Poster
P1 - ANTIPROLIFERATIVE AND PROAPOPTOTIC EFFECTS OF NEW REVERSIBLE AND IRREVERSIBLE INHIBITORS OF EGFR ON NON SMALL CELL LUNG CANCER CELL LINES
Alfieri R.1, Cavazzoni A.1, Galetti M.1, Carmi C.2, Lodola A.2, Mor M. 2, Petronini P.G.1
1Department of Experimental Medicine, University of Parma
2Pharmaceutical Department, University of Parma
Introduction: Epidermal Growth Factor Receptor (EGFR) is an established new target for the treatment of epithelial tumors, including non-small cell lung cancer (NSCLC). Small molecules inhibitors, such as erlotinib and gefitinib, have been proven to be a useful addition to standard therapy in advanced NSCLC. Although these drugs have been effective, the accumulating clinical experience indicates that most patients develop resistances. Therefore, the main challenge in the treatment of NSCLC is the design of a new generation of inhibitors effective in the treatment of tumors become resistant to traditional reversible kinase inhibitors.
Aim: In this study we have investigated the effects of new reversible and irreversible EGFR inhibitors on a panel of NSCLC cell lines showing a wide range of sensitivity to gefitinib.
Results and Discussion: UPR1024 belongs to a new class of EGFR inhibitors, synthesized by our group, characterized by a hydantoin nucleus and a 5-benzylidene substituent. UPR1024 exerted antiproliferative and proapoptotic effects. The growth inhibitory effect was associated with an accumulation of the cells in the S phase of the cell cycle. Moreover, UPR1024 was capable of inducing DNA damage, associated with increased espression of p53, suggesting an additive mechanism of action. Treatment with UPR1024 resulted in an apoptotic cell death with the involvement of both the extrinsic and intrinsic pathways. UPR1024 features chemical properties that confer multiple mechanisms of action and can be considered a novel combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing DNA damage.
Combining knowledge from the field of cysteine protease inhibitors and EGFR-TK inhibitors, we have designed and synthesized new potentially irreversible EGFR inhibitors covalently binding the cysteine773 residue in the EGFR-TK catalytic site. The activity of a panel of these new potential irreversible inhibitors of EGFR tyrosine kinase activity have been tested on H1975 NSCLC cell line (carrying T790M point mutation which increases the affinity to ATP, causing resistance to reversible inhibitors). The most promising compound, UPR1206 was more effective than gefitinib in suppressing ligand-induced EGFR autophosphorylation and its downstream signaling such as PI3K/AKT/mTOR and MAPK pathways. Similarly, UPR1206 suppressed proliferation in this cell line and induced cell death by apoptosis. Our findings suggest that compounds which possess a new cystein-trap group can inhibit EGFR autophosphorylation and possess irreversible activity proving their role as new anti-EGFR tyrosine kinase inhibitors when gefitinib based therapy failed.
P2 - STUDY OF APOPTOTIC DOUBLE STRAND DNA CLEAVAGE: AN ULTRASTRUCTURAL APPROACH
Battistelli M.2, Burattini S.1, Ferri P.1, Sestili P.3, Rocchi M.B.1, Falcieri E.1,4
1Dip. di Scienze dell’ Uomo, dell’ Ambiente e della Natura e 3Dip. di Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo”; 2Lab. di Biologia Cellulare e Microscopia Elettronica e 1,4Ist. di Genetica Molecolare, CNR, Istituti Ortopedici Rizzoli, Bologna
Introduction.
Apoptosis is characterized by typical morphological changes and biochemical events. A common hallmark of apoptosis is considered DNA cleavage. Apoptotic DNA cleavage initially produces large fragments (50 kbp), followed by the formation of nucleosomic/oligonucleosomic ones. On the other hand, apoptosis without DNA fragmentation, at least the nucleosomic one, has been described (Zamai et al., 1996; Renò et al., 1998).
Materials and Methods.
To study the correlation between the DNA cleavage and the well known chromatin behaviour, we compared DNA gel electrophoresis analysis with the ultrastructural patterns of chromatin fragmentation revealed by the TUNEL technique applied to electron microscopy. Therefore, a modified TUNEL method, utilizing a gold-conjugated anti-digoxigenin antibody (Goping et al., 1999; Lossi et al., 2002), was carried out on UVB- or staurosporine-treated U937 monocytoid cells, in comparison to UVB- or staurosporine-treated Molt-4 T-lymphoid cells.
Results.
Gold particle density in the different domains of apoptotic nuclei was statistically evaluated. Gold labelling was more intense in dense apoptotic chromatin than in the diffuse one. U937 cells, which evidenced in vitro oligonucleosomic fragmentation after both treatments, revealed a significantly higher gold particle density, when compared with Molt-4, which did not, even if showing larger DNA fragments in vitro. DNA fragment sizes, characterized by gel electrophoresis and by FIGE, appeared closely correlated to gold particle density on apoptotic chromatin domains (Sestili et al., 1996).
Discussion.
TUNEL applied to electron microscopy is an useful tool to highlight mechanisms underlying apoptotic chromatin condensation and DNA cleavage patterns.
Finally, this technique is very useful to study apoptotic process, since it is able to demonstrate not only the presence of DNA fragmentation but also to detect a precise localization of DNA break points within the different chromatin domains (Burattini et al., 2009).
P3 - CARATTERIZZAZIONE DELLA CAPACITA’ DIFFERENZIATIVA DELLA LINEA CELLULARE MESENCHIMALE MURINA SR-4987
Bonomi A.1, Sisto F.1, Coccè V.1, Cavicchini L.1, Gribaldo L.2, Pessina A.1
1Dipartimento di Sanità Pubblica, Microbiologia, Virologia, Università degli Studi, via Pascal 36, Milano
2Molecular Biology & Genomics, IHCP, Joint Research Centre, Ispra (VA)
Abstract
Introduzione
I recenti progressi nell’utilizzo di cellule staminali mesenchimali (MSC) nell’ambito della medicina rigenerativa/riparativa hanno confermato la notevole versatilità di queste cellule e posto nuovi ed interessanti interrogativi circa la loro attività biologica, che può essere ulteriormente indagata e approfondita mediante modelli di MSC stabilizzate in vitro. La linea murina SR-4987, stabilizzata nel nostro laboratorio da colture a lungo termine di cellule stromali di midollo osseo, presenta caratteristiche tipiche delle MSC (morfologia, produzione di M-CSF, sensibilità a FGF-b), esprime markers B-linfocitari ed è in grado di produrre sarcomi in animali singenici.
Scopi
Scopo di questa ricerca preliminare è stata la raccolta sistematica dei dati biologici disponibili sulla linea e la verifica di alcuni importanti parametri (cinetica di crescita, capacità differenziativa in vitro e sensibilità a farmaci antitumorali e antinfiammatori).
Metodi
Determinazione della Population Doubling Time (PDT) mediante conta al Trypan Blue; valutazione della capacità differenziativa mediante stimolazione in vitro con terreni di coltura specifici per la differenziazione osteo-condro-adipogenica (in comparazione a MSC umane da midollo osseo); valutazione dell’effetto anti-proliferativo di farmaci con test MTT.
Risultati
I risultati mostrano che le cellule SR-4987 mantengono solo in parte la loro plasticità, differenziando in senso osteo-condrogenico ma non adipogenico. Di particolare interesse l’osservazione che queste cellule differenziano spontaneamente in osteoblasti, anche coltivate in comuni terreni di coltura. Le SR-4987 sono molto sensibili all’effetto antiproliferativo di 5-FU, Doxorubicina e Camptotecina mentre risultano resistenti a Paclitaxel. L’indice di resistenza a Paclitaxel riferito ai valori di IC50 di altre linee cellulari tumorali (carcinoma HT-29, leucemia MOLT-4 e glioma T98G) è di circa 20 volte.
Discussione
I dati preliminari confermano la peculiarità di questa linea, le cui caratteristiche sembrano indicare che la sua stabilizzazione/trasformazione sia avvenuta ad uno stadio differenziativo nel quale erano espressi sia recettori stromali che di cellule ematiche (supportando l’ipotesi dell’esistenza di un progenitore comune stromale-ematico). Partendo da queste osservazioni, sarà importante studiare più a fondo l’espressione genica di caratteri utili per comprendere sia la biologia di questa linea cellulare che, più in generale, delle MSC.
