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1.
Mol Cell Biochem ; 477(3): 689-699, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34973124

RESUMO

Bardoxolone methyl [methyl-2-cyano-3, 12-dioxooleana-1, 9(11)dien-28-oate (CDDO-Me)], an activator of the nuclear factor erythroid-derived 2-related factor2 pathway, is a potential therapeutic candidate for the treatment of kidney diseases. However, its effect against cellular senescence remains unclear. This study aimed to investigate whether CDDO-Me protects cells against cisplatin-induced cellular senescence using an in vitro model. The human renal proximal tubular epithelial cell line HK-2 was treated with cisplatin for 6 h, followed by treatment with or without CDDO-Me (0.1 or 0.2 µmol/L). Senescence markers were analyzed using western blotting and real-time PCR. Apoptosis was evaluated through TUNEL staining. Cisplatin induced changes in the levels of markers specific for proliferation, cell cycle, and senescence in a time- and dose-dependent manner. Furthermore, IL-6 and IL-8 levels in the culture medium increased markedly. These data suggested that cellular senescence-like alterations occurred in HK-2 cells exposed to cisplatin. CDDO-Me treatment reversed the cisplatin-mediated alterations in the levels of cellular senescence markers. The antioxidant enzymes, HO1, NQO1, GPX1, and CAT were upregulated by CDDO-Me treatment. Furthermore, CDDO-Me treatment induced apoptosis in cisplatin-exposed HK-2 cells. Pretreatment with Ac-DEVD-CHO, the caspase inhibitor, suppressed the reversal effect of CDDO-Me against cisplatin-induced cellular senescence-like alterations. This study showed that CDDO-Me attenuated cisplatin-induced premature senescence of HK-2 cells. This beneficial effect may be related to Nrf2 activation. Our findings also showed that CDDO-Me induced apoptosis in cisplatin-treated HK-2 cells, potentially protecting the kidneys from cellular senescence. CDDO-Me appears to be a candidate treatment for acute kidney injury.


Assuntos
Senescência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Túbulos Renais Proximais/metabolismo , Ácido Oleanólico/análogos & derivados , Linhagem Celular , Humanos , Ácido Oleanólico/farmacologia
2.
Am J Physiol Renal Physiol ; 314(3): F462-F470, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29187367

RESUMO

Megalin, an endocytic receptor expressed in proximal tubule cells, plays a critical role in renal tubular protein reabsorption and is associated with the albuminuria observed in diabetic nephropathy. We have previously reported increased oxidant production in the renal cortex during the normoalbuminuric stage of diabetes mellitus (DM); however, the relationship between oxidative stress and renal megalin expression during the normoalbuminuric stage of DM remains unclear. In the present study, we evaluated whether oxidative stress affects megalin expression in the normoalbuminuric stage of DM in a streptozotocin-induced diabetic rat model and in immortalized human proximal tubular cells (HK-2). We demonstrated that increased expression of renal megalin accompanies oxidative stress during the early stage of DM, before albuminuria development. Telmisartan treatment prevented the diabetes-induced elevation in megalin level, possibly through an oxidative stress-dependent mechanism. In HK-2 cells, hydrogen peroxide significantly increased megalin levels in a dose- and time-dependent manner; however, the elevation in megalin expression was decreased following prolonged exposure to severe oxidative stress induced by 0.4 mmol/l hydrogen peroxide. High-glucose treatment also significantly increased megalin expression in HK-2 cells. Concurrent administration of the antioxidant N-acetyl-cysteine blocked the effects of high glucose on megalin expression. Furthermore, the hydrogen peroxide-induced increase in megalin expression was blocked by treatment with phosphatidylinositol 3-kinase and Akt inhibitors. Increase of phosphorylated Akt expression was also seen in the renal cortex of diabetic rats. Taken together, our results indicate that mild oxidative stress increases renal megalin expression through the phosphatidylinositol 3-kinase-Akt pathway in the normoalbuminuric stage of DM.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Estresse Oxidativo , Animais , Antioxidantes/farmacologia , Linhagem Celular , Diabetes Mellitus Experimental/induzido quimicamente , Relação Dose-Resposta a Droga , Glucose/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Estreptozocina , Telmisartan/farmacologia , Fatores de Tempo , Regulação para Cima
3.
Sci Rep ; 6: 18971, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26739257

