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1.
Biomed Environ Sci ; 34(7): 520-527, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34353415

RESUMO

Objective: Although benzene is a confirmed environmental carcinogen, the mechanism of its carcinogenicity remains largely unclear. The suggested oncogene, miR-221, is elevated and plays important roles in various tumors, but its role in benzene-induced carcinogenesis remains unknown. Methods: In the present study, we constructed hydroquinone (HQ, a representative metabolite of benzene with biological activity)-transformed malignant cell line (16HBE-t) and analyzed the level of miR-221 in it with qRT-PCR. Exosomes from 16HBE-t cells incubated with or without an miR-221 inhibitor were isolated by ultracentrifugation, characterized by transmission electron microscopy and laser scanning confocal microscope, and then transfected into 16HBE cells. The effects of exosomal miR-221 on apoptosis induced by HQ in recipient cells were determined using flow cytometry. Results: The amount of miR-221 in 16HBE-t was significantly increased compared with controls. When recipient cells ingested exosomes derived from 16HBE-t, miR-221 was increased, and apoptosis induced by HQ was inhibited. Blocking miR-221 in 16HBE-t using an inhibitor did not significantly alter miR-221 or apoptosis in recipient cells. Conclusion: Exosomal miR-221 secreted by 16HBE-t inhibits apoptosis induced by HQ in normal recipient cells.


Assuntos
Apoptose , Exossomos , Hidroquinonas , MicroRNAs , Brônquios/citologia , Linhagem Celular Transformada , Células Epiteliais , Humanos
2.
Nat Struct Mol Biol ; 28(6): 487-500, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34117478

RESUMO

Fanconi anemia (FA) is a devastating hereditary disease characterized by bone marrow failure (BMF) and acute myeloid leukemia (AML). As FA-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), ICLs are widely assumed to be the lesions responsible for FA symptoms. Here, we show that FA-mutated cells are hypersensitive to persistent replication stress and that FA proteins play a role in the break-induced-replication (BIR)-like pathway for fork restart. Both the BIR-like pathway and ICL repair share almost identical molecular mechanisms of 53BP1-BRCA1-controlled signaling response, SLX4- and FAN1-mediated fork cleavage and POLD3-dependent DNA synthesis, suggesting that the FA pathway is intrinsically one of the BIR-like pathways. Replication stress not only triggers BMF in FA-deficient mice, but also specifically induces monosomy 7, which is associated with progression to AML in patients with FA, in FA-deficient cells.


Assuntos
Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Anemia de Fanconi/genética , Aneuploidia , Animais , Transtornos da Insuficiência da Medula Óssea/etiologia , Linhagem Celular Transformada , Galinhas , Quebra Cromossômica , Deleção Cromossômica , Cromossomos Humanos Par 7/genética , DNA Polimerase III/fisiologia , Replicação do DNA/genética , Progressão da Doença , Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/deficiência , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Células HCT116 , Células HEK293 , Humanos , Hidroxiureia/farmacologia , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Especificidade da Espécie , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia , Ubiquitina-Proteína Ligases/fisiologia
3.
Cell Physiol Biochem ; 55(3): 311-326, 2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34148309

RESUMO

BACKGROUND/AIMS: The skeleton is a metabolically active organ undergoing continuous remodelling initiated by mesenchymal progenitors present in bone and bone marrow. Under certain pathological conditions this remodelling balance shifts towards increased resorption resulting in weaker bone microarchitecture, and there is consequently a therapeutic need to identify pathways that could inversely enhance bone formation from stem cells. Metabolomics approaches recently applied to stem cell characterisation could help identify new biochemical markers involved in osteogenic differentiation. METHODS: Combined intra- and extracellular metabolite profiling was performed by liquid chromatography-mass spectrometry (LC-MS) on human mesenchymal stem cells (MSCs) undergoing osteogenic differentiation in vitro. Using a combination of univariate and multivariate analyses, changes in metabolite and nutrient concentration were monitored in cultures under osteogenic treatment over 10 days. RESULTS: A subset of differentially detected compounds was identified in differentiating cells, suggesting a direct link to metabolic processes involved in osteogenic response. CONCLUSION: These results highlight new metabolite candidates as potential biomarkers to monitor stem cell differentiation towards the bone lineage.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Metaboloma , Metabolômica , Osteogênese , Linhagem Celular Transformada , Humanos
4.
Life Sci ; 281: 119771, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34186051

