Assuntos
Caspase 8 , Proteína Supressora de Tumor p53 , Regulação para Cima , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Caspase 8/metabolismo , Caspase 8/genética , Regulação para Cima/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacosRESUMO
Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.
Assuntos
Distrofina/genética , Mutação da Fase de Leitura , Edição de Genes/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , RNA Guia de Cinetoplastídeos/genética , Animais , Modelos Animais de Doenças , Éxons , Feminino , Regulação da Expressão Gênica , Terapia Genética , Genoma , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Espectrometria de Massas , Músculo Esquelético/metabolismo , Músculos/metabolismo , Mioblastos/metabolismo , Miócitos Cardíacos/metabolismo , Proteoma , SuínosRESUMO
The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.
Assuntos
Leucemia Mieloide Aguda/terapia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Sequências de Repetição em Tandem/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoterapia/métodos , Leucemia Mieloide Aguda/metabolismo , Mutação/efeitos dos fármacos , Mutação/genética , Prognóstico , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaAssuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Leucemia/genética , Osteonectina/genética , Animais , Progressão da Doença , Edição de Genes/métodos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Xenoenxertos , Humanos , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Microambiente Tumoral/genéticaRESUMO
Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/imunologia , Evasão Tumoral/efeitos dos fármacos , Animais , Antígeno B7-H1/análise , Antígeno B7-H1/fisiologia , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Receptor de Morte Celular Programada 1/análise , Receptor de Morte Celular Programada 1/fisiologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/análiseRESUMO
Breast cancer is a prevalent neoplastic disease among women worldwide which treatments still present several side effects and resistance. Considering that cancer cells present derangements in their energetic homeostasis, and that peroxisome proliferator-activated receptor- gamma coactivator 1 (PGC-1) is crucial for cellular metabolism and redox signaling, the main objective of this study was to investigate whether there is a relationship between PGC-1 expression, the proliferation of breast cancer cells and the mechanisms involved. We initially assessed PGC-1ß expression in complementary DNA (cDNA) from breast tumor of patients bearing luminal A, luminal B, and HER2-overexpressed and triple negative tumors. Our data showed that PGC-1ß expression is increased in patients bearing HER2-overexpressing tumors as compared to others subtypes. Using quantitative PCR and immunoblotting, we showed that breast cancer cells with HER2-amplification (SKBR-3) have greater expression of PGC-1ß as compared to a non-tumorous breast cell (MCF-10A) and higher proliferation rate. PGC-1ß expression was knocked down with short interfering RNA in HER2-overexpressing cells, and cells decreased proliferation. In these PGC-1ß-inhibited cells, we found increased citrate synthase activity and no marked changes in mitochondrial respiration. Glycolytic pathway was decreased, characterized by lower intracellular lactate levels. In addition, after PGC-1ß knockdown, SKBR-3 cells showed increased reactive oxygen species production, no changes in antioxidant activity, and decreased expression of ERRα, a modulator of metabolism. In conclusion, we show an association of HER2-overexpression and PGC-1ß. PGC-1ß knockdown impairs HER2-overexpressing cells proliferation acting on ERRα signaling, metabolism, and redox balance.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes erbB-2 , Redes e Vias Metabólicas , Oxirredução , Idoso , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Espécies Reativas de Oxigênio , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
P38α/ß has been described as a tumor-suppressor controlling cell cycle checkpoints and senescence in epithelial malignancies. However, p38α/ß also regulates other cellular processes. Here, we describe a role of p38α/ß as a regulator of acute lymphoblastic leukemia (ALL) proliferation and survival in experimental ALL models. We also report first evidence that p38α/ß phosphorylation is associated with the occurrence of relapses in TEL-AML1-positive leukemia. First, in vitro experiments show that p38α/ß signaling is induced in a cyclical manner upon initiation of proliferation and remains activated during log-phase of cell growth. Next, we provide evidence that growth-permissive signals in the bone marrow activate p38α/ß in a novel avian ALL model, in which therapeutic targeting can be tested. We further demonstrate that p38α/ß inhibition by small molecules can suppress leukemic expansion and prolong survival of mice bearing ALL cell lines and primary cells. Knockdown of p38α strongly delays leukemogenesis in mice xenografted with cell lines. Finally, we show that in xenografted TEL-AML1 patients, ex vivo p38α/ß phosphorylation is associated with an inferior long-term relapse-free survival. We propose p38α/ß as a mediator of proliferation and survival in ALL and show first preclinical evidence for p38α/ß inhibition as an adjunct approach to conventional therapies.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Adolescente , Animais , Proliferação de Células , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Camundongos , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapiaRESUMO
In acute myeloid leukemia (AML), several signaling pathways such as the phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway are deregulated and constitutively activated as a consequence of genetic and cytogenetic abnormalities. We tested the effectiveness of PI3K/AKT/mTOR-targeting therapies and tried to identify alterations that associate with treatment sensitivity. By analyzing primary samples and cell lines, we observed a wide range of cytotoxic activity for inhibition of AKT (MK-2206), mTORC1 (rapamycin) and PI3K/mTORC1/2 (BEZ-235) with a high sensitivity of cells carrying an MLL rearrangement. In vivo PI3K/mTOR inhibition delayed tumor progression, reduced tumor load and prolonged survival in an MLL-AF9(+)/FLT3-ITD(+) xenograft mouse model. By performing targeted amplicon sequencing in 38 MLL-AF9(+) and 125 cytogenetically normal AML patient samples, we found a high additional mutation rate for genes involved in growth factor signaling in 79% of all MLL-AF9(+) samples, which could lead to a possible benefit of this cohort. PI3K/mTOR inhibition for 24 h led to the cross-activation of the ERK pathway. Further in vitro studies combining PI3K/mTOR and ERK pathway inhibition revealed highly synergistic effects in apoptosis assays. Our data implicate a possible therapeutic benefit of PI3K/mTOR inhibition in the MLL-mutated subgroup. Inhibiting rescue pathways could improve the therapeutic efficacy of PI3K-targeted therapies in AML.
