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1.
Signal Transduct Target Ther ; 6(1): 331, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471099

RESUMO

The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of ongoing global pandemic of COVID-19, may trigger immunosuppression in the early stage and overactive immune response in the late stage of infection; However, the underlying mechanisms are not well understood. Here we demonstrated that the SARS-CoV-2 nucleocapsid (N) protein dually regulated innate immune responses, i.e., the low-dose N protein suppressed type I interferon (IFN-I) signaling and inflammatory cytokines, whereas high-dose N protein promoted IFN-I signaling and inflammatory cytokines. Mechanistically, the SARS-CoV-2 N protein dually regulated the phosphorylation and nuclear translocation of IRF3, STAT1, and STAT2. Additionally, low-dose N protein combined with TRIM25 could suppress the ubiquitination and activation of retinoic acid-inducible gene I (RIG-I). Our findings revealed a regulatory mechanism of innate immune responses by the SARS-CoV-2 N protein, which would contribute to understanding the pathogenesis of SARS-CoV-2 and other SARS-like coronaviruses, and development of more effective strategies for controlling COVID-19.


Assuntos
COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Imunidade Inata , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Células A549 , COVID-19/patologia , Células CACO-2 , Células HEK293 , Células Hep G2 , Humanos , Interferon Tipo I/imunologia , Fosfoproteínas/imunologia
2.
Nat Commun ; 12(1): 5204, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471136

RESUMO

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.


Assuntos
Retículo Endoplasmático/metabolismo , Canais de Translocação SEC/metabolismo , Via Secretória/fisiologia , Animais , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteoma/metabolismo , Proteômica
3.
Talanta ; 235: 122817, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517673

RESUMO

High heterogeneity of hepatocellular carcinoma (HCC) tumor has become an obstacle to select effective therapy for the treatment of HCC patients. Methods that can guide the decision on therapy choice for HCC treatment are highly demanded. Evaluating the drug response of heterogeneous tumor cells at the molecular level can help to reveal the toxicity mechanism of anticancer drugs and provide more information than current cell-based chemosensitivity assays. In the present work, nanostructure-assisted laser desorption/ionization mass spectrometry (NALDI-MS) was used to investigate the lipid response of HCC cells to anticancer drugs. Three types of HCC cells (LM3, Hep G2, Huh7) were treated with sorafenib, doxorubicin hydro-chloride, and cisplatin. We found that the lipid profiles of HCC cells changed a lot after the drug treatment, and the degree of lipid changes was related to the cell viability. Two pairs of fatty acids C16:1/C16:0 and C18:1/C18:0 were found to be strongly related to the viability of HCC cells after drug treatment, and were more sensitive than Methyl-thiazolyl tetrazolium (MTT) assay. Accordingly, they can act as sensitive and comprehensive indexes to evaluate the drug susceptibility of HCC cells. In addition, the peak ratio of several neighboring phospholipids displayed high correlation with drug response of specific cell subtype to specific drug. The ratio of neighboring lipids may be traced back to the activity of enzyme and gene expression which regulate the lipidomic pathway. This method provides drug response of heterogenous tumor cells at molecular level and could be a potential candidate to precise tumor chemosensitivity assay.


Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Nanoestruturas , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Fosfolipídeos
4.
J Med Food ; 24(9): 997-1009, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34524027

RESUMO

Oxidative stress has been demonstrated to be associated with numerous aging-related diseases. Ethyl acetate fraction of Abelmoschus manihot (L.) Medic (EA) had been reported to possess strong radical-scavenging activity due to its rich content of flavonoids. This work aimed to determine the protective effects of EA against oxidative injuries in vivo and in vitro, as well as to explore the relevant mechanisms behind these effects. Pretreatment with EA significantly elevated cell viability of H2O2-induced HepG2 cells, reduced the reactive oxygen species level, decreased apoptotic cells, and inhibited activities of caspase 3/9. Meanwhile, EA pretreatment elevated the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while reduced malondialdehyde (MDA) generation and lactate dehydrogenase (LDH) release dose-dependently. In addition, EA modulated key marker genes expression of antioxidation and apoptosis-related signaling pathways at the messenger RNA (mRNA) and protein levels. In the animal studies, EA also significantly enhanced the antioxidant activity and reduced MDA generation in serum, liver, and brain of the D-galactose (D-gal)-induced mice. Furthermore, the histological analysis indicated that EA effectively alleviated liver and brain injury of mice induced by D-gal, dose-dependently. EA as a potential antioxidant agent promoted health and reduced the risk of aging-associated diseases.


