RESUMO
Selective inhibition of BCL-2 is expected to enhance therapeutic vulnerability in luminal estrogen receptor-positive breast cancers. We show here that the BCL-2 dependency of luminal tumor cells is nevertheless mitigated by breast cancer-associated fibroblasts (bCAFs) in a manner that defines MCL-1 as another critical therapeutic target. bCAFs favor MCL-1 expression and apoptotic resistance in luminal cancer cells in a IL-6 dependent manner while their own, robust, survival also relies on MCL-1. Studies based on ex vivo cultures of human luminal breast cancer tissues further argue that the contribution of stroma-derived signals to MCL-1 expression shapes BCL-2 dependency. Thus, MCL-1 inhibitors are beneficial for targeted apoptosis of breast tumor ecosystems, even in a subtype where MCL-1 dependency is not intrinsically driven by oncogenic pathways.
Assuntos
Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/deficiência , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Receptores de Estrogênio/metabolismo , Apoptose/fisiologia , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Feminino , Humanos , Interleucina-6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Since brain tumours are the primary candidates for treatment by Boron Neutron Capture Therapy, one major challenge in the selective drug delivery to CNS is the crossing of the blood-brain barrier (BBB). The present pilot study investigated (i) the transport of a conventional B-containing product (i.e., L-(4-(10)Boronophenyl)alanine, L-(10)BPA), already used in medicine but still not fully characterized regarding its CNS interactions, as well as (ii) the effects of the L-(10)BPA on the BBB integrity using an in vitro model, consisting of brain capillary endothelial cells co-cultured with glial cells, closely mimicking the in vivo conditions. The multi-step experimental strategy (i.e. Integrity test, Filter study, Transport assay) checked L-(10)BPA toxicity at 80 µg Boron equivalent/ml, and its ability to cross the BBB, additionally by characterizing the cytoskeletal and TJ's proteins by immunocytochemistry and immunoblotting. In conclusion, a lack of toxic effects of L-(10)BPA was demonstrated, nevertheless accompanied by cellular stress phenomena (e.g. vimentin expression modification), paralleled by a low permeability coefficient (0.39 ± 0.01 × 10(-3)cm min(-1)), corroborating the scarce probability that L-(10)BPA would reach therapeutically effective cerebral concentration. These findings emphasized the need for novel strategies aimed at optimizing boron delivery to brain tumours, trying to ameliorate the compound uptake or developing new targeted products suitable to safely and effectively treat head cancer. Thus, the use of in vitro BBB model for screening studies may provide a useful early safety assessment for new effective compounds.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Compostos de Boro/toxicidade , Encéfalo/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Fenilalanina/análogos & derivados , Animais , Barreira Hematoencefálica/fisiopatologia , Boro/farmacocinética , Boro/toxicidade , Compostos de Boro/farmacocinética , Encéfalo/fisiopatologia , Permeabilidade Capilar/fisiologia , Bovinos , Técnicas de Cocultura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Células Endoteliais/fisiologia , Neuroglia/fisiologia , Ocludina/metabolismo , Fenilalanina/farmacocinética , Fenilalanina/toxicidade , Projetos Piloto , Ratos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/fisiologia , Vimentina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Antimitotic agents such as microtubule inhibitors (paclitaxel) are widely used in cancer therapy while new agents blocking mitosis onset are currently in development. All these agents impose a prolonged mitotic arrest in cancer cells that relies on sustained activation of the spindle assembly checkpoint and may lead to subsequent cell death by incompletely understood molecular events. We have investigated the role played by anti-apoptotic Bcl-2 family members in the fate of mitotically arrested mammary tumor cells treated with paclitaxel, or depleted in Cdc20, the activator of the anaphase promoting complex. Under these conditions, a weak and delayed mitotic cell death occurs that is caspase- and Bax/Bak-independent. Moreover, BH3 profiling assays indicate that viable cells during mitotic arrest are primed to die by apoptosis and that Bcl-xL is required to maintain mitochondrial integrity. Consistently, Bcl-xL depletion, or treatment with its inhibitor ABT-737 (but not with the specific Bcl-2 inhibitor ABT-199), during mitotic arrest converts cell response to antimitotics to efficient caspase and Bax-dependent apoptosis. Apoptotic priming under conditions of mitotic arrest relies, at least in part, on the phosphorylation on serine 62 of Bcl-xL, which modulates its interaction with Bax and its sensitivity to ABT-737. The phospho-mimetic S62D-Bcl-xL mutant is indeed less efficient than the corresponding phospho-deficient S62A-Bcl-xL mutant in sequestrating Bax and in protecting cancer cells from mitotic cell death or yeast cells from Bax-induced growth inhibition. Our results provide a rationale for combining Bcl-xL targeting to antimitotic agents to improve clinical efficacy of antimitotic strategy in cancer therapy.
Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Pontos de Checagem do Ciclo Celular , Proteína bcl-X/metabolismo , Substituição de Aminoácidos , Compostos de Bifenilo/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutação de Sentido Incorreto , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Sulfonamidas/farmacologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genéticaRESUMO
1. The objective was to investigate the transport of an anticancer agent carboplatin across the blood-brain barrier in combination with hyperbaric oxygenation treatment. An in vitro well-validated model of bovine brain capillary endothelial cells was used. 2. A transendothelial transport of doxorubicin, a known P-glycoprotein substrate, was enhanced 1.5-fold by verapamil for 2-h incubation at 37 degrees C. A transendothelial permeability coefficient of carboplatin (1.29 x 10(-3)cm min-1) was also increased 1.8-fold by verapamil. 3. Under the hyperbaric oxygenation conditions (at 0.2 MPa for the first 10 min), the transendothelial transport for 2 h of doxorubicin and carboplatin were increased 1.3- to 1.8-fold by hyperbaric oxygenation, like the suppressive effects of verapamil on P-gp function, without increase of the transport of lucifer yellow, a non P-glycoprotein substrate. 4. Combination of hyperbaric oxygenation treatment and verapamil could not further increase the permeability coefficients of these drugs that were already enhanced by either treatment, implying the P-glycoprotein-mediated carboplatin efflux transport similarly as doxorubicin. 5. Together with our reported high efficacy of carboplatin combined with hyperbaric oxygenation therapy on brain tumours, the present results suggest that carboplatin could be transported by P-glycoprotein, but that this efflux mechanism would be reduced by the hyperbaric oxygenation with the consequences of clinical efficacy.
Assuntos
Antineoplásicos/farmacocinética , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar/fisiologia , Carboplatina/administração & dosagem , Carboplatina/farmacocinética , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Oxigenoterapia Hiperbárica , Animais , Antineoplásicos/administração & dosagem , Bovinos , Células Cultivadas , RatosAssuntos
Apoptose , Ciclo Celular , Animais , Proliferação de Células , França , Humanos , Neoplasias/patologia , Neoplasias/terapiaRESUMO
Survivin is a fascinating member of the inhibitor of apoptosis protein (IAP) family with its dual roles in mitosis and apoptosis, and emerges as an attractive target for cancer therapy. Multiple myeloma (MM) is a plasma cell malignancy, characterized by deregulated proliferation, cell-death processes and fatal outcome. We thus investigated survivin expression in myeloma cells and its role in MM biology to evaluate its potential interest as a target in MM treatment. Our results describe the cancer-specific overexpression of survivin in myeloma cells and show a significant correlation between survivin expression at protein level and clinical course of MM. Moreover, survivin knockdown by RNA interference led to growth rate inhibition of myeloma cells related to apoptosis induction and deep cell-cycle disruption. Finally, survivin knockdown sensitized myeloma cells to conventional anti-myeloma agents. Altogether, these data argue for the interest to evaluate survivin antagonists in MM treatment.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/fisiologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/antagonistas & inibidores , Interferência de RNA , SurvivinaRESUMO
