1,721,017 research outputs found
Proteasome inhibitor PS-341 blocks cell growth and induces apoptosis in anaplastic large cell lymphoma cell lines
No full textBlockade of the proteasome degradation pathway has emerged as a novel strategy to induce apoptosis in several tumor models, and PS-341 (Velcade) is among the newest compounds that show proteasome inhibitor activity that strictly correlates with antitumor effect. In this study, we investigated the antiproliferative activity of PS-341 in Anaplastic Large Cell Lymphomas (ALCLs), a high grade non-Hodgkin lymphoma characterized by the presence of specific chromosomal translocations involving the anaplastic lymphoma kinase (ALK) gene. Here we report that when administered for increasing time intervals PS-341 potently inhibited the growth of both ALK-positive (KARPAS, SUDHL1, SR786) and -negative (FE-PD) ALCL cell lines, irrespective of ALK-fusion proteins status and activity. The IC50s, as assayed by MTT following 24 h exposure, were 4.5-6 nM for SUDHL1, SR786 cells and the ALK-negative cell line FE-PD, whereas 17.8 nM for KARPAS. These cells resulted somehow more resistant to drug treatment, as time-dependent cleavage of PARP protein observed between 8 and 12 hours in SR786, SUDHL1 and FE-PD with 20 nM PS341, occurred only at higher concentrations (100 nM) in KARPAS cells. Because of the PS-341 pro-apoptotic activity, we assessed the activation of apoptotic caspase proteases and found that effector caspase-3 was strongly induced between 4 and 8 hours in ALCL lymphoma cells, and preceded the activation of caspase-8 and -9. Indeed, the caspase-8 inhibitor IETD-CHO prevented the formation of the active form of caspase-8 in the presence of PS-341, while it failed to block the procaspase-3 processing and PARP cleavage under the same experimental conditions. In contrast, the pan-caspase inhibitor ZVAD-fmk and the specific caspase-3 peptide inhibitor DEVD-CHO prevented both caspase-3 activation and PARP cleavage in all the cell lines. As expected, activation of caspases and mitochondrial cytochrome-c release was delayed in KARPAS cells, as well as up-regulation of the two cell-cycle inhibitors p27Kip and p21WAF. In contrast, the anti-apoptotic protein Mcl-1 was overexpressed in KARPAS cells and only slightly downregulated in the presence of PS-341, despite the potent inhibition of proteasome activity (90%) measured by fluorometric assay. Together, these data demonstrate the efficacy of PS-341 at inducing apoptosis in ALCL lymphoma cells, and suggest that induction of apoptosis may depend not solely on the inhibition of the 20S Proteasome, but also on the specific molecular profile of the individual cell line
Riduzione dell'espressione dell'oncoproteina NPM-ALK ed inibizione della fosforilazione tirosinica NPM-ALK dipendente, mediata dal benzochinone 17-allylamino-17-demethoxygeldanamycin (17-AAG) in linee cellulari di ALCL
No full textEFFETTO ANTITUMORALE DELLA COMBINAZIONE BORTEZOMIB E FLAVOPIRIDOLO NEL TRATTAMENTO DI CELLULE DI ALCL IN VITRO
No full textBortezomib è il capostipite di una nuova classe di farmaci
nota come inibitori del proteosoma con potente attività proapoptotica
e antiproliferativa. Per la sua attività antitumorale
Bortezomib è attualmente impiegato in diversi trials clinici
come agente singolo od in combinazione con chemioterapici
convenzionali e sinergismo è stato osservato se somministrato
in combinazione con modificatori della trascrizione cellulare.
Tra questi, Flavopiridolo, un inibitore delle chinasi ciclina
dipendenti (cdk), ha già dimostrato attività antitumorale
sinergica in combinazione con Bortezomib in cellule di leucemia,
CML. Lo scopo di questo studio è stato quello di analizzare
l’interazione tra Flavopiridolo e Bortezomib, su quattro
linee cellulari di linfoma anaplastico a grandi cellule
(ALCL), con espressione o meno della chinasi oncogena NPMALK.
