1,721,005 research outputs found
Role of BCL-2 associated athanogene - 1 (BAG-1) in Acute Myeloid Leukemia (AML): protein with hundred faces
Full text linkBcl-2 associated AthanoGene-1 (BAG-1) is a multifunctional protein competent to delay cell death by a synergistic action with Bcl-2. BAG-1, as well as Bcl-2, has been reported as deregulated in diverse cancer types. During PhD study, we confirmed BAG-1 protein as over-expressed in a cohort of leukemic cell lines and heterogeneously expressed in patients affected by de novo acute myeloid or lymphoid leukemia. Silencing approach, used for a determination of BAG-1’s role in AML, suggested a correlation between BAG-1 down-regulation and decreased expression of certain proteins, which contribution to a proliferative advantage of leukemic cells has already been documented.
BAG-1 expression in leukemic cells was demonstrated to be reciprocally regulated with the expression of BAG-3, gene from the same family which shows functional similarities with BAG-1. In correspondence, major impact on leukemic cell faith was verified by co-silencing both of this genes. The increased cell death, confirmed after co-silencing of leukemic cell lines and primary AML, was sustained by caspase-3 and PARP cleavage and release of cytochrome c and Smac/DIABLO. Key effect of BAG-1 and BAG-3 co-silencing was seen at the levels of anti-apoptotic proteins Bcl-2, Mcl-1 and Bcl-XL, survival protein ERK1/2 and cyclin D1, which all resulted as decreased. Our results indicated that BAG-1 might take an important place in a protection of native Bcl-2 and Mcl-1 proteins from a proteasome derived degradation, since proteins became removed from cell more intensively after transient BAG-1;3 silencing. In consequence, BAG-1, sustained by BAG-3, occurred to be a novel protein able to affect leukemic cells faith when highly expressed, in major part by provoking in these cells a block of apoptosis induction. It is most likely that described role BAG-1 accomplishes either directly or indirectly. In fact, BAG-1 interacts directly with Bcl-2 and most likely with Usp9X, influencing thereby indirectly the expression of Mcl-1, protein of great importance for leukemic blasts survival, and in consequence impairing the apoptosis induction.
Finally, BAG-1 and BAG-3 seem to be new molecules capable to sustain AML. Therefore, identification of their molecular targets, such as Bcl-2, Mcl-1 and Usp9X, significant for leukemia development, gives a meaning to the novel pathways which could be of importance for paediatric AML development and opens a possibility for the future studies for a new therapeutic targets.Bcl-2 associated AthanoGene-1 (BAG-1) è una proteina multifunzionale competente per ritardare la morte cellulare mediante un'azione sinergica principalmente con Bcl-2. BAG-1 così come Bcl-2, è stato segnalato essere molto spesso deregolato in diversi tipi di cancro. Durante il dottorato di ricerca, abbiamo trovato che la proteina BAG-1 è sovra-espressa in una coorte di linee cellulari leucemiche e si esprime in modo eterogeneo in pazienti affetti da leucemia acuta mieloide o linfoide all’esordio. L’approccio del silenziamento genico è stato utilizzato per determinare il ruolo che BAG-1 svolge principalmente nelle leucemie acute mieloidi (LAM), permettendoci di scoprire che l’indotta sotto-espressione di BAG-1 comporta una ridotta espressione di alcune proteine molto importanti per conferire un vantaggio proliferativo alle cellule tumorali.
