conjugated with another drug in hybrid molecules, led to the production of novel chemical entities in the panorama of cardioprotective drugs.

Phosphorylation of AKT and ERK1/2 and mutations of PIK3CA and PTEN are predictive of breast cancer cell sensitivity to everolimus in vitro.

BACKGROUND: Everolimus is the hydroxyethyl derivative of sirolimus and a strong inhibitor of mammalian target of rapamycin (mTOR). This drug has immunosuppressive and anticancer activities and the present in vitro study was aimed at identifying the cellular and molecular profiles of breast cancer cells predictive of sensitivity to everolimus. MATERIALS AND METHODS: MCF-7, T-47D, ZR-75-1, CAMA-1, HCC-1500 and MCF-10A cells were used and viability was assessed using WST-1 dye. Sensitivity to everolimus was correlated with phosphorylation of AKT (Ser473/Thr308), mTOR (Ser2448), and ERK1/2 (Thr202/Tyr204) and mutational profile of KRAS, NRAS, BRAF, PIK3CA, PTEN, TSC1, TSC2 and FRAP genes. Protein phosphorylation was evaluated by AlphaScreen SureFire, while the mutational status was examined by digital droplet PCR and Sanger sequencing. RESULTS: Everolimus showed a transient growth inhibition in non-tumorigenic cells, while in tumorigenic lines the drug suppressed the proliferation in a concentration-dependent manner but with different potency (IC50) and efficacy (Emax), being ZR-75-1 the most sensitive and T47D the least sensitive. MCF-7, T47D and HCC1500 had activating mutations in PIK3CA gene, while loss-of-activity PTEN mutations were detected in sensitive cell lines, including ZR-75-1, which showed no changes or minimal increase in the amount of p-AKT(Ser473/Thr308) and p-ERK1/2(Thr202/Tyr204) induced by everolimus compared to the resistant cell line T47D in which phosphorylation of AKT and ERK was increased. CONCLUSIONS: Cellular levels of p-AKT(Ser473/Thr308) and p-ERK1/2(Thr202/Tyr204), activating mutations of PIK3CA and inactivating mutations of PTEN may predict response to everolimus in breast cancer cells; these findings have potential applications for treatment personalization of everolimus in breast cancer patients.

In vivo adenosine A(2B) receptor desensitization in guinea-pig airway smooth muscle: implications for asthma.

This study was aimed at characterizing the role of adenosine receptor subtypes in the contractility modulation of guinea-pig airway smooth muscle in normal and pathological settings. In vitro and in vivo experiments were performed by testing selective agonists and antagonists on isolated tracheal smooth muscle preparations and pulmonary inflation pressure, respectively, under normal conditions or following ovalbumin-induced allergic sensitization. In normal and sensitized animals, the adenosine A(2A)/A(2B) receptor agonist, NECA, evoked relaxing responses of isolated tracheal preparations precontracted with histamine, and such an effect was reversed by the adenosine A(2B) antagonist, MRS 1706, in the presence or in the absence of epithelium. The expression of mRNA coding for adenosine A(2B) receptors was demonstrated in tracheal specimens. In vitro desensitization with 100 microM NECA markedly reduced the relaxing effect of the agonist. In vivo NECA or adenosine administration to normal animals inhibited histamine-mediated bronchoconstriction, while these inhibitory effects no longer occurred in sensitized guinea-pigs. Adenosine plasma levels were significantly higher in sensitized than normal animals. In conclusion, our data demonstrate that: (i) adenosine A(2B) receptors are responsible for the relaxing effects of adenosine on guinea-pig airways; (ii) these receptors can undergo rapid adaptive changes that may affect airway smooth muscle responsiveness to adenosine; (iii) ovalbumin-induced sensitization promotes a reversible inactivation of adenosine A(2B) receptors which can be ascribed to homologous desensitization. These findings can be relevant to better understand adenosine functions in airways as well as mechanisms of action of asthma therapies targeting the adenosine system.

Cholinesterase-like organocatalysis by imidazole and imidazole-bearing molecules.

