Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 79
Filtrar
1.
J Exp Clin Cancer Res ; 42(1): 272, 2023 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-37858248

RESUMO

BACKGROUND: Immunotherapy has significantly advanced cancer treatments, but many patients do not respond to it, partly due to immunosuppressive mechanisms used by tumor cells. These cells employ immunosuppressive ligands to evade detection and elimination by the immune system. Therefore, the discovery and characterization of novel immunosuppressive ligands that facilitate immune evasion are crucial for developing more potent anti-cancer therapies. METHODS: We conducted gain-of-function screens using a CRISPRa (CRISPR activation) library that covered the entire human transmembrane sub-genome to identify surface molecules capable of hindering NK-mediated cytotoxicity. The immunosuppressive role and mechanism of MUC21 were validated using NK and T cell mediated cytotoxicity assays. Bioinformatics tools were employed to assess the clinical implications of mucin-21 (MUC21) in cancer cell immunity. RESULTS: Our genetic screens revealed that MUC21 expression on cancer cell surfaces inhibits both the cytotoxic activity of NK cells and antibody-dependent cellular cytotoxicity, but not affecting complement-dependent cytotoxicity. Additionally, MUC21 expression hinders T cell activation by impeding antigen recognition, thereby diminishing the effectiveness of the immune checkpoint inhibitor, anti-PD-L1. Moreover, MUC21 expression suppress the antitumor function of both CAR-T cells and CAR-NK cells. Mechanistically, MUC21 facilitates immune evasion by creating steric hindrance, preventing interactions between cancer and immune cells. Bioinformatics analysis revealed elevated MUC21 expression in lung cancer, which correlated with reduced infiltration and activation of cytotoxic immune cells. Intriguingly, MUC21 expression was higher in non-small cell lung cancer (NSCLC) tumors that were non-responsive to anti-PD-(L)1 treatment compared to responsive tumors. CONCLUSIONS: These findings indicate that surface MUC21 serves as a potent immunosuppressive ligand, shielding cancer cells from NK and CD8+T cell attacks. This suggests that inhibiting MUC21 could be a promising strategy to improve cancer immunotherapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Imunidade Celular , Células Matadoras Naturais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo
2.
Sci Rep ; 13(1): 11731, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37474737

RESUMO

Neurodegenerative diseases are explained by progressive defects of cognitive function and memory. These defects of cognition and memory dysfunction can be induced by the loss of brain-derived neurotrophic factors (BDNF) signaling. Paeonia lactiflora is a traditionally used medicinal herb in Asian countries and some beneficial effects have been reported, including anti-oxidative, anti-inflammatory, anti-cancer activity, and potential neuroprotective effects recently. In this study, we found that suffruticosol A is a major compound in seeds of Paeonia lactiflora. When treated in a SH-SY5 cell line for measuring cell viability and cell survival, suffruticosol A increased cell viability (at 20 µM) and recovered scopolamine-induced neurodegenerative characteristics in the cells. To further confirm its neural amelioration effects in the animals, suffruticosol A (4 or 15 ng, twice a week) was administered into the third ventricle beside the brain of C57BL/6 mice for one month then the scopolamine was intraperitoneally injected into these mice to induce impairments of cognition and memory before conducting behavioral experiments. Central administration of suffruticosol A into the brain restored the memory and cognition behaviors in mice that received the scopolamine. Consistently, the central treatments of suffruticosol A showed rescued cholinergic deficits and BDNF signaling in the hippocampus of mice. Finally, we measured the long-term potentiation (LTP) in the hippocampal CA3-CA1 synapse to figure out the restoration of the synaptic mechanism of learning and memory. Bath application of suffruticosol A (40 µM) improved LTP impairment induced by scopolamine in hippocampal slices. In conclusion, the central administration of suffruticosol A ameliorated neuronal effects partly through elevated BDNF signaling.


Assuntos
Paeonia , Escopolamina , Camundongos , Animais , Escopolamina/farmacologia , Paeonia/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais , Hipocampo/metabolismo , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/metabolismo , Aprendizagem em Labirinto
5.
Brain ; 146(7): 2957-2974, 2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37062541

