In vitro effects of bioactive extracts of local Italian cultivars: from molecular mechanisms to potential nutraceutical applications for consumers’ well-being

2015 - 2016Phytochemicals are non-nutritional compounds, naturally present in food and beverages, which exert beneficial effects on human health. The present Doctorate Thesis evaluates the biological properties of bioactive extracts prepared from Italian cultivars/products on different in vitro models to test their ability to ameliorate conditions of cellular oxidative stress, often associated with the occurrence of degenerative pathologies, such as cancer and neurodegenerative processes. The two classes of biomolecules investigated, polyphenols and carotenoids, are largely present in the three different foods and beverage investigated, e.g. red wine, elderberries and pumpkin. The results obtained suggest the existence of novel mechanisms triggered by red wine polyphenols and resulting in the protection of erythrocytes from plasma oxidizing species. These compounds are able to increase antioxidant defences in erythrocytes by activating Plasma Membrane Redox System. Moreover, we hypothesized that red wine polyphenols (73 µg/ml gallic acid equivalents) trigger a slight, but significant increase of intracellular reactive oxygen species, which induces a cellular adaptive response, which, in turn, increases antioxidant defences in erythrocytes. This aspect highlights the preventive role of polyphenols, which, through this mechanism of cellular adaptation against oxidative stress, can prevent the onset of degenerative and age-depending diseases related to oxidative damage, including neurodegeneration. In fact, the present work also describes an in vitro neurodegenerative model aimed to investigate the protective role of a polyphenolic extract from Sambucus nigra berries against oxidative damage induced by neurotoxic agents. We demonstrated that the efficacy of S. nigra polyphenolic extract (500 µg/ml, w/v) to effectively protect neuronal cells from apoptosis is probably related to its ability to modulate the cellular antioxidant response. The data obtained provide an initial indication for future clinical trials addressed to prove the preventive role of these natural extracts against degenerative conditions. Finally, a different class of antioxidant compounds, carotenoids from Cucurbita moschata, showed a functional effects on the proliferation of malignant cell lines. The mechanism of action of a carotenoid-enriched extract (200-400 µg/ml) has been investigated on two malignant cell lines, Caco-2 and SAOs, derived from a colon adenocarcinoma and an osteosarcoma, respectively. The results obtained indicated that pumpkin carotenoids do not directly kill cancer cells, but slow down their proliferation. This effect was associated with an alteration of the energetic metabolism which resulted in a decreases intracellular concentration of ATP and the activation of autophagy. Probably, the “not-protective” autophagy detected directly or indirectly induces cellular differentiation in malignant cells, a possibility that deserves further investigations. [edited by author]I fitochimici sono composti non-nutrizionali, naturalmente presenti in alimenti e bevande, che esercitano effetti benefici sulla salute umana. La presente Tesi di Dottorato valuta le proprietà biologiche di estratti bioattivi preparati da cultivar/prodotti italiani su diversi modelli in vitro in relazione alla loro capacità di migliorare le condizioni di stress ossidativo, spesso associato con la comparsa di patologie degenerative, come il cancro e processi neurodegenerativi. Le due classi di biomolecole indagate, polifenoli e carotenoidi, sono largamente presenti nei tre diversi alimenti studiati, tra cui vino rosso, bacche di sambuco e zucca. I risultati ottenuti suggeriscono l'esistenza di nuovi meccanismi innescati dai polifenoli del vino rosso e risultanti nella conseguente protezione degli eritrociti da specie ossidanti del plasma. Questi composti sono in grado di aumentare le difese antiossidanti degli eritrociti attivando il sistema Membrane Plasma Redox. Inoltre, abbiamo ipotizzato che i polifenoli del vino rosso (73 µg/ml di acido gallico equivalenti) innescano un lieve, ma significativo aumento di specie reattive dell'ossigeno intracellulare, che induce una risposta adattativa cellulare, che, a sua volta, aumenta le difese antiossidanti negli eritrociti. Questo aspetto evidenzia il ruolo preventivo dei polifenoli, che, attraverso questo meccanismo di adattamento cellulare contro lo stress ossidativo, può prevenire l'insorgenza di malattie degenerative dipendenti dall'età relative al danno ossidativo, compresa la neurodegenerazione. Infatti, il presente lavoro si è focalizzato anche su un modello neurodegenerativo in vitro al fine di studiare il ruolo protettivo di un estratto polifenolico di bacche di Sambucus nigra contro il danno ossidativo indotto da agenti neurotossici. E’ stato dimostrato che l'efficacia dell’estratto polifenolico di S. nigra (500 µg/ml, w/v) nel proteggere efficacemente le cellule neuronali da apoptosi è probabilmente legata alla sua capacità di modulare la risposta antiossidante. I dati ottenuti forniscono una prima indicazione per futuri studi clinici rivolti a dimostrare il ruolo preventivo di questi estratti naturali contro le condizioni degenerative. Infine, una diversa classe di composti antiossidanti, i carotenoidi presenti in Cucurbita moschata, hanno mostrato un effetto funzionale sulla proliferazione di linee cellulari maligne. Il meccanismo di azione di un estratto arricchito in carotenoidi (200-400 µg/ml) è stato studiato su due linee di cellule maligne, Caco-2 e SAOs, derivate rispettivamente da un adenocarcinoma del colon e un osteosarcoma. I risultati ottenuti hanno indicato che i carotenoidi della zucca non inducono la morte delle cellule tumorali, ma rallentano la loro proliferazione. Questo effetto è stato associato ad un'alterazione del metabolismo energetico cellulare che porta a un decremento della concentrazione intracellulare di ATP e all'attivazione dell'autofagia. Probabilmente, l'autofagia di tipo “non-protettivo” che si attiva dopo trattamento con i carotenoidi, induce differenziamento cellulare nelle cellule maligne, una possibilità che merita ulteriori indagini. [a cura dell'autore]XV n.s

