Attività farmacologica di un oligonucleotide molecular beacon anti-survivina in cellule di melanoma cutaneo

La survivina è un membro della famiglia delle IAP (proteine in grado di inibire l’apoptosi), coinvolta nel controllo dell’inibizione dell’apoptosi, della divisione e della motilità cellulare e caratterizzata da una selettiva over-espressione nei tessuti tumorali. Risulta, inoltre, essere coinvolta nella farmacoresistenza innata e acquisita a livello tumorale ed è, per questo, considerata un possibile target per la terapia a bersaglio molecolare, in molte forme tumorali. Una nuova strategia che coniuga la tecnica di imaging con l’attività terapeutica antitumorale può essere realizzata tramite molecole oligonucleotidiche con funzione antisenso, concepite nella forma di molecular beacon (MB). In un precedente lavoro di tesi (Valutazione di nanoparticelle di polimetilmetacrilato per la veicolazione intracellulare di un oligonucleotide molecular beacon silenziante la survivina, Desirè Fina) è stato utilizzato un MB anti-survivina dimostrandone, tramite analisi al miscroscopio confocale in cellule vive di melanoma cutaneo, la capacità di fluorescere a livello intracellulare in maniera specifica. Il lavoro sperimentale svolto nel presente lavoro di tesi è stato finalizzato alla valutazione dell’attività farmacologica di tale MB in cellule di melanoma cutaneo umane (A375). Gli esperimenti sono stati condotti trasfettando le cellule A375 con MB 100 nM e i livelli di espressione della survivina sono stati valutati con real-time PCR e Western blot, mentre la morte cellulare per apoptosi è stata esaminata attraverso l’analisi della frammentazione internucleosomica (ELISA), del potenziale di dissipazione mitocondriale e dell’alterazione della morfologia nucleare (DAPI). L’attività del MB è stata valutata anche in associazione con cisplatino e docetaxel, al fine di evidenziare possibili sinergismi d’azione. I risultati ottenuti hanno mostrato che il MB induce una diminuzione tempo-dipendente dell'espressione dell'mRNA con un effetto importante già a 24 ore (79.3±7.6 %, P<0.01) e porta, inoltre, ad una diminuita espressione dei livelli proteici. Il MB risulta anche indurre apoptosi e ridurre la farmacoresistenza nei confronti di cisplatino e docetaxel. In conclusione, i dati ottenuti indicano che il MB, oltre alle già dimostrate proprietà di imaging, può rappresentare un nuovo agente terapeutico per contrastare la crescita tumorale e superare fenomeni di farmacoresistenza

Circulating MicroRNAs in Cutaneous Melanoma Diagnosis and Prognosis

Cutaneous melanoma represents a challenge for pharmacologists and clinicians due to their high degree of genetic and phenotypic heterogeneity. The identification of new non-invasive and informative biomarkers would therefore represent a substantial step to adequately treat melanoma patients. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play an important role as negative regulators of gene expression. Several studies have demonstrated their correlation with disease status in different types of cancer including melanoma. Extracellular miRNAs are released from both tumor cells and/or normal cells. MiRNAs do not circulate freely in biological fluids but they are incorporated into extracellular vesicles or form complexes with lipids and proteins. Circulating miRNAs may represent potential biomarkers of cutaneous melanoma diagnosis and patient prognosis. Longitudinal monitoring of cell-free miRNAs in biological fluids of melanoma patients could help clinicians to predict disease progression before the tumor becomes resistant to a given drug. However, to confirm their clinical utility it will be necessary to validate the best available technique for their detection and quantification and to test selected miRNAs in prospective clinical trials

The Interplay between Cannabinoid Receptors and Microglia in the Pathophysiology of Alzheimer's Disease

Alzheimer’s disease (AD) is characterized by massive neuronal death, brain atrophy, and loss of neurons and synapses, which all lead to a progressive cognitive decline. Neuroinflammation has been recently identified as one of the main causes of AD progression, and microglia cells are considered to have a central role in this process. Growing evidence suggests that cannabinoids may be used as preventive treatment for AD. An altered expression of the endocannabinoids (eCBs) and their receptors (CBRs) is reported in several neurodegenerative disorders, including AD. Moreover, the modulation of CBRs demonstrated neuroprotective effects in reducing aggregated protein de- position, suggesting the therapeutic potential of natural and synthetic CBR ligands in the treatment of neurodegenerative proteinopathies. Here, we review the current knowledge regarding the in- volvement of CBRs in the modulation of microglia activation phenotypes, highlighting the role of neuroinflammation in the pathogenesis of neurodegenerative diseases, like AD. We also provide an overview of recently developed candidate drugs targeting CBRs that may afford a new innovative strategy for the treatment and management of AD

