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1.
Haematologica ; 102(9): 1587-1593, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28619845

RESUMO

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In the study herein, the pro-apoptotic effect of fluorizoline was assessed in 34 primary samples from patients with chronic lymphocytic leukemia. Fluorizoline induced apoptosis in chronic lymphocytic leukemia cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespective of patients' clinical or genetic features, whereas normal T lymphocytes were less sensitive. Fluorizoline increased the protein levels of the pro-apoptotic B-cell lymphoma 2 family member NOXA in chronic lymphocytic leukemia cells. Furthermore, fluorizoline synergized with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax to induce apoptosis. These results suggest that targeting prohibitins could be a new therapeutic strategy for chronic lymphocytic leukemia.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hidrocarbonetos Fluorados/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas Repressoras/metabolismo , Ribonucleosídeos/farmacologia , Sulfonamidas/farmacologia , Tiazolidinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Aminoimidazol Carboxamida/agonistas , Aminoimidazol Carboxamida/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/agonistas , Sinergismo Farmacológico , Feminino , Humanos , Hidrocarbonetos Fluorados/agonistas , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pirazóis/agonistas , Pirimidinas/agonistas , Ribonucleosídeos/agonistas , Sulfonamidas/agonistas , Tiazolidinas/agonistas , Células Tumorais Cultivadas
2.
Oncotarget ; 7(40): 64987-65000, 2016 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-27542247

RESUMO

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins (PHBs). In this study, the pro-apoptotic effect of fluorizoline was assessed in two cell lines and 21 primary samples from patients with debut of acute myeloid leukemia (AML). Fluorizoline induced apoptosis in AML cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespectively of patients' clinical or genetic features. In addition, fluorizoline inhibited the clonogenic capacity and induced differentiation of AML cells. Fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. These results suggest that targeting PHBs could be a new therapeutic strategy for AML.


Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Apoptose , Benzotiazóis/química , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Repressoras/metabolismo , Células Tumorais Cultivadas , Regulação para Cima
3.
Oncotarget ; 6(39): 41750-65, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26497683

RESUMO

We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa(-/-)/Bim(-/-) MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway.


Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Tiazóis/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HT29 , Células HeLa , Humanos , Células Jurkat , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Regulação para Cima
4.
Angew Chem Int Ed Engl ; 53(38): 10150-4, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25196378

RESUMO

A new class of small molecules, with an unprecedented trifluorothiazoline scaffold, were synthesized and their pro-apoptotic activity was evaluated. With an EC50 in the low micromolar range, these compounds proved to be potent inducers of apoptosis in a broad spectrum of tumor cell lines, regardless of the functional status of p53. Fast structure-activity relationship studies allowed the preparation of the strongest apoptosis-inducing candidate. Using a high performance affinity purification approach, we identified prohibitins 1 and 2, key proteins involved in the maintenance of cell viability, as the targets for these compounds.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Hidrocarbonetos Fluorados/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Tiazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Células Jurkat , Estrutura Molecular , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química
5.
Apoptosis ; 18(8): 1008-16, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23605481

RESUMO

5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 (-/-) /α2 (-/-) MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/efeitos dos fármacos , Fibroblastos/citologia , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Regulação para Cima/efeitos dos fármacos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ribonucleotídeos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética
6.
Epigenomics ; 4(5): 491-501, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23130831

RESUMO

AIM: To analyze the methylation status of 35 tumor suppressor genes using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in chronic lymphocytic leukemia (CLL). MATERIALS & METHODS: The DNA of 37 samples from patients with CLL, six healthy donors, and Jurkat and Ramos cell lines was analyzed by MS-MLPA. RESULTS: Our results confirm that hypermethylation is a common and not randomly distributed event in CLL, and some genes, such as WT1, CDH13, IGSF4/TSLC1, GATA5, DAPK1 and RARB, are hypermethylated in more than 25% of the analyzed samples. Importantly, MS-MLPA also detected hypermethylation of some genes not reported previously in CLL, and their methylation status was confirmed by bisulfite sequencing. CONCLUSION: These results indicate that MS-MLPA is a useful technique for the detection of methylation in CLL samples. Selecting CLL-specific methylation targets in order to generate a CLL-specific MS-MLPA probe set could enhance its usefulness as a tool in studies of risk stratification and guiding the best therapeutic decision.


