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1.
PLoS One ; 15(7): e0235965, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701960

RESUMO

Secondary lymphedema (SL)is a frequent and devastating complication of modern oncological therapy and filarial infections. A lack of a reliable preclinical model to investigate the underlying mechanism of clinical stage progression has limited the development of new therapeutic strategies. Current first line treatment has shown to be merely symptomatic and relies on lifetime use of compression garments and decongestive physiotherapy. In this study, we present the development of a secondary lymphedema model in 35 rats using pre- and intraoperative fluorescence-guided mapping of the lymphatics and microsurgical induction. In contrast to the few models reported so far, we decided to avoid the use of radiation for lymphedema induction. It turned out, that the model is nearly free of complications and capable of generating a statistically significant limb volume increase by water displacement measurements, sustained for at least 48 days. A translational, accurate lymphatic dysfunction was visualized by a novel VIS-NIR X-ray ICG-Clearance-Capacity imaging technology. For the first-time SL stage progression was validated by characteristic histological alterations, such as subdermal mast cell infiltration, adipose tissue deposition, and fibrosis by increased skin collagen content. Immunofluorescence confocal microscopy analysis suggested that stage progression is related to the presence of a characteristic α SMA+/HSP-47+/vimentin+ fibroblast subpopulation phenotype. These findings demonstrate that the in-vivo model is a reliable and clinically relevant SL model for the development of further secondary lymphedema therapeutic strategies and the analysis of the veiled molecular mechanisms of lymphatic dysfunction.


Assuntos
Corantes Fluorescentes/química , Linfedema/patologia , Microcirurgia/efeitos adversos , Actinas/metabolismo , Animais , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Verde de Indocianina/química , Linfedema/etiologia , Microscopia de Fluorescência , Ratos , Ratos Endogâmicos Lew , Pele/patologia , Vimentina/metabolismo
2.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32630064

RESUMO

Vimentin is an intermediate filament protein that plays key roles in integration of cytoskeletal functions, and therefore in basic cellular processes such as cell division and migration. Consequently, vimentin has complex implications in pathophysiology. Vimentin is required for a proper immune response, but it can also act as an autoantigen in autoimmune diseases or as a damage signal. Although vimentin is a predominantly cytoplasmic protein, it can also appear at extracellular locations, either in a secreted form or at the surface of numerous cell types, often in relation to cell activation, inflammation, injury or senescence. Cell surface targeting of vimentin appears to associate with the occurrence of certain posttranslational modifications, such as phosphorylation and/or oxidative damage. At the cell surface, vimentin can act as a receptor for bacterial and viral pathogens. Indeed, vimentin has been shown to play important roles in virus attachment and entry of severe acute respiratory syndrome-related coronavirus (SARS-CoV), dengue and encephalitis viruses, among others. Moreover, the presence of vimentin in specific virus-targeted cells and its induction by proinflammatory cytokines and tissue damage contribute to its implication in viral infection. Here, we recapitulate some of the pathophysiological implications of vimentin, including the involvement of cell surface vimentin in interaction with pathogens, with a special focus on its role as a cellular receptor or co-receptor for viruses. In addition, we provide a perspective on approaches to target vimentin, including antibodies or chemical agents that could modulate these interactions to potentially interfere with viral pathogenesis, which could be useful when multi-target antiviral strategies are needed.


Assuntos
Vírus da SARS/fisiologia , Vimentina/metabolismo , Viroses/patologia , Anticorpos/imunologia , Anticorpos/metabolismo , Anticorpos/uso terapêutico , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/tratamento farmacológico , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Vimentina/química , Vimentina/imunologia , Viroses/tratamento farmacológico , Viroses/metabolismo , Replicação Viral/efeitos dos fármacos
3.
PLoS One ; 15(6): e0234375, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555682

