Raman-AFM-fluorescence-guided impact of linoleic and eicosapentaenoic acids on subcellular structure and chemical composition of normal and cancer human colon cells.

The regular overconsumption of high-energy food (rich in lipids and sugars) results in elevated nutrient absorption in intestine and consequently excessive accumulation of lipids in many organs e.g.: liver, adipose tissue, muscles. In the long term this can lead to obesity and obesity-associated diseases e.g. type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, inflammatory bowel disease (IBD). In the presented paper based on RI data we have proved that Raman maps can be used successfully for subcellular structures visualization and analysis of fatty acids impact on morphology and chemical composition of human colon single cells - normal and cancer. Based on Raman data we have investigated the changes related to endoplasmic reticulum, mitochondria, lipid droplets and nucleus. Analysis of ratios calculated based on Raman bands typical for proteins (1256, 1656 cm-1), lipids (1304, 1444 cm-1) and nucleic acids (750 cm-1) has confirmed for endoplasmic reticulum the increased activity of this organelle in lipoproteins synthesis upon FAs supplementation; for LDs the changes of desaturation of accumulated lipids with the highest unsaturation level for CaCo-2 cells upon EPA supplementation; for mitochondria the stronger effect of FAs supplementation was observed for CaCo-2 cells confirming the increased activity of this organelle responsible for energy production necessary for tumor development; the weakest impact of FAs supplementation was observed for nucleus for both types of cells and both types of acids. Fluorescence imaging was used for the investigations of changes in LDs/ER morphology. Our measurements have shown the increased area of LDs/ER for CaCo-2 cancer cells, and the strongest effect was noticed for CaCo-2 cells upon EPA supplementation. The increased participation of lipid structures for all types of cells upon FAs supplementation has been confirmed also by AFM studies. The lowest YM values have been observed for CaCo-2 cells including samples treated with FAs.

Repair kinetics of acrolein- and (E)-4-hydroxy-2-nonenal-derived DNA adducts in human colon cell extracts.

ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) play a role in the pathogenesis of colon cancer. Upon oxidation, PUFAs generate α,ß-unsaturated aldehydes or enals, such as acrolein (Acr) and (E)-4-hydroxy-2-nonenal (HNE), which can form cyclic adducts of deoxyguanosine (Acr-dG and HNE-dG, respectively) in DNA. Both Acr-dG and HNE-dG adducts have been detected in human and animal tissues and are potentially mutagenic and carcinogenic. In vivo levels of Acr-dG in DNA are at least two orders of magnitude higher than those of HNE-dG. In addition to the facile reaction with Acr, the higher levels of Acr-dG than HNE-dG in vivo may be due to a lower rate of repair. Previous studies have shown that HNE-dG adducts are repaired by the NER pathway (Choudhury et al. [42]). We hypothesize that Acr-dG adducts are repaired at a slower rate than HNE-dG and that HNE-dG in DNA may influence the repair of Acr-dG. In this study, using a DNA repair synthesis assay and a LC-MS/MS method, we showed that Acr-dG in a plasmid DNA is repaired by NER proteins, but it is repaired at a much slower rate than HNE-dG in human colon cell extracts, and the slow repair of Acr-dG is likely due to poor recognition/excision of the lesions in DNA. Furthermore, using a plasmid DNA containing both adducts we found the repair of Acr-dG is significantly inhibited by HNE-dG, however, the repair of HNE-dG is not much affected by Acr-dG. This study demonstrates that the NER repair efficiencies of the two major structurally-related in vivo cyclic DNA adducts from lipid oxidation vary greatly. More importantly, the repair of Acr-dG can be significantly retarded by the presence of HNE-dG in DNA. Therefore, this study provides a mechanistic explanation for the higher levels of Acr-dG than HNE-dG observed in tissue DNA.

Versicolorin A, a precursor in aflatoxins biosynthesis, is a food contaminant toxic for human intestinal cells.

