RESUMO
Prostate cancer (PCa) relies in part on AR-signaling for disease development and progression. Earlier, we developed drug candidate galeterone, which advanced through phase 2-clinical trials in treating castration-resistant PCa (CRPC). Subsequently, we designed, synthesized, and evaluated next-generation galeterone-analogs including VNPP433-3ß which is potently efficacious against pre-clinical models of PCa. This study describes the mechanism of action of VNPP433-3ß that promotes degradation of full-length AR (fAR) and its splice variant AR-V7 besides depleting MNK1/2 in in vitro and in vivo CRPC models that stably overexpresses fAR. VNPP433-3ß directly engages AR within the cell and promotes proteasomal degradation of fAR and its splice variant AR-V7 by enhancing the interaction of AR with E3 ligases MDM2/CHIP but disrupting AR-HSP90 binding. Next, VNPP433-3ß decreases phosphorylation of 4EBP1 and abates binding of eIF4E and eIF4G to 5' cap of mRNA by depleting MNK1/2 with consequent depletion of phosphorylated eIF4E. Finally, RNA-seq demonstrates modulation of multiple pathways that synergistically contribute to PCa inhibition. Therefore, VNPP433-3ß exerts its antitumor effect by imposing 1) transcriptional regulation of AR and AR-responsive oncogenes 2) translational regulation by disrupting mRNA-5'cap-dependent translation initiation, 3) reducing AR half-life through enhanced proteasomal degradation in vitro and AR-overexpressing tumor xenografts in vivo.
Assuntos
Antagonistas de Receptores de Andrógenos , Neoplasias de Próstata Resistentes à Castração , Humanos , Masculino , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/metabolismo , RNA Mensageiro/uso terapêuticoRESUMO
BACKGROUND: Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. METHODS: Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. RESULTS: Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. CONCLUSIONS: Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.
Assuntos
Acetanilidas/farmacologia , Azepinas/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: Medulloblastoma, the most common pediatric brain malignancy, has Sonic Hedgehog (SHH) and group 3 (Myc driven) subtypes that are associated with the activity of eukaryotic initiation factor 4E (eIF4E), a critical mediator of translation, and enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and master regulator of transcription. Recent drug repurposing efforts in multiple solid and hematologic malignancies have demonstrated that eIF4E and EZH2 are both pharmacologically inhibited by the FDA-approved antiviral drug ribavirin. Given the molecular overlap between medulloblastoma biology and known ribavirin activity, the authors investigated the preclinical efficacy of repurposing ribavirin as a targeted therapeutic in cell and animal models of medulloblastoma. METHODS: Multiple in vitro assays were performed using human ONS-76 (a primitive SHH model) and D425 (an aggressive group 3 model) cells. The impacts of ribavirin on cellular growth, death, migration, and invasion were quantified using proliferation and Cell Counting Kit-8 (CCK-8) assays, flow cytometry with annexin V (AnnV) staining, scratch wound assays, and Matrigel invasion chambers, respectively. Survival following daily ribavirin treatment (100 mg/kg) was assessed in vivo in immunodeficient mice intracranially implanted with D425 cells. RESULTS: Compared to controls, ribavirin treatment led to a significant reduction in medulloblastoma cell growth (ONS-76 proliferation assay, p = 0.0001; D425 CCK-8 assay, p < 0.0001) and a significant increase in cell death (flow cytometry for AnnV, ONS-76, p = 0.0010; D425, p = 0.0284). In ONS-76 cells, compared to controls, ribavirin significantly decreased cell migration and invasion (Matrigel invasion chamber assay, p = 0.0012). In vivo, ribavirin significantly extended survival in an aggressive group 3 medulloblastoma mouse model compared to vehicle-treated controls (p = 0.0004). CONCLUSIONS: The authors demonstrate that ribavirin, a clinically used drug known to inhibit eIF4E and EZH2, has significant antitumor effects in multiple preclinical models of medulloblastoma, including an aggressive group 3 animal model. Ribavirin may represent a promising targeted therapeutic in medulloblastoma.
Assuntos
Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Ribavirina/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for â¼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.This article is highlighted in the In This Issue feature, p. 995.