Assuntos
Metilação de DNA , Epigênese Genética , Genes Supressores de Tumor , Leucemia Linfocítica Crônica de Células B/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Idoso , Idoso de 80 Anos ou mais , Caderinas/genética , Estudos de Casos e Controles , Ilhas de CpG , Feminino , Genes do Tumor de Wilms , Variação Genética , Humanos , Células Jurkat , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/genética , Reprodutibilidade dos Testes
7.
Mol Endocrinol ; 26(9): 1508-20, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22771494

RESUMO

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in different cell types and therefore are widely used to treat a variety of diseases including autoimmune disorders and cancer. This effect is mediated by the GC receptor (GR), a ligand-activated transcription factor that translocates into the nucleus where it modulates transcription of target genes in a promoter-specific manner. Glycogen synthase kinase-3 (GSK3) regulates GR response by genomic and nongenomic mechanisms, although the specific role of each isoform is not well defined. We used GSK3 pharmacological inhibitors and isoform-specific small interfering RNA to evaluate the role of GSK3 in the genomic regulation induced by GC. GSK3 inhibition resulted in the reduction of GC-induced mRNA expression of GC-induced genes such as BIM, HIAP1, and GILZ. Knockdown of GSK3ß but not GSK3α reduced endogenous GILZ induction in response to dexamethasone and GR-dependent reporter gene activity. Chromatin immunoprecipitation experiments revealed that GSK3 inhibition impaired the dexamethasone-mediated binding of GR and RNA polymerase II to endogenous GILZ promoter. These results indicate that GSK3ß is important for GR transactivation activity and that GSK3ß inhibition suppresses GC-stimulated gene expression. Furthermore, we show that genomic regulation by the GR is independent of known GSK3ß phosphorylation sites. We propose that GC-dependent transcriptional activation requires functional GSK3ß signaling and that altered GSK3ß activity influences cell response to GC.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transcrição Genética/genética , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Imunoprecipitação da Cromatina , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Indóis/farmacologia , Maleimidas/farmacologia , Microscopia Confocal , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
8.
Epigenetics ; 6(10): 1228-35, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21931276

RESUMO

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.


Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , Pirróis/farmacologia , Apoptose/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Vorinostat
9.
Apoptosis ; 16(7): 660-70, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21505869

RESUMO

Chemical inhibitors of cyclin-dependent kinase (CDK), like roscovitine, are promising drugs in the context of new cancer therapies. Roscovitine and related compounds, like seliciclib and olomoucine, are effective inducers of apoptosis in many proliferating cells in culture. These compounds are known to activate the intrinsic or mitochondrial pathway of apoptosis. In order to better characterize this intrinsic pathway, a transcriptional analysis was performed using the reverse transcriptase-multiplex ligation-dependent probe amplification procedure (RT-MLPA). In five cell lines, we detected an early and marked reduction of most transcripts, which is consistent with the disruption of transcription that results from the inhibition of CDK7 and CDK9. However, the mRNA of p53-upregulated modulator of apoptosis (PUMA) gene escaped from this transcription inhibition in neuroblastoma cells with a functional p53 protein. The increase of PUMA mRNA was not found in roscovitine-treated cell lines defective in p53, which underwent apoptosis like their p53 proficient counterparts. In addition, in SH-SY5Y cells, sublethal and lethal concentrations of roscovitine produced equivalent increases of PUMA mRNA and protein. In conclusion, the increased expression of PUMA was not associated with apoptosis induction. On the contrary, mRNA and protein depletion of MCL-1 gene correlated the best with cell demise. Moreover, NOXA protein suffered a far minor decrease than MCL-1. Because of the selective neutralization of NOXA by MCL-1, we hypothesize that the disruption of this balance is a critical event in apoptosis induction by roscovitine and related compounds.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Purinas/farmacologia , Transcrição Genética/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Roscovitina , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/metabolismo
10.
Blood ; 116(16): 3023-32, 2010 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-20664053