Scavenger-receptor class A has been held responsible for the clearance of modified LDL from the blood circulation. However, in mice deficient in scavenger-receptor class A, the decay in vivo of acetylated LDL (t1/2 < 2 min), as well as tissue distribution and liver uptake (at 5 min 77.4 +/- 4.6% of the injected dose) are not significantly different from control mice. The degradation capacity of acetylated LDL with liver endothelial cells, Kupffer cells, and peritoneal macrophages from knock-out mice was 58%, 63%, and 17% of the control, respectively, indicating that scavenger-receptor class A is relatively more important for the degradation of acetylated LDL and foam cell formation in peritoneal macrophages as compared to the liver cell types. This might explain the 60% reduction in atherosclerotic lesion area in scavenger-receptor-deficient apoE knock-out mice as compared to control apoE knock-out mice. Scavenger-receptor BI can facilitate selective uptake of cholesterol esters from HDL. A high cholesterol diet for two weeks induced an 80% downregulation of scavenger-receptor BI in the liver parenchymal cells while expression in liver macrophages is increased fourfold. The in vivo kinetics for the selective uptake of (oxidized) cholesterol esters from HDL correlate with the changes in scavenger-receptor BI expression. It is suggested that scavenger-receptor BI is subject to different regulatory mechanisms in parenchymal liver cells and macrophages related to a difference in function in these cell types.
Assuntos
Arteriosclerose/fisiopatologia , Antígenos CD36/fisiologia , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas de Membrana , Receptores Imunológicos/fisiologia , Animais , Moléculas de Adesão Celular/fisiologia , Humanos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores de Lipoproteínas/fisiologia , Receptores Depuradores , Receptores Depuradores Classe A , Receptores Depuradores Classe BRESUMO
This study investigates the suitability of the trimeric apolipoprotein (apo)AI(145-183) peptide that we recently described, to serve as a model to probe the relationship between apoAI structure and function. Three copies of the apoAI(145-183) unit, composed each of two amphipathic alpha-helical segments, were branched onto a covalent core matrix and the construct was recombined with phospholipids. A similar construct was made with the apoAI(102-140) peptide and used as a comparison with dimyristoylglycerophosphocholine (DMPC)-apoAI complexes. The DMPC-trimeric-apoAI(145-183) complexes had similar immunological reactivity with monoclonal antibodies directed against the 149-186 apoAI sequence (A44), suggesting that the A44 epitope is exposed similarly in both the synthetic peptide and the native apoAI complexes. The complexes generated with the trimeric-apoAI(145-183) bind specifically to HeLa cells with comparable affinity to the DMPC apoAI complexes; they are a good competitor for binding of apoAI to both HeLa cells and Fu5AH rat hepatoma cells; finally, these complexes promote cholesterol efflux from Fu5AH cells with an efficiency comparable with the apo AI/lipid complexes. To study LCAT activation by the trimeric apo AI(145-183) construct, complexes were prepared with dipalmitoylphosphatidylcholine (DPPC), cholesterol (C) and either the trimeric construct or apoAI. LCAT activation by the trimeric construct was much lower than by apo AI, possibly because the conformation of the trimeric 145-183 peptide in DPPC/C/peptide complexes does not mimic that of apoAI in the corresponding complexes. In comparison, the complexes generated with the multimeric apoAI(102-140) construct had a poor capacity to mimic the physico-chemical and biological properties of apoAI. The apoAI(102-140) construct had low affinity for lipid compared with the (145-183) construct. After association with lipids, it was a poor competitor of DMPC-apoAI complexes for cellular binding and had only limited capacity to promote cholesterol efflux. These results suggest trimeric constructs can serve as an appropriate models for apoAI, enabling further investigations and new experimental approaches to determine the structure-function relationship of apoAI.
