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Objective and design: Epigenetic regulation is important in the activation of inflammatory cells. In the present study, we evaluated if DNA-methylation variations are involved in Interleukin-1β (IL-1β)-induced intestinal epithelial cells activation. Materials and methods: Differentiated Caco-2 cells were exposed to IL-1β or to 5-azadeoxycytidine (5-azadC) for 24 or 48 h. Genome-wide methylation status was evaluated, while DNA methylation status at the promoter region of the gene encoding interleukin-6, 8 and 10 (IL-6, 8 and 10) was estimated. The levels of the corresponding gene products as well as DNA methyltransferases (DNMTs) quantity were assessed. Results: IL-1β decreased genomic methylation of human intestinal epithelial cells and induced demethylation at cg-specific sites at the promoter of pro-inflammatory genes IL6 and IL8; conversely it did not change the methylation of the IL10 promoter. IL-1β also increased the release of IL-6 and IL-8 but did not change the IL-10 expression. Finally, cell exposure to IL-1β decreased the DNMT3b expression, increased DNMT3a and was not able to change DNMT1 expression. Conclusions: Our results suggest a potential role of IL-1β as modulator of DNA methylation in activated differentiated Caco-2 cell line.

Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5' and 3' ends and the existence of three different transcriptional promoters (P1, P2 and P3), two of which (P2 and P3) contain CpG islands. It is known that the expression of PTHrP isoforms may be differentially regulated in a developmental stage- and tissue-specific manner. To search for novel molecular markers of stemness/differentiation, here we have examined isoform expression in fat-derived mesenchymal stem cells both maintained in stem conditions and induced toward adipo- and osteogenesis. In addition, the expression of the splicing isoforms derived from P2 and P3 promoters was correlated to the state of methylation of the latter. Moreover, we also performed a quantitative evaluation of intracellular and secreted PTHrP protein product in undifferentiated stem cells and in parallel cultures at various differentiation stages. The data obtained indicate that from the stemness condition to that of osteo- and adipo-genic differentiated cells, the expression of isoforms becomes increasingly selective, thereby being a potential gene signature for the monitoring of cell stem or committed/differentiating state and that the switching-off of PTHrP isoform expression is mostly promoter methylation-dependent. Moreover, PTHrP intracellular retention is down-regulated in osteo-differentiating cells whereas the secretion of the protein in the extracellular medium is up-regulated with respect to stem cells, thereby suggesting that these variations of the intracellular and extracellular levels of PTHrP could potentially be enclosed in the list of the available protein signature of osteogenic differentiation.

We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus and recently, demonstrated the presence of high heterogeneity in functional 5S rRNA. In this paper, we show some important distinctive data on 5S rRNA transcription for this organism. Using single strand conformation polymorphism (SSCP) analysis, we demonstrate the existence of two classes of 5S rRNA, one which is embryo-specific and encoded by the smallest (700 bp) cluster and the other which is expressed at every stage and encoded by longer clusters (900 and 950 bp). We also demonstrate that the embryo-specific class of 5S rRNA is expressed in oocytes and embryonic stages and is silenced in adult tissue and that this phenomenon appears to be due exclusively to DNA methylation, as indicated by sensitivity to 5-azacytidine, unlike Xenopus where this mechanism is necessary but not sufficient to maintain the silenced status.