L’espressione di BAG-1 nelle cellule leucemiche è stata dimostrata essere inversamente proporzionale all’espressione di BAG-3, gene della stessa famiglia genica con funzione altamente simile a quella di BAG-1, e questo fenomeno di compensazione genica è stato dimostrato svolgere un ruolo di forte impatto sulla sopravivenza delle cellule leucemiche soprattutto se entrambi i geni venivano silenziati insieme. Un aumento della morte cellulare, confermata dopo il co-silenziamento di BAG-1;3 in linee leucemiche e in colture primarie di LAM è stata osservata, così come l’attivazione delle proteine caspasi-3 e PARP e il rilascio delle molecole pro-apoptotiche citocromo c e Smac/DIABLO. L’elemento chiave del co-silenziamento di BAG-1 e BAG-3 è stato individuato essere soprattutto a livello di repressione dell’espressione proteica di elementi anti-apoptotici, quali Bcl-2, Mcl-1 e Bcl-XL, così come di alcuni regolatori della proliferazione come ERK1/2 e ciclina D1. La nostra ipotesi è che BAG-1 possa rivestire un ruolo importante nella protezione della degradazione di alcune di queste proteine che normalmente vengono degradate via proteasoma. Infatti, dato che queste proteine risultavano diminuite dopo il silenzia
mento transitorio di BAG-1;3, la mancanza di questi fattori noti per regolare il turnover proteico, dunque supportava il loro contributo alla aumentata degradazione proteica oservata. E’ molto probabile che il ruolo di BAG-1/-3 avvenga direttamente o indirettamente. Infatti, BAG-1 è noto interagire direttamente con particolari fattori, e noi abbiamo dimostrato che così avviene per Bcl-2, fenomeno già descritto in altri tessuti, e con una proteina di recente interesse la Usp9X, mai prima identificata tra i target diretti di BAG-1. L’abbassamento di BAG-1;3, molto probabilmente tramite Usp9X, ha influenzato l’espressione di Mcl-1, un altro fattore importante nell’apoptosi delle LAM, dimostrando che silenziamento era in grado di influenzare le cellule leucemiche provocando un blocco di induzione dell’apoptosi.
Infine, BAG-1 e BAG-3 si candidano a nuove molecole con un potenziale ruolo nel mantenimento delle LAM. L’identificazione dei loro principali targets molecolari, quali Bcl-2, Mcl-1 e Usp9X, affetti particolarmente da BAG-1, portano novità sul ruolo di nuovi pathway che potrebbero essere considerati nelle LAM pediatriche e per futuri sviluppi di targets terapeutici
Porous collagen scaffolds enable endothelial lumen formation in vitro under both static and dynamic growth conditions
No full textAbstract Despite recent advances in the field of tissue engineering, the development of complex tissue‐like structures in vitro is compromised by the lack of integration of a functioning vasculature. In this study, we propose a mesoscale three‐dimensional (3D) in vitro vascularized connective tissue model and demonstrate its feasibility to prompt the self‐assembly of endothelial cells into vessel‐like structures. Moreover, we investigate the effect of perfusion on the organization of the cells. For this purpose, primary endothelial cells (HUVECs) and a cell line of human foreskin fibroblasts are cultivated in ECM‐like matrices made up of freeze‐dried collagen scaffolds permeated with collagen type I hydrogel. A tailored bioreactor is designed to investigate the effect of perfusion on self‐organization of HUVECs. Immunofluorescent staining, two‐photon microscopy, second‐harmonic generation imaging, and scanning electron microscopy are applied to visualize the spatial arrangement of the cells. The analyses reveal the formation of hollow, vessel‐like structures of HUVECs in hydrogel‐permeated collagen scaffolds under both static and dynamic conditions. In conclusion, we demonstrate the feasibility of a 3D porous collagen scaffolding system that enables and maintains the self‐organization of HUVECs into vessel‐like structures independent of a dynamic flow
The cAMP Response Element Binding Protein (CREB) Overexpression Induces Myeloid Transformation in Zebrafish
No full textBAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.
Full text linkBCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance
Setting up of a neuroblastoma cell invasion dynamic 3D model inside a millifluidic optically accessible biorector
Full text linkLAUREA MAGISTRALEIl neuroblastoma è una forma tumorale pediatrica che origina da cellule della cresta neurale (NCCs), caratterizzato da un’elevata eterogeneità che influenza le condizioni cliniche dei pazienti.
