RESUMO
PURPOSE: To investigate the potential link between C-reactive protein (CRP), a known biomarker of acute and chronic inflammation, and TRAIL, a cytokine which plays a key role in the immune-surveillance against tumors. EXPERIMENTAL DESIGN: Primary normal peripheral blood mononuclear cell (PBMC) and CD14(+) monocytes were exposed to recombinant CRP (1-10 µmol/L). TRAIL expression was analyzed by ELISA and/or by quantitative real-time PCR (qRT-PCR). In parallel, the potential role of the transcription factor Egr-1 was investigated by analyzing its modulation in response to CRP and by transfection experiments. RESULTS: In vitro CRP exposure induced downregulation of TRAIL expression, both at the mRNA and protein level, in unfractionated PBMC and in purified CD14(+) monocytes. TRAIL downregulation was not due to a specific toxicity or to contaminating lipopolysaccharide (LPS), as shown by the lack of induction of monocyte apoptosis and by the inability of the inhibitor of LPS polymyxin B to interfere with CRP activity. Of note, CRP downregulated TRAIL expression/release in CD14(+) monocytes also in response to IFN-α, the most potent inducer of TRAIL. At the molecular level, the downmodulation of TRAIL by CRP was accompanied by a significant increase of Egr-1. Consistently, Egr-1 overexpression reduced the baseline levels of TRAIL mRNA, whereas knocking down Egr-1 counteracted the ability of CRP to downregulate TRAIL. CONCLUSIONS: Our findings suggest that a chronic elevation of CRP, which occurs during systemic inflammation and often in patients with cancer, might contribute to promote cancer development and/or progression by downregulating TRAIL in immune cells.
Assuntos
Proteína C-Reativa/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Monócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Western Blotting , Proteína C-Reativa/genética , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Interferência de RNA , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
The effect of Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction, was investigated on the steady-state mRNA levels of the transcription factors E2F1 and E2F7 in a cohort of primary B-chronic lymphocytic leukemia (B-CLL) patient samples (n = 15) and normal peripheral blood mononuclear cells (PBMC). A 24-h treatment with Nutlin-3 significantly down-regulated E2F1 and promoted the concomitant up-regulation of E2F7 in both leukemic and normal cells. Our data suggest that the ability of Nutlin-3 to up-regulate E2F7 likely represents an important molecular determinant in the anti-proliferative activity of Nutlin-3.
Assuntos
Fator de Transcrição E2F1/genética , Fator de Transcrição E2F7/genética , Regulação Leucêmica da Expressão Gênica , Imidazóis/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucócitos Mononucleares/metabolismo , Piperazinas/metabolismo , Proteína Supressora de Tumor p53/genética , Estudos de Casos e Controles , Células Cultivadas , Humanos , Hibridização in Situ Fluorescente , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The inflammatory properties of vein endothelium in relation to chronic venous disease (CVD) have been poorly investigated. Therefore, new insights on the characteristics of large vein endothelium would increase our knowledge of large vessel physiopathology. METHODOLOGY/PRINCIPAL FINDINGS: Surgical specimens of veins were obtained from the tertiary venous network (R3) and/or saphenous vein (SF) of patients affected by CVD and from control individuals. Highly purified venous endothelial cell (VEC) cultures obtained from CVD patients were characterized for morphological, phenotypic and functional properties compared to control VEC. An increase of CD31/PECAM-1, CD146 and ICAM-1 surface levels was documented at flow cytometry in pathological VEC with respect to normal controls. Of note, the strongest expression of these pro-inflammatory markers was observed in VEC obtained from patients with more advanced disease. Similarly, spontaneous cell proliferation and resistance to starvation was higher in pathological than in normal VEC, while the migratory response of VEC showed an opposite trend, being significantly lower in VEC obtained from pathological specimens. In addition, in keeping with a higher baseline transcriptional activity of NF-kB, the release of the pro-inflammatory cytokines osteoprotegerin (OPG) and vascular endothelial growth factor (VEGF) was higher in pathological VEC cultures with respect to control VEC. Interestingly, there was a systemic correlation to these in vitro data, as demonstrated by higher serum OPG and VEGF levels in CVD patients with respect to normal healthy controls. CONCLUSION/SIGNIFICANCE: Taken together, these data indicate that large vein endothelial cells obtained from CVD patients exhibit a pro-inflammatory phenotype, which might significantly contribute to systemic inflammation in CVD patients.