Assuntos
Neoplasias Colorretais/genética , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Inibidores de MTOR/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Animais , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Fragile X syndrome (FXS) is the leading monogenic cause of autism spectrum disorders (ASD). Trinucleotide repeat expansions in FMR1 abolish FMRP expression, leading to hyperactivation of ERK and mTOR signaling upstream of mRNA translation. Here we show that metformin, the most widely used drug for type 2 diabetes, rescues core phenotypes in Fmr1-/y mice and selectively normalizes ERK signaling, eIF4E phosphorylation and the expression of MMP-9. Thus, metformin is a potential FXS therapeutic.
Assuntos
Comportamento Animal/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Hipoglicemiantes/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metformina/farmacologia , Comportamento Social , Animais , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/fisiopatologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Expansão das Repetições de TrinucleotídeosRESUMO
The aim of the present study was to demonstrate that ribavirin, a known inhibitor of eIF4E and inosine 5'-phosphate dehydrogenase (IMPDH), also inhibits histone methyltransferase zeste homolog 2 (EZH2). A computational searching revealed that ribavirin has a high structural similarity to 3-deazaneplanocin A (DZNep). The growth inhibitory effects of ribavirin as well as its effects upon epigenetic enzymes were evaluated in various cancer cell lines. siRNA assays were used to downregulate eIF4E, EZH2 and IMPDH to determine the contribution of these targets to the growth inhibitory effects of ribavirin. Ribavirin decreased EZH2 expression, inhibited histone methyltransferase activity and decreased H3K27 trimethylation. Ribavirin induced variable growth inhibition in a number of cell lines and downregulation of the targets, EZH2, eIF4E and IMPDH1 and 2 by siRNA led to comparable growth inhibition while no significant further reduction in viability was observed when siRNA transfected cells were treated with ribavirin. The results showed that ribavirin inhibits these cancer targets and should thus be studied for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Proliferação de Células/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , IMP Desidrogenase/efeitos dos fármacos , Neoplasias/genética , Complexo Repressor Polycomb 2/efeitos dos fármacos , Ribavirina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Simulação por Computador , Reposicionamento de Medicamentos , Proteína Potenciadora do Homólogo 2 de Zeste , Fator de Iniciação 4E em Eucariotos/genética , Células HeLa , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/efeitos dos fármacos , Humanos , IMP Desidrogenase/genética , Células MCF-7 , Neoplasias/metabolismo , Complexo Repressor Polycomb 2/genética , RNA Interferente PequenoRESUMO
In eukaryotic cells, the rate-limiting component for cap-dependent mRNA translation is the translation initiation factor eIF4E. eIF4E is overexpressed in a variety of human malignancies, but whether it has a role in melanoma remains obscure. We hypothesized that eIF4E promotes melanoma cell proliferation and facilitates the development of acquired resistance to the BRAF inhibitor vemurafenib. We show that eIF4E is overexpressed in a panel of melanoma cell lines, compared with immortalized melanocytes. Knockdown of eIF4E significantly repressed the proliferation of a subset of melanoma cell lines. Moreover, in BRAF(V600E) melanoma cell lines, vemurafenib inhibits 4E-BP1 phosphorylation, thus promoting its binding to eIF4E. Cap-binding and polysome profiling analysis confirmed that vemurafenib stabilizes the eIF4E-4E-BP1 association and blocks mRNA translation, respectively. Conversely, in cells with acquired resistance to vemurafenib, there is an increased dependence on eIF4E for survival; 4E-BP1 is highly phosphorylated and thus eIF4E-4E-BP1 associations are impeded. Moreover, increasing eIF4E activity by silencing 4E-BP1/2 renders vemurafenib-responsive cells more resistant to BRAF inhibition. In conclusion, these data suggest that therapeutically targeting eIF4E may be a viable means of inhibiting melanoma cell proliferation and overcoming vemurafenib resistance.
Assuntos
Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/fisiologia , Indóis/farmacologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Sulfonamidas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/genética , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas B-raf , RNA Interferente Pequeno/farmacologia , VemurafenibRESUMO
Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Recently, HIF-1α has been recognized as the critical target of cardiac glycosides for cancer therapy, but the molecular mechanism of cardiac glycosides' inhibition of HIF-1α is still poorly understood. In the present study, we observed that neither HIF-1α mRNA levels nor HIF-1α protein degradation are affected by Ouabain. However, Ouabain was found to be associated with the regulation of HIF-1α translation. Basing on in silico, in vitro and ex vivo models of translation processing, further studies revealed that eIF4E plays a critical role in the inhibitory effect of Ouabain on HIF-1α protein synthesis, rather than mTORC1, eIF2α signaling or Na(+)/K(+)-ATPase inhibition. Mechanistically, Ouabain directly binds eIF4E, disrupts eIF4E/eIF4G association (200 µM, Inhibit rate =61 ± 3%) but not the eIF4E/mRNA complex formation (200 µM, Inhibit rate =18 ± 5%) both in vitro and in cells, thereby inhibiting the intracellular cap-dependent translation. The association between Ouabain and eIF4E not only raises the hope of using cardiac glycosides for cancer therapeutics more rational, but also offers a pharmacologic means for developing novel anti-cancer HIF-1α antagonists.
