RESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL.
Asunto(s)
Apoptosis , Proteínas Relacionadas con la Autofagia/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Apoptosis/efectos de los fármacos , Citoprotección/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Células Jurkat , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Análisis de Secuencia de ARN , Tretinoina/farmacologíaRESUMEN
Dasatinib is an ABL1 tyrosine kinase inhibitor (TKI) with a short in vivo plasmatic half-life but with good efficiency, which is not fully understood. We investigated the possibility that circulating erythrocytes store and then provide dasatinib to target cells. In vitro coincubation of dasatinib-treated cells with naïve leukemic cells followed by analysis of kinase inhibition, apoptosis induction, fluorescent molecule exchanges, and dasatinib dosage were performed. Cells incubated with clinically relevant concentrations of dasatinib for a short time retained, after a washout procedure, an intracellular pool of dasatinib which was transferable to naïve BCR-ABL1 expressing cells and induced their apoptosis. This was verified in total blood where the huge cellular volume of erythrocytes constituted a large reservoir of dasatinib able to induce apoptosis in naïve BCR-ABL1 cell lines and primitive chronic myeloid leukemia (CML) CD34+ cells. This dasatinib transfer necessitated a contact between donor and acceptor cells. A component exchange occurred during this contact, carrying dasatinib and other TKIs such as nilotinib or the fluorescent sunitinib. An active pool of dasatinib could be buried inside the circulating erythrocytes, out of reach of detoxifying mechanisms, but still available for target cells and thus extending the acute effect of the plasmatic pool of the drug.
RESUMEN
Tyrosine kinase inhibitors (TKI) constitute the frontline treatment for chronic myeloid leukemia patients. Dasatinib, a second-generation TKI, was developed to overcome TKI resistances. However, dasatinib resistances are also described but remain less characterized. To mimic in vivo acquired dasatinib resistance, the BCR-ABL1-positive cell line K562 was transiently treated with a pharmacological concentration of dasatinib, for a short time in the presence of stem cell factor. A dasatinib resistant counterpart (K562 RES) was developed. Investigation of resistance mechanisms using kinase substrate arrays revealed that FYN was overactivated in K562 RES. The FYN inhibitor KX2-391 cooperated with dasatinib to block K562 RES proliferation. Cell tracking experiments showed that activated FYN support cell proliferation independently of BCR-ABL1 in K562 RES cells. Moreover, the MEK-ERK pathway was found hyper-phosphorylated in K562 RES cells even in the presence of dasatinib. Actually, ERK1/2 activity supported viability in K562 RES only in the absence of BCR-ABL1 activity. Finally, BCR-ABL1 and MEK inhibitor combination was sufficient to induce cell death even in non-proliferating resistant cells. Considering the conditions used to generate this dasatinib resistant cell line, such a resistance mechanism could be found in dasatinib treated patients. Consequently, it is valuable to know that inhibition of the MEK-ERK1/2 axis can overcome this resistance.
Asunto(s)
Antineoplásicos/farmacología , Dasatinib/farmacología , Resistencia a Antineoplásicos/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Proteínas de Fusión bcr-abl/genética , Humanos , Hibridación Fluorescente in Situ , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Reacción en Cadena de la Polimerasa , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-fyn/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fyn/metabolismoRESUMEN
In spite of intensive research to improve treatment of acute myeloid leukemia (AML) more than half of all patients continue to develop a refractory disease. Therefore there is need to improve AML treatment. The overexpression of the BCL-2 family anti-apoptotic members, like BCL-2 or BCL-xL has been largely reported in lymphoid tumors but also in AML and other tumors. To counteract the anti-apoptotic effect of BCL-2, BH3 mimetics have been developed to target cancer cells. An increase in activity of ERK1/2 mitogen activated protein (MAP) kinase has also been reported in AML and might be targeted by MEK1/2 inhibitors. Hence, in the current work, we investigated whether the association of a BH3 mimetic such ABT-263 and the MEK1/2 inhibitor pimasertib (MEKI), was efficient to target AML cells. A synergistic increasing of apoptosis was observed in AML cell lines and in primary cells without affecting normal bone marrow cells. Such cooperation was confirmed on tumor growth in a mouse xenograft model of AML. In addition we demonstrated that MEKI sensitized the cells to apoptosis through its ability to promote a G1 cell cycle arrest. So, this combination of a MAP Kinase pathway inhibitor and a BH3 mimetic could be a promising strategy to improve the treatment of AML.
