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1.
Nat Commun ; 12(1): 863, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558489

RESUMO

A concept of polyclonal metastasis has recently been proposed, wherein tumor cell clusters break off from the primary site and are disseminated. However, the involvement of driver mutations in such polyclonal mechanism is not fully understood. Here, we show that non-metastatic AP cells metastasize to the liver with metastatic AKTP cells after co-transplantation to the spleen. Furthermore, AKTP cell depletion after the development of metastases results in the continuous proliferation of the remaining AP cells, indicating a role of AKTP cells in the early step of polyclonal metastasis. Importantly, AKTP cells, but not AP cells, induce fibrotic niche generation when arrested in the sinusoid, and such fibrotic microenvironment promotes the colonization of AP cells. These results indicate that non-metastatic cells can metastasize via the polyclonal metastasis mechanism using the fibrotic niche induced by malignant cells. Thus, targeting the fibrotic niche is an effective strategy for halting polyclonal metastasis.


Assuntos
Metástase Neoplásica/patologia , Neoplasias/genética , Neoplasias/patologia , Animais , Agregação Celular , Proliferação de Células , Células Clonais , Fibrose , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/irrigação sanguínea , Fígado/patologia , Camundongos Endogâmicos NOD , Organoides/patologia , Fenótipo , Baço/transplante , Fator de Crescimento Transformador beta/farmacologia
2.
Anal Chem ; 93(4): 2694-2705, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33397101

RESUMO

Glycoproteins secreted by cells play essential roles in the regulation of extracellular activities. Secreted glycoproteins are often reflective of cellular status, and thus glycoproteins from easily accessible bodily fluids can serve as excellent biomarkers for disease detection. Cultured cells have been extensively employed as models in the research fields of biology and biomedicine, and global analysis of glycoproteins secreted from these cells provides insights into cellular activities and glycoprotein functions. However, comprehensive identification and quantification of secreted glycoproteins is a daunting task because of their low abundances compared with the high-abundance serum proteins required for cell growth and proliferation. Several studies employed serum-free media to analyze secreted proteins, but it has been shown that serum starvation, even for a short period of time, can alter protein secretion. To overcome these issues, we developed a method to globally characterize secreted glycoproteins and their N-glycosylation sites from cultured cells by combining selective enrichment of secreted glycoproteins with a boosting approach. The results demonstrated the importance of the boosting sample selection and the boosting-to-sample ratio for improving the coverage of secreted glycoproteins. The method was applied to globally quantify secreted glycoproteins from THP-1 monocytes and macrophages in response to lipopolysaccharides (LPS) and from Hep G2 cells treated with TGF-ß without serum starvation. We found differentially secreted glycoproteins in these model systems that showed the cellular response to the immune activation or the epithelial-to-mesenchymal transition. Benefiting from the selective enrichment and the signal enhancement of low-abundance secreted glycoproteins, this method can be extensively applied to study secreted glycoproteins without serum starvation, which will provide a better understanding of protein secretion and cellular activity.


Assuntos
Glicoproteínas/química , Técnicas de Cultura de Células , Química Click , Glicoproteínas/metabolismo , Células Hep G2 , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Peptídeos/química , Fator de Crescimento Transformador beta/farmacologia
3.
Int J Nanomedicine ; 15: 8345-8356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154637

RESUMO

Purpose: In this study, chitosan/alginate nanoparticles are prospected as a carrier for controlled release of recombinant human bone morphogenetic protein-2 (rhBMP-2). Materials and Methods: The rhBMP-2-loaded chitosan/alginate nanoparticles (Cs/Alg/B NPs) were prepared using the ionic gelation (IG) method. The current research was conducted to optimize the effective factors for entrapping rhBMP-2 in Cs/Alg NPs using response surface methodology (RSM) and the Box-Behnken design (BBD). The variables were the Cs/Alg molecular weight (Mw) ratios (1-3), pH (4.8-5.5), stirring rates (900-1300 rpm) and the responses included size, ζ-potential, polydispersity index (PDI), loading efficacy (LE), cumulative release (CR), and morphological degradation time (MDE). Then, the morphological properties of optimum formulation were studied for post-characterization. In the next step, the MTT assay for the optimized run was done for 24 and 48 hours. Results: The results revealed that the optimum conditions for the mentioned variables were stirring rate=1100 rpm, pH=5.15, and Cs/Alg Mw ratio=1.75 based on numerical optimization. It was shown that the average particle size and loading efficacy at optimum conditions were 253 nm and 67%, respectively. Other responses were as follows: CR=66%, ζ-potential=+35mV, PDI=0.5, and MDT=7 days. Conclusion: The results have suggested that the statistical optimization of rhBMP-2 offers the possibility of preparing Cs/Alg/B NPs with a favorable size, controlled release characteristics, and high loading efficiency. It is expected that the acquired optimum conditions will be useful for efficient rhBMP-2 delivery.