Assuntos
Células Endoteliais/citologia , Doenças Vasculares/patologia , Adulto , Índice de Massa Corporal , Antígeno CD146/biossíntese , Técnicas de Cultura de Células , Feminino , Citometria de Fluxo/métodos , Humanos , Inflamação , Molécula 1 de Adesão Intercelular/biossíntese , Cinética , Masculino , Microscopia Eletrônica de Varredura/métodos , Pessoa de Meia-Idade , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Veia Safena/fisiopatologiaRESUMO
BACKGROUND: Both the multi-kinase inhibitor sorafenib and the small molecule inhibitor of the MDM2/p53 interaction, nutlin-3, used alone, have shown promising anti-leukemic activity in acute myeloid leukemia cells. Thus, in this study we investigated the effect of the combination of sorafenib plus nutlin-3 in acute myeloid leukemia. DESIGN AND METHODS: Primary acute myeloid leukemia blasts (n=13) and FLT3(wild-type)/p53(wild-type) (OCI-AML3), FLT3(mutated)/p53(wild-type) (MOLM), FLT3(mutated)/p53(mutated) (MV4-11), FLT3(wild-type)/p53(deleted) (HL60) or FLT3(wild-type)/p53(mutated) (NB4) acute myeloid cell lines were exposed to sorafenib, used alone or in association with nutlin-3 at a 1:1 ratio, in a range of clinically achievable concentrations (1-10 µM). Induction of apoptosis and autophagy was evaluated by transmission electron microscopy and by specific flow cytometry analyses. The levels of Mcl-1, p53 and Bak proteins were analyzed by western blotting. Knock-down of Bax and Bak gene expression was performed in transfection experiments with specific short interfering RNA. RESULTS: The sorafenib+nutlin-3 drug combination exhibits synergistic cytotoxicity in primary acute myeloid leukemia blasts and in acute myeloid leukemia cell lines with maximal cytotoxicity in FLT3(mutated) MV4-11 and MOLM, followed by the FLT3(wild-type) OCI-AML3, HL60 and NB4 cell lines. The cytotoxic activity of sorafenib+nutlin-3 was characterized by an increase of both apoptosis and autophagy. Moreover, Bax and Bak showed prominent roles in mediating the decrease of cell viability in response to the drug combination in p53(wild-type) OCI-AML3 and p53(deleted) HL-60 cells, respectively, as demonstrated in transfection experiments performed with specific short interfering RNA. CONCLUSIONS: Our data demonstrate that acute myeloid leukemia cells show a variable but overall good susceptibility to the innovative therapeutic combination of sorafenib+nutlin-3, which differentially involves the pro-apoptotic Bcl-2 family members Bax and Bak in p53(wild-type) and p53(deleted) cells.
Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Leucemia Mieloide Aguda , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Piperazinas/farmacologia , Proteína Supressora de Tumor p53 , Tirosina Quinase 3 Semelhante a fms , Antineoplásicos/agonistas , Sinergismo Farmacológico , Feminino , Células HL-60 , Humanos , Imidazóis/agonistas , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Niacinamida/agonistas , Niacinamida/farmacologia , Compostos de Fenilureia/agonistas , Piperazinas/agonistas , SorafenibeRESUMO
The basal SOCS1 mRNA levels were significantly lower in p53(mutated) BJAB and MAVER leukemic cell lines with respect to p53(wild-type) SKW6.4 and JVM-2 leukemic cell lines, p53(wild-type) primary B chronic lymphocytic leukemia (B-CLL) cells and primary normal peripheral blood mononuclear cells (PBMC). Moreover, the MDM2 small molecule inhibitor Nutlin-3 significantly increased the levels of SOCS1 mRNA in both primary p53(wild-type) B-CLL cells as well as in p53(wild-type) B leukemic cell lines, but not in p53(mutated) B leukemic cell lines nor in primary PBMC. Of note, a significant inverse correlation was observed between SOCS1 mRNA and miR-155 levels in Nutlin-3-treated primary B-CLL cells and PBMC, suggesting that the miRNA-155/SOCS1 axis represents a potentially important therapeutic target of Nutlin-3 in B-CLL.