Ameliorating a Complex Urban Ecosystem Through Instrumental Use of Softscape Buffers: Proposal for a Green Infrastructure Network in the Metropolitan Area of Naples

Green Infrastructure (GI) definition, deriving from the United States green infrastructure for hydro-geological rebalancing through imitating the nature stormwater management, was consolidated in Europe by GI Planning Guide. Nowadays GI can be considered a valid and meaningful approach for ameliorating urban complex ecosystems, and could also be considered as mitigation action of land consumption, according to the guidelines on the soil sealing of the European Commission (2012). The metropolitan area of Naples located in south Italy is characterized by an unauthorized and chaotic urban development. The land-use map reported an average of 30% of urbanization in the metropolitan area, rising up to 50–60% and as high as 98% in the north core area of the city. This high level of urbanization is directly related to the habitat fragmentation. The National Biodiversity Conservation Strategy defines several challenges and targets to counteract the biodiversity loss in Italy, identifying urban areas as places exposed to the greatest pressures on ecosystems. Therefore, the integration of different policies limiting habitat fragmentation, heat island effect and natural soil hydro-geological degradation into spatial planning, especially through green corridors and ecosystem enhancement in urban areas is an urgent need for the society. Spatial planning has to be renewed in metropolitan areas, where threats and weaknesses to biodiversity conservation are stronger than in any other place, according to the Law n. 56/2014, (Gazzetta Ufficiale della Repubblica Italiana, 2014) committing metropolitan cities to the enactment of General Territorial Plan. In the current paper, we aim at designing an ecological network for the metropolitan area of Naples one of the biggest city of southern Italy. The analyses include the adopted methodological procedure, i.e., ecological network analysis and design, and the introductory elements of a spatial analysis on a pilot ecological network of several patches. Finally, the paper illustrates the network analysis conceived as a monitoring system and also in future perspective, as a planning support system

Micro-Computed Tomography Evaluation of Minimally Invasive Shaping Systems in Mandibular First Molars

