Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

Variation in peach fruit susceptibility to Monilinia laxa and gene expression

Brown rot caused by Monilinia laxa and Monilinia fructigena is considered one of the most important diseases affecting Prunus species. Although some losses can result from the rotten fruits in the orchard, most of the damage is caused to fruits during the post-harvest phase. Several studies reported that brown rot incidence during fruit development highly varies; it was found that at a period corresponding to the the pit hardening stage, fruit susceptibility drastically decreases, to be quickly restored afterwards. However the molecular basis of this phenomenon is still not well understood. Furthermore, no difference in the rot incidence was found between wound and un-wound fruits, suggesting that resistance associated more to a specifc biochemical response of the fruit, rather than to a higher mechanical resistance. So far, the interaction Monilinia-peach was analyzed through chemical approaches. In this study, a bio-molecular approach was undertaken in order to reveal alteration in gene expression associated to the variation of susceptibility. In this thesis three different methods for gene expression analysis were used to analyze the alterations in gene expression occurring in peach fruits during the pit hardening stage, in a period encompassing the temporary change in Monilinia susceptibility: real time PCR, microarray and cDNA AFLP techniques. In 2005, peach fruits (cv.K2) were weekly harvested during a 19-week long-period, starting from the fourth week after full bloom, until full maturity. At each sampling time, three replicates of 5 fruits each were dipped in the M.laxa conidial suspension or in distilled water, as negative control. The fruits were maintained at room temperature for 3 hours; afterwards, they were peeled with a scalpel; the peel was immediately frozen in liquid nitrogen and transferred to -80 °C until use. The degree of susceptibility of peach fruit to the pathogen was determined on 3 replicates of 20 fruits each, as percentage of infected fruits, after one week at 20 °C. Real time PCR analysis was performed to study the variation in expression of those genes encoding for the enzymes of the phenylpropanoid pathway (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), cinnamate 4-hydroxylase (C4H), leucoanthocyanidine reductase (LAR), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and of the jasmonate pathway, such as lipoxygenase (LOX), both involved in the production of important defense compounds. Alteration in gene expression was monitored on fruit samples of a period encompassing the pit hardening stage and the corresponding temporary resistance to M.laxa infections, weekly, from the 6thto the 12th week after full bloom (AFB) inoculated with M. laxa or mock-inoculated. The data suggest a critical change in the expression level of the phenylpropanoid pathway from the 7th to the 8th week AFB; such change could be directly physiologically associated to the peach growth and it could indirectly determine the decrease of susceptibility of peach fruit to Monilinia rot during the subsequent weeks. To investigate on the transcriptome variation underneath the temporary loss of susceptibility of peach fruits to Monilinia rot, the microarray and the cDNA AFLP techniques were used. The samples harvested on the 8th week AFB (named S, for susceptible ones) and on the 12th week AFB (named R, for resistant ones) were compared, both inoculated or mock-inoculated. The microarray experiments were carried out at the University of Padua (Dept. of Environmental Agronomy and Crop Science), using the μPEACH1.0 microarray together with the suited protocols. The analysis showed that 30 genes (corresponding to the 0.6% of the total sequences (4806) contained in the μPeach1.0 microarray) were found up-regulated and 31 ( 0.6%) down regulated in RH vs. SH fruits. On the other hand, 20 genes (0.4%) were shown to be up-regulated and 13 (0.3%) down-regulated in the RI vs. SI fruit. No genes were found differentially expressed in the mock-inoculated resistant fruits (RH) vs. the inoculated resistant ones (RI). Among the up-regulated genes an ATP sulfurylase, an heat shock protein 70, the major allergen Pru P1, an harpin inducing protein and S-adenosylmethionine decarboxylase were found, conversely among the down-regulated ones, cinnamyl alcohol dehydrogenase, an histidine- containing phosphotransfer protein and the ferritin were found. The microarray experimental results and the data indirectly derived, were tested by Real Time PCR analysis. cDNA AFLP analysis was also performed on the same samples. 339 transcript derived fragments considered significant for Monilinia resistance, were selected, sequenced and classified. Genes potentially involved in cell rescue and defence were well represented (8%); several genes (12.1%) involved in the protein folding, post-transductional modification and genes (9.2%) involved in cellular transport were also found. A further 10.3% of genes were classified as involved in the metabolism of aminoacid, carbohydrate and fatty acid. On the other hand, genes involved in the protein synthesis (5.7%) and in signal transduction and communication (5.7%) were found. Among the most interesting genes found differentially expressed between susceptible and resistant fruits, genes encoding for pathogenesis related (PR) proteins were found. To investigate on the association of Monilinia resistance and PR biological function, the major allergen Pru P1 (GenBank accession AM493970) and its isoform (here named Pru P2), were expressed in heterologous system and in vitro assayed for their anti-microbial activity. The ribonuclease activity of the recombinant Pru P1 and Pru P2 proteins was assayed against peach total RNA. As the other PR10 proteins, they showed a ribonucleolytic activity, that could be important to contrast pathogen penetration. Moreover Pru P1 and Pru P2 recombinant proteins were checked for direct antimicrobial activity. No inhibitory effect of Pru P1 or Pru P2 was detected against the selected fungi.