Assuntos
Abelmoschus , Acetatos , Envelhecimento , Animais , Antioxidantes/farmacologia , Flores , Galactose , Células Hep G2 , Humanos , Peróxido de Hidrogênio , Malondialdeído , Camundongos , Estresse Oxidativo , Superóxido Dismutase/metabolismo
5.
J Med Food ; 24(9): 978-986, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34524028

RESUMO

Epigenetic regulation by histone acetyltransferase (HAT) is associated with various biological processes and the progression of diseases, including nonalcoholic fatty liver disease (NAFLD). The objective of this study was to investigate whether the hypolipidemic properties of black mulberry (Morus atropurpurea Roxb.) fruit extract (BME) contribute toward protection against NAFLD by HAT inhibition. HepG2 cells were treated with oleic and palmitic acids to induce lipid accumulation, which was significantly attenuated by the treatment with BME at 50 and 100 µg/mL. BME also markedly reduced the expression of proteins associated with lipogenesis, which was attributed to the BME-mediated downregulation of lipogenic genes in HepG2 cells. BME significantly inhibited in vitro total HAT and p300 activities. In addition, BME suppressed total acetylated lysine as well as specific histone acetylation of proteins H3K14 and H3K27 in HepG2 cells. Mice were then fed with either a chow diet or western diet (WD), with or without BME (1%, w/w) supplementation, for 12 weeks to confirm hypolipidemic activity of BME. BME attenuated serum nonesterified fatty acids and low-density lipoprotein (LDL) cholesterol levels, which was likely associated with the downregulation of hepatic lipogenic gene expression in WD-fed obese mice. Taken together, the hypolipidemic activity of BME was observed in HepG2 cells treated with fatty acids as well as in livers of obese mice, and the hepatoprotection of BME is likely associated with the inhibition of acetylation. Further investigation is warranted to determine whether BME can be developed into an efficacious dietary intervention to attenuate the progression of NAFLD by epigenetic regulation in clinical settings.


Assuntos
Morus , Hepatopatia Gordurosa não Alcoólica , Acetilação , Animais , Dieta Hiperlipídica/efeitos adversos , Epigênese Genética , Frutas/metabolismo , Células Hep G2 , Histonas/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia
6.
Anal Chem ; 93(32): 11159-11166, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34347435

RESUMO

Phenotypic plasticity is an emerging paradigm for providing biological and clinical insights into cancer initiation, progression, and resistance to therapy. However, it is a great challenge to track phenotypic information on live cells with high levels of sensitivity, specificity, and simplicity, when a specific cancer-cell subset is being targeted. In this work, we have successfully achieved cascade assembly of nanoparticles on the surface of specific cancer cells by designing a dual-aptamer-weaved molecular AND logic system. Taking advantage of spatial addressability, precise controllability, and targeting recognition of the nanostructure assemblies, we can precisely label the target-cell subset in a large population of similar cells and rapidly obtain phenotypic information in response to the surface changes of captured cancer cells. Without sophisticated instruments, we can know the phenotypic information on HepG2 cells in whole blood with a high level of sensitivity and rapid naked-eye tracking of on-cell phenotype changes of HepG2 cells undergoing epithelial-mesenchymal transition.