Organocatalysis, which is mostly explored for its new potential industrial applications, also represents a chemical event involved in endogenous processes. In the present study, we provide the first evidence that imidazole and imidazole derivatives have cholinesterase-like properties since they can accelerate the hydrolysis of acetylthiocholine and propionylthiocholine in a concentration-dependent manner. The natural imidazole-containing molecules as L-histidine and histamine show a catalytic activity, comparable to that of imidazole itself, whereas synthetic molecules, as cimetidine and clonidine, were less active. In the experimental conditions used, the reaction progress curves were sigmoidal and the rational of such unexpected behavior as well as the mechanism of catalysis is discussed. Although indirectly, findings of the present study suggests that imidazolic compounds may interfere with the homeostasis of the cholinergic system in vivo.

Iminothioethers as Hydrogen Sulfide Donors: From the Gasotransmitter Release to the Vascular Effects.

The gasotransmitter hydrogen sulfide (H2S) is an important tuner of the cardiovascular homeostasis, and its deficiency is etiologically associated with a number of cardiovascular diseases. Therefore, the research of original moieties able to release H2S represents a timely issue for drug discovery. In this work, we developed a collection of iminothioethers (ITEs), exhibiting H2S-releasing properties and producing vasorelaxing effects on rat aortic rings. Derivatives 4 and 11, selected as representative of slow and fast rate H2S donors, respectively, produced a complete recovery of the basal coronary flow, reverting the AngII-induced effects in isolated rat hearts. In addition, studies on human aortic smooth muscle cells (HASMCs) demonstrated membrane hyperpolarizing effects, well related to the intracellular generation of H2S. Taken together, the results obtained support ITEs 4 and 11 as new pharmacological tools, as well as effective and innovative H2S donors for cardiovascular drug discovery.

The Citrus Flavanone Naringenin Produces Cardioprotective Effects in Hearts from 1 Year Old Rat, through Activation of mitoBK Channels.

Background and Purpose: Incidence of cardiovascular disorders increases with age, because of a dramatic fall of endogenous self-defense mechanisms and increased vulnerability of myocardium. Conversely, the effectiveness of many cardioprotective drugs is blunted in hearts of 1 year old rat. The Citrus flavanone naringenin (NAR) was reported to promote cardioprotective effects against ischemia/reperfusion (I/R) injury, through the activation of mitochondrial large conductance calcium-activated potassium channel (mitoBK). These effects were observed in young adult rats, but no data are available about the possible cardioprotective effects of NAR in aged animals. Experimental Approach: This study aimed at evaluating the potential cardioprotective effects of NAR against I/R damage in 1 year old rats, and the possible involvement of mitoBK. Key Results: Naringenin protected the hearts of 1 year old rats in both ex vivo and in vivo I/R protocols. Noteworthy, these effects were antagonized by paxilline, a selective BK-blocker. The cardioprotective effects of NAR were also observed in senescent H9c2 cardiomyoblasts. In isolated mitochondria from hearts of 1 year old, NAR exhibited the typical profile of a mitoBK opener. Finally, Western Blot analysis confirmed a significant (albeit reduced) presence of BK-forming alpha and beta subunits, both in cardiac tissue of 1 year old rats and in senescent H9c2 cells. Conclusion and Implications: This is the first work reporting cardioprotective effects of NAR in 1 year old rats. Although further studies are needed to better understand the whole pathway involved in the NAR-mediated cardioprotection, these preliminary data represent a promising perspective for a rational nutraceutical use of NAR in aging.

Tumor-promoting effects of cannabinoid receptor type 1 in human melanoma cells.

The role of endocannabinoid system in melanoma development and progression is actually not fully understood. This study was aimed at clarifying whether cannabinoid-type 1 (CB1) receptor may function as tumor-promoting or -suppressing signal in human cutaneous melanoma. CB1 receptor expression was measured in human melanoma cell lines by real-time PCR. A genetic deletion of CB1 receptors in selected melanoma cells was carried out by using three different short hairpin RNAs (shRNAs). Performance of target gene silencing was verified by real-time PCR and Western blot. The effects of CB1 receptor silencing on cell growth, clonogenicity, migration capability, cell cycle progression, and activation of mitogenic signals was tested. Lentiviral shRNAs vectors targeting different regions of the human CB1 gene led to a significant reduction in CB1 receptor mRNA and a near complete loss of CB1 receptor protein, compared to control vector (LV-c). The number of viable cells, the colony-forming ability and cell migration were significantly reduced in cells transduced with CB1 lentiviral shRNAs compared to LV-c. Cell cycle analyses showed arrest at G1/S phase. p-Akt and p-ERK expression were decreased in transduced versus control cells. Findings of this study suggest that CB1 receptor might function as tumor-promoting signal in human cutaneous melanoma.