Assuntos
Alginatos/química , Proteína Morfogenética Óssea 2/farmacologia , Quitosana/química , Portadores de Fármacos/química , Nanopartículas/química , Estatística como Assunto , Fator de Crescimento Transformador beta/farmacologia , Alginatos/toxicidade , Animais , Quitosana/toxicidade , Liberação Controlada de Fármacos , Humanos , Camundongos , Células NIH 3T3 , Nanopartículas/ultraestrutura , Tamanho da Partícula , Proteínas Recombinantes/farmacologia , Eletricidade Estática
4.
Int J Nanomedicine ; 15: 8465-8478, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33149587

RESUMO

Introduction: Decellularized matrix from porcine small intestinal submucosa (SIS) endows scaffolds with an ECM-like surface, which enhances stem cell self-renewal, proliferation, and differentiation. Mesoporous bioactive glass (MBG) is extensively recognized as an excellent bio-ceramic for fabricating bone grafts. Materials and Methods: In the current study, SIS was doped on an MBG scaffold (MBG/SIS) using polyurethane foam templating and polydopamine chemistry method. To mimic the bony environment of a natural bone matrix, an ECM-inspired delivery system was constructed by coupling the BMP2-related peptide P28 to a heparinized MBG/SIS scaffold (MBG/SIS-H-P28). The release of P28 from MBG/SIS-H-P28 and its effects on the proliferation, viability, and osteogenic differentiation of bone marrow stromal stem cells were investigated in vitro and in vivo. Results: Our research indicated that the novel tissue-derived ECM scaffold MBG/SIS has a hierarchical and interconnected porous architecture, and superior biomechanical properties. MBG/SIS-H-P28 released P28 in a controlled manner, with the long-term release time of 40 d. The results of in vitro experiments showed improvements in cell proliferation, cell viability, alkaline phosphatase activity, and mRNA expression levels of osteogenesis-related genes (Runx-2, OCN, OPN, and ALP) compared to those of MBG/SIS or MBG/SIS-P28 and MBG/SIS-H-P28. The in vivo results demonstrated that MBG/SIS-H-P28 scaffolds evidently increased bone formation in rat calvarial critical-sized defect compared to that in controls. Conclusion: MBG/SIS-H-P28 scaffolds show potential as ideal platforms for delivery of P28 and for providing a bony environment for bone regeneration.


Assuntos
Ácido Aspártico/química , Materiais Biocompatíveis/farmacologia , Proteína Morfogenética Óssea 2/farmacologia , Osso e Ossos/efeitos dos fármacos , Cerâmica/farmacologia , Matriz Extracelular/metabolismo , Osteoblastos/efeitos dos fármacos , Peptídeos/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Regeneração Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , Masculino , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Porosidade , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Suínos , Tecidos Suporte/química
5.
PLoS One ; 15(9): e0239396, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32966314

