RESUMO
T-LAK-originated protein kinase (TOPK), a dual specificity serine/threonine kinase, is up-regulated and related to poor prognosis in many types of cancers. Y-box binding protein 1 (YB1) is a DNA/RNA binding protein and serves important roles in multiple cellular processes. Here, we reported that TOPK and YB1 were both highly expressed in esophageal cancer (EC) and correlated with poor prognosis. TOPK knockout effectively suppressed EC cell proliferation and these effects were reversible by rescuing YB1 expression. Notably, TOPK phosphorylated YB1 at Thr 89 (T89) and Ser 209 (S209) amino acid residues, then the phosphorylated YB1 bound with the promoter of the eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) to activate its transcription. Consequently, the AKT/mTOR signal pathway was activated by up-regulated eEF1A1 protein. Importantly, TOPK inhibitor HI-TOPK-032 suppressed the EC cell proliferation and tumor growth by TOPK/YB1/eEF1A1 signal pathway in vitro and in vivo. Taken together, our study reveals that TOPK and YB1 are essential for the growth of EC, and TOPK inhibitors may be applied to retard cell proliferation in EC. This study highlights the promising therapeutic potential of TOPK as a target for treatment of EC.
Assuntos
Neoplasias Esofágicas , Quinases de Proteína Quinase Ativadas por Mitógeno , Humanos , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Esofágicas/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/farmacologia , Transdução de SinaisRESUMO
Eicosapentaenoic acid (EPA) shows antioxidant activity, which may be attributed to its regulatory effect on microRNA expression. Our preliminary study indicated that EPA upregulated miR-494-5p, which was possibly involved in the regulation of cellular stress responses. The current study aimed to address whether miR-494-5p was targeted by EPA to regulate cellular oxidative stress and its possible functional mechanism. The results showed that miR-494-5p mediated the antioxidant effect of EPA and miR-494-5p reduction deteriorated EPA-induced increase in the cellular antioxidant capacity of HepG2 cells. Moreover, the mitochondrial elongation factor 1 (MIEF1) gene was a target gene of miR-494-5p. Both miR-494-5p overexpression and MIEF1 knockdown significantly enhanced cellular antioxidant capacity, as indicated by a reduction in the reactive oxygen species level and an increase in the total cellular antioxidant capacity, along with enhancing antioxidant enzymes. Thus, miR-494-5p and MIEF1 had opposite effects on cellular antioxidant capacity. Furthermore, their regulatory effects on oxidative stress may have been attributed to modulation of mitochondrial function, biogenesis and homeostasis. Taken together, the findings indicated that miR-494-5p mediated EPA activity and promoted cellular antioxidant capacity by inhibiting the expression of MIEF1, which further modulated mitochondrial structure and activity. This study may provide novel insights into the post-translational regulation of antioxidation reactions, which involves the coordinated control of mitochondria.
Assuntos
Antioxidantes , MicroRNAs , Humanos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Ácido Eicosapentaenoico/farmacologia , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/farmacologia , Células Hep G2 , Estresse Oxidativo , MicroRNAs/metabolismoRESUMO
BACKGROUND: Eukaryotic translation elongation factors 1 δ (EEF1D), has garnered much attention with regards to their role in the drug resistance of cancers. In this paper, we investigated the effects and mechanisms of increasing the sensitivity of ovarian cancer cells to cisplatin or cis-dichlorodiammine platinum (DDP) by knockdown and knockout of EEF1D gene in cellular and animal models. METHODS: The EEF1D gene was knocked-down or -out by siRNA or CRISPR/Cas9 respectively in human ovarian cancer cell SKOV3, DDP-resistant subline SKOV3/DDP, and EEF1D gene in human primary ovarian cancer cell from 5 ovarian cancer patients with progressive disease/stable disease (PD/SD) was transiently knocked down by siRNA interference. The mice model bearing xenografted tumor was established with subcutaneous inoculation of SKOV3/DDP. RESULTS: The results show that reducing or removing EEF1D gene expression significantly increased the sensitivity of human ovarian cancer cells to DDP in inhibiting viability and inducing apoptosis in vitro and in vivo, and also boosted DDP to inhibit xenografted tumor growth. Interfering with EEF1D gene expression in mice xenografted tumor significantly affected the levels of OPTN, p-Akt, Bcl-2, Bax, cleaved caspase-3 and ERCC1 compared to DDP treated mice alone, and had less effect on PI3K, Akt and caspase-3. CONCLUSIONS: The knocking down or out EEF1D gene expression could enhance the sensitivity of ovarian cancer cells to DDP partially, which may be achieved via inactivating the PI3K/AKT signaling pathway, thus inducing cell apoptosis and decreasing repairment of DNA damage. Our study provides a novel therapeutic strategy for the treatment of ovarian cancer.