RESUMO

Renal dysplasia, the major cause of childhood renal failure, is characterized by defective branching morphogenesis and nephrogenesis. Beta-catenin, a transcription factor and cell adhesion molecule, is markedly increased in the nucleus of kidney cells in human renal dysplasia and contributes to its pathogenesis by altering target genes that are essential for kidney development. Quercetin, a naturally occurring flavonoid, reduces nuclear beta-catenin levels and reduces beta-catenin transcriptional activity. In this study, we utilized wild type and dysplastic mouse kidney organ explants to determine if quercetin reduces beta-catenin activity during kidney development and whether it improves the severity of renal dysplasia. In wild type kidney explants, quercetin treatment resulted in abnormal branching morphogenesis and nephrogenesis in a dose dependent manner. In wild type embryonic kidneys, quercetin reduced nuclear beta-catenin expression and decreased expression of beta-catenin target genes Pax2, Six2, and Gdnf, which are essential for kidney development. Our RDB mouse model of renal dysplasia recapitulates the overexpression of beta-catenin and histopathological changes observed in human renal dysplasia. RDB kidneys treated with quercetin resulted in improvements in the overall histopathology, tissue organization, ureteric branching morphogenesis, and nephrogenesis. Quercetin treatment also resulted in reduced nuclear beta-catenin and reduced Pax2 expression. These improvements were associated with the proper organization of vimentin, NCAM, and E-cadherin, and a 45% increase in the number of developing and maturing nephrons. Further, our results show that in human renal dysplasia, beta-catenin, vimentin, and e-cadherin also have abnormal expression patterns. Taken together, these data demonstrate that quercetin treatment reduces nuclear beta-catenin and this is associated with improved epithelial organization of developing nephrons, resulting in increased developing nephrons and a partial rescue of renal dysplasia.


Assuntos
Rim/anormalidades , Rim/efeitos dos fármacos , Quercetina/farmacologia , beta Catenina/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Vimentina/metabolismo , beta Catenina/química , beta Catenina/genética
4.
Adv Exp Med Biol ; 1257: 169-178, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32483739

RESUMO

This chapter discusses a novel target of osteosarcoma (OS), cell-surface vimentin (CSV), and a novel generation of interleukin-12 (IL12), CSV-targeted IL12, for treating OS tumor metastasis. Vimentin is a known intracellular structural protein for mesenchymal cells but is also documented in tumor cells. Our recent study definitively revealed that vimentin can be translocated to the surface of very aggressive tumor cells, such as metastatic cells. This CSV property allows investigators to capture circulating tumor cells (CTCs) across any type of tumor, including OS. CTCs are known as the seeds of metastasis; therefore, targeting these cells using CSV is a logical approach for use in a metastatic OS setting. Interestingly, we found that the peptide VNTANST can bind to CSV when fused to the p40 subunit encoding the DNA of IL12. Systemic delivery of this CSV-targeted IL12 immune therapy inhibited OS metastasis and relapse in a mouse tumor model as detailed in this chapter. This CSV-targeted delivery of IL12 also reduced toxicity of IL12. In summary, this chapter details a novel approach for safe IL12 immune therapy via targeting CSV.


Assuntos
Neoplasias Ósseas , Imunoterapia , Interleucina-12 , Sarcoma , Vimentina , Animais , Neoplasias Ósseas/terapia , Imunoterapia/tendências , Interleucina-12/administração & dosagem , Camundongos , Recidiva Local de Neoplasia , Sarcoma/terapia , Vimentina/metabolismo
5.
Life Sci ; 256: 117893, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502539

