RESUMO
Advanced mesothelioma is considered an incurable disease and new treatment strategies are needed. Previous studies have demonstrated that mitochondrial antioxidant defense proteins and the cell cycle may contribute to mesothelioma growth, and that the inhibition of these pathways may be effective against this cancer. We demonstrated that the antioxidant defense inhibitor auranofin and the cyclin-dependent kinase 4/6 inhibitor palbociclib could decrease mesothelioma cell proliferation alone or in combination. In addition, we determined the effects of these compounds on colony growth, cell cycle progression, and the expression of key antioxidant defense and cell cycle proteins. Auranofin and palbociclib were effective in decreasing cell growth and inhibiting the above-described activity across all assays. Further study of this drug combination will elucidate the contribution of these pathways to mesothelioma activity and may reveal a new treatment strategy.
Assuntos
Mesotelioma Maligno , Mesotelioma , Humanos , Antioxidantes/farmacologia , Auranofina/farmacologia , Mesotelioma/tratamento farmacológico , Proliferação de Células , Quinase 4 Dependente de Ciclina , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular TumoralRESUMO
Deregulation of cap-dependent translation has been implicated in the malignant transformation of numerous human tissues. 4EGI-1, a novel small-molecule inhibitor of cap-dependent translation, disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex. The effects of 4EGI-1-mediated inhibition of translation initiation in malignant pleural mesothelioma (MPM) were examined. 4EGI-1 preferentially inhibited cell viability and induced apoptosis in MPM cells compared to normal mesothelial (LP9) cells. This effect was associated with hypophosphorylation of 4E-binding protein 1 (4E-BP1) and decreased protein levels of the cancer-related genes, c-myc and osteopontin. 4EGI-1 showed enhanced cytotoxicity in combination with pemetrexed or gemcitabine. Translatome-wide polysome microarray analysis revealed a large cohort of genes that were translationally regulated upon treatment with 4EGI-1. The 4EGI-1-regulated translatome was negatively correlated to a previously published translatome regulated by eIF4E overexpression in human mammary epithelial cells, which is in agreement with the notion that 4EGI-1 inhibits the eIF4F complex. These data indicate that inhibition of the eIF4F complex by 4EGI-1 or similar translation inhibitors could be a strategy for treating mesothelioma. Genome wide translational profiling identified a large cohort of promising target genes that should be further evaluated for their potential significance in the treatment of MPM.
Assuntos
Genoma Humano , Hidrazonas/farmacologia , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Capuzes de RNA/metabolismo , Tiazóis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/deficiência , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mesotelioma Maligno , Pemetrexede/farmacologia , Pemetrexede/uso terapêutico , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Neoplasias Pleurais/patologia , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica , Proteoma/metabolismo , Reprodutibilidade dos Testes , GencitabinaRESUMO
Gulf War illness (GWI) is a chronic disease of unknown etiology affecting over 200,000 veterans with symptoms including neurocognitive problems. We previously demonstrated GWI serum toxicity on neural cell cultures manifested by compromised neural network function, decreased cell spreading, and enhanced cell apoptosis. These patients lacked six human leukocyte antigen (HLA) class II alleles, resulting in an inability to form antibodies. Therefore, we hypothesized that GWI patients have vaccine-derived, persistent pathogens, which contribute to the development of the disease. Here, we examined whether individual vaccines were toxic in cultured N2A cells. Moreover, we used antibodies against each of the 20 vaccines administered to Gulf War (GW) veterans, to examine the effects of these antibodies on cell spreading and apoptosis in N2A cells. Antibodies against cholera toxin, hepatitis B, hemagglutinin H1N1, H3N2, and B from influenza A and B strains, measles, and Salmonella Typhi polysaccharide Vi had a remarkable protective effect on both cell spreading and apoptosis, whereas none of the other antibodies administered to GW veterans had an effect. The in vitro observed adverse effects of GWI serum may be due in part to vaccine-derived pathogens, antibodies against which had a protective effect in N2A cell cultures.
