Trasduzione del segnale e poliamine nell'apoptosi di cellule cardiache

Introduction: Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, thus representing a potential therapeutic target. Apoptosis of cardiac cells can be induced experimentally by several stimuli including hypoxia, serum withdrawal or combination of both. Several lines of research suggest that neurohormonal mechanisms play a central role in the progression of heart failure. In particular, excessive activation of the sympathetic nervous system or the renin-angiotensin-aldosterone system is known to have deleterious effects on the heart. Recent studies report that norepinephrine (NE), the primary transmitter of sympathetic nervous system, and aldosterone (ALD), which is actively produced in failing human heart, are able to induce apoptosis of rat cardiomyocytes. Polyamines are biogenic amines involved in many cellular processes, including apoptosis. Actually it appears that these molecules can act as promoting, modulating or protective agents in apoptosis depending on apoptotic stimulus and cellular model. We have studied the involvement of polyamines in the apoptosis of cardiac cells induced in a model of simulated ischemia and following treatment with NE or ALD. Methods: H9c2 cardiomyoblasts were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation. Cardiomyocyte cultures were prepared from 1-3 day-old neonatal Wistar rat hearts. Polyamine depletion was obtained by culturing the cells in the presence of α-difluoromethylornithine (DFMO). Polyamines were separated and quantified in acidic cellular extracts by HPLC after derivatization with dansyl chloride. Caspase activity was measured by the cleavage of the fluorogenic peptide substrate. Ornithine decarboxylase (ODC) activity was measured by estimation of the release of 14C-CO2 from 14C-ornithine. DNA fragmentation was visualized by the method of terminal transferase-mediated dUTP nick end-labeling (TUNEL), and DNA laddering on agarose gel electophoresis. Cytochrome c was detected by immunoflorescent staining. Activation of signal transduction pathways was investigated by western blotting. Results: The results indicate that simulated ischemia, NE and ALD cause an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of caspase activity, a family of proteases that execute the death program and induce cell death. This effect was prevented in the presence of DFMO, an irreversible inhibitor of ODC, thus suggesting that polyamines are involved in the execution of the death program activated by these stimuli. In H9c2 cells DFMO inhibits several molecular events related to apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, down-regulation of Bcl-xL, and DNA fragmentation. The anti-apoptotic protein survivin is down-regulated after ALD or NE treatement and polyamine depletion obtained by DFMO partially opposes survivin decrease. Moreover, a study of key signal transduction pathways governing cell death and survival, revealed an involvement of AMP activated protein kinase (AMPK) and AKT kinase, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact polyamine depleted cells show an altered pattern of AMPK and AKT activation that may contrast apoptosis and appears to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. Conclusions: These results indicate that polyamines are involved in the execution of the death program activated in cardiac cells by heart failure-related stimuli, like ischemia, ALD and NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.

Interazioni tra Glicoproteine che mediano la penetrazione del Virus Herpes Simplex nella cellula e disegno razionale di Peptidi Inibitori

Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of gD, gB and heterodimer gH/gL. gD serves as receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in a N-terminal domain carrying the receptor-binding sites, and a C-terminal domain carrying the profusion domain, required for fusion but not receptor-binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other we searched for biochemical defined complexes in infected and transfected cells, and in virions. We report that gD formed complexes with gB in absence of gH/gL, and with gH/gL in absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry, and did not increase at virus fusion with cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the aa 240-260 portion and downstream, with T306P307 as critical residues, and of the binding site to gH/gL at aa 260-310 portion, with P291P292 as critical residues. The results indicate that gD carries composite independent binding sites for gB and gH/gL, both of which partly located in the profusion domain. The second part of the project dealt with rational design of peptides inhibiting virus entry has been performed. Considering gB and gD, the crystal structure is known, so we designed peptides that dock in the structure or prevent the adoption of the final conformation of target molecule. Considering the other glycoproteins, of which the structure is not known, peptide libraries were analyzed. Among several peptides, some were identified as active, designed on glycoprotein B. Two of them were further analyzed. We identified peptide residues fundamental for the inhibiting activity, suggesting a possible mechanism of action. Furthermore, changing the flexibility of peptides, an increased activity was observed,with an EC50 under 10μM. New approaches will try to demonstrate the direct interaction between these peptides and the target glycoprotein B.