The aim of this study was to compare the shaping ability of a modified ProTaper Next technique (PTNm) with that of TruNatomy (TN) in lower molars mesial curved canals using micro-computed tomography (Micro-CT). Sixty mesial canals of first mandibular molars were randomly assigned between two groups (n = 30). After canal scouting with K-File #10, glide path and shaping were carried out with TN or PTNm systems. The PTNm sequence consists of ProGlider, followed by ProTaper Next X1 and apical finishing with NiTiFlex #25 up to working length (WL) to ensure adequate apical cleaning. Samples were scanned using micro-CT and pre- and post-shaping volumes were matched to analyse geometric parameters: the volume of removed dentin; the difference of canal surface; centroid shift, minimum and maximum root canal diameters; cross-sectional areas; the ratio of diameter ratios (RDR) and the ratio of cross-sectional areas (RA). Measurements were assessed 2 mm from the apex and in relation to the middle and coronal root canal thirds. Data were analysed using ANOVA (p 0.05). Both PTNm and TN sequences demonstrated similar maintenance of original anatomy during the shaping of lower molar mesial curved canals

Extra Virgin Olive Oil Phenols induce authophagy and apoptosis in human bladder cancer cell lines depending on tumor progression

Epidemiological evidences indicate that there is an inverse association between olive oil intake and bladder cancer risk and several data suggest that a key role to support these beneficial effects is played by its phenolic fraction. Bladder cancer is one of the most common cancers in Western countries. In particular, the transitional cell carcinoma histotype shows an aggressive behavior and the current therapies are ineffective. The anti-proliferative effects of an Extra Virgin Olive Oil phenolic Extract (EVOOE) has been investigated on RT112 and J82, two human bladder cancer cell lines employed as models of superficial and invasive bladder cancer, respectively.EVOOE reduces cell viability in both cell lines triggering different processes. In RT112 cells, EVOOE triggers a non-protective autophagic response, evidenced by vacuoles formation and LC-3 lipidation (45%) causing a delay in cell growth (132microg/ml induces 30% reduction). Instead, in J82, the invasive transitional cell carcinoma, EVOOE treatment induces a rapid and remarkable decrease of cell viability (33 microg/ml for 24 h induces 40% reduction) triggering an apoptotic process, evidenced with Annexin V positivity and the increased activity of caspases 3 and 9. EVOOE exerts an antioxidant activity in both cell lines reducing ROS (30% in J82 and 15% in RT112) and increasing GSH level (20% in J82 and 40% in RT112). However, comparing the effects of EVOOE with those of other well-known antioxidants, the absence of correlation between antioxidant effects and reduced cell viability was evidenced. Moreover, using an inhibitor of GSH synthesis, we demonstrated that EVOOE acts independently from GSH modulation.Data presented show that EVOOE possesses pleiotropic activities that intercept different pathways resulting in anti-proliferative effects independently of its antioxidant propert

A new role of red wine in modulating erythrocytes antioxidant defense

Dealcoholated red wine has been shown to exert protective effects, reducing the risk of cardiovascular events by improving endothelium-dependent vasodilation and inhibiting platelet aggregation These biological activities have been associated with the polyphenolic components of red wine, suggesting that the pool of phenolic compounds, including flavonoids and anthocyanins, could be responsible for its functional effects. The concept that antioxidant properties of polyphenols could explain the beneficial effects of red wine has been carried forward. Here, we hypothesize a new role of red wine in modulating oxidative stress, leading to the alteration of the antioxidant potential in humans. We previously demonstrated that red wine polyphenols (RWp) protect human erythrocytes from oxidative stress by the activation of an important enzymatic system involved in neutralizing plasma free radicals, namely Plasma Membrane Redox System (PMRS). The present work investigates the underlying mechanism triggered by RWp in the activation of PMRS via the involvement of intracellular GSH. Hence, the increase of GSH intracellular concentration results from the activation of GSH-dependent enzymes, namely glutathione reductase and glucose-6-phosphate dehydrogenase, of about 30% and 50% respectively after 2 min of incubation of human erythrocytes in the presence of RWp (73 ug/ml Gallic Acid Equivalents). Changes in GSH pathway induced by RW were associated with a slight but significant increase (about 15%) of ROS (reactive oxygen species) concentration. We conclude that the pro-oxidant effect of RWp promotes an adaptive stress response in human erythrocytes, which improves their antioxidant defense protecting them from oxidative stress