RESUMO

5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1(-/-) mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa(-/-) or Bim(-/-) mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa(-/-)/Bim(-/-) mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Apoptose/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Ribonucleosídeos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminoimidazol Carboxamida/farmacologia , Animais , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Regulação para Cima/efeitos dos fármacos
12.
Apoptosis ; 15(2): 219-29, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19936928

RESUMO

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in most cell types. In this study we examined the mechanism of aspirin-induced apoptosis in human leukemia cells. We analyzed the role of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPKs) pathways. Furthermore, we studied the changes induced by aspirin in some genes involved in the control of apoptosis at mRNA level, by performing reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), and at protein level by Western blot. Our results show that aspirin induced apoptosis in leukemia Jurkat T cells independently of NF-kappaB. Although aspirin induced p38 MAPK and c-Jun N-terminal kinase activation, selective inhibitors of these kinases did not inhibit aspirin-induced apoptosis. We studied the regulation of Bcl-2 family members in aspirin-induced apoptosis. Aspirin increased the mRNA levels of some pro-apoptotic members, such as BIM, NOXA, BMF or PUMA, but their protein levels did not change. In contrast, aspirin decreased the protein levels of Mcl-1. Interestingly, in the presence of aspirin the protein levels of Noxa remained high. This alteration of the Mcl-1/Noxa balance was also found in other leukemia cell lines and primary chronic lymphocytic leukemia cells (CLL). Furthermore, in CLL cells aspirin induced an increase in the protein levels of Noxa. Knockdown of Noxa or Puma significantly attenuated aspirin-induced apoptosis. These results indicate that aspirin induces apoptosis through alteration of the Mcl-1/ Noxa balance.


Assuntos
Apoptose/efeitos dos fármacos , Aspirina/farmacologia , Leucemia Linfocítica Crônica de Células B/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Citocromos c/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , MAP Quinase Quinase Quinases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas/metabolismo
13.
Haematologica ; 94(12): 1698-707, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19815839

RESUMO

BACKGROUND: The phosphatidylinositol-3-kinase/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia cells. In this study we analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) on the survival of chronic lymphocytic leukemia cells. DESIGN AND METHODS: Using cytometry we studied the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with chronic lymphocytic leukemia and from healthy donors. We studied the changes induced by Akti-1/2 and A-443654 at the mRNA level by performing reverse transcriptase multiplex ligation-dependent probe amplification. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on chronic lymphocytic leukemia cells by western blotting. Moreover, we analyzed the cytotoxic effect of Akt inhibitors in patients' cells with deleted/mutated TP53. RESULTS: Both inhibitors induced apoptosis in chronic lymphocytic leukemia cells in a dose-dependent manner. Moreover, B cells from patients with chronic lymphocytic leukemia were more sensitive to Akt inhibitors than T cells from leukemic patients, and B or T cells from healthy donors. Survival factors for chronic lymphocytic leukemia cells, such as interleukin-4 and stromal cell-derived factor-1alpha, were not able to block the apoptosis induced by either Akt inhibitor. Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. CONCLUSIONS: These results demonstrate that Akt inhibitors induce apoptosis of chronic lymphocytic leukemia cells and might be a new therapeutic option for the treatment of chronic lymphocytic leukemia.