Assuntos
Imidazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , MicroRNAs/metabolismo , Piperazinas/farmacologia , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Linhagem Celular Tumoral , Células Cultivadas , Regulação para Baixo , Humanos , Leucócitos Mononucleares/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteína Supressora de Tumor p53 , Regulação para CimaAssuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
A range of cell types of mesenchymal origin express α-smooth muscle actin (α-SMA), a protein that plays a key role in controlling cell motility and differentiation along the fibrocyte and myofibroblast lineages. Although α-SMA is often expressed in stromal cells associated to a variety of cancers including hematological malignancies, up to now the role of anti-cancer drugs on α-SMA has not been deeply investigated. In this study, we demonstrated that Nutlin-3, the small molecule inhibitor of the MDM2/p53 interactions, significantly up-regulated the mRNA and protein levels of α-SMA in normal macrophages as well as in p53(wild-type) but not in p53(mutated/null) myeloid leukemic cells. The p53-dependence of α-SMA up-regulation induced by Nutlin-3 was demonstrated in experiments performed with siRNA for p53. Of note, Nutlin-3 mediated up-regulation of α-SMA in OCI leukemic cells was accompanied by cell adhesion to plastic substrate and by reduced cell migratory response in transwell assays. Notably, the role of α-SMA induction in the modulation of myeloid cell migration was clearly documented in α-SMA gene knockdown experiments. In addition, Nutlin-3 significantly up-regulated α-SMA expression in primary endothelial cells, but not in fibroblasts and mesenchymal stem cells (MSC). Conversely, transforming growth factor-ß1 up-regulated α-SMA in fibroblasts and MSC, but not in macrophages and endothelial cells. Taken together, these data indicate that Nutlin-3 is a potent inducer of α-SMA in both normal and leukemic myeloid cells as well as in endothelial cells.
Assuntos
Actinas/metabolismo , Imidazóis/farmacologia , Leucemia Mieloide/metabolismo , Macrófagos/metabolismo , Piperazinas/farmacologia , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Actinas/genética , Movimento Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Leucemia Mieloide/patologia , Macrófagos/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/genéticaAssuntos
Regulação para Baixo/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Sulfeto de Hidrogênio/farmacologia , Osteoprotegerina/metabolismo , Morte Celular/efeitos dos fármacos , Citostáticos/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Osteoprotegerina/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
PURPOSE: The oncogene TCL1 plays a key role in the development of B chronic lymphocytic leukemia (B-CLL), but it is not known whether TCL1 could be modulated by therapeutic approaches. EXPERIMENTAL DESIGN: B-CLL patient samples (n = 35) and B leukemic cell lines (EHEB, JVM2, JVM3, MEC1, MEC2, and BJAB) with different p53 status were exposed to Nutlin-3, a small-molecule inhibitor of the p53-MDM2 interaction. Modulations of the steady-state mRNA levels of TCL1 were analyzed by quantitative real-time PCR and Western blotting in both primary B-CLL samples and leukemic cell lines. In addition, transfection experiments with either p53 siRNA or with a TCL1 expression plasmid were carried out in the EHEB B-CLL cell line. RESULTS: Upon ex vivo treatment with Nutlin-3, TCL1 was significantly (P < 0.05) decreased in 23 of 28 B-CLL p53(wild-type). The functionality of the p53 pathway in the same leukemic cell samples was underscored by the concomitant ability of Nutlin-3 to significantly (P < 0.05) upregulate the p53 target gene MDM2 in the p53(wild-type) leukemic cells. The dependence of TCL1 downregulation by a functional p53 pathway was confirmed in a panel of B lymphoblastoid cell lines and by p53 knockdown experiments with p53 siRNA. The importance of TCL1 in promoting leukemic cell survival was underscored in transfection experiments, in which TCL1 overexpression significantly counteracted the Nutlin-3-mediated induction of apoptosis in EHEB. CONCLUSIONS: Our data indicate that the Nutlin-3 downregulates TCL1 mRNA and protein, which likely represents an important molecular determinant in the proapoptotic activity of Nutlin-3.