Assuntos
Fator de Iniciação 4E em Eucariotos/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Ouabaína/farmacologia , Sequência de Bases , Primers do DNA , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genéticaRESUMO
Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. Here, we evaluated the role of PFD in regulation of the extracellular matrix. In mouse mesangial cells, PFD decreased TGF-beta promoter activity, reduced TGF-beta protein secretion, and inhibited TGF-beta-induced Smad2-phosphorylation, 3TP-lux promoter activity, and generation of reactive oxygen species. To explore the therapeutic potential of PFD, we administered PFD to 17-wk-old db/db mice for 4 wk. PFD treatment significantly reduced mesangial matrix expansion and expression of renal matrix genes but did not affect albuminuria. Using liquid chromatography with subsequent electrospray ionization tandem mass spectrometry, we identified 21 proteins unique to PFD-treated diabetic kidneys. Analysis of gene ontology and protein-protein interactions of these proteins suggested that PFD may regulate RNA processing. Immunoblotting demonstrated that PFD promotes dosage-dependent dephosphorylation of eukaryotic initiation factor, potentially inhibiting translation of mRNA. In conclusion, PFD is renoprotective in diabetic kidney disease and may exert its antifibrotic effects, in part, via inhibiting RNA processing.
Assuntos
Antineoplásicos/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Piridonas/uso terapêutico , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Albuminúria/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Proteômica , Piridonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Hypoxia-inducible factor-1 (HIF-1) is the central mediator of cellular responses to low oxygen and vital to many aspects of cancer biology. In a search for HIF-1 inhibitors, we identified a quassinoid 6alpha-tigloyloxychaparrinone (TCN) as an inhibitor of HIF-1 activation from Ailantus altissima. We here demonstrated the effect of TCN on HIF-1 activation induced by hypoxia or CoCl2. TCN showed the potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1alpha protein dose-dependently, whereas it did not affect the expressions of HIF-1beta and topoisomerase-I. Furthermore, TCN prevented hypoxia-induced expression of HIF-1 target genes for vascular endothelial growth factor (VEGF) and erythropoietin. Further analysis revealed that TCN strongly inhibited HIF-1alpha protein synthesis, without affecting the expression level of HIF-1alpha mRNA or degradation of HIF-1alpha protein. Moreover, the levels of phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), mitogen-activated protein (MAP) kinase-interacting protein kinase-1 (MNK1) and eukaryotic initiation factor 4E (eIF4E) were significantly suppressed by the treatment of TCN, without changing the total levels of these proteins. Our data suggested that TCN may exhibit anticancer activity by inhibiting HIF-1alpha translation through the inhibition of eIF4E phosphorylation pathway and thus provide a novel mechanism for the anticancer activity of quassinoids. TCN could be a new HIF-1-targeted anticancer agent and be effective on mammalian target of rapamycin (mTOR)-targeted cancer therapy, in which mTOR inhibition increases eIF4E phosphorylation.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Quassinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Genes Reporter/genética , Humanos , Luciferases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In experimental animals, including rats, MK-801 produces characteristic behavioural changes that model schizophrenia. It has been hypothesized that these changes accompany long-term synaptic changes, which require protein neosynthesis. We observed the effect of MK-801 on the "mammalian target of rapamycin" (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway that regulates protein synthesis in the rat frontal cortex. A single injection of MK-801 (0.5, 1, or 2mg/kg) induced an acute increase in the phosphorylation of Akt (Ser-473) eIF4E-binding protein (4E-BP1) (Thr-37/46) and p70S6K (Thr-389). In contrast, after repeated treatment with MK-801 (1mg/kg for 5 or 10 days), the phosphorylation of Akt (Ser-473), mTOR (Ser-2481), 4E-BP1 (Thr-37/46), p70S6K (Thr-389), and S6 (Ser-240/244) increased. Thus, proteins in the mTOR/p70S6K pathway are modulated in chronic MK-801 animal models. These findings may suggest that repeated MK-801 treatment activates the signal transduction pathways involved in the initiation of protein synthesis in the rat frontal cortex.
