RESUMO
Hepatocellular carcinoma (HCC), the most prevalent liver cancer, is considered one of the most lethal malignancies with a dismal outcome mainly due to frequent intrahepatic and distant metastasis. In the present study, we demonstrated that oroxylin A, a natural product extracted from Scutellaria radix, significantly inhibits transforming growth factor-beta1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) and metastasis in HCC. Oroxylin A blocked the TGF-ß1/Smad signaling via upregulating the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression. Oroxylin A promoted NAG-1 transcription by regulating the acetylation of CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor that binds to the NAG-1 promoter. In terms of the underlying mechanism, oroxylin A may interact with histone deacetylase 1 (HDAC1) by forming hydrogen bonds with GLY149 residue and induce proteasome-mediated degradation of HDAC1 subsequently impairing HDAC1-mediated deacetylation of C/EBPß and promoting the expression of NAG-1. Taken together, our findings revealed a previously unknown tumor-suppressive mechanism of oroxylin A. Oroxylin A should be further investigated as a potential clinical candidate for inhibiting HCC metastasis.
Assuntos
Carcinoma Hepatocelular/patologia , Flavonoides/farmacologia , Fator 15 de Diferenciação de Crescimento/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Fator de Ligação a CCAAT/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Histona Desacetilase 1/efeitos dos fármacos , Humanos , Proteínas Smad/efeitos dos fármacos , Fator de Crescimento Transformador beta1/efeitos dos fármacosRESUMO
Cadmium (Cd) is considered as a carcinogenic chemical with potential to endanger normal cellular functioning. The present study was aimed to investigate the impact of Cd on the expression of two oncogenic epigenetic regulators, viz., protein arginine methyltransferase 5 (PRMT5) and the polycomb repressive complex 2 (PRC2) member enhancer of Zeste homolog 2 (EZH2). Our results indicate that Cd at 1⯵M concentration increases the viability of HepG2 and MCF7 cells and significantly upregulates the expression of PRMT5 and EZH2, leading to an increased global level of symmetric dimethylarginine (SDMA), H4R3me2s, and H3K27me3. The luciferase reporter assay showed that the promoter activity of PRMT5 and EZH2 is significantly enhanced in both cell lines. Furthermore, Cd exposure induces global DNA hypomethylation due to a decrease in DNA methyltransferases (DNMTs) expression. Methylation-specific and bisulfite sequencing PCR reveal that the proximal promoters of PRMT5 and EZH2, which harbour CpG islands, are almost demethylated when exposed to Cd. The Cd exposure also increases the protein level of transcription factors NFYA and E2F1; consistently, the two transcription factors are found to be enriched at the PRMT5 and EZH2 promoter in chromatin immunoprecipitation experiments. The alterations induced by Cd in the two cancer cell lines were also observed in a non-cancerous cell line (HEK-293). In conclusion, we propose that Cd increases the expression of two oncogenic methyltransferases, possibly with a DNA methylation-dependent mechanism. Further studies focused on the epigenetic alterations induced by Cd would provide mechanistic insights on the carcinogenicity of this metal toxicant at the molecular level.
Assuntos
Cádmio/farmacologia , Metilação de DNA/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores de Transcrição , Fator de Ligação a CCAAT/efeitos dos fármacos , Fator de Ligação a CCAAT/metabolismo , Cádmio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ilhas de CpG , Fator de Transcrição E2F1/efeitos dos fármacos , Fator de Transcrição E2F1/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Células HEK293 , Humanos , Células MCF-7 , Metiltransferases/efeitos dos fármacos , Metiltransferases/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/genética , Fatores de Transcrição/metabolismoRESUMO
Butyric acid (sodium butyrate; BA) is an extracellular metabolite secreted from periodontopathic bacteria present in subgingival plaque. BA induces apoptosis of T and B cells, and acts as a potent inhibitor of histone deacetylases. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone, and may be crucial for osteoblast differentiation, bone matrix mineralization and tumor metastasis. In the present study we investigated the regulation of BSP transcription by BA in rat osteoblast-like ROS17/2.8 cells. At 12 h, BA (10(-4) M) increased the level of BSP mRNA, and enhanced the luciferase activity of the construct pLUC3, which includes the promoter sequence between nucleotides -116 and +60. Transcriptional stimulation by BA was abrogated in the pLUC3 construct which containing a 2-bp mutation in the fibroblast growth factor 2 response element (FRE). Gel shift analyses showed that BA increased the binding of nuclear protein to FRE. These data suggest that BA increases the transcription of the BSP gene mediated through FRE in the rat BSP gene promoter, and induces osteoblast activity in the early stage of bone formation.
