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Dysregulation of epigenetic mechanisms has been recognized to play a crucial role in cancer development, but these mechanisms vary between sexes. Therefore, we focused on sex-specific differences in the context of cancer-based data from a recent study. A total of 12 cell-free DNA methylation targets in CpG-rich promoter regions and 48 miRNAs were analyzed by qPCR in plasma samples from 8 female and 7 male healthy controls as well as 48 female and 80 male subjects with solid tumors of the bladder, brain, colorectal region (CRC), lung, stomach, pancreas, and liver. Due to the small sample size in some groups and/or the non-balanced distribution of men and women, sex-specific differences were evaluated statistically only in healthy subjects, CRC, stomach or pancreas cancer patients, and all cancer subjects combined (n female/male-8/7, 14/14, 8/15, 6/6, 48/80, respectively). Several miRNAs with opposing expressions between the sexes were observed for healthy subjects (miR-17-5p, miR-26b-5p); CRC patients (miR-186-5p, miR-22-3p, miR-22-5p, miR-25-3p, miR-92a-3p, miR-16-5p); stomach cancer patients (miR-133a-3p, miR-22-5p); and all cancer patients combined (miR-126-3p, miR-21-5p, miR-92a-3p, miR-183-5p). Moreover, sex-specific correlations that were dependent on cancer stage were observed in women (miR-27a-3p) and men (miR-17-5p, miR-20a-5p). Our results indicate the complex and distinct role of epigenetic regulation, particularly miRNAs, depending not only on the health status but also on the sex of the patient. The same miRNAs could have diverse effects in different tissues and opposing effects between the biological sexes, which should be considered in biomarker research.
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Background: Regular, especially sustained exercise plays an important role in the prevention and treatment of multiple chronic diseases. Some of the underlying molecular and cellular mechanisms behind the adaptive response to physical activity are still unclear, but recent findings suggest a possible role of epigenetic mechanisms, especially miRNAs, in the progression and management of exercise-related changes. Due to the combination of the analysis of epigenetic biomarkers (miRNAs), the intake of food and supplements, and genetic dispositions, a "fitness score" was evaluated to assess the individual response to nutrition, exercise, and metabolic influence. Methods: In response to a 12-week sports intervention, we analyzed genetic and epigenetic biomarkers in capillary blood from 61 sedentary, healthy participants (66.1% females, 33.9% males, mean age 33 years), including Line-1 methylation, three SNPs, and ten miRNAs using HRM and qPCR analysis. These biomarkers were also analyzed in a healthy, age- and sex-matched control group (n, 20) without intervention. Food frequency intake, including dietary supplement intake, and general health questionnaires were surveyed under the supervision of trained staff. Results: Exercise training decreased the expression of miR-20a-5p, -22-5p, and -505-3p (p < 0.02) and improved the "fitness score," which estimates eight different lifestyle factors to assess, nutrition, inflammation, cardiovascular fitness, injury risk, regeneration, muscle and hydration status, as well as stress level. In addition, we were able to determine correlations between individual miRNAs, miR-20a-5p, -22-5p, and -101-3p (p < 0.04), and the genetic predisposition for endurance and/or strength and obesity risk (ACE, ACTN3, and FTO), as well as between miRNAs and the body composition (p < 0.05). MiR-19b-3p and -101-3p correlated with the intake of B vitamins. Further, miR-19b-3p correlated with magnesium and miR-378a-3p with iron intake (p < 0.05). Conclusions: In summary, our results indicate that a combined analysis of several biomarkers (miRNAs) can provide information about an individual's training adaptions/fitness, body composition, nutritional needs, and possible recovery. In contrast to most studies using muscle biopsies, we were able to show that these biomarkers can also be measured using a minimally invasive method.
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MicroARNs , Actinina/metabolismo , Adulto , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Biomarcadores , Composición Corporal , Dieta , Ejercicio Físico/fisiología , Femenino , Humanos , Masculino , MicroARNs/genéticaRESUMEN
Healthy mitochondria and their epigenetic control are essential to maintaining health, extending life expectancy, and improving cardiovascular performance. Strategies to maintain functional mitochondria during aging include training; cardiovascular exercise has been suggested as the best method, but strength training has also been identified as essential to health and healthy aging. We therefore investigated the effects of concurrent exercise training and dietary habits on epigenetic mechanisms involved in mitochondrial (mt) functions and biogenesis. We analyzed epigenetic biomarkers that directly target the key regulator of mitochondrial biogenesis, PGC-1α, and mtDNA content. Thirty-six healthy, sedentary participants completed a 12-week concurrent training program. Before and after the intervention, dried blood spot samples and data on eating habits, lifestyle, and body composition were collected. MiR-23a, miR-30e expression, and mtDNA content were analyzed using real-time quantitative polymerase chain reaction (qPCR) analysis. PGC-1α methylation was analyzed using bisulfite pyrosequencing. MiR-23a, miR-30e expression, and PGC-1α methylation decreased after the intervention (p < 0.05). PGC-1α methylation increased with the consumption of red and processed meat, and mtDNA content increased with the ingestion of cruciferous vegetables (p < 0.05). Our results indicate that concurrent training could improve mitochondrial biogenesis and functions by altering the epigenetic regulation. These alterations can also be detected outside of the skeletal muscle and could potentially affect athletic performance.