Assuntos
Imidazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Linfócitos B/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Humanos , Imidazóis/uso terapêutico , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The soluble member of the TNF-R superfamily osteoprotegerin (OPG) is abundantly released under basal conditions by both mesenchymal stem cells (MSC) and fibroblasts and by endothelial cells upon stimulation with inflammatory cytokines. Since MSC, fibroblasts and endothelial cells represent key elements of the normal and tumor microenvironment and express detectable levels of surface TRAIL receptors, we investigated the effect of TRAIL on OPG release. Unexpectedly, recombinant TRAIL decreased the spontaneous OPG release in all cell types examined. Moreover, TRAIL decreased OPG release also in stromal cells co-cultured with lymphoma cells and counteracted the OPG induction by TN-alpha in HUVEC and MSC. Such down-regulation was not due to a masking effect in the ELISA quantification of the OPG released in the culture supernatants due to binding of OPG to its ligands (TRAIL and RANKL), as demonstrated by competition experiments with recombinant TRAIL and by the lack of RANKL release/induction. In addition, OPG down-regulation was not due to induction of cytotoxic effects by TRAIL, since the degree of apoptosis in response to TRAIL was negligible in all primary cell types. With regards to the possible molecular mechanism accounting for the down-regulation of OPG release by TRAIL, we found that treatment of MSC with TRAIL significantly decreased the phosphorylation levels of p38/MAPK. There is a suggestion that this pathway is involved in the stabilization of OPG mRNA. In this respect, the ability of TRAIL to decrease the release of OPG, in the absence of cell cytotoxicity, was mimicked by the p38/MAPK inhibitor SB203580.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Osteoprotegerina/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/enzimologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Osteoprotegerina/genética , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: To elucidate new molecular mechanisms able to downregulate the mRNA levels of key oncogenes, such as B-Myb and E2F1, in a therapeutic perspective. EXPERIMENTAL DESIGN: B-Myb and E2F1 mRNA levels were evaluated in primary B chronic lymphocytic leukemia (B-CLL, n = 10) and acute myeloid leukemia (AML, n = 5) patient cells, in a variety of p53(wild-type) and p53(mutated/deleted) leukemic cell lines, as well as in primary endothelial cells and fibroblasts. Knockdown experiments with siRNA for p53 and E2F1 and overexpression experiments with miR34a were conducted to elucidate the role of these pathways in promoting B-Myb downregulation. RESULTS: In vitro exposure to Nutlin-3, a nongenotoxic activator of p53, variably downregulated the expression of B-Myb in primary leukemic cells and in p53(wild-type) myeloid (OCI, MOLM) and lymphoblastoid (SKW6.4, EHEB) but not in p53(mutated) (NB4, BJAB, MAVER) or p53(deleted) (HL-60) leukemic cell lines. The transcriptional repression of B-Myb was also observed in primary normal endothelial cells and fibroblasts. B-Myb downregulation played a critical role in the cell-cycle block in G(1) phase induced by Nutlin-3, as shown by transfection experiments with specific siRNA. Moreover, we have provided experimental evidence suggesting that miR-34a is a central mediator in the repression of B-Myb both directly and through E2F1. CONCLUSIONS: Owing to the role of B-Myb and E2F1 transcription factors in controlling cell-cycle progression of leukemic cells, the downregulation of these oncogenes by miR-34a suggests the usefulness of therapeutic approaches aimed to modulate the levels of miR-34a.