Cannabinoid derivatives induce cell death in pancreatic MIA PaCa-2 cells via a receptor-independent mechanism.

Cannabinoids (CBs) are implicated in the control of cell survival in different types of tumors, but little is known about the role of CB system in pancreatic cancer. Herein, we investigated the in vitro antitumor activity of CBs and the potential role of their receptors in human pancreatic cancer cells MIA PaCa-2. Characterization tools used for this study included growth inhibition/cell viability analyses, caspase 3/7 induction, DNA fragmentation, microarray analysis and combination index-isobologram method. Our results demonstrate that CBs produce a significant cytotoxic effect via a receptor-independent mechanism. The CB1 antagonist N-(piperidin-1-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) was the most active compound with an IC50 of 8.6 +/- 1.3 microM after 72 h. AM251 induces apoptosis, causes transcriptional changes of genes in janus kinase/signal transducers and activators of transcription signaling network and synergistically interacts with the pyrimidine analogue, 5-fluorouracil. These findings exclude the involvement of CB receptors in the regulation of MIA PaCa-2 cell growth and put AM251 forward as a candidate for the development of novel compounds worthy to be tested in this type of neoplasia.

Voltage-operated potassium (Kv) channels contribute to endothelium-dependent vasorelaxation of carvacrol on rat aorta.

OBJECTIVES: Carvacrol, a monoterpene widely present in nature, is commonly used in the food industry and in cosmetics, besides to possess a plethora of pharmacological properties, among these also in vitro vasorelaxing effects and in vivo hypotensive responses. Although in rat aortic rings carvacrol evoked a vasodilatation both in the presence and in the absence of endothelium, in preparations with intact endothelial layer its vasoactive response markedly improved. METHODS: This study aimed at investigating the mechanism of action responsible for the endothelial component of the carvacrol-induced vasorelaxing response observed in rat isolated aortic rings. KEY FINDINGS: Pharmacological characterization led us to exclude the involvement of NO pathway (neither L-NAME, NO biosynthesis inhibitor, nor ODQ, guanylate cyclase inhibitor, was able to modify the vascular effects of carvacrol) and of arachidonic acid cascade (no inhibitor intercepting the cascade influenced the endothelial-dependent vasodilatation of the monoterpene). Moreover, endothelial TRP channels were also not involved, as capsazepine did not antagonize vasorelaxing effect. Finally, endothelial potassium channels were considered as possible targets of carvacrol; indeed, two voltage-operated potassium (Kv) channel blockers, 4-aminopyridine and quinine, significantly reduced carvacrol potency and efficacy indices. CONCLUSIONS: Kv channels seem to be responsible for vascular effects of the monoterpene typical of Labiatae family.

Application of a pharmacokinetic/pharmacogenetic approach to assess the nicotine metabolic profile of smokers in the real-life setting.

The nicotine metabolite ratio, i.e., the ratio 3-hydroxycotinine/cotinine, is used to assess the nicotine metabolic status and has been proven to predict the response to smoking cessation treatments in randomized clinical trials. In the current study, a pharmacokinetic-pharmacogenetic integrated approach is described, based on the development of a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for nicotine metabolite ratio assay in plasma and a real-time PCR analysis for fast genotyping of CYP2A6. The pharmacokinetic-pharmacogenetic approach was validated in 66 subjects with different smoking status. The LC/MS/MS assay was rapid and sensitive enough to detect plasma cotinine levels also in second-hand exposed abstainers. In the cohort of patients of the present study the following results were obtained: (i) the frequencies of CYP2A6 genetic variants were comparable with those from clinical trials carried out in Caucasian populations; (ii) all the subjects carrying the CYP2A6 deficient allele also had a slow metabolizer phenotype; (iii) slow metabolizers had mean nicotine metabolite ratio approximately 50% of that of the normal/fast metabolizers; (iv) women had higher nicotine metabolite ratio than men; and (v) salivary nicotine metabolite ratio measures were comparable to plasma levels. Overall, the findings of the current study demonstrate that the simultaneous assessment of nicotine metabolite ratio and CYP2A6 genotype from human blood samples is feasible and accurate and could be used in a smoking cessation program to optimize treatments and identify those smokers who inherit metabolically deficient CYP2A6 alleles.