RESUMEN
BACKGROUND: Cancer is an outcome of uncontrolled cell division eventually associated with dysregulated epigenetic mechanisms, including DNA methylation. DNA methyltransferase 1 is ubiquitously expressed in the proliferating cells and is essential for the maintenance of DNA methylation. It causes the abnormal silencing of tumor suppressor genes in human cancer which is necessary for proliferation, cell cycle progression, and survival. DNMT1 is involved in tumorigenesis of several cancers, its upregulation potentially upscale the promoter level inactivation of transcription of a tumor inhibitory gene by introducing repressive methylation marks on the CpG islands. This epigenetic perturbation caused by DNMT is targeted for cancer therapeutics. PURPOSE: To demonstrate the proliferative inhibitory potential of brazilin in human breast cancer cell line (MCF-7) with concurrent mitigation of DNMT1 functional expression and to understand its effect on downstream targets like cell cycle inhibitor p21. STUDY DESIGN/ METHODS: The impact of brazilin on the growth and proliferation of the MCF-7 cells was determined using the XTT assay. The global DNA 5-methyl cytosine methylation pattern was analyzed upon brazilin treatment. The gene and protein expression of DNMTs were determined with quantitative RTPCR and western blots respectively. The potential binding sites of transcription factors in the human DNMT1 promoter were predicted using the MatInspector tool on the Genomatix software. The chromatin immunoprecipitation (ChIP) assay was performed to demonstrate the transcription factors occupancy at the promoter. Methylation of promoter CpG islands was determined by the methylation-specific PCR (MSP) upon brazilin treatment. The molecular docking of the human DNMT1 with brazilin (ligand) was performed using the Schrödinger suite. RESULTS: The heterotetracyclic compound brazilin, present in the wood of Caesalpinia sappan, inhibited the proliferation of the human breast cancer cell line (MCF-7) and reduced the DNMT1 expression with a decrease in global DNA methylation. Brazilin, by activating p38 MAPK and elevating p53 levels within the exposed cells. The elevated level of p53 enriched the occupancy at binding sites within 200 bp upstream to the transcription start site in the DNMT1 promoter, resulting in reduced DNMT1 gene expression. Furthermore, the brazilin restored the p21 levels in the exposed cells as the CpGs in the p21 promoter (-128 bp/+17 bp) were significantly demethylated as observed in the methylation-specific PCR (MSP). CONCLUSION: Highly potential anti-proliferative molecule brazilin can modulate the DNMT1 functional expression and restore the cell cycle inhibitor p21expression. We propose that brazilin can be used in therapeutic interventions to restore the deregulated epigenetic mechanisms in cancer.
Asunto(s)
Benzopiranos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ADN (Citosina-5-)-Metiltransferasa 1 , Epigénesis Genética , Proteína p53 Supresora de Tumor , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Simulación del Acoplamiento Molecular , Fitoquímicos , Regiones Promotoras Genéticas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Maritime pine bark is a rich source of polyphenolic compounds and is commonly employed as a herbal supplement worldwide. This study was designed to check the potential of maritime pine tannin extract (MPTE) in anticancer therapy and to determine the underlying mechanism of action. Our results showed that MPTE, containing procyanidin oligomers and lanostane type terpenoids, has an inhibitory effect on cancer cell proliferation through cell cycle arrest in the G2/M phase. Treatment with MPTE also induced apoptosis in a concentration-dependent manner in human cancer cell lines (HeLa and U2OS), as evidenced by the enhanced activation of caspase 3 and the cleavage of PARP along with the downregulation of the antiapoptotic protein Bcl-2. Interestingly, human non-cancerous fibroblasts are much less sensitive to MPTE, suggesting that it preferentially targets cancer cells. MPTE played a pro-oxidant role in cancer cells and promoted the expression of the p73 tumor suppressor gene in p53-deficient cells. It also downregulated the protooncogenic proteins UHRF1 and DNMT1, mediators of the DNA methylation machinery, and reduced the global methylation levels in HeLa cells. Overall, our results show that maritime pine tannin extract can play a favorable role in cancer treatment, and can be further explored by the pharmaceutical industry.