EPIGENETIC CHANGES IN MALIGNANT UVEAL MELANOMA AND POSSIBILITIES OF THEIR THERAPEUTIC TARGETING.

Uveal melanoma (UM) is a deadly cancer that leads to metastatic disease in more than 50 % of the patients. Despite the improvement in the treatment of primary disease, there is still no effective therapy to prevent the development of metastases. Therefore, the disease requires intensive research to identify new treatment strategies. In preclinical UM models, epigenetic drugs have been shown to increase the sensitivity of resistant tumour cells to treatment. The successful use of histone deacetylase inhibitors, which induced cell cycle arrest, reprogramming consistent with melanocyte differentiation and inhibition of tumour growth in preclinical models, demonstrates the role of epigenetic regulation in UM metastasis. Identification of epigenetic changes associated with UM development an progression could contribute to the discovery of more effective drugs that, in combination with traditional approaches, may yield better therapeutic results for high-risk patients.

Role of epigenetic deregulation in hematogenous dissemination of malignant uveal melanoma.

It has become increasingly clear that epigenetic deregulation plays a fundamental role in cancer. Although the understanding of molecular, genetic and transcriptional alterations involved in the initiation and progression of uveal melanoma (UM) has grown significantly in recent years, little attention has been paid to the role of epigenetic changes. In cancer, epithelial-to-mesenchymal transition (EMT) enables trans-differentiation of epithelial tumor cells, endowing them with migratory and invasive properties. EMT-inducing transcription factors have been shown to interact with multiple epigenetic modifiers, thus reflecting the reversible nature of EMT. Therefore, the epigenetic therapy targeting these interactions may provide a promising therapeutic option, especially since no improvement in survival of patients with metastatic UM has been achieved using traditional approaches. This review summarizes current knowledge of epigenetic regulation of EMT in UM and emphasizes the need for deeper understanding of these highly dynamic and reversible processes. The potential for targeting individual members of the epigenetic machinery is also addressed.

ALDH1A inhibition sensitizes colon cancer cells to chemotherapy.

BACKGROUND: Recent evidence in cancer research, developed the notion that malignant tumors consist of different subpopulations of cells, one of them, known as cancer stem cells, being attributed many important properties such as enhanced tumorigenicity, proliferation potential and profound multidrug resistance to chemotherapy. Several key stem cells markers were identified in colon cancer. In our study we focused on the aldehyde dehydrogenase type 1 (ALDH1) expression in colon cancer-derived cell lines HT-29/eGFP, HCT-116/eGFP and LS-180/eGFP, and its role in the chemoresistance and tumorigenic potential. METHODS: The effect of pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde (DEAB) and also effect of molecular inhibition by specific siRNA was evaluated in vitro in cultures of human colorectal cell lines. The expression level of different isoenzymes of aldehyde dehydrogenase was determined using qPCR. Changes in cell biology were evaluated by expression analysis, western blot and apoptosis assay. The efficiency of cytotoxic treatment in the presence of different chemotherapeutic drugs was analyzed by fluorimetric assay. Tumorigenicity of cells with specific ALDH1A1 siRNA was tested in xenograft model in vivo. RESULTS: Treatment by DEAB partially sensitized the tested cell lines to chemotherapeutics. Subsequently the molecular inhibition of specific isoforms of ALDH by ALDH1A1 or ALDH1A3 siRNA led to sensitizing of cell lines HT-29/eGFP, HCT-116/eGFP to capecitabine and 5-FU. On the model of athymic mice we observed the effect of molecular inhibition of ALDH1A1 in HT-29/eGFP cells by siRNA. We observed inhibition of proliferation of subcutaneous xenografts in comparison to control cells. CONCLUSION: This research, verifies the significance of the ALDH1A isoforms in multidrug resistance of human colorectal cancer cells and its potential as a cancer stem cell marker. This provides the basis for the development of new approaches regarding the treatment of patients with colorectal adenocarcinoma and potentially the treatment of other tumor malignancies.

