RESUMO
The oncogenic epidermal growth factor receptor (EGFR) pathway triggers downstream phosphatidylinositol 3-kinase (PI3K)/RAS-mediated signaling cascades. In transgenic mice, glioblastoma cannot develop on single but only on simultaneous activation of the EGFR signaling mediators RAS and AKT. However, complete blockade of EGFR activation does not result in apoptosis in human glioblastoma cells, suggesting additional cross-talk between downstream pathways. Based on these observations, we investigated combination therapies using protein kinase inhibitors against EGFR, platelet-derived growth factor receptor, and mammalian target of rapamycin, assessing glioblastoma cell survival. Clinically relevant doses of AEE788, Gleevec (imatinib), and RAD001 (everolimus), alone or in combinations, did not induce glioblastoma cell apoptosis. In contrast, simultaneous inactivation of the EGFR downstream targets mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and PI3K by U0126 and wortmannin triggered rapid tumor cell death. Blocking EGFR with AEE788 in combination with sublethal concentrations of the microtubule stabilizer patupilone also induced apoptosis and reduced cell proliferation in glioblastoma cells, accompanied by reduced AKT and ERK activity. These data underline the critical role of the PI3K/AKT and the RAS/RAF/mitogen-activated protein/ERK kinase/ERK signaling cascades in the cell-intrinsic survival program of sensitive glioblastoma cell lines. We conclude that drug combinations, which down-regulate both ERK and protein kinase B/AKT activity, may prove effective in overcoming cell resistance in a subgroup of glioblastoma.
Assuntos
Apoptose/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Benzamidas , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Epotilonas/farmacologia , Receptores ErbB/metabolismo , Everolimo , Glioblastoma/metabolismo , Humanos , Mesilato de Imatinib , Imunossupressores/farmacologia , MAP Quinase Quinase 1/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ésteres de Forbol/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Purinas/farmacologia , Pirimidinas/farmacologia , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The nuclear receptor PPARgamma is implicated in the control of cell proliferation and apoptosis. However, the molecular mechanisms by which it controls these processes remain largely elusive. We show here that PPARgamma activation in the presence of the retinoblastoma protein (RB) results in the arrest of cells at the G1 phase of the cell cycle, whereas in the absence of RB, cells accumulate in G2/M, endoreduplicate, and undergo apoptosis. Through the use of HDAC inhibitors and coimmunoprecipitations, we furthermore demonstrate that the effects of RB on PPARgamma-mediated control of the cell cycle and apoptosis depend on the recruitment of histone deacetylase 3 (HDAC3) to PPARgamma. In combination, these data hence demonstrate that the effects of PPARgamma on cell proliferation and apoptosis are dependent on the presence of an RB-HDAC3 complex.
Assuntos
Apoptose , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína do Retinoblastoma/metabolismo , Tiazolidinedionas , Fatores de Transcrição/metabolismo , Células 3T3 , Adipócitos/metabolismo , Animais , Northern Blotting , Western Blotting , Bromodesoxiuridina/farmacologia , Ciclo Celular , Divisão Celular , Núcleo Celular/metabolismo , Citometria de Fluxo , Fase G1 , Fase G2 , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Marcação In Situ das Extremidades Cortadas , Luciferases/metabolismo , Camundongos , Microscopia de Fluorescência , Mitose , Neoplasias/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , RNA Interferente Pequeno/metabolismo , Rosiglitazona , Tiazóis/farmacologia , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
The retinoblastoma protein (RB) has previously been shown to facilitate adipocyte differentiation by inducing cell cycle arrest and enhancing the transactivation by the adipogenic CCAAT/enhancer binding proteins (C/EBP). We show here that the peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor pivotal for adipogenesis, promotes adipocyte differentiation more efficiently in the absence of RB. PPARgamma and RB were shown to coimmunoprecipitate, and this PPARgamma-RB complex also contains the histone deacetylase HDAC3, thereby attenuating PPARgamma's capacity to drive gene expression and adipocyte differentiation. Dissociation of the PPARgamma-RB-HDAC3 complex by RB phosphorylation or by inhibition of HDAC activity stimulates adipocyte differentiation. These observations underscore an important function of both RB and HDAC3 in fine-tuning PPARgamma activity and adipocyte differentiation.