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2.
Cell Prolif ; 53(8): e12858, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32592435

RESUMO

OBJECTIVES: Traditional cancer therapy and regular immunotherapy are ineffective for treating triple-negative breast cancer (TNBC) patients. Recently, chimeric antigen receptor-engineered natural killer cells (CAR NK) have been applied to target several hormone receptors on different cancer cells to improve the efficacy of immunotherapy. Furthermore, epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. Here, we demonstrated that EGFR-specific CAR NK cells (EGFR-CAR NK cells) could be potentially used to treat patients with TNBC exhibiting enhanced EGFR expression. MATERIALS AND METHODS: We investigated the cytotoxic effects of EGFR-CAR NK cells against TNBC cells in vitro and in vivo. The two types of EGFR-CAR NK cells were generated by transducing lentiviral vectors containing DNA sequences encoding the single-chain variable fragment (scFv) regions of the two anti-EGFR antibodies. The cytotoxic and anti-tumor effects of the two cell types were examined by performing cytokine release and cytotoxicity assays in vitro, and tumor growth assays in breast cancer cell line-derived xenograft (CLDX) and patient-derived xenograft (PDX) mouse models. RESULTS: Both EGFR-CAR NK cell types were activated by TNBC cells exhibiting upregulated EGFR expression and specifically triggered the lysis of the TNBC cells in vitro. Furthermore, the two EGFR-CAR NK cell types inhibited CLDX and PDX tumors in mice. CONCLUSIONS: This study suggested that treatment with EGFR-CAR NK cells could be a promising strategy for TNBC patients.


Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/imunologia , Humanos , Camundongos , Receptores de Antígenos Quiméricos/imunologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
4.
J Biomed Nanotechnol ; 16(3): 304-314, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32493541

RESUMO

We propose that nanogels (HLGs) prepared by simply blending an epidermal growth factor (EGF)-loaded hyaluronan (HA)-based nanoformulation and poloxamers can be efficient transdermal drug carriers. In particular, due to the thermogelling behavior of poloxamer, when the HLGs, which are liquid at room temperature, are applied to the skin's surface, they form a gel at skin temperature. First, lipid-based nanoformulations (EGF-LNs) were fabricated by the lipid thin film method and then chemically conjugated with HA on the surface of the films to prepare EGF-loaded HA-based nanoformulations (EGF-HLNs). Both EGF-LNs and EGF-HLNs exhibited a uniform size and spherical lamellar structure. The EGF-HLN was added to a poloxamer solution to form EGF-HLG, which is a liquid at room temperature and a gel at skin temperature. HLGs have been shown to be able to deliver and permeate EGF well into the skin using both in vitro and in vivo systems, thus serving as an effective transdermal delivery system. In addition, it has been confirmed that this system could be a possible implantable drug carrier. Therefore, HLGs, which are uncomplicated and easily prepared, are expected to be easily used not only in the pharmaceutical field but also in the cosmetic field.


Assuntos
Nanogéis , Cicatrização , Administração Cutânea , Portadores de Fármacos , Fator de Crescimento Epidérmico , Pele
5.
Cancer Sci ; 111(6): 2004-2015, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32227417

RESUMO

Epidermal growth factor receptor (EGFR) expression and activation are the major causes of metastasis in cancers such as head and neck squamous cell carcinoma (HNSCC). However, the reciprocal effect of EGF-induced COX-2 and angiopoietin-like 4 (ANGPTL4) on HNSCC metastasis remains unclear. In this study, we revealed that the expression of ANGPTL4 is essential for COX-2-derived prostaglandin E2 (PGE2 )-induced tumor cell metastasis. We showed that EGF-induced ANGPTL4 expression was dramatically inhibited with the depletion and inactivation of COX-2 by knockdown of COX-2 and celecoxib treatment, respectively. Prostaglandin E2 induced ANGPTL4 expression in a time- and dose-dependent manners in various HNSCC cell lines through the ERK pathway. In addition, the depletion of ANGPTL4 and MMP1 significantly impeded the PGE2 -induced transendothelial invasion ability of HNSCC cells and the binding of tumor cells to endothelial cells. The induction of molecules involved in the regulation of epithelial-mesenchymal transition was also dependent on ANGPTL4 expression in PGE2 -treated cells. The depletion of ANGPTL4 further blocked PGE2 -primed tumor cell metastatic seeding of lungs. These results indicate that the EGF-activated PGE2 /ANGPTL4 axis enhanced HNSCC metastasis. The concurrent expression of COX-2 and ANGPTL4 in HNSCC tumor specimens provides insight into potential therapeutic targets for the treatment of EGFR-associated HNSCC metastasis.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Ciclo-Oxigenase 2/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias de Cabeça e Pescoço/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Invasividade Neoplásica/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Regulação para Cima
6.
PLoS One ; 15(4): e0231466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298294