Impact of interleukin 13 (IL13) genetic polymorphism Arg130Gln on total serum immunoglobulin (IgE) levels and interferon (IFN)-γ gene expression.

This cross-sectional study was designed to investigate the extent of genetic susceptibility by targeting variants in interleukin (IL)-4/IL-13 signalling pathways leading to atopic disease in early childhood. We evaluated involvement of five single nucleotide polymorphisms IL4 C-590T, IL13 C-1055T, IL13 Arg130Gln, IL4RA Ile50Val and IL4RA Gln576Arg, in the control of serum total and antigen-specific immunoglobulin (Ig)E levels. Furthermore, we analysed their association with changes in gene expression of five cytokines having key roles in inflammatory and anti-inflammatory immune response [IL-4, IL-13, interferon (IFN)-γ, IL-8 and IL-10]. Total and antigen-specific IgE levels in serum and gene expression of selected cytokines in peripheral blood were measured in 386 children aged 1-8 years. TaqMan allelic discrimination, amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and restriction fragment length polymorphisms (RFLP) methods validated by sequencing were used for genotyping. All genotypes for children with total and antigen-specific IgE levels in the normal range were in Hardy-Weinberg equilibrium. Gene expression analyses were carried out using TaqMan gene expression assays. We found elevated total IgE levels in carriers of IL13 Arg130Gln variant allele [odds ratio (OR) = 1·84; 95% confidence interval (CI) = 1·16-2·93]. This effect was more apparent for boys (OR = 2·31; 95% CI = 1·25-4·28). However, no significant association was observed for the other four variants examined. We found up-regulation of IFN-γ in children with elevated serum total IgE levels carrying the Arg130 allele (P = 0·005). No differences were found for IL4, IL8 or IL10, while IL13 gene expression was under the detection limit. IL13 Arg130Gln genotypes can play a role in genetic susceptibility to allergy via regulation of serum total IgE levels and affecting IFN-γ gene expression.

[THE BRAF MUTATION AND THE POSSIBILITIES OF UVEAL MELANOMA METASTASING PROGNOSTIC MARKERS IDENTIFICATION]. / MUTÁCIA BRAF A MOZNOSTI IDENTIFIKÁCIE PROGNOSTICKÝCH MARKEROV METASTÁZOVANIA UVEÁLNEHO MELANÓMU.

AIM: The aim is to assess the BRAF gene mutations in patients with posterior uveal melanoma. MATERIAL AND METHODS: Retrospective analysis of the group of patients with malignant melanoma of the uvea, who were indicated to enucleation between 1.1 2015 to 1.3.2016. We analyzed stage of uveal melanoma, volume, cell type and BRAF gene mutations. RESULTS: In clinical study of 20 patients after enucleation due to uveal melanoma at the Department of Ophthalmology in Bratislava, patient age was ranged from 22 to 89 years with a median of 62 years. In 14 patients (70 %) enucleation was the primary treatment and in 6 patients (30 %) enucleation was after irradiation (brachytherapy, Leksell gama knife, linear accelerator). In 17 cases (85 %) the mutation of the BRAF gene was negative and in 3 cases the sample was not assessable for the BRAF mutation. CONCLUSION: BRAF gene mutation is confirmed by several studies found in malignant melanoma of the skin. The histopathology findings in our group did not confirmed our theory, that since the uveal melanoma itself has the similar origin as skin melanoma, should also contain a BRAF mutation.Key words: malignant melanoma of the uvea, mutation of the BRAF gene, chromosomal abnormalities as a prognostic factor.

Global and gene specific DNA methylation in breast cancer cells was not affected during epithelial-to-mesenchymal transition in vitro.