RESUMO

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD). However, a clinically validated neuroimaging probe to visualize the reactive astrogliosis is yet to be discovered. Here, we show that PET imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. To investigate the alterations of acetate and glucose metabolism in the diseased brains and their impact on the AD pathology, we adopted multifaceted approaches including microPET imaging, autoradiography, immunohistochemistry, metabolomics, and electrophysiology. Two AD rodent models, APP/PS1 and 5xFAD transgenic mice, one adenovirus-induced rat model of reactive astrogliosis, and post-mortem human brain tissues were used in this study. We further curated a proof-of-concept human study that included 11C-acetate and 18F-FDG PET imaging analyses along with neuropsychological assessments from 11 AD patients and 10 healthy control subjects. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1) in rodent models of both reactive astrogliosis and AD. The elevated acetate uptake is associated with reactive astrogliosis and boosts the aberrant astrocytic GABA synthesis when amyloid-ß is present. The excessive astrocytic GABA subsequently suppresses neuronal activity, which could lead to glucose uptake through decreased glucose transporter-3 in the diseased brains. We further demonstrate that 11C-acetate uptake was significantly increased in the entorhinal cortex, hippocampus and temporo-parietal neocortex of the AD patients compared to the healthy controls, while 18F-FDG uptake was significantly reduced in the same regions. Additionally, we discover a strong correlation between the patients' cognitive function and the PET signals of both 11C-acetate and 18F-FDG. We demonstrate the potential value of PET imaging with 11C-acetate and 18F-FDG by visualizing reactive astrogliosis and the associated neuronal glucose hypometablosim for AD patients. Our findings further suggest that the acetate-boosted reactive astrocyte-neuron interaction could contribute to the cognitive decline in AD.


Assuntos
Doença de Alzheimer , Camundongos , Humanos , Ratos , Animais , Doença de Alzheimer/metabolismo , Fluordesoxiglucose F18/metabolismo , Astrócitos/metabolismo , Radioisótopos de Carbono/metabolismo , Gliose/diagnóstico por imagem , Encéfalo/patologia , Tomografia por Emissão de Pósitrons/métodos , Ácido gama-Aminobutírico/metabolismo
6.
J Microbiol Biotechnol ; 32(11): 1382-1389, 2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-36330743

RESUMO

Asterias pectinifera, a species of starfish and cause of concern in the aquaculture industry, was recently identified as a source of non-toxic and highly water-soluble collagen peptides. In this study, we investigated the antioxidant and anti-photoaging functions of compounds formulated using collagen peptides from extracts of Asterias pectinifera and Halocynthia roretzi (AH). Our results showed that AH compounds have various skin protective functions, including antioxidant effects, determined by measuring the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radicals, as well as anti-melanogenic effects, determined by measuring tyrosinase inhibition activity. To determine whether ethosome-encapsulated AH compounds (E(AH)) exert ultraviolet (UV)-protective effects, human dermal fibroblasts or keratinocytes were incubated with E(AH) before and after exposure to UVA or UVB. E(AH) treatment led to inhibition of photoaging-induced secretion of matrix metalloproteinase-1 and interleukin-6 and -8, which are associated with inflammatory responses during UV irradiation. Finally, the antibacterial effects of AH and E(AH) were confirmed against both gram-negative and gram-positive bacteria. Our results indicate that E(AH) has the potential for use in the development of cosmetics with a range of skin protective functions.


Assuntos
Asterias , Envelhecimento da Pele , Dermatopatias , Animais , Humanos , Raios Ultravioleta , Colágeno , Pele/efeitos da radiação , Fibroblastos , Extratos Vegetais/farmacologia , Peptídeos/farmacologia , Antibacterianos/farmacologia
7.
J Korean Med Sci ; 37(37): e276, 2022 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-36163475

RESUMO

Autophagy is critically involved in the maintenance of intracellular nutrient homeostasis and organelle function. Dysregulated autophagy is likely to play a role in the development of metabolic disorders and diabetes because autophagy is critical in the rejuvenation of dysfunctional or stressed endoplasmic reticulum and mitochondria that play a crucial role in the development of diabetes. Indeed, systemic autophagy insufficiency led to the increased tissue lipid content, aggravated metabolic and finally more severe diabetes when metabolic stress was imposed, suggesting that autophagy insufficiency of dysfunction of lysosome, an effector organelle of autophagy, due to aging, genetic predisposition or environmental factors could be an underlying cause of diabetes. Conversely, autophagy enhancer could improve metabolic profile of obese mice by reducing tissue lipid content and ameliorating metabolic inflammation. Furthermore, clearance of human islet amyloid polypeptide (hIAPP) oligomer and amyloid that accumulate in pancreatic islets of > 90% of diabetes patients was also dependent on autophagy. Consistently, autophagy enhancer could improve glucose profile and ß-cell function of transgenic mice expressing amyloidogenic hIAPP in pancreatic ß-cells, which was accompanied by reduced accumulation of hIAPP oligomer or amyloid, ameliorated ß-cell apoptosis and increased ß-cell mass. These results suggest that autophagy enhancer could be a novel therapeutic modality against diabetes associated with lipid overload and human diabetes characterized by islet amyloid accumulation.