Assuntos
Antineoplásicos , Neoplasias Ovarianas , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Carcinoma Epitelial do Ovário/tratamento farmacológico , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.
Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Expressão Gênica , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/farmacologia , Peptídeos/genética , Peptídeos/farmacologia , Pichia/genética , Proteínas Recombinantes , Anti-Infecciosos/isolamento & purificação , Bactérias/metabolismo , Bactérias/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/isolamento & purificação , Peptídeos/isolamento & purificaçãoRESUMO
An archaeal phenylalanyl-tRNA synthetase (FRS) has been purified from the hyperthermophile Sulfolobus solfataricus (Ss). This enzyme is a heterotetramer made of two different subunits whose molecular mass is 56 kDa and 64 kDa, respectively. As thought, SsFRS is essential for the in vitro poly(Phe) synthesis. Interestingly, the enzyme is able to aminoacylate only endogenous tRNA but it does not seem to be a strictly ATP-dependent synthetase. SsFRS interacts with the elongation factor 1alpha isolated from the same source; this caused a significant enhancement of the SstRNA aminoacylation efficiency, thus indicating that, as well as in eukarya, in this archaeon a tRNA channelling mechanism should occur. The overall results presented in this paper show that the archaeal SsFRS behaves as the analogous enzymes isolated from eukaryal sources rather than those from eubacterial organisms.
Assuntos
Fenilalanina-tRNA Ligase/isolamento & purificação , Sulfolobus/enzimologia , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Estabilidade Enzimática , Guanosina Trifosfato/farmacologia , Dados de Sequência Molecular , Peso Molecular , Fator 1 de Elongação de Peptídeos/farmacologia , Fenilalanina/química , Fenilalanina-tRNA Ligase/química , Fenilalanina-tRNA Ligase/metabolismo , Temperatura , TrítioRESUMO
Effects of the active aldehyde group of ribose C1' at position 4324 of rat 28S rRNA, in the inactivated ribosome generated by RNA N-glycosidases (trichosanthin, A-chain of cinnamomin and ricin), on peptide elongation have been studied. The aldehyde group inhibits the activities of eEF1A-dependent aminoacyl-tRNA binding to the inactivated ribosome and eEF1A-dependent GTPase, but increases eEF2-dependent activity. At a high concentration of RNA N-glycosidase, the generated aldehyde group also inhibits aminoacyl-tRNA binding to the inactivated ribosome in the absence of elongation factor and translocation activity. When the aldehyde group is reduced into a hydroxyl group by sodium borohydride or blocked with an amino acid through nucleophilic addition, the activities of eEF1A-dependent aminoacyl-tRNA binding and eEF1A-dependent GTPase of the inactivated ribosome are partially restored, but the altered activities of eEF2-dependent GTPase, translocation and aminoacyl-tRNA binding in the absence of elongation factor are not normalized. Thus, reduction or blockage of the aldehyde group with sodium borohydride or amino acids might change the conformation of the S/R domain in rat 28S ribosomal RNA to meet the requirement for eEF1A-dependent reactions, but not eEF2-involved reactions.