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PURPOSE: To investigate whether the DNA methylation profiles of GNAS(20q13.32), MEST(7q32.2), MESTIT1(7q32.2), IGF2(11p15.5), H19 (7q32.2), and CEP41(7q32.2) genes are related to the transcriptomic and epigenomic etiology of male infertility. METHODS: The DNA methylation levels of spermatozoa were obtained from fertile (n = 30), oligozoospermic (n = 30), and men with normal sperm count (n = 30). The methylation status of each CpG site was categorized as hypermethylated or hypomethylated. Expression levels of target gene transcripts were determined using real-time PCR. RESULTS: The oligozoospermia showed a higher frequency of hypermethylation at GNASAS 1st, 3rd, and 5th CpG dinucleotides (66.7%, 73.3%, 73.3%) compared to the fertile group (33.3%, 33.3%, 40%, respectively). The normal sperm count exhibited a higher frequency of hypermethylation at the 3rd CpG of CEP41 (46.7%) than the fertile group (16.7%). Normal sperm count was predicted by CEP41 hypermethylation (OR = 1.750, 95%CI 1.038-2.950) and hypermethylation of both CEP41 and GNASAS (OR = 2.389, 95%CI 1.137-5.021). Oligozoospermia was predicted solely by GNASAS hypermethylation (OR = 2.460, 95%CI 1.315-4.603). In sperms with decreased IGF2 expression in the fertile group, we observed hypomethylation in the 2nd CpG of IGF2 antisense (IFG2AS), and hypermethylation in the 1st, 2nd, and 4th CpGs of H19. No significant relationship was found between IGF2 expression and methylation status of IGF2AS and H19 in infertile groups. CONCLUSION: The disappearance of the relationship between IGF2 expression and IGF2AS and H19 methylations in the infertile group provides new information regarding the disruption of epigenetic programming during spermatogenesis. A better understanding of sperm GNASAS and CEP41 hypermethylation could advance innovative diagnostic markers for male infertility.
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Recently, aberrant DNA methylation of the HIST1H4F gene (encodes Histone 4 protein) has been shown in many types of cancer, which may serve as a promising biomarker for early cancer diagnosis. However, the correlation between DNA methylation of the HIST1H4F gene and its role in gene expression is unclear in bladder cancer. Therefore, the first objective of this study is to explore the DNA methylation pattern of the HIST1H4F gene and then further elucidate its effects on HIST1H4F mRNA expression in bladder cancer. To this end, the methylation pattern of the HIST1H4F gene was analyzed by pyrosequencing and the effects of the methylation profiles of this gene on HIST1H4F mRNA expression in bladder cancer were examined by qRT-PCR. Sequencing analysis revealed significantly higher methylation frequencies of the HIST1H4F gene in bladder tumor samples compared to normal samples (p < 0,0001). However, when we evaluated the correlations between hypermethylation of HIST1H4F and the clinicopathological parameters (tumor stage, tumor grade, lymph node metastasis, muscle-invasion), no significant difference was found between the groups (p > 0.05). In addition, we examined the role of hypermethylation of the HIST1H4F gene on HIST1H4F mRNA expression. We found that hypermethylation of HIST1H4F in the exon have no effect HIST1H4F mRNA expression in bladder cancer (p > 0.05). We also confirmed our finding in cultured T24 cell line which HIST1H4F gene is hypermethylated. Our results suggest that hypermethylation of the HIST1H4F seems to be a promising early diagnostic biomarker in bladder cancer patients. However, further studies are needed to determine the role of HIST1H4F hypermethylation in tumorigenesis.
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Histonas , Neoplasias de la Vejiga Urinaria , Humanos , Histonas/genética , Histonas/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Metilación de ADN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Biomarcadores de Tumor/metabolismoRESUMEN
Background/aim: Thermopsisturcica is a perennial species endemic to Turkey and different extracts of T. turcica have an antiproliferative effect on cancer cells, but there has not been any report on HeLa (human cervical cancer) cells. Materials and methods: To get a better understanding of the molecular mechanism of anticancer activity of methanolic extracts of leaves (LE) and flowers (FE) of T. turcica, we employed 2-DE-based proteomics to explore the proteins involved in anticancer activity in HeLa cells. Results: T. turcica extracts showed a potent cytotoxic effect on HeLa cells with the IC50 values of 1.75 mg/mL for LE and 3.25 mg/mL for FE. The induction of apoptosis by LE and FE was also consistent with increased expression of caspase mRNAs and DNA fragmentation. In terms of the proteomic approach, 27 differentially expressed proteins were detected and identified through MALDI-TOF/TOF mass spectrometry. These altered proteins were involved in cytoskeleton organization and movement, protein folding, proteolysis and translation, cell cycle and proliferation, signal transduction, cell redox homeostasis, and metabolism. Conclusion: Up-regulation of protein disulfide isomerases and down-regulation of Rho GDP-dissociation inhibitor, heterogeneous nuclear ribonucleoproteins, and heat shock proteins may contribute to the induction of apoptosis and arresting of the cell cycle in HeLa cells.