Messa a punto di procedure per la semi-sintesi di composti ad attività antitumorale partendo da prodotti naturali. Studi di struttura-attività

Camptothecin, (CPT) is a pentacyclic alkaloid isolated for the first time from the Chinese tree Camptotheca acuminata, and which has soon attracted the attention of medicinal chemists and pharmacologists due to its promising anti-cancer activity against the most aggressive histo-types. So far, most of the synthesized camptothecin analogues are A and B ring modified compounds, which have been prepared via synthetic or semi-synthetic routes. To the best of our knowledge, a very limited number of C, D, or E ring modified analogues of CPT have been reported; moreover, the few derivatives known from the literature showed a reduced or no biological activity. This dissertation presents synthetic studies on camptothecin new derivatives along with the development of a new and general semi-synthetic methodology to obtain a large variety of analogues. We report here the semi-synthesis of a new family of 5-substituted CPT's, along with their biological activity evaluation, which will be compared with reference compounds. The use of carrier-linked prodrugs has emerged as a useful strategy to overcome some of the drawbacks related with the use of the parent drug, such as low solubility, membrane permeability properties, low oral absorption, instability, toxicity, and nontargeting. Herein we report CPT-prodrugs synthesized via ring opening of the lactone moiety as 17-O-acyl camptothecin tripartate conjugates, which bear a polyamine side chain with different architectures, as the carriers. Moreover, we found that the replacement of the oxygen atom with sulphur on the piridone D-ring, dramatically improves the potency of the novel 16a-thio-camptothecin derivatives, opening new possibilities in the modelling of this class of compounds.

Valutazione preclinica di nuovi potenziali bersagli terapeutici per la cura del medulloblastoma.

Il medulloblastoma (MB) è il tumore più comune del sistema nervoso centrale. Attualmente si riesce a curare solo il 50-60% dei pazienti che tuttavia presentano gravi effetti collaterali dovuti alle cure molto aggressive. Una recente classificazione molecolare ha ridistribuito le varianti più comuni di MB in quattro distinti gruppi. In particolare, il gruppo SHH e D sono caratterizzati da un’ alta espressione di MYCN ed associati a prognosi sfavorevole. MYCN è coinvolto nella regolazione della proliferazione cellulare, differenziazione, apoptosi, angiogenesi e metastasi. L’obiettivo di questo lavoro è la valutazione dell’attività antitumorale di oligonucleotidi diretti contro MYCN, sia in vitro su diverse linee cellulari di MB che in vivo in modelli murini xenograft ortotopici di MB. I risultati hanno dimostrato un’ottima inibizione della crescita in linee cellulari di MB, accompagnata da una riduzione della trascrizione genica e dei livelli proteici di MYCN. Inoltre, sono stati confermati tramite RT-PCR alcuni dei geni trovati significativamente variati nell’esperimento di microarray , dopo il trattamento. Molto interessanti sono stati geni quali BIRC5, che risulta down regolato mentre il gene p21 risulta up regolato in tutte le linee cellulari di MB utilizzate. Inoltre, sono stati generati modelli murini di MB utilizzando cellule precedentemente trasfettate per esprimere il gene della luciferasi e valutarne così la crescita tumorale tramite imaging bioluminescente in vivo (BLI), al fine di poter testare l’attività antitumorale data dagli oligonucleotidi. I risultati hanno dimostrato una buona risposta al trattamento come rilevato dalla tecnica di BLI. I dati preliminari prodotti, dimostrano che MYCN potrebbe esser un buon target per la terapia del MB nei casi in cui è overespresso. In particolare, una sua inibizione potrebbe presentare effetti indesiderati moderati in quanto è poco espresso dopo la nascita.