RESUMO

Despite recent progress in the treatment of rheumatoid arthritis (RA), many patients still fail to achieve remission or low disease activity. An imbalance between auto-reactive effector T cells (Teff) and regulatory T cells (Treg) may contribute to joint inflammation and damage in RA. Therefore, restoring this balance is a promising approach for the treatment of inflammatory arthritis. Accordingly, our group has previously shown that the combination of TGF-ß-releasing microparticles (MP), rapamycin-releasing MP, and IL-2-releasing MP (TRI MP) can effectively increase the ratio of Tregs to Teff in vivo and provide disease protection in several preclinical models. In this study TRI MP was evaluated in the collagen-induced arthritis (CIA) model. Although this formulation has been tested previously in models of destructive inflammation and transplantation, this is the first model of autoimmunity for which this therapy has been applied. In this context, TRI MP effectively reduced arthritis incidence, the severity of arthritis scores, and bone erosion. The proposed mechanism of action includes not only reducing CD4+ T cell proliferation, but also expanding a regulatory population in the periphery soon after TRI MP administration. These changes were reflected in the CD4+ T cell population that infiltrated the paws at the onset of arthritis and were associated with a reduction of immune infiltrate and inflammatory myeloid cells in the paws. TRI MP administration also reduced the titer of collagen antibodies, however the contribution of this reduced titer to disease protection remains uncertain since there was no correlation between collagen antibody titer and arthritis score.


Assuntos
Artrite Experimental/prevenção & controle , Interleucina-2/farmacologia , Microesferas , Sirolimo/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Autoanticorpos/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Contagem de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Liberação Controlada de Fármacos , Interleucina-2/química , Masculino , Camundongos , Sirolimo/química , Fator de Crescimento Transformador beta/química
6.
PLoS One ; 15(9): e0239188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946467

RESUMO

Epithelial-to-mesenchymal transition (EMT) and maturation of a fibrillar tumor microenvironment play important roles in breast cancer progression. A better understanding of how these events promote cancer cell migration and invasion could help identify new strategies to curb metastasis. The nucleus and Golgi affect migration in a microenvironment-dependent manner. Nucleus size and mechanics influence the ability of a cell to squeeze through confined tumor microenvironments. Golgi positioning determines front-rear polarity necessary for migration. While the roles of individual attributes of nucleus and Golgi in migration are being clarified, how their manifold features are inter-related and work together remains to be understood at a systems level. Here, to elucidate relationships among nucleus and Golgi properties, we quantified twelve morphological and positional properties of these organelles during fibrillar migration of human mammary epithelial cells. Principal component analysis (PCA) reduced the twelve-dimensional space of measured properties to three principal components that capture 75% of the variations in organelle features. Unexpectedly, nucleus and Golgi properties that co-varied in a PCA model built with data from untreated cells were largely similar to co-variations identified using data from TGFß-treated cells. Thus, while TGFß-mediated EMT significantly alters gene expression and motile phenotype, it did not significantly affect the relationships among nucleus size, aspect ratio and orientation with migration direction and among Golgi size and nucleus-Golgi separation distance. Indeed, in a combined PCA model incorporating data from untreated and TGFß-treated cells, scores of individual cells occupy overlapping regions in principal component space, indicating that TGFß-mediated EMT does not promote a unique "Golgi-nucleus phenotype" during fibrillar migration. These results suggest that migration along spatially-confined fiber-like tracks employs a conserved nucleus-Golgi arrangement that is independent of EMT state.


Assuntos
Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular , Movimento Celular , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Fator de Crescimento Transformador beta/farmacologia , Microambiente Tumoral
7.
Phytomedicine ; 78: 153294, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32771890

RESUMO

BACKGROUND: Hepatic fibrosis is considered integral to the progression of chronic liver diseases, as it leads to the development of cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) is the dominant event in hepatic fibrogenesis. The transforming growth factor-ß1 (TGF-ß1) and Yes-associated protein (YAP) pathways play a pivotal role in HSC activation, hepatic fibrosis and cirrhosis progression. Therefore, targeting the TGF-ß/Smad and YAP signaling pathways is a promising strategy for antifibrotic therapy. PURPOSE: The present study investigated the protective effects of Physalin D (PD), a withanolide isolated from Physalis species (Solanaceae), against liver fibrosis and further elucidated the mechanisms involved in vitro and in vivo. STUDY DESIGN/METHODS: We conducted a series of experiments using carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced fibrotic mice and cultured LX-2 cells. Serum markers of liver injury, and the morphology, histology and fibrosis of liver tissue were investigated. Western blot assays and quantitative real-time PCR were used to investigate the mechanisms underlying the antifibrotic effects of PD. RESULT: PD decreased TGF-ß1-induced COL1A1 promoter activity. PD inhibited TGF-ß1-induced expression of Collagen I and α-smooth muscle actin (α-SMA) in human hepatic stellate LX-2 cells. PD significantly ameliorated hepatic injury, including transaminase activities, histology, collagen deposition and α-SMA, in CCl4- or BDL-induced mice. Moreover, PD markedly decreased the expression of phosphorylated Smad2/3 in vitro and in vivo. Furthermore, PD significantly decreased YAP protein levels, and YAP knockdown did not further enhance the effects of PD, namely α-SMA inhibition, Collagen I expression and YAP target gene expression in LX-2 cells. CONCLUSION: These results clearly show that PD ameliorated experimental liver fibrosis by inhibiting the TGF-ß/Smad and YAP signaling pathways, indicating that PD has the potential to effectively treat liver fibrosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Secoesteroides/farmacologia , Proteínas Smad/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Tetracloreto de Carbono/toxicidade , Células Cultivadas , Colágeno Tipo I/genética , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1
8.
Phytomedicine ; 78: 153298, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32781391