Asunto(s)
Compuestos de Anilina/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Niacinamida/análogos & derivados , Sulfonamidas/farmacología , Enfermedad Aguda , Compuestos de Anilina/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Células HL-60 , Humanos , Inmunohistoquímica , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Niacinamida/administración & dosificación , Niacinamida/farmacología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/administración & dosificación , Carga Tumoral/efectos de los fármacos , Células U937 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: BIM is essential for the response to tyrosine-kinase inhibitors (TKI) in chronic myeloid leukaemia (CML) patients. Recently, a deletion polymorphism in intron 2 of the BIM gene was demonstrated to confer an intrinsic TKI resistance in Asian patients. The present study aimed at identifying mutations in the BIM sequence that could lead to imatinib resistance independently of BCR-ABL mutations. EXPERIMENTAL DESIGN: BIM coding sequence analysis was performed in 72 imatinib-treated CML patients from a French population of our centre and in 29 healthy controls (reference population) as a case-control study. Real-time quantitative PCR (RT qPCR) was performed to assess Bim expression in our reference population. RESULTS: No mutation with amino-acid change was found in the BIM coding sequence. However, we observed a silent single nucleotide polymorphism (SNP) c465C>T (rs724710). A strong statistical link was found between the presence of the T allele and the high Sokal risk group (pâ=â0.0065). T allele frequency was higher in non responsive patients than in the reference population (pâ=â0.0049). Similarly, this T allele was associated with the mutation frequency on the tyrosine kinase domain of BCR-ABL (p<0.001) and the presence of the T allele significantly lengthened the time to achieve a major molecular response (MMR). Finally, the presence of the T allele was related to a decreased basal expression of the Bim mRNA in the circulating mononuclear cells of healthy controls. CONCLUSION: These results suggest that the analysis of the c465C>T SNP of BIM could be useful for predicting the outcome of imatinib-treated CML patients.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas Reguladoras de la Apoptosis/genética , Benzamidas/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas de la Membrana/genética , Piperazinas/uso terapéutico , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas/genética , Pirimidinas/uso terapéutico , ARN Mensajero/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Biomarcadores Farmacológicos/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Expresión Génica , Frecuencia de los Genes , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Tasa de Mutación , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismoRESUMEN
Putative cancer stem cells have been identified in glioblastoma (GBM), associated with resistance to conventional therapies. Overcoming this resistance is a major challenge to manage this deadly brain tumor. Epidermal growth factor receptor (EGFR) is commonly amplified, over-expressed, and/or mutated in GBM, making it a compelling target for therapy. This study investigates the behavior of 3 primary neurosphere (NS) cell lines and their adherent counterparts originated from human GBM resections, when treated with EGFR-tyrosine kinase inhibitor erlotinib, associated or not with cyclopamine, a hedgehog pathway inhibitor. Adherent cells cultured in the presence of serum expressed the glial fibrillary acidic protein, whereas NS-forming cells cultured in serum-free medium expressed CD133, nestin, and Oct-4, markers of neural stem and progenitor cells. For the 3 adherent cell lines, erlotinib has a moderate effect (50% inhibitory concentration [IC50], >10 µM). Conversely, erlotinib induced a strong cell growth inhibition (IC50, <1 µM) on NS-forming cells, related to the EGFR gene amplification and EGFR protein expression. A short exposure to erlotinib reduced nestin-positive cell proliferation, but NS-initiating activity and self-renewal were not altered. EGFR pathway seems essential for GBM progenitor cell proliferation but dispensable for cancer stem-like cell self-renewal. Inhibition of hedgehog pathway with cyclopamine was evaluated in association with erlotinib on NS growth. Although each drug separately had no effect on sphere initiation, their combination significantly decreased the sphere number (P < .001). Our findings show synergic efficiency for erlotinib-cyclopamine association and provide a suitable in vitro model to explore drug combinations on GBM cells.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/patología , Diferenciación Celular/efectos de los fármacos , Glioblastoma/patología , Células Madre Neoplásicas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Hibridación Genómica Comparativa , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación Fluorescente in Situ , Células Madre Neoplásicas/patología , Quinazolinas/administración & dosificación , Alcaloides de Veratrum/administración & dosificaciónRESUMEN
