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.

Indicaxanthin Induces Autophagy in Intestinal Epithelial Cancer Cells by Epigenetic Mechanisms Involving DNA Methylation.

Autophagy is an evolutionarily conserved process critical in maintaining cellular homeostasis. Recently, the anticancer potential of autophagy inducers, including phytochemicals, was suggested. Indicaxanthin is a betalain pigment found in prickly pear fruit with antiproliferative and pro-apoptotic activities in colorectal cancer cells associated with epigenetic changes in selected methylation-silenced oncosuppressor genes. Here, we demonstrate that indicaxanthin induces the up-regulation of the autophagic markers LC3-II and Beclin1, and increases autophagolysosome production in Caco-2 cells. Methylomic studies showed that the indicaxanthin-induced pro-autophagic activity was associated with epigenetic changes. In addition to acting as a hypermethylating agent at the genomic level, indicaxanthin also induced significant differential methylation in 39 out of 47 autophagy-related genes, particularly those involved in the late stages of autophagy. Furthermore, in silico molecular modelling studies suggested a direct interaction of indicaxanthin with Bcl-2, which, in turn, influenced the function of Beclin1, a key autophagy regulator. External effectors, including food components, may modulate the epigenetic signature of cancer cells. This study demonstrates, for the first time, the pro-autophagic potential of indicaxanthin in human colorectal cancer cells associated with epigenetic changes and contributes to outlining its potential healthy effect in the pathophysiology of the gastrointestinal tract.

An In Vitro Model of Glioma Development.

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

Nutritional epigenomic and DNA-damage modulation effect of natural stilbenoids.

The aim of the present work is the evaluation of biological effects of natural stilbenoids found in Vitis vinifera, with a focus on their activity as epigenetic modulators. In the present study, resveratrol, pterostilbene and for the first time their dimers (±)-trans-δ-viniferin, (±)-trans-pterostilbene dehydrodimer were evaluated in Caco-2 and HepG-2 cell lines as potential epigenetic modulators. Stilbenoids were added in a Caco-2 cell culture as a model of the intestinal epithelial barrier and in the HepG-2 as a model of hepatic environment, to verify their dose-dependent toxicity, ability to interact with DNA, and epigenomic action. Resveratrol, pterostilbene, and (±)-trans-pterostilbene dehydrodimer were found to have no toxic effects at tested concentration and were effective in reversing arsenic damage in Caco-2 cell lines. (±)-trans-δ-viniferin showed epigenomic activity, but further studies are needed to clarify its mode of action.

Nutrigenetics, nutrigenomics and phenotypic outcomes of dietary low-dose alcohol consumption in the suppression and induction of cancer development: evidence from in vitro studies.

It is known that the intake of alcoholic beverages may impair genetic and epigenetic regulatory events with consequent crucial effects on cell phenotypes and that its association with selected genotypes can lead to a different risk of cancer in the population. The aim of this review is to pick up selected studies on this topic and recapitulate some of the biochemical and nutrigenetic/nutrigenomic aspects involved in the impact of dietary low-dose alcohol consumption on the switching-on or -off of tumorigenic pathways. These include i) the existence of predisposing or protective human genotypes and the relationship between dietary compounds and alcohol in the promotion or inhibition of carcinogenesis; ii) the effects of other components of alcoholic drinks in the modulation of the expression of oncogenes and oncosuppressors, the autophagic flux and the onset of apoptosis, with examples of their cytospecificity; and iii) the role of alcoholic beverage consumption within particular dietary regimens, including the Mediterranean diet. Taking all the data into account, several alcohol-associated bioactive dietary compounds appear capable to modulate peculiar intracellular pathways predisposing to or protecting from cancer. Advances in the nutrigenetic, nutrigenomic and nutriepigenetic knowledge complementing the biochemical and molecular approaches will help in unveiling their impact on health outcome.

Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on MCF-7 Breast Cancer Cells.

The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damage-inducing and DNA methylation-changing abilities. We demonstrate that small modifications in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, with PK9320 and PK9323 being eligible candidates as "anticancer compounds" and "anticancer epi-compounds" and PK083 a "damage-corrective" compound on human breast adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents and chemical probes.

Establishment and Preliminary Characterization of Three Astrocytic Cells Lines Obtained from Primary Rat Astrocytes by Sub-Cloning.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.

Melatonin reduces inflammatory response in human intestinal epithelial cells stimulated by interleukin-1ß.

Melatonin is the main secretory product of the pineal gland, and it is involved in the regulation of periodic events. A melatonin production independent of the photoperiod is typical of the gut. However, the local physiological role of melatonin at the intestinal tract is poorly characterized. In this study, we evaluated the anti-inflammatory activities of melatonin in an in vitro model of inflamed intestinal epithelium. To this purpose, we assessed different parameters usually associated with intestinal inflammation using IL-1ß-stimulated Caco-2 cells. Differentiated monolayers of Caco-2 cells were preincubated with melatonin (1 nmol/L-50 µmol/L) and then exposed to IL-1ß. After each treatment, different inflammatory mediators, DNA-breakage, and global DNA methylation status were assayed. To evaluate the involvement of melatonin membrane receptors, we also exposed differentiated monolayers to melatonin in the presence of luzindole, a MT1 and MT2 antagonist. Our results showed that melatonin, at concentrations similar to those obtained in the lumen gut after ingestion of dietary supplements for the treatment of sleep disorders, was able to attenuate the inflammatory response induced by IL-1ß. Anti-inflammatory effects were expressed as both a decrease of the levels of inflammatory mediators, including IL-6, IL-8, COX-2, and NO, and a reduced increase in paracellular permeability. Moreover, the protection was associated with a reduced NF-κB activation and a prevention of DNA demethylation. Conversely, luzindole did not reverse the melatonin inhibition of stimulated-IL-6 release. In conclusion, our findings suggest that melatonin, through a local action, can modulate inflammatory processes at the intestinal level, offering new opportunities for a multimodal management of IBD.

