Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects

We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 microg/ml) the number of cells in the G2/M phase increased to 51.82+/-2.69% relative to control cells (15.1+/-2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 microg/ml) to exert rapid inhibition of p38 (38.7+/-4.7%) and CREB (28.6+/-5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9+/-9.3%). Our data suggest that olive oil polyphenols may exert chemopreventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development.     

Effect of consumption of phenols from olives and extra virgin olive oil on LDL oxidizability in healthy humans

A high intake of olive oil has been proposed as an explanation for the low incidence of coronary heart disease in Mediterranean countries, but it is unclear whether olive oil offers specific benefits beyond a low content of saturated fat. Some types of extra virgin olive oil are rich in non-polar phenols, which might be taken up by plasma LDL particles and protect these from becoming atherogenic by oxidative modification. In a pilot study we found that consumption of 47 g fortified olive oil containing 31 mg phenols significantly increased the lag time of LDL oxidation from 112 ± 5 min before to 130 ± 7 min 2h after the meal. However, this study was not controlled, and in the current study we therefore investigated whether olive oil phenols increase the lag time of LDL oxidation in postprandial samples when compared with a control group.

Twelve healthy men and women consumed four different olive oil supplements with a meal on four separate occasions: one similar to the supplement in the pilot study (positive control); one containing mainly non-polar olive oil phenols; one containing mainly polar olive oil phenols; and one without phenols (placebo). Lag time significantly increased 2 h after the meals with the positive control (8 ± 2 min), the polar phenols (8 ± 2 min), and the placebo (8 ± 2 min), but not after the non-polar phenols (-0.4 ± 3 min). Increases were not statistically different between supplements.

These results indicate that the lag time of LDL-oxidation is increased after consumption of a meal. This increase is probably due to non-specific meal or time effects and not to phenols from olives or olive oil. Furthermore, these findings stress the need for adequate controlled studies to avoid misinterpretations of the data.     

氯化铬和吡啶羧酸铬在Caco-2细胞中的摄取和转运

采用一种体外培养的人小肠上皮细胞模型Caco-2研究了时间、浓度、温度对氯化铬和吡啶羧酸铬细胞摄取和跨细胞转运的影响。旨在探讨氯化铬和吡啶羧酸铬在小肠上皮细胞中的摄取和转运特点。结果表明:Caco-2细胞对氯化铬和吡啶羧酸铬的摄取和转运随浓度、时间而成线性增加,当温育温度从37℃降到4℃时,摄取和转运有下降趋势(P>0.05)。氯化铬和吡啶羧酸铬从肠腔侧(AP侧)到基底侧(BL侧)的表观通透系数(Papp)近似于BL侧到AP侧(分别为0.95～1.41倍和0.84～1.07倍)。氯化铬和吡啶羧酸铬的摄取率分别为(0.88±0.08)%和(4.73±0.60)%,转运率分别为(2.11±0.05)%和(9.08±0.25)%。结果提示氯化铬和吡啶羧酸铬是以被动扩散为主要方式被Caco-2细胞摄取和转运。     

Uptake of 3,4-methylenedioxymethamphetamine and its related compounds by a proton-coupled transport system in Caco-2 cells

3,4-Methylenedioxymethamphetamine (MDMA) is an illegal amphetamine-type stimulant (ATS) that is abused orally in the form of tablets for recreational purposes. The aim of this work is to investigate the absorption mechanism of MDMA and other related compounds that often occur together in ATS tablets, and to determine whether such tablet components interact with each other in intestinal absorption. The characteristics of MDMA uptake by the human intestinal epithelial Caco-2 cell line were investigated. The Michaelis constant and the maximal uptake velocity at pH 6.0 were 1.11 mM and 13.79 nmol/min/mg protein, respectively, and the transport was electroneutral. The initial uptake rate was regulated by both intra- and extracellular pH. MDMA permeation from the apical to the basolateral side was inferior to that in the reverse direction, and a decrease in apical pH enhanced MDMA permeation from the basolateral to the apical side. These facts indicate that this transport system may be an antiporter of H⁺. However, under physiological conditions, the proton gradient cannot drive the MDMA uptake because it is inwardly directed. Large concentration differences of MDMA itself drive this antiporter. Various compounds with similar amine moieties inhibited the uptake, but substrates of organic cation transporters (OCT1-3) and an H⁺-coupled efflux antiporter, MATE, were not recognized.     

