The Novel Antitubulin Agent TR-764 Strongly Reduces Tumor Vasculature and Inhibits HIF-1α Activation

Tubulin binding agents (TBAs) are commonly used in cancer therapy as antimitotics. It has been described that TBAs, like combretastatin A-4 (CA-4), present also antivascular activity and among its derivatives we identified TR-764 as a new inhibitor of tubulin polymerization, based on the 2-(alkoxycarbonyl)-3-(3',4',5'-trimethoxyanilino)benzo[b]thiophene molecular skeleton. The antiangiogenic activity of TR-764 (1-10 nM) was tested in vitro on human umbilical endothelial cells (HUVECs), and in vivo, on the chick embryo chorioallantoic membrane (CAM) and two murine tumor models. TR-764 binding to tubulin triggers cytoskeleton rearrangement without affecting cell cycle and viability. It leads to capillary tube disruption, increased cell permeability, and cell motility reduction. Moreover it disrupts adherens junctions and focal adhesions, through mechanisms involving VE-cadherin/β-catenin and FAK/Src. Importantly, TR-764 is active in hypoxic conditions significantly reducing HIF-1α. In vivo TR-764 (1-100 pmol/egg) remarkably blocks the bFGF proangiogenic activity on CAM and shows a stronger reduction of tumor mass and microvascular density both in murine syngeneic and xenograft tumor models, compared to the lead compound CA-4P. Altogether, our results indicate that TR-764 is a novel TBA with strong potential as both antivascular and antitumor molecule that could improve the common anticancer therapies, by overcoming hypoxia-induced resistance mechanisms