Chronic myeloid leukemia (CML) tumorigenicity is driven by the oncogenic BCR-ABL tyrosine kinase. Specific tyrosine kinase inhibitors (TKI) have been designed and are now used for the treatment of CML. These TKI induce apoptosis in leukemic cells in a BIM-dependent mechanism. We hypothesized that an increase in BIM activity could sensitize CML cells to TKI. We blocked the anti-apoptotic proteins of the Bcl-2 family by using ABT-737, a Bcl-2 and Bcl-XL inhibitor. ABT-737 modified Bcl-2 protein interactions toward a pro-apoptotic phenotype. Its combination with TKI resulted in a strong synergism in CML cell lines. The association also induced a large decrease in X-linked inhibitor of apoptosis (XIAP), followed by caspase-3 activation. This XIAP decrease was due to post-translational events. The mitochondrial serine protease HtrA2/Omi was identified as being responsible for this off-target effect. Then, ABT-737 and TKI cooperate at several levels to induce apoptosis of CML cells lines, and the benefit of this association was also observed in CML hematopoietic progenitors. Interestingly, a lethal effect was also observed in the more immature CD34(+)CD38(-) TKI-insensitive population. Combination therapy might by an interesting strategy for the treatment of CML patients.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de Neoplasias/biosíntesis , Células Madre Neoplásicas/efectos de los fármacos , Nitrofenoles/farmacología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pirimidinas/farmacología , Sulfonamidas/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/biosíntesis , Antígenos CD34/análisis , Proteínas Reguladoras de la Apoptosis/fisiología , Proteína 11 Similar a Bcl2 , Benzamidas , Línea Celular Tumoral/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Células Madre Hematopoyéticas/enzimología , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Mesilato de Imatinib , Células K562/efectos de los fármacos , Proteínas de la Membrana/fisiología , Proteínas Mitocondriales/fisiología , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/enzimología , Proteínas Proto-Oncogénicas/fisiología , Serina Endopeptidasas/fisiología , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Gliomas are the most common malignant primary brain tumors in adults. The median survival never exceeds 12 months, owing to inherent resistance to both radio and chemotherapies. Epidermal Growth Factor Receptor (EGFR) is amplified, overexpressed, and/or mutated in glioblastomas (GBM), making it a rational for therapy. Erlotinib, an EGFR kinase inhibitor is strongly associated with clinical response in several cancers. Inhibition of cell proliferation and induction of apoptosis by erlotinib were investigated in U87-MG and DBTRG-05MG, two human glioblastoma cell lines. The expression of several apoptosis-related proteins was investigated in these cell lines and in tumoral tissue from glioblastomas. Both cell lines expressed wild-type EGFR but were deficient for PTEN. Erlotinib induced a marked accumulation of the BIM protein, but the activation of caspase-3 machinery was missing, regardless of the decrease in XIAP. Moreover, in U87-MG, erlotinib promoted accumulation of αB-crystallin a small heat shock protein capable to impair caspase activation. DBTRG-05MG was found deficient for procaspase 3 and constitutively overexpressed αB-crystallin. Similarly, deficiencies in PTEN and procaspase 3 were constantly found in samples from glioblastoma samples, while αB-crystallin expression was inconsistent. In cell lines, high concentrations of erlotinib induced cell death through a caspase independent process and an autophagic process was evidenced in U87-MG. Inhibition of autophagy induced a marked increase in the death-inducing activity of erlotinib. These results confirm that glioblastoma cell lines exhibit several anti-apoptotic mechanisms, and underline that EGFR targeted therapy must be associated to other inhibitors to achieve an antitumoral effect.