RESUMO

AIMS: To investigate the effect and underlying mechanism of melittin and tripartite motif (TRIM) family in human embryonic lung fibroblast (HELF). MATERIALS AND METHODS: Lentiviral RNA interference vector and lentiviral overexpression vector were constructed and packaged by transfecting 293T cells; the proliferation of HELF was examined using Cell Counting Kit 8; Western blot and qRT-PCR were performed to examine protein and mRNA expression; the interaction with protein phosphatase magnesium-dependent 1A (PPM1A) was examined by Co-immunoprecipitation. KEY FINDINGS: Compared with the control group, the mRNA expression of the TRIM6, TRIM8 and TRIM47 in the IPF group significantly increased. Melittin inhibited the mRNA expression and protein expression levels of TRIM47, the HELF proliferation, the hydroxyproline levels, and the phosphorylation of Smad2/3; the interference of TRIM47 inhibited the protein expression of Vimentin, α-SMA, CTGF, the phosphorylation of Smad2/3 and the synthesis of hydroxyproline; TRIM47 overexpression elevated the phosphorylation of Smad2/3, induced ubiquitination of PPM1A and decreased the expression level of PPM1A, while TRIM47 RNA interference reversed this result. SIGNIFICANCE: Melittin has anti-fibrotic effect in HELF by directly reducing the phosphorylation of Smad2/3 or indirectly reducing the phosphorylation of Smad2/3 by decreasing the expression levels of TRIM47 whose overexpression induces ubiquitination of PPM1A.


Assuntos
Proteínas de Transporte/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Pulmão/embriologia , Meliteno/farmacologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Actinas/metabolismo , Proteínas de Transporte/sangue , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiprolina/metabolismo , Proteínas de Neoplasias/sangue , Proteínas Nucleares/sangue , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2C/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação/efeitos dos fármacos , Vimentina/metabolismo
6.
Invest Ophthalmol Vis Sci ; 61(5): 22, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32413125

RESUMO

Purpose: Glial fibrillary acid protein (GFAP) and vimentin are type III intermediate filament proteins, ubiquitously expressed in retinal glial cells. Under retinal stress, both GFAP and vimentin are well-known sensitive markers for retinal gliosis. However, little is known about whether these proteins are released into the vitreous body in response to retinal gliosis or are related to the severity of retinal gliosis seen in proliferative vitreoretinopathy (PVR). Methods: Vitreous fluids were collected from 44 patients who underwent pars plana vitrectomy for macular hole (Group 1; n = 8), epiretinal membrane (Group 2; n = 8), or retinal detachment (RD) with various degrees of PVR (Group 3; n = 28). The severity of PVR was determined by cumulative scores using PVR classification. GFAP, vimentin, and total protein levels from the vitreous samples were measured. Results: Both GFAP and vimentin levels were significantly elevated in vitreous fluid from Group 3 (RD) compared with Groups 1 and 2 (P < 0.01). GFAP levels (ng/mL) were 12.4 ± 9.8, 17.5 ± 17.7, and 572.0 ± 11659.7, and vimentin levels (ng/mL) were 40.8 ± 61.9, 88.6 ± 86.8, and 3952.8 ± 8179.5 in Groups 1, 2, and 3, respectively. Total protein levels were not significantly different among the three groups. Elevated GFAP and vimentin levels in Group 3 were positively correlated with the areas of RD (P < 0.01, r = 0.53 in GFAP and P < 0.05, r = 0.46 in vimentin) and PVR scores (P < 0.05, r = 0.46 in GFAP and P < 0.00001, r = 0.76 in vimentin). Conclusions: Our data suggest that human vitreous GFAP and vimentin are protein biomarkers for PVR, and reactive gliosis may play a part in PVR formation.


Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Vimentina/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Corpo Vítreo/metabolismo , Idoso , Biomarcadores/metabolismo , Feminino , Gliose , Humanos , Masculino , Pessoa de Meia-Idade
7.
Environ Toxicol ; 35(10): 1070-1081, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32420661