Epithelial-to-mesenchymal transition (EMT) significantly affects the risk of metastasising in breast cancer. Plasticity and reversibility of EMT suggest that epigenetic mechanisms could be the key drivers of these processes, but little is known about the dynamics of EMT-related epigenetic alterations. We hypothesised that EMT, mediated by autocrine and paracrine signals, will be accompanied by changes in DNA methylation profiles. Therefore, conditioned medium from adipose tissue-derived mesenchymal stromal cells was used for induction of EMT in human breast cancer SK-BR-3 cell line. EMT-related morphological alterations and changes in gene expression of EMT-associated markers were assessed. To reverse EMT, 20 nm size gold nanoparticles (AuNPs) synthesized by the citrate reduction method were applied. Finally, DNA methylation of LINE-1 sequences and promoter methylation of TIMP3, ADAM23 and BRMS1 genes were quantitatively evaluated by pyrosequencing. Despite the presence of EMT-associated morphological and gene expression changes in tumour cells, EMT induced by adipose tissue-derived mesenchymal stromal cells had almost no effect on LINE-1 and gene-specific DNA methylation patterns of TIMP3, ADAM23 and BRMS1 genes. Although treatment for 24, 48 or 72 hours with 20 nm AuNPs at a concentration of 3 µg/ml slightly decreased gene expression of EMT-associated markers in SK-BR-3 cells, it did not alter global or gene-specific DNA methylation. Our results suggest that changes in DNA methylation are not detectable in vitro in early phases of EMT. Previously published positive findings could represent rather the sustained presence of potent EMT-inducing signals or the synergistic effect of various epigenetic mechanisms. Treatment with AuNPs slightly attenuated EMT, and their therapeutic potential needs to be further investigated.

Evaluation of protein expression and DNA methylation profiles detected by pyrosequencing in invasive breast cancer.

Breast carcinoma is the most common cancer with high mortality caused by metastatic disease. New molecular biomarkers predicting the tumour's metastatic potential would therefore improve metastasis prevention and personalised care. The aim of the study was to investigate the relationship between DNA methylation levels in invasivity and metastasising associated genes with aberrant protein expression and also to evaluate whether a similar DNA methylation level is present in the tumour and circulating cell-free DNA for utilising plasma DNA methylation as prognostic biomarker. By using pyrosequencing, we analysed DNA methylation levels of 11 genes, namely APC, ADAM23, CXCL12, ESR1, PGR B, CDH1, RASSF1A, SYK, TIMP3, BRMS1 and SOCS1 in tumour, plasma and peripheral blood cells from 34 patients with primary breast cancer, as well as plasma and peripheral blood cells from 50 healthy controls. Simultaneously, the expression of related proteins in paraffin-embedded tumour samples was evaluated by immunohistochemistry. Statistical analysis was performed by SPSS statistics 15.0 software. Tumour DNA hypermethylation was found in most commonly methylated RASSF1A (71.9%), APC (55.9%), ADAM23 (38%) and CXCL12 (34.4%) genes with methylation levels up to 86, 86, 53 and 64 %, respectively. In tumours, significantly higher methylation levels were found in nine genes, compared with the patients´ peripheral blood cell DNA. Furthermore, in patients methylation levels in peripheral blood cell DNA were significantly higher than in controls in CXCL12, ESR1 and TIMP3 genes, but the values did not exceed 15%. On the other hand, no correlations were observed in patients between DNA methylation in tumours and cell-free plasma DNA. Moreover, in patients and controls nearly identical values of cumulative DNA methylation (43.6 % ± 20.1 vs. 43.7 % ± 15.0) were observed in plasma samples. A variable spectrum from high to none expressions presented in tumour tissues in all of the proteins evaluated, however in APC and CXCL12 genes a visible decreasing trend of mean DNA methylation level with increasing expression of the corresponding protein was observed. The DNA methylation profiles manifested in our group of breast carcinomas are cancer specific, but they are not the only cause that affects the silencing of evaluated genes and the decrease of relevant protein products. The clinical utility of DNA methylation testing in peripheral blood cell DNA for cancer diagnosis and therapy need to be further investigated.

Antioxidant supplementation reduces inter-individual variation in markers of oxidative damage.