RESUMO

We previously showed that H2 acts as a novel antioxidant to protect cells against oxidative stress. Subsequently, numerous studies have indicated the potential applications of H2 in therapeutic and preventive medicine. Moreover, H2 regulates various signal transduction pathways and the expression of many genes. However, the primary targets of H2 in the signal transduction pathways are unknown. Here, we attempted to determine how H2 regulates gene expression. In a pure chemical system, H2 gas (approximately 1%, v/v) suppressed the autoxidation of linoleic acid that proceeds by a free radical chain reaction, and pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC), one of the major phospholipids, was autoxidized in the presence or absence of H2. H2 modified the chemical production of the autoxidized phospholipid species in the cell-free system. Exposure of cultured cells to the H2-dependently autoxidized phospholipid species reduced Ca(2+) signal transduction and mediated the expression of various genes as revealed by comprehensive microarray analysis. In the cultured cells, H2 suppressed free radical chain reaction-dependent peroxidation and recovered the increased cellular Ca(2+), resulting in the regulation of Ca(2+)-dependent gene expression. Thus, H2 might regulate gene expression via the Ca(2+) signal transduction pathway by modifying the free radical-dependent generation of oxidized phospholipid mediators.


Assuntos
Radicais Livres/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hidrogênio/farmacologia , Fosfatidilcolinas/metabolismo , Sinalização do Cálcio , Linhagem Celular , Humanos , Ácido Linoleico/metabolismo , Fatores de Transcrição NFATC/metabolismo , Oxirredução , Fosfatidilcolinas/química , Transcriptoma/efeitos dos fármacos
4.
Int J Mol Med ; 27(6): 865-72, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21369691

RESUMO

The signal regulatory protein (SIRP) α1 is a cell surface receptor expressed predominantly in monocytes, granulocytes, dendritic cells, as well as hematopoietic stem cells. In contrast, SIRPα1 expression is significantly reduced in the majority of myeloid malignancies. SIRPα1 is a negative regulator of signaling and its reduced expression is considered to play a role in the pathogenesis of these diseases through aberrant signaling. To identify SIRPα1 downstream target genes, we established SIRP α1-knockdown chronic myeloid leukemia K562 (K562SIRPα1KD) cells expressing reduced levels of SIRPα1 by stably transfecting SIRPα1 siRNAs. Microarray analysis demonstrated that several genes, including ß-catenin, were significantly induced in K562SIRPα1KD cells. Real-time PCR and Western blot analyses, confirmed the induction of this gene. Phosphorylation of Ser9 of glycogen synthesis kinase (GSK) -3ß, results in the inactivation of GSK-3ß, leading to the induction of ß-catenin. We found significant phosphorylation of extracellular signal-regulated kinase (ERK), Akt, as well as of GSK-3ß-Ser9, which may play a role in the up-regulation of ß-catenin in K562SIRPα1KD cells. To our knowledge, this is a first report demonstrating the relationships between SIRPα1 and ß-catenin in leukemia cells.


