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I FLAVONOIDI COME FONTE DI SOSTANZE ANTIOSSIDANTI: STUDI SU COLTURE CELLULARI UMANE
Get PDFUV radiation and in particular its UVB component, is an important environmental factor in the pathogenesis of skin aging and cancer.
One of the features of UVB-caused DNA damage is the formation of cyclobutane pyrimidine dimers and (6-4) photoproducts. Further, indirect DNA damage is also caused by increase in the level of reactive oxygen species (ROS) that cause oxidative damage reacting with DNA, proteins, fatty acids and saccharides. Such injuries result in a number of harmful effects: disturbance of cell metabolism, morphological and ultrastructural changes, alterations in the regulation pathways in differentiation, proliferation and apoptosis of skin cells. These processes can lead to photoaging and skin cancer development. In order to avoid UVB radiation damage, phytocompounds and antioxidants as photoprotectives has been considered. In recent years naturally occurring herbal compounds such as phenolic acids, flavonoids, and high molecular weight polyphenols have gained remarkable attention as strong protective agents. In particular different natural compounds prevent the occurrence and reduced the severity of UV-induced photoaging and diseases of the skin.
In the present study we have investigated the anti-oxidant and anti-apoptotic effect of forty-five phytochemicals in UVB-irradiated normal human keratinocytes and melanocytes.
In the first part of our study we assessed the activity of these compounds on UVB-irradiated normal human keratinocytes and melanocytes. These substances were submitted to a primary in vitro screening by MTT test in order to valuate proliferation rate. Subsequently, we examined in cultured epidermal cells the effects of UVB doses at 50mJ/cm2.
In particular 2’7’- dichlorodihydrofluorescein diacetate (DCF) assay was performed to determine formation of intracellular ROS. The results showed that kaempferol, 18β-glycyrrhetinic acid and glabridin presented interesting properties in both cell lines tested.
In the second part of our study we investigated the antiapoptotic effects of these three compounds in UVB-irradiated normal human keratinocytes and melanocytes by western blot and cell cycle analysis. The results showed that pre-treatment of human keratinocytes and melanocytes with these phytocompounds inhibited UVB mediated cell cycle arrest. This result was confirmed by western blot analysis of p53 and p21 protein levels. To further elucidate the molecular mechanism of phytocompounds, we have decided to investigate the extrinsic and intrinsic apoptotic pathways.
The results showed that pre-treatment of human keratinocytes and melanocytes with these phytocompounds inhibited UVB mediated apoptosis through involvement of bcl-2 and bid protein, caspase-8 and -9, inhibition of PARP cleavage.
Human skin is constantly exposed to the UV radiation present in sunlight. This may induce a number of phatobiological cellular changes. The development of novel preventive and therapeutic strategies depends on our understanding of the molecular mechanism of UV-damage. Phytochemical that were identified may be candidates for prevention of adverse effects of UV radiation on the skin and evaluation of there clinical efficacy is awaited.La radiazione UV, in particolare la sua componente dovuta agli UVB, è un importante fattore implicato nella patogenesi dell’invecchiamento cutaneo e del cancro. Uno degli aspetti legato al danno indotto dagli UVB è la formazione dei dimeri di ciclobutano pirimidina e dei fotoprodotti pirimidina (6-4) pirimidone. Inoltre, le radiazioni UV sono in grado di indurre un danno indiretto al DNA attraverso un incremento dei livelli delle specie reattive dell’ossigeno (ROS) che di conseguenza causano danno ossidativo reagendo con DNA, proteine, acidi grassi e zuccheri. Tali danni inducono diversi effetti avversi: disturbo del metabolismo cellulare, cambiamenti morfologici ed ultrastrutturali, attacco delle vie di regolazione e alterazioni nella differenziazione e proliferazione cellulare, apoptosi delle cellule cutanee.