P4 - CHLOROQUINE POTENTIATES APOPTOTIC CELL DEATH INDUCED BY ENZYMATIC SPERMINE METABOLITES ON HUMAN CANCER CELLS
Condello M.1, Molinari A.1, Arancia G.1, Tempera G.2, Viceconte N.2, Saccoccio S.2, Agostinelli E.2
1Department of Technology and Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy 2Department of Biochemical Sciences ‘A. Rossi Fanelli’, University of Rome ‘La Sapienza’ and CNR, Biology and Molecular Pathology Institutes, Piazzale Aldo Moro 5, 00185 Rome, Italy
Introduction
Previous results demonstrated that the oxidation products, H2O2 and aldheyde, formed from the enzymatic system bovine serum aminoxidase (BSAO) and spermine, induced cytotoxicity on several tumor cell lines, in particular on multidrug resistant (MDR) ones (Agostinelli et al., 2006). Chloroquine (CQ) is a derivative of quinine and has been widely used as an anti-malarial and anti-inflammatory drug since 1950s. Some studies suggested that CQ, in addition to numerous other biological effects, is also able to sensitize cancer cells to a wide spectrum of cytotoxic drugs by inhibiting autophagic cell survival mechanism (Kim et al., 2009).
Aim
On these bases, aim of the present study was to investigate the possible therapeutic efficacy of an innovative combined treatment based on the use of CQ in association with BSAO/spermine. The study has been carried out in vitro on two tumor cell lines of different hystotype, melanoma M14 and LoVo colon adenocarcinoma and their MDR counterparts.
Methods
Both sensitive wild type (WT) and resistant counterpart of M14 and LoVo cells were cultured, pre-treated with CQ and then treated with BSAO/spermine, at various concentrations and incubation times. Cell survival was evaluated by the cloning efficiency assay. Morphological and ultrastructural changes were analyzed by transmission electron microscopy (TEM) and laser scanning confocal microscopy (LSCM). The expression of the apoptotic markers was evaluated by flow cytometry.
Results and discussion
The treatment with CQ alone (5-50 µM for 6-48 h, at 37°C) induced only a slightly inhibition of cell growth without appreciable signs of cell damage. When tumor cells were pre-treated with subcytotoxic concentration of CQ (5 µM for M14 and 20 µM for LoVo cells) and then incubated with 6 mM spermine in the presence of BSAO, in PBS-1% BSA, for 60 minutes at 37°C, the clonogenic assay showed that CQ was able to sensitize both WT and MDR cells to spermine metabolites. It was observed greater cytotoxicity on cells pre-treated with CQ than on those treated with BSAO/spermine alone. Transmission electron microscopy observations revealed that the treatment with CQ induced the appearance of numerous vacuoles and lysosomal structures inside the cytoplasm. The lysosomotropic property of CQ, confirmed by LSCM observations after acridine orange staining, seems to be the main reason for its sensitizing effect (Agostinelli and Seiler, 2007). Flow cytometric analysis indicated that the enhancement of the cytotoxic effect was accompanied by an increase of the apoptotic cells ratio in both cell lines. Further studies are in progress to verify if CQ is capable of exerting such an action by modulating the autophagic pathway.
The present study shows that CQ is able to potentiate the cytotoxic effect of the enzymatic oxidation products of spermine, and might represent a new and promising approach in anticancer therapy, particularly against MDR cancer cells (Agostinelli and Seiler, 2007).
References
Agostinelli E. et al. Biochem Biophys Acta 1763: 1040-1050 (2006).
Agostinelli E and Seiler N. Int J Oncol 31: 473-484 (2007).
Kim et al. Autophagy 5: 567-568 (2009).
P5 - PROTECTIVE ROLE OF ALDEHYDE DEHYDROGENASE 7A1 (ALDH7A1) IN HYPEROSMOTIC AND OXIDATIVE STRESS
Di Cesare Mannelli L., Cantore M., Ghelardini C., Failli P.
Dept. of Pharmacology - Viale Pieraccini, 6 - University of Florence
Introduction
Osmolalities exceeding the physiological range of 280-300 mosm/kg H2O have severe consequences as they induce cell shrinkage, oxidative stress, DNA damage, cell cycle delay, and ultimately apoptosis in the absence of protective mechanisms.
Aim: Since at least in the plants aldehyde dehydrogenase 7 (ALDH7) is involved in osmotic stress survival, in this project we have investigated: 1) if ALDH7A1, the human form of this ALDH7, protects mammalian cells from the toxic effect of hyperosmotic stress; 2) the mechanism of this protection.
Methods
The SV-40-transformed Chinese hamster ovarian cells were stably transfected with the mammalian expression vector pCEP4 alone (CHO-Vector) or with the human ALDH7A1 cDNA (CHO-ALDH7A1). Cell viability was measured by methyltetrazolium (MTT) assay, caspase 3 activity was evaluated by fluorescence measure, superoxide anion level and thiobarbituric acid reactive substances by spectrophotometric assays.
Results
In CHO-Vector and naïve cells, hyperosmotic stress induced by adding 400 mM NaCl (4 h) to isosmotic culture medium determined 1) cell shrinking; 2) production of superoxide anion; 3) lipid peroxidation; 4) increase of caspase 3 activity and 5) cell death. High expression of ALDH7A1 protected CHO cells from damages induced by hyperosmosis. In particular, ALDH7A1 expression reduced 1) cell shrinking; 2) superoxide anion level and lipid peroxidation by 50%; 4) caspase activity of about 85% and 5) increased cell viability from 40 to 60%. Moreover, cells high expressing ALDH7A1 were less exposed to lipid peroxidation induced by hypoxantine/xantine oxidase.
Discussion
Our data demonstrate that ALDH7A1 behaves as protector against hyperosmotic stress induced by high concentrations of NaCl in mammalian cells. This protection seems to be dependent on a reduction of oxidative attack due to superoxide anion. Moreover, ALDH7A1 protects cells from the oxidative stress induced independently from hyperosmosis, identifying a new antioxidant pathway in mammalian cells.
P6 - L’INIBIZIONE DELL’ATTIVITÀ DI HIF-1 CON OLIGONUCLEOTIDI ANTISENSO MIGLIORA LA RISPOSTA APOPTOTICA DI CELLULE DI CARCINOMA DEL COLON A 5FU E OXPT
Gariboldi M., Ravizza R., Molteni R., Monti E..
Dipartimento di Biologia Strutturale e Funzionale – Sezione di Farmacologia, Università dell’Insubria.
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I carcinomi colorettali rappresentano la seconda causa di morte fra tutti i tipi di tumore. La loro refrattarietà alla terapia è ascrivibile a diversi fattori, tra cui la presenza di aree ipossiche nella massa tumorale e i meccanismi adattativi che le cellule tumorali attivano in queste condizioni. Strategie terapeutiche in grado di colpire o sfruttare tali meccanismi potrebbero quindi manifestare un effetto antitumorale selettivo e trovare impiego anche sui tumori colorettali. Il grado di ipossia intratumorale è stato correlato positivamente all’espressione del fattore indotto da ipossia-1 (HIF-1), un fattore di trascrizione formato da due subunità, HIF-1α, regolabile, e HIF-1β, espressa costitutivamente. La riduzione della pressione parziale di ossigeno nel microambiente cellulare determina una stabilizzazione dei livelli di HIF-1a , cui fa seguito la sua traslocazione nel nucleo, dove avviene la formazione del dimero HIF-1a/b che lega le sequenze HRE (hypoxia response element) sui promotori dei geni bersaglio, attivandone la trascrizione. I geni target per HIF-1 sono coinvolti nella sopravvivenza e proliferazione cellulare, nella resistenza all’apoptosi, nell’aumento dell’angiogenesi e quindi nella progressione del tumore verso un fenotipo più aggressivo e maligno.
Nel presente studio abbiamo investigato gli effetti della modulazione di HIF-1 sulla risposta di due linee cellulari di adenocarcinoma del colon al 5-fluorouracile (5FU) e all’oxaliplatino (oxPt), in condizioni di ipossia; abbiamo inoltre valutato la possibilità di migliorare la risposta pro-apoptotica ai tre farmaci inibendo HIF-1a mediante la transfezione delle cellule con uno specifico oligonucleotide antisenso.
I risultati ottenuti mostrano che l’incubazione delle cellule in ipossia induce un aumento dei livelli di HIF-1a, valutato mediante analisi western blot, e dell’attività trascrizionale di HIF-1, valutata mediante analisi citofluorimetrica di cellule HCT116 e H630 transfettate con un vettore plasmidico contenente il gene della GFP posto a valle della sequenza HRE. . L’aumento dell’attività di HIF-1 è associato alla diminuzione della risposta apoptotica delle due linee cellulari al 5FU e oxPt e l’inibizione dell’espressione di HIF-1 a induce un aumento della percentuale di cellule apoptotiche in seguito al trattamento con i due farmaci, in ipossia.