Per valutare l’effetto antiproliferativo sinergico, saggi in
vitro di citotossicità sono stati condotti su linee di ALCL trattate
per tempi variabili con concentrazioni nanomolari di flavopiridolo
(50-200nM) e/o bortezomib (2.5-20 nM); mentre
tecniche di Western blotting e microscopia a fluorescenza
sono state impiegate per identificare le proteine coinvolte nei
meccanismi molecolari responsabili dell’attività antiproliferativa
sinergica. I dati ottenuti dimostrano che, quando usate in
combinazione, dosi sub-tossiche o citostatiche di bortezomib
(2,5-5nM) e flavopiridolo (50-100 nM), inducono una marcata
inibizione della proliferazione cellulare in maniera direttamente
proporzionale alla durata di somministrazione (25-
50% a 24h; 70-90% 48h). In particolare, il nostro studio ha
dimostrato che la contemporanea somministrazione di flavopiridolo
e bortezomib è responsabile della condensazione e
frammentazione della cromatina della maggior parte delle cellule
di ALCL trattate, dell’accumulo di proteine coinvolte nell’arresto
del ciclo cellulare (p27, p21), e della degradazione di
proteine fondamentali per la sopravvivenza della cellula linfomatosa
(PARP), attraverso l’attivazione di meccanismi apoptotici
caspasi- dipendenti. In conclusione, questo studio dimostra
per la prima volta l’efficacia della concomitante somministrazione
di bortezomib e flavopiridolo nell’inibire la proliferazione
e indurre apoptosi in cellule di ALCL
Villard 21 : From social housing to social habitat
No full textVillard è un seminario itinerante di progettazione, a cadenza annuale, che vede coinvolte Facoltà di Architettura, italiane ed estere. Il Seminario è rivolto a circa dieci studenti di ogni Facoltà partecipante, iscritti agli ultimi anni di corso, selezionati in base al merito. Il programma prevede la messa a punto di un progetto su un tema, in genere proposto da amministrazioni comunali o altre istituzioni legate alle diverse realtà territoriali. Il tema viene presentato all’inizio del seminario e sviluppato nel corso dell’anno nelle diverse tappe. Il viaggio costituisce la struttura portante del Seminario quale strumento di conoscenza delle città. Durante ogni tappa, con l’apporto dei docenti delle Facoltà partecipanti, sono organizzati incontri, lezioni, conferenze, visite guidate e mostre. Ogni tappa dura 3-4 giorni. Il lavoro di progettazione viene svolto principalmente durante gli orari che le diverse sedi dedicano al workshop. L’itineranza del seminario fa sì che gli studenti entrino in contatto con lughi fisici e culturali diversi, incrociando esperienze e conoscenze con docenti e studenti provenienti da altre sedi. Il seminario ha la sua conclusione in un evento finale: la mostra, con la presentazione e premiazione dei progetti migliori, a cui seguirà la pubblicazione del catalogo con i lavori degli studenti e degli apporti critici raccolti durante il seminario. Il tema della casa è recentemente ricomparso nell’agenda pubblica, sotto la spinta del crescente disagio abitativo di quella parte di popolazione esclusa tanto dal mercato quanto da un’offerta insufficiente di abitazioni pubbliche; le differenti esigenze di spazialità che la pandemia impone, sono tutte motivazioni che impongono di dare soluzioni adeguate al problema dell’abitare. Una questione nuovamente centrale nel dibattito contemporaneo che consiste, da un lato, nell’individuazione di politiche e strategie efficaci per la riorganizzazione dello spazio urbano, l’adeguamento dei servizi e la gestione del patrimonio edilizio; dall’altro, nella definizione delle nuove esigenze abitative di una società in rapida evoluzione. L’accessibilità alla casa e ai servizi essenziali è una delle questioni urgenti segnalate dalla nuova Agenda Urbana delle Nazioni Unite, insieme al tema del recupero di suoli e immobili abbandonati e a quello dell’integrazione sociale e della mobilità delle popolazioni. L’obiettivo, in Italia come nel resto del mondo, è quello di inquadrare il problema degli alloggi in uno scenario più ampio di soluzioni sostenibili dal punto di vista economico ed ambientale, capaci di ridefinire gli insediamenti per renderli resilienti. L’ecosistema tarantino, stravolto da scelte di localizzazione industriale e di edilizia residenziale pubblica disattente ai luoghi, offre un interessante caso di studio perché necessita di un ripensamento che, rigenerando lo spazio urbano per renderlo più resiliente, ricicli il patrimonio edilizio obsoleto o dismesso, riqualificando i vuoti urbani e, dove occorre, aumentando la densità abitativa
Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), a novel Hsp90-client tyrosine kinase: down-regulation of NPM-ALK expression and tyrosine phosphorylation in ALK(+) CD30(+) lymphoma cells by the Hsp90 antagonist 17-allylamino,17-demethoxygeldanamycin.