LIN28B, un gene importante per lo sviluppo embrionale, ha un ruolo chiave nella formazione e progressione di diverse tipologie tumorali quando non espresso fisiologicamente. Infatti, una overespressione di LIN28B blocca il differenziamento dei precursori simpatoadrenergici promuovendo in questo modo la formazione di neuroblastoma, oltre a favorire la migrazione e invasività cellulare. Tra i nuovi dispositivi di bioingegneria che potrebbero superare i limiti delle colture cellulari in 2D, il gold standard per lo studio in oncologia di neuroblastoma, si può annoverare il miniaturized optically accessible bioreactor (MOAB), un bioreattore capace di replicare in vitro entrambe le colture 2D e 3D mantenendo una condizione dinamica con un costante flusso di medium di crescita alle cellule. In questo studio abbiamo indagato le migliori condizioni per poter effettuare una coltura 2D e 3D in nichoid di linee cellulari umane di neuroblastoma SH-SY5Y all’interno del MOAB per confermare in tempo reale il fenotipo migratorio e invasivo delle cellule SH-SY5YLIN28B overesprimenti LIN28B in confronto alle cellule SH-SY5YCTRL di controllo. Questo lavoro è stato supportato dal laboratorio di Neuroblastoma, Fondazione Istituto di Ricerca Pediatrica Città della Speranza (Padova, Italia), supervisionato dalla Dott. Aveic Sanja e assistito dal team di Neuroblastoma (Dr. Corallo Diana e Sig.ra Pantile Marcella). Ci siamo concentrati sul determinare il numero di cellule più idoneo da seminare, un flusso (e rispettivi shear stress) adeguato a fornire il giusto quantitativo di medium di crescita alle cellule, un colorante ideale per l’analisi live-cell in fluorescenza. Infine, dopo aver confermato la sopravvivenza cellulare in coltura con un’analisi LIVE/DEAD, abbiamo acquisito delle immagini in Time Lapse per lo studio della migrazione cellulare. I risultati ottenuti in condizione dinamica hanno confermato un aumento significativo della motilità cellulare di neuroblastoma dovuta a una overespressione di LIN28B, sia in termini di distanza che di velocità. Non sono state riscontrate invece delle differenze significative tra i dati raccolti dalle culture dinamiche 2D e 3D in nichoid, nonostante siano stati evidenziati dei pattern di migrazioni diversi. Si sottolinea quindi la necessità in futuro di effettuare ulteriori valutazioni per poter comprendere e sfruttare al massimo le potenzialità della coltura tridimensionale in una condizione dinamica, fornendo così un modello tumorale per lo studio di neuroblastoma.Neuroblastoma is a pediatric tumor that originates from neural crest cells (NCCs), characterized by a high heterogeneity that affects the clinical conditions of patients. LIN28B, an important gene in embryonic development, has a key role in the formation and progression of several diseases when not physiologically expressed. Indeed, an overexpression of LIN28B leads to an impaired differentiation of sympathoadrenal precursors leading to neuroblastoma genesis in addition to enhancing the cell migration and invasion. Among the new bioengineered devices that could overcome the limitations of the gold standard 2D culture for neuroblastoma tumor cells, we count the miniaturized optically accessible bioreactor (MOAB), able to replicate in vitro both 2D and 3D cultures provided with a constant flow of growth medium. In this study we investigated a suitable protocol for human neuroblastoma cancer cell line SH-SY5Y in MOAB, in 2D and 3D in nichoid to confirm in real-time the migratory and invasive phenotypes of SH-SY5YLIN28B overexpressing LIN28B.
This work was supported by the Neuroblastoma laboratory, Fondazione Istituto di Ricerca Pediatrica Città della Speranza (Padua, Italy), supervised by Dott. Aveic Sanja and assisted by the neuroblastoma team (Dr. Corallo Diana and Miss Pantile Marcella). We focused on determine the most suitable number of cells to be plated, flow rate (respective shear stress) and amount of growth medium to be provided to cells, choice of dye for fluorescent live-cell analysis. Finally, after assessing the viability of cell cultures by LIVE/DEAD assay, we performed a Time Lapse imaging for migratory analysis.
The results obtained in dynamic conditions statistically confirmed an increase of cell motility of neuroblastoma cells due to overexpression of LIN28B, both in terms of distance and speed. There was no significant difference between SH-SY5YLIN28B cells cultured in 2D and 3D in nichoid, even though different patterns of migration were observed. Hence, further investigations will lead in future to a better understanding of 3D structures in dynamic conditions, permitting to exploit all its potentiality as disease model
The zebrafish as a model for studying neuroblastoma
Full text linkNeuroblastoma is a tumor arising in the peripheral sympathetic nervous system and is the most common cancer
in childhood. Since most of the cellular and molecular mechanisms underlying neuroblastoma onset and progression
remain unknown, the generation of new in vivo models might be appropriate to better dissect the peripheral
sympathetic nervous system development in both physiological and disease states. This review is focused on the use
of zebrafish as a suitable and innovative model to study neuroblastoma development. Here, we briefly summarize the
current knowledge about zebrafish peripheral sympathetic nervous system formation, focusing on key genes and cellular
pathways that play a crucial role in the differentiation of sympathetic neurons during embryonic development.