I dati presentati confermano un ruolo causale di HIF-1 nella scarsa risposta apoptotica in ipossia al 5FU e oxPt ed evidenziano come interventi volti a ridurne l’attivazione potrebbero migliorare la risposta dei carcinomi colorettali alla chemioterapia.
P7 - PIRAZOLCARBOSSIAMIDI E MORTE CELLULARE: UNA NUOVA ARMA PER LA CHEMIOTERAPIA
Giansanti V.1, Camboni T.1, Tillhon M.1, Parks M.1, Prosperi E.1, Santin G.2, Piscitelli F.3, La Regina G.3, Silvestri R.3, Scovassi A.S.1
1Via abbiategrasso 207, 27100 Pavia, Istituto di Genetica Molecolare CNR, 2P.zza Botta 10, 27 100 Pavia, Dipartimento di Biologia Animale, Università di Pavia; 3 P.le Aldo Moro 5 00185 Roma, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma Italy.
Abstract:
Introduzione e scopo del lavoro.
Numerosi composti avente attività anti-tumorale sono caratterizzati dalla presenza del pirazolo come nucleo centrale. Dopo aver sintetizzato un pannello di pirazolcarbossiamidi, ci siamo concentrati sul nuovo composto RS 2780 (N-2-feniletil 1-(4-clorofenil)-3-metil-5-pirrolilpirazole-4-carbossiamide) i cui effetti biologici sono stati valutati su cellule umane tumorali e non, allo scopo di valutarne l’eventuale citotossicità e il meccanismo d’azione.
Metodi.
Saggi di proliferazione (LD50) e di citotossicità (MTT), citofluorimetria a flusso, immunocitochimica, western blotting, elettroforesi in campo pulsato e convenzionale su gel d’agarosio. Linee cellulari umane utilizzate: HeLa (carcinoma della cervice uternina), SW613-B3 (coloncarcinoma), FO46 (fibroblasti non tumorali).
Risultati e discussione.
Un trattamento di 24 ore con concentrazioni crescenti di RS 2780 (da 0.1 a 100 <M) è in grado di inibire la proliferazione cellulare e di ridurre la vitalità. Il nuovo composto è risultato particolarmente attivo anche sulla linea cellulare SW613-B3, notoriamente resistente a molti chemioterapici, mentre non lo è sulla linea di fibroblasti non tumorali FO46. I nostri esperimenti ci hanno dimostrato che RS 2780 interferisce con le proprietà funzionali e strutturali dei mitocondri, portando così all’attivazione della via apoptotica mitocondrio-dipendente. L’induzione del meccanismo apoptotico è stata dimostrata dalla presenza di alcuni marcatori tipici come la condensazione della cromatina, la frammentazione internucleosomica del DNA, la proteolisi della PARP-1 e l’attivazione delle caspasi-3 e -9. In conclusione, i nostri risultati dimostrano per la prima volta le capacità antiproliferative del nuovo composto RS 2780 sulle cellule HeLa e SW613-B3, indicando che esso è in grado di indurre selettivamente la via mitocondriale dell’apoptosi. Per tale motivo, RS 2780 è stato scelto come capostipite per una nuova generazione di composti; alcuni suoi derivati si sono già mostrati più attivi del composto madre su varie linee cellulari tumorali umane.
P8 - EXPRESSION OF THE MAIN ANTIAPOPTOTIC BCL-2 GENE IS MODULATED AT POST-TRANSCRIPTIONAL LEVEL BY TINO/MEX-3D PROTEIN: UNRAVELLING ITS FUNCTION ACTIVITIES BY MEANS OF ITS C. ELEGANS ORTHOLOGOUS ceMEX-3
Loffredo R., Donnini M., Witort E., Granucci I., Palterer B., Capaccioli S. and Lulli M.
Department of Experimental Pathology and Oncology, University of Florence and Phoenix ONLUS Stem Cell Foundation for Human Life
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Introduction
Tino is an RNA-binding protein that belongs to the novel, evolutionarily conserved family of hMex-3 proteins involved in RNA metabolism. The functions of Tino/hMex-3, firstly identified in our lab as a post-transcriptional regulator able to bind to the AU-Rich Element (ARE) of bcl-2 mRNA, are largely unknown. Instead, its C. elegans ortholog ceMex-3 is known to be involved in RNA metabolism during nematode oogenesis/embryogenesis, and affects the spatial/temporal expression of the pal-1 gene. In turn, PAL-1 protein inhibits ceMex-3 expression in the early embryo, thereby specifying the fate of posterior blastomers. Though correlation between ceMex-3 and PAL-1 is definite, PAL-1 can be expressed even in the presence of high levels of ceMex-3. The lack of ceMex-3 and GLD-1, a general translational repressor during oogenesis, induces transdifferentiation of germ cells into neuronal and muscle cells, which leads to the onset of “worm teratomas”. Therefore, ceMex-3 and GLD-1 cooperate to maintain totipotency in nematode germ line by modulating common RNAs at the post-transcriptional level and suggests that their altered cooperation could impact on early phases of cell differentiation. The high conservation between the nematode ceMex-3 and the human Tino/hMex-3 suggests that also Tino/hMex-3 could impact on mammalian embryogenesis, cell polarity and cancer.
Aims
Our aims were to disclose further Tino/hMex-3 biological functions, by: 1) identification of bound transcripts and verification of its effect on their fate; 2) exploration of its role in the early phases of mammal embryogenesis; 3) analysis of Tino/hMex-3 knockout in mouse model.
Methods
The Tino target mRNAs were identified by bioinformatic analysis and the effect of recombinant Tino (Tino-his) on steady-state levels of a subset of them was assessed by quantitative RT-PCR. The levels of Tino mRNA or protein on several heterogeneous tissues and cell types of adult and embryo mouse as well of human have been evaluated by in situ hybridization or immunohistochemistry analysis, respectively.
Results. Results of in situ hybridization gave a map of Tino mRNA levels in different tissues of adult (8 weeks) and embryo (8,5 days) mouse, giving fundamental clues to Tino function in development. In particular, the positive detection of Tino in testicles and some areas of embryo, such as somites and brain mesenchymal cells strongly suggests its involvement on spermatogenesis and embryogenesis. Results of immunohistochemistry indicate that Tino is a cytoplasmic protein restricted to cells of mesenchymal origin. Furthermore, although transient transfection of HEK293 cells with pQE-Tino-his did not affect Bcl-2 mRNA levels, it markedly lowered the levels of BCL-2 protein, which suggested Tino could be a translational repressor in analogy to ceMex-3, its ortholog in C. elegans.
Discussion
The detection of Tino mRNA and protein in mouse embryo tissues suggests that it could play a pivotal role in early embryogenesis, while its presence in human adult tissues of mesenchymal origin is in keeping with the role of ceMex-3 in muscle development.
Acknowledgements
We are thankful to AIRC, MUR, ECR Firenze, FCR Lucca, Agenzia Spaziale Italiana (ASI).
P9 - THE EFFECT OF SENESCENCE AND CRYOPRESERVATION ON ADIPOSE-DERIVED MESENCHYMAL STEM CELLS
Martinello T.1, Bronzini I.1, Maccatrozzo L.1, Mollo A.2, Mascarello F.1, Sampaolesi M.3 and Patruno M.1
1Department of Experimental Veterinary Sciences, University of Padova, Italy. 2Department of Clinical Veterinary Sciences, University of Padova, Italy. 3Stem Cell Research Institute, University Hospital Gasthuisberg, Leuven, Belgium.
Canine adipose tissue represents an ideal source of autologous mesenchymal stem cells because of its wide distribution and availability by means of simple surgical procedures. Canine adipose tissue-derived mesenchymal stem cells (cA-MSC) have been shown to possess the capacity to differentiate into mesenchymal lineages although their full characterization is still at an early stage. During in vitro expansion, stem cells are damaged due to intracellular and extracellular influences becoming replicatively senescent. This fact restricts their proliferation and differentiation efficiency and limits the yield of numbers of cells needed for regenerative therapy.
In this study, we aimed to determine the effect of senescence and cryopreservation on “stemness” and differentiative potential of cA-MSC. The latter cells were serially passaged until reached the maximal life span. Cell growth, morphology, telomerase activity, senescence and apoptotic markers were determined and compared between early and late passages.