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Antineoplásicos/farmacología , Fabaceae , Extractos Vegetales/farmacología , Plantas Medicinales , Proteómica/métodos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Flores , Humanos , Hojas de la Planta , TurquíaRESUMEN
The present study was considered to assess the protective effects of boron (B) on gentamicin-induced oxidative stress, proinflammatory cytokines, and histopathological changes in rat kidneys. Rats were split into eight equal groups which were as follows: control (fed with low-boron diet); gentamicin group (100 mg/kg, i.p.); B5, B10, and B20 (5, 10, and 20 mg/kg B, i.p.) groups; gentamicin (100 mg/kg, i.p.) plus B5, B10, and B20 (5, 10, and 20 mg/kg B, i.p.) groups. B was given to rats 4 days before the gentamicin treatment and B administration was completed on the 14th day. Gentamicin administration was started on the 4th day and finished on the 12th day. Gentamicin increased malondialdehyde levels, while reduced glutathione levels in the blood and kidney. Furthermore, superoxide dismutase and catalase activities of erythrocyte were decreased. Besides, serum and kidney nitric oxide and 8-dihydroxyguanidine levels were increased by gentamicin. Additionally, serum levels and kidney mRNA expressions of TNF-α, NFκB, IL-1ß, and IFN-γ were found to be the highest in the gentamicin group. Histopathologically, interstitial hemorrhage and tubular necrosis were detected in the kidneys of the gentamicin group. Nonetheless, B administration reversed gentamicin-induced lipid peroxidation, antioxidant status, and inflammation. In conclusion, B has a preventive effect against gentamicin-induced nephrotoxicity and ameliorates kidney tissues of the rat.
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Boro/farmacología , Gentamicinas/antagonistas & inhibidores , Riñón/efectos de los fármacos , Animales , Boro/administración & dosificación , Daño del ADN , Gentamicinas/administración & dosificación , Inyecciones Intraperitoneales , Riñón/metabolismo , Riñón/patología , Masculino , Ratas , Ratas WistarRESUMEN
Background/aim: Age-related cataract is the most important visual impairment all over the world. Epigenetic modifications, especially overexpression of histone deacetylases, have become the focus of interest for cataract development in recent years. Sirtuin 1 (SIRT1), a class II histone deacetylase and a member of the sirtuin family, is one of the best-characterized histone deacetylases and has a pivotal role in age-related diseases. However, the association of SIRT1 with age-related cataracts has not yet been fully elucidated. Therefore, we aimed to determine the expression of SIRT1 in age-related cataract patients. Materials and methods: Expressions of SIRT1 were evaluated by quantitative polymerase chain reaction (qPCR) in patients and healthy controls. RNA samples were collected from the anterior capsule and peripheral blood samples of age-related cataract patients. Human lens epithelial cell line B3 and peripheral blood samples of healthy subjects were used as controls. Results: We determined that the expression of SIRT1 in blood and anterior capsule samples increased significantly compared to the control group (P < 0.05). Conclusion: The expression level of SIRT1 plays a vital role in the development of age-related cataract and it can be used as a biomarker. Thus, SIRT1 inhibitors can be used in the treatment of age-related cataract disease.