RESUMO

DaHuangWan (DHW) is a traditional herbal medicine used by Mongolian to treat liver cancer for many years. Clinical application of the drug has been shown to help control tumor progression, prolong survival and improve quality of life. However, the underlying mechanisms and side effects of this drug remain unclear, which greatly limits the clinical application and further optimization of DHW. In this study, we found that DHW inhibits the proliferation of hepatoma cells by modulating the epithelial growth factor (EGF) signaling pathway. Berberine and Costunolide are the main active ingredients in DHW. Interestingly, the combination of Berberine and Costunolide has a dramatic synergistic effect on inhibiting the proliferation of hepatoma cells. Neither Berberine nor Costunolide directly block EGFR phosphorylation. Berberine promotes endocytosis of activated EGFR, while as Costunolide increases ubiquitination of EGFR and reduces EGFR recycling to cell membrane distribution, thereby inhibiting EGF signaling. Berberine and Costunolide target two different steps in regulating the EGF signaling, which explains the synergistic anti-cancer effect of DHW. Since Berberine and Costunolide do not directly target EGFR phosphorylation, DHW could be a supplementary medicine to tyrosine kinase inhibitors in cancer therapy.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Fator de Crescimento Epidérmico/antagonistas & inibidores , Medicina Herbária/métodos , Neoplasias Hepáticas/tratamento farmacológico , Medicina Tradicional do Leste Asiático/métodos , Apoptose/efeitos dos fármacos , Berberina/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Mongólia , Plantas Medicinais , Sesquiterpenos/uso terapêutico
7.
Scand J Immunol ; 91(6): e12885, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32248557

RESUMO

Autosomal dominant hyper IgE syndrome (AD-HIES) caused by STAT3 gene mutation is a rare primary immunodeficiency disease. To better understand the disease, we described the clinical characteristics of 20 AD-HIES patients in Chongqing, China and explored the effect of mutations in different domains of STAT3 gene on the function of STAT3 protein by Western blot and confocal microscopy. The mean age at onset was 0.12 years. The mean age at diagnosis was 5.31 years. The most common presentation was eczema, pneumonia, skin abscesses and chronic mucocutaneous candidiasis. Seven patients suffered from BCG complications. R382W/Q were identified in 12 patients, V637M mutation in three patients. Three patients have died. The phosphorylated STAT3 was expressed more in wild-type(WT) and R382W mutant STAT3 in the cytoplasm of COS7 cells with epidermal growth factor(EGF) stimulation, less in the V637M mutation and T620S mutation. Dynamic observation showed that STAT3 cytoplasmic accumulation and nuclear translocation occurred rapidly after EGF stimulation in WT-STAT3-GFP, the time of accumulation and nuclear translocation was later and the expression was less in R382W-STAT3-GFP compared with WT-STAT3-GFP, followed by V637M and T620S mutation. These results suggested that our patients had earlier onset, diagnostic age and higher rate of BCG complications. However, our patients had higher incidence of mortality though the earlier diagnostic age. We did not find a significant genotype/phenotype correlation, but Src homology 2 domain mutations (V637M and T620S) had a greater effect on STAT3 phosphorylation and nuclear translocation than DNA-binding domain mutation (R382W) in vitro.


