The degree of global DNA hypomethylation in peripheral blood correlates with that in matched tumor tissues in several neoplasia.

There are no good blood and serum biomarkers for detection, follow up, or prognosis of brain tumors. However, they are needed for more detailed tumor classification, better prognosis estimation and selection of an efficient therapeutic strategy. The aim of this study was to use the epigenetic changes in DNA of peripheral blood samples as a molecular marker to diagnose brain tumors as well as other diseases. We have applied a very precise thin-layer chromatography (TLC) analysis of the global amount of 5-methylcytosine (m(5)C) in DNA from brain tumors, colon and breast cancer tissues and peripheral blood samples of the same patients. The m(5)C level in tissue DNA from different brain tumor types, expressed as R coefficient, changes within the range of 0.2-1.6 and overlaps with R of that of blood samples. It negatively correlates with the WHO malignancy grade. The global DNA hypomethylation quantitative measure in blood, demonstrates a big potential for development of non-invasive applications for detection of a low and a high grade brain tumors. We have also used this approach to analyze patients with breast and colon cancers. In all these cases the m(5)C amount in DNA cancer tissue match with data of blood. This study is the first to demonstrate the potential role of global m(5)C content in blood DNA for early detection of brain tumors and others diseases. So, genomic DNA hypomethylation is a promising marker for prognosis of various neoplasms as well as other pathologies

The list of brain tumor types identified in 183 patients for whom DNA from brain tumor tissue and peripheral blood samples was isolated and analyzed for the content of m⁵C in DNA.

<p>Specific R coefficient was calculated as (m5dC/m5dC+dC+dT)×100 on the basis of analysis TLC plate exposed to Phosphoimager. Histopathological analysis revealed the WHO grade. Sex is also mentioned.</p

Effect of brain tumor tissue handling on content of m⁵C in DNA.

<p>The level of m⁵C content (R) in DNA isolated from resected meningioma tissue (WHO grade I) stored at −80°C (grey bar), formalin-fixed paraffin-embedded (FFPE) (empty bar) and exposed to room temperature for 3 h (black bar). Analysis was done for 5 samples in each conditions. Standard deviations for R is shown.</p

Comparison of content of m⁵C in DNA from tissue and blood.

<p>5-methylcytosine content (R) in DNA from 183 patients with brain tumors: cancer tissues (upper panel) and blood (bottoms panel). The figure clearly shows that m⁵C content, expressed as R [ = (m5dC/m5dC+dC+dT)×100] coefficient, in DNA from tumor tissue matches the results from blood <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092599#pone.0092599-Barciszewska2" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092599#pone.0092599-Barciszewska3" target="_blank">[26]</a>.</p

The diagram showing the relation of m⁵C content (R values with deviations errors) in DNA isolated from peripheral blood of patients with different brain tumors, breast and colon cancers and arterial hypertension.

<p>As one can see there is strict relation of R with different diseases. R decreases as malignancy increases. The amount of m⁵C suggests a possibility of a disease occurrence. As a control samples from healthy patients of age group 19–50 were used.</p

Patients with brain tumors analyzed in this study classified according to age.

<p>The 183 patients were divided into 6 groups of different age. The largest group consisted of patients within the age range of 51–60 years.</p

Comparison of m⁵C content in DNA in tissue and blood with malignancy of brain tumors.

<p><b>A</b>. Pearson r correlation (0.9; p<0.0001) of genomic m⁵C contents of DNA from peripheral blood and from brain tumor tissues of the same subjects. <b>B</b>. Amounts of m⁵C in DNA (R) from blood and in tumor tissues of the same subjects with brain tumors (astrocytoma, anaplastic astrocytoma and glioblastoma) of different malignancy. Data on human brain metastasis and meningioma were also analyzed. Data were evaluated with ANOVA test.</p

Juglone in Combination with Temozolomide Shows a Promising Epigenetic Therapeutic Effect on the Glioblastoma Cell Line

Glioblastoma (GBM) is the most common and aggressive primary brain tumor and one of the human malignancies with the highest mortality. Standard approaches for GBM, including gross total resection, radiotherapy, and chemotherapy, cannot destroy all the cancer cells, and despite advances in its treatment, the prognosis for GBM remains poor. The problem is that we still do not understand what triggers GBM. Until now, the most successful chemotherapy with temozolomide for brain gliomas is not effective, and therefore new therapeutic strategies for GBM are needed. We found that juglone (J), which exhibits cytotoxic, anti-proliferative, and anti-invasive effects on various cells, could be a promising agent for GBM therapy. In this paper, we present the effects of juglone alone and in combination with temozolomide on glioblastoma cells. In addition to the analysis of cell viability and the cell cycle, we looked at the epigenetics effects of these compounds on cancer cells. We showed that juglone induces strong oxidative stress, as identified by a high increase in the amount of 8-oxo-dG, and decreases m5C in the DNA of cancer cells. In combination with TMZ, juglone modulates the level of both marker compounds. Our results strongly suggest that a combination of juglone and temozolomide can be applied for better GBM treatment

A New Epigenetic Mechanism of Temozolomide Action in Glioma Cells.

Temozolomide (TMZ) is an oral alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma (GBM). Despite that high TMZ potential, progression of disease and recurrence are still observed. Therefore a better understanding of the mechanism of action of this drug is necessary and may allow more durable benefit from its anti-glioma properties. Using nucleotide post-labelling method and separation on thin-layer chromatography we measured of global changes of 5-methylcytosine (m5C) in DNA of glioma cells treated with TMZ. Although m5C is not a product of TMZ methylation reaction of DNA, we analysed the effects of the drug action on different glioma cell lines through global changes at the level of the DNA main epigenetic mark. The first effect of TMZ action we observed is DNA hypermethylation followed by global demethylation. Therefore an increase of DNA methylation and down regulation of some genes expression can be ascribed to activation of DNA methyltransferases (DNMTs). On the other hand hypomethylation is induced by oxidative stress and causes uncontrolled expression of pathologic protein genes. The results of brain tumours treatment with TMZ suggest the new mechanism of modulation epigenetic marker in cancer cells. A high TMZ concentration induced a significant increase of m5C content in DNA in the short time, but a low TMZ concentration at longer time hypomethylation is observed for whole range of TMZ concentrations. Therefore TMZ administration with low doses of the drug and short time should be considered as optimal therapy

Epigenetic Integrity of Orthodox Seeds Stored under Conventional and Cryogenic Conditions

The level of 5-methylcytosine (m5C) in DNA has been observed to change in plants in response to biotic and abiotic stress factors. Little information has been reported on alterations in DNA methylation in orthodox tree seeds in response to storage conditions. In the current study, epigenetic integrity was analyzed in seeds of Pyrus communis L. in response to conventional and cryogenic storage. The results indicate that conventional storage under optimal conditions resulted in a significant increase in m5C. In contrast, a decrease in m5C level after cryostorage at high water content (WC) was observed, not only in seeds but also in 3-month-old seedlings which were smaller than seedlings obtained from seeds cryostored at optimal WC. This shows that non-optimal cryostorage conditions increase epigenetic instability in seeds and seedlings. Optimal procedures for germplasm conservation are very important for germplasm banking since they have serious implications for the quality of stored collections. Maintaining epigenetic integrity during WC adjustment and optimal storage is a characteristic feature of orthodox seeds. The current results underline the importance of proper protocols and techniques for conventional storage and particularly cryopreservation as a method for conservation of true-to-type germplasm for long periods