RESUMO

AIMS: Medical treatment for lower urinary tract symptoms secondary to benign prostatic hyperplasia is characterized by an unfavorable balance between limited efficacy and pronounced side effects. We recently reported, that thalidomide reduces prostate smooth muscle contraction and inhibits cell growth. Like thalidomide, its analogs lenalidomide and pomalidomide are also in clinical use. Therefore, we investigated the effects of lenalidomide and pomalidomide on human prostate smooth muscle contraction, cytoskeletal organization, and growth-related functions in stromal cells. MATERIALS AND METHODS: Proliferation was assessed by EdU assay and colony formation, cytoskeletal organization by phalloidin staining, cell viability by CCK8, and apoptosis and cell death by flow cytometry in cultured prostate stromal cells (WPMY-1). Contractions of human prostate tissues from radical prostatectomy were induced by methoxamine, noradrenaline, phenylephrine, endothelin-1, U46619, or electric field stimulation (EFS) in an organ bath. KEY FINDINGS: Proliferation of WPMY-1 cells was significantly reduced by lenalidomide (5-200 µM) and pomalidomide (2.5-5 µM). In parallel, organization of actin filaments collapsed after treatment with lenalidomide and pomalidomide. Lenalidomide and pomalidomide inhibited both adrenergic contractions and non-adrenergic contractions as well as neurogenic contractions induced by EFS. Neither reduction in viability, nor increase in cell death or apoptosis was observed in WPMY-1 cells. SIGNIFICANCE: Thalidomide-derivatives impair growth of human prostate stromal cells, without showing a decrease in cell viability and, in parallel, inhibit adrenergic, neurogenic, and non-adrenergic contractions by breakdown of the actin cytoskeleton. Urodynamic effects in vivo appear possible.


Assuntos
Lenalidomida/farmacologia , Músculo Liso/efeitos dos fármacos , Próstata/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Talidomida/análogos & derivados , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Humanos , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Próstata/citologia , Próstata/fisiologia , Talidomida/farmacologia
5.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071138

RESUMO

Schwann cells play an important role in peripheral nerve function, and their dysfunction has been implicated in the pathogenesis of diabetic neuropathy and other demyelinating diseases. The physiological functions of insulin in Schwann cells remain unclear and therefore define the aim of this study. By using immortalized adult Fischer rat Schwann cells (IFRS1), we investigated the mechanism of the stimulating effects of insulin on the cell proliferation and expression of myelin proteins (myelin protein zero (MPZ) and myelin basic protein (MBP). The application of insulin to IFRS1 cells increased the proliferative activity and induced phosphorylation of Akt and ERK, but not P38-MAPK. The proliferative potential of insulin-stimulated IFRS1 was significantly suppressed by the addition of LY294002, a PI3 kinase inhibitor. The insulin-stimulated increase in MPZ expression was significantly suppressed by the addition of PD98059, a MEK inhibitor. Furthermore, insulin-increased MBP expression was significantly suppressed by the addition of LY294002. These findings suggest that both PI3-K/Akt and ERK/MEK pathways are involved in insulin-induced cell growth and upregulation of MPZ and MBP in IFRS1 Schwann cells.


Assuntos
Insulina/farmacologia , Células de Schwann/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Cromonas/farmacologia , Neuropatias Diabéticas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos F344 , Receptor de Insulina/biossíntese , Receptor de Insulina/genética , Transdução de Sinais/efeitos dos fármacos
6.
Mol Immunol ; 135: 304-311, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33964631

RESUMO

The RIP family plays a key role in mediating cell inflammation, oxidative stress and death. Among them, RIPK1, as an important regulatory factor in the upstream of the NF-κB pathway, is involved in multiple pathways of cell inflammation and death. Epidermal cells constitute the outermost barrier of the human body. Radiation can induce epidermal cell death, inflammation and oxidative stress to cause damage. Therefore, this paper selected HaCaT cell and used CRISPR/Cas technology to construct a cell model of stable knockout of RIPK1 gene, to analyze the effect and regulation of RIPK1 knockout on the function and death of HaCaT cells induced by UVB or TNF-α. The results showed that knockout of RIPK1 had no significant effect on the morphology of HaCaT cells at rest, but it led to slowing cell proliferation and blocking the G2M phase of cell cycle. Compared with HaCaTWT, HaCaTRIP1KO was abnormally sensitive to TNF-α-induced cell death and apoptosis, and may be associated with inhibition of NF-κB pathway. Knocking out RIPK1 led to a more significant inhibition of cell growth by UVB, and up-regulation of the expression of the inflammatory factor IL-1α. P38 MAPK and NF-κB pathways may be involved this process. This study further found that RIPK1 in epidermal cell has a regulatory function on pro-survival signals.


Assuntos
Apoptose/fisiologia , Células Epidérmicas/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Raios Ultravioleta/efeitos adversos , Sistemas CRISPR-Cas/genética , Linhagem Celular Transformada , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Técnicas de Inativação de Genes , Células HaCaT , Humanos , Interleucina-1alfa/metabolismo , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/fisiologia , Pele/lesões , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Toxicology ; 457: 152811, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33971260

RESUMO

Preterm infants are at greater risk for adverse drug effects due to hepatic immaturity. Multiple interventions during intensive care increases potential for drug interactions. In this setting, high-dose caffeine used for apnea in premature infants may increase acetaminophen toxicity by inhibiting ataxia telangiectasia mutated (ATM) gene activity during DNA damage response. To define caffeine and acetaminophen interaction, we modeled infantile prematurity in late-gestation fetal stage through human immortalized hepatocytes and liver organoids. The acute toxicity studies included assays for cell viability, mitochondrial dysfunction and ATM pathway-related DNA damage. Fetal cells expressed hepatobiliary properties, albeit with lower metabolic, synthetic and antioxidant functions than more mature hepatocytes. Acetaminophen in IC50 amount of 7.5 millimolar caused significant oxidative stress, mitochondrial membrane potential impairments, and DNA breaks requiring ATM-dependent repair. Caffeine markedly exacerbated acetaminophen toxicity by suppressing ATM activity in otherwise nontoxic 2.5 millimolar amount. Similarly, the specific ATM kinase antagonist, KU-60019, reproduced this deleterious interaction in 5 micromolar amount. Replicative stress from combined acetaminophen and caffeine toxicity depleted cells undergoing DNA synthesis in S phase and activated checkpoints for G0/G1 or G2/M restrictions. Synergistic caffeine and acetaminophen toxicity in liver organoids indicated these consequences should apply in vivo. The antioxidant, N-acetylcysteine, decreased oxidative damage, mitochondrial dysfunction and ATM pathway disruption to mitigate caffeine and acetaminophen toxicity. We concluded that hepatic DNA damage, mitochondrial impairment and growth-arrest after combined caffeine and acetaminophen toxicity will be harmful for premature infants. Whether caffeine and acetaminophen toxicity may alter outcomes in subsequently encountered hepatic disease needs consideration.


Assuntos
Acetaminofen/toxicidade , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cafeína/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Acetaminofen/administração & dosagem , Cafeína/administração & dosagem , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Estimulantes do Sistema Nervoso Central/administração & dosagem , Estimulantes do Sistema Nervoso Central/toxicidade , Feto , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo
8.
Biochem Pharmacol ; 188: 114575, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33887260

RESUMO

Hyperactivation of signal transducer and activator of transcription 3 (STAT3) is strongly associated with cancer initiation, progression, metastasis, chemoresistance, and immune evasion; thus, STAT3 has been intensely studied as a therapeutic target for cancer treatment. Berberine (BBR), an active component extracted from Coptis chinensis, has shown anti-tumor effects in multiple tumors. However, its underlying mechanisms have not yet been fully elucidated. In this study, we investigated the effects and the underlying mechanisms of BBR on bladder cancer (BCa) cells. We found that BBR showed significant cytotoxic effects against BCa cell lines both in vivo and in vitro, with much lower cytotoxic effects on the human normal urothelial cell line SV-HUC-1. BBR treatment induced DNA replication defects and cell cycle arrest, resulting in apoptosis or cell senescence, depending on p53 status, in BCa cells. Mechanistically, BBR exerted anti-tumor effects on BCa cells by inhibiting Janus kinase 1 (JAK1)-STAT3 signaling through the upregulation of miR-17-5p, which directly binds to the 3'UTR of JAK1 and STAT3, downregulating their expressions. Collectively, our results demonstrate that BBR exerts anti-tumor effects by perturbing JAK1-STAT3 signaling through the upregulation of miR-17-5p in BCa cells, and that BBR may serve as a potential therapeutic option for BCa treatment.


Assuntos
Berberina/farmacologia , Proliferação de Células/fisiologia , Janus Quinase 1/metabolismo , MicroRNAs/biossíntese , Fator de Transcrição STAT3/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Animais , Berberina/uso terapêutico , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/genética , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
9.
Acta Biochim Biophys Sin (Shanghai) ; 53(6): 719-728, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-33856026

RESUMO

Acute kidney injury (AKI) is a complex syndrome with an abrupt decrease of kidney function, which is associated with high morbidity and mortality. Sepsis is the common cause of AKI. Mounting evidence has demonstrated that long non-coding RNAs (lncRNAs) play critical roles in the development and progression of sepsis-induced AKI. In this study, we aimed to illustrate the function and mechanism of lncRNA SNHG14 in lipopolysaccharide (LPS)-induced AKI. We found that SNHG14 was highly expressed in the plasma of sepsis patients with AKI. SNHG14 inhibited cell proliferation and autophagy and promoted cell apoptosis and inflammatory cytokine production in LPS-stimulated HK-2 cells. Functionally, SNHG14 acted as a competing endogenous RNA (ceRNA) to negatively regulate miR-495-3p expression in HK-2 cells. Furthermore, we identified that HIPK1 is a direct target of miR-495-3p in HK-2 cells. We also revealed that the SNHG14/miR-495-3p/HIPK1 interaction network regulated HK-2 cell proliferation, apoptosis, autophagy, and inflammatory cytokine production upon LPS stimulation. In addition, we demonstrated that the SNHG14/miR-495-3p/HIPK1 interaction network regulated the production of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) via modulating NF-κB/p65 signaling in LPS-challenged HK-2 cells. In conclusion, our findings suggested a novel therapeutic axis of SNHG14/miR-495-3p/HIPK1 to treat sepsis-induced AKI.


Assuntos
Injúria Renal Aguda/sangue , Injúria Renal Aguda/induzido quimicamente , Células Epiteliais/metabolismo , Lipopolissacarídeos/efeitos adversos , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Longo não Codificante/sangue , Sepse/sangue , Transdução de Sinais/genética , Apoptose/genética , Autofagia/genética , Estudos de Casos e Controles , Linhagem Celular Transformada , Proliferação de Células/genética , Citocinas/biossíntese , Células Epiteliais/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Túbulos Renais/citologia , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , RNA Longo não Codificante/genética , Sepse/complicações , Transfecção
10.
Mol Immunol ; 135: 21-27, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33857815

RESUMO

Ultraviolet A (UVA) irradiation caused skin keratinocytes to accumulate reactive oxygen species (ROS) leading to the skin injury. Thymoquinone (TQ) was identified as the prominent bioactive ingredient in Nigella sativa seeds which was applied in therapying various human diseases. This study aimed to illustrate the role and mechanism of TQ in UVA-induced skin injury. We pre-treated HaCaT cells with TQ and irradiated them by UVA. MTT and Elisa assays were used to evaluate cell viability and apoptosis, as well as cytokine levels. To detect the related parameters of oxidative stress and mitochondrial function, colorimetry, spectrophotometry, bioluminescence, and dual-luciferase reporter methods were used. RT-qPCR and western blotting were performed for expressions of related mRNAs and proteins. TQ significantly improved the UVA-induced cytotoxicity on HaCaT cells. TQ treatment alleviated the oxidative stress and inflammation in UVA-irradiated keratinocytes. Besides, UVA irradiation promoted mitochondrial dysregulation in HaCaT cells leading to cell apoptosis, which could be reversed by TQ treatment. More importantly, NrF2/ARE pathway was activated in TQ-treated cells, while COX-2 was depressed, and inhibiting the pathway or activating COX-2 blocked the therapeutic effect of TQ on UVA-induced skin cell injury. Our study suggested that TQ treatment attenuated the UVA-induced oxidative and inflammatory responses, as well as mitochondrial apoptosis in keratinocytes by COX-2 inhibition via activating NrF2/ARE pathway. This might be a novel sight for preventing the solar radiation damage to the skin.


Assuntos
Benzoquinonas/farmacologia , Queratinócitos/efeitos da radiação , Mitocôndrias/efeitos da radiação , Nigella sativa/metabolismo , Substâncias Protetoras/farmacologia , Raios Ultravioleta/efeitos adversos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Citocinas/metabolismo , Células HaCaT , Humanos , Inflamação/prevenção & controle , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos da radiação , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sementes/metabolismo , Pele/lesões
11.
J Biochem Mol Toxicol ; 35(6): 1-13, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33851497

RESUMO

BPDE (benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide), a metabolite of environmental carcinogenic BaP, weakens the migration and invasion of human villous trophoblast cells and may further induce miscarriage. However, the underlying mechanisms remain largely unknown. In this study, we identified that in trophoblast Swan 71 and HTR-8/SVneo cells, miR-hz02 upregulates the level of lnc-HZ02, which inhibits the expression of an RNA-binding protein HuR. HuR could interact with FAK mRNA and promote its mRNA stability, thus upregulating the FAK level and the FAK/SRC/PI3K/AKT pathway, and finally maintaining the normal migration and invasion of trophoblast cells. If trophoblast cells are exposed to BPDE, both miR-hz02 and lnc-HZ02 are upregulated, which reduce the level of HuR, weaken the interactions of HuR with FAK mRNA, downregulate FAK level and the FAK/SRC/PI3K/AKT pathway, and finally inhibit cell migration and invasion. This study provides a novel scientific understanding of the dysfunctions of human trophoblast cells.


Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/toxicidade , Regulação para Baixo/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , MicroRNAs/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , RNA Longo não Codificante/biossíntese , Trofoblastos/metabolismo , Regulação para Cima/efeitos dos fármacos , Linhagem Celular Transformada , Humanos , Trofoblastos/patologia
12.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803156

RESUMO

The objective of this study was to investigate the effects of supplementing with L-tryptophan (L-Trp) on milk protein synthesis using an immortalized bovine mammary epithelial (MAC-T) cell line. Cells were treated with 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM of supplemental L-Trp, and the most efficient time for protein synthesis was determined by measuring cell, medium, and total protein at 0, 24, 48, 72, and 96 h. Time and dose tests showed that the 48 h incubation time and a 0.9 mM dose of L-Trp were the optimal values. The mechanism of milk protein synthesis was elucidated through proteomic analysis to identify the metabolic pathway involved. When L-Trp was supplemented, extracellular protein (medium protein) reached its peak at 48 h, whereas intracellular cell protein reached its peak at 96 h with all L-Trp doses. ß-casein mRNA gene expression and genes related to milk protein synthesis, such as mammalian target of rapamycin (mTOR) and ribosomal protein 6 (RPS6) genes, were also stimulated (p < 0.05). Overall, there were 51 upregulated and 59 downregulated proteins, many of which are involved in protein synthesis. The results of protein pathway analysis showed that L-Trp stimulated glycolysis, the pentose phosphate pathway, and ATP synthesis, which are pathways involved in energy metabolism. Together, these results demonstrate that L-Trp supplementation, particularly at 0.9 mM, is an effective stimulus in ß-casein synthesis by stimulating genes, proteins, and pathways related to protein and energy metabolism.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Triptofano/farmacologia , Animais , Bovinos , Linhagem Celular Transformada , Meios de Cultura , Feminino , Glândulas Mamárias Animais/citologia
13.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802220

RESUMO

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac4ManNAz) and N-alkyneacetylmannosamine (Ac4ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac4ManNAz was detectable for up to six days while Ac4ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.


Assuntos
Glicocálix , Hexosaminas , Células-Tronco Mesenquimais/metabolismo , Engenharia Metabólica , Modelos Biológicos , Mioblastos Esqueléticos/metabolismo , Linhagem Celular Transformada , Glicocálix/química , Glicocálix/metabolismo , Hexosaminas/química , Hexosaminas/metabolismo , Humanos
14.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809456

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case.


Assuntos
Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/imunologia , Linfócitos/imunologia , Mutação/genética , Medicina de Precisão , Superóxido Dismutase-1/genética , Ácidos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Esclerose Amiotrófica Lateral/genética , Autofagia/genética , Linhagem Celular Transformada , Metabolismo Energético , Feminino , Heterozigoto , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Consumo de Oxigênio , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
15.
Biomed Res Int ; 2021: 6657206, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33860049

RESUMO

The study is aimed at investigating the effects of Ginkgo biloba extract EGB761 on renal tubular damage and endoplasmic reticulum stress (ERS) in diabetic kidney disease (DKD). A total of 50 C57BL/6 N mice were randomly divided into the normal group, DKD group, DKD+EGB761 group (36 mg/kg), and DKD+4-phenylbutyrate (4-PBA) group (1 g/kg). The DKD model was replicated by high-fat diet combined with intraperitoneal injection of streptozotocin (STZ). Renal tubular epithelial cells (HK-2) were divided into the control group, high-glucose group (30 mmol/L), EGB761 group (40 mg/L, 20 mg/L, 10 mg/L), TM group, and TM+4-PBA group. After 8 weeks of administration, expressions of serum creatinine (Scr), blood urea nitrogen (BUN), 24 h urinary protein (24 h Pro), fasting blood glucose (FBG), ß 2-microglobulin (ß 2-MG), and retinol binding protein 4 (RBP4) of mice were tested. The pathological changes of renal tissue were observed. The expressions of extracellular matrix (ECM) accumulation and epithelial-mesenchymal transition (EMT) markers α-smooth muscle actin (α-SMA), E-cadherin, fibronectin, and collagen IV, as well as the ERS markers GRP78 and ATF6, were tested by Western blot, qPCR, immunohistochemistry, or immunofluorescence. EGB761 could decrease the Scr, BUN, 24 h Pro, and FBG levels in the DKD group, alleviate renal pathological injury, decrease urine ß 2-MG, RBP4 levels, and decrease the expression of α-SMA, collagen IV, fibronectin, and GRP78, as well as ATF6, while increase the expression of E-cadherin. These findings demonstrate that EGB761 can improve renal function, reduce tubular injury, and ameliorate ECM accumulation and EMT in DKD kidney tubules, and the mechanism may be related to the inhibition of ERS.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Estresse do Retículo Endoplasmático , Matriz Extracelular/metabolismo , Mesoderma/patologia , Extratos Vegetais/uso terapêutico , Animais , Linhagem Celular , Linhagem Celular Transformada , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Humanos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/lesões , Túbulos Renais/fisiopatologia , Túbulos Renais/ultraestrutura , Masculino , Mesoderma/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Extratos Vegetais/farmacologia , Proteínas Plasmáticas de Ligação ao Retinol/urina , Microglobulina beta-2/urina
16.
Biochem Biophys Res Commun ; 555: 190-195, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33823365

RESUMO

Primary cilia are microtubule-based organelles that are involved in sensing micro-environmental cues and regulating cellular homeostasis via triggering signaling cascades. Hypoxia is one of the most common environmental stresses that organ and tissue cells may often encounter during embryogenesis, cell differentiation, infection, inflammation, injury, cerebral and cardiac ischemia, or tumorigenesis. Although hypoxia has been reported to promote or inhibit primary ciliogenesis in different tissues or cultured cell lines, the role of hypoxia in ciliogenesis is controversial and still unclear. Here we investigated the primary cilia change under cobalt chloride (CoCl2)-simulated hypoxia in immortalized human retina pigment epithelial-1 (hTERT RPE-1) cells. We found CoCl2 treatment elongated primary cilia in a time- and dose-dependent manner. The prolonged cilia recovered back to near normal length when CoCl2 was washed out from the cell culture medium. Under CoCl2-simulated hypoxia, the protein expression levels of HIF-1/2α and acetylated-α-tubulin (cilia marker) were increased, while the protein expression level of Rabaptin-5 is decreased during hypoxia. Taken together, our results suggest that hypoxia may elongate primary cilia by downregulating Rabaptin-5 involved endocytosis. The coordination between endocytosis and ciliogenesis may be utilized by cells to adapt to hypoxia.


Assuntos
Hipóxia Celular/efeitos dos fármacos , Cílios/efeitos dos fármacos , Cobalto/toxicidade , Epitélio Pigmentado da Retina/citologia , Hipóxia Celular/fisiologia , Linhagem Celular Transformada , Cobalto/administração & dosagem , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Humanos , Prolil Hidroxilases/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Telomerase/genética , Proteínas de Transporte Vesicular/metabolismo
17.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916959

RESUMO

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased ß-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.


Assuntos
Fibroblastos/metabolismo , Recombinação Homóloga , Integrases/metabolismo , Queratinócitos/metabolismo , Telomerase/genética , Transgenes , Adolescente , Adulto , Biomarcadores , Linhagem Celular Transformada , Proliferação de Células , Senescência Celular/genética , Criança , Epidermólise Bolhosa Distrófica/etiologia , Epidermólise Bolhosa Distrófica/metabolismo , Feminino , Fibroblastos/patologia , Imunofluorescência , Técnicas de Silenciamento de Genes , Ordem dos Genes , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Cultura Primária de Células , Proteômica/métodos , Telomerase/metabolismo , Adulto Jovem
18.
Biomed Pharmacother ; 138: 111463, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33725591

RESUMO

Centromeres are chromosomal loci where kinetochores assemble to ensure faithful chromosome segregation during mitosis. CENP-A defines the loci by serving as an epigenetic marker that recruits other centromere components for a functional structure. However, the mechanism that controls CENP-A regulation of centromeric chromatin integrity remains to be explored. Separate studies have shown that loss of CENP-A or the Cdk5 regulatory subunit associated protein 2 (Cdk5rap2), a key player in mitotic progression, triggers the occurrence of lagging chromosomes. This prompted us to investigate a potential link between CENP-A and Cdk5rap2 in the maintenance of centromeric chromatin integrity. Here, we demonstrate that loss of Cdk5rap2 causes reduced CENP-A expression while exogenous Cdk5rap2 expression in cells depleted of endogenous Cdk5rap2 restores CENP-A expression. Indeed, we show that Cdk5rap2 is a nuclear protein that acts as a positive transcriptional regulator of CENP-A. Cdk5rap2 interacts with the CENP-A promoter and upregulates CENP-A transcription. Accordingly, loss of Cdk5rap2 causes reduced level of centromeric CENP-A. Exogenous CENP-A expression partially inhibits the occurrence of lagging chromosomes in Cdk5rap2 knockdown cells, indicating that lagging chromosomes induced by loss of Cdk5rap2 is due, in part, to loss of CENP-A. Aside from manifesting lagging chromosomes, cells depleted of Cdk5rap2, and thus CENP-A, show increased micronuclei and chromatin bridge formation. Altogether, our findings indicate that Cdk5rap2 serves to maintain centromeric chromatin integrity partly through CENP-A.