RESUMO

Non-small cell lung cancer (NSCLC) is often complicated by pulmonary infection, which affects treatment and prognosis. Bacterial lipopolysaccharide (LPS) is an effective stimulator of inflammatory cytokine production, and previous studies have reported that LPS promotes tumor invasion and metastasis. Mangiferin is a plant-derived C-glucosylxanthone with many biological activities, such as antioxidation and anti-inflammation. This research mainly explored the mechanism of its antitumor activities on LPS-induced A549, NCI-H460, and NCI-H520 NSCLC cells. We determined that mangiferin exhibits growth inhibiting activity against LPS-induced NSCLC cells through the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. In addition, mangiferin reversed the LPS-induced downregulation of E-cadherin (epithelial marker); conversely, it significantly inhibited the expression of raised vimentin (mesenchymal markers). Moreover, the ability of NSCLC cells to migrate, as evidenced by the wound healing and transwell migration assays, and the expression of CXCR4 increased by LPS were significantly repressed by mangiferin. In addition, mangiferin markedly mediated protein levels of PER1 and NLRP3 in LPS-induced NSCLC cells and reduced the secretion of IL-1ß. These results indicate that mangiferin is not only a remarkable anti-inflammatory compound but also an antitumor agent; thus, it has the potential for being developed into anti-inflammatory and antitumor drugs in the future.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas Circadianas Period/genética , Xantonas/farmacologia , Células A549 , Antígenos CD/genética , Caderinas/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Humanos , Lipopolissacarídeos/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , RNA Interferente Pequeno/genética , Receptores CXCR4/genética , Transdução de Sinais , Vimentina/metabolismo
8.
Nat Commun ; 11(1): 1943, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327648

RESUMO

Kidney fibrosis is a highly deleterious process and a final manifestation of chronic kidney disease. Alpha-(α)-synuclein (SNCA) is an actin-binding neuronal protein with various functions within the brain; however, its role in other tissues is unknown. Here, we describe the expression of SNCA in renal epithelial cells and demonstrate its decrease in renal tubules of murine and human fibrotic kidneys, as well as its downregulation in renal proximal tubular epithelial cells (RPTECs) after TGF-ß1 treatment. shRNA-mediated knockdown of SNCA in RPTECs results in de novo expression of vimentin and α-SMA, while SNCA overexpression represses TGF-ß1-induced mesenchymal markers. Conditional gene silencing of SNCA in RPTECs leads to an exacerbated tubulointerstitial fibrosis (TIF) in two unrelated in vivo fibrotic models, which is associated with an increased activation of MAPK-p38 and PI3K-Akt pathways. Our study provides an evidence that disruption of SNCA signaling in RPTECs contributes to the pathogenesis of renal TIF by facilitating partial epithelial-to-mesenchymal transition and extracellular matrix accumulation.


Assuntos
Nefropatias/patologia , Rim/patologia , alfa-Sinucleína/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/genética , Vimentina/metabolismo , alfa-Sinucleína/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Clin Exp Metastasis ; 37(3): 413-424, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32335861

RESUMO

Patient-derived orthotopic xenograft (PDOX) models have been verified as a useful method for studying human cancers in mice. Previous studies on the extent of metastases in these models have been limited by the necessity of welfare euthanasia (primary tumors reaching threshold size), at which point metastases may only be micrometers in diameter, few in number, and solely identified by step-sectioning of formalin-fixed paraffin-embedded tissue. These small micro-metastases are less suitable for many downstream molecular analyses than macro-metastases. Resection of the primary tumor by survival surgery has been proven to allow further time for metastases to grow. Although PDOX models of triple-negative breast cancer (TNBC) shed circulating tumor cells (CTCs) into the bloodstream and metastasize, similar to human TNBC, little data has been collected in these TNBC PDOX models regarding the association between CTC characteristics and distant metastasis following excision of the primary tumor xenograft. This study assembles a timeline of PDOX tumor shedding and metastatic tumor progression before and after tumor excision surgery. We report the ability to use tumorectomies to increase the lifespan of TNBC PDOX models with the potential to obtain larger metastases. CTC clusters and CTCs expressing a mesenchymal marker (vimentin) were associated with metastatic burden in lung and liver. The data collected through these experiments will guide the further use of PDOX models in studying metastatic TNBC.