Per evitare i danni indotti dalla radiazione UVB, sono stati presi in considerazione come agenti fotoprotettivi, fotocomposti e molecole antiossidanti. Negli ultimi anni molte ricerche hanno rivolto la loro attenzione alle molecole naturali estraibili dalle piante e caratterizzate da una elevata azione antiossidante. Si tratta di acidi fenolici, i flavonoidi e i polifenoli ad alto peso molecolare. In particolare è stato dimostrato che differenti composti naturali sono in grado di prevenire gli effetti e ridurre la severità del fotoinvecchiamento e le patologie cutanee indotte dagli UV. Nel presente studio sono stati investigati l’effetto antiossidante ed antiapoptotico di 45 fitoprodotti naturali in colture di cheratinociti e melanociti umani normali irradiati con raggi di tipo UVB.
Nella prima parte dello studio è stata valutata l’attività di queste molecole sui cheratinociti e melanociti umani normali. Le 45 sostanze sono state dapprima sottoposte in vitro al test MTT, per valutare l’eventuale attività citotossica. In seguito, sono stati esaminati gli effetti delle medesime molecole sulle cellule esposte radiazione UVB (50mJ/cm2).
In particolare è stato effettuato il saggio della 2’7’- diclorodiidrofluoresceina diacetato per determinare la formazione intracellulare di specie reattive dell’ossigeno (ROS). I risultati hanno evidenziato 3 molecole: il kampferolo, l’acido 18β-glicirretinico e la glabridrina caratterizzate da interessante attività protettiva in entrambe le linee cellulari testate.
Nella seconda parte di questo studio è stato valutato mediante western-blot e analisi del ciclo cellulare, il potenziale effetto antiapoptotico di questi tre composti nei cheratinociti e melanociti umani normali irradiati con raggi UVB.
I risultati mostrano che il pre-trattamento di cheratinociti e melanociti con questi fotocomposti inibisce l’arresto del ciclo cellulare normalmente mediato dagli UVB. Inoltre questo risultato è stato confermato attraverso l’analisi wester-blot dei livelli delle proteine p53 e p21.
Per meglio delucidare il meccanismo molecolare con cui i fotocomposti mediano l’apoptosi, sono state studiate le principali vie apoptotiche, estrinseca ed intrinseca. I risultati hanno confermato che il pre-trattamento dei cheratinociti e dei melanociti con questi fotocomposti inibisce l’apoptosi mediata dagli UVB coinvolgendo la down-regolazione di bcl-2 e di bid, il clivaggio delle caspasi -8 e -9 e di PARP.
La cute umana è costantemente esposta alle radiazioni UV emesse dai raggi solari. Queste possono indurre diversi cambiamenti cellulari con la possibilità di insorgenza di patologie anche gravi. Lo sviluppo di nuove strategie terapeutiche e di prevenzione dipende sicuramente dall’approfondimento delle nostre conoscenze sul meccanismo molecolare del danno indotto dai raggi UV. I fitoprodotti identificati in questo lavoro presentano valide caratteristiche per un loro ulteriore sviluppo come molecole utilizzabili nella prevenzione degli effetti avversi indotti dalle radiazioni UV sulla pelle; tuttavia la conferma della suddetta attività richiede una scrupolosa valutazione clinica
A new role of phase I and phase II enzyme in keratinocytes UVB induced apoptosis.