In order to avoid negative consequences of senescence we analyzed the effect of cryopreservation on cA-MSC. Each sample was analyzed immediately and after being frozen in liquid nitrogen for 10-12 months. After cryopreservation, cells conserved their fibroblast-like morphology and alkaline posphatase positivity but showed lower proliferation, evaluated during 40 days, and lower telomerase activity. The cryopreservation did not alter the CD expression since both fresh and frozen cA-MSC expressed CD44, CD90, CD117 and CD140a, but not CD34 and CD45. Moreover, freezing and storing cA-MSC did not change the adipogenic, osteogenic and myogenic differentiative potential, as evidenced by histochemistry, immunofluorescence and PCR expression analysis. Our data demonstrate that cA-MSC may represent, in veterinary medicine , a promising type of progenitors cells in autologous cellular-based therapies even after a long term storage.
P10 - SELECTIVE ASCORBATE TOXICITY TO MALIGNANT MESOTHELIOMA
Martinotti S., Ranzato E., Burlando B.
Department of Environment and Life Sciences, DiSAV,
University of Piemonte Orientale “Amedeo Avogadro”, viale T. Michel 11, 15121 Alessandria, Italy
Introduction
Malignant mesothelioma (MMe) is a lethal tumor arising from the mesothelium of serous cavities. This cancer shows a close relationship with asbestos exposure and its incidence has been increasing in several countries as a result of widespread use of asbestos. The treatment of MMe is extremely problematic and there is an urgent need to find new therapeutic approaches.
Aims
Ascorbate is an essential nutrient to the human diet, but it is also widely used by people as a medicinal product. About 50 years ago, it was hypothesized that ascorbate could be used in cancer therapy. In this study, we explored the possibility of using ascorbate as a chemotherapeutic agent in the treatment of MMe.
Methods
We evaluated the cytotoxicity of ascorbate on different human MMe cell lines by using neutral red uptake, and compared these results with those obtained on normal mesothelial cells. By using confocal imaging of reactive oxygen species production and the cytochrome c superoxide assay, we also tried to disclose the mechanism of ascorbate toxicity and of its possible selectivity towards tumor cells.
Results
Our results showed that ascorbate is more toxic to MMe cells than to normal mesothelial cells, and revealed that ascorbate selective toxicity is due to a redox mechanism involving extracellular H2O2 production combined to higher rates of intracellular superoxide production in MMe cells than in mesothelial cells.
Perspectives
Since best results in chemotherapy are generally obtained with combinations of different compounds, we are focusing on the evaluation of the cytotoxic effect of some common chemotherapeutic drugs used in MM therapy such as cis-platin, etoposide, gemcitabine, imatinib and paclitaxel. Effective concentration values of these drugs will be used to evaluate ascorbate-drug interactions through isobologram analysis. This method will allow to point out possible synergistic interactions to be further tested in pre-clinical studies.
P11 - IL BLOCCO IN G2/M INDOTTO DAL TASSOLO SENSIBILIZZA LE CELLULE DI EPATOCARCINOMA UMANO HUH7 ALL’APOPTOSI DA TNFa
Minero V.G., De Stefanis D., Sponton L., Costelli P., Bonelli G., Baccino F.M.
Dipartimento di Medicina ed Oncologia Sperimentale, Università di Torino, Italia
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Introduzione
Il tassolo (TAX) è un potente agente antiproliferativo che altera l'equilibrio dei microtubuli bloccando il ciclo cellulare in fase G2/M, con conseguente morte per apoptosi attraverso meccanismi diversi. Per questa ragione il TAX viene utilizzato nella terapia oncologica, e acluni studi condotti su topi nudi portatori di tumori solidi hanno proposto che l’effetto antineoplastico del TAX potrebbe essere rafforzato dall’associazione con la citochina pro-infiammatoria TNFα.
Scopo
Questo studio era volto a valutare se l’apoptosi indotta da TAX aumenti in seguito all’associazione con il TNFα.
Metodi
Le cellule Huh7 sono state trattate con TAX (range: 0,1-5 µM), Colchicina (5 µM) e Nocodazolo (0,2 µg/ml) in presenza o in assenza di TNFα (15 ng/ml) e, dove indicato, pretrattate con diversi inibitori delle caspasi (20 µM). La distribuzione nelle diverse fasi del ciclo cellulare è stata analizzata mediante citofluorimetria a flusso. L’espressione di caspasi-3 è stata valutata con western blotting e l’attività delle proteasi tramite saggio enzimatico. L’espressione in membrana del TNF-R1 è stata analizzata con immunofluorescenza.
Risultati
Il trattamento con TAX-TNFα induce alterazioni morfologiche decisamente più marcate del solo TAX. I risultati ottenuti dimostrano che l’inibitore ad ampio spettro delle caspasi (z-VAD-fmk) e l’inibitore specifico di caspasi-8 (IETD-CHO) proteggono le cellule Huh7 dall’apoptosi indotta da TAX-TNFα. In particolare, la morte è associata ad attivazione delle caspasi-8 e -3. Quest’ultima, tuttavia, non sembra contribuire significativamente all’apoptosi, in quanto il trattamento delle colture con DEVD-CHO non modifica la percentuale di cellule morte. L’immunofluorescenza per il TNF-R1 di membrana ha dimostrato che le cellule bloccate in fase G2/M emettono un segnale molto più intenso rispetto ai controlli.
Conclusioni
Questi risultati dimostrano che il TAX sensibilizza le cellule di epatocarcinoma Huh7 all’azione del TNFα. Questo effetto sembra dovuto al blocco in G2/M indotto dal TAX, e potrebbe essere associato all’aumento dell’espressione in membrana del TNF-R1. L’apoptosi indotta dal trattamento con TAX-TNFα è caspasi-dipendente, anche se non è chiaro quale sia la caspasi effettrice. Il potenziamento dell’apoptosi ottenuto combinando il TAX al TNFα costituisce un risultato promettente, che indirizza lo studio verso una sperimentazione in vivo su tumori epatici murini.
P12 - RESTORING DRUG-INDUCED APOPTOSIS IN HYPOXIA-TREATED TUMOR CELLS BY MODULATING HIPK2/P53 AND HIF-1a PATHWAYS
Nardinocchi L.1,2, Puca R.1,2, Sacchi A.1, D’Orazi G.1,2
1Department of Experimental Oncology, Molecular Oncogenesis Laboratory, National Cancer Institute Regina Elena, Rome, Italy; 2Department of Oncology and Neurosciences, University “G. d’Annunzio”, Chieti, Italy
Introduction
Solid tumors can survive hypoxic condition by using protective mechanisms including the activation of hypoxia-inducible factor 1a (HIF-1a), a transcription factor involved in cell proliferation, angiogenesis, and chemoresistance. Moreover, hypoxia attenuates the pro-apoptotic response of oncosuppressor p53 to cellular damage and drug treatment. The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) by phosphorylating serine 46 (Ser46) and neutralizing MDM2 inhibition is a crucial regulator of p53 pro-apoptotic function. We have recently shown that HIPK2 is also a transcriptional co-repressor of HIF-1a and that, inhibition of HIF-1a activity by HIPK2 stimulates drug-induced apoptosis in p53-dependent and-independent ways. Given its central role in the targeting of cells towards apoptosis upon genotoxic stress, all the conditions that lead to HIPK2 deregulation would end in a multifactorial response leading to tumor chemoresistance by strongly affecting p53 transcriptional activity and apoptosis on one hand and HIF-1 activity on the other hand. HIPK2 can be deregulated in tumors by several mechanisms. Recent studies have shown that HIPK2 is degraded via the proteasome pathway by p53-induced MDM2 and by hypoxia-induced proteins.
Aim
The purpose of our investigation was to evaluate whether hypoxia could deregulate the HIPK2-induced p53 dependent apoptotic transcriptional activity in response to drug and therefore contribute to chemoresistance and whether zinc could counteract the HIPK2/p53Ser46 inhibition, in order to provide evidence for the involvement of both HIPK2 and p53 in counteracting hypoxia-induced chemoresistance.
Results
Upon exposure of colon and lung cancer cells to hypoxia, by either low oxygen or cobalt, HIPK2 function was impaired allowing for increased HIF-1a expression and inhibiting the p53-apoptotic response to drug. Hypoxia induced expression of the p53 target MDM2 that downregulates HIPK2, thus MDM2 inhibition by siRNA restored the HIPK2/p53Ser46 apoptotic response to drug. Surprisingly, zinc supplementation to hypoxia-treated cells increased HIPK2 protein stability and nuclear accumulation, leading to restoration of HIPK2 binding to HIF-1a promoter, repression of HIF-1 pathway, and activation of the p53 pro-apoptotic response to drug. Combination of zinc and ADR strongly suppressed tumor growth in vivo by inhibiting HIF-1 pathway and upregulating p53 apoptotic target genes.
