Antiproliferative and pro-apoptotic effects of the phytochemical Indicaxanthin on human intestinal (Caco-2) and hepatic (Ha 22T) cancer cell lines

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 \uf06dM, 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 \uf06dM, 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 \uf06dM 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 \uf06dM 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

Vanadium Toxicity Is Altered by Global Warming Conditions in Sea Urchin Embryos: Metal Bioaccumulation, Cell Stress Response and Apoptosis

In recent decades, the global vanadium (V) industry has been steadily growing, together with interest in the potential use of V compounds as therapeutics, leading to V release in the marine environment and making it an emerging pollutant. Since climate change can amplify the sensitivity of marine organisms already facing chemical contamination in coastal areas, here, for the first time, we investigated the combined impact of V and global warming conditions on the development of Paracentrotus lividus sea urchin embryos. Embryo-larval bioassays were carried out in embryos exposed for 24 and 48 h to sodium orthovanadate (Na3VO4) under conditions of near-future ocean warming projections (+3 °C, 21 °C) and of extreme warming at present-day marine heatwave conditions (+6 °C, 24 °C), compared to the control temperature (18 °C). We found that the concomitant exposure to V and higher temperature caused an increased percentage of malformations, impaired skeleton growth, the induction of heat shock protein (HSP)-mediated cell stress response and the activation of apoptosis. We also found a time- and temperature-dependent increase in V bioaccumulation, with a concomitant reduction in intracellular calcium ions (Ca2+). This work demonstrates that embryos’ sensitivity to V pollution is increased under global warming conditions, highlighting the need for studies on multiple stressors

An alternative approach of TUNEL assay to specifically characterize DNA fragmentation in cell model systems

DNA damage is one of the most important effects induced by chemical agents. We report a comparative analysis of DNA fragmentation on three different cell lines using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, generally applied to detect apoptosis. Our approach combines cytogenetic techniques and investigation in detached cellular structures, recovered from the culture medium with the aim to compare the DNA fragmentation of three different cell line even beyond the cells adherent to substrate. Consequently, we detect any fragmentation points on single chromosomes, whole nuclei and other cellular structures. Cells were exposed to resveratrol (RSV) and doxorubicin (Doxo), in single and combined treatments. Control and treated astrocytes showed DNA damage in condensed nuclei and detached structures. Caco-2 cells showed fragmented DNA only after Doxo-treatment, while controls showed fragmented chromosomes, indicating DNA damage in replicating cells. MDA-MB-231 cells showed nuclear condensation and DNA fragmentation above all after RSV-treatment and related to detached structures. This model proved to perform a grading of genomic instability (GI). Astrocytes show a hybrid level of GI. Caco-2 cells showed fragmented metaphase chromosomes, proving that the DNA damage was transmitted to the daughter cells probably due to an absence of DNA repair mechanisms. Instead, MDA-MB-231 cells showed few or no fragmented metaphase, suggesting a probable activation of DNA repair mechanisms. By applying this alternative approach of TUNEL test, we obtained data that can more specifically characterize DNA fragmentation for a suitable application in various fields

IL-1β maintains the DNA hypermethylation of anti-inflammatory IL-10 gene in a human intestinal epithelial cell line

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

Methylation decrease of BECN1 gene induced by phytochemical Indicaxantin in Caco2 cells: an epigenetic hypothesis of autophagy

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

Seasonal water level fluctuations: Implications for reservoir limnology and management

With the purpose of finding out whether seasonal water level fluctuations could affect water quality in a reservoir subjected to those changes, trends in environmental variables and in phytoplankton and zooplankton assemblages were analysed. The reservoir’s hydrological cycle was characterized by three regimes. The maximum level phase lasted from January to the beginning of June, the emptying phase existed between mid-June to the beginning of September and the minimum level phase lasted from mid-September to the beginning of the first autumn/winter rain events. The highest values of total phosphorus, soluble reactive phosphorus, nitrate, water colour and chlorophyll a were found during the minimum level phase. The phytoplankton assemblage was dominated by taxa typical of meso-eutrophic environments during the emptying and minimum level phases. However, during the maximum level phase, taxa generally found in more oligotrophic systems were observed here also. Similar to other disturbed systems, the zooplankton assemblage was dominated by Rotifera, except in summer and autumn when the cladoceran Ceriodaphnia quadrangula and/or the copepod Tropocyclops prasinus became dominant. Although those shifts seem to be related to water level variations, further research is needed to evaluate to what extent they might also be induced by other seasonal factors acting independently of water fluctuations. Based upon the obtained data, suggestions for reservoir management are proposed

Enhancing the quality and safety of Nocellara del Belice green table olives produced using the Castelvetrano method

The Castelvetrano method is the most widely used among the various table olive processing styles in Sicily. After debittering, the product is stored at low temperatures to prevent the growth of undesirable microorganisms. In an effort to enhance the production process, yeast isolates underwent genotypic characterization and technological screening. The screening process identified two yeast strains Candida norvegica OC10 and Candida boidinii LC1, which can grow at low temperatures and tolerate high pH values (up to 10) and salinity [10% (w/v)]. During the monitoring period, the inoculated trials showed limited presence of spoilage/pathogenic microorganisms. Additionally, the yeasts limited oxidative phenomena and softening of the drupes. The organic compounds detected were higher in the inoculated trials than in the control, and cold storage induced aromatic decay, which was less pronounced in the trial inoculated with C. norvegica. Sensory analysis revealed that the inoculated trials scored higher in sweetness, hardness and crispness

Genome-wide analysis reveals no evidence of trans chromosomal regulation of mammalian immune development.

It has been proposed that interactions between mammalian chromosomes, or transchromosomal interactions (also known as kissing chromosomes), regulate gene expression and cell fate determination. Here we aimed to identify novel transchromosomal interactions in immune cells by high-resolution genome-wide chromosome conformation capture. Although we readily identified stable interactions in cis, and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including previously described interactions. We suggest that advances in the chromosome conformation capture technique and the unbiased nature of this approach allow more reliable capture of interactions between chromosomes than previous methods. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that lineage identity is governed by cis, not trans chromosomal interactions