Amino acid facilitates absorption of copper in the Caco-2 cell culture model

Aim

Copper deficiency could cause fatal hematological and neurological disorders or other diseases. Amino acids are involved in the absorption of copper ions. The purpose of this study is to evaluate the absorption of copper in amino acid complex forms and determine its mechanism in the Caco-2 cell culture model.

Main methods

The human colonic adenocarcinoma cell line Caco-2 culture model was used to determine the permeability of copper ions in inorganic form (CuSO₄) and the amino acid complex forms. Lysine and methionine, as well as carboplatin were used to determine the possible involvement of amino acid transporters or copper transporter 1 (CTR1).

Key findings

The results showed that all of the amino acid complex forms facilitated copper absorption. The apparent permeabilities of copper ions in these complex forms were at least 7.6 fold higher than those in the CuSO₄ form. The permeability rank order of copper in various amino acid complex forms was Cu-glutamate < Cu-lysine = Cu-aspartic acid = Cu methionine < Cu-arginine < Cu-(lysine/glutamate). Mechanistic studies revealed that the enhanced absorption of copper in copper amino acid complexes could be the result of enhanced uptake (as in Cu-methionine complex) or enhanced basolateral efflux (as in Cu-lysine complex). Copper transporter 1 (or CTR1) inhibitor carboplatin did not affect the absorption of copper in Cu-methionine complex, suggesting that the dominant pathway for copper amino acid complexes is not CTR1.

Significance

Enhanced absorption of copper ions in amino acid complex appears to be mediated by amino acid transporters.     

Inhibition of adhesion of Clostridium difficile to Caco-2 cells

Abstract For many microorganisms, including Clostridium difficile , mucosal association is an important factor influencing intestinal colonisation and subsequent infection. Inhibition of adhesion of C. difficile to intestinal mucosa could be a new promising strategy for prevention and treatment of antibiotic-associated diarrhoea. We investigated the possibilities of influencing the adhesion of C. difficile by xylitol and bovine colostrum.whey. Caco-2 cells and C. difficile cells were incubated with 1%, 5% and 10% solutions of xylitol and colostrum. Our study revealed that both xylitol and colostrum inhibited the adhesion of C. difficile to Caco-2 cells. Inhibition by xylitol was dose-dependent. When compared to the control, the count of adherent C. difficile decreased 3.4 times when treated with 1% xylitol, 12 times when 5% xylitol was applied, and 18.7 times when treated with 10% xylitol. The inhibition of adherence by colostrum was partially dose-dependent: 3.1 times in the case of 1%, and 5.5 times in the cases of 5% and 10% colostrum. Further experimental and clinical studies are needed for the application of xylitol and colostrum in the treatment and prophylaxis of pseudomembraneous colitis.     

Effects of Food Lectins on the Transport System of Human Intestinal Caco-2 Cell Monolayers

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 µg/mL) decreased the TER value by 20–40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.     

Structural Effects of Phenolic Acids on the Transepithelial Transport of Fluorescein in Caco-2 Cell Monolayers

The structural specificity of the monocarboxylic acid transporter (MCT) for the transport of phenolic acids was investigated by measuring the inhibitory effect on the fluorescein transport in Caco-2 cell monolayers. Although most of the monohydroxylated derivatives had an inhibitory effect, the di- and tri-hydroxylated ones did not. The methoxylated derivatives were more inhibitory than the hydroxylated ones in all the meta-substituted derivatives, suggesting that meta-hydroxylation of the substrate would decrease the affinity for MCT.     