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Catarata , Sirtuina 1 , Adulto , Anciano , Anciano de 80 o más Años , Cápsula Anterior del Cristalino/química , Cápsula Anterior del Cristalino/citología , Cápsula Anterior del Cristalino/metabolismo , Catarata/epidemiología , Catarata/genética , Catarata/metabolismo , Células Cultivadas , Epigénesis Genética/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Sirtuina 1/análisis , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Heat shock protein 90 (HSP90), an ATP-dependent molecular chaperone required for the stability and function of numerous oncogenic signaling, is one of the hallmarks of cancer. Recent years, the studies showed that HSP90 plays a pivotal role in epigenetic pathways. Epigenetic regulation plays an important role in the etiology of bladder cancer. The aim of the present study was to investigate the effect of HSP90 proteins on DNA methylation and the levels of inactivated histone methylation markers in bladder cancers. The cytotoxic effect of geldanamycin (GA), a HSP90-specific inhibitor, in human bladder cancer cell line, T24, was studied by using WST1 (both time and dose-dependent), qPCR for the expression aberration of target genes DNMT1 and WIF-1 and western blot for the protein levels of DNMT1, Histone H4, Histone 4 lysine monomethylation (H4K20me1), Histone 4 lysine trimethylation (H4K20me3), Akt1, pAkt1 (S473) and Lysine methyltransferase 5C (KMT5C). High-dose GA treatment decreased cell proliferation. After the GA treatment, DNMT1 decreased at both transcriptional and translational levels due to Akt1 and pAkt1 (S473) inhibition. Following the GA-induced decrease in DNMT1, re-expression of WIF-1 gene was found at mRNA. In addition, the GA treatment resulted in dose- and time-dependent upregulation/downregulation of histone post-translational modifications (H4K20me1 and H4K20me3) and the KMT5C enzyme responsible for these modifications. There was no significant change in the H4 protein level. These findings may offer a new approach for the determination of the molecular effect of HSP90 on epigenetic regulation and the identification of new molecular targets (HSP90 client proteins) for bladder cancer treatment.
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Bisphenol A (BPA) is one of the most produced chemicals in the world and has been widely employed in the food industry. Continuous and widespread exposure to BPA through drinking water and food leads to health concerns for humans. This study evaluated the effects of boron (B) on BPA-mediated oxidative stress in male Wistar albino rats. Rats were equally divided into 5 groups; corn oil was given orally to the control group; 25 mg kg-1 of BPA dissolved in corn oil was given orally to the second group. All other groups received the same dose of BPA and different doses of B (5, 10, and 20 mg kg-1 per day, respectively) orally for 30 days. The administration of BPA significantly decreased glutathione levels and increased malondialdehyde levels in rat tissues. Furthermore, BPA treatment reduced the catalase and superoxide dismutase activities in tissues and erythrocytes. Also, mRNA expression levels of TNF-α, IL-1ß, and IL-6 in the brain, liver, and testes of rats were augmented, whereas IL-10 was decreased with BPA treatment. Besides, BPA treatment adversely altered biochemical parameters and caused damage to the cell integrity of rat tissues. However, B administration reversed BPA-induced alterations in rat tissues in a dose-dependent manner. Furthermore, B exhibited antioxidant and anti-inflammatory effects and regulated metabolic and histopathological alterations in male Wistar albino rats exposed to BPA.
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Arsenic, an element found in nature, causes hazardous effects on living organisms. Meanwhile, natural compounds exhibit protective effects against hazardous substances. This study evaluated the effects of boron against arsenic-induced genotoxicity and altered biochemical parameters in rats. Thirty-five male Wistar albino rats were equally divided into five groups, and the experimental period lasted 30 days. One group was used as the control, and another group was treated with 100 mg/L arsenic in drinking water. The other groups were orally treated with 5, 10, and 20 mg/kg boron plus arsenic (100 mg/L via drinking water). Arsenic caused changes in biochemical parameters, total oxidant/antioxidant status, and DNA damage in mononuclear leukocytes. Moreover, it increased IFN-γ, IL-1ß, TNF-α, and NFκB mRNA expression levels in rat tissue. However, boron treatment improved arsenic-induced alterations in biochemical parameters and increases in DNA damage and proinflammatory cytokine gene expressions.