MICROTUBULE DEPOLYMERIZING COMPOUNDS AS NOVEL POTENTIAL ANTIVASCULAR AGENTS IN CANCER THERAPY

In the last years, biomedical research was focused on cancer, since it is a leading cause of death in the developed countries. However several molecular targets were identified thus making necessary to find new strategies to improve the traditional chemotherapy. Aberrant tumors grow and spread, and they require their own blood supply. Some approaches of anticancer therapy utilize antiangiogenic drugs, including tubulin binding agents (TBAs). Here we focused our studies on several combretastatin-like molecules (TR-644, TR-764, 3b) which bind to tubulin with high affinity. We report the activity of our TBAs on endothelial cell cytoskeleton, cell motility, permeability, and other processes involved in angiogenesis. Our compounds strongly induce microtubules derangement, and alter the actin microfilaments organization. These effects result in focal adhesions and adherens junctions impairment, by affecting FAK/Src and VE-cadherin/β-catenin molecular pathways. We also evaluated the antivascular activity in vivo both in the chick embryo chorioallantoic membrane (CAM) and in an allograft mouse tumor model. These compounds induce a significant decrease in microvascular density, and strongly inhibit the tumor growth. Additionally, compound TR-764 is endowed with the ability to counteract in vitro hypoxic stimuli on endothelial cells, which usually give rise to resistance mechanisms. Thus we propose our molecules as potential single agents against highly vascularized tumors. A subtype of non-toxic TBAs is represented by noscapines. Some derivatives were synthesized and we investigated their antimitotic effect, on cancer cell lines. They induce cell cycle arrest in G2/M phase and consequently they stimulate apoptosis, following the mitochondrial way. Radial organization of microtubules is altered and multipolar spindles occur after treatment. These modifications trigger DNA damage, and caspase-9/PARP activation. Here we report a new effective method for chemical synthesis (Suzuki cross-coupling), even though the biological activity of our compounds is comparable to other known noscapine derivatives. Another part of the study concern the pharmacological evaluation of a series of small molecules as potential inhibitors of the Wnt/β-catenin pathway. The most potent compounds induce Wnt repression, in a colon cancer cell line (HT-29). They impair β-catenin activity as transcription factor, by downregulating its target genes cyclin D1 and c-myc and altering TCF-1/4 co-factors. Therefore the treatment leads to a reduction of cell proliferation in vitro. Finally we confirmed the inhibitory effects on β-catenin transcriptional activity in vivo, in Wnt-reporter zebrafish models. All these pharmacological studies could be helpful to improve potential approaches for an effective anticancer therapy.Negli ultimi anni la ricerca biomedica si è interessata principalmente di patologie oncologiche, le quali causano un’elevata incidenza di morte nei Paesi sviluppati. Sebbene siano stati identificati numerosi target molecolari, risulta ancora necessario trovare nuove strategie per migliorare la chemioterapia tradizionale. I tumori crescono in maniera aberrante, e la loro espansione necessita dell’apporto di nuovi vasi sanguigni. Alcuni approcci terapeutici nella lotta contro i tumori prevedono l’utilizzo di farmaci antiangiogenici, tra i quali sono comprese le molecole che legano la tubulina (TBA). Questo studio riguarda la valutazione farmacologica di molecole con struttura simile alla combretastatina (TR-644, TR-764, 3b) che sono in grado di legarsi alla tubulina con alta affinità. E’ stata studiata l’attività di questi composti sul citoscheletro di cellule endoteliali, e su processi legati all’angiogenesi, quali la motilità cellulare o la permeabilità. Essi alterano la struttura dei microtubuli e inducono un’elevata disorganizzazione dei microfilamenti di actina. Tali effetti danneggiano le proteine che costituiscono le adesioni focali o le giunzioni aderenti, compromettendo le vie di segnale di FAK/Src e VE-caderina/β-catenina. È stata valutata l’attività antivascolare anche in modelli in vivo, sulla membrana corioallantoidea di pollo (CAM) e in modelli di allotrapianto tumorale nel topo. I composti testati agiscono in maniera significativa sia riducendo la densità microvascolare, sia inibendo la crescita tumorale. Inoltre, in cellule endoteliali, il composto TR-764 è in grado di contrastare stimoli ipossici, che sono stati descritti come possibili responsabili dell’insorgenza di meccanismi di resistenza del tumore. Questi nuovi TBA vengono quindi proposti come potenziali agenti terapeutici per la monoterapia di tumori altamente vascolarizzati. Oltre a derivati della combretastatina è stato studiato un altro sottotipo di TBA non tossici, le noscapine. Una serie di derivati della noscapina è stata sintetizzata ed è stato analizzato il l’effetto antimitotico su linee cellulari tumorali. Questi composti bloccano il ciclo cellulare in fase G2/M, e di conseguenza inducono l’apoptosi, attivando la via mitocondriale. Essi agiscono riorganizzando la struttura radiale dei microtubuli e portano alla formazione di fusi mitotici multipolari. Queste modificazioni stimolano l’insorgenza di danni al DNA, e l’attivazione di caspasi-9 e PARP, effetti già dimostrati per altri derivati della noscapina. Tuttavia in questo studio viene proposto un nuovo metodo molto efficace per la sintesi chimica dei composti (Suzuki cross-coupling). Infine, è stata svolta una valutazione farmacologica di una serie di piccole molecole, con potenziale attività inibitoria della via di segnale di Wnt/β-catenina in una linea cellulare di cancro al colon (HT-29). I composti più attivi inibiscono questa via di segnale, compromettendo l’attività del fattore di trascrizione β-catenina, inibendo i suoi geni target, quali la ciclina D1 e c-myc, e alterando l’attività dei suoi cofattori TCF-1/4. Il trattamento quindi comporta la riduzione della proliferazione cellulare in vitro. Gli effetti di inibizione dell’attività trascrizionale della β-catenina sono stati confermati anche in vivo, tramite saggi reporter di Wnt, in modelli di zebrafish. Gli studi farmacologici riportati in questa tesi possono essere utili per migliorare gli approcci terapeutici per il trattamento di alcuni tumori