RESUMO

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a progressive inflammatory disorder driven by a fibrotic cascade of events such as epithelial to mesenchymal transition, extracellular matrix production and collagen formation in the lungs in a sequential manner. IPF incidences were raising rapidly across the world. FDA approved pirfenidone and nintedanib (tyrosine kinase inhibitors) are being used as a first-line treatment drugs for IPF, however, neither the quality of life nor survival rates have been improved because of patient noncompliance due to multiple side effects. Thus, the development of novel therapeutic approaches targeting TGF-ß mediated cascade of fibrotic events is urgently needed to improve the survival of the patients suffering from devastating disease. PURPOSE: The aim of this study was to investigate and validate the anti-fibrotic properties of Biochanin-A (isoflavone) against TGF-ß mediated fibrosis in in vitro, ex vivo, in vivo models and to determine the molecular mechanisms that mediate these anti-fibrotic effects. METHODS: The therapeutic activity of BCA was determined in in vitro/ex vivo models. Cells were pre-treated with BCA and incubated in presence or absence of recombinant-TGF-ß to stimulate the fibrotic cascade of events. Pulmonary fibrosis was developed by intratracheal administration of bleomycin in rats. BCA treatment was given for 14 days from post bleomycin instillation and then various investigations (collagen content, fibrosis gene/protein expression and histopathological changes) were performed to assess the anti-fibrotic activity of BCA. RESULTS: In vitro/ex vivo (Primary normal, IPF cell line and primary IPF cells/ Precision cut mouse lung slices) experiments revealed that, BCA treatment significantly (p < 0.001) reduced the expression of TGF-ß modulated fibrotic genes/protein expressions (including their functions) which are involved in the cascade of fibrotic events. BCA treatment significantly (p < 0.01) reduced the bleomycin-induced inflammatory cell-infiltration, inflammatory markers expression, collagen deposition and expression of fibrotic markers in lung tissues equivalent or better than pirfenidone treatment. In addition, BCA treatment significantly (p < 0.001) attenuated the TGF-ß1/BLM-mediated increase of TGF-ß/Smad2/3 phosphorylation and resulted in the reduction of pathological abnormalities in lung tissues determined by histopathology observations. CONCLUSION: Collectively, BCA treatment demonstrated the remarkable therapeutic effects on TGF-ß/BLM mediated pulmonary fibrosis using IPF cells and rodent models. This current study may offer a novel treatment approach to halt and may be even rescue the devastating lung scarring of IPF.


Assuntos
Colágeno/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Genisteína/farmacologia , Miofibroblastos/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Animais , Bleomicina/toxicidade , Diferenciação Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Humanos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Ratos Wistar , Reprodutibilidade dos Testes , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-32384749

RESUMO

Over-induction of epithelial to mesenchymal transition (EMT) by tumor growth factor beta (TGFß) in keratinocytes is a key feature in keloid scar. The present work seeks to investigate the effect of Kelulut honey (KH) on TGFß-induced EMT in human primary keratinocytes. Image analysis of the real time observation of TGFß-induced keratinocytes revealed a faster wound closure and individual migration velocity compared to the untreated control. TGFß-induced keratinocytes also have reduced circularity and display a classic EMT protein expression. Treatment of 0.0015% (v/v) KH reverses these effects. In untreated keratinocytes, KH resulted in slower initial wound closure and individual migration velocity, which sped up later on, resulting in greater wound closure at the final time point. KH treatment also led to greater directional migration compared to the control. KH treatment caused reduced circularity in keratinocytes but displayed a partial EMT protein expression. Taken together, the findings suggest the therapeutic potential of KH in preventing keloid scar by attenuating TGFß-induced EMT.