Assuntos
Diabetes Mellitus Tipo 2 , Diabetes Mellitus , Células Secretoras de Insulina , Ilhotas Pancreáticas , Síndrome Metabólica , Amiloide/genética , Amiloide/metabolismo , Animais , Autofagia/fisiologia , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/patologia , Diabetes Mellitus Tipo 2/patologia , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Lipídeos , Síndrome Metabólica/complicações , Camundongos , Camundongos Transgênicos
8.
Mol Psychiatry ; 2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35902630

RESUMO

There is a compelling need to develop disease-modifying therapies for Alzheimer's disease (AD), the most common neuro-degenerative disorder. Together with recent progress in vector development for efficiently targeting the central nervous system, gene therapy has been suggested as a potential therapeutic modality to overcome the limited delivery of conventional types of drugs to and within the damaged brain. In addition, given increasing evidence of the strong link between glia and AD pathophysiology, therapeutic targets have been moving toward those addressing glial cell pathology. Nurr1 and Foxa2 are transcription/epigenetic regulators that have been reported to cooperatively regulate inflammatory and neurotrophic response in glial cells. In this study, we tested the therapeutic potential of Nurr1 and Foxa2 gene delivery to treat AD symptoms and pathologies. A series of functional, histologic, and transcriptome analyses revealed that the combined expression of Nurr1 and Foxa2 substantially ameliorated AD-associated amyloid ß and Tau proteinopathy, cell senescence, synaptic loss, and neuro-inflammation in multiple in vitro and in vivo AD models. Intra-cranial delivery of Nurr1 and Foxa2 genes using adeno-associated virus (AAV) serotype 9 improved the memory and cognitive function of AD model mice. The therapeutic benefits of gene delivery were attained mainly by correcting pathologic glial function. These findings collectively indicate that AAV9-mediated Nurr1 and Foxa2 gene transfer could be an effective disease-modifying therapy for AD.

9.
Front Cell Infect Microbiol ; 12: 850744, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35558099

RESUMO

The endemic and pandemic caused by respiratory virus infection are a major cause of mortality and morbidity globally. Thus, broadly effective antiviral drugs are needed to treat respiratory viral diseases. Small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (U-exo) have recently gained attention as a cell-free therapeutic strategy due to their potential for safety and efficacy. Anti-viral activities of U-exo to countermeasure respiratory virus-associated diseases are currently unknown. Here, we tested the antiviral activities of U-exo following influenza A/B virus (IFV) and human seasonal coronavirus (HCoV) infections in vitro. Cells were subject to IFV or HCoV infection followed by U-exo treatment. U-exo treatment significantly reduced IFV or HCoV replication and combined treatment with recombinant human interferon-alpha protein (IFN-α) exerted synergistically enhanced antiviral effects against IFV or HCoV. Interestingly, microRNA (miR)-125b, which is one of the most abundantly expressed small RNAs in U-exo, was found to suppress IFV replication possibly via the induction of IFN-stimulated genes (ISGs). Furthermore, U-exo markedly enhanced RNA virus-triggered IFN signaling and ISGs production. Similarly, human nasal epithelial cells cultured at the air-liquid interface (ALI) studies broadly effective anti-viral and anti-inflammatory activities of U-exo against IFV and HCoV, suggesting the potential role of U-exo as a promising intervention for respiratory virus-associated diseases.


Assuntos
Coronavirus , Exossomos , Vesículas Extracelulares , Células-Tronco Mesenquimais , Antivirais/metabolismo , Antivirais/farmacologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical
10.
Mol Ther ; 30(8): 2800-2816, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35526096