Assuntos
Aptâmeros de Nucleotídeos , Nanoestruturas , Tecnologia de Rastreamento Ocular , Células Hep G2 , Humanos , Fenótipo
7.
J Agric Food Chem ; 69(33): 9671-9683, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34388345

RESUMO

Binge alcohol consumption is a serious health concern. Ferroptosis is an iron-dependent lipid peroxidation mediated cell death. Activation of the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway has been shown to exert a protective effect by blunting the responses to ferroptosis inducers. The autophagy substrate p62 was demonstrated to modulate Nrf2 and contribute to the suppression of ferroptosis. Furthermore, autophagy inhibition resulted in the accumulation of p62, which is a specific substrate for this process. Therefore, we aimed to explore the protective effect of autophagy inhibition against alcohol-induced ferroptosis through activating the p62-Keap1-Nrf2 pathway. Our results demonstrated that alcohol induced ferroptosis, which could be significantly reduced by ferrostatin-1. Additionally, we found that autophagy inhibition could protect HepG2 cells against alcohol-induced ferroptosis by activating the p62-Keap1-Nrf2 pathway. Furthermore, inhibition of autophagy increased the expression of p62, which interacted with Keap1 to promote Nrf2 translocation into the nucleus and upregulation its target proteins ferritin heavy (FTH), ferroportin (FPN), and heme oxygenase-1 (HO-1). This study provides a theoretical basis for further elucidation of the relationship between autophagy and ferroptosis and lays a preliminary foundation for further research concerning dietary guidance in the prevention and treatment of diseases related to alcohol-induced ferroptosis.


Assuntos
Ferroptose , Fator 2 Relacionado a NF-E2 , Autofagia , Células Hep G2 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
8.
J Enzyme Inhib Med Chem ; 36(1): 1938-1951, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34459690

RESUMO

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC50: 7.54 ± 1.10 µM), 5e (IC50: 9.00 ± 0.97 µM), and 5 h (IC50: 9.57 ± 0.62 µM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC50: 32.18 ± 1.66 µM), 5 h (IC50: 31.47 ± 1.42 µM), and 5 s (IC50: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.HighlightsA series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.Compound 5g exhibited promising activity (IC50 = 7.54 ± 1.10 µM) against α-glucosidase.Compound 5s exhibited promising activity (IC50 = 30.91 ± 0.86 µM) against α-amylase.In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.


Assuntos
Inibidores de Glicosídeo Hidrolases/síntese química , Indóis/síntese química , Metano/síntese química , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismo , Células 3T3 , Acarbose/química , Animais , Domínio Catalítico , Inibidores de Glicosídeo Hidrolases/metabolismo , Células Hep G2 , Humanos , Cinética , Metano/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
9.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 37(3): 266-271, 2021 May.
Artigo em Chinês | MEDLINE | ID: mdl-34374239

RESUMO

Objective: To investigate the effect of TGF-ß1/Smad signaling pathway on the apoptosis of HepG2 cells under endoplasmic reticulum stress (ERS). Methods: An ERS model was established firstly. Human hepatocellular carcinoma HepG2 cells were treated with 3 µmol/L tunicamycin (TM) for 24 h to induce ERS. Cells were divided into 6 groups, each with 3 replicate holes, and the experiment was repeated 3 times. The 6 groups included untreated group, TM group (3 µmol/L TM treatment group), TM + NC group(3 µmol/L TM + si-TGF-ß1 negative control group), TM + si-TGF-ß1 group(3 µmol/L TM + si-TGF-ß1 group), TM + pEX-3 group(3 µmol/L TM + plasmid control group), and TM + TGF-ß1 pEX-3 group(3 µmol/L TM + TGF-ß1 overexpressed plasmid group). HepG2 cells were transfected with TGF-ß1 small interfering RNA (TGF-ß1 si-RNA) and TGF-ß1 overexpressed plasmids (TGF-ß1 pEX-3) by Lipofectamine. Twenty-four hours after transfection, RT-qPCR and Western blot were used to detect the expression of TGF-ß1 and p-Smad2 in HepG2 cells of each group. CCK-8 and flow cytometry were used to analyze changes in the proliferation inhibition rate and apoptosis rate of HepG2 cells in each group. Results: Compared with the untreated group, the expressions of TGF-ß1 and p-Smad2 in TM group were significantly reduced (P<0.05). Compared with the TM group, the expressions of TGF-ß1 and p-Smad2, as well as the cell proliferation inhibition rate and apoptosis rate in TM + si-TGF-ß1 group were obviously decreased (P< 0.01), while the expressions of TGF-ß1 and p-Smad2, cell proliferation inhibition rate and apoptosis rate of TM + TGF-ß1 pEX-3 group were significantly increased (P<0.01). Conclusion: The TGF-ß1/Smad signaling pathway was inhibited in hepatocellular carcinoma HepG2 cells under ERS, when this pathway was activated, the apoptosis rate of HepG2 cells under ERS was increased significantly.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Apoptose , Estresse do Retículo Endoplasmático , Células Hep G2 , Humanos , Transdução de Sinais , Fator de Crescimento Transformador beta1
10.
Nutrients ; 13(7)2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-34371959

