Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

Analisi funzionale dei recettori per le neurotrofine p75NTR e Trka in neuroblastoma

The biological complexity of NGF action is achieved by binding two distinct Neurotrophin receptors, TrkA and p75NTR. While several reports have provided lines of evidence on the interaction between TrkA and p75NTR at the plasma membrane, much fewer data are available on the consequence of such an interaction in terms of intracellular signaling. In this study, we have focused on how p75NTR may affect TrkA downstream signaling with respect to neuronal differentiation. Here, we have shown that cooperation between p75NTR and TrkA results in an increased NGF-mediated TrkA autophosphorylation, leads to a sustained activation of ERK1/2 and accelerates neurite outgrowth. Interestingly, neurite outgrowth is concomitant with a selective enhancement of the AP-1 activity and the transcriptional activation of genes such as GAP-43 and p21(CIP/WAF), known to be involved in the differentiation process. Collectively, our results unveil a functional link between the specific expression profile of neurotrophin receptors in neuronal cells and the NGF-mediated regulation of the differentiation process possibly through a persistent ERKs activation and the selective control of the AP-1 activity. In our studies we discuss the functional role of the neurotrophin receptor p75NTR and TrkA in a ligand-dependent signal transduction. It is known that p75NTR is also involved in the mediation of cell death ligand dependent. Here we show for the first time that the membrane receptor p75NTR, upon binding to b- Amyloid (Ab) peptide, is able to transduce a cytotoxic signal through a mechanism very similar to the one adopted by Tumor Necrosis Factor Receptor 1 (TNFR1), when activated by TNFa. We define that in neuroblastoma cell line Ab cytotoxicity signals through a pathway depending on p75NTR death domain (DD), mostly through some specific conserved residues. We identified that TRADD is the first interactor recruiting to the membrane and activates JNK and NF-kB transcription factors. Since Ab is defined as the most important aetiologic element associated with the Alzheimer’s Disease (AD), characterization of the mechanism involved in the mediation of the neurodegeneration can suggest also new therapeutic approaches.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Roles of Ecdysone signaling in cell survival and epithelium morphogenesis during Drosophila melanogaster development

In Drosophila the steroid hormone ecdysone regulates a wide range of developmental and physiological responses, including reproduction, embryogenesis, postembryonic development and metamorphosis. Drosophila provides an excellent system to address some fundamental questions linked to hormone actions. In fact, the apparent relative simplicity of its hormone signaling pathways taken together with well-established genetic and genomic tools developed to this purpose, defines this insect as an ideal model system for studying the molecular mechanisms through which steroid hormones act. During my PhD research program I’ve analyzed the role of ecdysone signaling to gain insight into the molecular mechanisms through which the hormone fulfills its pleiotropic functions in two different developmental stages: the oogenesis and the imaginal wing disc morphogenesis. To this purpose, I performed a reverse genetic analysis to silence the function of two different genes involved in ecdysone signaling pathway, EcR and ecd.

adaptive capabilities of the pi3k/akt/mtor pathway in acute myeloid leukemia revealed by the use of selective inhibitors

Because of its aberrant activation, the PI3K/AKT/mTOR signaling pathway represents a pharmacological target in blast cells from patients with acute myelogenous leukemia (AML). Using Reverse Phase Protein Microarrays (RPMA), we have analyzed 20 phosphorylated epitopes of the PI3K/Akt/mTor signal pathway of peripheral blood and bone marrow specimens of 84 patients with newly diagnosed AML. Fresh blast cells were grown for 2 h, 4 h or 20 h untreated or treated with a panel of phase I or phase II Akt allosteric inhibitors, either alone or in combination with the mTOR kinase inhibitor Torin1 or the broad RTK inhibitor Sunitinib. By unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. Remarkably, however, we observed that inhibition of Akt phosphorylation, as well as of its substrates, was transient, and recovered or even increased far above basal level after 20 h in 60% samples. We demonstrated that inhibition of Akt induces FOXO-dependent insulin receptor expression and IRS-1 activation, attenuating the effect of drug treatment by reactivation of PI3K/Akt. Consistent with this model we found that combined inhibition of Akt and RTKs is much more effective than either alone, revealing the adaptive capabilities of signaling networks in blast cells and highliting the limations of these drugs if used as monotherapy.

