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1.
Sci Rep ; 7(1): 18099, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29273814

RESUMO

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.


Assuntos
Ameloblastos/metabolismo , Amelogênese/fisiologia , Diferenciação Celular/fisiologia , Canais de Cátion TRPM/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Camundongos , Camundongos Transgênicos , Odontoblastos/metabolismo , Fosforilação , Canais de Cátion TRPM/genética
3.
Biochem Biophys Res Commun ; 457(2): 206-12, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25562654

RESUMO

Cell-penetrating peptides (CPPs) as a novel biomedical delivery system have been highly anticipated, since they can translocate across biological membranes and are capable of transporting their cargo inside live cells with minimal invasiveness. However, non-selective internalization in various cell types remains a challenge in the clinical application of CPPs, especially in cancer treatment. In this study, we attempted to identify novel cancer-homing CPPs to target glioblastoma multiforme (GBM), which is often refractory and resistant to treatment. We screened for CPPs showing affinity for the human GBM cell line, U87MG, from an mRNA display random peptide library. One of the candidate peptides which amino-acid sequence was obtained from the screening showed selective cell-penetrating activity in U87MG cells. Conjugation of the p16(INK4a) functional peptide to the GBM-selective CPP induced cellular apoptosis and reduced phosphorylated retinoblastoma protein levels. This indicates that the CPP was capable of delivering a therapeutic molecule into U87MG cells inducing apoptosis. These results suggest that the novel CPP identified in this study permeates with high affinity into GBM cells, revealing it to be a promising imaging and therapeutic tool in the treatment of glioblastoma.


Assuntos
Neoplasias Encefálicas/metabolismo , Peptídeos Penetradores de Células/farmacologia , Glioblastoma/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/uso terapêutico , Humanos , Dados de Sequência Molecular
4.
PLoS One ; 9(1): e86639, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489756

RESUMO

Cell-penetrating peptides (CPPs) have gained attention as promising tools to enable the delivery of various molecules in a non-invasive manner. Among the CPPs, TAT and poly-arginine have been extensively utilized in numerous studies for the delivery of functional proteins, peptides, and macromolecules to analyze cellular signaling. However, the molecular mechanisms of cellular entry remain largely unknown. Here, we applied siRNA library screening to identify the regulatory genes for the cellular entry of poly-arginine peptide based on microscopic observation of the entry of fluorescent peptides in siRNA-treated cells. In this screening, we identified the cell membrane gene SLC4A4 and the trafficking regulator gene COPA, which also plays an important role in early endosome maturation. These results demonstrated that cellular entry of poly-arginine requires at least two different steps, probably binding on the cell surface and endosomal entry. The identification of genes for cellular entry of poly-arginine provides insights into its mechanisms and should further aid in the development of highly efficient cell-penetrating peptides.


Assuntos
Arginina/metabolismo , Peptídeos Penetradores de Células/metabolismo , Proteína Coatomer/metabolismo , Endocitose , Simportadores de Sódio-Bicarbonato/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Células HeLa , Humanos , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Simportadores de Sódio-Bicarbonato/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
5.
Nat Commun ; 3: 951, 2012 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-22805558

RESUMO

Cell-penetrating peptides have gained attention owing to their promise in noninvasive delivery systems. Among the identified cell-penetrating peptides, the TAT peptide has been preferentially used for transduction into cells of diverse origins. However, this activity is nonselective between neoplastic and non-neoplastic cells. Here we describe artificial cell-penetrating peptides that are selectively and efficiently incorporated into human tumour cells, according to their lineage. Ten representative tumour lineage-homing cell-penetrating peptides were obtained by screening of a random peptide library constructed using messenger RNA display technology, and some of the isolates were further modified by amino-acid substitution. Their advantageous tumour cell-targeting ability is corroborated in an in vivo mouse model for imaging and growth suppression of metastatic xenoplant tumours. These cell-penetrating peptides are potentially useful for the efficient targeting of human neoplasms in a tumour origin-dependent manner, and provide a framework for the development of peptide-based anti-tumour technologies.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/química , Sistemas de Liberação de Medicamentos/métodos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Linhagem Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Immunoblotting , Camundongos , Biblioteca de Peptídeos , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ressonância de Plasmônio de Superfície , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Biochem Pharmacol ; 81(6): 713-22, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21219881

RESUMO

We evaluated the anti-adult T-cell leukemia (ATL) effects of hippuristanol, an eukaryotic translation initiation inhibitor from the coral Isis hippuris. Hippuristanol inhibited proliferation of HTLV-1-infected T-cell lines and ATL cells, but not normal peripheral blood mononuclear cells. It induced cell cycle arrest during G1 phase by reducing the expression of cyclin D1, cyclin D2, CDK4 and CDK6, and induced apoptosis by reducing the expression of Bcl-x(L), c-IAP2, XIAP and c-FLIP. The induced apoptosis was associated with activation of caspase-3, -8 and -9. Hippuristanol also suppressed IkappaBalpha phosphorylation and depleted IKKalpha, IKKgamma, JunB and JunD, resulting in inactivation of NF-kappaB and AP-1. It also suppressed carbonic anhydrase type II expression. In addition to its in vitro effects, hippuristanol suppressed tumor growth in mice with severe combined immunodeficiency harboring tumors induced by inoculation of HTLV-1-infected T cells. These preclinical data suggest that hippuristanol could be a potentially useful therapeutic agent for patients with ATL.


Assuntos
RNA Helicases DEAD-box/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Esteróis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Inibidores Enzimáticos/uso terapêutico , Fator de Iniciação 4A em Eucariotos , Feminino , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Esteróis/uso terapêutico
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