Assuntos
Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Micrometástase de Neoplasia/patologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Contagem de Células , Progressão da Doença , Feminino , Humanos , Camundongos , Células Neoplásicas Circulantes/patologia , Neoplasias de Mama Triplo Negativas/cirurgia , Vimentina/metabolismo
10.
Genes Cells ; 25(6): 413-426, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32243065

RESUMO

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin, classified as the type III intermediate filament family, maintain the integrity and architecture of various cell types. Recently, we reported their cell surface expression and binding to multivalent N-acetylglucosamine-conjugated polymers. Furthermore, the presence of vimentin on the surface of various cell types including malignant tumor cells and fibroblasts has been demonstrated. Type III intermediate filament proteins are traditionally considered intracellular proteins and do not possess signal peptides for cell membrane recruitment. Therefore, the mechanism of their transport to the cell surface is unclear. In the current study, we aimed to elucidate this mechanism by focusing on the relationship between their multimeric structure and lipid bilayer affinity. Blue native polyacrylamide gel electrophoresis demonstrated that cell surface-expressed type III intermediate filament proteins formed a multimeric mostly including 4-12-mers but not filamentous structure. Moreover, surface plasmon resonance analysis revealed that the multimeric structure of these recombinant proteins had high affinity to lipid bilayers, whereas their filament-like large multimeric structure did not. Our results suggest that type III intermediate filaments are incorporated into the cell membrane through alteration from a filamentous to a multimeric structure.


Assuntos
Membrana Celular/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Bicamadas Lipídicas/metabolismo , Acetilglucosamina/metabolismo , Animais , Linhagem Celular Tumoral , Desmina/genética , Desmina/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Proteínas de Filamentos Intermediários/genética , Cinética , Camundongos , Microscopia de Força Atômica , Periferinas/genética , Periferinas/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Vimentina/genética , Vimentina/metabolismo
11.
Life Sci ; 249: 117503, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32142767

RESUMO

AIMS: To investigate the role and mechanism of insulin-like growth factor 1(IGF-1)-mediated EMT on multiple myeloma (MM) growth and metastasis. MATERIALS AND METHODS: The expression data from GEO datasets were utilized to explore the expression levels of IGF-1 and epithelial-mesenchymal transition (EMT) markers in MM. Western blotting and flow cytometry analysis were performed to detect the protein levels of EMT markers as well as key components of the PI3K/Akt pathway. Cell proliferation ability was assessed using colony formation assay and EdU incorporation assays. Transwell migration and invasion assays were performed to assess cell metastasis properties. Vimentin was knocked down by using electro-transfection with small interfering RNA (siRNA) to detect the effect of IGF-1-mediated EMT on MM cell growth and metastasis. KEY FINDINGS: First of all, the analysis of GEO database revealed that IGF-1 was excessively expressed and closely correlated with the expression of the EMT markers in MM patients. Furthermore, we demonstrated that IGF-1 enhanced the acquisition of mesenchymal features in a time-dependent manner. Additionally, in vitro studies revealed that IGF-1-mediated mesenchymal phenotype promoted MM migration, invasion and colony formation. Finally, the mechanism study showed PI3K/Akt signaling pathway was involved in the IGF-1-induced EMT in MM cells. SIGNIFICANCE: IGF-1-induced mesenchymal phenotype contributed to MM progression via the PI3K/Akt pathway regulation.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Mieloma Múltiplo/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Progressão da Doença , Regulação para Baixo , Humanos , Mieloma Múltiplo/metabolismo , Metástase Neoplásica , Transdução de Sinais , Regulação para Cima , Vimentina/metabolismo
12.
PLoS One ; 15(3): e0230230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214328

RESUMO

OBJECTIVE: To investigate the effect and mechanism of SB525334 on self-renewal, migration and invasion of ovarian cancer stem cells. METHODS: ALDHhigh-expressing cancer stem cells (CSCs) were isolated from human ovarian cancer cell line SKOV-3 by flow cytometry and treated with 2µg/mL SB525334 for 6h. The sphere forming assay was used to detect the ability of self-renewal of CSCs and the colony formation assay was used to detect the tumorigenicity in vitro. Transwell migration and invasion assay were used to detect the migration and invasion ability of CSCs. To further explore the mechanism, real-time quantitative PCR and flow cytometry were used to detect the mRNA and protein expression of TGF-ß, Smad2, Smad3, phosphorylated Smad2, phosphorylated Smad3 and Smad4, respectively. Expressions of epithelial-mesenchymal transition (EMT)-related genes E-cadherin, Snail, Vimentin were also assessed. RESULTS: The self-renewal ability, tumorigenicity in vitro, migration and invasion ability of CSCs were significantly attenuated after SB525334 treatment. The expressions of TGF-ß, phosphorylated Smad2, phosphorylated Smad3, Snail, and Vimentin were decreased, while Smad4 and E-cadherin expressions were increased. CONCLUSION: SB525334 may inhibit the self-renewal, invasion and migration of ovarian CSCs by blocking the TGF-ß/Smad/EMT pathway.