Ruolo della fosfolipasi C-β1 nel differenziamento miogenico: identificazione di nuovi target nucleari

The expression of phospholipase C-β1 (PLC-β1) and cyclin D3 is highly induced during skeletal myoblast differentiation. We have previously shown that PLC-β1 activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-β1 is a crucial regulator of mouse cyclin D3 gene. Here we report that PLC-β1 catalytic activity plays a role in the increase of cyclin D3 levels and in the induction of differentiation of C2C12 skeletal muscle cells. PLC-β1 mutational analysis revealed the importance of His331 and His378 for the catalytic activity. We show that following insulin administration, cyclin D3 mRNA levels are lower in cells overexpressing the PLC-β1 catalytically inactive form, as compared to wild type cells. We describe a novel signaling pathway elicited by PLC-β1 that modulates Activator Protein-1 (AP-1) activity. Indeed, gel mobility shift assays indicate that there is a c-jun binding site located in cyclin D3 promoter region specifically regulated by PLC-β1 and that c-jun binding activity is significantly increased by insulin stimulation and PLC-β1 overexpression. Moreover, mutation of c-jun/AP-1 binding site decreases the basal cyclin D3 promoter activity and eliminates its induction by insulin and PLC-β1 overexpression. Interestingly, we observed that the ectopic expression of the Inositol Polyphosphate Multikinase (IPMK) in C2C12 myoblasts enhances cyclin D3 gene expression and that the mutation of c-jun site in cyclin D3 promoter determines an impairment of IPMK-dependent promoter induction. These results indicate that PLC-β1 activates a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation through IPMK signaling.

Identificazione delle Lamine di tipo A come nuovi substrati della protein chinasi Akt/PKB

Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

Molecular bases, pathogenic mechanisms and possible therapeutic approach in Leber's Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by a rapid loss of central vision and optic atrophy, due to the selective degeneration of retinal ganglion cells. The age of onset is around 20, and the degenerative process is fast and usually the second eye becomes affected in weeks or months. Even if this pathology is well known and has been well characterized, there are still open questions on its pathophysiology, such as the male prevalence, the incomplete penetrance and the tissue selectivity. This maternally inherited disease is caused by mutations in mitochondrial encoded genes of NADH ubiquinone oxidoreductase (complex I) of the respiratory chain. The 90% of LHON cases are caused by one of the three common mitochondrial DNA mutations (11778/ND4, 14484/ND6 and 3460/ND1) and the remaining 10% is caused by rare pathogenic mutations, reported in literature in one or few families. Moreover, there is also a small subset of patients reported with new putative pathogenic nucleotide changes, which awaits to be confirmed. We here clarify some molecular aspects of LHON, mainly the incomplete penetrance and the role of rare mtDNA mutations or variants on LHON expression, and attempt a possible therapeutic approach using the cybrids cell model. We generated novel structural models for mitochondrial encoded complex I subunits and a conservation analysis and pathogenicity prediction have been carried out for LHON reported mutations. This in-silico approach allowed us to locate LHON pathogenic mutations in defined and conserved protein domains and can be a useful tool in the analysis of novel mtDNA variants with unclear pathogenic/functional role. Four rare LHON pathogenic mutations have been identified, confirming that the ND1 and ND6 genes are mutational hot spots for LHON. All mutations were previously described at least once and we validated their pathogenic role, suggesting the need for their screening in LHON diagnostic protocols. Two novel mtDNA variants with a possible pathogenic role have been also identified in two independent branches of a large pedigree. Functional studies are necessary to define their contribution to LHON in this family. It also been demonstrated that the combination of mtDNA rare polymorphic variants is relevant in determining the maternal recurrence of myoclonus in unrelated LHON pedigrees. Thus, we suggest that particular mtDNA backgrounds and /or the presence of specific rare mutations may increase the pathogenic potential of the primary LHON mutations, thereby giving rise to the extraocular clinical features characteristic of the LHON “plus” phenotype. We identified the first molecular parameter that clearly discriminates LHON affected individuals from asymptomatic carriers, the mtDNA copy number. This provides a valuable mechanism for future investigations on variable penetrance in LHON. However, the increased mtDNA content in LHON individuals was not correlated to the functional polymorphism G1444A of PGC-1 alpha, the master regulator of mitochondrial biogenesis, but may be due to gene expression of genes involved in this signaling pathway, such as PGC-1 alpha/beta and Tfam. Future studies will be necessary to identify the biochemical effects of rare pathogenic mutations and to validate the novel candidate mutations here described, in terms of cellular bioenergetic characterization of these variants. Moreover, we were not able to induce mitochondrial biogenesis in cybrids cell lines using bezafibrate. However, other cell line models are available, such as fibroblasts harboring LHON mutations, or other approaches can be used to trigger the mitochondrial biogenesis.