Assuntos
Genótipo , Síndrome de Job/diagnóstico , Mutação/genética , Fator de Transcrição STAT3/genética , Candidíase Cutânea , Criança , Pré-Escolar , China , Análise Mutacional de DNA , Eczema , Fator de Crescimento Epidérmico/metabolismo , Estudos de Associação Genética , Humanos , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Lactente , Síndrome de Job/mortalidade , Masculino , Pneumonia , Análise de Sobrevida
8.
Medicine (Baltimore) ; 99(17): e19456, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32332599

RESUMO

To explore the association between epidermal growth factor (EGF) 61A/G polymorphism and lung cancer.All eligible case-control studies published up to August, 2019 were identified by searching PubMed, The excerpta medica database, China Academic Journals Full-text Database, China Biology Medicine, China National Knowledge Infrastructure, China Science and Technology Journal Database, and Wanfang databases. Two researchers independently identified the literature, extracted data, and evaluated quality according to inclusion and exclusion criteria. Meta-analysis was performed by Stata 15.0.A total of 6 studies is included, including 1487 cases and 2044 control subjects. Compared with allele A, allele G was considered to have no association with the risk of lung cancer, odds ratio = 1.07 (95% confidence interval: 0.98-1.15). GG recessive genotype, GG + GA dominant genotype, GG homozygote genotype and GA heterozygote genotype were found out that all of them are not associated with the risk of lung cancer. No association between EGF 61A/G polymorphism and lung cancer was found out by ethnical subgroup analysis. However, in view of the limitations of this study, such as the results of quantitative and sensitivity analysis may be lack of accuracy, so the conclusions of allele model and recessive gene model should be made carefully.It suggested that there was no association between polymorphism of EGF 61A/G and susceptibility of lung cancer.


Assuntos
Fator de Crescimento Epidérmico/genética , Neoplasias Pulmonares/genética , Alelos , Predisposição Genética para Doença , Genótipo , Humanos , Estudos Observacionais como Assunto , Polimorfismo de Nucleotídeo Único , Fatores de Risco
9.
PLoS One ; 15(4): e0231250, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275673

RESUMO

Single-cell expression analysis is an effective tool for studying the dynamics of cell population profiles. However, the majority of statistical methods are applied to individual profiles and the methods for comparing multiple profiles simultaneously are limited. In this study, we propose a nonparametric statistical method, called Decomposition into Extended Exponential Family (DEEF), that embeds a set of single-cell expression profiles of several markers into a low-dimensional space and identifies the principal distributions that describe their heterogeneity. We demonstrate that DEEF can appropriately decompose and embed sets of theoretical probability distributions. We then apply DEEF to a cytometry dataset to examine the effects of epidermal growth factor stimulation on an adult human mammary gland. It is shown that DEEF can describe the complex dynamics of cell population profiles using two parameters and visualize them as a trajectory. The two parameters identified the principal patterns of the cell population profile without prior biological assumptions. As a further application, we perform a dimensionality reduction and a time series reconstruction. DEEF can reconstruct the distributions based on the top coordinates, which enables the creation of an artificial dataset based on an actual single-cell expression dataset. Using the coordinate system assigned by DEEF, it is possible to analyze the relationship between the attributes of the distribution sample and the features or shape of the distribution using conventional data mining methods.


Assuntos
Biomarcadores/metabolismo , Análise de Célula Única , Estatísticas não Paramétricas , Adulto , Simulação por Computador , Bases de Dados como Assunto , Fator de Crescimento Epidérmico/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Tempo
10.
Zhonghua Yi Xue Za Zhi ; 100(16): 1230-1234, 2020 Apr 28.
Artigo em Chinês | MEDLINE | ID: mdl-32344494