No full textBortezomib-mediated 26S proteasome inhibition causes cell-cycle arrest and induces apoptosis in CD-30(+) anaplastic large cell lymphoma
No full textThe 26S proteasome has a direct impact on cellular transcription regulation, cell-cycle progression, oncogenesis and apoptosis, since the turnover of the vast majority of intracellular proteins involved in the aforementioned mechanisms is regulated through the ubiquitin–proteasome pathway.1 Proteasome inhibitors have entered single-agent or combination Phase I and Phase II trials in solid tumors and hematologic malignancies because of the high susceptibility of cancer cells to proteasome inhibitor-induced cell death.2 Here, we have investigated the antiproliferative and pro-apoptotic activity of proteasome inhibitor bortezomib in anaplastic large cell lymphomas (ALCLs), and demonstrate that bortezomib potently inhibits the in vitro growth of ALCL cells and induces apoptosis at nanomolar concentrations in a time- and dose-dependent fashion, suggesting that targeting 26S proteasome may represent a novel therapeutic strategy for ALCLs. ALCLs can be subdivided into two clinically significant subtypes based on expression and constitutive activation of anaplastic lymphoma kinase (ALK), a tyrosine kinase that impacts proliferation, drug resistance, apoptosis and cellular transcription of ALCL cells. Activated NPM-ALK causes transcription silencing of p27KIP, via induction of the PI3K/Akt pathway, as well as upregulation of Bcl-xL, Mcl-1, survivin and cyclin D3, through phosphorylation of STAT3 transcription factor. This promotes cell survival over apoptosis in ALCL cells, given that upregulation of p27KIP expression, pharmacological impairment of PI3K/Akt pathway, or inhibition of STAT3 transcription activity has been shown to cause apoptosis of ALCL cells.3 Targeting ALK kinase or interfering with ALK-dependent signaling are promising strategies for the treatment of ALCLs, but have some limitations: ALK-negative ALCLs, which are characterized by a dismal prognosis, also express at high levels both phosphorylated Akt and STAT3, and these two proteins in some cases may be dispensable for NPM-ALK transforming capability. To determine the antiproliferative activity of bortezomib, ALK-positive (KARPAS299, SR786 and SUDHL1) and ALK-negative ALCL cells (FE-PD) were cultivated in the presence or absence of increasing concentrations (0.0005–0.02 M) of the proteasome inhibitor, and viability was assessed by MTT assay at 24 and 48 h. As shown in Figure 1a, in vitro growth of all ALCL cells was strongly inhibited at submicromolar concentrations in a dose- and time-dependent manner, irrespective of NPM-ALK status and activity. Cytotoxicity profile measured after 24 h exposure demonstrated that KARPAS299 cells had a higher IC50 (0.018 M) compared to the other cell lines (SR786, 0.0045 M; SUDHL1, 0.0071 M; FE-PD, 0.0067 M), as confirmed by the degree of cleavage of PARP protein shown in Figure 1b. Bortezomib almost completely inhibited the growth of SR786, SUDHL1 and FE-PD cells at 0.02 M for 24 h, whereas a significant growth inhibition was observed in KARPAS299 cells at later time points. To rule out any difference in the uptake of the drug, proteasome activity of ALCL cells was assessed by measuring the release of 7-amino-4-methylcoumarin (AMC) fluorophore from the proteasome peptide substrate N-succinyl-Leu-Leu-Val-Tyr-AMC. On the basis of the dose-dependent cleavage of PARP described above, a bortezomib concentration of 0.02 M was used to inhibit herein the cellular proteasome activity and to characterize the molecular events responsible for the growth inhibition of SR786 and FE-PD in all the subsequent experiments, whereas 0.1 M bortezomib was used for KARPAS299 and SUDHL1 cells. At these equal toxic concentrations, bortezomib was shown to penetrate cells and cause up to 80% inhibition of proteasome chimotryptic activity after 1 h in all four cell lines (Figure S1A, inset), maintaining such an inhibitory activity for as long as 24 h (Figure S1A). Chymotryptic inhibitory activity of bortezomib was confirmed by the comparable accumulation level of the proteasome substrate -catenin, later on substituted by a 70–75 kDa apoptotic fragment (Figure S1B, arrowheads). Given that bortezomib causes cell-cycle arrest