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Potenciadoras de Unión a CCAAT , Epigénesis Genética/efectos de los fármacos , Pinus/química , Taninos/farmacología , Ubiquitina-Proteína Ligasas , Apoptosis/efectos de los fármacos , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Células HeLa , Humanos , Corteza de la Planta/química , Extractos Vegetales/farmacología , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Diabetic retinopathy (DR) is one of the severe microvascular complications of diabetes. The protective effects of FA on retinal vascular endothelial cells against high glucose levels involve in multiple aspects in DR; however, the underlying mechanism is not fully elucidated. In present study, we investigated the transcriptome as well as genome-wide DNA methylation and hydroxymethylation signature in human retinal microvascular endothelial ACBRI 181 cells cultured within high glucose (HG) medium supplemented with or without FA by RNA-seq, MeDIP-seq, and hMeDIP-seq. Total 3308 differential expressed genes (DEGs) were involved in multiple biological processes and molecular functions containing angiogenesis, inflammation, S-adenosyl methionine metabolism, and hypoxia response. Moreover, the global DNA methylation and hydroxymethylation in ACBRI 181 cells with FA treatment were both compromised compared to HG. Combined with transcriptome data, four subclusters of DEGs with hyper- or hypomethylated promoters were further verified. Unexpectedly, promoters of these 487 genes all displayed a pattern of increased DNA hydroxymethylation. Furthermore, hyperglycemia rat model was established and administered with FA. The DNA methylation and hydroxymethylation changes of selected target genes COL1A1, ITGA7, MMP-14, and VEGFB confirmed by MeDIP-qPCR were consistent with the results in human ACBRI 181 cells. Finally, the presence of activated DNMT1 and TET2 induced by FA was determined in ACBRI 181 cells and hyperglycemia rat. Taken together, this research provided a resource of expression and epigenetic profiles in retinal microvascular endothelial cell, emphasizing a pharmacological mechanism of FA on DNA methylation and hydroxymethylation regulation in retinal microvessel cells of DR.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Retinopatía Diabética/tratamiento farmacológico , Ácido Fólico/farmacología , Microvasos/efectos de los fármacos , Retina/efectos de los fármacos , Animales , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1/genética , Diabetes Mellitus Experimental/genética , Retinopatía Diabética/genética , Células Endoteliales/efectos de los fármacos , Humanos , Masculino , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Ratas , Ratas Sprague-Dawley , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Dedos de Zinc/genéticaRESUMEN
Liver fibrosis is the consequence of chronic liver injury and is a major challenge to global health. However, successful therapy for liver fibrosis is still lacking. Sennoside A (SA), a commonly used clinical stimulant laxative, is reported to improve hepatic disease, but the underlying mechanisms remain largely elusive. Here, we show for the first time that SA enhanced suppressor of cytokine signaling 1 (SOCS1) expression in a DNA methyltransferase 1 (DNMT1)-dependent manner and thereby attenuated liver fibrosis. Consistently, SA inhibited the expression of the liver fibrogenesis markers α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) and suppressed inflammatory responses in vivo and in vitro. Coculture experiments with macrophages/hepatic stellate cells (HSCs) revealed that SA suppressed HSC proliferation by downregulating proinflammatory cytokines in macrophages. Mechanically, SA promoted the aberrant expression of SOCS1 in liver fibrosis. However, blocking SOCS1 expression weakened the inhibitory effect of SA on HSC proliferation, indicating that SOCS1 may play an important role in mediating the antifibrotic effect of SA. Furthermore, SA inhibited DNMT1-mediated SOCS1 and reduced HSC proliferation by inhibiting inflammatory responses in carbon tetrachloride (CCl4) -induced liver fibrosis.
Asunto(s)
Antiinflamatorios/uso terapéutico , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Cirrosis Hepática/tratamiento farmacológico , Senósidos/uso terapéutico , Proteína 1 Supresora de la Señalización de Citocinas/genética , Animales , Antiinflamatorios/farmacología , Tetracloruro de Carbono , Línea Celular , Proliferación Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratas , Senósidos/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.