Discussion
We show here for the first time that hypoxia-induced HIPK2 deregulation was counteracted by zinc that restored HIPK2 suppression of HIF-1 pathway and reactivated p53 apoptotic response to drug, underscoring the potential use of zinc supplementation in combination with chemotherapy to address hypoxia and improve tumor treatment.
P13 - ACTIVATION OF THE HISTAMINERGIC H3 RECEPTOR INDUCES PHOSPHORYLATION OF THE AKT/GSK-3B PATHWAY IN CULTURED CORTICAL NEURONS AND PROTECTS AGAINST NEUROTOXIC INSULTS
Passani M.B., Scartabelli T., Blandina P., Pellegrini-Giampietro D. and Mariottini C.*
Dipartimento di Farmacologia Preclinica e Clinica, Universita’ di Firenze, Viale Pieraccini 6, 50139 Firenze
*Present address: Dept. of Pharmacology and System Therapeutics, Mount Sinai School of Medicine, One Gustave Levy Place, 10029 New York NY (USA)
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Stimulation of histamine H3 receptors (H3R) activates Gi/o-proteins that inhibit adenylyl cyclase and triggers several intracellular pathways such as MAPK and phospholipase A2. In a previous study we showed that H3R-mediated phosphorylation of Akt at Ser473 occurs in primary cultures of rat cortical neurons1, but neither the physiological significance of H3R-induced Akt activation nor the intracellular events associated with Akt phosphorylation were studied. In this report we address these questions. Using Western blot analysis we characterized the H3R-dependent activation of the Akt/GSK-3b axis in primary rat cortical neurons. We also investigated the ability of the H3R activation to modulate the activity of antiapoptotic pathways and to prevent neuronal damage in two distinct models of neurotoxicity.
Western blotting experiments showed that H3R-mediated activation of Akt in cultured rat cortical neurons depends on phosphoinositide-3-kinase and mitogen-activated-protein-kinase-kinase (MEK). H3R activation phosphorylated, hence inactivated, the Akt downstream effector glycogen-synthase kinase-3b (GSK-3b), increased the expression of the antiapoptotic protein Bcl-2 and protected cultured rat and mouse cortical neurons from neurotoxic, NMDA-mediated insults in a dose-dependent manner. All these effects were inhibited by the H3R antagonist inverse/agonist thioperamide. Mouse cortical cells expressed H3R as revealed by immunostaining experiments, and stimulation of H3R phoshorylated Akt and decreased caspase-3 activity.
In the CNS, the Akt/GSK-3b axis plays a prominent role in several brain functions. Evidence links increased GSK-3b activity with Alzheimer’s disease2; in a mouse model of this neurodegenerative disease, downregulation of GSK-3b-conditional-overexpression diminishes neuronal death and cognitive deficits3 and reduced Akt function has been reported in schizophrenic patients4.
Hence, we uncovered a yet unexplored neuroprotective action of the H3R. Our results suggest that H3R stimulation may have relevance in the treatment of, e.g., ischemia or neurodegenerative diseases such as schizophrenia or Alzheimer’s disease.
1Bongers et al. 2007 J Pharmacol Exp Ther 323, 888–898
2Jope and Johnson 2004 Trends Biochem Sci 29, 95–102
3Engel et al. 2006 J Neurosci 26, 5083–5090
4Emamian et al. 2004 Nat Genet 36, 131–137
P14 - COINVOLGIMENTO DELLO STRESS DEL RETICOLO E DEL PROCESSO AUTOFAGICO NELL’APOPTOSI INDOTTA DAL CANNABINOIDE SINTETICO WIN IN CELLULE DI EPATOMA UMANO IN COLTURA
Pellerito O., Portanova P., Notaro A., Calvaruso G., Giuliano M., Tesoriere G.
Dipartimento di Scienze Biochimiche, Università degli Studi di Palermo, Policlinico, Via del Vespro 129, Palermo
Introduzione
Studi da noi condotti precedentemente hanno dimostrato la capacità del cannabinoide sintetico WIN di indurre apoptosi in cellule di epatocarcinoma umano HepG2 attraverso un meccanismo, dipendente dal fattore trascrizionale PPARg, che prevede riduzione dei livelli di alcuni fattori di sopravvivenza e attivazione di fattori pro-apoptotici della famiglia Bcl-2 (M. Giuliano et al. Biochimie. 2009). Recentemente è, inoltre, emerso che in cellule di glioma i cannabinoidi possono stimolare l’apoptosi attraverso induzione di stress del reticolo endoplasmatico seguito da autofagia.
Scopo
L'obiettivo del presente studio è stato quello di valutare il coinvolgimento dell’autofagia nel percorso di morte indotto dal WIN in cellule HepG2 e la dipendenza dell’apoptosi da tale evento.
Metodi
I livelli dei fattori coinvolti nella risposta all’ER stress e nel meccanismo autofagico sono stati analizzati mediante Western blotting e RT-PCR. La formazione dei vacuoli autofagici è stata valutata marcando le cellule con il composto autofluorescente monodansylcadaverina (MDC) e successiva osservazione al microscopio a fluorescenza.
Risultati
Il trattamento delle cellule HepG2 con 10 mM WIN determina, già a tempi precoci (8-16 ore), up-regulation di p8 e CHOP, due fattori con attività pro-apoptotica associati alla risposta allo stress del reticolo. Parallelamente, WIN induce la marcata caduta dei livelli di AKT e della sua forma fosforilata attiva. Poichè è noto che l’inibizione di AKT può promuovere l’autofagia causata dalla mancata attivazione di mTORC1 AKT-dipendente, abbiamo investigato la formazione dei vacuoli autofagici. WIN determina in cellule HepG2, dopo 8 ore di trattamento, la comparsa di un elevato numero di vacuoli autofagici, visibili come granuli brillanti dopo colorazione con MDC. Parallelamente si osserva, dopo trattamento, un marcato incremento nei livelli della forma lipidata attiva di LC3 (LC3-II), un marker della formazione dell’autofagosoma. Per confermare la relazione tra induzione dell’ER stress, autofagia e apoptosi WIN-dipendente, sono stati condotti esperimenti di silenziamento genico su CHOP. Tali studi hanno indicato che la down-regulation di CHOP contrasta la riduzione dei livelli di AKT e parallelamente la citotossicità indotta dal WIN.
Conclusioni
I risultati riportati, sebbene preliminari, sembrano indicare che l’autofagia sia parte del meccanismo attraverso il quale WIN induce apoptosi nelle cellule di epatoma.
P15 - PARP INHIBITION INCREASES THE DNA DAMAGE AT TELOMERES INDUCED BY THE G-QUADRUPLEX LIGAND RHPS4 AND IMPROVES THE THERAPEUTIC EFFICACY OF IRINOTECAN/RHPS4 COMBINATION ON COLON CANCER XENOGRAFT
Porru M.1, Salvati E.1, Rizzo A.1, Scarsella M.1, Tentori L.2, Graziani G.2, D’Incalci M.3, Stevens M.F.G.4, Zupi G.1, Biroccio A.1 and Leonetti C.1
1Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute and 2Department of Neuroscience, University of Rome “Tor Vergata, Rome, Italy; 3Department of Oncology, Pharmacological Research Institute “Mario Negri”, Milan, Italy; 4Center for Biomolecular Sciences, School of Pharmacy, the University of Nottingham, Nottingham UK
Background and Aim
The G-quadruplex ligand RHPS4 is known to rapidly induce an ATR driven DNA damage response at telomeres, specifically interfering with telomere replication. Poly (ADP-ribosyl)ation is involved in the regulation of many cellular processes including DNA replication and damage repair. In the last years a number of PARP inhibitors were generated and the synergistic effect of some of those in combination with classic chemiotherapeutic agents was described. We recently showed that the combination of RHPS4 with camptothecins has a strong synergistic interaction in vitro on colon cancer lines and produced a marked antitumor activity on xenografts, which is in agreement with the proposed model in which RHPS4 interferes with telomere replication. Unfortunately, this combination failed to cure animals as no complete remissions were reported. So, based on the key role of PARP in DNA replication and on DNA damage response activation and on the observation that PARP inhibitors sensitize tumor cells to camptothecins, we evaluated a multicomponent strategy based on the addition of the PARP-1 inhibitor GPI 15427 treatment to the previously established camptothecins followed by RHPS4 combination, with the aim to search for a more effective anticancer therapy.