In addition, we include examples of how genetic changes known to be associated with aggressive neuroblastoma
can mimic this malignancy in zebrafish. Thus, we note the value of the zebrafish model in the field of neuroblastoma
research, showing how it can improve our current knowledge about genes and biological pathways that contribute
to malignant transformation and progression during embryonic life
ZNF521 Is a Zinc Finger Protein That Prevents Differentiation Of Human MLL-AF9-Positive Myeloid Leukemic Cells
No full textTargeting BAG-1: A novel strategy to increase drug efficacy in acute myeloid leukemia
No full textOverexpression of antiapoptotic proteins occurs frequently in cancer, resulting in defective apoptosis that may contribute to a poor chemosensitivity of tumor cells. B-cell lymphoma (BCL) 2-associated AthanoGene-1 (BAG-1) is a prosurvival chaperone recently found involved in the maintenance of acute myeloid leukemia (AML) cells survival in vitro. Here we reported BAG-1 upregulation in 87 of 99 analyzed AML patients with respect to healthy control samples applying reverse phase protein assay. Silencing of BAG-1 expression confirmed a decreased BCL-2 protein level but, in addition, provoked the increased transcription of GADD34 stress sensor. Furthermore, a dephosphorylation of eIF2α, as well as alteration of expression of IRE-1 and CHOP proteins, were documented, suggesting that a disruption of the endoplasmic reticulum stress/unfolded protein response was provoked by downregulation of BAG-1. A similar phenomenon was triggered after addition of Thioflavin S, which was shown to block BAG-1/BCL-2 interaction and to increase cell death, enforcing a prosurvival role of the BAG-1 protein in AML. Interestingly, synergic cytotoxic effects of doxorubicin, VP16 drugs, and ABT-737 compound were observed when Thioflavin S was coupled with these drugs. Taken together, our results gave further proof that upregulation of BAG-1 plays a critical role in AML and that BAG-1 targeting might be considered for a combined therapeutic strategy with conventional chemotherapy drugs in the treatment of AML patients
Autophagic flux inhibition enhances cytotoxicity of the receptor tyrosine kinase inhibitor ponatinib
Full text linkBACKGROUND: Despite reported advances, acquired resistance to tyrosine kinase inhibitors still represents a serious problem in successful cancer treatment. Among this class of drugs, ponatinib (PON) has been shown to have notable long-term efficacy, although its cytotoxicity might be hampered by autophagy. In this study, we examined the likelihood of PON resistance evolution in neuroblastoma and assessed the extent to which autophagy might provide survival advantages to tumor cells.METHODS: The effects of PON in inducing autophagy were determined both in vitro, using SK-N-BE(2), SH-SY5Y, and IMR-32 human neuroblastoma cell lines, and in vivo, using zebrafish and mouse models. Single and combined treatments with chloroquine (CQ)-a blocking agent of lysosomal metabolism and autophagic flux-and PON were conducted, and the effects on cell viability were determined using metabolic and immunohistochemical assays. The activation of the autophagic flux was analyzed through immunoblot and protein arrays, immunofluorescence, and transmission electron microscopy. Combination therapy with PON and CQ was tested in a clinically relevant neuroblastoma mouse model.RESULTS: Our results confirm that, in neuroblastoma cells and wild-type zebrafish embryos, PON induces the accumulation of autophagy vesicles-a sign of autophagy activation. Inhibition of autophagic flux by CQ restores the cytotoxic potential of PON, thus attributing to autophagy a cytoprotective nature. In mice, the use of CQ as adjuvant therapy significantly improves the anti-tumor effects obtained by PON, leading to ulterior reduction of tumor masses.CONCLUSIONS: Together, these findings support the importance of autophagy monitoring in the treatment protocols that foresee PON administration, as this may predict drug resistance acquisition. The findings also establish the potential for combined use of CQ and PON, paving the way for their consideration in upcoming treatment protocols against neuroblastoma
Screening of novel genetic aberrations in pediatric acute myeloid leukemia: a report from the AIEOP AML-2002 study group
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