No full textA new role of Phase I and Phase II enzymes in keratinocytes UVB induced apoptosis.UVB is the major cause for cutaneous malignancies in the human population. The skin is able to activate anti-oxidants and enzymatic detoxification reactions to neutralize reactive photochemical products. Apoptosis, removing irreversibly DNA-damaged and potentially neoplastic cells, represents a major defence mechanism towards malignant transformation. Very little is known about the role of cutaneous cytochrome P450 (CYP450) isoenzymes and glutathione S-transferase (GST) in UVB response. Although not yet fully investigated, it has been studied a possible relationship between CYP450 inductors and UVB initiated apoptosis in rat hepatocytes. Altered high levels of GST has been directly correlated to resistance to chemotherapeutic drugs suggesting that GST plays a role in prevention of apoptosis. Our study is focused on phase I and phase II enzyme activities in normal human keratinocytes. In the first part of the study we demonstrated that CYP450 (1A1 and 2B1) and GST are induced not only by classical inducers such as β-naphthoflavone, 3-methylcholanthrene, phenobarbital but also by UVB radiations (50 mJ/cm2). Differentiated keratinocytes were employed for all the experiments as confirmed by immunoblotting with Cytokeratin 10, a specific marker of the basal spinous transition. In the second part we evaluated a possible involvement of these enzyme in UVB mediated apoptosis process. Western blot analysis of Bcl2 expression and PARP cleavage showed that inhibition of CYP450 by Proadifen prevented UVB induced apoptotic cell death. In contrast, the diuretic drug, ethacrinic acid, a GST inhibitor, was able to improve UVB induced apoptosis. In order to confirm the anti-apoptotic activity displayed by GST, when GST activity was increased by Phenobarbital, UVB apoptosis was prevented.These results suggest that UVB radiations may play a critical role as tumour promoters even through the regulation of CYP450 and GST metabolising enzymes
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
CYP450 and GST are induced by xenobiotics in normal human keratinocytes and play a different role in UVB-mediated apoptosis.
No full textSkin enzymatic detoxification systems may provide a barrier for protection against the external environment. UVB is the major cause for cutaneous malignancies in the human population. The skin is able to activate anti-oxidants and enzymatic detoxification reactions to neutralize reactive photochemical products. Very little is known about the behaviour of cutaneous metabolizing enzymes after ultraviolet B exposure. In the first part of the present study we analysed the induction and expression of cytocrome P450 (CYP450) phase I and gluthatione S transferase (GST) phase II enzymes in normal human keratinocytes in culture after exposure to classical inducers such as: β-naphthoflavone (BNF), 3-methylcholanthrene (MC), phenobarbital (PB) and also to ultraviolet B (UVB) radiation. Phase I enzymes (CYP450 1A1, 1A2, 1B2 and 2B1, 2B2) were investigated through 7-ethoxyresorufìn O-deethylase (EROD) and 7-pentoxyresorufìn O-depenthylase activities (PROD), while GST was analysed throught the coniugation of 1-chloro-2,4 dinitrobenzene with reduced gluthatione.MC, BNF and UVB exposure resulted in a dose-dependent and time dependent induction of CYP450 confirmed by immunoblotting assay. GST was induced by PB and inhibited by UVB. According to these results in the second part of this study we analysed the role of these enzymes in UVB induced apoptotic process. Apoptosis is a key mechanism in the cellular homeostasis and can be triggered by exogenous insults and in particular by genotoxic agents. The exact function of these enzymes in carcinogenesis remains unclear. Western blot analysis of Bcl2 expression showed that inhibition of CYP450 prevented UVB induced apoptotic cell death. On the contrary inhibition of GST, by ethacrynic acid, showed to improve UVB induced apoptosis. These experimental findings stress the value that CYP450 and GST enzymes may play an important role in keratinocytes apoptosis
Mepacrine antagonises tumour cell growth induced by natural polyamines.
No full textBACKGROUND: Mepacrine is an antiproliferative agent, characterised by an aliphatic chain similar to that of natural polyamines whose activation is closely associated with cell proliferation and may lead to malignant transformation and neurodegenerative diseases. This study aims to investigate a possible antagonism between mepacrine and polyamines in tumour proliferation. MATERIALS AND METHODS: MCF-7 and Vero cells were cultured in Eagle's minimum essential medium and then subjected to graded concentrations of putrescine, spermine and spermidine alone and in combination with mepacrine. Methyl thiazole tetrazolium test and Western-blotting were performed. RESULTS: Putrescine and spermidine at 0.5 mg/l significantly stimulated cell growth, whereas mepacrine treatment confirmed the enhanced p21 expression previously reported by a recent study and growth inhibition. When used in combination, mepacrine antagonized MCF-7 growth induced by polyamines. CONCLUSION: Our results suggest that mepacrine may represent a choice in the treatment of tumours induced by the modified concentration of polyamines
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