Assuntos
Apoptose/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Benzilaminas/farmacologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Indazóis/farmacologia , Indóis/farmacologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinoxalinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologia , Proteína Supressora de Tumor p53/genética
14.
Br J Haematol ; 142(5): 793-801, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18564355

RESUMO

Chronic lymphocytic leukaemia (CLL) is the commonest form of leukaemia in adults in Western countries. We performed multiplex ligation-dependent probe amplification (MLPA) analysis in 50 CLL patients to identify multiple genomic CLL-specific targets, including genes located at 13q14, 17p13 (TP53), 11q23 (ATM) and chromosome 12, and compared the results with those obtained with fluorescence in situ hybridization (FISH). There was a good correlation between MLPA and FISH results, as most alterations (89%) were detected by both techniques. Only three cases with a low percentage (<25%) of cells carrying the alterations were not detected by MLPA. On the other hand, as MLPA uses multiple probes it identified intragenic or small alterations undetected by FISH in three cases. MLPA also detected alterations in 8q24 (MYC) and 6q25-26. In summary, unlike interphase FISH, MLPA enabled the simultaneous analysis of many samples with automated data processing at a low cost. Therefore, the combination of robust multiplexing and high throughput makes MLPA a useful technique for the analysis of genomic alterations in CLL.


Assuntos
Hibridização in Situ Fluorescente/métodos , Leucemia Linfocítica Crônica de Células B/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Aberrações Cromossômicas , Deleção Cromossômica , Cromossomos Humanos Par 12 , Proteínas de Ligação a DNA , Amplificação de Genes , Dosagem de Genes , Genes p53 , Genômica/métodos , Humanos , Proteínas Serina-Treonina Quinases , Espanha , Proteínas Supressoras de Tumor
15.
Haematologica ; 92(12): 1631-8, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18055986

RESUMO

BACKGROUND AND OBJECTIVES: The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein (18KDa) (TSPO) ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents. Primary chronic lymphocytic leukemia (CLL) cells overexpress TSPO. The aim of this study was to examine the effects of PK11195 on CLL cells. Table 1. Characteristics of the patients with chronic lymphocytic leukemia. DESIGN AND METHODS: Using cytometric analysis, we studied the cytotoxic effects of PK11195 on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Western blot and cytometric analyses were used to study the mitochondrial effects of PK11195 on CLL cells. Moreover, we analyzed the cytotoxic effect of PK11195 in patients' cells with mutated p53 or ATM. RESULTS: PK11195 induces apoptosis and had additive effects with chemotherapeutic drugs in primary CLL cells. Other TSPO ligands such as RO 5-4864 and FGIN-1-27 also induce apoptosis in CLL cells. PK11195 induces mitochondrial depolarization and cytochrome c release upstream of caspase activation, and dithiocyana-tostilbene-2,2- disulfonic acid (DIDS), a voltage-dependent anion channel (VDAC) inhibitor, inhibits PK11195-induced apoptosis, demonstrating a direct involvement of mitochondria. CLL cells and normal B cells are more sensitive than T cells to PK11195-induced apoptosis. Interestingly, PK11195 induced apoptosis in CLL cells irrespective of their p53 or ATM status. INTERPRETATION AND CONCLUSIONS: These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Isoquinolinas/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/metabolismo , Benzodiazepinonas/farmacologia , Inibidores de Caspase , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Citocromos c/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Hipolipemiantes/farmacologia , Ácidos Indolacéticos/farmacologia , Isoquinolinas/uso terapêutico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de GABA/genética , Receptores de GABA/metabolismo , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Canais de Ânion Dependentes de Voltagem/antagonistas & inibidores , Canais de Ânion Dependentes de Voltagem/metabolismo
16.
Exp Hematol ; 34(12): 1663-9, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17157163