Results and conclusions
We observed that RHPS4 treatment produced a rapid induction of PARP activity and that the ADP ribose polymers induced by RHPS4 are completely localized at telomeres. Furthermore RHPS4 treated cells showed an increased recruitment of the PARP protein at telomeres as an increased number and the intensity of PARP spots colocalized with TRF1 thus providing a strong rational for the combination between RHPS4 and PARP inhibitors. The RHPS4 treatment rapidly induces the formation of TIFs and the combination with GPI 15427 a huge increase of the number of TIFs in TIF positive cells, thus suggesting a cooperative effect between the two molecules. As a consequence of, the combination with GPI 15427 was able to increase the ability of RHPS4 in inhibiting the number of colonies of HT29 cells compared to the treatment with RHPS4 alone. The in vivo experiments demonstrated the high efficacy of the combination of GPI 15427 with Irinotecan and RHPS4 as this treatment produced an impressive inhibition of tumor weight and a marked tumor growth delay. Notably, after the triple combination a complete regression of tumors was observed in most of the mice treated accompanied by a significant increase of overall survival. In conclusion, these data suggest that the targeting of PARP is a promising strategy to improve the response of solid tumors to antitumoral agents.
P16 - PLATELET LYSATE-DRIVEN WOUND HEALING: MECHANISMS OF ACTION
Ranzato E.1, Martinotti S.1, Mazzucco L.2, Patrone M.1, Burlando B.1
1Department of Environment and Life Sciences, DiSAV, University of Piemonte Orientale “Amedeo Avogadro”,
viale Teresa Michel 11, 15121 Alessandria, Italy
2Department of Haematology & Blood Transfusion Medicine, Azienda Ospedaliera Nazionale SS. Antonio e Biagio e C. Arrigo, via Venezia 16, 15121 Alessandria, Italy
Introduction
Wound healing requires a number of interactions among various cells and their mediators, resulting in an overlapping series of events such as coagulation, inflammation, epithelialization, matrix formation and remodelling.
Aims
Underhealing or hyperhealing sores are a significant challenge to public health service, since conventional treatments are poorly effective and the patient’s quality of life is impaired. Hence, great interest has been attracted by new-generation therapies providing healing acceleration and reducing wound complications.
Methods
Growth factors are known to promote wound healing, and the delivering of concentrated amounts of growth factors to the wound site can be obtained by the use of platelet derivatives. We have studied the effects of a platelet lysate (PL) obtained from repeated freezing-thawing of platelet-enriched blood samples.
Results
By using in vitro scratch wound models of skin, endothelial, and myoblast cells we have shown that PL promotes wound closure by stimulating cell proliferation and motility. By studying the activation of cell signalling, we have found that the mechanism of action of PL involves cell Ca2+, MAP kinases and PI3K/Akt pathways, although with different patterns in different cell types. By means of chromatographic separation and mass spectrometry we have distinguished PL fractions that induce strong wound-promoting effects from others that are almost uneffective.
Perspectives
Taken together, our results demonstrate that the mechanism of PL-induced wound healing consists in a complex activation involving different cell types and cellular activities, such as proliferation, locomotion and matrix remodelling. These data provide a scientific basis for the development of more accurate clinical applications of platelet derivatives.
P17 - HIV AND APOPTOSIS OF CANCER CELLS: THE KILLER’S PROMISES
Ruggiero M., Punzi T., Morucci G., Pacini S.
Dipartimento di Patologia e Oncologia Sperimentali, Viale Morgagni 50, 50134, Firenze
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INTRODUCTION
It is estimated that HIV has been in humans for more than 100 years, thus establishing a delicate survival balance (Curr Opin HIV AIDS. 2009; 4: 247-52). In fact, HIV-produced Vpr protein is cytotoxic against a number of different tumor cells, and in vivo studies have indicated an anti-cancer effect mediated by Vpr (Curr HIV Res. 2009; 7: 144-52).
OBJECTIVE
To demonstrate that the anti-tumor properties of HIV are responsible for establishing a symbiotic relationship in humans. METHODS. Meta-analysis of studies showing anti-tumor activity mediated by HIV proteins and peptides.
RESULTS
Vpr induces selective killing of rapidly dividing cells (Curr Drug Deliv. 2004; 1: 335-44), and Vpr-mediated apoptosis was observed in all tumor cell lines tested (Cancer Cell Int. 2009; 12; 9:20). In vivo, a dramatic example of anti-tumor activity is the Vpr-induced inhibition of melanoma growth and the induction of complete tumor regression coupled with long-term survival of mice in a highly aggressive and metastatic solid tumor model (Mol Ther. 2006;14: 647-55). It is worth noting that free Vpr is detectable in the serum of HIV patients, and in vitro studies implicate extracellular forms of Vpr as an effector of cellular responses mediated through its ability to transduce through intact cytoplasmic membranes (DNA Cell Biol. 2002; 21: 679-88). These results suggest that HIV infection could be associated with reduction of the risk of developing neoplasms, provided that the patient does not assume toxic drugs. In fact, HAART increases the risk of developing cancer and its potential oncogenicity is under investigation (Curr HIV/AIDS Rep. 2008 5: 140-9. Curr Opin Oncol. 2008 20: 534-40). DISCUSSION. These data could be interpreted as follows: on one hand, HAART itself might be involved in the development of cancer, in particular lung cancer, and may not have a beneficial effect on either the incidence or outcome of the lung cancer (Curr Opin Oncol. 2008; 20: 529-33). On the other hand, HAART-induced reduction of viremia could decrease HIV-associated anti-tumor activity. Thus, HIV-associated anti-tumor activity could be responsible for its symbiotic relationship with humans that has led to its persistence; anti-tumor activity could also be responsible for the fact that, despite the potential for different divergent viruses to spread, surprisingly few viruses successfully expanded in humans (Curr Opin HIV AIDS. 2009; 4: 247-52).
We thank Professor Henry Bauer for constructive, helpful discussions. This study was supported by grants of the University of Firenze.
P18 - APOPTOSIS AND DIFFERENTIATION IN TRANSFORMED ENDOTHELIAL CELLS GM7373
Tanganelli E., Caldini R., Barletta E., Papucci L., Magnelli L. and Chevanne M.
Department of Experimental Pathology and Oncology; University of Florence, Firenze, Italy
Poly(ADP-Ribose) polymerase (PARPs) is a superfamily of at least 18 enzymes that catalyzes poly(ADP-ribosyl)ation reaction on a variety of proteins, among which PARP itself. Poly(ADP-ribose) is a branched negative-charged polyanion produced by the polymerization of ADP-ribose moieties from NAD+ and is covalently but transiently bound to acceptor proteins. PARP-1 is a highly conserved DNA-binding protein, the most abundant member of the PARP family. PARP-1 and -2 are activated by single- or double-strand breaks of DNA and are involved in DNA repair and cell death induction upon DNA damages. For this reason PARPs is considered a sort of “guardian of DNA integrity”, and a molecular switch between life and death. On the other hand, poly(ADP-ribosyl)ation of proteins affects the local chromosome organization and consequently alters many gene expressions, because of the accumulation of negative charges and conformational changes on acceptor proteins, such as transcription factors. Thus, PARPs are implicated in the regulation of proliferation, and differentiation, both important in tumorigenesis. Moreover, PARPs include the centrosomal PARP-3, the vault-particle associated PARP-4 and the telomeric and Golgi tankyrases-1 and -2, that display complex patterns of subcellular localization, extending the biological relevance of poly(ADP-ribosyl)ation in cell life organization. Recently it was demonstrated that neoplastic cells undergo differentiation in vitro in the presence of PARP inhibitors. However, the chromatin-mediated molecular and cellular events involved remain elusive.
In this study we investigated the effect of PARP inhibitor 3-aminobenzamide (3ABA) on a morphological feature of differentiation in transformed endothelial cells GM7373 with particular regard to the programmed cell death. In fact, apoptosis is a general mechanism in angiogenesis, perhaps to eliminate superfluous cells not included in the vascular network.
Exposed to 3ABA, the cells displayed a 40% growth inhibition due to apoptosis. At the same time, survival cell population showed enhanced motility and cytoskeleton rearrangement. Cells cultured on Matrigel plus 3ABA, began to organize a capillary network, in a PI3K/Akt pathway activation dependent manner. Moreover, in these experimental conditions, cells displayed NF-kB nuclear translocation, and significantly increased expression of Cox-2 and iNOS, all index of angiogenetic differentiation.
