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1.
Cell Mol Life Sci ; 81(1): 297, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38992309

RESUMO

Muse cells, identified as cells positive for the pluripotent surface marker SSEA-3, are pluripotent-like endogenous stem cells located in the bone marrow (BM), peripheral blood, and organ connective tissues. The detailed characteristics of SSEA-3(+) cells in extraembryonic tissue, however, are unknown. Here, we demonstrated that similar to human-adult tissue-Muse cells collected from the BM, adipose tissue, and dermis as SSEA-3(+), human-umbilical cord (UC)-SSEA-3(+) cells express pluripotency markers, differentiate into triploblastic-lineage cells at a single cell level, migrate to damaged tissue, and exhibit low telomerase activity and non-tumorigenicity. Notably, ~ 20% of human-UC-SSEA-3(+) cells were negative for X-inactive specific transcript (XIST), a naïve pluripotent stem cell characteristic, whereas all human adult tissue-Muse cells are XIST-positive. Single-cell RNA sequencing revealed that the gene expression profile of human-UC-SSEA-3(+) cells was more similar to that of human post-implantation blastocysts than human-adult tissue-Muse cells. The DNA methylation level showed the same trend, and notably, the methylation levels in genes particularly related to differentiation were lower in human-UC-SSEA-3(+) cells than in human-adult tissue-Muse cells. Furthermore, human-UC-SSEA-3(+) cells newly express markers specific to extraembryonic-, germline-, and hematopoietic-lineages after differentiation induction in vitro whereas human-adult tissue-Muse cells respond only partially to the induction. Among various stem/progenitor cells in living bodies, those that exhibit properties similar to post-implantation blastocysts in a naïve state have not yet been found in humans. Easily accessible human-UC-SSEA-3(+) cells may be a valuable tool for studying early-stage human development and human reproductive medicine.


Assuntos
Blastocisto , Diferenciação Celular , Antígenos Embrionários Estágio-Específicos , Cordão Umbilical , Humanos , Antígenos Embrionários Estágio-Específicos/metabolismo , Cordão Umbilical/citologia , Blastocisto/citologia , Blastocisto/metabolismo , Antígenos Glicosídicos Associados a Tumores/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Célula Única , Telomerase/metabolismo , Telomerase/genética , Feminino
2.
Mol Pharmacol ; 92(1): 57-66, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28461586

RESUMO

In brain disorders, astrocytes change phenotype to reactive astrocytes and are involved in the induction of neuroinflammation and brain edema. The administration of glucocorticoids (GCs), such as dexamethasone (Dex), reduces astrocytic activation, but the mechanisms underlying this inhibitory action are not well understood. Endothelins (ETs) promote astrocytic activation. Therefore, the effects of Dex on ET receptor expressions were examined in cultured rat astrocytes. Treatment with 300 nM Dex for 6-48 hours reduced the mRNA expression of astrocytic ETA and ETB receptors to 30-40% of nontreated cells. Levels of ETA and ETB receptor proteins became about 50% of nontreated cells after Dex treatment. Astrocytic ETA and ETB receptor mRNAs were decreased by 300 nM hydrocortisone. The effects of Dex and hydrocortisone on astrocytic ET receptors were abolished in the presence of mifepristone, a GC receptor antagonist. Although Dex did not decrease the basal levels of matrix metalloproteinase (MMP) 3 and MMP9 mRNAs, pretreatment with Dex reduced ET-induced increases in MMP mRNAs. The effects of ET-1 on the release of MMP3 and MMP9 proteins were attenuated by pretreatment with Dex. ET-1 stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in cultured astrocytes. Pretreatment with Dex reduced the ET-induced increases in ERK1/2 phosphorylation. In contrast, pretreatment with Dex did not affect MMP production or ERK1/2 phosphorylation induced by phorbol myristate acetate, a protein kinase C activator. These results indicate that Dex downregulates astrocytic ET receptors and reduces ET-induced MMP production.