MICROTUBULE DEPOLYMERIZING COMPOUNDS AS NOVEL POTENTIAL ANTIVASCULAR AGENTS IN CANCER THERAPY

In the last years, biomedical research was focused on cancer, since it is a leading cause of death in the developed countries. However several molecular targets were identified thus making necessary to find new strategies to improve the traditional chemotherapy. Aberrant tumors grow and spread, and they require their own blood supply. Some approaches of anticancer therapy utilize antiangiogenic drugs, including tubulin binding agents (TBAs). Here we focused our studies on several combretastatin-like molecules (TR-644, TR-764, 3b) which bind to tubulin with high affinity. We report the activity of our TBAs on endothelial cell cytoskeleton, cell motility, permeability, and other processes involved in angiogenesis. Our compounds strongly induce microtubules derangement, and alter the actin microfilaments organization. These effects result in focal adhesions and adherens junctions impairment, by affecting FAK/Src and VE-cadherin/β-catenin molecular pathways. We also evaluated the antivascular activity in vivo both in the chick embryo chorioallantoic membrane (CAM) and in an allograft mouse tumor model. These compounds induce a significant decrease in microvascular density, and strongly inhibit the tumor growth. Additionally, compound TR-764 is endowed with the ability to counteract in vitro hypoxic stimuli on endothelial cells, which usually give rise to resistance mechanisms. Thus we propose our molecules as potential single agents against highly vascularized tumors. A subtype of non-toxic TBAs is represented by noscapines. Some derivatives were synthesized and we investigated their antimitotic effect, on cancer cell lines. They induce cell cycle arrest in G2/M phase and consequently they stimulate apoptosis, following the mitochondrial way. Radial organization of microtubules is altered and multipolar spindles occur after treatment. These modifications trigger DNA damage, and caspase-9/PARP activation. Here we report a new effective method for chemical synthesis (Suzuki cross-coupling), even though the biological activity of our compounds is comparable to other known noscapine derivatives. Another part of the study concern the pharmacological evaluation of a series of small molecules as potential inhibitors of the Wnt/β-catenin pathway. The most potent compounds induce Wnt repression, in a colon cancer cell line (HT-29). They impair β-catenin activity as transcription factor, by downregulating its target genes cyclin D1 and c-myc and altering TCF-1/4 co-factors. Therefore the treatment leads to a reduction of cell proliferation in vitro. Finally we confirmed the inhibitory effects on β-catenin transcriptional activity in vivo, in Wnt-reporter zebrafish models. All these pharmacological studies could be helpful to improve potential approaches for an effective anticancer therapy.Negli ultimi anni la ricerca biomedica si è interessata principalmente di patologie oncologiche, le quali causano un’elevata incidenza di morte nei Paesi sviluppati. Sebbene siano stati identificati numerosi target molecolari, risulta ancora necessario trovare nuove strategie per migliorare la chemioterapia tradizionale. I tumori crescono in maniera aberrante, e la loro espansione necessita dell’apporto di nuovi vasi sanguigni. Alcuni approcci terapeutici nella lotta contro i tumori prevedono l’utilizzo di farmaci antiangiogenici, tra i quali sono comprese le molecole che legano la tubulina (TBA). Questo studio riguarda la valutazione farmacologica di molecole con struttura simile alla combretastatina (TR-644, TR-764, 3b) che sono in grado di legarsi alla tubulina con alta affinità. E’ stata studiata l’attività di questi composti sul citoscheletro di cellule endoteliali, e su processi legati all’angiogenesi, quali la motilità cellulare o la permeabilità. Essi alterano la struttura dei microtubuli e inducono un’elevata disorganizzazione dei microfilamenti di actina. Tali effetti danneggiano le proteine che costituiscono le adesioni focali o le giunzioni aderenti, compromettendo le vie di segnale di FAK/Src e VE-caderina/β-catenina. È stata valutata l’attività antivascolare anche in modelli in vivo, sulla membrana corioallantoidea di pollo (CAM) e in modelli di allotrapianto tumorale nel topo. I composti testati agiscono in maniera significativa sia riducendo la densità microvascolare, sia inibendo la crescita tumorale. Inoltre, in cellule endoteliali, il composto TR-764 è in grado di contrastare stimoli ipossici, che sono stati descritti come possibili responsabili dell’insorgenza di meccanismi di resistenza del tumore. Questi nuovi TBA vengono quindi proposti come potenziali agenti terapeutici per la monoterapia di tumori altamente vascolarizzati. Oltre a derivati della combretastatina è stato studiato un altro sottotipo di TBA non tossici, le noscapine. Una serie di derivati della noscapina è stata sintetizzata ed è stato analizzato il l’effetto antimitotico su linee cellulari tumorali. Questi composti bloccano il ciclo cellulare in fase G2/M, e di conseguenza inducono l’apoptosi, attivando la via mitocondriale. Essi agiscono riorganizzando la struttura radiale dei microtubuli e portano alla formazione di fusi mitotici multipolari. Queste modificazioni stimolano l’insorgenza di danni al DNA, e l’attivazione di caspasi-9 e PARP, effetti già dimostrati per altri derivati della noscapina. Tuttavia in questo studio viene proposto un nuovo metodo molto efficace per la sintesi chimica dei composti (Suzuki cross-coupling). Infine, è stata svolta una valutazione farmacologica di una serie di piccole molecole, con potenziale attività inibitoria della via di segnale di Wnt/β-catenina in una linea cellulare di cancro al colon (HT-29). I composti più attivi inibiscono questa via di segnale, compromettendo l’attività del fattore di trascrizione β-catenina, inibendo i suoi geni target, quali la ciclina D1 e c-myc, e alterando l’attività dei suoi cofattori TCF-1/4. Il trattamento quindi comporta la riduzione della proliferazione cellulare in vitro. Gli effetti di inibizione dell’attività trascrizionale della β-catenina sono stati confermati anche in vivo, tramite saggi reporter di Wnt, in modelli di zebrafish. Gli studi farmacologici riportati in questa tesi possono essere utili per migliorare gli approcci terapeutici per il trattamento di alcuni tumori