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Liquid biopsy-based tests emerge progressively as an important tool for cancer diagnostics and management. Currently, researchers focus on a single biomarker type and one tumor entity. This study aimed to create a multi-analyte liquid biopsy test for the simultaneous detection of several solid cancers. For this purpose, we analyzed cell-free DNA (cfDNA) mutations and methylation, as well as circulating miRNAs (miRNAs) in plasma samples from 97 patients with cancer (20 bladder, 9 brain, 30 breast, 28 colorectal, 29 lung, 19 ovarian, 12 pancreas, 27 prostate, 23 stomach) and 15 healthy controls via real-time qPCR. Androgen receptor p.H875Y mutation (AR) was detected for the first time in bladder, lung, stomach, ovarian, brain, and pancreas cancer, all together in 51.3% of all cancer samples and in none of the healthy controls. A discriminant function model, comprising cfDNA mutations (COSM10758, COSM18561), cfDNA methylation markers (MLH1, MDR1, GATA5, SFN) and miRNAs (miR-17-5p, miR-20a-5p, miR-21-5p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-92a-3p, miR-101-3p, miR-133a-3p, miR-148b-3p, miR-155-5p, miR-195-5p) could further classify healthy and tumor samples with 95.4% accuracy, 97.9% sensitivity, 80% specificity. This multi-analyte liquid biopsy-based test may help improve the simultaneous detection of several cancer types and underlines the importance of combining genetic and epigenetic biomarkers.
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Obesity- or diabetes-induced oxidative stress is discussed as a major risk factor for DNA damage. Vitamin E and many polyphenols exhibit antioxidative activities with consequences on epigenetic regulation of inflammation and DNA repair. The present study investigated the counteraction of oxidative stress by vitamin E in the colorectal cancer cell line Caco-2 under normal (1 g/l) and high (4.5 g/l) glucose cell culture condition. Malondialdehyde (MDA) as a surrogate marker of lipid peroxidation and reactive oxygen species (ROS) was analyzed. Gene expression and promoter methylation of the DNA repair gene MutL homolog 1 (MLH1) and the DNA methyltransferase 1 (DNMT1) as well as global methylation by LINE-1 were investigated. Results revealed a dose-dependent counteracting effect of vitamin E on H2O2-induced oxidative stress. Thereby, 10 µM vitamin E proved to be more efficient than did 50 µM in reducing MDA. Further, an induction of MLH1 and DNMT1 gene expression was noticed, accompanied by an increase in global methylation. Whether LINE-1 hypomethylation is a cause or effect of oxidative stress is still unclear. In conclusion, supplementation of exogenous antioxidants like vitamin E in vitro exhibits beneficial effects concerning oxidative stress as well as epigenetic regulation involved in DNA repair.
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ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/efectos de los fármacos , Homólogo 1 de la Proteína MutL/genética , Estrés Oxidativo/efectos de los fármacos , Vitamina E/farmacología , Células CACO-2 , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/biosíntesis , Relación Dosis-Respuesta a Droga , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucosa/administración & dosificación , Glucosa/metabolismo , Humanos , Elementos de Nucleótido Esparcido Largo , Homólogo 1 de la Proteína MutL/biosíntesis , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Therapy of malignant glioma relies on treatment with the O6 -methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6 -alkylguanine. Since this damage is repaired by O6 -methylguanine-DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high-grade gliomas. Although MGMT has been shown to be transcriptionally up-regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up-regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up-regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up-regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level.
