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BACKGROUND: The phosphatase chronophin (CIN/PDXP) has been shown to be an important regulator of glioma cell migration and invasion. It has two known substrates: p-Ser3-cofilin, the phosphorylated form of the actin binding protein cofilin, and pyridoxal 5'-phosphate, the active form of vitamin B6. Phosphoregulation of cofilin, among other functions, plays an important role in cell migration, whereas active vitamin B6 is a cofactor for more than one hundred enzymatic reactions. The role of CIN has yet only been examined in glioblastoma cell line models derived under serum culture conditions. RESULTS: We found that CIN is highly expressed in cells cultured under non-adherent, serum-free conditions that are thought to better mimic the in vivo situation. Furthermore, the substrates of CIN, p-Ser3-cofilin and active vitamin B6, were significantly reduced as compared to cell lines cultured in serum-containing medium. To further examine its molecular role we stably knocked down the CIN protein with two different shRNA hairpins in the glioblastoma cell lines NCH421k and NCH644. Both cell lines did not show any significant alterations in proliferation but expression of differentiation markers (such as GFAP or TUBB3) was increased in the knockdown cell lines. In addition, colony formation was significantly impaired in NCH644. Of note, in both cell lines CIN knockdown increased active vitamin B6 levels with vitamin B6 being known to be important for S-adenosylmethionine biosynthesis. Nevertheless, global histone and DNA methylation remained unaltered as was chemoresistance towards temozolomide. To further elucidate the role of phosphocofilin in glioblastoma cells we applied inhibitors for ROCK1/2 and LIMK1/2 to our model. LIMK- and ROCK-inhibitor treatment alone was not toxic for glioblastoma cells. However, it had profound, but antagonistic effects in NCH421k and NCH644 under chemotherapy. CONCLUSION: In non-adherent glioblastoma cell lines cultured in serum-free medium, chronophin knockdown induces phenotypic changes, e.g. in colony formation and transcription, but these are highly dependent on the cellular background. The same is true for phenotypes observed after treatment with inhibitors for kinases regulating cofilin phosphorylation (ROCKs and LIMKs). Targeting the cofilin phosphorylation pathway might therefore not be a straightforward therapeutic option in glioblastoma.
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Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Fosfoproteínas Fosfatases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Vitamina B 6/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Meios de Cultura Livres de Soro , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Quinases Lim/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
BACKGROUND: Seventy percent of all bladder tumours tend to recur and need intensive surveillance, and a subset of tumours progress to muscle-invasive and metastatic disease. However, it is still difficult to find the adequate treatment for every individual patient as it is a very heterogeneous disease and reliable biomarkers are still missing. In our study we searched for new target genes in the critical chromosomal region 8p and investigated the potential tumour suppressor gene candidate MTUS1/ATIP in bladder cancer. METHODS: MTUS1 was identified to be the most promising deleted target gene at 8p in aCGH analysis with 19 papillary bladder tumours. A correlation with bladder cancer was further validated using immunohistochemistry of 85 papillary and 236 advanced bladder tumours and in functional experiments. Kaplan-Meier analysis and multivariate Cox-regression addressed overall survival (OS) and disease-specific survival (DSS) as a function of MTUS1/ATIP expression. Bivariate correlations investigated associations between MTUS1/ATIP expression, patient characteristics and histopathology. MTUS1 expression was analysed in cell lines and overexpressed in RT112, where impact on viability, proliferation and migration was measured. RESULTS: MTUS1 protein expression was lost in almost 50% of all papillary and advanced bladder cancers. Survival, however, was only influenced in advanced carcinomas, where loss of MTUS1 was associated with adverse OS and DSS. In this cohort, there was also a significant correlation of MTUS1 expression and histological subtype: positive expression was detected in all micropapillary tumours and aberrant nuclear staining was detected in a subset of plasmocytoid urothelial carcinomas. MTUS1 was expressed in all investigated bladder cell lines and overexpression in RT112 led to significantly decreased viability. CONCLUSIONS: MTUS1 is a tumour suppressor gene in cultured bladder cancer cells and in advanced bladder tumours. It might represent one new target gene at chromosome 8p and can be used as an independent prognostic factor for advanced bladder cancer patients. The limitation of the study is the retrospective data analysis. Thus, findings should be validated with a prospective advanced bladder tumour cohort.