RESUMO

Polycystic ovary syndrome (PCOS) increases type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) with insulin resistance. We hypothesized that a 35 g whey preload would improve insulin sensitivity and glucose handling while reducing biomarkers associated with NAFLD. Twenty-nine age-matched women (CON = 15, PCOS = 14) completed oral glycemic tolerance tests following baseline (Day 0) as well as an acute (Day 1) and short-term whey supplementation (Day 7). Whey had an interaction effect on glucose (p = 0.02) and insulin (p = 0.03), with glucose remaining stable and insulin increasing with whey supplementation. Insulin sensitivity (p < 0.01) improved with whey associated with increased glucagon secretion (p < 0.01). Alanine aminotransferase (ALT), and aspartate aminotransferase (AST) remained unchanged, but "day" had an effect on the AST:ALT ratio (p = 0.04), whereas triglycerides and sex hormone binding globulin overall were greater in the PCOS group (p < 0.05). Total cholesterol decreased in PCOS (by 13%) and CON (by 8%) (NS). HepG2 cells treated with plasma from participants before and after whey decreased lipid accumulation in the PCOS group after whey (p < 0.05). Whey provided an insulinogenic and glycemic homeostatic effect in women with PCOS with the potential to combat NAFLD-consequences.


Assuntos
Glicemia/análise , Suplementos Nutricionais , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/complicações , Proteínas do Soro do Leite/administração & dosagem , Adulto , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Colesterol/sangue , Feminino , Células Hep G2 , Humanos , Insulina/sangue , Resistência à Insulina , Metabolismo dos Lipídeos , Síndrome do Ovário Policístico/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Triglicerídeos/sangue , Adulto Jovem
11.
Nat Chem Biol ; 17(9): 947-953, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34413525

RESUMO

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.


Assuntos
Receptor de Asialoglicoproteína/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Dinitrofenóis/química , Dinitrofenóis/metabolismo , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química
12.
FASEB J ; 35(9): e21788, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34425031

RESUMO

Hypoxia increases fetal hepatic insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR-mediated IGFBP-1 hyperphosphorylation in response to hypoxia requires hypoxia-inducible factor-1α (HIF-1α) and regulated in development and DNA-damage responses-1 (REDD-1) signaling. Western blotting (n = 6) and immunohistochemistry (n = 3) using fetal liver indicated greater expression of HIF-1α, REDD-1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2 ) (n = 3) induced REDD-1, inhibited mTOR complex-1 (mTORC1) activity and increased IGFBP-1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF-1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia-mediated inhibition of mTORC1 and induction of IGFBP-1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF-1α and also REDD-1 expression, inhibited mTORC1 and increased IGFBP-1 secretion/phosphorylation. Induction of HIF-1α (DMOG) and REDD-1 by Compound 3 inhibited mTORC1, increased IGFBP-1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF-1α induction, increased REDD-1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP-1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia-responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.