Assuntos
Transição Epitelial-Mesenquimal , Mel/análise , Queratinócitos/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Cicatrização , Movimento Celular , Humanos , Masculino
10.
Cancer Sci ; 111(7): 2385-2399, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32385953

RESUMO

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer-associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF-ß, which induces endothelial-to-mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor-infiltrating inflammatory cells secrete various cytokines, including TNF-α. However, the role of TNF-α in TGF-ß-induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF-α on TGF-ß-induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF-ß and TNF-α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF-ß and TNF-α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF-ß type I receptor, TGF-ß2, activin A, and integrin αv, suggesting that TNF-α enhanced TGF-ß-induced EndMT by augmenting TGF-ß family signals. Furthermore, oral squamous cell carcinoma-derived cells underwent epithelial-to-mesenchymal transition (EMT) in response to humoral factors produced by TGF-ß and TNF-α-cultured ECs. This EndMT-driven EMT was blocked by inhibiting the action of TGF-ßs. Collectively, our findings suggest that TNF-α enhances TGF-ß-dependent EndMT, which contributes to tumor progression.


Assuntos
Transição Epitelial-Mesenquimal , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Biomarcadores , Fibroblastos Associados a Câncer/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Células Cultivadas , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , NF-kappa B/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Microambiente Tumoral/genética , Fator de Necrose Tumoral alfa/farmacologia
11.
Environ Toxicol ; 35(9): 942-951, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32270919

RESUMO

Long noncoding RNA (lncRNA) AC026904.1 has been confirmed to be necessary for breast cancer metastasis. This work aims to investigate the effects of lncRNA AC026904.1 on lung cancer metastasis. We found that lncRNA AC026904.1 displayed a higher level in metastatic lung cancer tissues than adjacent tissues and nonmetastatic lung cancer tissues, and lung cancer cells treated with TGF-ß. The expression of AC026904.1 was increased by the non-canonical TGF-ß signaling. Additionally, AC026904.1 acts as an enhancer of the key metastatic factor Slug in the nucleus. This AC026904.1/Slug axis is necessary for TGF-ß-mediated migration and epithelial-mesenchymal transition in lung cancer cells. This work firstly uncovers that AC026904.1 increases Slug expression at transcriptional level and subsequently plays critical effects in lung cancer metastasis, providing novel evidences that AC026904.1 holds great potential to be used as a marker for metastatic lung cancer.


Assuntos
Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/patologia , Pulmão/patologia , RNA Longo não Codificante/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metástase Neoplásica , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail/genética , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima
12.
Sci Rep ; 10(1): 6550, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32300237

RESUMO

Human γδ T cells are potent cytotoxic effector cells, produce a variety of cytokines, and can acquire regulatory activity. Induction of FOXP3, the key transcription factor of regulatory T cells (Treg), by TGF-ß in human Vγ9 Vδ2 T cells has been previously reported. Vitamin C is an antioxidant and acts as multiplier of DNA hydroxymethylation. Here we have investigated the effect of the more stable phospho-modified Vitamin C (pVC) on TGF-ß-induced FOXP3 expression and the resulting regulatory activity of highly purified human Vγ9 Vδ2 T cells. pVC significantly increased the TGF-ß-induced FOXP3 expression and stability and also increased the suppressive activity of Vγ9 Vδ2 T cells. Importantly, pVC induced hypomethylation of the Treg-specific demethylated region (TSDR) in the FOXP3 gene. Genome-wide methylation analysis by Reduced Representation Bisulfite Sequencing additionally revealed differentially methylated regions in several important genes upon pVC treatment of γδ T cells. While Vitamin C also enhances effector functions of Vγ9 Vδ2 T cells in the absence of TGF-ß, our results demonstrate that pVC potently increases the suppressive activity and FOXP3 expression in TGF-ß-treated Vγ9 Vδ2 T cells by epigenetic modification of the FOXP3 gene.