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

miRNA Modulation and Antitumor Activity by the Extra-Virgin Olive Oil Polyphenol Oleacein in Human Melanoma Cells

Extra-virgin olive oil (EVOO) polyphenols contribute to Mediterranean diet health- promoting properties. One of the most abundant secoiridoid present in EVOO, Oleacein (OA), demonstrated anticancer activity against several tumors. Nevertheless, its role against melanoma has not still investigated. This study aimed at determining in vitro the antimelanoma activity of OA and the relative mechanism of action. OA induced cell growth inhibition in 501Mel melanoma cells with an IC50 in the low micromolar range of concentrations. Moreover, an OA concentration approximating the IC50 induced G1/S phase arrest, DNA fragmentation, and downregulation of genes encoding antiapoptotic (BCL2 and MCL1) and proproliferative (c-KIT, K-RAS, PIK3R3, mTOR) proteins, while increased transcription levels of the proapoptotic protein BAX. Concordantly, OA increased the levels of miR-193a-3p (targeting MCL1, c-KIT and K-RAS), miR-193a-5p (targeting PIK3R3 and mTOR), miR-34a-5p (targeting BCL2 and c-KIT) and miR-16-5p (miR-16-5p targeting BCL2, K-RAS and mTOR), while decreased miR-214-3p (targeting BAX). These modulatory effects might contribute to the inhibition of 501Mel melanoma cell growth observed after treatment with an olive leaves-derived formulation rich in OA, with potential application against in situ cutaneous melanoma. Altogether, these results demonstrate the ability of OA to contrast the proliferation of cutaneous melanoma cells through the transcriptional modulation of relevant genes and microRNAs, confirming the anticancer potential of EVOO and suggesting OA as a chemopreventive agent for cancer disease therapy

Понятие внешней среды в современном менеджменте

Анализируются изменения, произошедшие в определении понятия "внешняя среда". Приводятся различные подходы к определению факторов, входящих во внешнюю среду предприятия. В процессе рассмотрения особое внимание уделяется изменениям, происходящим в определении факторов данной среды в современном менеджменте. По результатам исследования определен современный состав факторов внешней среды, определена их роль в становлении концепта "современный менеджмент" в России

Intake of Natural Compounds and Circulating microRNA Expression Levels: Their Relationship Investigated in Healthy Subjects With Different Dietary Habits

Diet has a strong influence on many physiological processes, which in turn have important implications on a variety of pathological conditions. In this respect, microRNAs (miRNAs), a class of small non-coding RNAs playing a relevant epigenetic role in controlling gene expression, may represent mediators between the dietary intake and the healthy status. Despite great advances in the field of nutri-epigenomics, it remains unclear how miRNA expression is modulated by the diet and, specifically, the intake of specific nutrients. We investigated the whole circulating miRNome by small RNA-sequencing performed on plasma samples of 120 healthy volunteers with different dietary habits (vegans, vegetarians, and omnivores). Dietary intakes of specific nutrients were estimated for each subject from the information reported in the food-frequency questionnaire previously validated in the EPIC study. We focused hereby on the intake of 23 natural compounds (NCs) of the classes of lipids, micro-elements, and vitamins. We identified 78 significant correlations (rho &gt; 0.300, p-value &lt; 0.05) among the estimated daily intake of 13 NCs and the expression levels of 58 plasma miRNAs. Overall, vitamin D, sodium, and vitamin E correlated with the largest number of miRNAs. All the identified correlations were consistent among the three dietary groups and 22 of them were confirmed as significant (p-value &lt; 0.05) by age-, gender-, and body-mass index-adjusted Generalized Linear regression Model analysis. miR-23a-3p expression levels were related with different NCs including a significant positive correlation with sodium (rho = 0.377) and significant negative correlations with lipid-related NCs and vitamin E. Conversely, the estimated intake of vitamin D was negatively correlated with the expression of the highest number of circulating miRNAs, particularly miR-1277-5p (rho = −0.393) and miR-144-3p (rho = −0.393). Functional analysis of the targets of sodium intake-correlated miRNAs highlighted terms related to cardiac development. A similar approach on targets of those miRNAs correlated with vitamin D intake showed an enrichment in genes involved in hormone metabolisms, while the response to chronic inflammation was among the top enriched processes involving targets of miRNAs negatively related with vitamin E intake. Our findings show that nutrients through the habitual diet influence circulating miRNA profiles and highlight that this aspect must be considered in the nutri-epigenomic research

