RESUMO
PURPOSE: Over a range roughly C5-C6, sopranos usually tune their first vocal tract resonance (R1) to the fundamental frequency (fo) of the note sung: R1:fo tuning. Those who sing well above C6 usually adjust their second vocal tract resonance (R2) and use R2:fo tuning. This study investigated these questions: Can singers quickly learn R2:fo tuning when given suitable feedback? Can they subsequently use this tuning without feedback? And finally, if so, does this assist their singing in the high range? METHODS: New computer software for the technique of resonance estimation by broadband excitation at the lips was used to provide real-time visual feedback on fo and vocal tract resonances. Eight sopranos participated. In a one-hour session, they practised adjusting R2 whilst miming (i.e. without phonating), and then during singing. RESULTS: Six sopranos learned to tune R2 over a range of several semi-tones, when feedback was present. This achievement did not immediately extend their singing range. When the feedback was removed, two sopranos spontaneously used R2:fo tuning at the top of their range above C6. CONCLUSIONS: With only one hour of training, singers can learn to adjust their vocal tract shape for R2:fo tuning when provided with visual feedback. One additional participant who spent considerable time with the software, acquired greater skill at R2:fo tuning and was able to extend her singing range. A simple version of the hardware used can be assembled using basic equipment and the software is available online.
Assuntos
Canto , Voz , Retroalimentação Sensorial , Feminino , Humanos , Vibração , Qualidade da VozRESUMO
OBJECTIVE: Triptolide has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro. MATERIALS AND METHODS: The THP1 cell line and primary acute myeloid leukemia (AML) cells were cultured with triptolide alone or in association with AraC or idarubicin in increasing concentrations. Apoptosis was measured by flow cytometry using DiOC6(3) for the cell line and fluorescein isothiocyanateAnnexin-V and CD45 labeling for fresh blast cells. Protein expression was measured by Western blot. Cell cycle distribution of apoptotic cells was measured by flow cytometry. RESULTS: A synergistic effect was observed when triptolide was added to idarubicin or to AraC to induce apoptosis of THP-1 leukemic cells. The triptolide/AraC association was also investigated in vitro on primary blast cells from 25 AML patients. This combination induced significantly higher percentages of apoptosis vs treatment with each drug separately (p<0.005). The IkappaB and X-linked inhibitor of apoptosis protein contents, which were altered by triptolide in idarubicin-treated cells, were not modified in AraC-treated cells. The association of AraC with triptolide increased the number of cells blocked in the S phase and most underwent apoptosis. CONCLUSION: These results suggest that, by modifying the cell cycle kinetics, AraC sensitizes AML cells to apoptosis induced by low concentration triptolide. The in vitro proapoptotic effect of triptolide associated with the antiproliferative activity of AraC warrants further clinical investigation for treatment of AML patients, especially elderly patients for whom low-dose AraC treatment could be improved by the addition of triptolide.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Citarabina/farmacologia , Diterpenos/farmacologia , Idarubicina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Fenantrenos/farmacologia , Anexina A5/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Citarabina/agonistas , Citarabina/uso terapêutico , Diterpenos/agonistas , Diterpenos/uso terapêutico , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Compostos de Epóxi/agonistas , Compostos de Epóxi/farmacologia , Compostos de Epóxi/uso terapêutico , Humanos , Proteínas I-kappa B/metabolismo , Idarubicina/agonistas , Idarubicina/uso terapêutico , Leucemia Mieloide Aguda/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Fenantrenos/agonistas , Fenantrenos/uso terapêutico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
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.
Assuntos
Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Niacinamida/análogos & derivados , Sulfonamidas/farmacologia , Doença Aguda , Compostos de Anilina/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Células HL-60 , Humanos , Imuno-Histoquímica , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Niacinamida/administração & dosagem , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/biossíntese , Células-Tronco Neoplásicas/efeitos dos fármacos , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Antígenos CD34/análise , Proteínas Reguladoras de Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2 , Benzamidas , Linhagem Celular Tumoral/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Células-Tronco Hematopoéticas/enzimologia , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Mesilato de Imatinib , Células K562/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Proteínas Mitocondriais/fisiologia , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/enzimologia , Proteínas Proto-Oncogênicas/fisiologia , Serina Endopeptidases/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Glioblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Caspases/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Glioblastoma/ultraestrutura , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
It is an important challenge to better understand the mechanisms of tyrosine kinase inhibitors-induced apoptosis in CML cells. Thus, we have investigated how this apoptosis can be modulated by extracellular factors. Apoptosis induced by imatinib and nilotinib was determined in BCR-ABL expressing cell lines and primary CML CD34+ cells. Both molecules induced apoptosis of BCR-ABL expressing cells. This apoptosis was inhibited by protein synthesis inhibition in both K562 and CML CD34+ cells. In K562, 80% inhibition of the BCR-ABL auto-phosphorylation by either imatinib or nilotinib induced a two fold increase in Bim-EL expression and induction of apoptosis in 48 h. Bim accumulation preceded apoptosis induction which was completely abolished by depletion in Bim using shRNA. However, the anti-proliferative effect of imatinib was preserved in Bim-depleted cells. When K562 cells were cultured in a cytokine containing medium, the pro-apoptotic effect of nilotinib was decreased by 68% and this was related to a decrease in Bim-EL dephosphorylation and accumulation. Similarly, the presence of a combination of cytokines inhibited 88% of NIL- and 39% of IMA-induced apoptosis in primary CML CD34+ cells. In conclusion, both nilotinib and imatinib induce apoptosis through Bim accumulation independently of cell cycle arrest. However, the pro-apoptotic effect of both molecules can be attenuated by the presence of cytokines and growth factors, particularly concerning nilotinib. Thus BCR-ABL inhibition restores the cytokine dependence but is not sufficient to induce apoptosis when other signaling pathways are activated.