“VEGF induces human endothelial progenitor cells proliferations by eliciting oscillations in intracellular Ca2+ concentration”

Endothelial progenitor cells (EPCs) traffic from the bone marrow to the site of tissue regeneration and sustain neo-vascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell-based therapy in regenerative medicine and provide a novel promising target in the fight against cancer. The main stimulus responsible for EPC egression from the bone marrow and engraftment within neovessels is vascular endothelial growth factor (VEGF). Intracellular Ca2+ signals regulate numerous endothelial functions, such as proliferation, migration, and differentiation, and underpin VEGF effect on mature endothelium. We have recently shown that EPC growth is governed by a store-dependent Ca2+ entry (SOCE) pathway on the plasma membrane, which is activated by depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools1. The present study aimed at investigating the nature and the role of VEGF-elicited Ca2+ signals in EPCs. All the putative SOCE mediators (i.e. TRPC1, TRPC4, Orai1 and Stim1) were present in EPCs. VEGF induced long lasting Ca2+ oscillations, however, removal of external Ca2+ (0Ca2+) and SOCE inhibition with BTP-2 reduced the number of Ca2+ spikes. Blockade of phospholipase C-? (PLC-?) with U73122 and emptying the InsP3-sensitive Ca2+ pools with cyclopiazonic acid (CPA) prevented the Ca2+ response to VEGF. Accordingly, the Ca2+ response to VEGF was inhibited by superfusing CPA during the ongoing oscillations. Notably, VEGF induced EPC was abrogated by SOCE inhibition with BTP-2. Similarly, VEGF promoted NF-kB translocation into the nucleus in a BTP-2-sensitive manner. Thus, VEGF causes an initial InsP3-dependent Ca2+ discharge followed by SOCE-mediated Ca2+ entry in cEPCs. SOCE, in turn, controls store refilling and induces cell proliferation by recruiting NF-kB

Phenotypic and functional characterization of endothelial progenitor cells isolated from peripheral blood of renal cell carcinoma patients

Endothelial progenitor cells (EPCs) are mobilized from either bone marrow or arterial walls to restore blood perfusion to ischemic organs and establish the vascular network within growing tumors [1]. The Ca2+ machinery plays a key role in EPC activation and might serve a molecular target for novel therapies of highly angiogenic tumors, such as renal cell carcinoma (RCC) [1]. The Ca2+ toolkit is remodelled in EPCs isolated from RCC patients (RCC-EPCs) as respect to healthy donors [2]. The present study was undertaken to evaluate for the first time the functional properties of EPCs isolated from tumor patients by focusing on RCC-EPCs. We extended our analysis at microscopic level by monitoring the sub-cellular structure of RCC-EPCs relative to their Ca2+ signalling fingerprint. Our results showed a striking functional and ultrastructural difference between RCC-EPCs and their normal counterparts, which might be the basis for designing novel, more specific anti-angiogenic treatments

Ultrastructural and functional differences between normal and tumor endothelial progenitor cells

Endothelial progenitor cells (EPCs) may be released from bone marrow to sustain the angiogenic switch that promotes tumor growth and metastatization of several solid cancers (Moccia et al., 2014). It has long been thought that tumor endothelium represents a rather stable structure, devoid of the genetic heterogeneity featuring neoplastic cells; however, more recent studies showed that tumor endothelial cells (TECs) present with an altered gene expression profile that bestows massive morphological and functional differences on them as compared to normal cells (Aird, 2012). Similarly, circulating EPCs isolated from individuals suffering from metastatic renal cellular carcinoma (mRCC) undergo a significant remodelling of their Ca2+ machinery, which is a master regulator of both angiogenesis and vasculogenesis. The present study clearly indicate that EPCs isolated from RCC (RCC-EPCs) and breast carcinoma (BC-EPCs) patients display ultrastructural and functional differences as compared to normal cells (N-EPCs)