Assuntos
Antineoplásicos/farmacologia , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Sinergismo Farmacológico , Rearranjo Gênico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Imidazóis/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolinas/farmacologia , Transdução de Sinais , Sirolimo/farmacologia , Análise de Sobrevida , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Resting tumor cells represent a huge challenge during anticancer therapy due to their increased treatment resistance. TNF-related apoptosis-inducing ligand (TRAIL) is a putative future anticancer drug, currently in phases I and II clinical studies. We recently showed that TRAIL is able to target leukemia stem cell surrogates. Here, we tested the ability of TRAIL to target cell cycle-arrested tumor cells. Cell cycle arrest was induced in tumor cell lines and xenografted tumor cells in G0, G1 or G2 using cytotoxic drugs, phase-specific inhibitors or RNA interference against cyclinB and E. Biochemical or molecular arrest at any point of the cell cycle increased TRAIL-induced apoptosis. Accordingly, when cell cycle arrest was disabled by addition of caffeine, the antitumor activity of TRAIL was reduced. Most important for clinical translation, tumor cells from three children with B precursor or T cell acute lymphoblastic leukemia showed increased TRAIL-induced apoptosis upon knockdown of either cyclinB or cyclinE, arresting the cell cycle in G2 or G1, respectively. Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts enhanced antitumor activity against cell cycle-arrested tumor cells. Therefore, TRAIL might represent an interesting drug to treat static-tumor disease, for example, during minimal residual disease.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular , Pontos de Checagem da Fase G2 do Ciclo Celular , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Ciclina B/genética , Ciclina B/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Dexametasona/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Metotrexato/farmacologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: In polychemotherapy protocols, that is for treatment of neuroblastoma and Ewing sarcoma, Vinca alkaloids and cell cycle-arresting drugs are usually administered on the same day. Here we studied whether this combination enables the optimal antitumour effects of Vinca alkaloids to be manifested. EXPERIMENTAL APPROACH: Vinca alkaloids were tested in a preclinical mouse model in vivo and in vitro in combination with cell cycle-arresting drugs. Signalling pathways were characterized using RNA interference. KEY RESULTS: In vitro, knockdown of cyclins significantly inhibited vincristine-induced cell death indicating, in accordance with previous findings, Vinca alkaloids require active cell cycling and M-phase transition for induction of cell death. In contrast, anthracyclines, irradiation and dexamethasone arrested the cell cycle and acted like cytostatic drugs. The combination of Vinca alkaloids with cytostatic therapeutics resulted in diminished cell death in 31 of 36 (86%) tumour cell lines. In a preclinical tumour model, anthracyclines significantly inhibited the antitumour effect of Vinca alkaloids in vivo. Antitumour effects of Vinca alkaloids in the presence of cytostatic drugs were restored by caffeine, which maintained active cell cycling, or by knockdown of p53, which prevented drug-induced cell cycle arrest. Therapeutically most important, optimal antitumour effects were obtained in vivo upon separating the application of Vinca alkaloids from cytostatic therapeutics. CONCLUSION AND IMPLICATIONS: Clinical trials are required to prove whether Vinca alkaloids act more efficiently in cancer patients if they are applied uncoupled from cytostatic therapies. On a conceptual level, our data suggest the implementation of polychemotherapy protocols based on molecular mechanisms of drug-drug interactions. LINKED ARTICLE: This article is commented on by Solary, pp 1555-1557 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12101.