Asunto(s)
Antiparkinsonianos/farmacología , Antiparkinsonianos/uso terapéutico , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Extractos de Tejidos/química , alfa-Sinucleína/metabolismo , Animales , ADN (Citosina-5-)-Metiltransferasa 1/efectos de los fármacos , Hidroxidopaminas , Masculino , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Células PC12 , Enfermedad de Parkinson Secundaria/inducido químicamente , Ratas , Ratas Sprague-Dawley , Estearatos/farmacología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , alfa-Sinucleína/efectos de los fármacosRESUMEN
Glioblastoma multiforme (GBM) is a deadly malignant brain tumor that is resistant to most clinical treatments. Novel therapeutic agents that are effective against GBM are required. Antrodia cinnamomea has shown antiproliferative effects in GBM cells. However, the exact mechanisms and bioactive components remain unclear. Thus, the present study aimed to investigate the effect and mechanism of 4-acetylantrocamol LT3 (4AALT3), a new ubiquinone from Antrodia cinnamomeamycelium, in vitro. U87 and U251 cell lines were treated with the indicated concentration of 4AALT3. Cell viability, cell colony-forming ability, migration, and the expression of proteins in well-known signaling pathways involved in the malignant properties of glioblastoma were then analyzed by CCK-8, colony formation, wound healing, and western blotting assays, respectively. We found that 4AALT3 significantly decreased cell viability, colony formation, and cell migration in both in vitro models. The epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), Hippo/yes-associated protein (YAP), and cAMP-response element binding protein (CREB) pathways were suppressed by 4AALT3. Moreover, 4AALT3 decreased the level of DNA repair enzyme O6-methylguanine-DNA methyltransferase and showed a synergistic effect with temozolomide. Our findings provide the basis for exploring the beneficial effect of 4AALT3 on GBM in vivo.
Asunto(s)
4-Butirolactona/análogos & derivados , Ciclohexanonas/farmacología , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Reparación del ADN/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Guanina/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclohexanonas/química , Regulación hacia Abajo , Guanina/metabolismo , Humanos , Ubiquinona/farmacologíaRESUMEN
Ethanol (EtOH) has been linked to neurotoxic effects on the fetus and prenatal alcohol exposure (PAE) has a negative impact on brain neurodevelopment. Therefore, the present study was aimed to focus on the underlying mechanisms of alcohol-induced oxidative stress and apoptotic cell death in addition to shedding the light on the modulatory effect of nanocurcumin in rats' offspring prefrontal cortices. The current study investigated the effects of prenatal maternal exposure to EtOH intragastric (i.g.) administration of 0.015 mL/g of a 10 % v/v ethanol solution throughout gestation and the concomitant use of nanocurcumin, on 21-day-old offspring Wistar rat prefrontal cortex parameters. CYP2E1, DBN1, DNMT1, miRNA-335, miRNA-21, c-Fos and Cox-2 gene expression as well as the accompanying histological and ultrastructural alterations were assessed. The implemented experimental setting has revealed that ethanol exposure caused significant alterations in the above mentioned parameters. Changes observed in nanocurcumin-treated animals were significantly different to the ethanol-treated group when nanocurcumin was concomitantly administered.