The aim of this study was to examine the effect of antioxidant supplementation on oxidative damage and chromosome stability in middle-aged men, smokers and non-smokers. A total of 124 men aged 48+/-6 years from Bratislava and from the rural population near Bratislava were investigated; 64 men (22 smokers and 42 non-smokers) were supplemented for 12 weeks with antioxidants, while 60 (25 smokers and 35 non-smokers) were given placebo. The daily antioxidant supplementation consisted of vitamin C (100 mg), vitamin E (100 mg), ss-carotene (6 mg), and selenium (50 microg). Samples of blood were taken on two occasions: At the beginning and at the end of the supplementation trial. Concentrations of dietary antioxidants, ferric reducing ability, malondialdehyde as an indicator of lipid peroxidation in plasma, micronuclei and chromosome aberrations in lymphocytes were measured. Antioxidant supplementation significantly increased the levels of vitamin C, ss-carotene, a-tocopherol and selenium in plasma. The overall antioxidant status of plasma measured as ferric reducing ability of plasma (FRAP) increased significantly (p<0.001) after antioxidant supplementation as well. The increase in antioxidant parameters after supplementation were consistently more pronounced in non-smokers than in smokers. There was a significant decrease of malondialdehyde concentration in the non-smokers, while in smokers the decrease of malondialdehyde concentration was not significant. Antioxidant supplementation did not affect the proportion of lymphocytes with micronuclei or the total number of micronuclei; however, there was a significant positive correlation (p<0.001) between the malondialdehyde concentration at the beginning of the supplementation trial and the difference in number of cells with micronuclei before and after the supplementation. The percent of cells with chromosome aberrations decreased significantly after antioxidant supplementation in smokers. These results indicate that a combined antioxidant supplementation (a) is effective even at very moderate doses; (b) significantly diminishes oxidative damage to lipids when it is high initially; and (c) is effective in decreasing chromosomal instability in lymphocytes of middle-aged men.

Lipid peroxidation in men after dietary supplementation with a mixture of antioxidant nutrients.

Antioxidants and antioxidant enzymes protect living organisms against the attack of reactive oxygen species. An adequate daily intake of the individual antioxidants is therefore important to prevent the cells against oxidative damage. We investigated the effect of a modest dietary supplementation with a mixture of antioxidant nutrients (100 mg vitamin E, 100 mg vitamin C,6 mg beta-carotene and 50 microg of selenium per day) for 3 months on the plasma antioxidant capacity and indices of oxidative stress. Two groups of middle-age men were selected: group 1 with survivors of myocardial infarction (MI), and group 2 with clinically normal controls. The values of total antioxidant capacity of plasma (FRAP) significantly increased after supplementation with antioxidants in the both groups. Markers of in vivo lipid peroxidation, plasma malondialdehyde (MDA) and conjugated diene (CD) levels significantly decreased in the both supplemented groups. MDA and CD values were significantly higher at baseline in the group of survivors of myocardial infarction when compared with the group of healthy men. The results demonstrate that short-term and modest supplementation with a mixture of antioxidant nutrients improves antioxidative capacity and reduces products of lipid peroxidation in plasma. Since a more pronounced effect was observed within the group of survivors of myocardial infarction, a recommendation of antioxidant supplements seems appropriate for patients with a history of cardiovascular disease. (Fig. 1, Ref. 19.)

Folate levels determine effect of antioxidant supplementation on micronuclei in subjects with cardiovascular risk.

