RESUMO
PURPOSE: Human Apo2-Ligand/TRAIL secreted by natural killer cells and cytotoxic T lymphocytes plays an important role immunosurveillance controlling tumor growth and metastasis. Moreover, the fact that Apo2L/TRAIL is capable of inducing cell death in tumor cells but not in normal cells makes this death ligand a promising anti-tumor agent. Previous data from our group demonstrated that Apo2L/TRAIL was physiologically released as transmembrane protein inserted in lipid vesicles, called exosomes. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) resembling the natural exosomes, greatly improved Apo2L/TRAIL activity and were able to induce apoptosis in hematological malignancies. In this study, we have deepened in the underlying mechanism of action of LUV-TRAIL in hematologic cells. METHODS/PATIENTS: Cytotoxic ability of LUV-TRAIL was assessed on Jurkat cells either over-expressing the anti-apoptotic protein Mcl1 or down-regulating the pro-apoptotic protein Bim previously generated in our laboratory. We also tested LUV-TRAIL cytotoxic ability against primary human leukemic cells from T-cell ALL patient. RESULTS: Silencing Bim but not Mcl-1 over-expression partially protects Jurkat cells from apoptosis induced by sTRAIL. LUV-TRAIL induced caspase-8 and caspase-3 activation and killed Jurkat-Mcl1 and Jurkat-shBim more efficiently than sTRAIL independently of the mitochondrial pathway. On the other hand, LUV-TRAIL were clearly more cytotoxic against primary leukemic cells from a T-cell ALL patient than sTRAIL. CONCLUSION: Tethering Apo2L/TRAIL to the surface of lipid nanoparticles greatly increases its bioactivity and could be of potential use in anti-tumor therapeutics.
Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose , Imunoterapia , Leucemia/patologia , Leucemia/terapia , Lipossomos , Proteínas de Membrana/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Humanos , Técnicas Imunoenzimáticas , Leucemia/imunologia , Leucemia/metabolismo , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de SinaisRESUMO
PURPOSE: Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells. METHODS: Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA. RESULTS: Cell death was characterized by phosphatidylserine exposure, ΔΨm loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL50 at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-xL and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis. CONCLUSION: Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Mitocôndrias/patologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Inibidores de Caspase/farmacologia , Caspases/química , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mieloma Múltiplo/metabolismo , Necrose , Niacinamida/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorafenibe , Células Tumorais CultivadasRESUMO
Lipoprotein lipase (LPL) in the arterial wall has been proposed to enhance the retention of apoB-containing lipoproteins, an early event in atherosclerosis. As the neointima is considered the primary site of lipid accumulation in atherogenesis, the arterial expression and location of LPL was investigated in distinct experimental models of neointimal formation in normolipidemic rabbits and rats. Neointima elicited by balloon aortic denudation or raised beneath an anatomically intact endothelial layer by placing a silastic collar around the common carotid artery, both showed a striking LPL immunostaining that mostly co-localized with neointimal smooth muscle cells. Besides, increased LPL protein and mRNA in deendothelialized aortas was demonstrated by Western and Northern blot analysis, respectively, suggesting an enhanced expression of LPL in injured arteries. It was concluded that LPL is increased in neointima developed in either denuded vessels or arteries with a preserved endothelium, a finding which suggests that LPL abundance may be an attribute of the neointima, whatever the stimulus that promotes its formation. On the basis of former evidence concerning the role of LPL in lipid retention, this study provides a possible explanation for the injury-induced vessel susceptibility to atherosclerosis, and the particular proneness of the neointimal layer to lipid accretion.
Assuntos
Aorta/enzimologia , Arteriosclerose/etiologia , Artérias Carótidas/enzimologia , Lipase Lipoproteica/metabolismo , Túnica Íntima/enzimologia , Animais , Aorta/metabolismo , Aorta Torácica/patologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Endotélio Vascular/fisiologia , Lipase Lipoproteica/genética , Masculino , RNA Mensageiro/metabolismo , Coelhos , Ratos , Ratos Wistar , Fatores de RiscoRESUMO
Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.
Assuntos
Genes de Protozoários , RNA Helicases/genética , Trypanosoma cruzi/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , DNA Complementar , Perfilação da Expressão Gênica , Biblioteca Genômica , Dados de Sequência Molecular , RNA Helicases/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Regulação para CimaRESUMO
A peptide from hindguts of the Triatoma hematophagous Chagas insect vector activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes to metacyclic trypomastigotes. Hindguts were obtained from insects fed 2 days earlier with chicken blood. Purification was performed by gel filtration and HPLC on C18 and C4 columns. SDS/PAGE of the purified peptide showed a single band of about 10 kDa. The following sequence was determined for the 20 amino-terminal residues of this peptide: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln- Gln-Ala-Trp-Glu-Lys-Ala-Ala-Ser-His. This sequence is identical to the amino terminus of chicken alpha D-globin. On a Western blot, the peptide immunoreacted with a polyclonal antibody against chicken globin D. A synthetic peptide corresponding to residues 1-40 of the alpha D-globin amino terminus also stimulated adenylyl cyclase activity and promoted differentiation. This 125I-labeled synthetic peptide bound specifically to T. cruzi epimastigote cells. Activation of epimastigote adenylyl cyclase by the hemoglobin-derived peptide may play an important role in T. cruzi differentiation and consequently in the transmission of Chagas disease.
Assuntos
Adenilil Ciclases/metabolismo , Globinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Triatoma/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Galinhas , Cromatografia Líquida de Alta Pressão , Fenômenos Fisiológicos do Sistema Digestório , Eletroforese em Gel de Poliacrilamida , Globinas/química , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Hemoglobinas/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/síntese química , Peptídeos/farmacologia , Homologia de Sequência de AminoácidosRESUMO
A peptide from hindguts of the Triatoma infestans, the hematophagous Chagas' insect vector, activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes (proliferative and non-infectious forms) to metacyclic trypomastigotes (non-proliferative and infectious forms). The peptide was purified from hindguts of insects fed two days before with chicken blood. After purification, the peptide showed upon SDS-PAGE a single band of about 10 kDa. The sequence for 20 residues of the amino terminus of this peptide was: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln-Gln-Ala-Trp-Glu-Lys-Ala- Ala-Ser-His. This sequence corresponds to the amino terminus of chicken alpha D-globin. A synthetic peptide with the sequence of the 40 amino acids corresponding to the amino terminus of alpha D-globin, also stimulated T. cruzi adenylyl cyclase activity and promoted metacyclogenesis.
Assuntos
Adenilil Ciclases/metabolismo , Globinas/fisiologia , Fragmentos de Peptídeos/fisiologia , Triatoma/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Galinhas , Fenômenos Fisiológicos do Sistema Digestório , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
A guanosine 5'-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.