Assuntos
Proteínas de Ciclo Celular/genética , Fator de Transcrição E2F1/genética , Leucemia/genética , MicroRNAs/fisiologia , Transativadores/genética , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Fator de Transcrição E2F1/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/genética , Células HCT116 , Células HL-60 , Humanos , Imidazóis/farmacologia , Leucemia/patologia , MicroRNAs/genética , Modelos Biológicos , Oncogenes/genética , Piperazinas/farmacologia , Homologia de Sequência do Ácido Nucleico , Transativadores/metabolismo , TransfecçãoRESUMO
PURPOSE: To characterize the role of the oncogene DEK in modulating the response to either Nutlin-3, a small-molecule inhibitor of the MDM2/p53 interaction, or chlorambucil in primary B-chronic lymphocytic leukemia (B-CLL) cells. EXPERIMENTAL DESIGN: DEK mRNA and protein levels were evaluated in primary B-CLL samples (n = 21), p53(wild-type) SKW6.4, p53(mutated) BJAB lymphoblastoid cell lines, and normal CD19(+) B lymphocytes-treated Nutlin-3 or chlorambucil (10 micromol/L, each). Knocking down experiments with either p53 or DEK small interfering RNA (siRNA) were done to investigate the potential role of p53 in controlling the expression of DEK and the role of DEK in leukemic cell survival/apoptosis. RESULTS: Both Nutlin-3 and chlorambucil downregulated DEK in primary B-CLL samples (n = 21) and SKW6.4 but not in BJAB cells. Knocking down p53 attenuated the effect of Nutlin-3 on DEK expression, whereas knocking down DEK significantly increased both spontaneous and Nutlin-3-induced apoptosis. Conversely, counteracting DEK downmodulation by using p53 small interfering RNA reduced Nutlin-3-mediated apoptosis. On the other hand, Nutlin-3 potently induced p53 accumulation, but it did not affect DEK levels in normal CD19(+) B lymphocytes. CONCLUSIONS: These data show that the downregulation of DEK in response to either Nutlin-3 or chlorambucil represents an important molecular determinant in the cytotoxic response of leukemic cells, and suggest that strategies aimed to downregulate DEK might improve the therapeutic potential of these drugs.
Assuntos
Linfócitos B/patologia , Clorambucila/farmacologia , Proteínas Cromossômicas não Histona/fisiologia , Imidazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Oncogênicas/fisiologia , Piperazinas/farmacologia , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Alquilantes/farmacologia , Apoptose , Linfócitos B/metabolismo , Western Blotting , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Técnicas Imunoenzimáticas , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estereoisomerismo , Proteína Supressora de Tumor p53/fisiologiaRESUMO
OBJECTIVE: To evaluate the potential therapeutic effect of recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) treatment in a model of type 1 diabetes. RESEARCH DESIGN AND METHODS: Recombinant TRAIL was added in vitro to primary human and mouse peripheral blood mononuclear cells (PBMCs) and isolated human islets to evaluate the expression of the immunoregulatory gene SOCS1. Diabetes was induced by five consecutive daily injections of low-concentration (50 mg/kg) streptozotocin (STZ) in C57 black mice (n = 24). A group of these mice (n = 12) was co-injected with recombinant TRAIL (20 microg/day) for 5 days, and the diabetic status (glycemia and body weight) was followed over time. After 6 weeks, circulating levels of insulin, TNF-alpha, and osteoprotegerin (OPG) were measured, and animals were killed to perform the histological analysis of the pancreas. RESULTS: The in vitro exposure of both PBMCs and human islets to recombinant TRAIL significantly upregulated the expression of SOCS1. With respect to STZ-treated animals, mice co-injected with STZ+TRAIL were characterized by 1) lower levels of hyperglycemia, 2) higher levels of body weight and insulinemia, 3) a partial preservation of pancreatic islets with normal morphology, and 4) a lower expression of both systemic (TNF-alpha and OPG) and pancreatic (vascular cell adhesion molecule [VCAM]-1) inflammatory markers. CONCLUSIONS: Overall, these data demonstrate that the administration of recombinant TRAIL ameliorates the severity of STZ-induced type 1 diabetes, and this effect was accompanied by the upregulation of SOCS1 expression.