Assuntos
Antraciclinas/farmacologia , Antineoplásicos/farmacologia , Dexametasona/farmacologia , Vincristina/farmacologia , Animais , Antraciclinas/uso terapêutico , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Linhagem Celular Tumoral , Dexametasona/uso terapêutico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Antagonismo de Drogas , Feminino , Raios gama , Humanos , Camundongos , Camundongos Nus , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Transplante de Neoplasias , Vincristina/uso terapêuticoRESUMO
Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor-proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD's ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.
Assuntos
Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , NF-kappa B/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ativação Transcricional , Caspase 8/genética , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas/genética , Humanos , NF-kappa B/metabolismo , Ligação Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de SinaisRESUMO
During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug-drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.
Assuntos
Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Criança , Doxorrubicina/farmacologia , Combinação de Medicamentos , Quimioterapia Combinada , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Triterpenos Pentacíclicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Triterpenos/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Vincristina/farmacologia , Ácido BetulínicoRESUMO
Evasion of apoptosis may contribute to poor treatment response in pediatric acute lymphoblastic leukemia (ALL), calling for novel treatment strategies. Here, we report that inhibitors of apoptosis (IAPs) at subtoxic concentrations cooperate with various anticancer drugs (that is, AraC, Gemcitabine, Cyclophosphamide, Doxorubicin, Etoposide, Vincristine and Taxol) to induce apoptosis in ALL cells in a synergistic manner as calculated by combination index and to reduce long-term clonogenic survival. Importantly, we identify RIP1 as a critical regulator of this synergism of IAP inhibitors and AraC that mediates the formation of a RIP1/FADD/caspase-8 complex via an autocrine/paracrine loop of tumor necrosis factor-α (TNFα). Knockdown of RIP1 abolishes formation of this complex and subsequent activation of caspase-8 and -3, mitochondrial perturbations and apoptosis. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 or blockage of TNFα by Enbrel inhibits IAP inhibitor- and AraC-triggered interaction of RIP1, FADD and caspase-8 and apoptosis. In contrast to malignant cells, IAP inhibitors and AraC at equimolar concentrations are non-toxic to normal peripheral blood lymphocytes or mesenchymal stromal cells. Thus, our findings provide first evidence that IAP inhibitors present a promising strategy to prime childhood ALL cells for chemotherapy-induced apoptosis in a RIP1-dependent manner. These data have important implications for developing apoptosis-targeted therapies in childhood leukemia.
Assuntos
Apoptose , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas de Ligação a RNA/fisiologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Criança , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Proteínas Inibidoras de Apoptose/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas de Ligação a RNA/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Spongistatin 1 is a new experimental chemotherapeutic agent isolated from marine sponges. Here we show that spongistatin 1 potently induces cell death in patient primary acute leukemic cells with higher efficiency than 8/10 clinically used cytotoxic drugs and prevents long-term survival of leukemic cell lines. Spongistatin 1 triggers caspase-dependent apoptosis in Jurkat T cells by the release of cytochrome c, Smac/DIABLO and Omi/HtrA2. As caspase-9 acts as an initiator caspase and Bcl-2 and Bcl-xL overexpression suppress spongistatin 1-induced apoptosis, cell death is mediated through the mitochondrial apoptosis pathway. Importantly, spongistatin 1 leads to the degradation of the antiapoptotic X-linked inhibitor of apoptosis protein. In apoptosis-resistant leukemic tumor cells overexpressing XIAP, spongistatin 1 effectively causes cell death and potentiates cell death induction by other apoptosis-promoting factors that might be caused by spongistatin 1-mediated degradation of XIAP. Our data show that spongistatin 1 represents a promising novel therapeutic agent for the treatment of leukemic tumor cells especially in the clinically highly relevant situation of chemoresistance due to overexpression of XIAP.