Analisi molecolare dei linfomi cutanei aggressivi

I linfomi primitivi cutanei riconosciuti nella classificazione della WHO/EORTC si presentano come “entità cliniche distinte” su base clinica, morfologica, immunofenotipica e molecolare. Il fenotipo linfocitario T helper CD4+ caratterizza i CTCL, ma alcune entità a prognosi aggressiva presentano un immunofenotipo citotossico CD8+. Numerosi studi di citogenetica (CGH) e gene-expression profiling (GEP) sono stati condotti negli ultimi anni sui CTCL e sono state riscontrate numerose aberrazioni cromosomiche correlate ai meccanismi di controllo del ciclo cellulare. Scopo del nostro studio è la valutazione delle alterazioni genomiche coinvolte nella tumorigenesi di alcuni CTCL aggressivi: il linfoma extranodale NK/T nasal-type, il linfoma primitivo cutaneo aggressivo epidermotropo (AECTCL) e il gruppo dei PTCL/NOS pleomorfo CD8+. Il materiale bioptico dei pazienti è stato sottoposto alla metodica dell’array-CGH per identificare le anomalie cromosomiche; in alcuni casi di AECTCL è stata applicata la GEP, che evidenzia il profilo di espressione genica delle cellule neoplastiche. I dati ottenuti sono stati valutati in modo statistico, evidenziando le alterazioni cromosomiche comuni significative di ogni entità. In CGH, sono state evidenziate alcune aberrazioni comuni fra le entità studiate, la delezione di 9p21.3, l’amplificazione di 17q, 19p13, 19q13.11-q13.32 , 12q13 e 16p13.3, che determinano la delezione dei geni CDKN2A e CDKN2B e l’attivazione del JAK/STAT signaling pathway. Altre alterazioni definiscono l’amplificazione di c-MYC (8q24) e CCND1/CDK4-6 (11q13). In particolare, sono state evidenziate numerose anomalie genomiche comuni in casi di AECTCL e PTCL/NOS pleomorfo. L’applicazione della GEP in 5 casi di AECTCL ha confermato l’alterata espressione dei geni CDKN2A, JAK3 e STAT6, che potrebbero avere un ruolo diretto nella linfomagenesi. Lo studio di un numero maggiore di casi in GEP e l’introduzione delle nuove indagini molecolari come l’analisi dei miRNA, della whole-exome e whole genome sequences consentiranno di evidenziare alterazioni molecolari correlate con la prognosi, definendo anche nuovi target terapeutici.

Studio del ruolo della Protein chinasi B/Akt nella migrazione di Cellule Staminali Mesenchimali umane