Assuntos
Movimento Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Imidazóis/farmacologia , Invasividade Neoplásica/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Quinoxalinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Smad/metabolismo , Vimentina/metabolismo
13.
Nat Commun ; 11(1): 1435, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188843

RESUMO

Regeneration of corneal stroma has always been a challenge due to its sophisticated structure and keratocyte-fibroblast transformation. In this study, we fabricate grid poly (ε-caprolactone)-poly (ethylene glycol) microfibrous scaffold and infuse the scaffold with gelatin methacrylate (GelMA) hydrogel to obtain a 3 D fiber hydrogel construct; the fiber spacing is adjusted to fabricate optimal construct that simulates the stromal structure with properties most similar to the native cornea. The topological structure (3 D fiber hydrogel, 3 D GelMA hydrogel, and 2 D culture dish) and chemical factors (serum, ascorbic acid, insulin, and ß-FGF) are examined to study their effects on the differentiation of limbal stromal stem cells to keratocytes or fibroblasts and the phenotype maintenance, in vitro and in vivo tissue regeneration. The results demonstrate that fiber hydrogel and serum-free media synergize to provide an optimal environment for the maintenance of keratocyte phenotype and the regeneration of damaged corneal stroma.


Assuntos
Substância Própria/fisiologia , Gelatina/farmacologia , Hidrogéis/farmacologia , Metacrilatos/farmacologia , Poliésteres/farmacologia , Polietilenoglicóis/farmacologia , Regeneração , Animais , Substância Própria/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Limbo da Córnea/citologia , Masculino , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Estresse Mecânico , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Suínos , Tecidos Suporte/química , Vimentina/metabolismo
14.
Am J Physiol Renal Physiol ; 318(5): F1160-F1166, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174141

RESUMO

Renal fibrosis is a major contributor to the development and progression of chronic kidney disease. A low-protein diet can reduce the progression of chronic kidney disease and reduce the development of renal fibrosis, although the mechanism is not well understood. Urea reabsorption into the inner medulla is regulated by inner medullary urea transporter (UT)-A1 and UT-A3. Inhibition or knockout of UT-A1/A3 will reduce interstitial urea accumulation, which may be beneficial in reducing renal fibrosis. To test this hypothesis, the effect of unilateral ureteral obstruction (UUO) was compared in wild-type (WT) and UT-A1/A3 knockout mice. UUO causes increased extracellular matrix associated with increases in transforming growth factor-ß, vimentin, and α-smooth muscle actin (α-SMA). In WT mice, UUO increased the abundance of three markers of fibrosis: transforming growth factor-ß, vimentin, and α-SMA. In contrast, in UT-A1/A3 knockout mice, the increase following UUO was significantly reduced. Consistent with the Western blot results, immunohistochemical staining showed that the levels of vimentin and α-SMA were increased in WT mice with UUO and that the increase was reduced in UT-A1/A3 knockout mice with UUO. Masson's trichrome staining showed increased collagen in WT mice with UUO, which was reduced in UT-A1/A3 knockout mice with UUO. We conclude that reduced UT activity reduces the severity of renal fibrosis following UUO.