Asunto(s)
Curcumina/uso terapéutico , Etanol/farmacología , Expresión Génica/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Corteza Prefrontal/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/genética , Animales , Curcumina/farmacología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Femenino , MicroARNs/genética , MicroARNs/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Fármacos Neuroprotectores/farmacología , Corteza Prefrontal/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas WistarRESUMEN
Triple-negative breast cancer (TNBC) is an aggressive cancer, and rapidly progresses following relapse in advanced stage. This cancer is usually associated with worse overall survival, so the carcinogenesis of TNBC needs to be further explored to find more effective therapies. In this study, we intended to identify the roles of YY1-mediated long non-coding RNA Kcnq1ot1 in TNBC. First, the paired samples of tumor tissues and adjacent tissues were collected to determine YY1, lncRNA Kcnq1ot1, and PTEN expression using RT-qPCR and Western blot analysis followed by analysis of the relationship between them and patient survival. The results revealed that YY1 and lncRNA Kcnq1ot1 were upregulated in TNBC tissues, and high expression of YY1 and lncRNA Kcnq1ot1 was associated with poor patient survival. Then, ChIP and MSP assays were employed to explore interactions between YY1, lncRNA Kcnq1ot1, and PTEN gene. We obtained that YY1 upregulated lncRNA Kcnq1ot1, which mediated PTEN methylation via DNMT1, thus decreasing PTEN expression. Afterward, TNBC cells were examined for their viability using functional assays with the results displaying that overexpression of YY1 facilitated TNBC cell proliferation, invasion, and migration. Mechanistically, upregulated YY1 repressed tumor growth by inhibiting PTEN via upregulation of lncRNA Kcnq1ot1. Mouse models were also constructed, and the above effects of YY1, lncRNA Kcnq1ot1, and PTEN on TNBC were also established in vivo. Taken together, this study demonstrates that the silencing of YY1 exerted tumor-suppressive effects on TNBC by modulating lncRNA Kcnq1ot1/DNMT1/PTEN pathway, in support of further investigation into anti-tumor therapy for TNBC.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Factor de Transcripción YY1/metabolismo , Adulto , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Persona de Mediana Edad , Canales de Potasio con Entrada de Voltaje/metabolismoRESUMEN
Although the mechanism of DNA demethylating drugs has been understood for many years, the direct effect of these drugs on methylation of the complementary strands of DNA has not been formally demonstrated. By using hairpin-bisulphite sequencing, we describe the kinetics and pattern of DNA methylation following treatment of cells by the DNA methyltransferase 1 (DNMT1) inhibitor, decitabine. As expected, we demonstrate complete loss of methylation on the daughter strand following S-phase in selected densely methylated genes in synchronized Jurkat cells. Thereafter, cells showed a heterogeneous pattern of methylation reflecting replication of the unmethylated strand and restoration of methylation.
Asunto(s)
Desmetilación del ADN , Metilación de ADN , Azacitidina , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Decitabina , Humanos , SulfitosRESUMEN
Current plant-derived anticancer therapeutics aim to reach higher effectiveness, to potentiate chemosensitivity and minimize the toxic side effects compared to conventional chemotherapy. Cotinus coggygria Scop. is a herb with high pharmacological potential, widely applied in traditional phytotherapy. Our previous study revealed that leaf aqueous ethanolic extract from C. coggygria exerts in vitro anticancer activity on human breast, ovarian and cervical cancer cell lines. The objective of the present research was to investigate possible molecular mechanisms and targets of the antitumor activity of the extract in breast cancer MCF7 cells through analysis of cell cycle and apoptosis, clonogenic ability assessment, evaluation of the extract genotoxic capacity, characterization of cells thermodynamic properties, and analysis on the expression of genes involved in cellular epigenetic processes. The obtained results indicated that in MCF7 cells C. coggygria extract causes S phase cell cycle arrest and triggers apoptosis, reduces colony formation, induces DNA damage, affects cellular thermodynamic parameters, and tends to inhibit the relative expression of DNMT1, DNMT3a, MBD3, and p300. Further studies on the targeted molecules and the extract anti-breast cancer potential on animal experimental model system, need to be performed in the future.