Assuntos
Receptores Imunológicos/antagonistas & inibidores , beta Catenina/genética , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Células K562 , Análise em Microsséries , Fosforilação , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/genética , Transfecção , beta Catenina/metabolismo
5.
J Biol Chem ; 285(14): 10300-9, 2010 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-20139074

RESUMO

PU.1 is a key transcription factor for hematopoiesis and plays important roles in various hematological malignancies. To clarify the molecular function of PU.1, we initially tried to identify bona fide target genes regulated by PU.1. Dual microarrays were employed for this study to compare PU.1-knockdown K562 cells (K562PU.1KD) stably expressing PU.1 short inhibitory RNAs versus control cells and PU.1-overexpressing K562 cells (K562PU.1OE) versus control cells. In these analyses, we found that several genes, including metallothionein (MT)-1 isoforms (MT-1G and MT-1A) and vimentin (VIM), were markedly induced while Jun dimerization protein (JDP) 2 was suppressed in K562PU.1KD cells. Furthermore, the mRNA expressions of the MT-1 and VIM genes were inversely correlated and the mRNA expression of JDP2 was positively correlated with PU.1 mRNA expression in 43 primary acute myeloid leukemia specimens (MT-1G: R = -0.50, p < 0.001; MT-1A: R = -0.58, p < 0.0005; VIM: R = -0.39, p < 0.01; and JDP2: R = 0.30, p < 0.05). Next, we analyzed the regulation of the MT-1 and VIM genes. We observed increased associations of acetylated histones H3 and H4 with the promoters of these genes in K562PU.1KD cells. Sequence analyses of the regions approximately 1 kb upstream from the transcription start sites of these genes revealed numerous CpG sites, which are potential targets for DNA methylation. Chromatin immunoprecipitation assays revealed that methyl CpG-binding protein 2 (MeCP2) and PU.1 bound to the CpG-rich regions in the MT-1 and VIM promoters. Bisulfite sequencing analyses of the PU.1-bound regions of these promoters revealed that the proportions of methylated CpG sites were tightly related to the PU.1 expression levels.


Assuntos
Leucemia/metabolismo , Metalotioneína/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Vimentina/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Imunoprecipitação da Cromatina , Ilhas de CpG , Metilação de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Células K562 , Leucemia/genética , Leucemia/patologia , Luciferases/metabolismo , Metalotioneína/genética , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Vimentina/genética
7.
Leuk Res ; 33(12): 1658-63, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19428102

RESUMO

To identify PU.1 downstream target genes, we first established PU.1-knockdown K562 (K562PU.1KD) cells expressing reduced levels of PU.1 by stably transfected PU.1 siRNAs. From microarray analysis, we found that several genes including annexin 1 were markedly induced in K562PU.1KD cells. Annexin 1 is a calcium- and phospholipid-binding protein and increased expression leads to the constitutive activation of extracellular signal-regulated kinase (ERK). Consistent with this, we observed constitutive activation of ERK in K562PU.1KD cells. Furthermore, we revealed the mRNA expression of annexin 1 was negatively correlated with PU.1 mRNA expression in 43 primary AML specimens (R=-0.31, p<0.042).


Assuntos
Anexina A1/genética , Leucemia Eritroblástica Aguda/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Sequência de Bases , Citometria de Fluxo , Humanos , Células K562 , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno
9.
Leuk Res ; 33(5): 693-9, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-18995898

RESUMO

In this study, the biological effects of signal transduction inhibitors on leukemia cells were examined. We found that the p38 inhibitor SB202190 enhanced the growth of THP-1 and MV4-11 cells. To determine the pathway affected by SB202190, we examined the 50% effective dose (ED(50)) values for THP-1 cell growth in combination with several inhibitors. In the presence of SB202190, the ED(50) values for the farnesyltransferase inhibitor FPT inhibitor II and MEK inhibitor U0126 were significantly decreased. Western blot analysis revealed that SB202190 increased the phosphorylation of C-Raf and extracellular regulated kinase (ERK), suggesting that Ras-Raf-MEK-mitogen-activated protein kinase (MAPK) pathway activation is involved in the leukemia cell growth induced by SB202190.


Assuntos
Divisão Celular/efeitos dos fármacos , Imidazóis/farmacologia , Leucemia/patologia , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Ativação Enzimática , Humanos , Leucemia/enzimologia , Fosforilação
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