RESUMO

OBJECTIVE: Antiapoptotic Bcl-2 is overexpressed in most cases of chronic lymphocytic leukemia (CLL). The inhibition of the antiapoptotic Bcl-2 proteins is an attractive strategy for either restoring normal apoptotic process in cancer cells or making these cells more susceptible to conventional chemotherapy. We studied the effect of Bcl-2 inhibitors on the viability of cells from CLL and other mature B-cell neoplasms. MATERIALS AND METHODS: We studied the cytotoxic effects of four nonpeptidic cell-permeable Bcl-2 inhibitors (HA14-1, antimycin A, GX15-003, and GX15-070) on B cells from patients with CLL, mantle cell lymphoma (MCL), and splenic marginal zone lymphoma (SMZL). Moreover, we analyzed the effect of these inhibitors in combination with fludarabine or chlorambucil. RESULTS: HA14-1 induced apoptosis with an EC50 lower than 50 microM in 26 of the 36 CLL samples analyzed. The mean EC50 for these sensitive patients was 23 +/- 2 microM. Antimycin A induced apoptosis in 13 of the 18 CLL samples analyzed. Both HA14-1 and antimycin A induced cytochrome c release from mitochondria and caspase-3 activation. Moreover, HA14-1 induced apoptosis in peripheral cells from MCL and SMZL. HA14-1 also induced apoptosis in CLL samples with alterations in p53 or ATM. Finally, GX compounds induced apoptosis in B cells from 9 of the 11 CLL samples tested. The combination of either HA14-1, antimycin A, or GX compounds with fludarabine or chlorambucil had additive cytotoxic effects on CLL cells. CONCLUSION: Bcl-2 inhibitors induce apoptosis in CLL cells ex vivo and could be used in CLL as monotherapy or given in combination with current chemotherapy.


Assuntos
Antimicina A/farmacologia , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfoma de Célula do Manto/metabolismo , Nitrilos/farmacologia , Pirróis/farmacologia , Neoplasias Esplênicas/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma de Célula do Manto/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Neoplasias Esplênicas/tratamento farmacológico
17.
J Leukoc Biol ; 80(6): 1473-9, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16940331

RESUMO

Apoptosis of B cell chronic lymphocytic leukemia (B-CLL) cells is regulated by the PI-3K-Akt pathway. In the present work, we have analyzed the mechanisms of Akt phosphorylation in B-CLL cells. Freshly isolated cells present basal Akt phosphorylation, which is PI-3K-dependent, as incubation with the PI-3K inhibitor LY294002 decreased Ser-473 and Thr-308 phosphorylation in most samples analyzed (seven out of 10). In three out of 10 cases, inhibition of protein kinase C (PKC) inhibited basal Akt phosphorylation. Stromal cell-derived factor-1alpha, IL-4, and B cell receptor activation induced PI-3K-dependent Akt phosphorylation. PMA induced the phosphorylation of Akt at Ser-473 and Thr-308 and the phosphorylation of Akt substrates, independently of PI-3K in B-CLL cells. In contrast, PKC-mediated phosphorylation of Akt was PI-3K-dependent in normal B cells. Finally, a specific inhibitor of PKCbeta blocked the phosphorylation and activation of Akt by PMA in B-CLL cells. Taken together, these results suggest a model in which Akt could be activated by two different pathways (PI-3K and PKCbeta) in B-CLL cells.


Assuntos
Apoptose , Leucemia Linfocítica Crônica de Células B/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Carcinógenos/farmacologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Biológicos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C beta , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores de Citocinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas
18.
Blood ; 107(10): 4109-14, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16439685

RESUMO

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. Several drugs currently used in the therapy of B-CLL act, at least partially, through activation of the p53 pathway. Recently, nongenotoxic small-molecule activators of p53, the nutlins, have been developed that inhibit p53-MDM2 binding. We have investigated the antitumor potential of nutlin-3 in B-CLL and find that it can activate the p53 pathway and effectively induce apoptosis in cells with wild-type p53, including cells with dysfunctional ataxia telangiectasia mutated, but not mutant p53. Nutlin-3 stabilized p53 and induced p53 target genes, including MDM2, p21(CIP1), PUMA, BAX, PIG3, and WIG1. Nutlin-3 synergized with the genotoxic drugs doxorubicin, chlorambucil, and fludarabine, but not with acadesine, which induces p53-independent apoptosis. Normal human T cells showed lower sensitivity to nutlin-3 than B-CLL cells and no synergism with the genotoxic drugs. These results suggest that MDM2 antagonists alone or in combination with chemotherapeutic drugs may offer a new treatment option for B-CLL.


Assuntos
Antineoplásicos/farmacologia , Linfoma de Células B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Antígenos CD , Apoptose , Antígenos CD5 , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia
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