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Background: The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis. This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM. Methods: Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients. Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles. Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm. The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses. Results: Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients. Patients with a "favorable" methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis. Median OS of stage IIIC patients with a "favorable" vs. "unfavorable" methylation profile were 31.5 and 10.4 months, respectively. The 5 year OS for stage IIIC patients with a "favorable" methylation profile was 41.2% as compared to 0% for patients with an "unfavorable" methylation profile. Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for "unfavorable" methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045). A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified. Conclusions: A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients. Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients. [ABSTRACT FROM AUTHOR]

Background: The prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established. Genome-wide alterations in DNA methylation are a common event in cancer. This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients.Methods: Overall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients. The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis.Results: Hypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association. Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis. Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively. The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences. Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01).Conclusion: LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC. Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients. [ABSTRACT FROM AUTHOR]

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of posttranslational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects. [ABSTRACT FROM AUTHOR]

Background: Perinatal exposure to hyperhomocysteinemia might disturb neurogenesis during brain development and growth. Also, high levels of homocysteine trigger neurodegeneration in several experimental models. However, the putative mechanisms of homocysteine-induced toxicity in the developing nervous system have poorly been elucidated. This study was aimed to investigate homocysteine effects in undifferentiated neuroblastoma cells, Neuro2a., Results: A 4 h exposure to homocysteine in a concentration range of 10-100 microM did not affect cell viability and ROS production in Neuro2a cell cultures. Instead, ROS levels were increased by two-three folds in cells treated with 250 microM and 500 microM homocysteine, respectively, in comparison with control cells. Also, the highest homocysteine dose significantly reduced the viable cell number by 40%. Notably, the treatment with homocysteine (250 microM-500 microM) in the presence of antioxidants, such as N-acetylcysteine and IRFI 016, a synthetic alpha-tocopherol analogue, recovered cell viability and significantly reduced homocysteine-evoked increases in ROS production. Moreover, antioxidants, particularly IRFI 016, were able to counteract NF-kappaB activation induced by 250 microM homocysteine. Cell treatment with 250 microM homocysteine also triggered the onset of apoptosis, as demonstrated by the increased expression of early apoptotic markers such as Bax, caspase-3 and p53. In contrast, Bcl2 expression was not affected by homocysteine exposure. Interestingly, the specific inhibition of NF-kappaB nuclear translocation by the synthetic peptide SN50 was able to almost completely suppress the homocysteine-evoked rises in pro-apoptotic protein expression as well as in caspase-3 activity. Further, also IRFI 016 and N-acetylcysteine were able to significantly reduce caspase-3 activation induced by homocysteine treatment., Conclusion: These observations suggest an involvement of redox state alterations and activated NF-kappaB in apoptosis onset triggered by homocysteine in neuroblastoma cells Neuro2a. However, further investigations are needed to characterize molecular events involved in the NF-kappaB activation induced by homocysteine.