Asunto(s)
Anacardiaceae/química , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Extractos Vegetales/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Daño del ADN/efectos de los fármacos , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/genética , Epigénesis Genética/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Extractos Vegetales/química , Hojas de la Planta/químicaRESUMEN
Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl4 -induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, in vivo and in vitro. SA decreased the expression of Cyclin D1, CDK, and C-myc, indicating that SA may inhibit the activation and proliferation of TGF-ß1-induced HSC-T6. Moreover, SA significantly promoted the expression of PTEN and remarkably inhibited the expression of p-AKT and p-ERK in vitro. Blocking PTEN or overexpressing DNMT1 could reduce the effect of SA on liver fibrosis. These data suggest that SA directly binds and inhibits the activity and that attenuated DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the inhibition of the AKT and ERK pathways and prevented the development of liver fibrosis. Hence, SA might be employed as a promising natural supplement for liver fibrosis drug therapy.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática/tratamiento farmacológico , Fosfohidrolasa PTEN/genética , Senósidos/farmacología , Actinas/genética , Actinas/metabolismo , Animales , Línea Celular , Proliferación Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/fisiología , Cirrosis Hepática/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfohidrolasa PTEN/metabolismo , Unión Proteica , Senósidos/uso terapéutico , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
We evaluated if chronic consumption of quercetin (Q) with green tea extract (GTE) enhances the bioavailability of GT polyphenols (GTPs) and reduces methylation activity as previously observed in mouse xenograft tumors. In this prospective, randomized, parallel design, placebo controlled study, thirty-one men with prostate cancer consumed daily 1 gram of GTE (830 mg of GTP) with 800 mg of Q (GT + Q) or placebo (GT + PL) for four weeks before prostatectomy. First morning voided urine was collected at baseline, 3 weeks and the day of surgery, and prostate tissue on the day of surgery. In week 3, plasma concentration of GTPs and Q was measured in blood collected before and 2 hours after the morning dose. Prostate tissue epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) were detected in 67 and 93% of participants in the GT + Q group and 75 and 94% of participants in the GT + PL group. Q was increased 14-fold, 12-fold and 4.5-fold in plasma, urine, and prostate tissue, respectively, in the GT + Q compared to the GT + PL-group. There was a trend for decreased EGC levels in urine collected prior to prostatectomy in the GT + Q compared to GT + PL-group (p = 0.053). Plasma epigallocatechin (EGC) showed a trend to increase (p = 0.066) two hours after capsule intake in the GT + Q vs. the GT + PL-group. There was no significant difference between the groups in GTP content or methylation activity in prostate tissue or RBCs. No liver toxicity was observed. Although our findings are suggestive, further studies are warranted evaluating if Q alters GTP metabolism.
Asunto(s)
Polifenoles/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Quercetina/metabolismo , Té/química , Anciano , Biomarcadores , Catecol O-Metiltransferasa/genética , Catecol O-Metiltransferasa/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Suplementos Dietéticos , Quimioterapia Combinada , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Polifenoles/química , Quercetina/administración & dosificación , Quercetina/químicaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The classical and traditional Chinese medicine prescription, Liuwei Dihuang (LWDH), has been commonly used to treat the menopausal syndrome. It has been reported that LWDH could improve estrogen receptor α (ERα) expression to prevent atherosclerosis (AS), while the mechanism of LWDH on regulating ERα expression was still unknown. AIM OF THE STUDY: To reveal the mechanism of LWDH on regulating the ERα expression. MATERIALS AND METHODS: The protective effect of LWDH on Hcy-induced apoptosis of human umbilical vein endothelial cells (HUVECs) was examined. The expression of ERα and DNA methyltransferases 1 (DNMT1) were detected by Western blot and real-time polymerase chain reaction (RT-PCR). The methylation rate of the ERα gene was assayed by the bisulfite sequencing PCR (BSP). High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was applied to determine the level of S-Adenosyl methionine (SAM) and S-Adenosyl homocysteine (SAH). In vivo, the ApoE-/- mice were ovariectomized to establish postmenopausal atherosclerosis (AS) model. RESULTS: In vitro study showed that LWDH protects HUVECs from Hcy-induced apoptosis. Treatment with LWDH significantly increased the ERα expression and reduced the methylation rate of the ERα gene by inhibiting the DNMT1 expression. The level of main methyl donor SAM and the ration of SAM/SAH were reduced by LWDH. In vivo, LWDH prevented the formation of plaque and reduced the concentration of Hcy. In addition, LWDH upregulated the ERα expression, as well as inhibiting the expression of DNMT1 in atherosclerotic mice. CONCLUSIONS: LWDH exerted protective effects on postmenopausal AS mice, and HUVECs treated with Hcy. LWDH increased of ERα expression via inhibiting DNMT1-dependent ERα methylation.