Assuntos
Nefropatias/metabolismo , Rim/patologia , Proteínas de Membrana Transportadoras/deficiência , Obstrução Ureteral/complicações , Actinas/metabolismo , Animais , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibrose , Rim/metabolismo , Nefropatias/etiologia , Nefropatias/patologia , Nefropatias/prevenção & controle , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/metabolismo
15.
Toxicol Appl Pharmacol ; 394: 114956, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32171571

RESUMO

Proper enterocytic proliferation/differentiation, besides providing adequate adherens junctions (AJ) integrity, are responsible for strengthening of the gut barrier that acts as a first line defense against endotoxemia. However, the preferential role of the underlying PI3K/Akt (PKB) axis in triggering enterocytic proliferation/differentiation signaling and AJ assembly is still obscure in sepsis. Additionally, the potential involvement of dipeptidyl peptidase (DPP)-IV in cholestatic sepsis has not yet been reported. Common bile duct ligation (CBDL) insult was performed in adult male Sprague-Dawley rats except for sham operated animals; three doses of vildagliptin (VLD3, 10 and 30 mg/kg/d; p.o) were administered for 10 consecutive days post CBDL. VLD3/10/30 dose-dependently decreased DPP-IV and elevated GLP-1, IGF-1, PI3K, pS473-Akt (PKB), pS9-GSK-3ß, pS133-CREB and cyclin-D1. VLD3/10 reduced fever, portal/aortic endotoxin and IgG, body weight loss as well as ileal NF-κB, TNF-α, MPO, TBARS, subepithelial/pericryptal and submucosal collagen deposition, vimentin immunoreactivity, N-cadherin, Zeb1 and pY654-ß-catenin but increased E-cadherin, NPSH and colon/spleen indices - effects that were quite the opposite of VLD30. Accordingly, maintaining proper enterocytic proliferation/differentiation and phosphorylation inputs consequent to adequate DPP-IV inhibition is integral to AJ assembly in cholestatic sepsis; however, perturbed signals by excessive suppression of the enzyme activity induce toxic effects manifested as AJ disassembly and EMT, hence gut leakage and overt endotoxemia.


Assuntos
Colestase/patologia , Inibidores da Dipeptidil Peptidase IV/farmacologia , Enterócitos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Sepse/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Colestase/tratamento farmacológico , Ducto Colédoco/lesões , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Íleo/patologia , Ligadura , Masculino , Ratos , Ratos Sprague-Dawley , Sepse/tratamento farmacológico , Vildagliptina/farmacologia , Vimentina/metabolismo , Perda de Peso/efeitos dos fármacos
16.
Proc Natl Acad Sci U S A ; 117(14): 8001-8012, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193336

RESUMO

The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Melanoma Experimental/patologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/genética , Feminino , Dosagem de Genes , Humanos , Masculino , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/mortalidade , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Prognóstico , Pele/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Vimentina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Commun ; 11(1): 834, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047143

RESUMO

The protein inhibitor of activated STAT1 (PIAS1) is an E3 SUMO ligase that plays important roles in various cellular pathways. Increasing evidence shows that PIAS1 is overexpressed in various human malignancies, including prostate and lung cancers. Here we used quantitative SUMO proteomics to identify potential substrates of PIAS1 in a system-wide manner. We identified 983 SUMO sites on 544 proteins, of which 62 proteins were assigned as putative PIAS1 substrates. In particular, vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, was SUMOylated by PIAS1 at Lys-439 and Lys-445 residues. VIM SUMOylation was necessary for its dynamic disassembly and cells expressing a non-SUMOylatable VIM mutant showed a reduced level of migration. Our approach not only enables the identification of E3 SUMO ligase substrates but also yields valuable biological insights into the unsuspected role of PIAS1 and VIM SUMOylation on cell motility.