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Carcinoma Papilar/metabolismo , Carcinoma de Células de Transição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Papilar/patologia , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 8 , Hibridização Genômica Comparativa , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Estudos Retrospectivos , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
Internal transformative qualities are essential contributing factors to sustainable behavior. Besides awareness, insight, purpose, and agency, connectedness is one of those inner qualities. In this study, we investigated the relationship between connectedness to oneself (self-love), towards the environment (connectedness to nature), towards other human beings (pro-socialness), and sustainable behavior towards clothes and food. One hundred thirty-nine mostly students participated. The results showed that self-love, connectedness to nature, and pro-socialness correlate. Sustainability behavior towards food was predicted by pro-socialness, the choice of diet, and environmental and ethical reasons for nutrition. Sustainable behavior towards clothes was predicted by connectedness to nature. This study hints that the factors of inner transformative qualities and the type of sustainable behavior must be investigated differently. It strengthens the multi-facet dimensions of sustainable behavior.
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We previously reported that DIRAS-3 is frequently inactivated in oligodendrogliomas due to promoter hypermethylation and loss of the chromosomal arm 1p. DIRAS-3 inactivation was associated with better overall survival. Consequently, we now investigated regulation and function of its family members DIRAS-1 and DIRAS-2. We found that DIRAS-1 was strongly downregulated in 65% and DIRAS-2 in 100% of analyzed glioma samples compared to non-neoplastic brain tissue (NNB). Moreover, a significant down-regulation of DIRAS-1 and -2 was detected in glioma data obtained from the TCGA database. Mutational analyses did not reveal any inactivating mutations in the DIRAS-1 and -2 coding regions. Analysis of the DIRAS-1 and -2 promoter methylation status showed significantly higher methylation in IDH-mutant astrocytic and IDH-mutant and 1p/19q-codeleted oligodendroglial tumors compared to NNB. Treatment of U251MG and Hs683 glioblastoma cells lines with 5-azacytidine led to significant re-expression of DIRAS-1 and -2. For IDH-wild-type primary gliomas, however, we did not observe significantly elevated DIRAS-1 and -2 promoter methylation levels, but still detected strong downregulation of both DIRAS family members. Additional analyses revealed that DIRAS-1 and -2 expression was also regulated by histone modifications. We observed a shift towards promoter heterochromatinization for DIRAS-1 and less promoter euchromatinization for DIRAS-2 in IDH-wild-type glioblastomas compared to controls. Treatment of the two glioblastoma cell lines with a histone deacetylase inhibitor led to significant re-expression of DIRAS-1 and -2. Functionally, overexpression of DIRAS-1 and -2 in glioblastoma cells translated into significantly higher sensitivity to lomustine treatment. Analyses of DNA damage markers revealed that DIRAS-1 and -2 may play a role in p53-dependent response to alkylating chemotherapy.
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OBJECTIVES: Mindfulness-based interventions in the context of sports have been shown to result in higher mindfulness scores and improved physiological and psychological parameters. The goal of this pilot study was to investigate the effects of a newly developed seven-session mindfulness-based intervention, mindful emotions, on German tennis players. METHODS: The study was conducted in a pre-post test design with an intervention and a control group. Before and after the mindfulness-based stress reduction training the following dependent measurements were assessed: athletic performance (serve accuracy) and psychological performance indicators (mindfulness, stress and competition anxiety). RESULTS: The results indicate a better performance of the intervention group for the difference scores between post-test and pre-test in one of the sport-related anxiety factors, the concentration disturbances index. CONCLUSION: In order to further investigate the effects of this mindfulness-based intervention in competitive sports, additional studies with a larger number of participants have to follow.
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It was the main goal of this study to investigate performance on the mental rotation test (MRT) in Brazilian and German adolescents. Mental rotation is the ability to mentally transform a three-dimensional stimulus in mind and relates to science education. 60 German and 60 Brazilian adolescents (76 males and 44 females, 11-17 years) completed the Mental Rotation Test, a physical activity and media use questionnaire and a Number Connection Test. The result showed no difference between Brazilian and German adolescents in the cognitive processing speed measurement. German adolescents are more active and show a less media use compared to the Brazilian adolescents. Furthermore, German adolescents demonstrate a better MRT performance than Brazilian ones, as well as boys show a better performance than girls do. A multiple regression analysis indicated that the MRT performance could be predicted by nationality, sex, and cognitive processing speed. Since cognitive processing speed did not differ between Brazilian and German adolescents, the worse MRT performance of the Brazilian adolescents could be explained by different educational systems. Further studies have to follow which investigate the reasons for the different nations in more detail.