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Intoxicación por Arsénico , Arsénico/toxicidad , Boro/farmacología , Citocinas/biosíntesis , Daño del ADN , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Intoxicación por Arsénico/tratamiento farmacológico , Intoxicación por Arsénico/metabolismo , Intoxicación por Arsénico/patología , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Masculino , Oxidación-Reducción/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Acrylamide (ACR) is a hazardous substance associated with the accumulation of excessive reactive oxygen species and causes oxidative stress. Presence of ACR in foods leads to public health concerns due to its known neurotoxic, genotoxic, and carcinogenic effects. The present study investigated the ameliorative effects of boron (B) against ACR exposed rats. Forty Wistar albino male rats, fed with low-boron diet, were randomly and equally allocated into 5 groups. The control group was orally treated with physiological saline as placebo, the second group was orally given 15â¯mg/kg ACR. The other groups were orally treated with 15â¯mg/kg ACR and B at the levels of 5, 10, and 20â¯mg/kg/day for 60 days, respectively. ACR-treatment significantly increased malondialdehyde levels whereas decreased glutathione levels in rat tissues. Also, ACR-treatment increased the activities of superoxide dismutase and catalase in erythrocytes and tissues. Meanwhile, mRNA expression levels of NFĸB, IFN-γ, IL-1ß, and TNF-α in liver and brain of rats were increased under ACR treatment. Additionally, ACR caused a significant decrease in the level of high-density lipoprotein, with increase in the levels of low-density lipoprotein, triglyceride, cholesterol, glucose, urea nitrogen, and creatinine. Lastly, B alleviated histopathological alterations induced by ACR in rat tissues.
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Acrilamida/toxicidad , Boro/farmacología , Inflamación/prevención & control , Estrés Oxidativo/efectos de los fármacos , Alimentación Animal/análisis , Animales , Antioxidantes/metabolismo , Boro/análisis , Catalasa/metabolismo , Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
The present study was considered to evaluate the protective effect of taurine on malathion-induced toxicity in rats. Totally, 48 male rats were divided into 6 equal groups: 0.5ml physiological salt solution was given orally to control rats. 0.5ml corn oil was given orally to rats in corn oil group. Malathion at dose of 27mg/kg (1/50 of LD50) was dissolved in 0.5ml corn oil and given to orally rats in malathion group. The other groups; malathion (27mg/kg) and taurine (dissolved in 0.5ml physiological salt solution) at dose of 50, 100, and 200mg/kg were given orally to rats for 30days, respectively. Malathion treatment decreased acetylcholinesterase levels in serum (30%) and liver (25%) compared to the control group. Malathion resulted in a significant increase in malondialdehyde levels whereas decreased glutathione levels, superoxide dismutase, and catalase activities in rats. Also, IF-γ, IL1-ß, TNF-α, and NFĸB mRNA expression levels were found to be increased 5, 1.7, 2.3, and 2.5 fold in malathion treated rats compared to control, respectively. However, treatment of taurine, in a dose-dependent manner, resulted in a reversal of malathion-induced lipid peroxidation, antioxidant enzyme activities, and mRNA expression levels of proinflammatory cytokines. Moreover, taurine demonstrated preventive action against malathion-induced histopathological changes in rat tissues. In conclusion, taurine exhibited a protective effect in rats against malathion-induced lipid peroxidation, besides it ameliorated antioxidant status, decreased mRNA expression levels of proinflammatory cytokine and repaired rat tissues.
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Citocinas/antagonistas & inhibidores , Mediadores de Inflamación/antagonistas & inhibidores , Peroxidación de Lípido/efectos de los fármacos , Malatión/toxicidad , Estrés Oxidativo/efectos de los fármacos , Taurina/farmacología , Animales , Inhibidores de la Colinesterasa/toxicidad , Citocinas/biosíntesis , Citocinas/genética , Expresión Génica , Mediadores de Inflamación/metabolismo , Peroxidación de Lípido/fisiología , Masculino , Estrés Oxidativo/fisiología , Ratas , Ratas WistarRESUMEN
Background/aim: Nasal polyposis is a chronic inflammatory disease affecting the paranasal sinuses and nasal mucosae. It is thought that genetic and molecular mechanisms in inflammatory and apoptotic pathways are the main factors in the etiopathogenesis of nasal polyposis. The aim of this study was to investigate the expression patterns of CD11b, galectin-1, beclin-1, and caspase-3 in nasal polyps.Materials and methods: The mRNA expression levels of CD11b, galectin-1, beclin-1, and caspase-3 protein and western blot analysis of caspase-3 protein were evaluated in inferior turbinate mucosae and nasal polyp tissues.Results: CD11b expression was markedly higher in nasal polyp tissues when compared to turbinate mucosae (5.5 times higher, P < 0.05). Expression of galectin-1 was not statistically higher in nasal polyp tissues when compared to the controls. Beclin-1 expression in nasal polyp tissues was lower than in controls (17 times lower, P < 0.05). Caspase-3 expression was significantly lower in nasal polyp tissues than in controls (5.5 times lower, P < 0.05).Conclusion: Inflammation, apoptosis, and hyperproliferation are the major cellular processes in nasal polyposis and these proteins may take part and play some important roles in formation of this disease and the targeting of new treatment protocols.