in transformed cells,4 we sought to extend these findings to ALCLs. SR786 and FE-PD cells were exposed to 0.02 M bortezomib, whereas KARPAS299 and SUDHL1 were treated with 0.1 M bortezomib, and cell-cycle profile was evaluated after 0, 8, 16 and 24 h (Figure 2). When maintained in the presence of bortezomib, the percentage of cells in the G2/M phase increased in a time-dependent manner from 14.3–20.6% at time 0 to 16.9–50.2% at 24 h post-treatment, along with an increase of the sub-G1 population (from 0.4–1.4% at time 0 to 10.2–43.4% at 24 h post-treatment), which indicated the ability of bortezomib to induce G2/M cell-cycle arrest and apoptosis. With respect to the intracellular levels of cyclins, treatment with bortezomib confirmed a marked upregulation of G2/M phase control proteins cyclin B1 or cyclin A, whereas intracellular levels of cyclin E, which controls G1–S transition, were unchanged (Figure S2A). The endogenous levels of cell-cycle regulators p21WAF and p27KIP, short-lived proteins regulated through the ubiquitin–proteasome pathway, were also significantly increased in all four cell lines. This occurred with different kinetics, as transient increase of p21WAF peaked at 8 h in SR786 and FE-PD, whereas at 16 h post-treatment in KARPAS299 and SUDHL1 cells. In contrast, the extent of p27KIP accumulation was similar in all four cell lines, and prolonged if compared to p21WAF (Figure S2A). Since low levels of p27KIP transcription are maintained in ALCL cells by Akt,5 we measured Akt steady state upon exposure to bortezomib and found that endogenous Akt was downregulated with kinetics that correlated with the cytotoxicity profile of each ALCL cell line but not with p27KIP accumulation pattern (Figure S2A). To assess whether depletion of Akt, p21WAF and p27KIP proteins were related to induction of apoptosis, ALCL cells were exposed to equal toxic concentrations of bortezomib for 16 h in the presence of the broad-range caspase inhibitor z-VAD-fmk. As expected, co-administration of z-VAD-fmk prevented the disappearance of Akt, p21WAF and p27KIP from the whole cell extracts of bortezomib-treated SR786 and FE-PD, whereas it did not affect p53, which is not a caspase target protein (Figure S2B). According to the downregulation kinetics of these proteins, this was not observed in KARPAS299 and SUDHL1 cells at this time point. We next investigated the processing and activation of initiator and of effector caspases. As shown in Figure 3, inhibition of proteasome activity in SR786 and FE-PD caused a time-dependent processing and activation of caspase-3, along with cleavage of apical caspase-8 and -9 (Figure 3, cleaved caspase-3, Cl. C-3; Figure S2C). We observed that proteolysis of caspase-8 and -9 did not precede caspase-3 activation, and all were found to happen with similar kinetics, along with PARP cleavage. Nevertheless, despite a less complete activation of the apoptotic machinery, shown by limited processing of pro-caspases-3, -8 and -9, the accumulation of active caspase-3 subunits p19/17 was also observed in bortezomib-treated KARPAS299 and SUDHL1 cells, and this was accompanied by the generation of C-terminal 89 kDa PARP fragment. Being XIAP (X-linked inhibitor of apoptosis) both regulator and target of caspase enzymes in cells undergoing apoptosis, we also measured the steady state of the protein as function of bortezomib exposure time in ALCL cells. As expected, intracellular XIAP was downregulated in drug-treated lymphoma cells (Figure S2C), and such a downregulation was prevented in the presence of the pan-caspase inhibitor z-VAD-fmk (data not shown). Next, we assessed the mitochondrial involvement in bortezomib-induced apoptosis, by evaluating the steady state of pro-apoptotic and anti-apoptotic factors involved in mitochondria integrity. Whereas no consistent changes were observed for Bak protein, pro-apoptotic Bax, which is thought to cause cytocrome-c release from mitochondria, was found to relocate into the mitochondrial fraction (Figure 4a). Because Bax activation occurs through the exposure of its buried BH3 domain at the N-terminus, followed by its oligomerization at the outer mitochondrial membrane, we confirmed Bax activation by immunostaining bortezomib-treated ALCL cells with the specific anti-Bax monoclonal antibody clone 