RESUMO
Protective antigen (PA) 63 (PA63) is a protein derived from the PA83 component contained in the anthrax vaccine. The anthrax vaccine ("Biothrax") was administered together with other vaccines to Gulf War veterans, about 35% of whom later developed a multisymptom disease (Gulf War Illness [GWI]), with prominent neurological/cognitive/mood symptoms, among others. The disease has been traditionally attributed to exposures to toxic chemicals during the war but other factors could be involved, including vaccines received. Of these, the anthrax vaccine is the most toxic. Here, we assessed directly the PA63 toxin's harmful effects on cultured neuroblastoma 2A (N2A) cells with respect to cell spreading, process formation, apoptosis, and integrity of cell membrane, cytoskeleton, and mitochondria. We found that, when added in N2A cultures, PA63 toxin led to decreased cell spreading and cell aggregation, leading to apoptosis. The mechanisms of PA63-induced cell damage included compromised cell membrane permeability indicated by enhanced access of propidium iodide in cells. In addition, signaling pathways leading to organization of N2A cytoskeleton were negatively affected, as both actin and microtubular networks were compromised. Finally, the mitochondrial membrane potential was impaired in specific assays. Altogether, these alterations led to apoptosis as a collective toxic effect of PA63 which was substantially reduced by the concomitant addition of specific antibodies against PA63.
RESUMO
Hyperactivation of eIF4F-mediated translation occurs in many if not all cancers. As a consequence, cancer cells aberrantly enhance expression of malignancy-related proteins that are involved in cell cycle progression, angiogenesis, growth, and proliferation. With this in mind eIF4F is a promising molecular target for therapeutics that counteract pathological eIF4F activity. Here we used 4EGI-1, a small-molecule inhibitor of cap-mediated translation that disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex to treat non-small cell lung cancer (NSCLC). Treatment of cells with 4EGI-1 reduced cell proliferation, decreased cap-dependent complex formation, induced apoptosis, enhanced sensitivity to gemcitabine, and altered global cellular translation. Suppression of cap-dependent translation by 4EGI-1 resulted in diminished expression of oncogenic proteins c-Myc, Bcl-2, cyclin D1, and survivin, whereas ß-actin expression was left unchanged. In light of these results, small-molecule inhibitors like 4EGI-1 alone or with chemotherapy should be further evaluated in the treatment of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Hidrazonas/metabolismo , Neoplasias Pulmonares/genética , Tiazóis/metabolismo , Apoptose , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Humanos , Neoplasias Pulmonares/patologiaRESUMO
UNLABELLED: Disruption of the 9p21 locus is common in mesothelioma and leads to loss of both the p16INK4a and the p14ARF gene products. This study tested the hypothesis that reexpression of p16INK4a carried out using the TAT delivery system that carries the protein transduction domain of the HIV TAT will result in mesothelioma cell death. MATERIALS AND METHODS: A synthetic TATp16INK4a peptide and a charge matched control were transduced into mesothelioma cells in vitro and in vivo. Cells were assayed for Cdk4 inhibition, cell cycle arrest, and cell death. RESULTS: Treatment of mesothelioma cells with TATp16INK4a for 48 hours resulted in cell death. Apoptosis and G1 cell cycle arrest was also observed. Following transduction of cells with TATp16INK4a there was complete but transient hypophosphorylation of pRb. Similar effects were observed in mesothelioma xenografts. CONCLUSION: Therapeutic strategies which introduce either TATp16INK4a peptide, or small molecule mimetic, could be an effective strategy for mesothelioma treatment.