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Beclina-1/metabolismo , Antígeno CD11b/metabolismo , Caspasa 3/metabolismo , Galectina 1/metabolismo , Mucosa Nasal/patología , Pólipos Nasales/metabolismo , Adulto , Apoptosis , Western Blotting , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pólipos Nasales/patología , ARN Mensajero , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The proteasome inhibitor bortezomib is a promising novel agent in bladder cancer therapy; however, inducible cytoprotective mechanisms may limit its potential efficacy. To date, the cellular and molecular effects of proteasome inhibitors on bladder cancer cells have been poorly characterized. Despite the consistent rate of initial responses, cisplatin treatment typically results in the development of chemoresistance, leading to therapeutic failure. Therefore, the present study aimed to characterize the molecular mechanisms underlying the anti-proliferative effects of cisplatin and bortezomib combination therapy on the human T24 bladder cancer cell line, by analyzing the protein expression levels of apoptotic genes. Cytotoxic effects were measured using a water-soluble tetrazolium salt-1 assay, and the apoptosis-associated molecules were examined using western blot analysis and ELISA. It was observed that combined administration of cisplatin and bortezomib induced upregulation of caspase-3, -8 and -9, B-cell lymphoma-2 (Bcl-2)-like 11 and Bcl-2-interacting killer, but downregulated Bcl-2 and Bcl-extra large protein expression levels in T24 cells in a dose-dependent manner. Furthermore, enhanced protein expression of caspase-8 and -9, in line with the significantly increased caspase-3 activation, was detected when the cells were treated with a combination of cisplatin and bortezomib, compared with that of either agent alone. Bortezomib appeared to synergize with cisplatin to promote apoptosis via the extrinsic and intrinsic apoptotic pathways. Taken together, the results of the current study provide the preclinical framework for additional evaluation of the effects of combining bortezomib with other agents to induce apoptosis in bladder cancer cells.
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OBJECTIVE: To understand whether cerebrovascular events, a major complication of atherosclerosis, are associated with any specific DNA methylation changes in the carotid plaque. APPROACH AND RESULTS: We profiled the DNA methylomes of human symptomatic carotid plaques obtained from patients who had cerebrovascular events (n=19) and asymptomatic counterparts (n=19) with a high-density microarray (≈485 000 CpG sites, Illumina), and crossed DNA methylation data with RNAseq-based expression data from an independent symptomatic carotid plaque set (n=8). Few (30) CpGs showed a significant (P<0.05; absolute Delta-Beta, >0.20) differential methylation between the 2 groups. Within symptomatic carotid plaques, DNA methylation correlated significantly with postcerebrovascular event time (range, 3-45 days; r-value range, -0.926 to 0.857; P<0.05) for ≈45 000 CpGs, the vast majority of which became hypomethylated with increasing postcerebrovascular event time. Hypomethylation was not due to erasure of the gene-body and CG-poor region hypermethylation that accompany the progression of stable lesions, but rather targeted promoters and CpG islands. Noticeably, promoter hypomethylation and increased expression of genes involved in the inhibition of the inflammatory response, defense against oxidative stress, and active DNA demethylation were observed with increasing postcerebrovascular event time. Concomitantly, histological changes consistent with phagocyte-driven plaque healing were observed. CONCLUSIONS: Weak changes in the DNA methylome distinguish symptomatic from asymptomatic plaques, but a widespread demethylation resulting in permissive transcriptional marks at atheroprotective gene promoters is established in plaques after a cerebrovascular event, thus mirroring previous observations that ruptured plaques tend to revert to a stable structure. The identified loci are candidate targets to accelerate the pace of carotid plaque stabilization.