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Expressão Gênica/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly up-regulated the steady-state mRNA and protein levels of Notch1 in TP53(wild-type) (OCI, SKW6.4) but not in TP53(deleted) (HL-60) or TP53(mutated) (BJAB) leukemic cell lines. A direct demonstration that NOTCH1 was a transcriptional target of p53 in leukemic cells was obtained in experiments carried out with siRNA for p53. Moreover, inhibition of Notch1 expression using Notch1-specific siRNA significantly increased cytotoxicity in TP53(wild-type) leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary TP53(wild-type) B-chronic lymphocytic leukemia (B-CLL) cells and the combined use of Nutlin-3 plus pharmacological gamma-secretase inhibitors of the Notch signaling showed a synergistic cytotoxicity in both TP53(wild-type) leukemic cell lines and primary B-CLL cells. A potential drawback of gamma-secretase inhibitors was their ability to enhance osteoclastic maturation of normal circulating preosteoclasts induced by RANKL + M-CSF. Notwithstanding, Nutlin-3 completely suppressed osteoclastogenesis irrespective of the presence of gamma-secretase inhibitors. Taken together, these data indicate that the p53-dependent up-regulation of Notch1 in response to Nutlin-3 represents an antiapoptotic feedback mechanism able to restrain the potential therapeutic efficacy of Nutlin-3 in hematologic malignancies. Therefore, therapeutic combinations of Nutlin-3 + gamma-secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53(wild-type) leukemic cells.
Assuntos
Apoptose , Retroalimentação Fisiológica , Imidazóis/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Piperazinas/farmacologia , Receptor Notch1/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Western Blotting , Carbamatos/farmacologia , Diferenciação Celular , Dipeptídeos/farmacologia , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Células HL-60 , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Osteoclastos/citologia , Osteoclastos/metabolismo , RNA Mensageiro/metabolismo , Receptor Notch1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estereoisomerismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
Adult multipotent stromal cells (MSCs), also known as mesenchymal stem cells, represent an important source of cells for the repair of a number of damaged tissues. Both bone marrow (BM)-derived and amniotic MSCs expressed detectable surface levels of two (tumor necrosis factor-related apoptosis-inducing ligand receptor 2 [TRAIL-R2] and TRAIL-R4) of four transmembrane TRAIL receptors. Although the best-characterized activity of TRAIL-R2 is the transduction of apoptotic signals, neither recombinant TRAIL (rTRAIL) nor infection with an adenovirus-expressing TRAIL induced cytotoxic effects on MSCs. Moreover, whereas rTRAIL did not affect proliferation or differentiation of MSCs along the osteogenic and adipogenic lineages, it significantly promoted the migration of human MSCs in range of concentrations comparable to that of soluble TRAIL in human plasma (100 pg/ml). Since rTRAIL induced the rapid phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in MSC cultures and pretreatment with pharmacological inhibitors of the ERK1/2 pathway efficiently counteracted the rTRAIL-induced human MSC migration, these data indicate that ERK1/2 is involved in mediating the ability of rTRAIL to stimulate MSC migration. Taking into consideration that the soluble factors able to induce MSC migration have not been extensively characterized, our current data indicate that the TRAIL/TRAIL-R system might play an important role in the biology of MSCs. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Células da Medula Óssea/fisiologia , Proliferação de Células , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Células-Tronco Mesenquimais/fisiologia , Fosforilação , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Receptores Chamariz do Fator de Necrose Tumoral/metabolismoRESUMO
At variance to solid tumors, which show percentage of p53 deletions and/or mutations close to 50%, more than 80% of haematological malignancies express wild-type p53 at diagnosis. Therefore, activation of the p53 pathway by antagonizing its negative regulator murine double minute 2 (MDM2) might offer a new therapeutic strategy for the great majority of haematological malignancies. Recently, potent and selective small-molecule MDM2 inhibitors, the Nutlins, have been identified. Studies with these compounds have strengthened the concept that selective, non-genotoxic p53 activation might represent an alternative to the current cytotoxic chemotherapy. Interestingly, Nutlins not only are able to induce apoptotic cell death when added to primary leukemic cell cultures, but also show a synergistic effect when used in combination with the chemotherapeutic drugs commonly used for the treatment of haematological malignancies. Of interest, Nutlins also display non-cell autonomous biological activities, such as inhibition of vascular endothelial growth factor, stromal derived factor-1/CXCL12 and osteprotegerin expression and/or release by primary fibroblasts and endothelial cells. Moreover, Nutlins have a direct anti-angiogenic and anti-osteoclastic activity. Thus, Nutlins might have therapeutic effects by two distinct mechanisms: a direct cytotoxic effect on leukemic cells and an indirect non-cell autonomous effect on tumor stromal and vascular cells, and this latter effect might be therapeutically relevant also for treatment of haematological malignancies carrying p53 mutations.