Assuntos
Ácido Butírico/farmacologia , Sialoglicoproteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Pareamento de Bases/genética , Northern Blotting , Fator de Ligação a CCAAT/efeitos dos fármacos , Proteína de Ligação a CREB/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/genética , Proteínas de Homeodomínio/efeitos dos fármacos , Sialoproteína de Ligação à Integrina , Mutação/genética , Proteínas Nucleares/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos , Ratos , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/genética , Sialoglicoproteínas/genética , Fatores de Tempo , Fator de Transcrição Pit-1/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
FSH is a heterodimeric glycoprotein hormone secreted from the gonadotrope cell population of the anterior pituitary. Despite its crucial role in mammalian reproduction, very little is known about regulation of the FSH beta-subunit gene at the molecular level. In this report, we examine the basis for cell-specific expression of FSH beta using the mouse L beta T2 and alpha T3-1 gonadotrope-derived cell lines. Characterization of the hormonal content of L beta T2 and alpha T3-1 cells at the protein level classifies these cells as relatively mature and immature gonadotropes, respectively. We studied L beta T2 cell-specific expression of FSH beta using 398 bp of the mouse FSH beta regulatory region linked to a luciferase reporter gene in transient transfection assays. This mouse FSH beta promoter can direct reporter gene expression specifically to L beta T2 cells when compared with other pituitary- and non-pituitary-derived cell lines, including alpha T3-1 cells. Furthermore, it is induced by activin, and interruption of the autocrine activin loop in L beta T2 cells by the addition of follistatin reduces its expression. Truncation analysis indicates that several regions of the promoter are involved in this specificity and that these can be dissociated from activin regulation. We identify binding sites for the orphan nuclear receptor steroidogenic factor-1 and the heterotrimeric transcription factor nuclear factor Y and show that these elements functionally interact to regulate FSH beta gene expression in an L beta T2 cell-specific manner. Moreover, steroidogenic factor-1 and nuclear factor Y are shown to physically interact with each other. This study is the first to demonstrate the presence of basal FSH beta protein in L beta T2 cells and to identify specific elements within the FSH beta promoter that contribute to basal and cell-specific expression of the gene.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Subunidade beta do Hormônio Folículoestimulante/genética , Adeno-Hipófise/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3/metabolismo , Ativinas/farmacologia , Animais , Sítios de Ligação , Fator de Ligação a CCAAT/efeitos dos fármacos , Fator de Ligação a CCAAT/genética , Células Cultivadas , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Subunidade beta do Hormônio Folículoestimulante/efeitos dos fármacos , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Folistatina/farmacologia , Fatores de Transcrição Fushi Tarazu , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Proteínas de Homeodomínio , Luciferases/genética , Luciferases/metabolismo , Camundongos , Especificidade de Órgãos , Adeno-Hipófise/citologia , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fator Esteroidogênico 1 , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Advanced stage neuroblastomas (NB) exhibit a tissue inhibitor of metalloproteinase (TIMP)-2/matrix metalloproteinase (MMP) imbalance, considered a prerequisite for MMP involvement in tumor progression in vivo. Human SH-SY5Y NB cells exhibit a similar TIMP-2/MMP imbalance that promotes in vitro invasive behavior that is inhibited by exogenous TIMP-2. The DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) redresses this TIMP-2/MMP imbalance, reconstituting TIMP-2 expression, without effecting that of MMP-2, by stimulating TIMP-2 transcription and inhibiting in vitro invasivity of SH-SY5Y cells. 5-AzaC stimulated transcription from a nonmethylated TIMP-2 promoter reporter gene construct consistent with regulation of a TIMP-2 transactivator. Promoter deletion and point-mutation analysis localized this effect to an inverted CCAAT element at position -73. This element bound specific complexes containing NF-YA and NF-YB proteins in SH-SY5Y nuclear extracts, the binding of which was augmented by 5-AzaC in association with enhanced levels of NF-YB protein and the function of which was confirmed by inhibition using dominant-negative NF-YA. The data highlight a novel indirect methylation-mediated mechanism for regulating the TIMP/MMP equilibrium in NB cells, involving repression of TIMP-2 relative to MMP-2 expression, dependent upon suboptimal NF-Y transcription factor function, which can be reversed by methyltransferase inhibition.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Reguladores/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Fator de Ligação a CCAAT/efeitos dos fármacos , Fator de Ligação a CCAAT/genética , Criança , Metilação de DNA/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reguladores/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Mutação/efeitos dos fármacos , Mutação/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Células Tumorais CultivadasRESUMO
Expression of the human DNA topoisomerase IIalpha (topo IIalpha) gene is positively regulated by the binding of the nuclear factor Y (NF-Y) transcription factor to four of five inverted CCAAT boxes (ICBs) located in its promoter. We have demonstrated previously that expression of the p53 tumor suppressor inhibits human topo IIalpha promoter activity in murine (10)1 cells. In this report, we demonstrate that the inhibition of topo IIalpha gene expression by wild-type p53 correlates with the decreased binding of the transcription factor NF-Y to the first four ICBs of the topo IIalpha promoter. The expression of mutant p53 does not affect the binding of NF-Y. In NIH3T3 cells, we show that topo II-targeted drugs inhibit the binding of NF-Y to ICB sites in the topo IIalpha promoter. This effect is seen not only with drugs that result in DNA strand breaks but also with drugs that inhibit the catalytic activity of topo II, and even with the mitotic spindle inhibitor, vinblastine. Further experiments with p53-null (10)1 cells treated with these same drugs also demonstrate decreased NF-Y binding to the topo IIalpha ICBs. The data presented points to the existence of both p53-dependent and -independent mechanisms for regulating NF-Y binding to ICBs in the topo IIalpha promoter and thus the modulation of topo IIalpha gene expression.