Assuntos
Astrócitos/metabolismo , Dexametasona/farmacologia , Endotelinas/farmacologia , Metaloproteinase 3 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Receptores de Endotelina/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Astrócitos/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Endotelinas/antagonistas & inibidores , Ratos , Ratos Wistar
4.
Int J Nanomedicine ; 8: 1989-99, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23717042

RESUMO

This study describes the applications of a naturally occurring small heat shock protein (Hsp) that forms a cage-like structure to act as a drug carrier. Mutant Hsp cages (HspG41C) were expressed in Escherichia coli by substituting glycine 41 located inside the cage with a cysteine residue to allow conjugation with a fluorophore or a drug. The HspG41C cages were taken up by various cancer cell lines, mainly through clathrin-mediated endocytosis. The cages were detected in acidic organelles (endosomes/lysosomes) for at least 48 hours, but none were detected in the mitochondria or nuclei. To generate HspG41C cages carrying doxorubicin (DOX), an anticancer agent, the HspG41C cages and DOX were conjugated using acid-labile hydrazone linkers. The release of DOX from HspG41C cages was accelerated at pH 5.0, but was negligible at pH 7.2. The cytotoxic effects of HspG41C-DOX against Suit-2 and HepG2 cells were slightly weaker than those of free DOX, but the effects were almost identical in Huh-7 cells. Considering the relatively low release of DOX from HspG41C-DOX, HspG41C-DOX exhibited comparable activity towards HepG2 and Suit-2 cells and slightly stronger cytotoxicity towards Huh-7 cells than free DOX. Hsp cages offer good biocompatibility, are easy to prepare, and are easy to modify; these properties facilitate their use as nanoplatforms in drug delivery systems and in other biomedical applications.


Assuntos
Antineoplásicos/química , Doxorrubicina/química , Nanocápsulas/química , Proteínas Recombinantes/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Endocitose , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/genética , Células Hep G2 , Humanos , Espaço Intracelular/metabolismo , Tamanho da Partícula , Proteínas Recombinantes/genética
5.
Int J Nanomedicine ; 7: 4353-62, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22927755

RESUMO

Protein nanocages are self-organized complexes of oligomers whose three-dimensional architecture can been determined in detail. These structures possess nanoscale inner cavities into which a variety of molecules, including therapeutic or diagnostic agents, can be encapsulated. These properties yield these particles suitable for a new class of drug delivery carrier, or as a bioimaging reagent that might respond to biochemical signals in many different cellular processes. We report here the design, synthesis, and biological characterization of a hepatocyte-specific nanocage carrying small heat-shock protein. These nanoscale protein cages, with a targeting peptide composed of a preS1 derivative from the hepatitis B virus on their surfaces, were prepared by genetic engineering techniques. PreS1-carrying nanocages showed lower cytotoxicity and significantly higher specificity for human hepatocyte cell lines than other cell lines in vitro. These results suggested that small heat-shock protein-based nanocages present great potential for the development of effective targeted delivery of various agents to specific cells.


Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Hepatócitos/metabolismo , Nanocápsulas/química , Precursores de Proteínas/metabolismo , Sobrevivência Celular/fisiologia , Células HeLa , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Hepatócitos/citologia , Humanos , Microscopia Confocal , Nanomedicina , Tamanho da Partícula , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Bioconjug Chem ; 23(7): 1494-501, 2012 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-22621213

RESUMO

We described herein a human hepatocellular carcinoma (HCC) cell-targeted protein cage for which the HCC-binding peptide termed SP94 was modified at the surface of a naturally occurred heat shock protein (Hsp) cage. Six types of HCC-targeted Hsp cages were chemically synthesized using two types of heterobifunctional linker (SM(PEG)(n)) with different lengths and two types of SP94 peptide, which contained a unique Cys residue at the N- or C-terminus of the peptide. These Hsp cages were characterized using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analyses, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and dynamic light scattering (DLS) measurement. Fluorescence microscopic observations revealed that all the engineered protein cages bind selectively to HCC cells but not to the other cell lines tested (including normal liver cell). Moreover, the number of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide had important effects upon the binding of engineered Hsp cages to HCC cells. An engineered Hsp cage conjugated to the N-terminus of SP94 peptide via a longer linker molecule and containing high SP94 peptide levels showed greater binding toward HCC cells. Surprisingly, through optimization of these three factors, up to 10-fold greater affinity toward HCC cells was achieved. These results are critically important not only for the development of HCC cell-targeting devices using SP94 peptide, but also to create other cell-targeting materials that utilize other peptide ligands.


Assuntos
Carcinoma Hepatocelular/metabolismo , Sistemas de Liberação de Medicamentos , Neoplasias Hepáticas/metabolismo , Peptídeos/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/farmacocinética , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Estrutura Molecular , Peptídeos/química , Peptídeos/farmacocinética , Polietilenoglicóis/química , Propriedades de Superfície
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