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Inibidor p16 de Quinase Dependente de Ciclina/farmacologia , Mesotelioma/terapia , Fragmentos de Peptídeos/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Sequência de Aminoácidos , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/genética , Humanos , Mesotelioma/tratamento farmacológico , Mesotelioma/genética , Mesotelioma/patologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto , Produtos do Gene tat do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Aberrant hyperactivation of the cap-dependent protein synthesis apparatus has been documented in a wide range of solid tumors, including epithelial carcinomas, but causal linkage has only been established in breast carcinoma. In this report, we sought to determine if targeted disruption of deregulated cap-dependent translation abrogates tumorigenicity and enhances cell death in non-small cell lung cancer (NSCLC). NSCLC cell lines were stably transfected with either wild-type 4E-BP1 (HA-4E-BP1) or the dominant-active mutant 4E-BP1(A37/A46) (HA-TTAA). Transfected NSCLC cells with enhanced translational repression showed pronounced cell death following treatment with gemcitabine. In addition, transfected HA-TTAA and HA-4E-BP1wt proteins suppressed growth in a cloning efficiency assay. NSCLC cells transduced with HA-TTAA also show decreased tumorigenicity in xenograft models. Xenograft tumors expressing HA-TTAA were significantly smaller than control tumors. This work shows that hyperactivation of the translational machinery is necessary for maintenance of the malignant phenotype in NSCLC, identifies the molecular strategy used to activate translation, and supports the development of lung cancer therapies that directly target the cap-dependent translation initiation complex.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Biossíntese de Proteínas , Capuzes de RNA , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Fosfoproteínas/genética , Transfecção , GencitabinaRESUMO
STUDY OBJECTIVE: We investigated the gene expression profiles of malignant pleural mesothelioma (MPM) specimens to identify novel genes that are potentially involved in the oncogenic transformation of human pleural cells. DESIGN: Complementary DNA (cDNA) microarray transcriptional profiling studies of 10 MPM cell lines and 4 MPM primary tumor specimens were performed using hierarchic clustering. To confirm microarray data, we used real-time polymerase chain reaction and immunoblotting. RESULTS: Cluster analysis differentiated among epithelial (E), sarcomatoid, and biphasic MPM variants. Expression profiling identified common overexpressed or underexpressed genes in MPM. Notably, matriptase messenger RNA was found to be overexpressed by 826-fold in E MPM, with protein expression subsequently confirmed by immunoblot analysis. This recently characterized trypsin-like serine protease has been implicated in tumor invasion and metastasis of E-derived cancers, but has not been described until now in MPM. We also identified other novel genes, such as insulin-like growth factor binding protein 5 and a cDNA clone similar to proteolipid MAL2. CONCLUSIONS: Thus, further large-scale profiling of MPM may elucidate previously unrecognized molecular mechanisms by identifying novel genes that are involved in malignant transformation. Our study has now found matriptase to be one of these mesothelioma-associated genes, with potential pathogenic and therapeutic significance.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mesotelioma/enzimologia , Mesotelioma/genética , Neoplasias Pleurais/enzimologia , Neoplasias Pleurais/genética , Serina Endopeptidases/biossíntese , Linhagem Celular Tumoral , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
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
INTRODUCTION: The molecular mechanisms of oncogenesis in mesothelioma involve the loss of negative regulators of cell growth including p16INK4a. Absence of expression of the p16INK4a gene product is exhibited in virtually all mesothelioma tumors and cell lines examined to date. Loss of p16INK4a expression has also been frequently observed in more common neoplasms such as lung cancer as well. In a wide variety of these malignancies, including lung cancer, p16INK4a expression is known to be inactivated by hypermethylation of the first exon. This project (CALGB 159904) intended to test the hypothesis that in mesothelioma loss of p16INK4a via methylation would correlate with response to the cytidine analog and methylation inhibitor dihydro-5-azacytidine (DHAC). METHODS: Using tissue samples from CALGB 8833 and 9031, two clinical studies which used DHAC based therapy in mesothelioma, this study tested the hypothesis that tumors possessing methylation of p16INK4a would have a better response and survival following DHAC treatment than their nonmethylated counterparts. RESULTS: Methylation of p16INK4a was identified in 4 of the 20 specimens. Although there was a trend towards improved survival the result was not statistically significant. CONCLUSIONS: There was no significant correlation between the presence of p16INK4a methylation and response to DHAC therapy or overall survival.