Topical application of silymarin enhances cutaneous wound healing in rats

Abstract Wound healing in a short period with minimum side effects is one of the major goals of medical sciences. Silymarin, an extract from Silybum marianum, has been shown to have antioxidant and anti-inflammatory properties. This study investigates the wound healing activity of silymarin topical formulation in an in experimental model. A 875 mm2 (25 × 35 mm) full-thickness excision was made on the abdominal region of each rat by a surgical blade and the day on which the wound was made considered as day 0. Each rat was treated two times each day. On days 1,4, 8 and 12, the wound area was measured using precise caliber and camera imaging. On day 12, blood samples were collected for the analysis of antioxidant, malondialdehyde and estradiol levels. After 12 days of treatment, rats were sacrificed and abdominal region tissues used for histological analyses. The study showed that topical application of silymarin on wound in rats improved wound healing correlating with less redness, exudates and swelling. Furthermore, in serum of rats treated with silymarin ointment improved antioxidant and estradiol levels, while decreased malondialdehyde levels, a marker of oxidative stress. Histological analyses showed also an improve of novel blood vessels. This effect on angiogenesis correlated with improve nitric oxide synthase expression and epithelial cells after treatment with silymarin. Silymarin ointment represents a promising therapeutic agent for the treatment of wounds through its antioxidant and anti-inflammatory properties

Oleocanthal and oleacein contribute to the in vitro therapeutic potential of extra virgin oil-derived extracts in non-melanoma skin cancer

Although the anticancer properties of extra virgin olive oil (EVOO) extracts have been recognized, the role of single compounds in non-melanoma skin cancer is still unknown. The in vitro chemopreventive and anticancer action of EVOO extracts and oil-derived compounds in non-melanoma skin cancer models were evaluated on cutaneous squamous cell carcinoma cells and on immortalized human keratinocytes stimulated with epidermal growth factor. Preparation of EVOO extracts and isolation of single compounds was carried out by chromatographic methods. Antitumor activity was assessed by cell-based assays (cell viability, migration, clonogenicity, and spheroid formation) and apoptosis documented by internucleosomal DNA fragmentation. Finally, inhibition of key oncogenic signaling nodes involved in the progression from actinic keratosis to cutaneous squamous cell carcinoma was studied by western blot. EVOO extracts reduced non-melanoma skin cancer cell viability and migration, prevented colony and spheroid formation, and inhibited proliferation of atypical keratinocytes stimulated with epidermal growth factor. Such a pharmacological activity was promoted by oleocanthal and oleacein through the inhibition of Erk and Akt phosphorylation and the suppression of B-Raf expression, whereas tyrosol and hydroxytyrosol did not have effect. The current study provides in vitro evidence for new potential clinical applications of EVOO extracts and/or single oil-derived compounds in the prevention and treatment of non-melanoma skin cancers

Tailoring of silica-based nanoporous pod by spermidine multi-activity

Ubiquitous in nature, polyamines (PAs) are a class of low-molecular aliphatic amines critically involved in cell growth, survival and differentiation. The polycation behavior is validated as a successful strategy in delivery systems to enhance oligonucleotide loading and cellular uptake. In this study, the chemical features and the functional roles of the PA spermidine are synergistically exploited in the synthesis and bioactive functionalization of SiO2-based structures. Inspired by biosilicification, the role of spermidine is assessed both as catalyst and template in a biomimetic one-pot synthesis of dense silica-based particles (SPs) and as a competitive agent in an interfacial reassembly strategy, to empty out SPs and generate spermidine-decorated hollow silica nanoporous pods (spd-SNPs). Spermidine bioactivity is then employed for targeting tumor cell over-expressed polyamine transport system (PTS) and for effective delivery of functional miRNA into melanoma cells. Spermidine decoration promotes spd-SNP cell internalization mediated by PTS and along with hollow structure enhances oligonucleotide loading. Accordingly, the functional delivery of the tumor suppressor miR-34a 3p resulted in intracellular accumulation of histone-complexed DNA fragments associated with apoptosis. Overall, the results highlight the potential of spd-SNP as a multi-agent anticancer therapy