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Amaurosis Fugax/genética , Arterias Carótidas/patología , Estenosis Carotídea/genética , Metilación de ADN , Epigénesis Genética , Ataque Isquémico Transitorio/genética , Placa Aterosclerótica , Accidente Cerebrovascular/genética , Amaurosis Fugax/diagnóstico , Enfermedades Asintomáticas , Estenosis Carotídea/complicaciones , Estenosis Carotídea/diagnóstico , Islas de CpG , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Ataque Isquémico Transitorio/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Regiones Promotoras Genéticas , Rotura Espontánea , Accidente Cerebrovascular/diagnóstico , Transcripción GenéticaRESUMEN
BACKGROUND: Atherosclerosis severity-independent alterations in DNA methylation, a reversible and highly regulated DNA modification, have been detected in aortic atheromas, thus supporting the hypothesis that epigenetic mechanisms participate in the pathogenesis of atherosclerosis. One yet unaddressed issue is whether the progression of atherosclerosis is associated with an increase in DNA methylation drift in the vascular tissue. The purpose of the study was to identify CpG methylation profiles that vary with the progression of atherosclerosis in the human aorta. METHODS: We interrogated a set of donor-matched atherosclerotic and normal aortic samples ranging from histological grade III to VII, with a high-density (>450,000 CpG sites) DNA methylation microarray. RESULTS: We detected a correlation between histological grade and intra-pair differential methylation for 1,985 autosomal CpGs, the vast majority of which drifted towards hypermethylation with lesion progression. The identified CpG loci map to genes that are regulated by known critical transcription factors involved in atherosclerosis and participate in inflammatory and immune responses. Functional relevance was corroborated by crossing the DNA methylation profiles with expression data obtained in the same human aorta sample set, by a transcriptome-wide analysis of murine atherosclerotic aortas and from available public databases. CONCLUSIONS: Our work identifies for the first time atherosclerosis progression-specific DNA methylation profiles in the vascular tissue. These findings provide potential novel markers of lesion severity and targets to counteract the progression of the atheroma.
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Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Metilación de ADN , Animales , Análisis por Conglomerados , Islas de CpG , Bases de Datos Genéticas , Progresión de la Enfermedad , Epigénesis Genética , Humanos , Sistema Inmunológico , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Placa Aterosclerótica/patología , Factores de Transcripción/metabolismoRESUMEN
DNA methylation is considered as one of the most important epigenetic mechanisms and it is catalyzed by DNA methyltransferases (DNMTs). DNMT1 abundance has been frequently seen in urogenital system tumors but the reasons for this abundance are not well understood. We aimed to look into the effects of Wnt/ß-catenin signaling pathway on overexpression of DNMT1 and aberrant expression of UHRF1 and HAUSP which are responsible for stability of DNMT1 at transcriptional and protein levels in urogenital cancers. In this context, firstly, Wnt/ß-catenin signaling pathway was activated by using SB216763 which is a glycogen synthase kinase-3 (GSK3) ß inhibitor. Cell proliferation levels in bladder cancer cells, renal cell carcinoma, and prostate cancer cells treated with GSK3ß inhibitor (SB216763) were detected by WST-1 reagent. WIF-1 gene methylation profile was determined by methylation-specific PCR (MSP); expression levels of target genes ß-catenin and WIF-1 by real-time PCR; and protein levels of ß-catenin, DNMT1, pGSK3ß(Ser9), HAUSP, and UHRF1 by Western Blot. Our results indicated that treatment with SB216763 caused an increased cell proliferation at low dose. mRNA levels of ß-catenin increased after treatment with SB216273 and protein levels of pGSK3ß(Ser9), ß-catenin, and DNMT1 increased in comparison to control. HAUSP and UHRF1 were either up-regulated or down-regulated at the same doses depending on the type of cancer. Also, we showed that protein levels of DNMT1, ß-catenin, HAUSP, and UHRF1 decreased after re-expression of WIF-1 following treatment with DAC. In Caki-2 cells, ß-catenin pathway might have accounted for the stability of DNMT1 expression, whereas such relation is not valid for T24 and PC3 cells. Our findings may offer a new approach for determination of molecular effects of Wnt/ß-catenin signal pathway on DNMT1. This may allow us to identify new molecular targets for the treatment of urogenital cancers.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Humanos , Indoles/farmacología , Maleimidas/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas , Peptidasa Específica de Ubiquitina 7 , Neoplasias de la Vejiga Urinaria/enzimología , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Death-associated protein kinase-1 (DAPK1) is a pro-apoptotic gene that induces cellular apoptosis in response to internal and external apoptotic stimulants. The silencing of DAPK1 can result in uncontrolled cell proliferation, indicating that it may have a role in tumor suppression. DAPK1 activity can be inhibited by the cytosine methylation that occurs in its promoter region. These methylation changes in the promoter region of DAPK1 have been reported in a range of solid and hematological malignancies. In the present study, DAPK1 methylation was investigated in chronic myeloid leukemia patients (n=43) using bisulfite conversion followed by methylation-specific polymerase chain reaction. The present study included a number of patients who were identified to be resistant to the common chemotherapeutic agent imatinib (STI571, Gleevec®, Glivec®), exhibiting at least one mutation in the breakpoint cluster region-Abelson murine leukemia (BCR-ABL) gene. Thus, the patients in the present study were divided into two groups according to their response to imatinib therapy: Non-resistant (n=26) and resistant (n=17) to imatinib. Resistant patients were characterized by the presence of single or multiple mutations of the BCR-ABL gene: i) T315I, ii) M351T, iii) E255K, iv) T315I and M351T or v) T315I, M351T and E255K. The present study identified that: i) The incidence of DAPK1 methylation was significantly higher in the resistant patients compared with the non-resistant patients; ii) the extent of resistance varied between mutation types; and iii) there was no DAPK1 methylation in any of the healthy controls. These findings indicate that DAPK1 methylation may be associated with a signaling pathway for imatinib resistance in chronic myeloid leukemia.