Assuntos
Ácido Ascórbico/farmacologia , Epigênese Genética/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Desmetilação , Humanos , Fosforilação/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia
13.
Arch Biochem Biophys ; 687: 108384, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32343974

RESUMO

Epithelial mesenchymal transition (EMT) is a well-known and important step in metastasis and thus can be a key target in cancer treatment. Here, we tested the EMT inhibitory actions of Selaginella tamariscina and its active component, amentoflavone (AF). EMT was examined in vitro using wound-healing and invasion assays and by monitoring changes in the expression of the EMT-related proteins, E-cadherin, Snail, and Twist. Metastasis was examined in vivo using SCID mice injected with luciferase-labeled A549 cells. We confirmed that aqueous extracts of S. tamariscina (STE) and AF inhibited EMT in human cancer cell lines. We found that STE and AF at nontoxic concentrations exerted remarkable inhibitory effects on migration (wound healing assay) and invasion (Transwell assay) in tumor necrosis factor (TGF)-ß-treated cancer cells. Western blotting and immunofluorescence imaging show that AF treatment also restored E-cadherin expression in these cells compared to cells treated with TGF-ß only. Suppression of metastasis by AF was investigated by monitoring migration of tail-vein-injected, circulating A549-luc cells to the lungs in mice. After 3 wk, fewer nodules were observed in mice co-treated with AF compared with those treated with TGF-ß only. Our findings indicate that STE and AF are promising EMT inhibitors and, ultimately, potentially potent antitumor agents.


Assuntos
Antineoplásicos/uso terapêutico , Biflavonoides/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Metástase Neoplásica/prevenção & controle , Selaginellaceae/química , Células A549 , Animais , Antígenos CD/metabolismo , Antineoplásicos/farmacologia , Biflavonoides/farmacologia , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Camundongos SCID , Proteínas Nucleares/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteína 1 Relacionada a Twist/metabolismo
14.
Nat Commun ; 11(1): 1749, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273499

RESUMO

Transforming growth factor beta (TGFß) is a multipotent immunosuppressive cytokine. TGFß excludes immune cells from tumors, and TGFß inhibition improves the efficacy of cytotoxic and immune therapies. Using preclinical colorectal cancer models in cell type-conditional TGFß receptor I (ALK5) knockout mice, we interrogate this mechanism. Tumor growth delay and radiation response are unchanged in animals with Treg or macrophage-specific ALK5 deletion. However, CD8αCre-ALK5flox/flox (ALK5ΔCD8) mice reject tumors in high proportions, dependent on CD8+ T cells. ALK5ΔCD8 mice have more tumor-infiltrating effector CD8+ T cells, with more cytotoxic capacity. ALK5-deficient CD8+ T cells exhibit increased CXCR3 expression and enhanced migration towards CXCL10. TGFß reduces CXCR3 expression, and increases binding of Smad2 to the CXCR3 promoter. In vivo CXCR3 blockade partially abrogates the survival advantage of an ALK5ΔCD8 host. These data demonstrate a mechanism of TGFß immunosuppression through inhibition of CXCR3 in CD8+ T cells, thereby limiting their trafficking into tumors.


Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Neoplasias/genética , Receptores CXCR3/genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptores CXCR3/metabolismo , Proteína Smad2/metabolismo
15.
Phytomedicine ; 69: 153192, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32200292