Aflatoxin B1 (AFB1) is the most potent carcinogen among mycotoxins. Its biosynthesis involves the formation of versicolorin A (VerA), whose chemical structure shares many features with AFB1. Our data revealed significant levels of VerA in foodstuff from Central Asia and Africa. Given this emerging food risk, it was of prime interest to compare the toxic effects of the two mycotoxins against cells originating from the intestinal tract. We used human colon cell lines (Caco-2, HCT116) to investigate the cytotoxic process induced by the two mycotoxins. Contrary to AFB1, a low dose of VerA (1 µM) disturbed the expression level of thousands of genes (18 002 genes). We show that the cytotoxic effects of low doses of VerA (1-20 µM) were stronger than the same low doses of AFB1 in both Caco-2 and HCT116 cell lines. In Caco-2 cells, VerA induced DNA strand breaks that led to apoptosis and reduced DNA replication of dividing cells, consequently inhibiting cell proliferation. Although VerA was able to induce the p53 signaling pathway in p53 wild-type HCT116 cells, its toxicity process did not mainly rely on p53 expression since similar cytotoxic effects were also observed in HCT116 cells that do not express p53. In conclusion, this study provides evidence of the risk of food contamination by VerA and shed light on its toxicological effect on human colon cells.

Supplement comprising of laccase and citric acid as an alternative for antibiotics: In vitro triggers of melanin production.

An indiscriminate use of antibiotics in humans and animals has led to the widespread selection of antibiotic-resistance, thus constricting the use of antibiotics. A possible solution to counter this problem could be to develop alternatives that can boost the host immunity, thus reducing the quantity and frequency of antibiotic use. In this work, for the first time, citric acid and laccase were used as extracellular inducers of melanin production in yeast cells and human cell lines. It is proposed that the formulation of laccase and citric acid together could further promote melatonin-stimulated, melanocyte-derived melanin production. Melanization as a probe of immunity described in this study, is an easy and a rapid test compared to other immunity tests and it allows performing statistical analyses. The results showed the synergistic effect of citric acid and laccase on melanin production by yeast cells, with significant statistical differences compared to all other tested conditions (p: 0.0005-0.005). Laccase and citric acid together boosted melanin production after 8 days of incubation. An increase in melanin production by two human colon cells lines (Cacao-2/15 and HT-29) was observed on supplementation with both laccase and citric acid in the cell growth medium. Produced melanin showed antimicrobial properties similar to antibiotics. Therefore, a formulation with citric acid and laccase may prove to be an excellent alternative to reduce the antibiotic use in human and animal subjects.

Biphasic regulation of spindle assembly checkpoint by low and high concentrations of resveratrol leads to the opposite effect on chromosomal instability.

Resveratrol (RSV) is a naturally occurring polyphenolic phytoalexin possessing numerous health-promoting effects. Chromosomal instability (CIN), usually results from defective spindle assembly checkpoint (SAC), is a major contributor to many diseases. While it's recently recognized that RSV exhibits a nonlinear dose response for disease prevention, whether it's the case for its role in CIN remains unknown. Here, we investigated the potential of a broad range of RSV concentrations (0.01-100µM) on CIN and the underlying mechanisms in human normal colon epithelial NCM460 cells. CIN was measured by cytokinesis-block micronucleus assay; mitotic fidelity was determined by aberrant mitosis analysis; SAC activity was assessed by nocodazole-challenge assay, and the expression of SAC genes was examined by RT-qPCR. We found that 0.1µM RSV significantly reduced CIN (P<0.01), while 100µM RSV significantly induced it (P<0.05). Mitotic infidelity was significantly prevented by 0.1µM RSV but promoted by 100µM RSV (P<0.05 for both). Moreover, the function of SAC was sustained and impaired by 0.1µM and 100µM RSV, respectively. Several SAC genes, including Aurora-B, Aurora-C, Plk-1 and CENP-E, were significantly up-regulated and down-regulated by 0.1µM and 100µM RSV, respectively (P<0.05). In conclusion, RSV exhibited a biphasic dose-dependent effect on CIN that was exerted via the regulation of mitotic fidelity through the SAC network. The health implications of these findings were summarized.

Targeted proteome analysis with isotope-coded protein labels for monitoring the influence of dietary phytochemicals on the expression of cytoprotective proteins in primary human colon cells.