This evidence confirms PARP involvement in tumorigenesis affecting cellular differentiation through apoptosis modulation of a part of the cell populations in the tumor.
Ringraziamenti: This work was supported in part by a grant of Ente Cassa di Risparmio di Firenze and “Ministero dell’Istruzione, dell’Università e della Ricerca”.
P19 - M2 RECEPTOR ACTIVATION AFFECTS CELL PROLIFERATION AND SURVIVAL INDUCING APOPTOSIS IN HUMAN GLIOBLASTOMA CELLS
Tata A.M., Ferretti M., #*Fabbiano C., Centofante G., #*Ruggeri P., #*Ponti D., #*Pacini L., §Castigli E., §Catacuzzeno L., § Fioretti B., çRicordy R., #Mancini P. and #*Calogero A.
Dip. Biol. Cellulare e dello Sviluppo, La Sapienza, University of Rome, Italy;
#Dip. Medicina Sperimentale, La Sapienza, University of Rome,
*Polo Pontino, Latina, Italy;
§Dip. Di Biologia Cellulare e Molecolare, Università di Perugia;
çIst. di Biologia e Patologia Molecolare, CNR, Rome, Italy
The potential involvement of acetylcholine and muscarinic receptors in different pathologies, among which cancer, emerged in recent years. Muscarinic receptors are expressed in several primary and metastatic tumors and appear involved in their growth and propagation. In the present work we have characterized the effects of muscarinic receptor activation on cell growth and survival in two glioblastoma lines (U251MG and U87MG) and in primary cultures of glioma biopsies. We have demonstrated by RT-PCR and immunocytochemistry that muscarinic receptor subtypes are expressed in glioblastoma cells. 3[H]-thymidine incorporation experiments and FACS analysis have demonstrated that the M2 agonist arecaidine causes a decrease of glioma cell proliferation and the clonogenic ability in U251MG and U87MG lines. In particular arecaidine causes in U87 cell an arrest in G1/S transition, and in G2/M transition in U251 cells. This different modulation appears dependent on arecaidine induced-p53 activation in U87 but not in U251 cells. Analysis of cell survival by trypan blue and Hoechst staining has showed that cell death is significantly enhanced in arecaidine treated cells. Apoptosis induction has been further analysed by ELISA detection of cytoplasmic nucleosomes and by FACS scattered analysis. Similar results have been obtained in primary cultures obtained from 5 glioma biopsies after 50 micromolar arecaidine treatment, independently on p53 and p16 levels or mutations. It is relevant that arecaidine at 50 micromolar concentration didn't affect survival of normal human and mouse astrocytes. Given the relevant role of K+ efflux in apoptotic cell death, we have found that arecaidine upregulates the expression and activity of IK channels, and that the selective IK channel inhibitor TRAM-34 prevented the arecaidine-induced apoptotic cell death.
Finally, preliminary analysis of cell migration performed by the wound healing test and by Boyden’s chamber has suggested that the arecaidine treatment does not modify cell migration in U251 but, on the contrary, it stimulates in the U87.
In conclusion we demonstrate that M2 receptor can play a relevant role in the inhibition of glioma cell growth and survival and that it may be an interesting tool for development of new strategies for cancer therapy.
This project has been supported by Cofin-Prin 2007
P20 - ESTABLISHMENT OF AN INTERLEUKIN-6 INDEPENDENT VARIANT (CMA-03/06) OF THE HUMAN MYELOMA CELL LINE CMA-03: BIOLOGICAL AND MOLECULAR CHARACTERIZATION BY A GENOMIC INTEGRATIVE ANALYSIS
Verdelli D., Nobili L., Todoerti K., Mosca L., Fabris S., Leone S., Lambertenghi Deliliers G., Lombardi L., Neri A..
Dipartimento di Scienze Mediche, Centro di Ricerca per lo Studio delle Leucemie, Università di Milano e U.O. Ematologia 1, Fondazione IRCCS Policlinico, Milano, Italy
Background
Interleukin-6 (IL-6) is the most important growth and survival factor for multiple myeloma (MM) cells. The novel CMA-03/06 human myeloma cell line is an IL-6-independent variant of CMA-03/06, previously established in our laboratory.
Aims and methods
To perform a biological and molecular characterization of the new cell line, and to provide insights into the signaling pathways and target genes involved in the growth and survival of CMA-03/06 using an integrative genomic analysis involving both gene expression and genome-wide profiling approaches. Results
The addition of IL-6 to the culture medium of CMA-03/06 cells or coculture with multipotent mesenchymal stromal cells did not induce an increase in their proliferation. The immunophenotypic analysis revealed that CD45 expression was considerably reduced in CMA-03/06 compared with CMA-03 cells, whereas they were found positive for both chains of IL-6 receptor, almost undetectable in CMA-03 cells. IL-6 was not detected in the supernatants from either CMA-03 or CMA-03/06 cell lines within 48 h using a high sensitivity IL-6 specific ELISA. Nevertheless, Western blot analysis revealed the IL-6 induced activation of STAT3 and STAT1 in both cell lines. Global gene expression profiling analysis of CMA-03/06 compared with CMA-03 cells identified 21 upregulated and 47 downregulated genes, many of which particularly relevant for MM biology, mainly involved in cellular signaling, cell cycle, cell adhesion, cell development, regulation of transcription, immunologic, inflammatory or defense activity, apoptosis. Comparison of genome-wide profiling analysis of CMA-03/06 and CMA-03 cells evidenced a different copy number in only 15 small chromosomal regions. None of the genes differentially expressed in CMA-03/06 compared with CMA-03 except one were positioned on these regions. Finally, CMA-03/06 cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA-03 cells.
Conclusions
Our data confirm the IL-6 independence of CMA-03/06 cell line and the absence of an autocrine IL-6 loop, even though the cells maintain the IL-6 signaling pathway responsiveness. Furthermore, CMA-03/06 cell line shows an increased resistance to apoptosis in comparison with CMA-03 cells. The novel CMA03/06 cell line may thus represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells.
P21 - COENZYME Q10 INHIBITS APOPTOSIS IN EXPERIMENTAL MODELS OF RETINAL DAMAGE BY PREVENTING OPENING OF MITOCHONDRIAL PERMEABILITY TRANSITION PORE
Witort E.1, Papucci L.1, Lulli M.1, Donnini M., Loffredo R., Di Gesualdo F.1, Piccini M., Carella G.2, Blasi M.A.3 and Capaccioli S.1,4
Department of Experimental Pathology and Oncology, University of Florence; 1Department of Ophthalmology, University-Hospital San Raffaele, Milano 2; Department of Ophthalmology, Catholic University of Rome3; Phoenix ONLUS Stem Cell Foundation for Human Life4
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Introduction
Apoptotic cell death is one of the main responses of the ophthalmologic districts to environmental damaging agents. Besides those present on earth, they include space irradiations and microgravity, to which astronauts will be subjected during the interplanetary flights programmed by international agencies such as ESA, ASI and NASA, in the next future. More importantly, excessive apoptotic cell death is one of the key pathogenetic events of the two more diffused and severe retinal degenerative diseases, which are glaucoma and age related macular degeneration. We have previously demonstrated that Coenzyme Q10 (CoQ10) prevented apoptosis of corneal keratocytes both in vitro and in vivo in response to excimer laser irradiation with a higher efficiency in respect to other antioxidants. Subsequently, our results were directly translated to pharmacology and produced a para-pharmacological drug in form of eye drops. Using a corneal keratocyte cell line as experimental model, we have than demonstrated that the ability of CoQ10 in preventing apoptosis is independent of its free radical scavenging ability being also consequent to its property to inhibit mitochondrial permeability transition pore (PTP), which is the main trigger of apoptotic machinery.
Aims
Aims of this study were to evaluate possible anti-apoptotic property of CoQ10 on cultured retinal pigmented epithelium (RPE) and retinal ganglion cells (RGCs) as inhibitor of mPTP opening, as well as its ability, if administered to cornea as eye drops, to reach retina and to prevent retinal cell apoptosis also in vivo.
Methods
Apoptotic stimuli were UVB (15mJ/m2) irradiation for in vitro treatments, UVC (2 J/m2) irradiations for in vivo treatments, or γ-irradiations (3H-thymidine, 20 µCi/ml), glutamate (50 µM) and respiratory chain inhibitor Antimycin A (200µM). The anti-apoptotic activity of CoQ10 (10 µM) dissolved in Lutrol was evaluated by Time-lapse video microscopy, phase contrast and confocal microscopy and WB analysis of cytosolic cytochrome c. Its ability to reach retina when administered to cornea as eye-drops was confirmed by HPLC analysis of rabbit’s retinal specimens.