We have investigated the effect of modest supplementation with alpha-tocopherol (100 mg/day), beta-carotene (6 mg/day), vitamin C (100 mg/day) and selenium (50 microg/day) on oxidative stress and chromosomal damage, and the influence of methylenetetrahydrofolate reductase (MTHFR) genotype on these end-points. Subjects were two groups of middle-aged men differing in cardiovascular risk; 46 survivors of myocardial infarction before age 50 and 60 healthy controls. They were randomly divided into equal groups to receive antioxidants or placebo for 12 weeks. Twenty-eight patients and 58 controls completed the intervention. Micronucleus levels in peripheral lymphocytes and changes seen after intervention were studied in relation to the MTHFR C677T genotype, basal homocysteine and plasma folate levels. Ferric reducing ability of plasma and concentration of malondialdehyde were measured to assess the antioxidant effect of supplementation. There was no association of micronuclei with folate, homocysteine or malondialdehyde levels before supplementation. Micronucleus frequencies and plasma folate levels did not vary significantly with MTHFR genotype. Homocysteine levels in subjects with the TT variant genotype were significantly higher compared with CT or CC (P = 0.001), especially in subjects with low folate (P = 0.012). In the placebo control group an increase in micronuclei (P = 0.04) was detected at the end of the intervention period. This effect was not seen in the supplemented group. In antioxidant-supplemented myocardial infarction survivors we found an increase in the ferric reducing ability of plasma (P < 0.001) and a decrease in malondialdehyde (P = 0.001). Micronucleus frequency showed a decrease, strongest in subjects with normal folate levels (P = 0.015). In subjects with low folate levels, a high correlation was found between micronuclei after supplementation and homocysteine, both before (r = 0.979, P = 0.002) and after supplementation (r = 0.922, P = 0.009). Thus, folate deficiency may amplify the effect of other risk factors such as elevated homocysteine levels or variant MTHFR genotype, as well as influencing the ability of antioxidant supplementation to protect against genetic damage.

Nutritional supplementation with antioxidants decreases chromosomal damage in humans.

In order to investigate the effects of antioxidant supplementation on chromosome damage, a 3 month antioxidant supplementation trial was conducted on groups of 28 myocardial infarction survivors and 57 rural controls, all male. The supplement consisted of vitamin C (100 mg/day), vitamin E (100 mg/day), beta-carotene (6 mg/day) and selenium (50 microg/day). Dietary antioxidants in plasma were measured, as well as the ferric reducing ability of plasma (a measure of total plasma antioxidant status) and the concentration of malondialdehyde as an indicator of oxidative stress. Lymphocytes collected at the beginning and end of the supplementation period were stimulated to proliferate and metaphases accumulated for scoring of chromosome aberrations: per cent aberrant cells and chromatid and chromosome breaks. Supplementation with antioxidants was associated with a decrease in the percentage of cells with chromosome aberrations in the group of rural controls (0.63% before compared with 0.27% after supplementation; P = 0.03). The largest effect of supplementation was seen in smokers in this group (0.12% aberrant cells in supplemented compared with 0.81% in placebo group; P > 0.001). The results support the hypothesis that antioxidants decrease genetic damage.

DNA damage and antioxidants; fluctuations through the year in a central European population group.

Dietary antioxidant levels in the blood depend on intake of fruits and vegetables and therefore might be expected to show seasonal variation. A group of healthy male subjects in Bratislava, Slovakia gave blood samples each month for 1 year. Vitamin C, alpha- and gamma-tocopherol and several carotenoids were measured in plasma, and concentrations of essential metals zinc, copper and selenium in serum. Oxidative DNA damage was assessed in lymphocytes using the comet assay. Seasonal variations in antioxidant levels did not follow a common pattern. beta-Cryptoxanthin was highest in the spring. Lycopene peaked in late summer. Lutein/zeaxanthin was higher in summer than in winter. The concentration of zinc in serum was higher in winter than in summer. DNA damage was lower in summer than in winter. Selenium as well as several antioxidants correlated negatively with indices of DNA damage, while zinc levels showed a positive correlation with DNA damage. These results provide some support for a link between consumption of antioxidants and protection against DNA oxidation.

Risk factors for atherosclerosis in survivors of myocardial infarction and their spouses: comparison to controls without personal and family history of atherosclerosis.