Cytoprotective effects by upregulating cellular expression levels of antioxidant proteins are attributed to a significant number of food ingredients. Evaluation of those cytoprotective effects and identification of the most active components requires reliable and comprehensive proteomic strategies. Thus, promising potential bioactive ingredients could be determined for the prevention of various diseases, including colon cancer formation. We established a multiplexed nanoLC-MS/MS targeted proteomic method, operated in scheduled selected reaction monitoring mode (sSRM), to record modulation of the expression levels of six major antioxidant proteins induced by dietary phytochemicals. Relative quantification was achieved by isotope-coded protein labels (ICPLs) and based on two to three proteotypic peptides per target protein. The assay provided accurate (mean relative error 6.4%) and precise (mean RSD 7.4%) quantification. Incubation experiments were carried out in primary human colon epithelial cells (HCoEpiCs) and revealed significant upregulation of NAD(P)H dehydrogenase [quinone] 1 (up to threefold) and thioredoxin reductase 1 (up to twofold) by 10µM sulforaphane (from broccoli), 5µM carnosol (rosemary), and 20µM cinnamaldehyde (cinnamon). The latter two substances additionally upregulated heme oxygenase-1 and were identified as the most active components in the test set. The results provide additional evidence for nutritive cytoprotection in human colon cells. SIGNIFICANCE: Targeted proteome analysis using LC coupled to scheduled selected reaction monitoring (sSRM)-MS is a highly flexible and reliable method to monitor protein expression profiles. The present study screened modulators occurring in food, which may be protective against colon cancer by inducing cytoprotective enzymes. Primary human colonic epithelial cells were used because they model the conditions in healthy gut tissue better than immortalized cells. Thus, an LC-MS/MS-sSRM protocol was established and validated including relative quantification of cytoprotective protein expression by isotope-coded protein labels, because metabolic labelling cannot be applied for primary cells. The present study demonstrated that the major components of cinnamon and rosemary, respectively, i.e. cinnamaldehyde and carnosol, are potent alimentary candidates to increase the expression of cytoprotective enzymes in the human colon. Among the investigated enzymes, NAD(P)H dehydrogenase [quinone] 1 (NQO1) was most susceptible towards modulation by phytochemicals. NQO1 exerts its cytoprotective activity by detoxifying electrophilic and oxidative xenobiotics with quinone structure.

Differential stress reaction of human colon cells to oleic-acid-stabilized and unstabilized ultrasmall iron oxide nanoparticles.

Therapeutic engineered nanoparticles (NPs), including ultrasmall superparamagnetic iron oxide (USPIO) NPs, may accumulate in the lower digestive tract following ingestion or injection. In order to evaluate the reaction of human colon cells to USPIO NPs, the effects of non-stabilized USPIO NPs (NS-USPIO NPs), oleic-acid-stabilized USPIO NPs (OA-USPIO NPs), and free oleic acid (OA) were compared in human HT29 and CaCo2 colon epithelial cancer cells. First the biophysical characteristics of NS-USPIO NPs and OA-USPIO NPs in water, in cell culture medium supplemented with fetal calf serum, and in cell culture medium preconditioned by HT29 and CaCo2 cells were determined. Then, stress responses of the cells were evaluated following exposure to NS-USPIO NPs, OA-USPIO NPs, and free OA. No modification of the cytoskeletal actin network was observed. Cell response to stress, including markers of apoptosis and DNA repair, oxidative stress and degradative/autophagic stress, induction of heat shock protein, or lipid metabolism was determined in cells exposed to the two NPs. Induction of an autophagic response was observed in the two cell lines for both NPs but not free OA, while the other stress responses were cell- and NP-specific. The formation of lipid vacuoles/droplets was demonstrated in HT29 and CaCo2 cells exposed to OA-USPIO NPs but not to NS-USPIO NPs, and to a much lower level in cells exposed to equimolar concentrations of free OA. Therefore, the induction of lipid vacuoles in colon cells exposed to OA utilized as a stabilizer for USPIO NPs is higly amplified compared to free OA, and is not observed in the absence of this lipid in NS-USPIO NPs.

Evaluation of uptake and transport of cationic and anionic ultrasmall iron oxide nanoparticles by human colon cells.

Nanoparticles (NPs) are in clinical use or under development for therapeutic imaging and drug delivery. However, relatively little information exists concerning the uptake and transport of NPs across human colon cell layers, or their potential to invade three-dimensional models of human colon cells that better mimic the tissue structures of normal and tumoral colon. In order to gain such information, the interactions of biocompatible ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) (iron oxide core 9-10 nm) coated with either cationic polyvinylamine (aminoPVA) or anionic oleic acid with human HT-29 and Caco-2 colon cells was determined. The uptake of the cationic USPIO NPs was much higher than the uptake of the anionic USPIO NPs. The intracellular localization of aminoPVA USPIO NPs was confirmed in HT-29 cells by transmission electron microscopy that detected the iron oxide core. AminoPVA USPIO NPs invaded three-dimensional spheroids of both HT-29 and Caco-2 cells, whereas oleic acid-coated USPIO NPs could only invade Caco-2 spheroids. Neither cationic aminoPVA USPIO NPs nor anionic oleic acid-coated USPIO NPs were transported at detectable levels across the tight CacoReady™ intestinal barrier model or the more permeable mucus-secreting CacoGoblet™ model.