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Glioblastoma (GBM) represents the most common and most malignant type of primary brain tumor and significantly contributes to cancer morbidity and mortality. Invasion into the healthy brain parenchyma is a major feature of glioblastoma aggressiveness. Reelin (RELN) is a large secreted extracellular matrix glycoprotein that regulates neuronal migration and positioning in the developing brain and sustains functionality in the adult brain. We here show that both RELN and its main downstream effector DAB1 are silenced in glioblastoma as compared to non-neoplastic tissue and mRNA expression is inversely correlated with malignancy grade. Furthermore, RELN expression is positively correlated with patient survival in two large, independent clinically annotated datasets. RELN silencing occurs via promoter hypermethylation as shown by both database mining and bisulfite sequencing of the RELN promoter. Consequently, treatment with 5'-Azacytidine and trichostatin A induced RELN expression in vitro. On the functional level, we found RELN to regulate glioblastoma cell migration both in a DAB1 (tyrosine phosphorylation)-dependent and -independent fashion, depending on the substrate provided. Moreover, stimulation of RELN signaling strongly reduced proliferation in glioblastoma cells. This phenotype depends on DAB1 stimulation by RELN, as a mutant that lacks all RELN induced tyrosine phosphorylation sites (DAB1-5F) failed to induce a growth arrest. Proteomic analyzes revealed that these effects are mediated by a reduction in E2F targets and dephosphorylation of ERK1/2. Taken together, our data establish a relevance of RELN signaling in glioblastoma pathology and thereby might unearth novel, yet unrecognized treatment options.
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Neoplasias Encefálicas/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Glioblastoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Simulação por Computador , Proteínas da Matriz Extracelular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Proteoma , RNA Mensageiro/metabolismo , Proteína Reelina , Serina Endopeptidases/genética , Transdução de Sinais , Adulto JovemRESUMO
Many glioblastoma patients suffer from seizures why they are treated with antiepileptic agents. Valproic acid (VPA) is a histone deacetylase inhibitor that apart from its anticonvulsive effects in some retrospective studies has been suggested to lead to a superior outcome of glioblastoma patients. However, the exact molecular effects of VPA treatment on glioblastoma cells have not yet been deciphered. We treated glioblastoma cells with VPA, recorded the functional effects of this treatment and performed a global and unbiased next generation sequencing study on the chromatin (ChIP) and RNA level. 1) VPA treatment clearly sensitized glioma cells to temozolomide: A protruding VPA-induced molecular feature in this context was the transcriptional upregulation/reexpression of numerous solute carrier (SLC) transporters that was also reflected by euchromatinization on the histone level and a reexpression of SLC transporters in human biopsy samples after VPA treatment. DNA repair genes were adversely reduced. 2) VPA treatment, however, also reduced cell proliferation in temozolomide-naive cells: On the molecular level in this context we observed a transcriptional upregulation/reexpression and euchromatinization of several glioblastoma relevant tumor suppressor genes and a reduction of stemness markers, while transcriptional subtype classification (mesenchymal/proneural) remained unaltered. Taken together, these findings argue for both temozolomide-dependent and -independent effects of VPA. VPA might increase the uptake of temozolomide and simultaneously lead to a less malignant glioblastoma phenotype. From a mere molecular perspective these findings might indicate a surplus value of VPA in glioblastoma therapy and could therefore contribute an additional ratio for clinical decision making.
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Neoplasias Encefálicas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Glioma/tratamento farmacológico , Ácido Valproico/farmacologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/química , Reparo do DNA/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Sistemas de Apoio a Decisões Clínicas , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Inibidores de Histona Desacetilases/farmacologia , Histonas/química , Humanos , RNA/análise , RNA Interferente Pequeno/metabolismo , Temozolomida , Transcrição Gênica , Resultado do TratamentoRESUMO
In this study, we determined the genotype distribution of two single nucleotide polymorphisms (SNPs) in secreted frizzled related protein 1 (SFRP1), rs3242 and rs921142, in a Caucasian bladder cancer case-control study. Allelic variants of the SNPs were determined using restriction fragment length polymorphism (RFLP) analysis and partly verified by sequencing analysis. Overall, DNA from 188 consecutive and 215 early-onset bladder cancer patients (≤45 years) as well as from 332 controls was investigated. Potential microRNA binding sites were determined for rs3242, and microRNA expression was analysed in cell lines and tumour specimens. We observed a remarkable distribution difference in rs3242 between bladder cancer patients and healthy controls (p=0.05). Additionally, we found a significant difference in genotype distribution (p=0.032), resulting from the difference of early-onset patients and the control group (p=0.007). The risk allele T showed increased frequency in the early-onset patient group (p=0.002). Genotype-dependent differences of microRNA binding capacity were predicted in SFRP1 mRNA for two microRNAs. Hsa-miR-3646 showed strong expression in cell lines and tumour tissue, whereas hsa-miR-603 exhibited weak expression. The rs921142 SNP showed no significant association with bladder cancer risk. This is the first study to describe an association of the SFRP1 SNP rs3242 and bladder cancer risk as well as the influence of rs3242 on genotype-dependent microRNA capacity on SFRP1 mRNA. The onset of bladder seems to be associated with the increased occurrence of the T-allele in rs3242.