RESUMO

Several preclinical studies demonstrate that antitumor efficacy of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade can be improved by combination with other checkpoint inhibitors. Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint receptor involved in T cell exhaustion and tumor immune escape. Here, we describe ABL501, a bispecific antibody targeting LAG-3 and PD-L1 in modulating immune cell responses against tumors. ABL501 that efficiently inhibits both LAG-3 and PD-L1 pathways enhances the activation of effector CD4+ and CD8+ T cells with a higher degree than a combination of single anti-LAG-3 and anti-PD-L1. The augmented effector T cell responses by ABL501 resulted in mitigating regulatory-T-cell-mediated immunosuppression. Mechanistically, the simultaneous binding of ABL501 to LAG-3 and PD-L1 promotes dendritic cell (DC) activation and tumor cell conjugation with T cells that subsequently mounts effective CD8+ T cell responses. ABL501 demonstrates its potent in vivo antitumor efficacy in a humanized xenograft model and with knockin mice expressing human orthologs. The immune profiling analysis of peripheral blood reveals an increased abundance of LAG-3hiPD-1hi memory CD4+ T cell subset in relapsed cholangiocarcinoma patients after gemcitabine plus cisplatin therapy, which are more responsive to ABL501. This study supports the clinical evaluation of ABL501 as a novel cancer immunotherapeutic, and a first-in-human trial has started (NCT05101109).


Assuntos
Anticorpos Biespecíficos , Antígenos CD , Antígeno B7-H1 , Neoplasias , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos , Células Dendríticas , Camundongos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1 , Evasão Tumoral , Proteína do Gene 3 de Ativação de Linfócitos
11.
CRISPR J ; 5(1): 95-108, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35191750

RESUMO

Mutations in specific genes, including synuclein alpha (SNCA) that encodes the α-synuclein protein, are known to be risk factors for sporadic Parkinson's disease (PD), as well as critical factors for familial PD. In particular, A53T-mutated SNCA (A53T-SNCA) is a well-studied familial pathologic mutation in PD. However, techniques for deletion of the mutated SNCA gene in vivo have not been developed. Here, we used the CRISPR-Cas9 system to delete A53T-SNCA in vitro as well as in vivo. Adeno-associated virus carrying SaCas9-KKH with a single-guide RNA targeting A53T-SNCA significantly reduced A53T-SNCA expression levels in vitro. Furthermore, we tested its therapeutic potential in vivo in a viral A53T-SNCA-overexpressing rat model of PD. Gene deletion of A53T-SNCA significantly rescued the overexpression of α-synuclein, reactive microgliosis, dopaminergic neurodegeneration, and parkinsonian motor symptoms. Our findings propose CRISPR-Cas9 system as a potential prevention strategy for A53T-SNCA-specific PD.


Assuntos
Edição de Genes , Doença de Parkinson , alfa-Sinucleína , Animais , Sistemas CRISPR-Cas/genética , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Ratos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
12.
ISME J ; 16(5): 1205-1221, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34972816

RESUMO

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.


Assuntos
Neoplasias Colorretais , Microbiota , Ubiquitina-Proteína Ligases/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Propionatos , Regulação para Cima
13.
Stem Cell Res Ther ; 12(1): 569, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34772451

RESUMO

BACKGROUND: Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. METHODS: We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. RESULTS: We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. CONCLUSION: This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.


Assuntos
Âmnio , Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos
14.
Sci Rep ; 11(1): 20169, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635730

RESUMO

Toll-like receptor (TLR)-4 and TLR9 are known to play important roles in the immune system, and several studies have shown their association with the development of rheumatoid arthritis (RA) and regulation of tumor necrosis factor alpha (TNF-α). However, studies that investigate the association between TLR4 or TLR9 gene polymorphisms and remission of the disease in RA patients taking TNF-α inhibitors have yet to be conducted. In this context, this study was designed to investigate the effects of polymorphisms in TLR4 and TLR9 on response to TNF-α inhibitors and to train various models using machine learning approaches to predict remission. A total of six single nucleotide polymorphisms (SNPs) were investigated. Logistic regression analysis was used to investigate the association between genetic polymorphisms and response to treatment. Various machine learning methods were utilized for prediction of remission. After adjusting for covariates, the rate of remission of T-allele carriers of TLR9 rs352139 was about 5 times that of the CC-genotype carriers (95% confidence interval (CI) 1.325-19.231, p = 0.018). Among machine learning algorithms, multivariate logistic regression and elastic net showed the best prediction with the area under the receiver-operating curve (AUROC) value of 0.71 (95% CI 0.597-0.823 for both models). This study showed an association between a TLR9 polymorphism (rs352139) and treatment response in RA patients receiving TNF-α inhibitors. Moreover, this study utilized various machine learning methods for prediction, among which the elastic net provided the best model for remission prediction.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/patologia , Predisposição Genética para Doença , Aprendizado de Máquina , Polimorfismo de Nucleotídeo Único , Receptor Toll-Like 9/genética , Fator de Necrose Tumoral alfa/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Feminino , Seguimentos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto Jovem
15.
Viruses ; 13(9)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34578425