6A7 that recognizes the exposed N-terminus portion of the protein. As shown in Figure 4b, most of bortezomib-treated SR786 and FE-PD cells were apoptotic (panels d and h) and positive for activated Bax (panels c and g), unlike KARPAS299 and SUDHL1 cells in which both Bax labeling and nuclear morphology were not affected by bortezomib at this time point (panels a and b and e and f, respectively). Nevertheless, unlike PARP cleavage, Bax activation was not prevented by the broad-range caspase inhibitor z-VAD-fmk (data not shown). Accordingly, bortezomib promoted the translocation of cytochrome-c from the membrane enriched to the cytoplasmic fraction of ALCL cells in a time-dependent manner (Figure 4a), and the release of such a death inducer occurred upstream of caspases activation, as cytoplasmic relocation of cytochrome c proceeded even in the presence of z-VAD-fmk inhibitor (Figure S3A). Disruption of the mitochondrial membrane potential was further assayed in KARPAS299 and FE-PD cells treated for 12 and 24 h with bortezomib by using as mitochondrial activity marker a fluorescent-tagged lipophilic cation (MitoLightTM), which accumulates in the mitochondria of living cells in a membrane potential-dependent manner (red fluorescence), whereas it remains in the cytoplasm of apoptotic cells in its monomeric form (green fluorescence). As shown by the ratio of green to red fluorescence measurements (Figure S3B, 530/590 nm), bortezomib caused a marked loss of membrane permeability in FE-PD cells in a time-dependent manner, which was at least in part prevented by z-VAD-fmk treatment. Yet, and consistent with the recent observation that caspase-3 may act on permeabilized mitochondria to disrupt further m and the permeability of the inner membrane,6 changes in membrane potential of mitochondria were modest in bortezomib-treated KARPAS299 cells, in which activation of the apoptotic program has been shown to be delayed. Among the anti-apoptotic members of the Bcl-2 family, Mcl-1 is consistently expressed in ALK-positive cell lines and tumors, and moderately in the ALK-negative lymphoma cells.7 Mcl-1 promotes cell survival in lymphoma and myeloma cells by binding to and inhibiting pro-apoptotic Bak activity, and the accumulation of Mcl-1 exerts a protective role in cancer cells exposed to proteasome inhibitors.8 We observed a significant accumulation of Mcl-1 in both cytosol and enriched-mitochondrial fraction in the presence of bortezomib (Figure S4A), owing to the inhibition of its ubiquitin-dependent proteasomal degradation, but this was followed by a delayed downregulation perhaps through the activity of executioner caspase proteases. Interestingly, time-dependent downregulation of Mcl-1 paralleled caspase-3 activation described previously, and when ALCL cells were exposed to bortezomib for 24 h, Mcl-1 was completely degraded unless the cells were pre-treated with the broad-range caspase-inhibitor z-VAD-fmk (Figure S4B, SR786 and FE-PD). Consistently, Mcl-1 associated to Bak in the mitochondria of untreated ALCL cells, but this complex was disrupted when the cells were cultivated in the presence of bortezomib, unless z-VAD-fmk caspase inhibitor was added before bortezomib (Figure S4C, FE-PD). These data support the hypothesis that late Mcl-1 downregulation observed in ALCL cell lines occurs downstream of caspase activation, and the inability of the proteasome inhibitor bortezomib to affect steady state and function of Mcl-1 in SUDHL1 or KARPAS299 cells would depend on the level of activated caspases. In conclusion, this study underlines for the first time the effectiveness of bortezomib to induce, at clinically achievable concentrations, growth inhibition and apoptosis in ALCL cells in vitro, regardless of the expression and activity of NPM-ALK protein, and suggests that targeting 26S proteasome may represent a novel therapeutic strategy for ALCLs, especially for those that do not express ALK and are associated with worse prognosis
Effects of mutational loss of nucleoside kinases on deoxyadenosine 5'-phosphate/deoxyadenosine substrate cycle in cultured CEM and V79 cells.
No full textGrowth inhibition and induction of apoptosis in anaplastic large cell lymphoma cells by the proteasome inhibitor bortezomib
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