Cellular and molecular effects of cross reacting material 197 (CRM197) on HT-29 colon cancer cell line

Cross Reacting Material 197(CRM197) is a Diphteria toxin non toxic mutant that had shown anti-tumor activity in mice and humans. CRM197 is utilized as a specific inhibitor of heparin-binding epidermal growth factor (HB-EGF), that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. We evaluated the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible effects on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment, or changes in cell cycle distribution, in EGFR localization or activation, they show a change in gene expression profile analyzed by microarray. This is the first study where the CRM197 treatment on HT-29 show the alteration of a specific and selected number of genes.

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.

Terapie sistemiche dell'epatocarcinoma (HCC): dalla preclinica alla clinica

Introduzione Attualmente i principali punti critici del trattamento dell’HCC avanzato sono: 1) la mancanza di predittori di risposta alla terapia con sorafenib, 2) lo sviluppo resistenze al sorafenib, 3) la mancanza di terapie di seconda linea codificate. Scopo della tesi 1) ricerca di predittori clinico-laboratoristici di risposta al sorafenib in pazienti ambulatoriali con HCC; 2) valutazione dell’impatto della sospensione temporanea-definitiva del sorafenib in un modello murino di HCC mediante tecniche ecografiche; 3) valutazione dell’efficacia della capecitabina metronomica come seconda linea dell’HCC non responsivo a sorafenib. Risultati Studio-1: 94 pazienti con HCC trattato con sorafenib: a presenza di metastasi e PVT-neoplastica non sembra inficiare l’efficacia del sorafenib. AFP basale <19 ng/ml è risultata predittrice di maggiore sopravvivenza, mentre lo sviluppo di nausea di una peggiore sopravvivenza. Studio -2: 14 topi con xenografts di HCC: gruppo-1 trattato con placebo, gruppo-2 trattato con sorafenib con interruzione temporanea del farmaco e gruppo-3 trattato con sorafenib con sospensione definitiva del sorafenib. La CEUS targettata per il VEGFR2 ha mostrato al giorno 13 valori maggiori di dTE nel gruppo-3 confermato da un aumento del VEGFR2 al Western-Blot. I tumori del gruppo-2 dopo 2 giorni di ritrattamento, hanno mostrato un aumento dell’elasticità tissutale all’elastonografia. Studio-3:19 pazienti trattati con capecitabina metronomica dopo sorafenib. Il TTP è stato di 5 mesi (95% CI 0-10), la PFS di 3,6 mesi (95% CI 2,8-4,3) ed la OS di 6,3 mesi (95% CI 4-8,6). Conclusioni Lo sviluppo di nausea ed astenia ed AFP basale >19, sono risultati predittivi di una minore risposta al sorafenib. La sospensione temporanea del sorafenib in un modello murino di HCC non impedisce il ripristino della risposta tumorale, mentre una interruzione definitiva tende a stimolare un “effetto rebound” dell’angiogenesi. La capecitabina metronomica dopo sorafenib ha mostrato una discreta attività anti-neoplastica ed una sicurezza accettabile.