Assuntos
Técnicas de Cultura de Células , Modelos Animais de Doenças , Retardo do Crescimento Fetal/metabolismo , Feto/metabolismo , Hipóxia/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Animais , Eritropoetina/metabolismo , Peso Fetal , Feto/química , Células Hep G2 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Microscopia de Fluorescência , Tamanho do Órgão , Papio , Fosforilação , Proteína Quinase C-alfa/metabolismo , Receptores da Eritropoetina/metabolismo , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445462

RESUMO

Liver cancer is the sixth most common cancer worldwide with high morbidity and mortality. Programmed death ligand 1 (PD-L1) is a major ligand of programmed death 1 receptor (PD1), and PD1/PD-L1 checkpoint acts as a negative regulator of the immune system. Cancers evade the host's immune defense via PD-L1 expression. This study aimed to investigate the effects of tumor-related cytokines, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) on PD-L1 expression in human hepatocellular carcinoma cells, HepG2. Furthermore, as atorvastatin, a cholesterol-lowering agent, is documented for its immunomodulatory properties, its effect on PD-L1 expression was investigated. In this study, through real-time RT-PCR, Western blot, and immunocytochemistry methods, PD-L1 expression in both mRNA and protein levels was found to be synergistically upregulated in HepG2 by a combination of IFNγ and TNFα, and STAT1 activation was mainly responsible for that synergistic effect. Next, atorvastatin can inhibit the induction of PD-L1 by either IFNγ alone or IFNγ/TNFα combination treatment in HepG2 cells. In conclusion, in HepG2 cells, expression of PD-L1 was augmented by cytokines in the tumor microenvironment, and the effect of atorvastatin on tumor immune response through inhibition of PD-L1 induction should be taken into consideration in cancer patients who have been prescribed atorvastatin.


Assuntos
Atorvastatina/farmacologia , Antígeno B7-H1/imunologia , Carcinoma Hepatocelular/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/imunologia , Proteínas de Neoplasias/imunologia , Antígeno B7-H1/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/genética
14.
Nat Commun ; 12(1): 5057, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417449

RESUMO

Argonaute is the primary mediator of metazoan miRNA targeting (MT). Among the currently identified >1,500 human RNA-binding proteins (RBPs), there are only a handful of RBPs known to enhance MT and several others reported to suppress MT, leaving the global impact of RBPs on MT elusive. In this study, we have systematically analyzed transcriptome-wide binding sites for 150 human RBPs and evaluated the quantitative effect of individual RBPs on MT efficacy. In contrast to previous studies, we show that most RBPs significantly affect MT and that all of those MT-regulating RBPs function as MT enhancers rather than suppressors, by making the local secondary structure of the target site accessible to Argonaute. Our findings illuminate the unappreciated regulatory impact of human RBPs on MT, and as these RBPs may play key roles in the gene regulatory network governed by metazoan miRNAs, MT should be understood in the context of co-regulating RBPs.


Assuntos
MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sítios de Ligação , Evolução Molecular , Células HeLa , Células Hep G2 , Humanos , MicroRNAs/genética , Conformação de Ácido Nucleico , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Especificidade por Substrato
15.
Ecotoxicol Environ Saf ; 223: 112575, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352575

RESUMO

Treatment of liver cancer has always been a challenge for clinicians and development of appropriate drug against hepatocellular carcinoma is the major focus for researchers working in the field. The synthesis of metal-based nanoparticles (NPs) by green method for pharmacological uses has attained considerable attention recently. In current study three different NPs (AgO2, CeO2, CuO2) were synthesized by using Trianthima portulacastrum and Chinopodium quinoa leaf extracts. These biogenic NPs were analyzed by High-tech. approaches including Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray (EDX) spectroscope, SEM-EDS spot analysis, elemental mapping and X-ray diffraction (XRD). The anticancer potential of these nanoparticles was estimated using MTT assay, against hepatic cancer cell line (HepG2). SEM secondary electron images presented the nano size of prepared particles in agglomerated form with few porous forms. Average size of Ag-, Ce-, and CuNPs was observed 19-24 nm, 8-12 nm, 13-15 nm respectively. Elemental mapping and EDS-spot analysis ratifies the formation of AgNPs, CeNPs, and CuNPs. These NPs have shown good anticancer activity at different concentrations against HepG2 cell line. Further studies are however needed to identify the molecular mechanisms of these anticancer activities.


Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Nanopartículas Metálicas , Antibacterianos , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas Metálicas/toxicidade , Extratos Vegetais/farmacologia , Prata , Difração de Raios X
16.
In Vivo ; 35(5): 2599-2608, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410947

RESUMO

BACKGROUND/AIM: Asian Traditional medicines are renowned for their antitumor properties and are efficacious in the clinical treatment of various cancer types. ERM210 is a Korean traditional medicine comprising nine types of medicinal plants. In the present study, we examined the pro-apoptotic effect and molecular mechanisms of the effects of ERM210 on HepG2 liver cancer cells. MATERIALS AND METHODS: The cytotoxicity of ERM210 on HepG2 cells was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and wound-healing assays, and apoptosis and signaling pathways by fluorescence microscopy flow cytometry and western blotting. RESULTS: ERM210 significantly impaired HepG2 cell viability and enhanced mitochondria-dependent cellular apoptosis in a time- and dose-dependent manner by up-regulating the expression of caspases 3, 7 and 9, and of BCL2 apoptosis regulator (BCL2)-associated X, apoptosis regulator (BAX) proteins, whilst down-regulating that of BCL2 protein. Furthermore, ERM210 treatment increased accumulation of cellular and mitochondrial reactive oxygen species (ROS) and significantly inhibited cell migration. Additionally, all these phenomena were reversed by treating with the ROS scavenger N-acetylcysteine. The analysis of signaling proteins revealed that ERM210 significantly up-regulated the phosphorylation of ROS-dependent mitogen-activated protein kinases (p38, extracellular-regulated kinase, and c-Jun N-terminal kinase in HepG2 liver cancer cells. CONCLUSION: ERM210 exerts anticancer effects in HepG2 liver cancer cells by up-regulating ROS/mitochondria-dependent apoptosis signaling, providing new insight into the possibility of employing this traditional medicine for the clinical treatment of liver cancer.


Assuntos
Apoptose , Neoplasias Hepáticas , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
17.
FASEB J ; 35(9): e21803, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34365685

RESUMO

Melatonin pleiotropically regulates physiological events and has a putative regulatory role in the circadian clock desynchrony-mediated Non-alcoholic fatty liver disease (NAFLD). In this study, we investigated perturbations in the hepatic circadian clock gene, and Nrf2-HO-1 oscillations in conditions of high-fat high fructose (HFHF) diet and/or jet lag (JL)-mediated NAFLD. Melatonin treatment (100 µM) to HepG2 cells led to an improvement in oscillatory pattern of clock genes (Clock, Bmal1, and Per) in oleic acid (OA)-induced circadian desynchrony, while Cry, Nrf2, and HO-1 remain oblivious of melatonin treatment that was also validated by circwave analysis. C57BL/6J mice subjected to HFHF and/or JL, and treated with melatonin showed an improvement in the profile of lipid regulatory genes (CPT-1, PPARa, and SREBP-1c), liver function (AST and ALT) and histomorphology of fatty liver. A detailed scrutiny revealed that hepatic mRNA and protein profiles of Bmal1 (at ZT6) and Clock (at ZT12) underwent corrective changes in oscillations, but moderate corrections were recorded in other components of clock genes (Per1, Per2, and Cry2). Melatonin induced changes in oscillations of anti-oxidant genes (Nrf2, HO-1, and Keap1) subtly contributed in the overall improvement in NAFLD recorded herein. Taken together, melatonin induced reprograming of hepatic core clock and Nrf2-HO-1 genes leads to an improvement in HFHF/JL-induced NAFLD.


Assuntos
Ritmo Circadiano/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Melatonina/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Relógios Circadianos/efeitos dos fármacos , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Circadianas Period/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
18.
FASEB J ; 35(9): e21814, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34369624

