Effect of beta-carotene-rich tomato lycopene beta-cyclase ( tlcy-b) on cell growth inhibition in HT-29 colon adenocarcinoma cells.

Lycopene beta-cyclase (tlcy-b) tomatoes, obtained by modulating carotenogenesis via genetic engineering, contain a large amount of beta-carotene, as clearly visible by their intense orange colour. In the present study we have subjected tlcy-b tomatoes to an in vitro simulated digestion and analysed the effects of digestate on cell proliferation. To this aim we used HT-29 human colon adenocarcinoma cells, grown in monolayers, as a model. Digested tomatoes were diluted (20 ml, 50 ml and 100 ml/l) in culture medium and added to the cells for different incubation times (24 h, 48 h and 72 h). Inhibition of cell growth by tomato digestate was dose-dependent and resulted from an arrest of cell cycle progression at the G0/G1 and G2/M phase and by apoptosis induction. A down-regulation of cyclin D1, Bcl-2 and Bcl-xl expression was observed. We also found that heat treatment of samples before digestion enhanced beta-carotene release and therefore cell growth inhibition. To induce with purified beta-carotene solubilised in tetrahydrofuran the same cell growth inhibition obtained with the tomato digestate, a higher amount of the carotenoid was necessary, suggesting that beta-carotene micellarised during digestion is utilised more efficiently by the cells, but also that other tomato molecules, reasonably made available during digestion, may be present and cooperate with beta-carotene in promoting cell growth arrest.

Novel approach for food safety evaluation. Results of a pilot experiment to evaluate organic and conventional foods.

There is evidence that organic food often contains relatively high amounts of natural toxic compounds produced by fungi or plants, whereas corresponding conventional food tends to contain more synthetic toxins such as pesticide residues, but only a few studies have evaluated the impact of their consumption on health. This study proposes a novel approach to evaluate the potential health risk of organic compared to conventional food consumption, that is, the assay of sensitive markers of cell function in vulnerable conditions. The markers utilized were intestinal and splenic lymphocyte proliferative capacity and liver acute-phase reaction, both responding to the presence of toxins. The vulnerable conditions in which body defenses can be less efficient were weaning and protein-energy malnutrition. This study reports the results of a pilot experiment on one sample of eight varieties of organically and conventionally grown wheat. Weaned rats were assigned to two groups fed conventional (CV) or organic (ORG) wheat for 30 days. Each group was divided in two subgroups of well-nourished (WN) or protein-energy-malnourished (PEM) rats. For each rat, the lymphocyte proliferation was assayed by [(3)H]thymidine incorporation after stimulation of cells with a mitogen, in a culture medium containing either commercial fetal calf serum (FCS) or the corresponding rat serum (RS) to mimic the in vivo proliferative response. The acute-phase proteins (albumin, transthyretin, transferrin, ceruloplasmin, retinol-binding protein) were measured in plasma by Western blotting and immunostaining with specific antibodies. The proliferative response of lymphocytes cultured with FCS and the amount of acute-phase proteins of rats fed the ORG wheat sample, either WN or PEM, did not differ from those of rats fed the CV wheat sample. However, the proliferative response of lymphocytes cultured with RS was inhibited in PEM-CV compared with PEM-ORG. The content of mycotoxins was highest in the organic sample, and therefore the immunotoxic effect was probably due to other contaminants in the CV wheat. In conclusion, these results indicate that the conventional wheat sample tested represented a higher risk for lymphocyte function than the wheat sample organically grown, at least in vulnerable conditions.

The new murine hepatic 3A cell line responds to stress stimuli by activating an efficient Unfolded Protein Response (UPR).

In the present study we have investigated the properties of a novel cell line (3A cells) obtained from the liver of 14.5 days post coitum (dpc) wild-type mouse embryo. 3A cells morphology was characterized by fluorescent localization of F-actin and ß-catenin. The expression of specific genes and proteins essential to liver function in these cells was comparable or even more efficient then in the differentiated hepatocytic cell line MMH-D6. 3A cells also showed the capability to excrete molecules in extracellular spaces resembling functional bile canaliculi, glycogen storage activity and the ability to control retinol-binding protein 4 secretion in response to retinol deprivation. Their response to the exogenous stress stimulus induced by tunicamycin was analysed by PCR Pathway Array containing 84 genes involved in the Unfolded Protein Response (UPR). 3A cells were shown to activate the UPR following a typical stressful event, indicating that this cellular model could be further exploited to investigate hepatic proteins secretion and specific reaction to different injuries.