Assuntos
Maleato de Dizocilpina/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Animais , Fator de Iniciação 4E em Eucariotos/biossíntese , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/genética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Alucinógenos/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Fosforilação/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Biossíntese de Proteínas/genética , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/biossíntese , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Assembly of the eIF4E/eIF4G complex has a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the 4E-BPs, which compete with eIF4G for binding to eIF4E and which have tumor-suppressor activity. To pharmacologically mimic 4E-BP function we developed a high-throughput screening assay for identifying small-molecule inhibitors of the eIF4E/eIF4G interaction. The most potent compound identified, 4EGI-1, binds eIF4E, disrupts eIF4E/eIF4G association, and inhibits cap-dependent translation but not initiation factor-independent translation. While 4EGI-1 displaces eIF4G from eIF4E, it effectively enhances 4E-BP1 association both in vitro and in cells. 4EGI-1 inhibits cellular expression of oncogenic proteins encoded by weak mRNAs, exhibits activity against multiple cancer cell lines, and appears to have a preferential effect on transformed versus nontransformed cells. The identification of this compound provides a new tool for studying translational control and establishes a possible new strategy for cancer therapy.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação Eucariótico 4G/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Nitrocompostos/isolamento & purificação , Nitrocompostos/farmacologia , Tiazóis/isolamento & purificação , Tiazóis/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/fisiologia , Imunoensaio de Fluorescência por Polarização/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Hidrazonas , Células Jurkat , Camundongos , Modelos Moleculares , Nitrocompostos/química , Oncogenes/efeitos dos fármacos , Oncogenes/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Tiazóis/químicaRESUMO
BACKGROUND AND OBJECTIVE: Inhibition of established left ventricular hypertrophy (LVH) and fibrosis may bring clinical benefits by reducing cardiac morbidity and mortality. The mammalian target of rapamycin, mTOR, is known to play a critical role in determining cell and organ size. We investigated whether mTOR inhibition can inhibit the chronic pressure-overload-induced LVH and fibrosis. METHODS: Male FVB/N mice underwent transverse aortic constriction (TAC) for 5 weeks to allow for establishment of LVH, followed by treatment with the mTOR inhibitor, Rapamune (2 mg/kg per day, gavage), for 4 weeks. Echocardiography was used to monitor changes in LVH and function. Haemodynamic, morphometric, histological and molecular analyses were conducted. RESULTS: Inhibition of mTOR by Rapamune was confirmed by a suppression of activated phosphorylation of ribosomal S6 protein and eukaryotic translation initiation factor-4E due to pressure overload. Despite a comparable degree of pressure overload between the vehicle- or Rapamune-treated TAC groups, Rapamune treatment for 4 weeks attenuated TAC-induced LVH by 46%, estimated by LV weight or myocyte size, and LV fractional shortening was also preserved versus vehicle-treated control (39 +/- 1 versus 32 +/- 2%, P < 0.05). Inhibition of established LVH by Rapamune was associated with a 38% reduction in collagen content. Moreover, altered gene expression due to pressure overload was largely restored. CONCLUSION: Despite sustained pressure overload, inhibition of mTOR by a 4-week period of Rapamune treatment attenuates chronically established LVH and cardiac fibrosis with preserved contractile function.
Assuntos
Pressão Sanguínea , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Proteínas Quinases/metabolismo , Análise de Variância , Animais , Fator Natriurético Atrial/efeitos dos fármacos , Fator Natriurético Atrial/metabolismo , Doença Crônica , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Fibrose , Frequência Cardíaca , Imunossupressores/farmacologia , Masculino , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Cadeias Pesadas de Miosina/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Proteína S6 Ribossômica/efeitos dos fármacos , Proteína S6 Ribossômica/metabolismo , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Volume Sistólico , Serina-Treonina Quinases TORRESUMO
The delayed therapeutic onset observed in response to chronic antidepressant drug treatment is little understood. While current theories emphasize effects on gene transcription, possible effects of antidepressant drugs on translation control pathways have not been explored. We examined the effect of the selective serotonergic reuptake inhibitor fluoxetine on regulation of two major determinants of mRNA translation, eukaryotic initiation factor 4E (eIF4E) and eukaryotic elongation factor 2 (eEF2). Chronic fluoxetine treatment induced hyperphosphorylation of eEF2 (Thr56) in prefrontal cortex, hippocampus and dentate gyrus of rats. By contrast, phosphorylation of eIF4E (Ser209) was observed specifically in the dentate gyrus. Acute fluoxetine treatment had no effect on translational factor activity. These findings suggest that region-specific regulation of translation contributes to the delayed action of antidepressant drugs such as fluoxetine.