RESUMO

Nonstructural protein 1 (NS1) of influenza virus (IFV) is essential for evading interferon (IFN)-mediated antiviral responses, thereby contributing to the pathogenesis of influenza. Mitophagy is a type of autophagy that selectively removes damaged mitochondria. The role of NS1 in IFV-mediated mitophagy is currently unknown. Herein, we showed that overexpression of NS1 protein led to enhancement of mitophagy. Mitophagy induction via carbonyl cyanide 3-chlorophenylhydrazone treatment in IFV-infected A549 cells led to increased viral replication efficiency, whereas the knockdown of PTEN-induced kinase 1 (PINK1) led to the opposite effect on viral replication. Overexpression of NS1 protein led to changes in mitochondrial dynamics, including depolarization of mitochondrial membrane potential. In contrast, infection with NS1-deficient virus resulted in impaired mitochondrial fragmentation, subsequent mitolysosomal formation, and mitophagy induction, suggesting an important role of NS1 in mitophagy. Meanwhile, NS1 protein increased the phosphorylation of Unc-51-like autophagy activating kinase 1 (ULK1) and the mitochondrial expression of BCL2- interacting protein 3 (BNIP3), both of which were found to be important for IFV-mediated mitophagy. Overall, these data highlight the importance of IFV NS1, ULK1, and BNIP3 during mitophagy activation.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Dinâmica Mitocondrial , Mitofagia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Potencial da Membrana Mitocondrial , Proteínas não Estruturais Virais/efeitos dos fármacos , Replicação Viral
16.
Sci Adv ; 7(23)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34078609

RESUMO

Advanced technologies are required for generating human intestinal epithelial cells (hIECs) harboring cellular diversity and functionalities to predict oral drug absorption in humans and study normal intestinal epithelial physiology. We developed a reproducible two-step protocol to induce human pluripotent stem cells to differentiate into highly expandable hIEC progenitors and a functional hIEC monolayer exhibiting intestinal molecular features, cell type diversity, and high activities of intestinal transporters and metabolic enzymes such as cytochrome P450 3A4 (CYP3A4). Functional hIECs are more suitable for predicting compounds metabolized by CYP3A4 and absorbed in the intestine than Caco-2 cells. This system is a step toward the transition from three-dimensional (3D) intestinal organoids to 2D hIEC monolayers without compromising cellular diversity and function. A physiologically relevant hIEC model offers a novel platform for creating patient-specific assays and support translational applications, thereby bridging the gap between 3D and 2D culture models of the intestine.


Assuntos
Citocromo P-450 CYP3A , Mucosa Intestinal , Células CACO-2 , Citocromo P-450 CYP3A/metabolismo , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Organoides/metabolismo
17.
Cancer Lett ; 510: 37-47, 2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-33872695

RESUMO

Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1 cells. More than 182 genes (p < 0.01) were found to suppress the cytokine-independent growth of TF-1 cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ-/-) mice injected with CMAS or SLC35A1-knockout TF-1 cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1 cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Leucemia Mieloide Aguda/genética , Animais , Carcinogênese , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Transdução de Sinais
18.
Molecules ; 26(7)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808438

RESUMO

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.


Assuntos
Acetanilidas , Adamantano/análogos & derivados , Antineoplásicos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Acetanilidas/sangue , Acetanilidas/metabolismo , Acetanilidas/farmacocinética , Adamantano/sangue , Adamantano/metabolismo , Adamantano/farmacocinética , Animais , Antineoplásicos/sangue , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Biotransformação , Masculino , Camundongos , Camundongos Endogâmicos ICR
19.
Cells ; 10(3)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33801464

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteína DEAD-box 58/metabolismo , Interferons/metabolismo , Receptores Imunológicos/metabolismo , SARS-CoV-2/metabolismo , Proteína DEAD-box 58/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interferons/genética , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Fosfoproteínas/metabolismo , Poli C/farmacologia , Poli I/farmacologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Imunológicos/genética , SARS-CoV-2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima , Zika virus/genética , Zika virus/metabolismo
20.
Molecules ; 26(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921487

RESUMO

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUClast values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes (t1/2 > 1 h) and serum (t1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.


Assuntos
Acetanilidas/farmacologia , Acetanilidas/farmacocinética , Adamantano/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Acetanilidas/sangue , Acetanilidas/metabolismo , Adamantano/sangue , Adamantano/metabolismo , Adamantano/farmacocinética , Adamantano/farmacologia , Animais , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Injeções Intravenosas , Masculino , Metaboloma , Camundongos Endogâmicos ICR , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA