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1.
Clin Sci (Lond) ; 134(12): 1305-1318, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32478397

RESUMO

Podocyte injury and loss contribute to proteinuria, glomerulosclerosis and eventually kidney failure. Recent studies have demonstrated that the loss of Kruppel-like factor 15 (KLF15) in podocytes increases the susceptibility to injury; however, the mechanism underlying the protective effects on podocyte injury remains incompletely understood. Herein, we showed that KLF15 ameliorates podocyte injury through suppressing NFAT signaling and the salutary effects of the synthetic glucocorticoid dexamethasone in podocyte were partially mediated by the KLF15-NFATc1 axis. We found that KLF15 was significantly reduced in glomerular cells of proteinuric patients and in ADR-, LPS- or HG-treated podocyets in vitro. Overexpression of KLF15 attenuated podocyte apoptosis induced by ADR, LPS or HG and resulted in decreased expression of pro-apoptotic Bax and increased expression of anti-apoptotic Bcl-2. Conversely, the flow cytometry analysis and TUNEl assay demonstrated that loss of KLF15 accelerated podocyte apoptosis and we further found that 11R-VIVIT, a specific NFAT inhibitor, and NFATc1-siRNA rescued KLF15-deficient induced podocyte apoptosis. Meanwhile, Western blot and RT-qPCR showed that the expression of NFATc1 was up-regulated in KLF15 silenced podocytes and reduced in KLF15 overexpressed podocytes. Mechanistically, ChIP analysis showed that KLF15 bound to the NFATc1 promoter region -1984 to -1861base pairs upstream of the transcription start site and the binding amount was decreased after treatment with LPS. The dual-luciferase reporter assay indicated that NFATc1 was a direct target of KLF15. In addition, we found that in vitro treatment with dexamethasone induced a decrease of NFATc1 expression in podocytes and was abrogated by knockdown of KLF15. Hence, our results identify the critical role of the KLF15-NFATc1 axis in podocyte injury and loss, which may be involved in mediating the salutary effects of dexamethasone in podocytes.


Assuntos
Glucocorticoides/uso terapêutico , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição NFATC/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/tratamento farmacológico , Proteinúria/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacologia , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Glucose/toxicidade , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Modelos Biológicos , Transdução de Sinais
2.
PLoS Biol ; 18(6): e3000722, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569301

RESUMO

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.


Assuntos
Macrófagos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Processamento Alternativo/genética , Animais , Cálcio/metabolismo , Espaço Extracelular/metabolismo , Inativação Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Íons , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Células RAW 264.7 , Cloreto de Sódio/farmacologia
3.
Int J Nanomedicine ; 15: 3347-3362, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32494134

RESUMO

Introduction: Temozolomide (TMZ) is the first-line chemotherapeutic option to treat glioma; however, its efficacy and clinical application are limited by its drug resistance properties. Polo-like kinase 1 (PLK1)-targeted therapy causes G2/M arrest and increases the sensitivity of glioma to TMZ. Therefore, to limit TMZ resistance in glioma, an angiopep-2 (A2)-modified polymeric micelle (A2PEC) embedded with TMZ and a small interfering RNA (siRNA) targeting PLK1 (siPLK1) was developed (TMZ-A2PEC/siPLK). Materials and Methods: TMZ was encapsulated by A2-PEG-PEI-PCL (A2PEC) through the hydrophobic interaction, and siPLK1 was complexed with the TMZ-A2PEC through electrostatic interaction. Then, an angiopep-2 (A2) modified polymeric micelle (A2PEC) embedding TMZ and siRNA targeting polo-like kinase 1 (siPLK1) was developed (TMZ-A2PEC/siPLK). Results: In vitro experiments indicated that TMZ-A2PEC/siPLK effectively enhanced the cellular uptake of TMZ and siPLK1 and resulted in significant cell apoptosis and cytotoxicity of glioma cells. In vivo experiments showed that glioma growth was inhibited, and the survival time of the animals was prolonged remarkably after TMZ-A2PEC/siPLK1 was injected via their tail vein. Discussion: The results demonstrate that the combination of TMZ and siPLK1 in A2PEC could enhance the efficacy of TMZ in treating glioma.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Sistemas de Liberação de Medicamentos , Glioma/tratamento farmacológico , Nanopartículas/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/administração & dosagem , Temozolomida/administração & dosagem , Temozolomida/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Temozolomida/farmacologia , Distribuição Tecidual/efeitos dos fármacos , Resultado do Tratamento
4.
Life Sci ; 256: 117897, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502543

RESUMO

Glioma is the most common brain malignancy and surgical resection is the primary option for patient with glioma. Anesthetics could be used to inhibit cancer dissemination and metastasis during surgery. This study aims to assess the function of volatile anesthetic sevoflurane in glioma migration and invasion and explore the potential mechanism. Twenty-five patients with glioma were recruited in this study. LN229 and U251 cells were used in vitro experiments. Cell viability was analyzed by MTT analysis. Cell migration and invasion were examined via transwell analysis. microRNA-34a-5p (miR-34a-5p) and matrix metalloproteinase-2 (MMP-2) levels were measured via quantitative real-time polymerase chain reaction. The relationship of miR-34a-5p and MMP-2 was tested via bioinformatics analysis, luciferase reporter analysis, RNA immunoprecipitation and RNA pull-down. Sevoflurane decreased glioma cell migration and invasion. In glioma cells, sevoflurane up-regulated miR-34a-5p abundance and down-regulated MMP-2 level. Overexpression of miR-34a-5p contributed to sevoflurane-caused suppression of migration and invasion, while its knockdown played an opposite effect. MMP-2 was targeted via miR-34a-5p and MMP-2 silence reversed the influence of miR-34a-5p knockdown under sevoflurane. Sevoflurane exposure represses cell migration and invasion, which might be related to inhibition of MMP-2 by up-regulating miR-34a-5p. This study provides a novel mechanism for understanding the pharmacological effects of sevoflurane on glioma.


Assuntos
Neoplasias Encefálicas/patologia , Movimento Celular/efeitos dos fármacos , Glioma/patologia , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/metabolismo , Sevoflurano/farmacologia , Sequência de Bases , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Glioma/enzimologia , Glioma/genética , Humanos , MicroRNAs/genética , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos
5.
Life Sci ; 256: 117964, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32534036

RESUMO

AIMS: Vascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. MATERIALS AND METHODS: Cell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. KEY FINDINGS: Recombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SIGNIFICANCE: In conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. CHEMICAL COMPOUNDS STUDIED IN THIS STUDY: HDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Osteoblastos/citologia , Animais , Biomarcadores/metabolismo , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Inativação Gênica/efeitos dos fármacos , Cinética , Miócitos de Músculo Liso/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Nat Commun ; 11(1): 2082, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350257

RESUMO

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited.


Assuntos
Células Endócrinas/citologia , Células Endócrinas/metabolismo , Elementos Facilitadores Genéticos/genética , Inativação Gênica , Histona Desmetilases/metabolismo , Ilhotas Pancreáticas/citologia , Transdução de Sinais , Tretinoína/metabolismo , Adulto , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Células Endócrinas/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ilhotas Pancreáticas/embriologia , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Adulto Jovem
7.
J Vis Exp ; (157)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32225152

RESUMO

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.


Assuntos
Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Ralstonia solanacearum/fisiologia , Transformação Genética , Agrobacterium/metabolismo , Antibacterianos/farmacologia , Arabidopsis/microbiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Lycopersicon esculentum/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Ralstonia solanacearum/efeitos dos fármacos , Ralstonia solanacearum/crescimento & desenvolvimento , Reprodutibilidade dos Testes , Solo , Transformação Genética/efeitos dos fármacos
8.
PLoS One ; 15(3): e0230362, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176712

RESUMO

Fungi in the genus Cercospora cause crop losses world-wide on many crop species. The wide host range and success of these pathogens has been attributed to the production of a photoactivated toxin, cercosporin. We engineered tobacco for resistance to Cercospora nicotianae utilizing two strategies: 1) transformation with cercosporin autoresistance genes isolated from the fungus, and 2) transformation with constructs to silence the production of cercosporin during disease development. Three C. nicotianae cercosporin autoresistance genes were tested: ATR1 and CFP, encoding an ABC and an MFS transporter, respectively, and 71cR, which encodes a hypothetical protein. Resistance to the pathogen was identified in transgenic lines expressing ATR1 and 71cR, but not in lines transformed with CFP. Silencing of the CTB1 polyketide synthase and to a lesser extent the CTB8 pathway regulator in the cercosporin biosynthetic pathway also led to the recovery of resistant lines. All lines tested expressed the transgenes, and a direct correlation between the level of transgene expression and disease resistance was not identified in any line. Resistance was also not correlated with the degree of silencing in the CTB1 and CTB8 silenced lines. We conclude that expression of fungal cercosporin autoresistance genes as well as silencing of the cercosporin pathway are both effective strategies for engineering resistance to Cercospora diseases where cercosporin plays a critical role.


Assuntos
Ascomicetos/genética , Resistência à Doença/genética , Farmacorresistência Fúngica/genética , Inativação Gênica , Genes Fúngicos , Engenharia Genética , Perileno/análogos & derivados , Tabaco/microbiologia , Ascomicetos/efeitos dos fármacos , Resistência à Doença/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Perileno/farmacologia , Plantas Geneticamente Modificadas , Transformação Genética , Transgenes
9.
Nat Commun ; 11(1): 1466, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193428

RESUMO

The positive or negative value (valence) of past experiences is normally integrated into neuronal circuits that encode episodic memories and plays an important role in guiding behavior. Here, we show, using mouse behavioral models, that glutamatergic afferents from the ventral tegmental area to the dorsal hippocampus (VTA→DH) signal negative valence to memory circuits, leading to the formation of fear-inducing context memories and to context-specific reinstatement of fear. To a lesser extent, these projections also contributed to opioid-induced place preference, suggesting a role in signaling positive valence as well, and thus a lack of dedicated polarity. Manipulations of VTA terminal activity were more effective in females and paralleled by sex differences in glutamatergic signaling. By prioritizing retrieval of negative and positive over neutral memories, the VTA→DH circuit can facilitate the selection of adaptive behaviors when current and past experiences are valence congruent.


Assuntos
Hipocampo/fisiologia , Memória/fisiologia , Rede Nervosa/fisiologia , Área Tegmentar Ventral/fisiologia , Animais , Condicionamento Clássico , Giro Denteado/efeitos dos fármacos , Giro Denteado/fisiologia , Medo/fisiologia , Feminino , Inativação Gênica/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Glutamatos/metabolismo , Hipocampo/efeitos dos fármacos , Cinética , Masculino , Memória/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Rede Nervosa/efeitos dos fármacos , Optogenética , Receptores de N-Metil-D-Aspartato/metabolismo , Caracteres Sexuais , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Área Tegmentar Ventral/efeitos dos fármacos , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo
10.
Life Sci ; 247: 117441, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32074481

RESUMO

OBJECTIVE: To study the effect of the circadian clock gene Clock on the biological behavior of trophoblasts and its role in the pathogenesis of preeclampsia. METHODS: Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of Clock mRNA. Western blot and immunohistochemistry were used to detect the expression and localization of Clock protein. CoCl2 was used to induce the hypoxic trophoblast cells. Cell invasion assay, wound healing assay and MTT assays were used to detect the invasion, migration, and proliferation ability. Reduced uterine perfusion pressure (RUPP) rat model was established by surgically clamping the abdominal aorta and uterine arteries. Transfection of si-Clock was used to silencing the expression of Clock. RESULTS: Clock mRNA expression was increased in placenta of preeclampsia and CoCl2-induced hypoxic trophoblasts, while protein was decreased. But the trend was opposite in RUPP rat models. Hypoxia can also change the expression rhythm of Clock. The proliferation, migration and invasion ability of trophoblasts decreased after hypoxia, while these abilities restored to near normal level after silencing Clock. CONCLUSION: The expression of Clock gene in human placenta tissue, hypoxia cell model and RUPP rat model suggests that it may regulate the biological behavior of trophoblast cells through hypoxia, and then participate in the pathogenesis of preeclampsia.


Assuntos
Hipóxia Celular/genética , Relógios Circadianos/genética , Hipóxia/metabolismo , Pré-Eclâmpsia/genética , Animais , Aorta Abdominal/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Cobalto/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Inativação Gênica/efeitos dos fármacos , Humanos , Placenta/citologia , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Gravidez , RNA Mensageiro , Ratos , Transdução de Sinais , Transfecção , Trofoblastos/citologia , Trofoblastos/metabolismo , Artéria Uterina/metabolismo
11.
Biochem Biophys Res Commun ; 524(3): 643-648, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32029272

RESUMO

Digoxin, a compound of the cardiac glycoside family, was originally prescribed for heart failure but has recently been rediscovered for its potent antitumor activity. However, it has a narrow therapeutic margin due to its cardiotoxicity, limiting its safe use as an antitumor agent in clinical practice. To widen its therapeutic margin, we investigated whether the antitumor effect of digoxin is potentiated by the depletion of BCL-2-interacting cell death suppressor (BIS) in A549 lung cancer cells. BIS is a multifunctional protein that is frequently overexpressed in most human cancers including lung cancer. Our results demonstrated that the inhibitory potential of digoxin on the migratory behavior of A549 cells is significantly enhanced by BIS depletion as assessed by transwell assay and collagen-incorporated 3D spheroid culture. Western blotting revealed that combination treatment significantly reduces p-STAT3 expression. In addition, a STAT3 inhibitor substantially suppressed the aggressive phenotypes of A549 cells. Thus, our results suggest that loss of STAT3 activity is a possible molecular mechanism for the synergistic effect of digoxin and BIS depletion. Our findings suggest the sensitizing role of BIS silencing to reduce the dose of digoxin for treatment of lung cancer with a high metastatic potential.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Movimento Celular/efeitos dos fármacos , Digoxina/farmacologia , Regulação para Baixo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica
12.
Toxicol Lett ; 322: 12-19, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31899212

RESUMO

Benzene exposure is a risk factor of acute myeloid leukemia (AML), during such carcinogenesis long non-coding RNAs (lncRNAs) are important epigenetic regulators. HOTAIRM1 (HOXA transcript antisense RNA, myeloid-specific 1) plays an indispensable role in the development of AML. Hydroquinone (HQ) is one major metabolite of benzene and its ideal replacement in toxicology research. But the influence of benzene or HQ on HOTAIRM1 expression in AML associated pathway is still unclear. In the TK6 cells with short-term exposure to HQ (HQ-ST cells) or long term HQ exposure induced malignant transformed TK6 cells (HQ-MT cells), the relationship between DNMT3b and HOTAIRM1 was explored. Comparing to counterparts, HOTAIRM1 expression was increased firstly and then decreased in HQ-ST cells, and definitely decreased in HQ-MT cells; while the expression change tendency of DNMT3b was in contrast to that of HOTAIRM1. Moreover, the average HOTAIRM1 expression of 17 paired workers being exposed to benzene within 1.5 years was increased, but that of the remaining 92 paired workers with longer exposure time was decreased. Furthermore, in 5-AzaC (DNA methyltransferase inhibitor) or TSA (histone deacetylation inhibitor) treated HQ-MT cells, the expression of HOTAIRM1 was restored by reduced DNA promoter methylation levels. HQ-MT cells with DNMT3b knockout by CRISPR/Cas9 displayed the promoter hypomethylation and the increase of HOTAIRM1, also confirmed in benzene exposure workers. These suggest that long term exposure to HQ or benzene might induce the increase of DNMT3b expression and the promoter hypermethylation to silence the expression of HOTAIRM1, a possible tumor-suppressor in the AML associated carcinogenesis pathway.


Assuntos
Benzeno/efeitos adversos , DNA (Citosina-5-)-Metiltransferases/biossíntese , Metilação de DNA/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Hidroquinonas/toxicidade , Leucemia Mieloide Aguda/induzido quimicamente , MicroRNAs/metabolismo , Doenças Profissionais/induzido quimicamente , Exposição Ocupacional/efeitos adversos , Estudos de Casos e Controles , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/genética , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Doenças Profissionais/enzimologia , Doenças Profissionais/genética , Regiões Promotoras Genéticas , Medição de Risco
13.
EBioMedicine ; 51: 102547, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31902674

RESUMO

BACKGROUND: Dysregulation of splicing variants (SVs) expression has recently emerged as a novel cancer hallmark. Although the generation of aberrant SVs (e.g. AR-v7/sst5TMD4/etc.) is associated to prostate-cancer (PCa) aggressiveness and/or castration-resistant PCa (CRPC) development, whether the molecular reason behind such phenomena might be linked to a dysregulation of the cellular machinery responsible for the splicing process [spliceosome-components (SCs) and splicing-factors (SFs)] has not been yet explored. METHODS: Expression levels of 43 key SCs and SFs were measured in two cohorts of PCa-samples: 1) Clinically-localized formalin-fixed paraffin-embedded PCa-samples (n = 84), and 2) highly-aggressive freshly-obtained PCa-samples (n = 42). FINDINGS: A profound dysregulation in the expression of multiple components of the splicing machinery (i.e. 7 SCs/19 SFs) were found in PCa compared to their non-tumor adjacent-regions. Notably, overexpression of SNRNP200, SRSF3 and SRRM1 (mRNA and/or protein) were associated with relevant clinical (e.g. Gleason score, T-Stage, metastasis, biochemical recurrence, etc.) and molecular (e.g. AR-v7 expression) parameters of aggressiveness in PCa-samples. Functional (cell-proliferation/migration) and mechanistic [gene-expression (qPCR) and protein-levels (western-blot)] assays were performed in normal prostate cells (PNT2) and PCa-cells (LNCaP/22Rv1/PC-3/DU145 cell-lines) in response to SNRNP200, SRSF3 and/or SRRM1 silencing (using specific siRNAs) revealed an overall decrease in proliferation/migration-rate in PCa-cells through the modulation of key oncogenic SVs expression levels (e.g. AR-v7/PKM2/XBP1s) and alteration of oncogenic signaling pathways (e.g. p-AKT/p-JNK). INTERPRETATION: These results demonstrate that the spliceosome is drastically altered in PCa wherein SNRNP200, SRSF3 and SRRM1 could represent attractive novel diagnostic/prognostic and therapeutic targets for PCa and CRPC.


Assuntos
Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Processamento de RNA/genética , Idoso , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Processamento de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Spliceossomos/metabolismo
14.
Biochem Biophys Res Commun ; 523(4): 987-992, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31973819

RESUMO

In previous studies we demonstrated that zinc stimulates iron uptake in intestinal Caco-2 cells via Zinc-PI3K-IRP2-DMT1 axis. In the current study we investigated the effect of zinc on basolateral iron release and characterized the associated mechanisms. In Caco-2 cells grown on permeable supports, zinc induced iron transport and expression of DMT1, HEPH mRNA and protein, but not that of FPN1. LY294002, an inhibitor of PI3K, inhibited the zinc-induced iron transport, DMT1, HEPH mRNA and protein expression. In addition, LY294002 also inhibited the basal expression of HEPH and FPN1 resulting in blockade of iron egress from cells. In addition, siRNA-silencing of HEPH led to inhibition of both zinc-induced and basal iron transport. Conversely, TPEN, a chelator of zinc, inhibited iron uptake, DMT1, HEPH and FPN1 mRNA and protein expression. These results suggest that intestinal cell zinc status is a critical determinant of iron absorption and effects are mediated via activation of PI3K. Further, PI3K pathway appears to selectively modulate the expression of iron transporters and iron absorption, therefore this might serve as a therapeutic target in iron overload disorders.


Assuntos
Absorção Intestinal , Intestinos/fisiologia , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Zinco/farmacologia , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Cromonas/farmacologia , Etilenodiaminas/farmacologia , Inativação Gênica/efeitos dos fármacos , Humanos , Intestinos/efeitos dos fármacos , Morfolinas/farmacologia
15.
Gene ; 727: 144245, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31715302

RESUMO

DEK involves in the modulation of cell proliferation, differentiation, apoptosis, migration and cell senescence. However, direct genetic evidence proving the functions of DEK in disease resistance against pathogens is still deficient. In the present study, four DEKs were identified in tomato genome and their roles in disease resistance in tomato were analyzed. The expression levels of DEKs were differently induced by Botrytis cinerea, Pseudomonas syringae pv. tomato (Pst) DC3000 and defense-related signaling molecules (such as jasmonic acid, aethylene precursor and salicylic acid). The DEKs' silencing by virus induced gene silencing led to decreased resistance against B. cinerea or Pst DC3000. The underlying mechanisms may be through the upregulation of the accumulation of reactive oxygen species (ROS) and the changed expression levels of defense-related genes by pathogen inoculation. These results indicate that DEKs involve in disease resistance against different pathogens and thus broaden the knowledge of DEK genes' function in tomato.


Assuntos
Resistência à Doença/genética , Inativação Gênica/efeitos dos fármacos , Lycopersicon esculentum/genética , Botrytis/patogenicidade , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Lycopersicon esculentum/metabolismo , Oxilipinas/farmacologia , Doenças das Plantas/genética , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Pseudomonas syringae/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/farmacologia , Transdução de Sinais/genética , Fatores de Transcrição/genética
16.
Drug Deliv ; 27(1): 15-25, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31830840

RESUMO

Small interfering RNA (siRNA) exhibits great potential as a novel therapeutic option due to its highly sequence-specific ability to silence genes. However, efficient and safe delivery carriers are required for developing novel therapeutic paradigms. Thus, the successful development of efficient delivery platforms for siRNA is a crucial issue for the development of siRNA-based drugs in cancer treatments. In this study, biocompatible selenium nanoparticles (SeNPs) were loaded with RGDfC peptide to fabricate tumor-targeting gene delivery vehicle RGDfC-SeNPs. Subsequently, RGDfC-SeNPs were loaded with Derlin1-siRNA to fabricate RGDfC-Se@siRNA, which are functionalized selenium nanoparticles. RGDfC-Se@siRNA showed greater uptake in HeLa cervical cancer cells in comparison with that in human umbilical vein endothelial cells (HUVECs), verifying the RGDfC-mediated specific uptake of RGDfC-Se@siRNA. RGDfC-Se@siRNA was capable of entering HeLa cells via clathrin-associated endocytosis, and showed faster siRNA release in a cancer cell microenvironment in comparison with a normal physiological environment. qPCR and western blotting assays both indicated that RGDfC-Se@siRNA exhibited an obvious gene silencing efficacy in HeLa cells. RGDfC-Se@siRNA suppressed the invasion, migration and the proliferation of HeLa cells, and triggered HeLa cell apoptosis. Moreover, RGDfC-Se@siRNA induced the disruption of mitochondrial membrane potentials. Meanwhile, RGDfC-Se@siRNA enhanced the generation of reactive oxygen species (ROS) in HeLa cell, suggesting that mitochondrial dysfunction mediated by ROS might play a significant role in RGDfC-Se@siRNA-induced apoptosis. Interestingly, RGDfC-SeNPs@siRNA exhibited significant antitumor activity in a HeLa tumor-bearing mouse model. Additionally, RGDfC-SeNPs@siRNA is nontoxic to main organ of mouse. The above results indicate that RGDfC-Se@siRNA provides a promising potential for cervical cancer therapy.


Assuntos
Proteínas de Membrana/efeitos dos fármacos , Nanopartículas/química , Oligopeptídeos/farmacologia , RNA Interferente Pequeno/administração & dosagem , Selênio/química , Apoptose/efeitos dos fármacos , Western Blotting , Inibição de Migração Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Inativação Gênica/efeitos dos fármacos , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacocinética , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral , Neoplasias do Colo do Útero/tratamento farmacológico
17.
Toxicol In Vitro ; 62: 104695, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31639451

RESUMO

Neuropathies caused by mitochondrial dysfunction are the most common and serious impediment of high glucose (HG)-induced toxicity. We have previously reported mitoprotective potency of Sirtuin1 (Sirt1) in diabetic neuropathy (DN) via targeting mitochondrial dysfunction but its nuclear control over mitochondrial bioenergetics remains unknown. Here, we studied the effect of SRT1720; a small molecule activator of Sirt1 in attenuating the HG mediated mitochondrial dysfunction in differentiated rat pheochromocytoma (PC12) cells and aiming to determine (1) whether SRT1720 can improve mitochondrial function in HG exposed PC12 cells (2) if yes then this effect is dependent or independent of mitochondrial Lon protease (LONP1) (3) and whether silencing of LONP1 affects the mitochondrial function or not. HG (30 mM) exposed PC12 cells demonstrated reduced mitochondrial complex activities and oxygen consumption rate (OCR), decreased the expressions of Sirt1, peroxisome proliferator-activated receptor coactivator-1α (PGC1α), nuclear respiratory factor-2 (NRF2), LONP1 and ATP synthase c. SRT1720 treatment (4 µM) significantly reversed these effects in hyperglycemia insulted PC12 cells but silencing the expression of LONP1 impeded this effect of SRT1720 on mitochondrial complex activities, OCR and mitochondrial membrane potential. Based on these findings, we inferred that SRT1720 might improve mitochondrial function in HG induced mitochondrial dysfunction in PC12 cells via stimulation of Sirt1-LONP1 axis.


Assuntos
Proteases Dependentes de ATP/sangue , Glucose/toxicidade , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Doenças Mitocondriais/tratamento farmacológico , Proteínas Mitocondriais/sangue , Síndromes Neurotóxicas/prevenção & controle , Protease La/biossíntese , Proteases Dependentes de ATP/genética , Animais , Inativação Gênica/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Doenças Mitocondriais/induzido quimicamente , Proteínas Mitocondriais/genética , Consumo de Oxigênio/efeitos dos fármacos , Células PC12 , Protease La/genética , Ratos , Sirtuína 1/biossíntese , Sirtuína 1/efeitos dos fármacos
18.
Biomed Pharmacother ; 120: 109527, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31629953

RESUMO

Excessive formation of advanced glycation end products (AGEs) impairs voltage-gated potassium (Kv) channels in rat coronary artery smooth muscle cells (CSMCs), resulting in weakened Kv-mediated coronary vasodilation. We hypothesized that induction of the nuclear factor-κB (NF-κB) signaling pathway by AGEs plays a significant role in the regulation of Kv channel-mediated vasodilation in Zucker diabetic fatty (ZDF) rats. Assays of mRNA transcripts, protein expression, and intracellular localization as well as patch-clamp experiments in cultured CSMCs revealed that AGEs significantly induced activation of the NF-κB signaling pathway, reduced Kv1.2/1.5 expression, and inhibited Kv currents. In addition, silencing of the receptor for AGEs (RAGE) or p65 with siRNA and treatment with alagrebrium (ALA) or pyrrolidine dithiocarbamate (PDTC) alleviated the AGE-induced impairment of Kv channels in CSMCs. Compared with Zucker lean (ZL) rats, the amount of AGEs, RAGE protein expression, and NF-κB activity in coronary arteries were higher in ZDF rats; whereas Kv1.2/1.5 expression was significantly lower in ZDF rats. Reduced Kv1.2/1.5 expression in coronary arteries and impaired Kv-mediated coronary relaxation tested by wire myography in ZDF rats were markedly improved by treatment with aminoguanidine (AG), ALA, or PDTC. These effects were accompanied by diminished NF-κB activity, inflammation, and oxidative stress. Taken together, these results indicate that an increased interaction between AGEs and RAGE in diabetic rats leads to impaired Kv channel-mediated coronary vasodilation. Moreover, activation of the NF-κB signaling pathway and a subsequent increase of inflammation and oxidative stress may play an important role in AGE-induced impairment of coronary vasodilation in diabetes.


Assuntos
Vasos Coronários/fisiopatologia , Produtos Finais de Glicação Avançada/toxicidade , NF-kappa B/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Animais , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Inativação Gênica/efeitos dos fármacos , Produtos Finais de Glicação Avançada/sangue , Testes de Função Cardíaca , Inflamação/sangue , Inflamação/patologia , Masculino , Modelos Biológicos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ratos Zucker , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Fator de Transcrição RelA/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
19.
Int J Nanomedicine ; 14: 6575-6585, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31616144

RESUMO

Background and purpose: In a past study, we developed and optimized a novel cationic PEGylated niosome containing anticancer drugs (doxorubicin or quercetin) and siRNA. This study intended to evaluate the anti-tumor effects of the combination therapy to target both the proteins and genes responsible for the development of gastric cancer. CDC20, known as an oncogene, is a good potential therapeutic candidate for gastric cancer. Methods: In order to increase the loading capacity of siRNA and achieve appropriate physical properties, we optimized the cationic PEGylated niosome in terms of the amount of the cationic lipids. Drugs (doxorubicin and quercetin) and CDC20siRNA were loaded into the co-delivery system, and physical characteristics, thermosensitive controlled-release, gene silencing efficiency, and apoptosis rate were determined. Results: The results showed that the designed co-delivery system for the drugs and gene silencer had an appropriate size and a high positive charge for loading siRNA, and also showed a thermosensitive drug release behavior, which successfully silenced the CDC20 expression when compared with the single delivery of siRNA or the drug. Moreover, the co-delivery of drugs and CDC20siRNA exhibited a highly inhibitory property for the cell growth of gastric cancer cells. Conclusion: It seems that the novel cationic PEGylated niosomes co-loaded with anticancer drug and CDC20siRNA has a promising application for the treatment of gastric cancer.


Assuntos
Proteínas Cdc20/metabolismo , Doxorrubicina/uso terapêutico , Polietilenoglicóis/química , Quercetina/uso terapêutico , RNA Interferente Pequeno/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Cátions , Proteínas Cdc20/genética , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Endocitose , Ácidos Graxos Monoinsaturados/química , Inativação Gênica/efeitos dos fármacos , Humanos , Lipossomos , Tamanho da Partícula , Compostos de Amônio Quaternário/química , Quercetina/farmacologia , RNA Interferente Pequeno/genética , Eletricidade Estática , Temperatura
20.
Mol Cell Biochem ; 462(1-2): 97-105, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31473882

RESUMO

Our previous study shows that high Chloride intracellular channel 1 (CLIC1) expression can efficiently enhance invasion and migration of gastric cancer (GC) cells in vitro. Growing evidences have found that exosomes are involved in chemotherapy resistance in several cancers including GC. We aimed to evaluate the effect of the exosome-mediated transfer of CLIC1 in the vincristine-resistance of GC. The effect of exosome-mediated transfer of CLIC1 on the development of resistance to vincristine in GC cell line SGC-7901 and the potential underlying mechanisms were investigated by Cell Counting Kit-8 (CCK8), RT-PCR, and Western blotting. Exosomes were isolated from cell supernatants by differential ultracentrifugation. Comparing with SGC-7901, the expression level of CLIC1 is higher in vincristine­resistant cell line SGC-7901/VCR (P < 0.05). After silencing the expression of CLIC1 by RNA interference, the half inhibition concentration (IC50) to vincristine decreased significantly in SGC-7901/VCR, and the expression of CLIC1 decreased significantly in exosomes from SGC-7901/VCR. After 48 h co-culturing with exosomes from SGC-7901/VCR, the IC50 to vincristine in SGC-7901 increased significantly, and the expression of CLIC1, P-gp, and Bcl-2 were significantly up-regulated. CLIC1 was closely associated with the resistance to vincristine in GC, and exosome-mediated transfer of CLIC1 could induce the development of resistance to vincristine in vitro. The possible mechanism was related to up-regulated P-gp and Bcl-2. However, in vivo study was needed to confirm the results in future.


Assuntos
Canais de Cloreto/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Exossomos/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Vincristina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Exossomos/ultraestrutura , Inativação Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Gástricas/ultraestrutura
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