RESUMO

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells is the main pathological alteration in diabetic nephropathy (DN). Traditional Chinese medicine (TCM) has been used for the treatment of DN in clinical practice and has been proven to be effective. PURPOSE: This aim of this study was to shed light on the efficacy of Shenxiao decoction (SXD) on the EMT of renal tubular epithelial cells and the molecular mechanisms of SXD in mice with DN, as well as on the high glucose (HG)- and TGF-ß1-induced EMT of NRK-52E and HK-2 cells. STUDY DESIGN AND METHODS: A bioinformatics and network pharmacology method were utilized to construct the active ingredient-target networks of SXD that were responsible for the beneficial effects against DN. The effects of RUNX3 were validated in HG- and TGF-ß1-induced EMT processes in NRK-52E and HK-2 cells. RESULTS: Bioinformatics analysis revealed that 122 matching targets were closely associated with the regulation of cell migration and the AGE-RAGE signaling pathway in diabetic complications. The results also revealed that, relative to the mice with DN, the mice in the treatment group had an improved general state and reduced blood glucose levels. The degradation of renal function was ameliorated by SXD. Moreover, the protective effects of SXD were also observed on renal structural changes. Furthermore, SXD suppressed the activation of the transforming growth factor (TGF)-ß1/Smad pathway and upregulated the RUNX3 and E-cadherin levels and downregulated the extracellular matrix (ECM) protein levels in mice with DN. SXD was further found to prevent the HG- and TGF-ß1-induced EMT processes in NRK-52E and HK-2 cells. Additionally, the overexpression of RUNX3 markedly inhibited the EMT and TGF-ß1/Smad pathway induced by HG and TGF-ß1 in NRK-52E and HK-2 cells. CONCLUSION: Taken together, these results suggest that SXD maybe alleviate EMT in DN via the inhibition of the TGF-ß1/Smad/RUNX3 signaling pathway under hyperglycemic conditions.


Assuntos
Biologia Computacional/métodos , Nefropatias Diabéticas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Animais , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas , Ratos , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima/efeitos dos fármacos
16.
Phytomedicine ; 69: 153185, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32120244

RESUMO

BACKGROUND: Dihydroquercetin (DHQ) is an antifibrotic agent. However, whether DHQ can prevent renal fibrosis remains unknown. PURPOSE: This study aimed to investigate the effects of DHQ on tubulointerstitial fibrosis and its underlying mechanisms in unilateral ureteral obstruction (UUO) mice in vivo and NRK-49F cells in vitro. METHODS: In vivo, UUO mice received vehicle or DHQ treatment. In vitro, NRK-49F cells were pretreated with DHQ and exposed to transforming growth factor-ß1 (TGF-ß1). Changes in fibroblast activation, collagen synthesis, oxidative stress, and related signaling pathways were assessed by immunohistochemical staining, Western blot analysis, real-time reverse transcription-PCR, and fluorescence microscopy. RESULTS: UUO induced tubular atrophy, inflammation, fibroblast differentiation into myofibroblast, and collagen deposition, whereas DHQ ameliorated these effects. UUO also resulted in decreased levels of nuclear factor-erythroid-2-related factor 2 (Nrf2), catalase, and heme oxygenase-1, but increased H2O2 and malondialdehyde levels. DHQ treatment corrected these changes. In vitro, the intracellular Nrf2 level of NRK-49F exposed to TGF-ß1 decreased. However, DHQ rescued intracellular Nrf2 level and promoted nuclear translocation of Nrf2. DHQ scavenged TGF-ß1-induced accumulation of reactive oxygen species, inhibited TGF-ß1-induced Smad3 phosphorylation, and prevented TGF-ß1-induced fibroblast activation and collagen synthesis in NRK-49F. Nrf2 knockdown could suppress the DHQ-mediated inhibitory effects on oxidative stress, Smad3 phosphorylation, fibroblast activation, and collagen deposition. Furthermore, DHQ ameliorated established renal fibrosis in UUO mice. CONCLUSIONS: DHQ posed remarkable preventive and therapeutic effects on UUO-induced renal fibrosis and suppressed fibroblast activation by reducing oxidative stress and Smad3 phosphorylation via Nrf2 signaling. This study provided a mechanistic basis for the clinical application of DHQ in renal fibrosis treatment.


Assuntos
Nefropatias/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Substâncias Protetoras/farmacologia , Quercetina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Animais , Fibrose , Peróxido de Hidrogênio/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Quercetina/química , Quercetina/farmacologia , Ratos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
17.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L852-L863, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32159970

RESUMO

Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40-60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-ß-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Fatores de Transcrição/genética , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Comunicação Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
18.
Genes Cells ; 25(6): 375-390, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32181976