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Aterosclerosis/tratamiento farmacológico , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Receptor alfa de Estrógeno/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Aterosclerosis/metabolismo , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADN/efectos de los fármacos , Dieta Alta en Grasa , Receptor alfa de Estrógeno/genética , Femenino , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Posmenopausia , Ratas Sprague-DawleyRESUMEN
Ochratoxin A (OTA) is a potent nephrotoxin. Selenium (Se) is an essential micronutrient for humans and animals, and plays a key role in antioxidant defense. To date, little is known about the effect of Se on OTA-induced DNA damage. In this study, the protective effects of Se (from selenomethionine) against OTA-induced cytotoxicity and DNA damage were investigated by using PK15 cells as a model. The results showed that OTA at 4.0 µg/mL induced cytotoxicity and DNA damage. Se at 0.5, 1, 2 and 4 µM significantly blocked OTA-induced cytotoxicity and DNA damage. Furthermore, Se blocked the increases of DNMT1, DNMT3a and HDAC1 mRNA and protein expression, reversed the decreases of glutathione peroxidase 1 (GPx1) mRNA and protein expression, and promoted the increases of SOCS3 mRNA and protein expression induced by OTA. Overexpression of GPx1 by pcDNA3.1-GPx1 inhibited the OTA-induced DNMT1 expression, promoted OTA-induced SOCS3 expression, and prevented the OTA-induced cytotoxicity and DNA damage. In contrast, knock-down of GPx1 by using a GPx1-specific siRNA had the opposite effects. The results suggest that GPx1-mediated DNMT1 expression is involved in the blocking effects of selenium on OTA-induced cytotoxicity and DNA damage.
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ADN (Citosina-5-)-Metiltransferasa 1/biosíntesis , Daño del ADN , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Ocratoxinas/toxicidad , Selenometionina/farmacología , Animales , Línea Celular , Selenio/farmacología , Porcinos , Glutatión Peroxidasa GPX1RESUMEN
SCOPE: The aim of the current study is to evaluate whether l-methionine supplementation (l-Met-S) improves type 2 diabetes-induced alterations in glucose and lipid metabolism by modulating one-carbon metabolism and methylation status. METHODS AND RESULTS: Diabetes is induced in male Sprague-Dawley rats using high-fat diet and low dose streptozotocin. At the end of study, various biochemical parameters, immunoblotting, qRT-PCR and ChIP-qPCR are performed. The first evidence that l-Met-S activates p-AMPK and SIRT1, very similar to "metformin," is provided. l-Met-S improves the altered key one-carbon metabolites in diabetic rats by modulating methionine adenosyl transferase 1A and cystathione ß synthase expression. qRT-PCR shows that l-Met-S alleviates diabetes-induced increase in Forkhead transcription factor 1 expression and thereby regulating genes involved in glucose (G6pc, Pdk4, Pklr) and lipid metabolism (Fasn). Interestingly, l-Met-S inhibits the increased expression of DNMT1 and also prevents methylation of histone H3K36me2 under diabetic condition. ChIP assay shows that persistent increase in abundance of histone H3K36me2 on the promoter region of FOXO1 in diabetic rats and it is recovered by l-Met-S. CONCLUSION: The first evidence that dietary supplementation of l-Met prevents diabetes-induced epigenetic alterations and regulating methionine levels can be therapeutically exploited for the treatment of metabolic diseases is provided.