Assuntos
Apolipoproteína A-I/metabolismo , Lipoproteínas HDL/metabolismo , Animais , Apolipoproteína A-I/química , Colesterol/metabolismo , Células HeLa , Humanos , Neoplasias Hepáticas Experimentais , Peptídeos/síntese química , Fosfatidilcolina-Esterol O-Aciltransferase/fisiologia , Conformação Proteica , Ratos , Células Tumorais CultivadasRESUMO
Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP). HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [3H]-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal. Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites.
Assuntos
Proteínas de Transporte , Glicosilfosfatidilinositóis/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA , Receptores de Lipoproteínas/metabolismo , Anticorpos/farmacologia , Sítios de Ligação , Humanos , Proteínas de Membrana/imunologia , Fosfatidilcolinas/metabolismo , Fosfolipases/antagonistas & inibidores , Receptores de Lipoproteínas/imunologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
In [3H]phosphatidylcholine (PC) prelabelled HepG2 cells, HDL3 stimulates a biphasic increase in 1.2-diacylglycerol (DAG). The early phase is mediated in part by a phospholipase C which is inhibited by 10 microM D 609, RHC-80267 or U-73122 and less by 100 microM propranolol. A phospholipase D is more likely involved in the late phase, as the DAG peak lags behind phosphatidic acid rise and is blocked by 100 microM propranolol. Cellular preincubation with 200 microg/ml antibodies against the inositolphosphoglycan (IPG) moiety of the GPI-anchor (Ab(IPG)), or depletion in GPI-anchored proteins by cellular pretreatment with 0.5 U/ml PI-PLC, 1 mM insulin and 2 HU/ml streptolysin-O, or depletion in membrane cholesterol content by filipin (5 microg/ml), digitonin (5 microg/ml) and cholesterol oxidase (0.5 U/ml) decreases the HDL3-signal, suggesting the involvement of a lipolytic cleavage of GPI-anchored proteins. Inhibition of proteases by 1 mM leupeptin/PMSF improves the response time to HDL3, with a DAG peak at 2-3 min. In the presence of protease-inhibitors, HDL3 releases in the culture medium several proteins with a residual IPG that binds Ab(IPG) after SDS-PAGE analysis and immunoblotting. HDL3-signalling pathways comprise tyrosine kinases, as preincubation with 100 microg/ml genistein or tyrphostin inhibits the HDL3-signal. HDL3 activates PC hydrolysis through a multistep pathway involving the cleavage of GPI-anchored proteins.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Anticorpos/imunologia , Meios de Cultura/análise , Diglicerídeos/metabolismo , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Fosfatidilcolinas/metabolismo , Inibidores de Proteases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
To elucidate further the binding of high-density-lipoprotein subfraction 3 (HDL3) to cells, the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-proteins) was studied. Treatment of cultured cells, such as fibroblasts or SK-MES-1 cells, with a phosphatidylinositol-specific phospholipase C (PI-PLC) significantly decreases specific HDL3 binding. Moreover, PI-PLC treatment of cultured cells or cellular plasma membrane fractions results in releasing proteins. These proteins have a soluble form and can also bind HDL3, as revealed by ligand blotting experiments with HDL3. In order to obtain enriched GPI-proteins, we used a detergent-free purification method to prepare a caveolar membrane fraction. In the caveolar fraction, we obtained, by ligand blotting experiments, the enrichment of two HDL3-binding proteins with molecular masses of 120 and 80 kDa. These proteins were also revealed in a plasma membrane preparation with two other proteins, with molecular masses of 150 and 104 kDa, and were sensitive to PI-PLC treatment. Electron microscopy also showed the binding of Au-labelled HDL3 inside the caveolar membrane invaginations. In SK-MES-1 cells, HDL3 are internalized into a particular structure, resulting in the accumulation and concentration of such specific membrane domains. To sum up, a demonstration has been made of the implication of GPI-proteins as well as caveolae in the binding of HDL3 to cells.