Assuntos
Proteínas de Ciclo Celular/deficiência , Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Cromatina/metabolismo , Proteínas do Tecido Nervoso/deficiência , Ativação Transcricional/fisiologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Centrômero/genética , Proteína Centromérica A/genética , Cromatina/genética , Segregação de Cromossomos/fisiologia , Células HEK293 , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Nucleossomos/genética , Nucleossomos/metabolismo
19.
Int J Biol Macromol ; 180: 36-50, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33727184

RESUMO

Composition of polymers and choosing the type of solvents in electrospinning systems is of great importance to achieve a mat with optimal properties. In this work, with emphasizing the influence of a novel solvent system, an electrospun wound dressing was designed in four steps. Firstly, to study the effect of polymer-solvent interactions and electrospinning distance, a constant amount of polycaprolactone (PCL) was dissolved at different compositions of formic acid (FA)/dichloromethane (DCM) and was electrospun at different distances. The composition of 80FA/20DCM and distance of 15 cm were selected as optimal conditions by lowest average diameter of fibers and simultaneously good surface uniformity. In the second step, the concentration of PCL was considered variable to achieve the lowest diameter of fibers. Finally, in the third and fourth steps, different concentrations of chitosan (CN) and constant dosage of Melilotus officinalis (MO) extract were added to the solution. The extract contained fibers had a mean diameter of 275 ± 41 nm which is in the required condition for wound caring. Eventually, the optimized PCL/CN and PCL/CN/MO specimens were evaluated by FTIR, DSC, Tensile, water contact angle, antibacterial assays, cell viability, and drug release analysis for determining their function and properties.


Assuntos
Antibacterianos/química , Bandagens , Quitosana/química , Melilotus/química , Nanofibras/química , Extratos Vegetais/química , Poliésteres/química , Cicatrização , Animais , Varredura Diferencial de Calorimetria , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Fibroblastos/efeitos dos fármacos , Formiatos/química , Interações Hidrofóbicas e Hidrofílicas , Cloreto de Metileno/química , Camundongos , Testes de Sensibilidade Microbiana , Solventes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Resistência à Tração
20.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673197

RESUMO

T cell immunotherapy is now a mainstay therapy for several blood-borne cancers as well as metastatic melanoma. Unfortunately, many epithelial tumors respond poorly to immunotherapy, and the reasons for this are not well understood. Cancer-associated fibroblasts (CAFs) are the most frequent non-neoplastic cell type in most solid tumors, and they are emerging as a key player in immunotherapy resistance. A range of immortalized CAF lines will be essential tools that will allow us to understand immune responses against cancer and develop novel strategies for cancer immunotherapy. To study the effect of CAFs on T cell proliferation, we created and characterized a number of novel immortalized human CAFs lines (Im-CAFs) from human breast, colon, and pancreatic carcinomas. Im-CAFs shared similar phenotypes, matrix remodeling and contraction capabilities, and growth and migration rates compared to the primary CAFs. Using primary isolates from breast carcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma, we report that CAFs across major tumor types are able to potently suppress T cell proliferation in vitro. Im-CAFs retained this property. Im-CAFs are a key tool that will provide important insights into the mechanisms of CAF-mediated T cell suppression through techniques such as CRISPR-Cas9 modification, molecular screens, and pipeline drug testing.


Assuntos
Fibroblastos Associados a Câncer/imunologia , Proliferação de Células , Neoplasias/imunologia , Linfócitos T/imunologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Transformada , Humanos , Neoplasias/patologia , Linfócitos T/patologia
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