All the cell lines were cultured as previously described29,30,31.
Caco-2 (ATCC, Palo Alto, CA, USA) represent a human colon epithelial cancer cell line used as a study model for the mechanisms underlying colon cancer development, toxicology and, above all, for the analysis of colon cancer processes, absorption and metabolism in food science, nutrition and drug discovery32.
Caco-2 cells, cultured as a monolayer, differentiate after 21 days and are a model for studying the paracellular movement of compounds across a monolayer. Furthermore, they express several morphological and biochemical characteristics typically shown by mature small intestinal normal enterocytes33,34. Numerous studies have revealed that differentiated Caco-2 cells constitute a polarized monolayer characterized by domes, with microvilli on the apical side and tight junctions between adjacent cells20. Thus, Caco-2 cells were allowed to grow to confluence and differentiated after 21 days for here-showed biological experiments.
The HepG-2 cell line (ATCC, Palo Alto, CA, USA), a hepatic cell line derived from a human hepatoblastoma, expresses a variety of liver-specific metabolic functions35. These cells have an epithelial-like morphology and secrete a variety of important plasma proteins. HepG-2 cells are a suitable model for studying the metabolic fate/effects of xenobiotic on metabolism.
Molecules for pre-treatments, treatments and positive controls
Resveratrol and pterostilbene are commercially available (Merck Life Science, Milano, Italy); dimers (±)-trans-δ-viniferin and (±)-trans-pterostilbene dehydrodimer (Fig. 1) were synthesized according to reported procedures36.
For treatments with (±)-trans-δ-viniferin and positive control molecules, we used both cell lines while for the combined treatments we used the Caco-2 cell line only. The determination of treatment times was established on a functional basis. Differentiated Caco-2 cells were treated for 3 h mimicking the average time of in vivo intestinal absorption. In parallel, for HepG-2 liver cancer cells, a time of 24 h was used simulating in vivo hepatic metabolism.
Bobcat339 hydrochloride (B339H), a selective inhibitor of TET1 and TET2 (at doses 33 and 75 μM, respectively), that does not inhibit DNMT3A was selected for DNA methylation studies37. The treatment was carried out for 72 h in order to evidence appreciable DNA methylation differences, after, at least, two or three cell cycles.
Moreover, as second DNA methylation-specific positive control, 5-Azacytidine (5-AzaC, 10 μM), a widely known molecule that acts preferentially on the complex DNMTs-S-adenosine methionine (SAM), was used for 48 h (with fresh adding at 24 h due to low half-life of this molecule).
For B339H and 5-AzaC concentration, literature data were used37,38.
To simulate a specific genotoxic insult, we pretreated the Caco-2 cells with sodium arsenite (SA) [10 µg/L] for 72 h and then incubated the cells with each stilbenoid for 3 h. This concentration of arsenic was used in accordance with current legislation in Europe and United States and according to the guidelines of the World Health Organization that defines a limit value of arsenic in drinking water39.
All stilbenoids and B339H were solubilized in dimethyl sulfoxide (DMSO) taking care that the vehicle volume is always less than 0.01% of the cell medium to avoid self-effect on the followed biological and epigenetic/molecular endpoints40,41. SA and 5-AzaC were dissolved in cell medium. With the aim to mimic the low concentrations occurring in the intestinal lumen after ingestion of foods or vegetables rich in these substances, we have chosen to treat the cells with two doses of stilbenoids, one corresponding to the respective IC50 concentration and the other one order of magnitude lower than the IC50.
For the treatments of Caco-2 cells with resveratrol (indicated as stilbenoid 1 in Fig. 1), we used one of the concentrations reported and used by Storniolo et al.42. HepG-2 cell line was treated with resveratrol at the same concentration used for Caco-2 cells, referring to the concentrations used by Akashina et al. and Aja et al.43,44.
In order to evaluate the IC50 (Fig. 9), Caco-2 cell line was treated with stilbenoids (indicated as 2–4 in Fig. 1) for 24 h (Table 4).
All the cell-cultures, pre-treatments and treatments are summarized in Table 5. A total amount of 19 cell-cultures was carried out, 7 of which were also pre-treated.
Morphological cell aspects were considered to evaluate the onset of changes induced by treatments. By respecting the same microscope magnification (Leica, 40 ×), the observations were aimed to evaluate: (i) cell density, (ii) adhesion to the substrate and (iii) presence of cell suffering (bubble and/or shriveled cells). With these bases, cellular carpets after treatments were photographed and compared with the control ones.
Genomic DNA isolation
Isolation of genomic DNA from cells was carried out with the PureLink Genomic DNA Kit (Invitrogen, UK) as previously described45 and DNAzol (Invitrogen®) following the manufacturer recommendations. The obtained DNA was quantified by NanoDrop® ND-1000.
Epigenomic assessment of DNA methylation
To assess the possible genomewide changes in DNA methylation, Methylation-Sensitive Arbitrarily-Primed PCR (MeSAP-PCR) was performed as previously described25 on cells treated with stilbenoids and DMSO vehicle only.
Quantitation of DNA damage by Comet Assay
Comet Assay method46, also called Single Cell Gel Electrophoresis (SCGE), is a micro-electrophoretic technique through which the nuclear DNA, possibly fragmented, can be visualized by observing the shape of the nuclei and the presence of a tail similar to a comet visible through observation under a epifluorescence microscopy. The extent of DNA damage is visually assessed by visualization of the comet’s tail (see Fig. 8b and c); an image analysis software is also available to measure different parameters related to the dimensions of the comet’s tail. With alkaline Comet assay, it is possible to detect DNA damage of different entities: single and double DNA strand breaks, presence of adducts, alterations convertible into breaks such as labile alkali sites or incisions caused by the repair mechanisms by excision of nucleotides. We performed alkaline Comet assay as previously described45,47 with some differences. Briefly, at the end of the incubation times of the different treatments, the treated and untreated cells were recovered to be analyzed using the OxiSelect™ Comet Assay kit (Cell Biolabs Inc, San Diego, CA), a rapid and sensitive kit for measuring DNA cellular damage. Once recovered, the cells were mixed with molten agarose and then placed on the slide. After treating the slides with a lysis and alkaline solution the samples on the slide were subjected to horizontal electrophoresis, in order to separate the intact DNA from the damaged DNA fragments. After electrophoresis, the samples were air dried, stained with a fluorescent DNA probe and visualized by epifluorescence microscopy (Nikon). Lastly, the images were analyzed with CASP software (Biotools) to obtain the “tail moment”, a quantitative parameter that express the length of the tail in function of the nucleus size.