SYK Targeting Represents a Potential Therapeutic Option for Relapsed Resistant Pediatric ETV6-RUNX1 B-Acute Lymphoblastic Leukemia Patients

The presence of the chromosomal rearrangement t(12;21)(ETV6-RUNX1) in childhood B-acute lymphoblastic leukemia (B-ALL) is an independent predictor of favorable prognosis, however relapses still occur many years later after stopping therapy, and patients often display resistance to current treatments. Since spleen tyrosine kinase (SYK), a cytosolic nonreceptor tyrosine kinase interacting with immune receptors, has been previously associated with malignant transformation and cancer cell proliferation, we aimed to assess its role in ETV6-RUNX1 cell survival and prognosis. We evaluated the effects on cell survival of three SYK inhibitors and showed that all of them, in particular entospletinib, are able to induce cell death and enhance the efficacy of conventional chemotherapeutics. By using reverse phase protein arrays we next revealed that activated SYK is upregulated at diagnosis in pediatric ETV6-RUNX1 patients who will experience relapse, and, importantly, hyperactivation is maintained at a high level also at relapse occurrence. We thus treated primary cells from patients both at diagnosis and relapse with the combination entospletinib + chemotherapeutics and observed that SYK inhibition is able to sensitize resistant primary cells to conventional drugs. Entospletinib could thus represent a new therapeutic option supporting conventional chemotherapy for relapsed ETV6-RUNX1 patients, and these evidences encourage further studies on SYK for treatment of other relapsed resistant acute lymphoblastic leukemia (ALL) subgroups

Synthesis and biological evaluation of imidazo[1,2-a]pyrimidines and imidazo[1,2-a]pyridines as new inhibitors of the Wnt/β-catenin signaling

Wnt/β-catenin signaling plays an important role in the regulation of embryonic development and tumorigenesis. Since its deregulation results in severe human diseases, especially cancer, the Wnt signaling pathway constitutes a promising platform for pharmacological targeting of cancer. In this study we synthesized a series of imidazo[1,2-a]pyrimidines and imidazo[1,2-a]pyridines and identified some derivatives that were able to inhibit the Wnt/β-catenin signaling pathway in a luciferase reporter assay and cell proliferation in selected cancer cell lines, endowed with APC or β-catenin gene mutations. The most active compounds significantly downregulate the expression of Wnt target genes such as c-myc and cyclin D1. Further studies indicated that these compounds function independently of GSK-3β activity. More importantly, in vivo experiments, carried out on a Wnt-reporter zebrafish model indicate, in particular for compounds 4c and 4i as the most active compounds, an activity comparable to that of the reference compound IWR1, suggesting their potential use not only as small molecule inhibitors of the Wnt/β-catenin signal in Wnt driven cancers, but also in other Wnt-related diseases

Identification of Homoharringtonine as a potent inhibitor of glioblastoma cell proliferation and migration