RESUMO

PMEPA1 (prostate transmembrane protein, androgen-induced 1)/TMEPAI (transmembrane prostate androgen-induced protein) is highly expressed in diverse cancers, including breast, lung and prostate cancers. It consists of four isoforms with distinct extracellular regions (isoforms a-d). The expression and function of these isoforms are still poorly understood. Hence, we aimed to identify the preferentially expressed isoforms in breast cancer cells and analyze possible differences in tumorigenic functions. In this study, we used 5' Rapid Amplification of cDNA Ends (RACE) and Western blot analyses to identify the mRNA variants and protein isoforms of TMEPAI and found that TMEPAI isoform d as the major isoform expressed by TGF-ß stimulation in breast cancer cells. We then generated CRISPR/Cas9-mediated TMEPAI knockout (KO) breast cancer cell lines and used a lentiviral expression system to complement each isoform individually. Although there were no clear functional differences between isoforms, double PPxY (PY) motifs and a Smad-interaction motif (SIM) of TMEPAI were both essential for colony and sphere formation. Collectively, our results provide a novel insight into TMEPAI isoforms in breast cancer cells and showed that coordination between double PY motifs and a SIM of TMEPAI are essential for colony and sphere formation but not for monolayer cell proliferation.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Proteínas Smad/metabolismo , Motivos de Aminoácidos , Animais , Neoplasias da Mama/genética , Células COS , Carcinogênese/genética , Proliferação de Células/genética , Chlorocebus aethiops , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Organoides/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Smad/genética , Fator de Crescimento Transformador beta/farmacologia
19.
Int J Mol Sci ; 21(4)2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32085622

RESUMO

Activating transcription factor-6 α (ATF6) is one of the three main sensors and effectors of the endoplasmic reticulum (ER) stress response and, as such, it is critical for protecting the heart and other tissues from a variety of environmental insults and disease states. In the heart, ATF6 has been shown to protect cardiac myocytes. However, its roles in other cell types in the heart are unknown. Here we show that ATF6 decreases the activation of cardiac fibroblasts in response to the cytokine, transforming growth factor ß (TGFß), which can induce fibroblast trans-differentiation into a myofibroblast phenotype through signaling via the TGFß-Smad pathway. ATF6 activation suppressed fibroblast contraction and the induction of α smooth muscle actin (αSMA). Conversely, fibroblasts were hyperactivated when ATF6 was silenced or deleted. ATF6 thus represents a novel inhibitor of the TGFß-Smad axis of cardiac fibroblast activation.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Miocárdio/patologia , Resposta a Proteínas não Dobradas , Animais , Biomarcadores/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Ventrículos do Coração/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Fator de Crescimento Transformador beta/farmacologia
20.
J Craniofac Surg ; 31(4): 912-915, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32068727

RESUMO

INTRODUCTION: The aim of this study was to assess the efficacy of using bone morphogenetic protein-2 with hydroxyapatite granules (BMP-2/hydroxyapatite) during augmentation of maxillary sinus floor, with respect to changes in volume, relative to conventional bone graft materials. METHOD AND MATERIALS: Twenty of 25 patients in the BMP-2/hydroxyapatite group, and 16 of 33 patients in the conventional materials group met the criteria for inclusion in this study. Computed tomography scans were performed preoperatively, immediately postoperatively, and at follow-up, approximately 6 months postoperatively. Changes in volume and height of both grafted materials were measured using 3-dimensional reconstruction software; these changes were compared between groups. RESULTS: The mean (standard deviation) volumetric changes were 0.25 (0.11) cc and -0.07 (0.35) cc, and the mean rates of volumetric changes were 26.44% (7.78%) and -2.92% (30.92%) in BMP-2/hydroxyapatite and conventional materials groups, respectively. The mean height changes were 0.34 (0.73) mm and -0.63 (1.07) mm, and the mean rates of height changes were 3.67% (7.57%) and -5.95% (9.98%) in BMP-2/hydroxyapatite and conventional materials groups, respectively. CONCLUSION: Compared with the conventional materials group, the BMP-2/hydroxyapatite group showed better maxillary sinus floor augmentation results in terms of volumetric changes and grafted material densities, and can provide predictably reliable outcomes.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Durapatita/farmacologia , Seio Maxilar , Levantamento do Assoalho do Seio Maxilar , Fator de Crescimento Transformador beta/farmacologia , Adulto , Feminino , Humanos , Masculino , Seio Maxilar/cirurgia , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia
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