Le cellule staminali/stromali mesenchimali umane (hMSC) sono attualmente applicate in diversi studi clinici e la loro efficacia è spesso legata alla loro capacità di raggiungere il sito d’interesse. Poco si sa sul loro comportamento migratorio e i meccanismi che ne sono alla base. Perciò, questo studio è stato progettato per comprendere il comportamento migratorio delle hMSC e il coinvolgimento di Akt, nota anche come proteina chinasi B. L’espressione e la fosforilazione della proteinchinasi Akt è stata studiata mediante Western blotting. Oltre al time-lapse in vivo imaging, il movimento cellulare è stato monitorato sia mediante saggi tridimensionali, con l’uso di transwell, che mediante saggi bidimensionali, attraverso la tecnica del wound healing. Le prove effettuate hanno rivelato che le hMSC hanno una buona capacità migratoria. E’ stato osservato che la proteinchinasi B/Akt ha elevati livelli basali di fosforilazione in queste cellule. Inoltre, la caratterizzazione delle principali proteine di regolazione ed effettrici, a monte e a valle di Akt, ha permesso di concludere che la cascata di reazioni della via di segnale anche nelle hMSC segue un andamento canonico. Specifici inibitori farmacologici sono stati utilizzati per determinare il potenziale meccanismo coinvolto nella migrazione cellulare e nell'invasione. L’inibizione della via PI3K/Akt determina una significativa riduzione della migrazione. L’utilizzo di inibitori farmacologici specifici per le singole isoforme di Akt ha permesso di discriminare il ruolo diverso di Akt1 e Akt2 nella migrazione delle hMSC. E’ stato infatti dimostrato che l'inattivazione di Akt2, ma non quella di Akt1, diminuisce significativamente la migrazione cellulare. Nel complesso i risultati ottenuti indicano che l'attivazione di Akt2 svolge un ruolo critico nella migrazione della hMSC; ulteriori studi sono necessari per approfondire la comprensione del fenomeno. La dimostrazione che l’isoforma Akt2 è necessaria per la chemiotassi diretta delle hMSC, rende questa chinasi un potenziale bersaglio farmacologico per modulare la loro migrazione.

Studio della via di segnale PI3K/Akt/mTOR nelle Cellule Dendritiche

Il trapianto allogenico di cellule staminali emopoietiche è spesso l’unica soluzione per la cura di diverse malattie ematologiche. La aGVHD è la complicanza più importante che si può avere a seguito del trapianto allogenico ed è causata dai linfociti T del donatore che riconoscono gli antigeni del ricevente presentati dalle APC. Eliminare o inattivare la APC del ricevente prima del trapianto potrebbe prevenire la aGVHD. Ad oggi non esistono farmaci specifici diretti contro le APC, sono però noti i meccanismi molecolari coinvolti nella sopravvivenza cellulare come la via di segnale di PI3K. In questo lavoro abbiamo testato l’attività di due farmaci, che colpiscono target molecolari della via di PI3K, la rapamicina e la perifosina, sul differenziamento dei monociti a differenti popolazioni di cellule dendritiche (DC), in vitro. La rapamicina riduceva il recupero cellulare delle DC derivate da monociti coltivate in presenza di IL-4 aumentando l’apoptosi, mentre i monociti coltivati in presenza di GM-CSF con o senza IFN-α risultavano resistenti alla rapamicina. Inoltre la rapamicina riduceva l’espressione della molecola costimolatoria CD86 e incrementava l’espressione della molecola CD1a solo nei monociti coltivati con GM-CSF e IL-4. Nelle DC derivate dai monociti in presenza di IL-4 la rapamicina bloccava la produzione di IL-12 e TNF-α e ne alterava la capacità allostimolatoria. La rapamicina non alterava la sopravvivenza e la funzione delle DC circolanti. Il trattamento con perifosina provocava un incremento di apoptosi nei monociti coltivati sia con GM-CSF che con GM-CSF e IL-4. La perifosina bloccava la produzione di TNF-α nelle DC derivate da monociti coltivati nelle diverse condizioni. Questi risultati dimostrano che l’azione della rapamicina è strettamente dipendente dalla presenza dell’IL-4 nel terreno di coltura, in vitro, rispetto alla perifosina e suggeriscono un possibile ruolo della perifosina nella prevenzione della GVHD prima del trapianto allogenico di cellule staminali.

Taste receptors in the gut: a chemosensitive mechanism from fish to human

The ingestion of a meal evokes a series of digestive processes, which consist of the essential functions of the digestive system: food transport, secretory activity, absorption of nutrients and the expulsion of undigested residues do not absorbed. The gastrointestinal chemosensitivity is characterized by cellular elements of the endocrine gastrointestinal mucosa and nerve fibers, in particular of vagal nature. A wide range of mediators endocrine and/or paracrine can be released from various endocrine cells in response to nutrients in the diet. These hormones, in addition to their direct activity, act through specific receptors activating some of the most important functions in the control of energy intake and energy homeostasis in the body. For integration of this complex system of control of gastrointestinal chemosensitivity, recent evidence demonstrates the presence of taste receptors (TR) belonging to the family of G proteins coupled receptor expressed in the mucosa of the gastrointestinal tract of different mammals and human. This thesis is divided into several research projects that have been conceived in order to clarify the relationship between TR and nutrients. To define this relationship I have used various scientific approaches, which have gone on to evaluate changes in signal molecules of TR, in particular of the α-transducin in the fasting state and after refeeding with standard diet in the gastrointestinal tract of the pig, the mapping of the same molecule signal in the gastrointestinal tract of fish (Dicentrarchus labrax), the signaling pathway of bitter TR in the STC-1 endocrine cell line and finally the involvement of bitter TR in particular of T2R38 in patients with an excessive caloric intake. The results showed how there is a close correlation between nutrients, TR and hormonal release and how they are useful both in taste perception but also likely to be involved in chronic diseases such as obesity.