Results
CoQ10 was shown to be highly effective in reduction of retinal cell apoptosis induced both by free radical generating (UVB, 3H-thymidine irradiation) and by non free radical generating (serum starvation, Antimycin A) stimuli. The eye drops containing CoQ10 administered to cornea reached the choroid-retina district in time- and dose dependent manner.
Discussion
The possibility that the topical administration of CoQ10 could countermeasure retinal lesions induced by environmental and space-related damaging agents by reducing apoptotic death of photoreceptor and ganglion cells is quite feasible. Furthermore, CoQ10 administered to cornea as eye drops could be evaluated as candidate drug to treat severe apoptosis excess-related retinopathies, including glaucoma and age-related macular degeneration
Acknowledgements
We are thankful to ECR Firenze, FCR Lucca, Agenzia Spaziale Italiana (ASI).
P22 - INTERFERONE-BETA E TROGLITAZONE: UNA NUOVA STRATEGIA TERAPEUTICA PER L’ADENOCARCINOMA PANCREATICO BASATA SULL’INDUZIONE DELL’AUTOFAGIA
Zappavigna S1, Vitale G2, Marra M1, Dicitore A2, Hofland L3, Giuberti G1, Misso G1, Lombardi A1, Arancia G4, Meschini S4, Meo G1, Abbruzzese A1, Caraglia M1
1Dipartimento di Biochimica e Biofisica, Seconda Università degli Studi di Napoli, Napoli, Italia; 2Dipartimento di Endocrinologia, Università di Milano, Istituto Auxologico Italiano IRCCS, Milano, Italia; 3Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands; 4Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, Roma, Italia
L’adenocarcinoma pancreatico rappresenta, ad oggi, la quarta causa di morte nel mondo occidentale per le caratteristiche di aggressività e resistenza alla chemioterapia convenzionale. Pertanto, nuove strategie terapeutiche sono richieste.
Abbiamo studiato la possibile interazione tra l’interferone-beta (IFNb) e il troglitazone (TGZ), agonista di PPAR-g, nelle cellule di adenocarcinoma pancreatico BxPC3.
Abbiamo osservato un forte sinergismo tra IFNb e TGZ nell’indurre inibizione proliferativa quando sono usati a concentrazioni equitossiche (CI50 0,6). Abbiamo, quindi, studiato gli effetti della combinazione di farmaci risultata sinergica sulla fosforilazione delle proteine STAT1 e STAT3, bersaglio dell’IFNb. L’IFNb da solo induce un aumento della fosforilazione di STAT3 già dopo 6 h di trattamento, che risulta antagonizzato dalla combinazione; la fosforilazione di STAT1, in seguito al trattamento combinato, aumenta in modo paragonabile a quella indotta dall’IFNb da solo. Inoltre, abbiamo studiato gli effetti della combinazione sui pathway di proliferazione e sopravvivenza cellulare. Abbiamo osservato una riduzione della fosforilazione di Erk1/2 e di Akt dopo 6h e 24h di trattamento combinato, rispettivamente mentre l’IFNb da solo ma non il TGZ aumenta sia l’attività di Erk1/2 che di Akt. Inoltre, il saggio EMSA dimostrava che l’IFNb riduce il legame di PPAR-g al DNA mentre il TGZ da solo aumenta di 3 volte l’attività trascrizionale di PPARg, che risulta potenziata (4,5 volte) dalla combinazione. L’analisi del ciclo cellulare al FACS con ioduro di propidio mostrava un blocco nella transizione delle cellule dalla fase G1 alla fase S, che correla con l’aumentata espressione delle proteine p21 e p27, più evidente dopo 24h di trattamento combinato. La combinazione IFNb/TGZ non era in grado di indurre apoptosi; su queste basi, abbiamo rivolto la nostra attenzione ad un meccanismo alternativo di morte cellulare, l’autofagia. Abbiamo osservato una notevole riduzione del complesso beclina1-bcl2 che suggerisce l’attivazione dell’autofagia beclina1-dipendente in cellule trattate con la combinazione, dato confermato dall’analisi al microscopio elettronico a trasmissione che mostra un significativo aumento della formazione di autofagosomi indotto dalla combinazione. Abbiamo, quindi, rivolto la nostra attenzione alla via mTOR-dipendente ed in particolare a due molecole chiave di tale via, 4EBP1 e eIF4E. Abbiamo osservato una riduzione della fosforilazione di 4EBP1 già dopo 6h di trattamento con la combinazione ed ancora più evidente dopo 24h, mentre la fosforilazione di eIF4E risulta ridotta a tempi più precoci (3h) per poi riprendere a 24h.
In conclusione, questi risultati rappresentano la prima dimostrazione del sinergismo esistente tra IFN-b e PPAR-g agonisti nell’indurre autofagia nell’adenocarcinoma pancreatico e potrebbero rappresentare il razionale molecolare per gli studi in vivo.
Ringraziamenti: Ringrazio di cuore il Dott. Michele Caraglia, la D.ssa Monica Marra e il Prof. Abbruzzese per il supporto scientifico e morale, il Prof. Hofland, il Dott. Giovanni Vitale e il Dott. Giuseppe Arancia per la gentile collaborazione.
P23 - BALSAMITA MAJOR: DALLA PIANTA ALLO SVILUPPO DI PRODOTTI COSMETICI E POTENZIALMENTE PRO-APOPTOTICI INNOVATIVI.
Alba Ena1, Manuela Nelli2, Cristina Pintucci1, Ewa Witort3*, Matteo Lulli3*, Sergio Capaccioli3*, Pietro Carlozzi1
1 CNR Istituto per lo Studio degli Ecosistemi sez. di Firenze.
Polo Scientifico via Madonna del Piano 10 Sesto Fiorentino FI.
2 Officina Profumo Farmaceutica Santa Maria Novella. Via Reginaldo Giuliani 71 b/r Firenze
3 Dipartimento di Patologia e Oncologia Sperimentali, Università di Firenze and
*PHOENIX ONLUS Stem Cell Foundation for Human Life
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P24 - RESPONSE OF SKELETAL MUSCLE CELLS TO DIFFERENT APOPTOTIC TRIGGERS
S. Salucci 1, M. Battistelli 1, S. Burattini 1, C. Squillace 1, B. Canonico 1,2 and E. Falcieri 1,3.
1 DISUAN and 2 Centro di Citometria e Citomorfologia, Università degli Studi di Urbino “Carlo Bo”, Urbino; Italy
3 IGM, CNR, Istituti Ortopedici Rizzoli, Bologna; Italy
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Introduction and aim
Apoptosis is considered essential for skeletal muscle development, where cells with defects are deleted during differentiation (Fujio et al., 1999). In addition, it plays a relevant role in muscle pathology (Ferreira et al., 2007). Apoptosis is characterized by cell volume decrease, chromatin condensation and biochemical markers, including degradation of genomic DNA into nucleosomal fragments (Burattini et al., 2009). Murine C2C12 myoblasts have been widely used as a model to study apoptosis in developing muscle (D’Emilio et al., 2009). Interestingly, DNA degradation patterns in C2C12 apoptosis differ depending on differentiation degree. Nucleosomal ladder is clearly detected when apoptosis is induced under differentiating conditions; differently, apoptosis under proliferating conditions does not produce DNA laddering (McArdle et al., 1999).
The aim of this study was to evaluate apoptotic sensitivity of C2C12 myoblasts treated with different apoptotic triggers.
Materials and Methods
C2C12 cells, a skeletal muscle murine cell line, was utilized at undifferentiated (myoblasts) stage, and grown as previously reported (Curci et al., 2008; Ferri et al., 2009). To induce apoptosis, they have been exposed to 500µM H2O2, 10µM cisplatinum, 25µM etoposide and 1µM staurosporine for 24 hours and monitoring cell response by reverted microscope. Then, cells have been analysed by transmission electron microscopy and cell cycle flow cytometry (D’Emilio et al., 2009).
Results
Cell cycle analysis does not reveal a sharp subdiploid peak. On the contrary, morphological analysis evidences that all triggers induce an apoptotic effect: in a number of myoblasts, the characteristic chromatin condensation is revealed. In particular, incubation with staurosporine and H2O2 increases dead cell number, and myoblasts in late apoptotic stages and in secondary necrosis are observed.
Conclusions
All these results indicate that all used triggers, acting through different mechanisms of action, are able to induce apoptosis in C2C12 myoblasts. Further studies are in progress to investigate the sensitivity of differentiated myotubes.