Assuntos
Movimento Celular/fisiologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteômica , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas Inibidoras de STAT Ativados/genética , Mapas de Interação de Proteínas , Proteína SUMO-1/genética , Análise de Sequência de Proteína , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Vimentina/metabolismo
18.
Gene ; 735: 144404, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32018013

RESUMO

Glucose uptake in adipocytes is crucial for regulating systemic metabolism. Long noncoding RNAs (lncRNAs), defined as being transcripts with lengths exceeding 200 nucleotides that are not translated, are recently identified regulators of cellular functions. Previously, we have shown that an lncRNA, "down-regulated expression by hepatitis B virus X" (dreh), is involved in glucose transport in skeletal muscle cells. Here, we aimed to examine the involvement of dreh in glucose transport in 3T3-L1 adipocytes. Expression analysis showed that dreh was expressed in 3T3-L1 fibroblasts and adipocytes. Knockdown of dreh expression using its specific siRNAs lowered the glucose concentration of the medium and facilitated [3H]-2-deoxyglucose transport in adipocytes. Additionally, dreh silencing enhanced the protein expression of glucose transporter (GLUT4) in the plasma membrane of adipocytes. Treatment with siRNA against vimentin attenuated the glucose-lowering effect of dreh depletion. These results suggest that the repression of dreh facilitates glucose transport via increased GLUT4 expression in the plasma membrane through the involvement of vimentin in 3T3-L1 adipocytes. In conclusion, dreh is the first observed lncRNA that regulates glucose transport in adipocytes and could serve as a novel therapeutic target for diabetes by modulating adipocyte function. Considering the new function of dreh, we propose that dreh be renamed "down-regulated expression-related hexose/glucose transport enhancer."


Assuntos
Adipócitos/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , RNA Longo não Codificante/genética , Vimentina/metabolismo , Animais , Linhagem Celular , Fibroblastos/metabolismo , Camundongos , RNA Longo não Codificante/metabolismo
19.
Life Sci ; 248: 117469, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32109485

RESUMO

AIMS: Histone deacetylases inhibitors have shown favorable antitumor activity in clinical investigations. In the present study, we assessed the effects of a novel hydroxamic acid-based HDAC inhibitor, SB939, on breast cancer metastasis and tumor growth and characterized the underlying molecular mechanisms. MAIN METHODS: MTS, Wound-healing, and Transwell chamber invasion assays were used to detect the inhibition effects of SB939 on proliferation, migration, and invasion of breast cancer cells. Western blot, cellular immunofluorescence, and EMSA were used to explore the molecular mechanism of SB939 in suppressing breast cancer metastasis. MDA-MB-231 subcutaneous tumor-bearing model of nude mice and the spontaneous metastasis model of breast cancer were both applied to verify in vivo anti-tumor growth and anti-metastatic effects. KEY FINDINGS: Our results demonstrated that SB939 at 0.5-1 µmol/L markedly impaired the chemotactic motility of breast cancer cells. SB939 reversed epithelial-mesenchymal transition (EMT) process, as evidenced by upregulation E-cadherin expression and downregulation expressions of N-cadherin and vimentin through increasing the levels of ac-histone H3 and H4 and drecreasing the expressiongs of HDAC 5 and 4. This cascade inhibition mediated by SB939 was well interpreted by inactivating phosphorylation of STAT3, blocking its DNA-binding activity, and decreasing the expressions of STAT3-dependent target genes, including MMP2 and MMP9. Furhtermore, we found that SB939 significantly inhibited breast cancer metastasis and tumor growth in vivo and showed superior anti-tumor properties compared with SAHA in two breast cancer animal models. SIGNIFICANCE: Our findings indicate that SB939 may be an effective therapeutic option for treating advanced breast cancer.


Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Vimentina/genética , Vimentina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Artigo em Chinês | MEDLINE | ID: mdl-32086930

RESUMO

Sarcomatoid carcinoma, a malignant tumor containing both epithelial-derived malignant cells and malignant mesenchymal cells. Microscopically, cancer cells and sarcoma cells migrate to each other, and CK and Vimentin are simultaneously expressed. A rare case of tonsillar sarcomatoid carcinoma is discussed in our department with dysphagia as the first symptom.


Assuntos
Carcinoma/diagnóstico , Tonsila Palatina/patologia , Sarcoma/diagnóstico , Neoplasias Tonsilares/diagnóstico , Transtornos de Deglutição/etiologia , Humanos , Vimentina/metabolismo
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