RESUMO

Alteration in glucose homeostasis during cancer metabolism is an important phenomenon. Though several important transcription factors have been well studied in the context of the regulation of metabolic gene expression, the role of epigenetic readers in this regard remains still elusive. Epigenetic reader protein transcription factor 19 (TCF19) has been recently identified as a novel glucose and insulin-responsive factor that modulates histone posttranslational modifications to regulate glucose homeostasis in hepatocytes. Here we report that TCF19 interacts with a non-histone, well-known tumor suppressor protein 53 (p53) and co-regulates a wide array of metabolic genes. Among these, the p53-responsive carbohydrate metabolic genes Tp53-induced glycolysis and apoptosis regulator (TIGAR) and Cytochrome C Oxidase assembly protein 2 (SCO2), which are the key regulators of glycolysis and oxidative phosphorylation respectively, are under direct regulation of TCF19. Remarkably, TCF19 can form different transcription activation/repression complexes which show substantial overlap with that of p53, depending on glucose-mediated variant stress situations as obtained from IP/MS studies. Interestingly, we observed that TCF19/p53 complexes either have CBP or HDAC1 to epigenetically program the expression of TIGAR and SCO2 genes depending on short-term high glucose or prolonged high glucose conditions. TCF19 or p53 knockdown significantly altered the cellular lactate production and led to increased extracellular acidification rate. Similarly, OCR and cellular ATP production were reduced and mitochondrial membrane potential was compromised upon depletion of TCF19 or p53. Subsequently, through RNA-Seq analysis from patients with hepatocellular carcinoma, we observed that TCF19/p53-mediated metabolic regulation is fundamental for sustenance of cancer cells. Together the study proposes that TCF19/p53 complexes can regulate metabolic gene expression programs responsible for mitochondrial energy homeostasis and stress adaptation.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mitocôndrias/genética , Chaperonas Moleculares/genética , Monoéster Fosfórico Hidrolases/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , Proteína Supressora de Tumor p53/genética , Adaptação Biológica/genética , Apoptose/genética , Linhagem Celular Tumoral , Metabolismo Energético/genética , Glucose/genética , Células Hep G2 , Homeostase/genética , Humanos , Potencial da Membrana Mitocondrial/genética , Estresse Fisiológico/genética , Ativação Transcricional/genética
19.
Nat Cell Biol ; 23(8): 905-914, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34354237

RESUMO

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.


Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Regulação da Expressão Gênica , Heterocromatina/fisiologia , Células Cultivadas , Sequência Conservada , Células Hep G2 , Histonas/metabolismo , Humanos
20.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361728

RESUMO

Glycyrrhetinic acid (GA) is one of many interesting pentacyclic triterpenoids showing significant anticancer activity by triggering apoptosis in tumor cell lines. This study deals with the design and synthesis of new glycyrrhetinic acid (GA)-amino acid peptides and peptide ester derivatives. The structures of the new derivatives were established through various spectral and microanalytical data. The novel compounds were screened for their in vitro cytotoxic activity. The evaluation results showed that the new peptides produced promising cytotoxic activity against the human breast MCF-7 cancer cell line while comparing to doxorubicin. On the other hand, only compounds 3, 5, and 7 produced potent activity against human colon HCT-116 cancer cell line. The human liver cancer (HepG-2) cell line represented a higher sensitivity to peptide 7 (IC50; 3.30 µg/mL), while it appeared insensitive to the rest of the tested peptides. Furthermore, compounds 1, 3, and 5 exhibited a promising safety profile against human normal skin fibroblasts cell line BJ-1. In order to investigate the mode of action, compound 5 was selected as a representative example to study its in vitro effect against the apoptotic parameters and Bax/BCL-2/p53/caspase-7/caspase-3/tubulin, and DNA fragmentation to investigate beta (TUBb). Additionally, all the new analogues were subjected to antimicrobial assay against a panel of Gram-positive and Gram-negative bacteria and the yeast candida Albicans. All the tested GA analogues 1-8 exhibited more antibacterial effect against Micrococcus Luteus than gentamicin, but they exhibited moderate antimicrobial activity against the tested bacterial and yeast strains. Molecular docking studies were also simulated for compound 5 to give better rationalization and put insight to the features of its structure.


Assuntos
Antibacterianos/síntese química , Antifúngicos/síntese química , Antineoplásicos/síntese química , Citotoxinas/síntese química , Ácido Glicirretínico/química , Peptídeos/química , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Caspase 3/química , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/farmacologia , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ácido Glicirretínico/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Células HCT116 , Células Hep G2 , Humanos , Células MCF-7 , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
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