Riparazione del danno ischemico ostruttivo del miocardio mediante cellule staminali mesenchimali sottoposte a stimolazione elettromeccanica in bioreattore

Nell’ambito dell’ingegneria dei tessuti, la possibilità di rigenerazione del miocardio post-infartuale è un argomento “caldo”, che suscita grandi speranze ma solleva altrettanto grandi interrogativi - sostenuti dal sussistere di dubbi di base sulle scelte operative praticabili. Esiste tuttavia concordanza nel considerare fondamentale l’utilizzo di un “supporto” che possa mantenere nella sede peri-infartuale le cellule competenti. Infatti, la semplice iniezione di cellule staminali per via endovenosa o direttamente nell’area infartuata non si è dimostrata particolarmente efficace, soprattutto a causa della cospicua perdita cellulare che si verifica rapidamente dopo il trapianto. Ci si orienta quindi verso la strategia di seminare cellule in grado di transdifferenziare in senso muscolare cardiaco su un materiale biocompatibile in vitro e di impiantare successivamente il costrutto ottenuto in vivo dove ci si attende il riassorbimento del biomateriale e l’integrazione delle cellule. Tuttavia, mentre in altri settori della medicina - quali ortopedia e dermatologia - l’impiego di pseudotessuti ingegnerizzati ha già permesso di conseguire ottimi risultati nella rigenerazione di tessuti danneggiati, allo stato attuale, i progressi ottenuti nell’ambito della rigenerazione del miocardio infartuato appaiono ancora aneddotici e distanti dall’ottenere protocolli condivisi per l’impiego in clinica. Il lavoro presentato in questa ricerca, condotto grazie alla sinergia di competenze interdisciplinari negli ambiti chimico, biologico e dell’ingegneria biomedica meccanica ed elettronica, è uno studio di fattibilità di una metodica standardizzata in grado di indirizzare cellule staminali mesenchimali (MSCs) indifferenziate verso l’acquisizione in vitro di caratteri fenotipici confrontabili con quelli delle cellule muscolari cardiache attraverso il paradigma della coltura dinamica in bioreattore. Il prototipo di bioreattore impiegato, in quanto sviluppato originalmente nel corso di questa attività di ricerca, presenta rispetto ad altri strumenti descritti l’innovazione e il vantaggio di non richiedere l’utilizzo di un incubatore, in quanto esso stesso permette di coltivare cellule al suo interno in condizioni controllate di temperatura, pH e concentrazione di CO2. La sua flessibilità operativa consente di impostare e controllare da personal computer leggi di moto di qualsiasi forma anche con cicliche molto veloci. Infine, la presenza di estensimetri in grado di misurare finemente la variazione di tensione esercitata sulla matrice polimerica utilizzata, posta in trazione tra due afferraggi, permette di applicare nel tempo una forza di stiramento costante, ottenendo deformazioni controllate e risultati riproducibili in termini di modificazioni cellulari. Il superamento delle problematiche sorte durante la fase di messa a punto del sistema, che deve essere ritenuto parte integrante del lavoro di sviluppo condotto, ha permesso di studiare l’adattamento di MSCs allo stiramento ciclico, mostrando che questo effettivamente determina alcune differenze fenotipiche rispetto al controllo statico. Inoltre le cellule hanno acquistato una disposizione orientata lungo l’asse longitudinale delle fibre, dato questo particolarmente importante se si considera la disposizione ordinata delle cellule del miocardio, le quali costituiscono un vero e proprio sincizio, indispensabile per una diffusione sincrona dell’impulso elettrico di contrazione. La creazione di uno pseudotessuto cardiaco ottimale richiederà ovviamente ulteriore lavoro, ma la metodica qui presentata si propone al tempo stesso come uno strumento di studio e come una strategia operativa per un approccio innovativo e standardizzabile alla medicina rigenerativa del miocardio.

Il cane come modello animale spontaneo di patologie neoplastiche dell'uomo: ene Ataxia-Telangiectasia Mutated (ATM) Nnella predisposizione al cancro ed importanza dei riarrangimanti genici dei geni Ig/TCR per la diagnosi e prognosi delle malattie linfoproliferative

“Naturally occurring cancers in pet dogs and humans share many features, including histological appearance, tumour genetics, molecular targets, biological behaviour and response to conventional therapies. Studying dogs with cancer is likely to provide a valuable perspective that is distinct from that generated by the study of human or rodent cancers alone. The value of this opportunity has been increasingly recognized in the field of cancer research for the identification of cancer-associated genes, the study of environmental risk factors, understanding tumour biology and progression, and, perhaps most importantly, the evaluation and development of novel cancer therapeutics”.(Paoloni and Khanna, 2008) In last years, the author has investigated some molecular features of cancer in dogs. The Thesis is articulated in two main sections. In section 1, the preliminary results of a research project aimed at investigating the role of somatic mutations of Ataxia-Telangiectasia mutated (ATM) gene in predisposing to cancer in boxer dogs, are presented. The canine boxer breed may be considered an unique opportunity to disclose the role of ATM somatic mutation since boxer dogs are known to be dramatically susceptible to cancer and since they may be considered a closed gene pool. Furthermore, dogs share with human the some environment. Overall, the abovementioned features could be considered extremely useful for our purposes. In the section 2, the results of our studies aimed at setting up accurate and sensitive molecular assays for diagnosing and assessing minimal residual disease in lymphoproliferative disorders of dogs, are presented. The results of those molecular assay may be directly translated in the field of Veterinary practice as well as the may be used to improve our objective evaluation of new investigational drugs effectiveness in canine cancer trials.

Il modello di Fister-Panetta per la crescita di cellule tumorali : analisi qualitativa e aspetti didattici

Si analizza il modello di Fister-Panetta per la crescita di cellule tumorali nella fase avascolare del tumore e si propone un percorso didattico di introduzione alla modellistica matematica in una quinta liceo. Si riporta poi una riflessione sulle motivazioni per cui l'introduzione degli aspetti modellistici e applicativi della matematica è importante anche nella scuola superiore.

Validazione e applicazioni cliniche della metodica NIRA (Near Infrared Reflectance Analysis) per la valutazione del contenuto nutrizionale del latte materno in terapia intensiva neonatale

Phase 1: To validate Near-Infrared Reflectance Analysis (NIRA) as a fast, reliable and suitable method for routine evaluation of human milk’s nitrogen and fat content. Phase 2: To determine whether fat content, protein content and osmolality of HM before and after fortification may affect gastroesophageal reflux (GER) in symptomatic preterm infants. Patients and Methods: Phase 1: 124 samples of expressed human milk (55 from preterm mothers and 69 from term mothers) were used to validate NIRA against traditional methods (Gerber method for fat and Kjeldhal method for nitrogen). Phase 2: GER was evaluated in 17 symptomatic preterm newborns fed naïve and fortified HM by combined pH/intraluminal-impedance monitoring (pH-MII). HM fat and protein content was analysed by a Near-Infrared-Reflectance-Analysis (NIRA). HM osmolality was tested before and after fortification. GER indexes measured before and after fortification were compared, and were also related with HM fat and protein content and osmolality before and after fortification. Results: Phase 1: · A strong agreement was found between traditional methods’ and NIRA’s results (expressed as g/100 g of milk), both for fat and nitrogen content in term (mean fat content: NIRA=2.76; Gerber=2.76; mean nitrogen content: NIRA=1.88; Kjeldhal =1.92) and preterm (mean fat content: NIRA=3.56; Kjeldhal=3.52; mean nitrogen content: NIRA=1.91; Kjeldhal =1.89) mother’s milk. · Nitrogen content of the milk samples, measured by NIRA, ranged from 1.18 to 2.71 g/100 g of milk in preterm milk and from 1.48 to 2.47 in term milk; fat content ranged from 1.27 to 6.23 g/100 g of milk in preterm milk and from 1.01 to 6.01 g/100 g of milk in term milk. Phase 2: · An inverse correlation was found between naïve HM protein content and acid reflux index (RIpH: p=0.041, rho=-0.501). · After fortification, osmolality often exceeded the values recommended for infant feeds; furthermore, a statistically significant (p<.05) increase in non acid reflux indexes was observed. Conclusions: NIRA can be used as a fast, reliable and suitable tool for routine monitoring of macronutrient content of human milk. Protein content of naïve HM may influence acid GER in preterm infants. A standard fortification of HM may worsen non acid GER indexes and, due to the extreme variability in HM composition, may overcome both recommended protein intake and HM osmolality. Thus, an individualized fortification, based on the analysis of the composition of naïve HM, could optimize both nutrient intake and feeding tolerance.

Studio immunologico per l'individuazione di pazienti trapiantati per cirrosi HBV relata che potrebbero sospedere la terapia con immunoglobine specifiche (HBIG)

Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT) is associated with poor graft and patient survival. Treatment with HBV-specific immunoglobulins (HBIG) in combination with nucleos(t)ide analogs is effective in preventing HBV reinfection of the graft and improving OLT outcome. However, the combined immunoprophylaxis has several limitations, mainly the high cost and the lack of standard schedules about duration. So far, the identification of markers able to predict the reinfection risk is needed. Although the HBV-specific immune response is believed to play an essential role in disease outcome, HBV-specific cellular immunity in viral containment in OLT recipients is unclear. To test whether or not OLT recipients maintain robust HBV-specific cellular immunity, the cellular immune response against viral nucleocapsid and envelope-protein of HBV was assessed in 15 OLT recipients and 27 individuals with chronic and 24 subjects with self-limited HBV infection, respectively. The data demonstrate that OLT recipients mounted fewer but stronger clusters of differentiation (CD)8 T cell responses than subjects with self-limited HBV infection and showed a preferential targeting of the nucleocapsid antigen. This focused response pattern was similar to responses seen in chronically infected subjects with undetectable viremia, but significantly different from patients who presented with elevated HBV viremia and who mounted mainly immune responses against the envelope protein. In conclusion, virus-specific CD4 T cell–mediated responses were only detected in subjects with self-limited HBV infection. Thus, the profile of the cellular immunity against HBV was in immune suppressed patients similar to subjects with chronic HBV infection with suppressed HBV-DNA.

Ruolo dell'Infliximab nella terapia della colite ulcerosa moderato-severa

Background: Almost 10-15% of patients with active Ulcerative Colitis are refractory to conventional therapy. Infliximab is a treatment of proven efficacy in this group of patients. Aims: To evaluate the role of Inliximab in inducing and maintaining remission in patients with chronically active moderate-severe Ulcerative Colitis. Materials and methods: 53 patients were enrolled, 47 patients entered the study and were treated with a dose of 5 mg/kg. The remission was evaluated through endoscopy and clinical criteria. (Mayo Score). The primary endpoint were clinical and endoscopic remission in moderate-severe Ulcerative Colitis refractory to standard therapy, the secondary out point was the maintenance of remission in the long period. Results: 47 patients started the study, 43 completed the study, 4 dropped out for worsening disease or adverse events; 27 patients were treated with 3 infusions, 9 patients with 4 infusions, 7 patients with > o = 5 infusions. 34 /47 patients (72.3%) were responders 12 (25.5%) improved their symptoms, 22 ( 46.8%) were in remission after the treatment. Among the responders, 21/34 (61.8%) stopped the steroid therapy after 3 infusions, the others reduced the dose or maintained just topic therapy. 13/47 patients (27.7%) were non responders (p <0.001). After 3 months all 22 patients which had reached remission maintained low Mayo Score; 10/12 (83.3%) patients with clinical response maintained their low score, 2 relapsed . Conclusions: Infliximab is a valid therapy for the treatment of Ulcerative Colitis and can avoid surgery in selected patients.

I linfociti T regolatori nella patologia epatica da HCV: dall'epatite cronica al trapianto

La storia naturale dell’epatopatia HCV-relata passa dall’epatite cronica alla cirrosi ed eventualmente all’epatocarcinoma fino ad arrivare alla possibile necessità del trapianto di fegato. HCV non esercita citolisi diretta, pertanto i fattori immunologici giocano il duplice ruolo di determinare l’evoluzione dell’infezione e il danno epatico. All’interno del sistema immunitario esistono linfociti in grado di inibire l’attivazione delle cellule effettrici modulando la risposta immunitaria; la popolazione regolatoria meglio conosciuta è costituita dai cosiddetti T-reg caratterizzati dal fenotipo CD4+CD35hiFoxp3+. Scopo di questo studio è stato determinare fenotipo e funzione dei T-reg, valutandone le correlazioni con caratteristiche cliniche e parametri biochimici e virologici, nelle diverse fasi della malattia epatica da HCV, a partire dall’epatite cronica, passando per la cirrosi, l’epatocarcinoma e terminando con il follow-up post-trapianto di fegato. Sono stati reclutati 80 pazienti con infezione cronica da HCV non in trattamento antivirale, di cui 52 con epatite cronica, 12 con cirrosi e 16 con epatocarcinoma. Di questi, 11 sono andati incontro a trapianto di fegato e sono stati poi seguiti fino a 36 mesi di follow up. Ventinove soggetti avevano transaminasi persistentemente nella norma e 28 mostravano ALT costantemente oltre 2.5x i valori normali. Quaranta donatori di sangue sono stati utilizzati come controlli sani. Marcatori di superficie (CD4, CD25) ed intracellulari (Foxp3) sono stati valutati in citofluorimetria su sangue intero periferico per tutti i soggetti al basale ed ogni 2-4 settimane dopo trapianto. In una quota di pazienti i T-reg sono stati estratti dai linfociti del sangue periferico con metodi immunomagnetici e la loro funzione valutata come percentuale di inibizione di proliferazione e produzione di IFN-γ da parte delle cellule bersaglio CD4+CD25- in esperimenti di co-coltura effettuati al basale e dopo 24-36 settimane dal trapianto. La percentuale di T-reg e l’espressione del Foxp3 sono risultate aumentate nei soggetti con HCV rispetto ai controlli sani, in particolare in coloro con cirrosi, HCC e nei pazienti con transaminasi normali indipendentemente dallo stadio di malattia, correlando inversamente con i livelli di transaminasi e direttamente con il punteggio MELD. La produzione di IFN-γ è incrementata in tutti i pazienti HCV ma efficacemente controllata solamente dai T-reg dei pazienti con transaminasi normali. Dopo il trapianto di fegato, si verifica una precoce e reversibile riduzione delle T-reg circolanti. Alla 24ma e 36ma settimana dal trapianto la percentuale dei T-reg circolanti è sovrapponibile al basale e i loro effetti, sia in termini di proliferazione che di produzione di IFN-γ, sulle cellule bersaglio, già dotate di una ridotta attività intrinseca, appaiono particolarmente incisivi. In conclusione, l’epatopatia cronica da HCV è caratterizzata da una popolazione di T-reg espansa che però, con l’eccezione dei soggetti con transaminasi normali, non appare in grado di limitare il danno epatico immuno-mediato e potrebbe favorire lo sviluppo e la crescita di lesioni tumorali nei pazienti con malattia avanzata. Il trapianto di fegato, probabilmente a causa della terapia immunosoppressiva, si associa ad un marcato e transitorio declino dei T-reg le cui numerosità e funzione vengono completamente recuperate a sei mesi dall’intervento. La migliore conoscenza dei meccanismi alla base delle cinetica e della funzione delle cellule regolatorie potrà fornire utili strumenti per il loro utilizzo come adiuvanti nella terapia dell’epatopatia cronica HCV relata.