A SMO inhibitor drug reduces the progenitor cell's maintenance in the Drosophila hematopoietic organ: a novel model to study Chronic Myelogenous Leukemia relapse

The chronic myeloid leukemia complexity and the difficulties of disease eradication have recently led to the development of drugs which, together with the inhibitors of TK, could eliminate leukemia stem cells preventing the occurrence of relapses in patients undergoing transplantation. The Hedgehog (Hh) signaling pathway positively regulates the self-renewal and the maintenance of leukemic stem cells and not, and this function is evolutionarily conserved. Using Drosophila as a model, we studied the efficacy of the SMO inhibitor drug that inhibit the human protein Smoothened (SMO). SMO is a crucial component in the signal transduction of Hh and its blockade in mammals leads to a reduction in the disease induction. Here we show that administration of the SMO inhibitor to animals has a specific effect directed against the Drosophila ortholog protein, causing loss of quiescence and hematopoietic precursors mobilization. The SMO inhibitor induces in L3 larvae the appearance of melanotic nodules generated as response by Drosophila immune system to the increase of its hemocytes. The same phenotype is induced even by the dsRNA:SMO specific expression in hematopoietic precursors of the lymph gland. The drug action is also confirmed at cellular level. The study of molecular markers has allowed us to demonstrate that SMO inhibitor leads to a reduction of the quiescent precursors and to an increase of the differentiated cells. Moreover administering the inhibitor to heterozygous for a null allele of Smo, we observe a significant increase in the phenotype penetrance compared to administration to wild type animals. This helps to confirm the specific effect of the drug itself. These data taken together indicate that the study of inhibitors of Smo in Drosophila can represent a useful way to dissect their action mechanism at the molecular-genetic level in order to collect information applicable to the studies of the disease in humans.

Study of novel natural compounds as potential drugs in the treatment of acute leukemias

Introduction: Among all cancer types leukemia represents the leading cause of cancer death in man younger than 40 years. Single-target drug therapy has generally been highly ineffective in treating complex diseases such as cancer. A growing interest has been directed toward multi-target drugs able to hit multiple targets. In this context, plant products, based on their intrinsic complexity, could represent an interesting and promising approach. Aim of the research followed during my PhD was to indentify and study novel natural compounds for the treatment of acute leukemias. Two potential multi-target drugs were identified in Hemidesmus indicus and piperlongumine. Methodology/Principal Findings: A variety of cellular assays and flow cytometry were performed on different cell lines. We demonstrated that Hemidesmus modulates many components of intracellular signaling pathways involved in cell viability and proliferation and alters gene and protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential, raise of [Ca2+]i, inhibition of Mcl-1, increasing Bax/Bcl-2 ratio, and ROS formation. Moreover, we proved that the decoction causes differentiation of HL-60 and regulates angiogenesis of HUVECs in hypoxia and normoxia, by the inhibition of new vessel formation and the processes of migration/invasion. Clinically relevant observations are that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemia (AML) patients. Moreover, both Hemidesmus and piperlongumine showed a selective action toward leukemic stem cell (LSC). Conclusions: Our results indicate the molecular basis of the anti-leukemic effects of Hemidesmus indicus and indentify the mitochondrial pathways, [Ca2+]i, cytodifferentiation and angiogenesis inhibition as crucial actors in its anticancer activity. The ability to selectively hit LSC showed by Hemidesmus and piperlongumine enriched the knowledge of their anti-leukemic activity. On these bases, we conclude that Hemidesmus and piperlongumine can represent a valuable strategy in the anticancer pharmacology.