RESUMO

Objective: To explore the changes and clinical value of urinary monocyte chemotactic protein 1 (MCP-1), epidermal growth factor (EGF) and their ratio in patients with idiopathic membranous nephropathy (IMN). Methods: A total of 67 IMN patients were enrolled according to kidney biopsy in the Department of Nephrology of the First Affiliated Hospital of Zhengzhou University from January 2017 to December 2018. The patients included 28 males and 39 females, with an average age of (47.6±14.2) years. Eighteen age-and sex-matched healthy controls were also enrolled. Clinical and pathological data, blood and urine samples of all subjects were collected. Urinary MCP-1 and EGF level were detected by enzyme-linked immunosorbent assay (ELISA). And then the levels of urinary MCP-1, EGF and the ratio of EGF/MCP-1 and their correlations with clinicopathology were analyzed. Results: There was no statistical difference of the urine EGF levels between the two groups [8.3(6.0,12.6) vs 8.4(6.5,10.2) ng/mg Cr, P=0.575]. The urine MCP-1 levels of IMN patients were 0.37 (0.21, 0.69) ng/mg Cr, which was statistical significantly higher than those of the control group [0.09 (0.02, 0.19) ng/mg Cr] (P<0.001), while the EGF/MCP-1 ratio was lower than that of the control group [22.2(15.1,36.6) vs 87.6(71.2,132.7), P<0.001]. Urine MCP-1 was negatively correlated with eGFR (r=-0.303, P=0.012), but positively correlated with the urinary ratio of albumin to creatinin (r=0.368, P=0.002). EGF was positively correlated with eGFR (r=0.722, P<0.001), but negatively correlated with the severity of interstitial fibrosis and renal tubular atrophy (IFTA) (r=-0.312, P=0.011). EGF/MCP-1 ratio was positively correlated with eGFR (r=0.693, P<0.001), but negatively correlated with the severity of the urinary ratio of albumin to creatinin and IFTA (r=-0.261, P=0.028 and r=-0.684, P<0.001, respectively). Further multivariate logistic regression analysis showed that EGF/MCP-1 was a protective factor for moderate-to-severe IFTA (OR=0.891, 95%CI: 0.844-0.949, P=0.008). Conclusion: Patients with IMN have elevated urine MCP-1 level and decreased EGF/MCP-1 ratio, which correlate with clinical indicators. In particular, EGF/MCP-1 ratio is independently related to moderate-severe IFTA, and may be a potential clinical biomarker for diagnosis of IMN.


Assuntos
Glomerulonefrite Membranosa , Adulto , Biomarcadores , Quimiocina CCL2 , Fator de Crescimento Epidérmico , Feminino , Taxa de Filtração Glomerular , Humanos , Rim , Masculino , Pessoa de Meia-Idade
11.
PLoS Negl Trop Dis ; 14(4): e0008236, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32302357

RESUMO

Bartonella are Gram-negative bacterial pathogens that trigger pathological angiogenesis during infection of humans. Bartonella bacilliformis (Bb) is a neglected tropical agent endemic to South America, where it causes Carrión's disease. Little is known about Bb's virulence determinants or how the pathogen elicits hyperproliferation of the vasculature, culminating in Peruvian warts (verruga peruana) of the skin. In this study, we determined that active infection of human umbilical vein endothelial cells (HUVECs) by live Bb induced host cell secretion of epidermal growth factor (EGF) using ELISA. Killed bacteria or lysates of various Bb strains did not cause EGF production, suggesting that an active infection was necessary for the response. Bb also caused hyperproliferation of infected HUVECs, and the mitogenic response could be inhibited by the EGF-receptor (EGFR) inhibitor, AG1478. Bb strains engineered to overexpress recombinant GroEL, evoked greater EGF production and hyperproliferation of HUVECs compared to control strains. Conditioned (spent) media from cultured HUVECs that had been previously infected by Bb were found to be mitogenic for naïve HUVECs, and the response could be inhibited by EGFR blocking with AG1478. Bb cells and cell lysates stimulated HUVEC migration and capillary-like tube formation in transmigration and Matrigel assays, respectively. To our knowledge, this is the first demonstration of EGF production by Bb-infected endothelial cells; an association that could contribute to hyperproliferation of the vascular bed during bartonellosis.


Assuntos
Infecções por Bartonella/patologia , Bartonella bacilliformis/crescimento & desenvolvimento , Células Endoteliais/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Interações Hospedeiro-Patógeno , Proliferação de Células , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Biológicos
12.
Nat Commun ; 11(1): 1711, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32249764

RESUMO

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk.