Noisy Oncology: applications of bounded noise transitions in modelling tumor growth

I tumori macroscopici e microscopici, dopo la loro prima fase di crescita, sono composti da un numero medio elevato di cellule. Così, in assenza di perturbazioni esterne, la loro crescita e i punti di equilibrio possono essere descritti da equazioni differenziali. Tuttavia, il tumore interagisce fortemente col macroambiente che lo circonda e di conseguenza una descrizione del tutto deterministica risulta a volte inappropriata. In questo caso si può considerare l'interazione con fluttuazioni statistiche, causate da disturbi esterni, utilizzando le equazioni differenziali stocastiche (SDE). Questo è vero in modo particolare quando si cerca di modellizzare tumori altamente immunogenici che interagiscono con il sistema immunitario, in quanto la complessità di questa interazione risulta in fenomeni di multistabilità. Così, il rumore può provocare disturbi e indurre transizioni di stato (Noise-Induced-Transitions). E' importante notare che una NIT può avere implicazioni profonde sulla vita di un paziente, dal momento che una transizione da uno stato di equilibrio piccolo, nelle dimensioni del tumore, ad uno stato di equilibrio macroscopico, nella maggior parte dei casi significa il passaggio dalla vita alla morte. Generalmente l'approccio standard è quello di modellizzare le fluttuazioni stocastiche dei parametri per mezzo di rumore gaussiano bianco o colorato. In alcuni casi però questa procedura è altamente inadeguata, a causa della illimitatezza intrinseca dei rumori gaussiani che può portare a gravi incongruenze biologiche: pertanto devono essere utilizzati dei rumori "limitati", che, tuttavia, sono molto meno studiati di quelli gaussiani. Inoltre, l'insorgenza di NIT dipende dal tipo di rumore scelto, che rivela un nuovo livello di complessità in biologia. Lo scopo di questa tesi è quello di studiare le applicazioni di due tipi diversi di "rumori limitati" nelle transizioni indotte in due casi: interazione tra tumore e sistema immunitario e chemioterapia dei tumori. Nel primo caso, abbiamo anche introdotto un nuovo modello matematico di terapia, che estende, in modo nuovo, il noto modello di Norton-Simon.

Effetti clinici e biologici della terapia epigenetica nelle sindromi mielodisplastiche a basso rischio

