RESUMO
BACKGROUND: Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO) is assessed as a therapy for mesothelioma. METHODS: Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and ß-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. RESULTS: eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. CONCLUSION: 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity.
Assuntos
Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4F em Eucariotos/antagonistas & inibidores , Mesotelioma/genética , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Antineoplásicos/farmacologia , Apoptose , Contagem de Células , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Expressão Gênica , Glutamatos/farmacologia , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Mesotelioma/metabolismo , Mesotelioma/patologia , Terapia de Alvo Molecular , Oligonucleotídeos Antissenso/metabolismo , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Pemetrexede , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , GencitabinaRESUMO
Cap-mediated translation is the default mechanism for the synthesis of proteins in eukaryotic cells. Increasingly, malignant cells have been found to have deregulation of this process. Return of normal translational control is associated with loss of tumorigenic potential in pre-clinical models. Currently, a variety of novel therapeutics are in development targeting this mechanism as a treatment for cancer.
Assuntos
Fator de Iniciação 4E em Eucariotos/fisiologia , Neoplasias/terapia , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Animais , Humanos , Neoplasias/genética , Neoplasias/patologia , Capuzes de RNARESUMO
INTRODUCTION: Mutations in Ras family genes are rare in malignant mesothelioma. The role of activation of the Ras signaling pathway in the pathogenesis of mesothelioma is not clear. METHODS: We studied the activation status of the Ras pathway and the status of other Ras-associated kinases in a panel of human mesothelioma cell lines. In addition, we tested the effect of inhibition of several kinase pathways on mesothelioma cell proliferation. The potential role of kinase signaling on the regulation of cap-dependent translation was also studied. RESULTS: In general, Ras-guanosine triphosphate (GTP) was higher in mesothelioma cell lines when compared with a nontransformed mesothelial cell line (LP9). Furthermore, known Ras effectors such as extracellular-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase were found to be active in most of the mesothelioma cell lines tested. Exposure to specific inhibitors of extracellular-regulated kinase 1/2 (U0126) and c-Jun N-terminal kinase (SP600125) significantly decreased the proliferation of H2596 and H2373 cells compared with mock-treated cells. SP600125-mediated c-Jun N-terminal kinase inhibition, but not extracellular-regulated kinase 1/2 inhibition, resulted in a decrease in phosphorylation of 4E-BP1, consequently decreasing cap-dependent activation. CONCLUSIONS: These experiments provide a rationale for targeting Ras and associated signaling pathways in mesothelioma and also suggest cap-dependent translation as one mechanism by which Ras induces proliferation in this disease.