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Antineoplásicos Alquilantes/farmacología , Enzimas Reparadoras del ADN/genética , Glucocorticoides/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/genética , Factor de Transcripción Sp1/genética , Temozolomida/farmacología , Enzimas Reparadoras del ADN/efectos de los fármacos , Enzimas Reparadoras del ADN/efectos de la radiación , Dexametasona/farmacología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de la radiación , Técnicas de Silenciamiento del Gen , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/efectos de los fármacos , O(6)-Metilguanina-ADN Metiltransferasa/efectos de la radiación , Regiones Promotoras Genéticas/genética , ARN Mensajero/farmacología , Factor de Transcripción Sp1/efectos de los fármacos , Factor de Transcripción Sp1/efectos de la radiación , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
XAF1 (X-linked inhibitor of apoptosis (XIAP)-associated factor 1) is a tumor suppressor that counteracts the anti-apoptotic effects of XIAP and can sensitize cells to cell death triggering events. XAF1 knockdown abrogated the temozolomide (TMZ)-induced G2-arrest and prevented TMZ-induced apoptosis in the glioblastoma (GB) cell line LN229. Promoter methylation of XAF1 was found to be inversely correlated with mRNA expression in GB cells. We analyzed XAF1 methylation in a panel of 16 GB cell lines and 80 patients with first-diagnosed WHO grade III/IV high-grade gliomas using methylation-sensitive high-resolution melt (MS-HRM) analysis. In those patients, XAF1 promoter methylation was strongly associated with enhanced progression free and overall survival. Interestingly, XAF1 promoter methylation was strictly correlated with the occurrence of IDH1 mutations, indicating a causal link to the IDH1 mutant phenotype. XAF1 methylation was observed in 18 grade III tumors all of which showed heterozygous mutations in the IDH1 gene. 17 harbored a mutation leading to an arginine > histidine (R132H) and one carried a mutation causing an arginine > glycine (R132G) substitution. Furthermore, six out of six recurrent and IDH1 mutated grade III tumors also showed XAF1 promoter methylation. The data demonstrate that XAF1 promoter methylation determined by MS-HRM is a robust and precise indicator of IDH1 mutations in grade III gliomas. It is useful for complementing the immunohistochemistry-based detection of mutant IDH, uncovering rare 2-HG-producing IDH1 and potentially IDH2 mutations. The MS-HRM-based detection of XAF1 methylation could therefore be a reliable tool in assisting the sub-classification of high-grade gliomas.
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Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/mortalidad , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Isocitrato Deshidrogenasa/genética , Mutación/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Anciano , Apoptosis , Proteínas Reguladoras de la Apoptosis , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Proliferación Celular , Metilación de ADN , Femenino , Silenciador del Gen , Glioma/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Clasificación del Tumor , Proteínas de Neoplasias/genética , Pronóstico , Regiones Promotoras Genéticas , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
BACKGROUND: The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. RESULTS: We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. CONCLUSIONS: Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.
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Metilación de ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Glioblastoma/terapia , Glioma/terapia , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Proteínas Supresoras de Tumor/genética , Anciano , Anciano de 80 o más Años , Femenino , Glioblastoma/genética , Glioblastoma/patología , Glioma/genética , Glioma/patología , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/economía , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/economía , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy (hypotonia, absent tendon reflexes etc.) from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes (MEST, SNRPN, LIT1, H19, GTL2 and PEG3) as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status.
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Elementos Alu , Errores Innatos del Metabolismo de los Aminoácidos/genética , Metilación de ADN , Impresión Genómica , Glicina N-Metiltransferasa/deficiencia , Elementos de Nucleótido Esparcido Largo , Errores Innatos del Metabolismo de los Aminoácidos/sangre , Creatina/sangre , Femenino , Glicina N-Metiltransferasa/sangre , Glicina N-Metiltransferasa/genética , Humanos , Lactante , Recién Nacido , MasculinoRESUMEN
BACKGROUND: Oxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1) gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS), we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2) and impaired fasting glucose (IFG). METHODS: In this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA) and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student's two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount. RESULTS: The intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate. CONCLUSION: This vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a result of the ROS scavenging antioxidant rich diet, leading to lower activity of DNA demethylating enzymes. Our results suggest the hypothesis of CpG demethylation via DNA repair enzymes under these circumstances. NIDDM2 and IFG patients benefit from this simple dietary intervention involving epigenetic and DNA repair mechanisms.
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BACKGROUND: We investigated whether chemotherapy with the presence or absence of antibiotics against different kinds of cancer changed the gastrointestinal microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Feces of 17 ambulant patients receiving chemotherapy with or without concomitant antibiotics were analyzed before and after the chemotherapy cycle at four time points in comparison to 17 gender-, age- and lifestyle-matched healthy controls. We targeted 16S rRNA genes of all bacteria, Bacteroides, bifidobacteria, Clostridium cluster IV and XIVa as well as C. difficile with TaqMan qPCR, denaturing gradient gel electrophoresis (DGGE) fingerprinting and high-throughput sequencing. After a significant drop in the abundance of microbiota (pâ=â0.037) following a single treatment the microbiota recovered within a few days. The chemotherapeutical treatment marginally affected the Bacteroides while the Clostridium cluster IV and XIVa were significantly more sensitive to chemotherapy and antibiotic treatment. DGGE fingerprinting showed decreased diversity of Clostridium cluster IV and XIVa in response to chemotherapy with cluster IV diversity being particularly affected by antibiotics. The occurrence of C. difficile in three out of seventeen subjects was accompanied by a decrease in the genera Bifidobacterium, Lactobacillus, Veillonella and Faecalibacterium prausnitzii. Enterococcus faecium increased following chemotherapy. CONCLUSIONS/SIGNIFICANCE: Despite high individual variations, these results suggest that the observed changes in the human gut microbiota may favor colonization with C. difficile and Enterococcus faecium. Perturbed microbiota may be a target for specific mitigation with safe pre- and probiotics.