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Metilación de ADN , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metionina/administración & dosificación , Animales , ADN (Citosina-5-)-Metiltransferasa 1/genética , Diabetes Mellitus Tipo 2/genética , Suplementos Dietéticos , Glutatión/metabolismo , Masculino , Metformina/farmacología , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Sprague-Dawley , Sirtuina 1/análisis , EstreptozocinaRESUMEN
BACKGROUND: Sulforaphane (SFN), a natural compound present in cruciferous vegetable, has been shown to possess anti-cancer activities. Cancer stem cell (CSC) in bulk tumor is generally considered as treatment resistant cell and involved in cancer recurrence. The effects of SFN on nasopharyngeal carcinoma (NPC) CSCs have not yet been explored. PURPOSE: The present study aims to examine the anti-tumor activities of SFN on NPC cells with CSC-like properties and the underlying mechanisms. METHODS: NPC cells growing in monolayer culture, CSCs-enriched NPC tumor spheres, and also the NPC nude mice xenograft were used to study the anti-tumor activities of SFN on NPC. The population of cells expressing CSC-associated markers was evaluated using flow cytometry and aldehyde dehydrogenase (ALDH) activity assay. The effect of DNA methyltransferase 1 (DNMT1) on the growth of NPC cells was analyzed by using small interfering RNA (siRNA)-mediated silencing method. RESULTS: SFN was found to inhibit the formation of CSC-enriched NPC tumor spheres and reduce the population of cells with CSC-associated properties (SRY (Sex determining Region Y)-box 2 (SOX2) and ALDH). In the functional study, SFN was found to restore the expression of Wnt inhibitory factor 1 (WIF1) and the effect was accompanied with the downregulation of DNMT1. The functional activities of WIF1 and DNMT1 were confirmed using exogenously added recombinant WIF1 and siRNA knockdown of DNMT1. Moreover, SFN was found to inhibit the in vivo growth of C666-1 cells and enhance the anti-tumor effects of cisplatin. CONCLUSION: Taken together, we demonstrated that SFN could suppress the growth of NPC cells via the DNMT1/WIF1 axis.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antineoplásicos Fitogénicos/farmacología , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Isotiocianatos/farmacología , Carcinoma Nasofaríngeo/tratamiento farmacológico , Neoplasias Nasofaríngeas/tratamiento farmacológico , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Brassicaceae/química , Línea Celular Tumoral , Cisplatino/administración & dosificación , ADN (Citosina-5-)-Metiltransferasa 1/genética , Humanos , Isotiocianatos/administración & dosificación , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Sulfóxidos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
During DNA replication, ubiquitin-like, containing PHD and RING fingers domainsâ 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferaseâ 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.
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Antraquinonas/química , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Inhibidores Enzimáticos/química , 5-Metilcitosina/química , Sitios de Unión , ADN (Citosina-5-)-Metiltransferasa 1/química , Evaluación Preclínica de Medicamentos/métodos , Células HeLa , Humanos , Cinética , Metilación , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Transfección/métodos , Ubiquitina-Proteína LigasasRESUMEN
Aberrant epigenetic modifications are responsible for tumor development and cancer progression; however, readily reversible. Bioactive molecules from diets are promising to cure cancer by modulating epigenetic marks and changing immune response. These compounds specifically target the activity of DNMTs and HDACs to cure various human cancers. In view of this, we investigated the anticancer and epigenetic regulatory activities of an edible-plant Paederia foetida. The efficacy of methanolic extract of P. foetida leaves (MEPL) was tested for the modulation of epigenetic factors in gene silencing, i.e. DNMT and HDAC and expression pattern of certain tumor-suppressor genes. After treatment of prostate cancer cells (PC-3 and DU-145) with MEPL, lupeol and ß-sitosterol; induction of apoptosis, decrease in cellular-viability and inhibition of cellular-migration were noticed. Simultaneously there was inhibition of DNMT1, HDACs and pro-inflammatory, IL-6, IL1-ß, TNF-α and anti-inflammatory, IL-10 genes in cancer and THP1 cell lines. The DNMT1 protein content, enzyme activity and Bcl2 expression decreased significantly; however, expression of E-cadherin (CDH1) and pro-apoptotic gene Bax increased significantly after the treatment of cells with drugs. We conclude plant-derived compounds can be considered to target epigenetic machineries involved with malignant transformation and can open new avenues for cancer therapeutics provoking immune response.