Background: Recently, we have shown anti-proliferative and pro-apoptotic effects of indicaxanthin associated with epigenetic modulation of the onco-suppressor p16INK4a in the human colon cancer cell line CACO2. In the present study, the epigenetic activity of indicaxanthin and the mechanisms involved were further investigated in other colorectal cancer cell lines. Methods: LOVO1, CACO2, HT29, HCT116, and DLD1 cells were used to evaluate the potential influence of consistent dietary concentrations of indicaxanthin on DNA methylation, and the epigenetic mechanisms involved were researched. Results: Indicaxanthin exhibited anti-proliferative activity in all cell lines but HT29, induced demethylation in the promoters of some methylation-silenced onco-suppressor genes involved in colorectal carcinogenesis (p16INK4a, GATA4, and ESR1), and left unchanged others which were basally hypermethylated (SFRP1 and HPP1). In apparent contrast, cell exposure to indicaxanthin increased DNMT gene expression, although indicaxanthin appeared to be an inhibitor of DNMT activity. Indicaxanthin also increased the expression of genes involved in DNA demethylation. Finally, an in silico molecular modelling approach suggested stable binding of indicaxanthin at the DNMT1 catalytic site. Conclusions: Our findings contribute to new knowledge in the field of phytochemicals and specifically suggest dietary indicaxanthin as a potential epigenetic agent to protect colon cells against tumoral alterations.

Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes [1]. The role of this process in tumorigenesis and tumor progression is controversial: in the early stages, it can block tumor growth and conversely it can promote its progression in the later stages [2]. The tumor suppressor BECN1 gene, encodes the protein Beclin 1, a marker of autophagy down-regulated in several types of cancer, such as colorectal cancer [3]. There are a lot of both genetic and environmental risk factors for colorectal cancer, including diet: for this reason, in accordance with epidemiological studies, consumption of foods rich in phytochemicals is widely promoted. The betalain indicaxantin (Ind) is a phytochemical from the Opuntia Ficus-Indica fruit having several biological activities, such as antioxidant, anti-inflammatory. It showed antiproliferative and proapoptotic effects in colorectal adenocarcinoma (Caco2) cells where was able to regulate gene expression through modulation of methylation state of DNA at CpG islands [4]. For the first time, using Methylation-Sensitive Restriction Endonuclease PCR (MSRE-PCR), we report that Ind (50 e 100 µM) decreases the methylation of BECN1 promoter in Caco2 cells, to the same extent as 5-azacytidine (Zcyd, positive control). Interestingly, colorimetric detection of DNA Methyltransferases activity, indicates that Ind reduced the activity of these enzymes, like Zcyd did. These preliminary data, indicating that Ind is able to decrease the methylation of BECN1 gene, allow us to propose an epigenetic hypothesis of autophagy regulation in Caco2 cells.

Intestinal inflammation is a natural process crucial to maintain gut integrity, but its deregulation is involved in the pathogenesis of severe intestinal disorders[1]. Intestinal epithelial cells play a crucial role in the inflammatory response, modulating the immune cell exposure to antigens and by their ability to secrete many inflammatory mediators. IL-1β represents a pivotal player: secreted by infiltrated leucocytes, it induces the expression of several pro-inflammatory genes. Also the anti-inflammatory IL-10, whose function is to terminate the inflammatory process, modulates the intestinal physiology[2]. Recent clinical reports showed that patients with ulcerative colitis in remission phase have significantly higher IL10 gene expression in mucosa compared with active patients and controls[3]. Moreover, in the latest years aberrant epigenetic mechanisms were put in binomial relationship with chronic inflammatory diseases[4]. Previously, we described a demethylation of pro-inflammatory IL6 and IL8 genes in human colonic Caco-2 cells differentiated into an enterocyte-like phenotype and exposed to the inflammatory action of IL-1β[5]. In the present study we evaluate whether the IL-1β treatment affected the methylation status of the anti-inflammatory IL10 gene, in the same in vitro model. Our results showed that IL-1β treatment did not change the hypermethylation status of the IL10 promoter. Moreover, in cell lysates from IL-1β-treated Caco-2 cells, we observed a dose-dependent increase of DNMTs activity and, surprisingly, a decrease of DNMT3b expression. These findings put in evidence the complexity of relationship between IL-1β and DNMTs, and may suggest a potential role of IL-1β as pleiotropic modulator of DNA methylation in Caco-2 cell line.

Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5' flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associated with the increase of enzyme expression. Interestingly, the Rsa/Pst haplotype differentially influenced CYP2E1 enzyme expression. In addition, regarding the Variable Number of Tandem Repeats polymorphism, cells with A4/A4 genotype showed a greater expression inhibition (ranging from 20% to 30%) compared with others carrying the A2/A2 one, while those cells bringing A2/A3 genotype showed an increase of expression (of 25%, about). Finally, we demonstrated for the first time that the A2 and A3 CYP2E1 alleles play a more important role in the expression of the enzyme, compared with other (epi)genetic factors, since they are binding sites for trans-acting proteins.

Inflammation is a complex physiological response that requires the activity of a sophisticated regulatory network involving the activation of specific genes for defense, tissue repair and remodeling. Although transcriptional activation has been shown to be critical in the regulation of inflammatory genes (1) the role of epigenetic phenomena in the modulation of the inflammatory response is now emerging (2). Specifically, it has been recently reported that proinflammatory stimuli induce DNA demethylation in the interleukin IL-1 promoter of human articular chondrocytes (3). IL-1 cytokine, among several proinflammatory agents, represents an essential player in the inflammatory conditions of the gut (4): functioning as the strongest signal transduction to NF-kB, IL-1 increases in intestinal paracellular permeability and over-expression of proinflammatory genes (5). In this tissue, moreover, inflammatory response is crucial to maintain its structural integrity and function, thus, alteration and deregulation of inflammatory pathways contribute to tissue damage and ulceration, and are pivotal factors in the pathogenesis of several inflammatory gut diseases. In the present study we evaluate both wide-ranging and gene-specific epigenetic changes in the inflammatory response of Caco-2 cells differentiated into intestinal epithelial cells and exposed to the inflammatory actions of IL-1β. Our results clearly show that IL-1induces changes in the DNA methylation either at genome and gene level and that the local methylation changes are induced in two pro-inflammatory genes that are IL-1-regulated. In particular, we show that a cell exposure to IL-1β for 24 h induces, in a dose-dependent manner, hypomethylation of genomic DNA in respect to untreated cells. We also observe a reduced DNA methyltransferases activity of cell lysates obtained from IL-1 treated cells. Finally our data show that IL-1 is able to induce hypomethylation of specific CpG sites in IL-6 and IL-8 genes. These preliminary results suggest that IL-1 in intestinal epithelial cells is able to act as an epigenetic modulator towards the entire genome and specific genes. Modulation of epigenetic changes in inflammation may provide a new “reading frame” of the inflammatory diseases molecular basis. Since epigenetic modifications are potentially reversible, a thorough understanding of these changes during inflammatory response opens opportunities to develop efficient agents for specific targets.

In the present study antiproliferative effects of Indicaxanthin (Ind), a highly bioavailable pigment from the fruits of Opuntia ficus-indica (1), were investigated on a number of human cancer cell lines including hepatocarcinoma cells (HepG2, Ha22T, HUH 7), breast cancer cells (MCF7), cervix epithelial carcinoma (HeLa), and colorectal carcinoma cells (Caco-2). Cytotoxicity of Ind, in a concentration range between 25 to 100 M, was evaluated by Trypan blue exclusion method and MTT assay. Ind caused a clear dose- and time-dependent decrease in the proliferation of Caco-2 and Ha 22T cells with an IC(50) of about 50 M, with minor effect on the other cell lines. Flow cytometric analysis after Annexin V-FITC and propidium iodure double staining, at 24, 48 and 72 h of treatment with 100 M Ind, showed a pro-apoptotic effect of the pigment at 48 and 72 h. Effect of Ind on DNA methylation investigated on DNA from Ha22T cells line and Caco2 cells line at 48 h of treatment with 10 M Ind, using MESAP-PCR (Methylation-Sensitive Arbitrarily-Primed Polymerase Chain Reaction) (3) showed that Ind induces a slight global demethylation. While antiproliferative effects of indicaxanthin add further value to the nutritional characteristics of the fruits of O. ficus-indica (2), our results also are consistent with the emerging role of dietary phytochemicals on the epigenetic regulation of gene expression.