To explore the hypothesis that an interplay between genetic and environmental factors contributes to the development of coronary atherosclerosis, we compared the prevalence of risk factors for atherosclerosis among survivors of myocardial infarction (MI) and their spouses and apparently healthy men and women (spousal pairs) with no personal and family history of atherosclerosis in three generations. There were no significant differences in life-style and dietary habits between the groups. The daily vegetable and/or fruit intake was generally low and did not differ between the groups. Thirty percent and 25% of men and women did not consume any vegetables or fruits, respectively. All differences found in the male MI survivors and control men were also found between the female groups: MI survivors and their spouses were significantly more obese and had higher systolic and diastolic blood pressure and more pathologic plasma lipid levels compared with control males and females, respectively. Compared with the control men and women, MI survivors and spouses had higher plasma homocysteine (Hcgamma) levels (15.3 +/- 10.5, 11.9 +/- 4.0, 16.9 +/- 5.5, and 14.3 +/- 4.0, micromol/L, respectively, P = .01). The frequency of the homozygous C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphism in MI survivors was twice that observed in their spouses and controls (12.1%, 4.8%, and 5.8%, respectively), but this difference did not reach statistical significance. A statistically significant association of the MTHFR genotype and Hcgamma concentration (multiple ANOVA) was shown. Neither the frequencies of apolipoprotein E (apoE) alleles nor Asp9Asn mutation of exon 2, Asn29lSer mutation of exon 6, and Ser447Ter of exon 9 of the lipoprotein lipase (LPL) gene varied significantly among the groups. A possible explanation for our findings is that individuals with a genetic predisposition for atherosclerosis and their spouses share a life-style that results in a higher body mass index (BMI) and waist to hip ratio (WHR). On the other hand, individuals with no family history of atherosclerosis, despite an unhealthy life-style similar to that in the affected families (diet and physical activity), had a lower BMI and WHR and more favorable metabolic parameters, including plasma Hcgamma. In conclusion, we have shown that a personal and/or family history of atherosclerosis corresponds to the prevalence and level of risk factors for atherosclerosis. A combination of life-style factors and inherited metabolic abnormalities, including high plasma Hcgamma, are the more likely explanation for our findings.

Effect of diet and 677 C-->T 5, 10-methylenetetrahydrofolate reductase genotypes on plasma homocyst(e)ine concentrations in slovak adolescent population.

The objective of this study was to evaluate the effect of diet and 677 C-->T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene on plasma homocyst(e)ine concentrations in an adolescent population (113 males, age: 14.2+/-2.4 years; 202 females, age: 14.9+/-2.1 years) from a region characterized by high cardiovascular mortality. Homocyst(e)ine levels did not differ between males and females (9.4+/-3.5 and 8.9+/-3.1 micromol/l, respectively). The homozygosity for the 677 C-->T MTHFR mutation was found in 4.6 % of subjects. No differences in homocyst(e)ine levels were found between MTHFR genotypes. Analysis of the diet composition which was performed on a 24-hour daily recall basis and a food frequency questionnaire showed a low daily intake of vitamin B6 (males: 1.13 mg/66% RDA; females: 0.92 mg/61% RDA). Daily folic acid intake was 0.21 g/105% RDA in males and 0.23 g/115% RDA in females. The results of our study show that the high homocyst(e)ine levels in the adolescent population were not affected by the 677 C-->T MTHFR mutation. We conclude that an insufficient dietary intake of vitamin B6 and folic acid is responsible for this finding. This is in accord with the recommendation that the dietary allowances for folate should be reset to the originally prescribed levels of 0.4 g/day which should be sufficient to control the homocysteine levels.

Apolipoprotein E genotypes in offspring with a positive and negative family history of premature myocardial infarction.

Apolipoprotein E (apo-E) allele and genotype frequencies were evaluated in offspring with positive (MI-offspring) and negative (control-offspring) parental history of myocardial infarction (MI). The apo-E allele frequencies in MI- and control-offspring were as follows: epsilon2: 9.04 and 2.08% (p < 0.02), epsilon3: 84.04 and 87.5%, epsilon4: 6.91 and 10.41%, respectively. The frequencies of the E2-genotypes were significantly lower in offspring of controls (4.2%, 17.0%, respectively, p < 0.03). The epsilon2-allele is associated with raised plasma triglyceride concentrations in subjects on a diet high in saturated fat. We therefore hypothesize that offspring carrying an epsilon2-allele are predisposed to develop disturbance of plasma triglyceride metabolism when exposed to a traditional Slovak high-fat diet and/or weight gain, resulting in altered lipid levels and increased predisposition to atherosclerosis.