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Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inflamación/metabolismo , Extractos Vegetales/farmacología , Neoplasias de la Próstata , Rubiaceae/química , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Humanos , Inflamación/genética , Masculino , Triterpenos Pentacíclicos , Fitoquímicos , Extractos Vegetales/química , Hojas de la Planta/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , SitoesterolesRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese herbal medicine Fuzheng Kang-Ai (FZKA) decoction obtained from Guangdong Kangmei Pharmaceutical Company, which contains 12 components with different types of constituents, has been used as part of the adjuvant treatment of lung cancer for decades. We previously showed that FZKA decoction enhances the growth inhibition of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant non-small cell lung cancer (NSCLC) cells by suppressing glycoprotein mucin 1 (MUC1) expression. However, the molecular mechanism underlying the therapeutic potential, particularly in sensitizing or/and enhancing the anti-lung cancer effect of EGFR-TKIs, remains unclear. MATERIALS AND METHODS: Cell viability was measured using 3-(4, 5-diMEThylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and 5-ethynyl -2'-deoxyuridine (EdU) assays. Western blot analysis was performed to examine the protein expressions of DNA methyltransferase 1 (DNMT1), specificity protein 1 (SP1), and MET, an oncogene encoding for a trans-membrane tyrosine kinase receptor activated by the hepatocyte growth factor (HGF). The expression of MET mRNA was measured by quantitative real-time PCR (qRT-PCR). Exogenous expression of DNMT1 and SP1, and MET were carried out by transient transfection assays. The promoter activity of MET was tested using Dual-luciferase reporter assays. A nude mouse xenografted tumor model further evaluated the effect of the combination of FZKA decoction and erlotinib in vivo. RESULTS: The combination of FZKA and erlotinib produced an even greater inhibition of NSCLC cell growth. FZKA decreased the expressions of DNMT1, SP1, and MET (c-MET) proteins, and the combination of FZKA and erlotinib demonstrated enhanced responses. Interestingly, there was a mutual regulation of DNMT1 and SP1. In addition, exogenously expressed DNMT1 and SP1 blocked the FZKA-inhibited c-MET expression. Moreover, excessive expressed MET neutralized FZKA-inhibited growth of NSCLC cells. FZKA decreased the mRNA and promoter activity of c-MET, which was not observed in cells with ectopic expressed DNMT1 gene. Similar findings were observed in vivo. CONCLUSION: FZKA decreases MET gene expression through the repression and mutual regulation of DNMT1 and SP1 in vitro and in vivo. This leads to inhibit the growth of human lung cancer cells. The combination of FZKA and EGFR-TKI erlotinib exhibits synergy in this process. The regulatory loops among the DNMT1, SP1 and MET converge in the overall effects of FZKA and EGFR-TKI erlotinib. This in vitro and in vivo study clarifies an additional novel molecular mechanism underlying the anti-lung cancer effects in response to the combination of FZKA and erlotinib in gefitinib-resistant NSCLC cells.
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Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Medicamentos Herbarios Chinos/farmacología , Clorhidrato de Erlotinib/farmacología , Neoplasias Pulmonares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Sinergismo Farmacológico , Medicamentos Herbarios Chinos/uso terapéutico , Clorhidrato de Erlotinib/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Factor de Transcripción Sp1/metabolismoRESUMEN
Lifestyle interventions, including exercise and dietary supplementation, can modify DNA methylation and exert health benefits; however, the underlying mechanisms are poorly understood. Here we investigated the impact of acute aerobic exercise and the supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) and extra virgin olive oil (EVOO) on global and gene-specific (PPARGC1A, IL6 and TNF) DNA methylation, and DNMT mRNA expression in leukocytes of disease-free individuals. Eight trained male cyclists completed an exercise test before and after a four-week supplementation of n-3 PUFA and EVOO in a double-blind, randomised, repeated measures design. Exercise triggered global hypomethylation (Pre 79.2%; Post 78.7%; p = 0.008), alongside, hypomethylation (Pre 6.9%; Post 6.3%; p < 0.001) and increased mRNA expression of PPARGC1A (p < 0.001). Associations between PPARGC1A methylation and exercise performance were also detected. An interaction between supplement and trial was detected for a single CpG of IL6 indicating increased DNA methylation following n-3 PUFA and decreased methylation following EVOO (p = 0.038). Global and gene-specific DNA methylation associated with markers of inflammation and oxidative stress. The supplementation of EVOO reduced DNMT1 mRNA expression compared to n-3 PUFA supplementation (p = 0.048), whereas, DNMT3a (p = 0.018) and DNMT3b (p = 0.046) mRNA expression were decreased following exercise. In conclusion, we demonstrate that acute exercise and dietary supplementation of n-3 PUFAs and EVOO induce DNA methylation changes in leukocytes, potentially via the modulation of DNMT mRNA expression. Future studies are required to further elucidate the impact of lifestyle interventions on DNA methylation.