Assuntos
Técnicas de Cultura de Células/métodos , Linhagem da Célula , Glândulas Mamárias Humanas/citologia , Organoides/citologia , Organoides/metabolismo , Células-Tronco/citologia , Adulto , Proteína BRCA1/genética , Neoplasias da Mama , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Feminino , Humanos , Glândulas Mamárias Humanas/química , Glândulas Mamárias Humanas/metabolismo , Pessoa de Meia-Idade , Organoides/química , Análise de Célula Única , Células-Tronco/química , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
14.
Proc Natl Acad Sci U S A ; 117(14): 7941-7949, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32179676

RESUMO

Late-onset sepsis (LOS) is a highly consequential complication of preterm birth and is defined by a positive blood culture obtained after 72 h of age. The causative bacteria can be found in patients' intestinal tracts days before dissemination, and cohort studies suggest reduced LOS risk in breastfed preterm infants through unknown mechanisms. Reduced concentrations of epidermal growth factor (EGF) of maternal origin within the intestinal tract of mice correlated to the translocation of a gut-resident human pathogen Escherichia coli, which spreads systemically and caused a rapid, fatal disease in pups. Translocation of Escherichia coli was associated with the formation of colonic goblet cell-associated antigen passages (GAPs), which translocate enteric bacteria across the intestinal epithelium. Thus, maternally derived EGF, and potentially other EGFR ligands, prevents dissemination of a gut-resident pathogen by inhibiting goblet cell-mediated bacterial translocation. Through manipulation of maternally derived EGF and alteration of the earliest gut defenses, we have developed an animal model of pathogen dissemination which recapitulates gut-origin neonatal LOS.


Assuntos
Translocação Bacteriana/imunologia , Receptores ErbB/metabolismo , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Microbioma Gastrointestinal/imunologia , Leite Humano/imunologia , Sepse Neonatal/imunologia , Animais , Animais Recém-Nascidos , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Aleitamento Materno , Colo/metabolismo , Colo/microbiologia , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Fezes/química , Fezes/microbiologia , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Transgênicos , Leite Humano/metabolismo , Sepse Neonatal/metabolismo , Sepse Neonatal/microbiologia , Transdução de Sinais/imunologia , Fatores de Tempo
15.
Int J Nanomedicine ; 15: 1421-1435, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32184596

RESUMO

Purpose: In this study, we aim to explore the effects of graphene oxide (GO), a derivative of graphene, nanoparticles of four different sizes on the cellular fate of mouse neural stem cells (mNSCs). Methods: GO NPs were characterized with transmission electron microscopy (TEM), scanning electron micrography (SEM), atomic force microscopy (AFM) and Raman Spectra analysis. The cytotoxic effects of the GO NPs of different sizes on the mNSCs were determined using CCK-8 assay, Annexin V-APC/ 7-AAD staining and EdU staining assays. We investigated the biological and the mechanisms of GO NPs on cells using immunofluorescence analysis and quantitative real-time PCR (qPCR). Results: The average hydrodynamic sizes of the GO NPs were 417 nm, 663 nm, 1047 nm, and 4651 nm, with a thickness of approximately 22.5 nm, 17.7 nm, 22.4 nm, and 13.4 nm, respectively. GO NPs of all sizes showed low cytotoxicity at a concentration of 20 µg/mL on the mNSCs. Immunostaining demonstrated that treatment with GO NPs, especially the 663 nm ones, enhanced the self-renewal ability of mNSCs in the absence of EGF and bFGF. Under differentiation medium conditions that are free of mitogenic factors, all the GO NPs, particularly the 4651 nm ones, increased the expression level of Tuj1 and GFAP. With regards to the migration ability, we found that 417 nm GO-NP-treated mNSCs migrated over a longer distance than the control group obviously. In addition, higher expression of Rap1, Vinculin and Paxillin was observed in the GO NP-treated groups compared to the control group. mRNA-Sequence analysis and Western blotting results suggested that the 4651 nm GO NPs triggered positive neuronal differentiation through phosphorylation of ERK1/2 by the downregulating of TRPC2. Conclusion: GO NPs play an important role in the applications of inducing self-renewal and differentiation of mNSC, and are promising in the future for further studies.