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BACKGROUND: Combination treatments of botulinum toxin type-A and other rejuvenation agents or instruments are gradually becoming more popular. After observing a high incidence of therapy failure following simultaneous applications of botulinum toxin type-A and platelet-rich plasma mesotherapy, we aimed to investigate whether PRP has an inhibitory effect on botulinum toxin type-A. METHODS: Twenty-four New Zealand white rabbits were divided into 4 groups, and the anterior auricular muscle and overlying skin were used for injections. Groups I and II both received onabotulinumtoxinA intramuscular injections. In addition, autologous platelet-rich plasma mesotherapy was performed in Group I while Group II received saline mesotherapy. Group III was designed as the in vitro mixture group in which onabotulinumtoxinA and platelet-rich plasma were mixed and then administered intramuscularly. Group IV received saline within the mixture instead of platelet-rich plasma. The contralateral ears of all the rabbits served as control and were only treated with onabotulinumtoxinA. Visual evaluation of ear positions and electroneuromyographic studies were done prior to all procedures and at day 14. Anterior auricular muscles were harvested at day 14 and were evaluated with quantitative real-time PCR. RESULTS: Visual and electroneuromyographic studies revealed less onabotulinumtoxinA activity in Groups I and III. When platelet-rich plasma was administered through skin mesotherapy, onabotulinumtoxinA activity failure was more severe in comparison with direct contact. No significant difference in SNAP-25 mRNA expression through quantitative real-time PCR was observed between groups. CONCLUSION: Although we could not explain the exact mechanism underlying this interaction, platelet-rich plasma applications result in less onabotulinumtoxinA muscle paralysis activity.
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Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Fármacos Neuromusculares/antagonistas & inhibidores , Plasma Rico en Plaquetas , Animales , Masculino , ConejosRESUMEN
BACKGROUND: Epigenetic alterations may contribute to the development of atherosclerosis. In particular, DNA methylation, a reversible and highly regulated DNA modification, could influence disease onset and progression because it functions as an effector for environmental influences, including diet and lifestyle, both of which are risk factors for cardiovascular diseases. METHODS AND RESULTS: To address the role of DNA methylation changes in atherosclerosis, we compared a donor-matched healthy and atherosclerotic human aorta sample using whole-genome shotgun bisulfite sequencing. We observed that the atherosclerotic portion of the aorta was hypermethylated across many genomic loci in comparison with the matched healthy counterpart. Furthermore, we defined specific loci of differential DNA methylation using a set of donor-matched aortic samples and a high-density (>450 000 CpG sites) DNA methylation microarray. The functional importance in the disease was corroborated by crossing the DNA methylation signature with the corresponding expression data of the same samples. Among the differentially methylated CpGs associated with atherosclerosis onset, we identified genes participating in endothelial and smooth muscle functions. These findings provide new clues toward a better understanding of the molecular mechanisms of atherosclerosis. CONCLUSIONS: Our data identify an atherosclerosis-specific DNA methylation profile that highlights the contribution of different genes and pathways to the disorder. Interestingly, the observed gain of DNA methylation in the atherosclerotic lesions justifies efforts to develop DNA demethylating agents for therapeutic benefit.