Assuntos
Neoplasias Hematológicas/tratamento farmacológico , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Proteína Supressora de Tumor p53/metabolismoRESUMO
It has been shown that the expression of osteoprotegerin (OPG) is up-regulated in tumor-associated endothelial cells as well as in the sera of patients affected by both solid tumors and hematologic malignancies. We now report that sera of p53(-/-) mice contain higher levels of OPG with respect to p53(+/+) mice and that endothelial cells, in which p53 was knocked down by siRNA, release increased levels of OPG with respect to mock-transfected cells. Conversely, activation of the p53 pathway by the MDM2 small molecule antagonist Nutlin-3 significantly attenuated both spontaneous and tumor necrosis factor-alpha (TNF-alpha)-induced OPG mRNA and protein release in endothelial cell cultures. OPG promoter functional assays and chromatin immunoprecipitation experiments revealed inhibitory effects of Nutlin-3 on the TNF-alpha-induced NF-kappaB DNA binding activity to the OPG promoter. Because OPG inhibits the pro-tumoricidal activity of TNF-related apoptosis-inducing ligand, our findings suggest that, besides its well-documented functions within the malignant cancer cells, the ability of p53 to down-modulate OPG production by endothelial cells may be an additional important mechanism whereby it exerts non-cell-autonomous tumor suppression function.
Assuntos
Regulação para Baixo , Células Endoteliais/metabolismo , Osteoprotegerina/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular , Células Cultivadas , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/citologia , Humanos , Imidazóis/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Piperazinas/metabolismo , Regiões Promotoras Genéticas/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Among 14 peripheral blood samples obtained from patients affected by B chronic lymphocytic leukemia (B-CLL) at initial stages (Rai 0-1) of the disease, 6 showed intermediate/high levels of Zap-70 while 8 displayed low/absent levels of Zap-70. Although Zap-70(high) and Zap-70(low) B-CLL samples displayed similar levels of surface death receptor TRAIL-R2, recombinant TRAIL induced cytotoxicity only in a subset of Zap-70(low) B-CLL samples while Zap-70(high) were completely resistant to TRAIL. The gene expression profiling was next analyzed in all B-CLL samples treated with either chlorambucil or recombinant TRAIL. While chlorambucil up-regulated the steady-state mRNA levels of known p53 target genes, such as PUMA, Fas/CD95 and MDM2 in all B-CLL samples examined, it significantly down-regulated survivin in Zap-70(low) but not in Zap-70(high). On the other hand, recombinant TRAIL up-regulated the expression of several cytokines (IL-1beta, IL-1alpha, IL-8), which have been involved in promoting B-CLL cell survival. In particular, TRAIL selectively up-regulated IL-1beta in Zap-70(low) B-CLL samples, while it markedly and selectively up-regulated its own mRNA and that of cyclooxigenase-2 (COX-2) in Zap-70(high). Taken together, our findings suggest that a significant expression of Zap-70 modulate the response of B-CLL to TRAIL, which might represents an initial step in the pathogenesis of B-CLL.