Assuntos
Grafite/farmacologia , Nanopartículas/química , Células-Tronco Neurais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Grafite/química , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Células-Tronco Neurais/fisiologia , Tamanho da Partícula , Fosforilação/efeitos dos fármacos , Análise Espectral Raman , Canais de Cátion TRPC/metabolismo , Tubulina (Proteína)/metabolismo
16.
Endocrinology ; 161(4)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32147716

RESUMO

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone that promotes intestinal growth and proliferation through downstream mediators, including epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1). EGF synergistically enhances the proliferative actions of IGF-1 in intestinal cell lines, and both of these factors are known to be essential for the trophic effects of GLP-2 in vivo. However, whether EGF and IGF-1 interact to mediate the proliferative actions of GLP-2 in vivo remains unknown. Normal and knockout (KO) mice lacking the intestinal epithelial IGF-1 receptor (IE-IGF-1R) were therefore treated chronically with EGF and/or long-acting human hGly2GLP-2, followed by determination of intestinal growth parameters. Intestines from control and IE-IGF-1R KO mice were also used to generate organoids (which lack the GLP-2 receptor) and were treated with EGF and/or IGF-1. Combination treatment with EGF and hGly2GLP-2 increased small intestinal weight and crypt-villus height in C57Bl/6 mice in an additive manner, whereas only hGly2GLP-2 treatment increased crypt cell proliferation. However, although combination treatment also increased small intestinal weight and crypt-villus height in IE-IGF-1R KO mice, the proliferative responses to hGly2GLP-2 alone or with EGF were diminished in these animals. Finally, IGF-1 treatment of organoids undergoing EGF withdrawal was not additive to the effect of EGF replacement on proliferation, but could restore normal proliferation in the absence of EGF. Together, these findings demonstrate that the intestinal proliferative effects of hGly2GLP-2 are augmented by exogenous EGF in a manner that is partially dependent upon IE-IGF-1R signaling.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Intestino Delgado/metabolismo , Camundongos , Camundongos Knockout , Receptor IGF Tipo 1/genética , Transdução de Sinais/efeitos dos fármacos
17.
J Neurosci ; 40(13): 2618-2632, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32079647

RESUMO

Sensory hair cell losses underlie the vast majority of permanent hearing and balance deficits in humans, but many nonmammalian vertebrates can fully recover from hearing impairments and balance dysfunctions because supporting cells (SCs) in their ears retain lifelong regenerative capacities that depend on proliferation and differentiation as replacement hair cells. Most SCs in vertebrate ears stop dividing during embryogenesis; and soon after birth, vestibular SCs in mammals transition to lasting quiescence as they develop massively thickened circumferential F-actin bands at their E-cadherin-rich adherens junctions. Here, we report that treatment with EGF and a GSK3 inhibitor thinned the circumferential F-actin bands throughout the sensory epithelium of cultured utricles that were isolated from adult mice of either sex. That treatment also caused decreases in E-cadherin, ß-catenin, and YAP in the striola, and stimulated robust proliferation of mature, normally quiescent striolar SCs. The findings suggest that E-cadherin-rich junctions, which are not present in the SCs of the fish, amphibians, and birds which readily regenerate hair cells, are responsible in part for the mammalian ear's vulnerability to permanent balance and hearing deficits.SIGNIFICANCE STATEMENT Millions of people are affected by hearing and balance deficits that arise when loud sounds, ototoxic drugs, infections, and aging cause hair cell losses. Such deficits are permanent for humans and other mammals, but nonmammals can recover hearing and balance after supporting cells regenerate replacement hair cells. Mammalian supporting cells lose the capacity to proliferate around the time they develop unique, exceptionally reinforced, E-cadherin-rich intercellular junctions. Here, we report the discovery of a pharmacological treatment that thins F-actin bands, depletes E-cadherin, and stimulates proliferation in long-quiescent supporting cells within a balance epithelium from adult mice. The findings suggest that high E-cadherin in those supporting cell junctions may be responsible, in part, for the permanence of hair cell loss in mammals.