Asunto(s)
Aorta/metabolismo , Aterosclerosis/genética , Mapeo Cromosómico/métodos , Metilación de ADN , Aorta/patología , Análisis por Conglomerados , Islas de CpG , Dieta , Progresión de la Enfermedad , Epigénesis Genética , Femenino , Regulación de la Expresión Génica , Humanos , Estilo de Vida , Factores de Riesgo , Sulfitos/químicaRESUMEN
To investigate the differences in the mRNA and protein expression levels of vascular endothelial growth factor (VEGF) in murine retina between mice subjected to conventional laser (AG) and those subjected to Pattern Scan Laser (PASCAL) system. Male C57BL/6 mice were randomly assigned to one of three groups: Group 1 (G1) receiving retinal scatter laser photocoagulation using with AG photocoagulator (n=16), Group 2 (G2) receiving retinal scatter laser photocoagulation using with PASCAL (n=16) and Group 3 (G3) served as an untreated control group (n=6). Molecular and morphological analyses of VEGF were performed on days 1, 2 and 5 by ELISA, real-time PCR and immuno-histochemical analysis. In samples which underwent AG (G1), when compared with the control group (G3), VEGF mRNA level increased 2.4 folds on day 2, whereas it decreased on day 5 (pâ¡0.001). In samples which underwent PASCAL (G2), on the other hand, VEGF mRNA level increased 1.8 folds on day 1 and 2.2 folds on day 5 when compared with the control group (G3). In samples which underwent AG (G1), when compared with the control group (G3), VEGF protein level increased significantly on day 2, whereas it decreased on day 5 (pâ¡0.001). In group G2, the VEGF levels in the sensory retina significantly increased as compared to control groups at both 2 and 5 days after laser photocoagulation using PASCAL laser (p=0.012, both time points). The peak expressions of VEGF protein in samples which underwent PASCAL and conventional laser were found on day 5 and day 2 respectively. In retinas of PASCAL-treated mice, VEGF immunoreactivity gradually increased during the 5-day follow-up. However, in argon laser group, the strongest VEGF immunoreactivity was detected on day 2, then started to decrease on day 5. In summary, the expression of VEGF protein and mRNA gradually increase during a 5-day follow-up period in PASCAL-treated mouse eyes, whereas in AG group they reach their peak levels on the second day of follow-up and started decreasing after then. These results may also explain why the PASCAL system is less effective in regressing neovascularization in the clinic.
Asunto(s)
Coagulación con Láser/efectos adversos , Coagulación con Láser/instrumentación , Retina/cirugía , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Argón , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Retina/metabolismo , Factores de TiempoRESUMEN
The epigenetic suppression of Wnt antagonists (sFRPs, DKKs, and WIF-1) causes the activation of both ß-catenin and target genes, which play an important role in cell proliferation, metastasis, and angiogenesis. This study is aimed to investigate, on transcriptional and protein levels, the synergic effects of unaccompanied and/or combined use of 5-aza-2'-deoxycytidine (DAC, 5-aza-dC), trichostatin A (TSA), and gemcitabine+cisplatin chemotherapeutic agents on the apoptotic pathway of human bladder cancer cell line T24. The anti-tumor effects of gemcitabine (0-500 nM), cisplatin (0-10 µM), DAC (10 µM), and TSA (300 nM) alone and/or together on T24 cells were determined by WST-1. ELISA method was used to analyze the effects of unaccompanied and combined use of gemcitabine+cisplatin, DAC, and TSA on cell proliferation and determine the cytotoxic and apoptotic dosages at the level of H3 histone acetylation. Methylation-specific PCR was used to evaluate methylation profiles of Wnt antagonist gene (WIF-1). In the case of unaccompanied and/or combined use of specified drugs, the variations in the expression levels of CTNNB1, GSK3ß, c-MYC, CCND1, CASP-3, CASP-8, CASP-9, BCL2L1, and WIF-1 genes were determined by quantitative real-time PCR. Our results indicate that through inhibition of DNA methylation, expression of ß-catenin and Wnt antagonist re-activation and expressions of canonical Wnt/ß-catenin pathway target genes, c-myc and cyclin D1 (CCND1), have decreased. In addition, DAC, TSA, and gemcitabine+cisplatin combination caused an increase in GSK3ß mRNA levels, which in turn significantly decreased CCND1 mRNA levels. Moreover, BCL2L1, an anti-apoptotic gene, was downregulated significantly. Meanwhile, both CASP-3 mRNA and active caspase-3 protein levels increased with respect to control (p<0.01). The results revealed that use of quadruplicate gemcitabine+cisplatin+DAC+TSA combination led to a reduced inhibition of canonical Wnt/ß-catenin pathway and reduced cell proliferation. Our findings may offer a new approach to consider in the treatment of bladder cancer.