Assuntos
Encéfalo/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fluoxetina/administração & dosagem , Fator 2 de Elongação de Peptídeos/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Animais , Western Blotting , Encéfalo/metabolismo , Esquema de Medicação , Eletroforese em Gel de Poliacrilamida , Fator de Iniciação 4E em Eucariotos/metabolismo , Imuno-Histoquímica , Masculino , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Mounting evidence links deregulated protein synthesis to tumorigenesis via the translation initiation factor complex eIF4F. Components of this complex are often overexpressed in a large number of cancers and promote malignant transformation in experimental systems. mTOR affects the activity of the eIF4F complex by phosphorylating repressors of the eIF4F complex, the eIF4E binding proteins. The immunosuppressant rapamycin specifically inhibits mTOR activity and retards cancer growth. Importantly, mutations in upstream negative regulators of mTOR cause hamartomas, haemangiomas, and cancers that are sensitive to rapamycin treatment. Such mutations lead to increased eIF4F formation and consequently to enhanced translation initiation and cell growth. Thus, inhibition of translation initiation through targeting the mTOR-signalling pathway is emerging as a promising therapeutic option.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Transformação Celular Neoplásica , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4F em Eucariotos/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Proteínas Quinases/efeitos dos fármacos , Sirolimo/uso terapêutico , Serina-Treonina Quinases TORRESUMO
Upon binding to the glycolipid receptor globotriaosylceramide, Shiga toxins (Stxs) undergo retrograde transport to reach ribosomes, cleave 28S rRNA, and inhibit protein synthesis. Stxs induce the ribotoxic stress response and cytokine and chemokine expression in some cell types. Signaling mechanisms necessary for cytokine expression in the face of toxin-mediated protein synthesis inhibition are not well characterized. Stxs may regulate cytokine expression via multiple mechanisms involving increased gene transcription, mRNA transcript stabilization, and/or increased translation initiation efficiency. We show that treatment of differentiated THP-1 cells with purified Stx1 resulted in prolonged activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) cascades, and lipopolysaccharides (LPS) rapidly triggered transient activation of JNK and p38 and prolonged activation of extracellular signal-regulated kinase cascades. Simultaneous treatment with Stx1 + LPS mediated prolonged p38 MAPK activation. Stx1 increased eukaryotic translation initiation factor 4E (eIF4E) activation by 4.3-fold within 4-6 h, and LPS or Stx1 + LPS treatment increased eIF4E activation by 7.8- and 11-fold, respectively, within 1 h. eIF4E activation required Stx1 enzymatic activity and was mediated by anisomycin, another ribotoxic stress inducer. A combination of MAPK inhibitors or a MAPK-interacting kinase 1 (Mnk1)-specific inhibitor blocked eIF4E activation by all stimulants. Mnk1 inhibition blocked the transient increase in total protein synthesis detected in Stx1-treated cells but failed to block long-term protein synthesis inhibition. The MAPK inhibitors or Mnk1 inhibitor blocked soluble interleukin (IL)-1beta and IL-8 production or release by 73-96%. These data suggest that Stxs may regulate cytokine expression in part through activation of MAPK cascades, activation of Mnk1, and phosphorylation of eIF4E.
Assuntos
Citocinas/biossíntese , Fator de Iniciação 4E em Eucariotos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Toxina Shiga I/farmacologia , Compostos de Anilina/farmacologia , Anisomicina/farmacologia , Antracenos/farmacologia , Linhagem Celular Tumoral , Citocinas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , Purinas/farmacologia , Piridinas/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/imunologia , Toxina Shiga I/antagonistas & inibidores , Fatores de TempoRESUMO
OBJECTIVE: To investigate the activity of Caspase-3 and the expression of eukaryotic initiation factor families, bFGF and VEGF after elemene on laryngeal carcinoma HEp-2 cells. METHOD: The HEp-2 cells after elemene treatment were analyzed utilizing Westernblot and reverse transcriptase polymerase chain reaction (RT-PCR). The activity of Caspase-3 was assessed by colorimetric assay. RESULT: The activity of Caspase-3 was enhanced after elemene treatment. The protein expression of eIF4E, eIF4G, bFGF and VEGF were significantly inhibited by elemene; and the mRNA expression of bFGF and VEGF were inhibited either. CONCLUSION: Elemene can effectively inhibit the growth of HEp-2 cells and result in the alteration of activity of Caspase-3. There were significant correlations between the decreased expression of protein eIF4E, eIF4G, bFGF and VEGF. The mechanism of eIF4E and eIF4G decrease the expression of bFGF and VEGF is post-transcriptional.