Assuntos
Caderinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células Ciliadas Auditivas/efeitos dos fármacos , Sáculo e Utrículo/efeitos dos fármacos , Actinas/metabolismo , Animais , Caderinas/genética , Células Ciliadas Auditivas/metabolismo , Camundongos , Piridinas/farmacologia , Pirimidinas/farmacologia , Sáculo e Utrículo/metabolismo , beta Catenina/metabolismo
18.
Nat Commun ; 11(1): 787, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034161

RESUMO

Quantitative phosphoproteomics has transformed investigations of cell signaling, but it remains challenging to scale the technology for high-throughput analyses. Here we report a rapid and reproducible approach to analyze hundreds of phosphoproteomes using data-independent acquisition (DIA) with an accurate site localization score incorporated into Spectronaut. DIA-based phosphoproteomics achieves an order of magnitude broader dynamic range, higher reproducibility of identification, and improved sensitivity and accuracy of quantification compared to state-of-the-art data-dependent acquisition (DDA)-based phosphoproteomics. Notably, direct DIA without the need of spectral libraries performs close to analyses using project-specific libraries, quantifying > 20,000 phosphopeptides in 15 min single-shot LC-MS analysis per condition. Adaptation of a 3D multiple regression model-based algorithm enables global determination of phosphorylation site stoichiometry in DIA. Scalability of the DIA approach is demonstrated by systematically analyzing the effects of thirty kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that specific protein kinases mediate EGF-dependent phospho-regulation.


Assuntos
Algoritmos , Biologia Computacional/métodos , Fosfopeptídeos/análise , Proteínas Quinases/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Fator de Crescimento Epidérmico/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Fosfopeptídeos/metabolismo , Fosfoproteínas/análise , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos Testes
19.
Adv Exp Med Biol ; 1218: 77-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32060872

RESUMO

Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt-and-pepper pattern formation during various biological processes. In many cases, Notch signaling acts together with other signaling systems. However, it is not clear what happens when Notch signaling is combined with other signaling systems. Mathematical modeling and the use of a simple biological model system will be essential to address this uncertainty. A wave of differentiation in the Drosophila visual center, the "proneural wave," accompanies the activity of the Notch and EGF signaling pathways. Although all of the Notch signaling components required for lateral inhibition are involved in the proneural wave, no salt-and-pepper pattern is found during the progression of the proneural wave. Instead, Notch is activated along the wave front and regulates proneural wave progression. How does Notch signaling control wave propagation without forming a salt-and-pepper pattern? A mathematical model of the proneural wave, based on biological evidence, has demonstrated that Notch-mediated lateral inhibition is implemented within the proneural wave and that the diffusible action of EGF cancels salt-and-pepper pattern formation. The results from numerical simulation have been confirmed by genetic experiments in vivo and suggest that the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a novel function of Notch signaling that regulates propagation of the proneural wave. Similar mechanisms may play important roles in diverse biological processes found in animal development and cancer pathogenesis.


Assuntos
Diferenciação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos
20.
PLoS One ; 15(2): e0229077, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32069320

RESUMO

Epidermal growth factor receptor (EGFR) is a pro-tumorigenic receptor tyrosine kinase that facilitates growth for cancer cells that overexpress the receptor. Monoclonal anti-EGFR antibody Cetuximab (CTX) provides significant clinical benefit in patients with head and neck squamous cell carcinoma (HNSCC). Missense mutations in the ectodomain (ECD) of EGFR can be acquired under CTX treatment and mimic the effect of large deletions on spontaneous untethering and activation of the receptor. Little is known about the contribution of EGFR ECD mutations to EGFR activation and CTX resistance in HNSCC. We identified two concurrent non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD in patient-derived HNSCC cells that were selected for CTX resistance through repeated exposure to the agent in an effort to mimic what may occur clinically. Structural modeling predicted that the G33S and N56K mutants would restrict adoption of a fully closed (tethered) and inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation, leading to persistent downstream AKT signaling. Our results demonstrate that HNSCC cells can select for EGFR ECD mutations under CTX exposure that converge to trap the receptor in an open, ligand-independent, constitutively activated state. These mutants impede the receptor's competence to bind CTX possibly explaining certain cases of CTX treatment-induced or de novo resistance to CTX.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Cetuximab/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Antineoplásicos Imunológicos/uso terapêutico , Cetuximab/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Ligantes , Modelos Moleculares , Mutação de Sentido Incorreto , Cultura Primária de Células , Domínios Proteicos/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Células Tumorais Cultivadas
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