Background: Nucleoside 5-Azacitidine (5-Aza) in high risk MDS patients (pts) at a dose of 75mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates (hematologic improvement (HI) 50-60%, complete remission (CR) 10-30%) and prolongation of survival (at 2 years 50,8%). Aim: The role of 5-Aza in low-risk MDS patients is not well defined but its use in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial a low-dose schedule of 5-Aza (75 mg/mq daily for 5 consecutive days every 28 days) was given to low-risk MDS pts in order to evaluate its efficacy and tolerability and to identify biological markers to predict the response. Methods: From September 2008 to February 2010, 34 patients were enrolled into the study. Fifteen patients had refractory anemia (RA), 5 patients refractory anemia with ringed sideroblasts (RARS), 7 patients refractory cytopenia with multilineage dysplasia (RCMD) and 7 patients refractory anemia with excess blasts-1 (RAEB-1). All patients failed previously EPO therapy and were in chronic red blood cell (RBC) supportive care with a median transfusions requirement of 4 units/monthly. The response treatment criteria was according to IWG 2006. Results: At present time 31 out of 34 pts are evaluable: 12/31 pts (39%) completed the treatment plan (8 courses), 7/31 pts (22%) performed the first 4 courses, 8/31 (26%) made 1 to 3 courses and 4/31 (13%) died during the treatment period. Out of 12 pts who completed the 8 courses of therapy 10 (83%) obtained an HI, 2/12 (17%) maintained a stable disease. Out of 10 pts who obtained HI, 4 pts (40%) achieved a CR. Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%) but they were transitory and usually no delay of treatment was necessary. 2/4 pts died early after the 1th cycle for septic shock and gastrointestinal hemorrage respectively whereas 2/4 pts died in a condition of stable disease after the 4th cycle for pneumonia and respiratory distress. Samples for biologic studies have been collected from the pts before starting the therapy and at the end of 4th and 8th course. Preliminary data on the lipid signalling pathways suggested a direct correlation between PI-PLC-β1 gene expression and 5-Aza responsiveness. Conclusion: Interim analysis of our study based on the small number of cases who completed the treatment program, shows that 83% of pts obtain an HI and 40% obtain a CR. 4 patients died during the treatment and even if the causes were reported as no related to the therapy it has been considered that caution has to be reserved in given 5-Aza in these pts who are elderly and frail. Preliminary data of PI-PLC-β1 gene expression suggest that this and probably other biological markers could help us to know a priori who are the patients who have more chances to respond.

Infiammazione e cancro: l'interazione COX-2/CA-IX promuove il potenziale invasivo e la sopravvivenza in ipossia delle cellule del cancro colon-rettale

Cyclooxygenase-2/Carbonic anhydrase-IX up-regulation promotes invasive potential and hypoxia survival in colorectal cancer cells Purpose: Cyclooxygenase-2 (COX-2) is a major mediator of inflammation, playing a pivotal role in colorectal carcinogenesis. Hypoxia is an universal hallmark of solid tumour in vivo. This investigation was prompted by the observation that in colorectal cancer cells the expression of COX-2 protein is positively correlated with that of the hypoxia survival gene Carbonic Anhydrase-IX (CA-IX). Experimental Design: Since COX-2 gene expression and activity is increased in hypoxia, and that CA-IX is expressed also in normoxia in colorectal cancer cells, we tested the hypothesis that COX-2 activity in normoxia, as well as in hypoxia may be functionally linked to that of CA-IX gene. We investigated the role of COX-2 and CA-IX in colorectal cancer cell lines. In this regard, we performed RNA interference to knockdown COX-2 gene in vitro and immunohistochemistry to evaluate the protein expression of COX-2 and CA-IX in human colon cancer tissue specimens ex vivo. Results: We found that COX-2, by PGE2 production, controls CA-IX gene expression in an ERK dependent manner. In line with this finding, we also showed that the COX-2 inhibition by a specific short harpin COX-2 RNA (shCOX-2) or by a specific drug (SC-236), down-regulated CA-IX expression in colon cancer cells. We then exposed colon cancer cells to hypoxia stimuli and found that COX-2/CA-IX interplay promoted hypoxia survival. Moreover, we also report that COX-2/CA-IX interplay triggers Matrix Metalloproteinase 2/9 (MMP-2/9) activation and enhances the invasiveness of colorectal cancer cells. Thus given our above observations, we found that CA-IX and COX-2 protein expressions correlate with more aggressive stage colorectal cancer tissues ex vivo. Conclusions: Taken together these data indicate that COX-2/CA-IX interplay promotes an aggressive phenotype (hypoxia survival and invasiveness) which can be modulated in vitro by COX-2 selective inhibition and which may play a role in determining the biological aggressiveness of colorectal tumours. Moreover, in vitro and ex vivo data also suggest that the signatures of inflammation (COX-2) and hypoxia (CA-IX) may be difficult to be disentangled in colon cancer, being both responsible for the up-regulation of the same pathways.