Assuntos
Caspases/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação Eucariótico 4G/efeitos dos fármacos , Neoplasias Laríngeas/tratamento farmacológico , Sesquiterpenos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/biossíntese , Linhagem Celular Tumoral , Fator de Iniciação 4E em Eucariotos/biossíntese , Fator de Iniciação Eucariótico 4G/biossíntese , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Humanos , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacosRESUMO
Regulation of the PHAS-1-eukaryotic initiation factor-4E (eIF4E) complex is the rate-limiting step in the initiation of protein synthesis. This study characterized the upstream signaling pathways that mediate ANG II-dependent phosphorylation of PHAS-1 and eIF4E in vascular smooth muscle. ANG II-dependent PHAS-1 phosphorylation was maximal at 10 min (2.47 +/- 0.3 fold vs. control). This effect was completely blocked by the specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase, LY-294002), mammalian target of rapamycin, and extracellular signal-regulated kinase 1/2 (ERK1/2, U-0126) or by a recombinant adenovirus encoding dominant-negative Akt. PHAS-1 phosphorylation was followed by dissociation of eIF4E. Increased ANG II-induced eIF4E phosphorylation was observed at 45 min (2.63 +/- 0.5 fold vs. control), was maximal at 90 min (3.38 +/- 0.3 fold vs. control), and was sustained at 2 h. This effect was blocked by inhibitors of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways, but not by PI3-kinase inhibition, and was dependent on PKC, intracellular Ca2+, and tyrosine kinases. Downregulation of proline-rich tyrosine kinase 2 (PYK2) by antisense oligonucleotides led to a near-complete inhibition of PHAS-1 and eIF4E phosphorylation in response to ANG II. Therefore, PYK2 represents a proximal signaling intermediate that regulates ANG II-induced vascular smooth muscle cell protein synthesis via regulation of the PHAS-1-eIF4E complex.
Assuntos
Proteínas de Transporte/fisiologia , Fator de Iniciação 4E em Eucariotos/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Músculo Liso Vascular/enzimologia , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/fisiologia , Angiotensina II/metabolismo , Animais , Proteínas de Transporte/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Quinase 2 de Adesão Focal , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Mutação , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas/efeitos dos fármacos , Fosforilação , Testes de Precipitina , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Fatores de TempoRESUMO
Previous work has shown that increased phosphorylation of eukaryotic initiation factor (eIF) 4E at Ser209 in the C-terminal loop of the protein is observed in response to cellular stress. SB203580, a cell permeable inhibitor of stress-activated protein kinase 2a (SAPK2a/p38), suppresses this response in a number of cell types. To validate the in vivo specificity of this inhibitor for the investigation of signalling pathways, which modulate the phosphorylation of eIF4E, we have used 293 cells which inducibly express either a wild-type form (WT-SAPK2a) or a drug-resistant mutant of SAPK2a (DR-SAPK2a). These data show that while the arsenite-induced increase in the phosphorylation of eIF4E and hsp25 was sensitive to SB203580 in cells expressing WT-SAPK2a, these responses to SB203580 were abrogated in cells expressing DR-SAPK2a. In addition, the phosphorylation of the eIF4E kinase, MAP kinase integrating kinase-1 (Mnk1), which is activated in response to growth factors or stress, was insensitive to SB203580 in DR-SAPK2a-expressing cells. However, a cell-permeable, specific inhibitor of Mnk1, CGP57380 and the phosphatidylinositol-3-kinase (PI3-K) inhibitor, LY294002, prevented eIF4E phosphorylation in 293 cells irrespective of SAPK2a expression. Therefore, this study validates the use of SB203580 for investigating signalling pathways modulating the phosphorylation of eIF4E in cultured cells.