We previously demonstrated that Annexin A2 (ANXA2) is a pivotal mediator of the pro-oncogenic features displayed by glioblastoma (GBM) tumors, the deadliest adult brain malignancies, being involved in cell stemness, proliferation and invasion, thus negatively impacting patient prognosis. Based on these results, we hypothesized that compounds able to revert ANXA2-dependent transcriptional features could be exploited as reliable treatments to inhibit GBM cell aggressiveness by hampering their proliferative and migratory potential. Transcriptional signatures obtained by the modulation of ANXA2 activity/levels were functionally mapped through the QUADrATiC bioinformatic tool for compound identification. Selected compounds were screened by cell proliferation and migration assays in primary GBM cells, and we identified Homoharringtonine (HHT) as a potent inhibitor of GBM cell motility and proliferation, without affecting their viability. A further molecular characterization of the effects displayed by HHT, confirmed its ability to inhibit a transcriptional program involved in cell migration and invasion. Moreover, we demonstrated that the multiple antitumoral effects displayed by HHT are correlated to the inhibition of a platelet derived growth factor receptor α (PDGFRα)-dependent intracellular signaling through the impairment of Signal transducer and activator of transcription 3 (STAT3) and Ras homolog family member A (RhoA) axes. Our results demonstrate that HHT may act as a potent inhibitor of cancer cell proliferation and invasion in GBM, by hampering multiple PDGFRα-dependent oncogenic signals transduced through the STAT3 and RhoA intracellular components, finally suggesting its potential transferability for achieving an effective impairment of peculiar GBM hallmarks

A novel copper(I) complex induces ER-stress-mediated apoptosis and sensitizes B-acute lymphoblastic leukemia cells to chemotherapeutic agents

none8noA phosphine copper(I) complex [Cu(thp)(4)][PF6] (CP) was recently identified as an efficient in vitro antitumor agent. In this study, we evaluated the antiproliferative activity of CP in leukemia cell lines finding a significant efficacy, especially against SEM and RS4; 11 cells. Immunoblot analysis showed the activation of caspase-12 and caspase-9 and of the two effector caspase-3 and -7, suggesting that cell death occurred in a caspase-dependent manner. Interestingly we did not observe mitochondrial involvement in the process of cell death. Measures on semipurified proteasome from RS4; 11 and SEM cell extracts demonstrated that chymotrypsin-, trypsin-and caspase-like activity decreased in the presence of CP. Moreover, we found an accumulation of ubiquitinated proteins and a remarkable increase of ER stress markers: GRP78, CHOP, and the spliced form of XBP1. Accordingly, the protein synthesis inhibitor cycloheximide significantly protected cancer cells from CP-induced cell death, suggesting that protein synthesis machinery was involved. In well agreement with results obtained on stabilized cell lines, CP induced ER-stress and apoptosis also in primary cells from B-acute lymphoblastic leukemia patients. Importantly, we showed that the combination of CP with some chemotherapeutic drugs displayed a good synergy that strongly affected the survival of both RS4;11 and SEM cells.noneR. Bortolozzi; G. Viola; E. Porcù; F. Consolaro; C. Marzano; M. Pellei; V. Gandin; G. BassoBortolozzi, Roberta; Viola, G.; Porcù, E.; Consolaro, Francesca; Marzano, Cristina; Pellei, M.; Gandin, Valentina; Basso, Giusepp

Newborn Screening for Fabry Disease: Current Status of Knowledge

Fabry disease is an X-linked progressive lysosomal disorder, due to α-galactosidase A deficiency. Patients with a classic phenotype usually present in childhood as a multisystemic disease. Patients presenting with the later onset subtypes have cardiac, renal and neurological involvements in adulthood. Unfortunately, the diagnosis is often delayed until the organ damage is already irreversibly severe, making specific treatments less efficacious. For this reason, in the last two decades, newborn screening has been implemented to allow early diagnosis and treatment. This became possible with the application of the standard enzymology fluorometric method to dried blood spots. Then, high-throughput multiplexable assays, such as digital microfluidics and tandem mass spectrometry, were developed. Recently DNA-based methods have been applied to newborn screening in some countries. Using these methods, several newborn screening pilot studies and programs have been implemented worldwide. However, several concerns persist, and newborn screening for Fabry disease is still not universally accepted. In particular, enzyme-based methods miss a relevant number of affected females. Moreover, ethical issues are due to the large number of infants with later onset forms or variants of uncertain significance. Long term follow-up of individuals detected by newborn screening will improve our knowledge about the natural history of the disease, the phenotype prediction and the patients’ management, allowing a better evaluation of risks and benefits of the newborn screening for Fabry disease

A synthetic BMP-2 mimicking peptide induces glioblastoma stem cell differentiation

Glioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. In this context, Bone Morphogenetic Proteins (BMPs) have been demonstrated to induce CSC differentiation and to sensitize GBM cells to treatments