Assuntos
Leucemia/tratamento farmacológico , Macrolídeos/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Humanos , Leucemia/patologia , Macrolídeos/uso terapêutico , Proteínas Mitocondriais , Poríferos , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Apoptosis resistance is crucially involved in cancer development and progression, represents the leading cause for failure of anticancer therapy and is caused, for example, by downregulation of proapoptotic intracellular signaling molecules such as caspase-8. We found that the cytotoxic drugs methotrexate (MTX) and 5-fluorouracil (5-FU) were both able to sensitize resistant tumor cells for induction of apoptosis by p53-mediated upregulation of caspase-8. Increase in caspase-8 messenger RNA and protein expression disabled tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced proliferation and restored sensitivity toward TRAIL-induced apoptosis which was inhibited by transfection of p53 decoy oligonucleotides, p53 shRNA and caspase-8 shRNA. Upregulation of caspase-8 and sensitization toward TRAIL-induced apoptosis was found both in a broad panel of tumor cell lines with downregulated caspase-8 and in TRAIL-resistant primary tumor cells of children with acute leukemia. Taken together, we have identified caspase-8 as an important p53 target gene regulated by cytotoxic drugs. These findings highlight a new drug-induced modulation of physiological apoptosis pathways, which may be involved in successful anticancer therapy using MTX and 5-FU in leukemia and solid tumors over decades.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Metotrexato/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Caspase 8/genética , Inibidores de Caspase , Linhagem Celular Tumoral , Humanos , RNA Mensageiro/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
BACKGROUND: Glioblastoma multiforme (WHO Grade IV, GBM) is the most malignant brain tumour with a mean survival time of less than one year. Betulinic acid, ceramide and TRAIL (TNF-related apoptosis inducing ligand) represent novel therapeutic agents for potential use in GBM. METHOD: Primary GBM cells of 21 patients with macroscopically complete tumour resection were tested in vitro for cell death induction by betulinic acid, ceramide, TRAIL and established therapeutics (BCNU, cisplatin, doxorubicin, vincristin and gamma-irradiation). FINDINGS: At peak plasma concentrations (PPC), Betulinic acid, ceramide and TRAIL induced cell death in primary GBM cells at higher rates than established cytotoxic drugs. Specific cell death > or =75% was observed in 43% (9/21), 38% (8/21), and 19% (4/21) for betulinic acid, ceramide, and TRAIL respectively, while this was only found in 5% (1/21) of gamma-irradiated and cisplatin-treated cells, and in none of the GBM cultures, where BCNU or vincristin were applied in PPC. CONCLUSION: Due to a markedly improved cell death of GBM cells as compared with established therapeutics, Betulinic acid, ceramide and TRAIL might represent potent substances for future treatment of GBM.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/patologia , Morte Celular/efeitos dos fármacos , Ceramidas/farmacologia , Glioblastoma/patologia , Glicoproteínas de Membrana/farmacologia , Triterpenos/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Idoso , Proteínas Reguladoras de Apoptose , Técnicas de Cultura de Células , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Triterpenos Pentacíclicos , Estudos Retrospectivos , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Ácido BetulínicoRESUMO
Betulinic acid (BA), a natural component isolated from Birch trees, effectively induces apoptosis in neuroectodermal and epithelial tumor cells and exerts little toxicity in animal trials. Here, we show that BA-induced marked apoptosis in 65% of primary pediatric acute leukemia cells and all leukemia cell lines tested. When compared for in vitro efficiency with conventionally used cytotoxic drugs, BA was more potent than nine out of 10 standard therapeutics and especially efficient in tumor relapse. No crossresistances were found between BA and any cytotoxic drug. Intracellular apoptosis signaling in leukemia tumor cells paralleled the pathway found in neuroectodermal cells involving caspases, but not death receptors. In isolated mitochondria, BA induced release of both cytochrome c and Smac. Taken together, BA potently induces apoptosis in leukemia cells and should be further evaluated as a future drug to treat leukemia.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Leucemia/patologia , Triterpenos/farmacologia , Adolescente , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Criança , Pré-Escolar , Citocromos c/metabolismo , Interações Medicamentosas , Feminino , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia/tratamento farmacológico , Masculino , Proteínas Mitocondriais/metabolismo , Triterpenos Pentacíclicos , Prevenção Secundária , Transdução de Sinais , Células Tumorais Cultivadas , Ácido BetulínicoRESUMO
BACKGROUND: The death of cardiac cells during ischemia and reperfusion is partially mediated by apoptosis, as seen, eg, in autopsy material of patients after acute myocardial infarction. METHODS AND RESULTS: To study the role of CD95/Fas/Apo1 for induction of postischemic cell death, we used an ischemia/reperfusion model of isolated rat and mouse hearts in Langendorff perfusion. In this model, caspase-dependent apoptosis occurred during postischemic reperfusion. Moreover, soluble CD95 ligand/Fas ligand was released by the postischemic hearts early after the onset of reperfusion. In addition, this ligand was synthesized de novo under these circumstances. Similar findings were observed for other "death-inducing" ligands, such as tumor necrosis factor (TNF)-alpha and TNF-related apoptosis-inducing ligand. In primary adult rat myocyte culture, hypoxia and reoxygenation caused a marked increase in sensitivity to the apoptotic effects of CD95 ligand. Isolated hearts from mice lacking functional CD95 (lpr) display marked reduction in cell death after ischemia and reperfusion compared with wild-type controls. CONCLUSIONS: These data suggest that CD95/Apo1/Fas is directly involved in cell death after myocardial ischemia. The CD95 system might thus represent a novel target for therapeutic prevention of postischemic cell death in the heart.