Cytotoxic Activity of the Histone Deacetylase 3-Selective Inhibitor Pojamide on MDA-MB-231 Triple-Negative Breast Cancer Cells.

We examined the effects of the ferrocene-based histone deacetylase-3 inhibitor Pojamide (N¹-(2-aminophenyl)-N8-ferrocenyloctanediamide) and its two derivatives N¹-(2-aminophenyl)-N6-ferrocenyladipamide and N¹-(2-aminophenyl)-N8-ferroceniumoctanediamide tetrafluoroborate on triple-negative MDA-MB-231 breast cancer cells. Viability/growth assays indicated that only the first two compounds at 70 µM concentration caused an approximate halving of cell number after 24 h of exposure, whereas the tetrafluoroborate derivative exerted no effect on cell survival nor proliferation. Flow cytometric and protein blot analyses were performed on cells exposed to both Pojamide and the ferrocenyladipamide derivative to evaluate cell cycle distribution, apoptosis/autophagy modulation, and mitochondrial metabolic state in order to assess the cellular basis of the cytotoxic effect. The data obtained show that the cytotoxic effect of the two deacetylase inhibitors may be ascribed to the onset of non-apoptotic cell death conceivably linked to a down-regulation of autophagic processes and an impairment of mitochondrial function with an increase in intracellular reactive oxygen species. Our work expands the list of autophagy-regulating drugs and also provides a further example of the role played by the inhibition of autophagy in breast cancer cell death. Moreover, the compounds studied may represent attractive and promising targets for subsequent molecular modeling for anti-neoplastic agents in malignant breast cancer.

Mid-region parathyroid hormone-related protein is a genome-wide chromatin-binding factor that promotes growth and differentiation of HB2 epithelial cells from the human breast.

Human parathyroid hormone-related protein (PTHrP) is a polyhormone that undergoes proteolytic cleavage producing smaller peptides which exert diversified biological effects. PTHrP signalization is prominently involved in breast development and physiology, but the studies have been focused onto either N-terminal species or full-length protein introduced by gene transfer techniques. Our present work investigates for the first time the effect of the mid-region PTHrP secretory form, that is, the fragment [38-94], on HB2 non-tumoral breast epithelial cells. We examined viability/proliferation of cells grown in PTHrP-containing media supplemented with different serum concentration and on different substrates, extending our investigation to check whether (a) by analogy with MDA-MB231 cells, also HB2 cell chromatin possesses genome-wide binding sites for mid-region PTHrP, and (b) the peptide is endowed with modulating properties toward the expression of proliferation/differentiation signatures by HB2 cells. Our results indicate that mid-region PTHrP acts as a cell growth/differentiation stimulator in routine and "nutrient stress" culture conditions, accordingly reprogramming gene expression, and is able to bind to cytogenetic preparations from HB2 cells. This supports the concept that the physiological mechanisms involving PTHrP during breast development may include mature processed forms of the protein different from the N-terminal fragment. © 2018 BioFactors, 45(2):279-288, 2019.

Methylation of cytokines gene promoters in IL-1ß-treated human intestinal epithelial cells.

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-1ß (IL-1ß)-induced intestinal epithelial cells activation. MATERIALS AND METHODS: Differentiated Caco-2 cells were exposed to IL-1ß 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-1ß 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-1ß also increased the release of IL-6 and IL-8 but did not change the IL-10 expression. Finally, cell exposure to IL-1ß decreased the DNMT3b expression, increased DNMT3a and was not able to change DNMT1 expression. CONCLUSIONS: Our results suggest a potential role of IL-1ß as modulator of DNA methylation in activated differentiated Caco-2 cell line.

Molecular Signatures Associated with Treatment of Triple-Negative MDA-MB231 Breast Cancer Cells with Histone Deacetylase Inhibitors JAHA and SAHA.

Jay Amin hydroxamic acid (JAHA; N8-ferrocenylN1-hydroxy-octanediamide) is a ferrocene-containing analogue of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA). JAHA's cytotoxic activity on MDA-MB231 triple negative breast cancer (TNBC) cells at 72 h has been previously demonstrated with an IC50 of 8.45 µM. JAHA's lethal effect was found linked to perturbations of cell cycle, mitochondrial activity, signal transduction, and autophagy mechanisms. To glean novel insights on how MDA-MB231 breast cancer cells respond to the cytotoxic effect induced by JAHA, and to compare the biological effect with the related compound SAHA, we have employed a combination of differential display-PCR, proteome analysis, and COMET assay techniques and shown some differences in the molecular signature profiles induced by exposure to either HDACis. In particular, in contrast to the more numerous and diversified changes induced by SAHA, JAHA has shown a more selective impact on expression of molecular signatures involved in antioxidant activity and DNA repair. Besides expanding the biological knowledge of the effect exerted by the modifications in compound structures on cell phenotype, the molecular elements put in evidence in our study may provide promising targets for therapeutic interventions on TNBCs.