Co-cultures of enterocytes and hepatocytes for retinoid transport and metabolism.

Dietary retinoid bioavailability involves the interplay of the intestine (transport and metabolism) and the liver (secondary metabolism). To reproduce these processes in vitro, differentiated human intestinal Caco-2/TC7 cells were co-cultured with two hepatocyte cell lines. Murine 3A cells and the more highly differentiated human HepaRG hepatocytes were both shown to respond to ß-carotene (BC) and retinol (ROH) treatment by secreting Retinol Binding Protein 4 (RBP4). In co-culture experiments, Caco-2/TC7 were differentiated on filter inserts and transferred for the time of the experiment to culture wells containing confluent 3A or differentiated HepaRG cells. Functionality of the co-cultures was assayed using as endpoints the retinol-dependent secretion of RBP4 and the retinoic acid-dependent induction of CYP26A1 in hepatocytes. BC and ROH added to intestinal Caco-2/TC7 induced a reduction in intracellular RBP4 levels in the underlying hepatocytes and its secretion into the medium. HepaRG hepatocytes were also shown to up-regulate the expression of CYP26A1 mRNA in response to retinoid treatment. This in vitro model represents a useful tool to analyze the absorption and metabolism of retinoids and could be further developed to investigate other dietary compounds and molecules of pharmacological interest.

The growth-inhibitory effects of tomatoes digested in vitro in colon adenocarcinoma cells occur through down regulation of cyclin D1, Bcl-2 and Bcl-xL.

In the present study, we utilised an in vitro digestion procedure to deliver molecules contained in tomatoes to cultured cells and to analyse potential mechanisms underlying the antitumoural effects of tomatoes reported in the literature. Ripe tomatoes underwent in vitro simulated digestion and the aqueous fraction obtained was delivered to HT-29 and HCT-116 colon adenocarcinoma cells. The amount of lycopene released during digestion and transferred to the aqueous fraction during digestion was 10-fold lower than that present in tomato homogenate before digestion. The carotenoid was accumulated by colon adenocarcinoma cells in a dose-dependent manner after the addition of tomato digestate (20-100 ml/l) for 24 h. Tomato digestate inhibited the growth of HT-29 and HCT-116 cells in a dose-dependent manner. Growth inhibition resulted from an arrest of cell cycle progression at the G0/G1 phase and by apoptosis induction. A down regulation of cyclin D1, Bcl-2 and Bcl-xL expression was also observed, without apparent changes in p53, p21, p27 and Bax. In conclusion, the present data demonstrate that the in vitro digestion procedure represents a useful approach to supply tomato to colon cultured cells. Moreover, we have shown that tomato digestate is able to inhibit the growth of colon cancer cells by modulating the expression of regulators of the cell cycle and apoptosis.

Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane.

Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH(2)-terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process.

Hepatic synthesis, maturation and complex formation between retinol-binding protein and transthyretin.

The retinol/retinol-binding protein/transthyretin complex, that carries and delivers hydrophobic retinol molecules to target cells, is assembled in the hepatocyte endoplasmic reticulum. In this paper, we review data related to events that lead to the formation of this complex, including transthyretin oligomerization and retinol-binding protein secretion. Our studies on transthyretin oligomerization have demonstrated that cleavage of signal peptide and the environment of endoplasmic reticulum influence transthyretin oligomerization. In vitro, mutated transthyretin without signal sequence fails to form dimers, while wild-type transthyretin is translocated into the microsomes where it forms dimers and small amounts of tetramers. In vivo, tetramers were detected in HepG2 cells but not in transfected Cos cells, suggesting that tissue-specific factors affect tetramer stability. In vitamin A deficiency, retinol-binding protein secretion is blocked and the protein accumulates in the endoplasmic reticulum, from where it is promptly released after retinol repletion. We use MMH cells to identify factors involved in complex formation, retention and secretion, the crucial steps to understand the molecular mechanisms underlying vitamin A homeostasis. In parallel, studies on vitamin A transport in fish are in progress; retinol-binding protein and transthyretin have already been characterized in different fish species.