Improvement of olive oil phenolics content by means of enzyme formulations: Effect of different enzyme activities and levels

The present work deals with the development of an enzymatic treatment aiming at producing high-quality olive oil with increased phenolics content and antioxidant activity. Three different enzyme formulations, specifically pectinase, hemicellulase and cellulase (A), pectinase and hemicellulase (B), and only pectinase (C), were used either at three different level of each or in ternary and binary mixtures at a constant level. All of them were added to the olive paste (Italian cultivar Coratina) at the beginning of the malaxation step. Results demonstrated that an increased enzyme level led to higher phenolics content in the oils, and such an effect was found to be enzyme dependent, being greater when using formulation A, followed by formulations B and C. Two significant correlations were obtained between total polyphenols and o-diphenols contents versus antiradical power (R² = 0.8743 and R² = 0.8635, respectively), which pointed out the effectiveness of the proposed enzymatic treatment to produce olive oils characterized by low susceptibility to oxidation. A synergistic effect between the different enzymatic activities contained in the single formulations was observed by combining enzymes A and B. The ternary mixture was selected as the most efficient enzymatic system ensuring the highest phenolics content increase (40 and 37% for total polyphenols and o-diphenols contents, respectively) compared to the other enzymes when used at the same level.     

Development and characterization of edible chitosan/olive oil emulsion films

The properties of plasticized chitosan-olive oil emulsion films prepared with increasing oil concentrations were investigated. Emulsifying nature of chitosan was enough to stabilize olive oil droplets in the film forming emulsions; hence homogeneous, thin and translucent films were obtained in all cases. The homogeneity of the lipid globules distribution in the films was confirmed by contact angle measurements and optical microscopy. All the tensile properties (Young Modulus, strength and maximum elongation) increased with olive oil concentration and were explained considering the interactions developed between lipid and carbohydrate phases in addition to the lubricant characteristics of the oil. Moisture sorption, water vapor permeation through the films and effective diffusion coefficients decreased as oil concentration increases, as a result of the non-polar nature of the lipid. Total soluble matter measurements were used to confirm the development of strong associations between chitosan and olive oil.     

Influence of surface modification on vitality and differentiation of Caco-2 cells

Abstract It is widely accepted that the functional and morphological differentiation of cells is initiated and determined by the interaction of molecules of the extracellular matrix and adhesion molecules of the cell membrane. To assess the influence of the underlying matrix on the characteristics of cells, enterocyte-like Caco-2 cells were cultivated on substrates commonly used for cell culture as well as on glass coated with hydrophobic layers. Providing the same starting conditions for growth, the parameters investigated on preconfluent Caco-2 cells were the number of adhering cells, the proliferative activity and the degree of differentiation indicated by the expression of three brush border enzymes. Whereas tissue culture treated polystyrene elicited highest rates of adhesion, proliferation, and differentiation, even glass altered the pattern of brush border enzyme expression. The hydrophobic surfaces strongly decreased the adhesion and the proliferation but the surviving cells exhibited a pronounced higher degree of differentiation. Interestingly, each sub-type of hydrophobic matrix triggered a different pattern of brush border enzyme expression. Thus, the development of a certain phenotype of a cell can not only be triggered by certain components of the extracellular matrix but also by artificially prepared surface coatings of the underlying matrix. In the future it seems to be feasible that cells can be programmed by tailoring the surface of the underlying substrate.     

Effect of erythromycin on Shigella infection of Caco-2 cells

Erythromycin (EM), one of the macrolides, shows a dose-dependent effect on Shigella flexneri invasion of Caco-2 cells even at concentrations less than the minimum inhibitory concentration (subMIC). LS13, a strain of S. flexneri 1b, invaded Caco-2 cells in vitro. When the strain was treated with subMIC of EM, the invasion efficiency decreased. The carrier rate of the invasion plasmid containing virulence genes was reduced by EM treatment, as determined by the colony pigmentation test on Congo red agar plates. Presence of the invasion plasmid was found to increase susceptibility of the organisms to EM. The growth of virulent organisms carrying the invasion plasmid was inhibited at 25 microg ml(-1) of EM, whereas the growth of organisms without the plasmid was inhibited at 100 microg ml(-1) of EM. This was supported by the finding that the MIC of EM for a virulent isolate of S. flexneri 2a YSH6000 (6.25 microg ml(-1)) and for the mutant strain del-17 (50 microg ml(-1)), carrying the type III apparatus, impaired plasmid. These findings suggested that EM passed through the type III apparatus and suppressed the growth of invasive organisms selectively. This mechanism may account for the clinical effect of EM on shigellosis.     

Transport of micro-opioid receptor agonists and antagonist peptides across Caco-2 monolayer

Milk is the source of β-casomorphins – biologically active peptides with opioid activity – which are suspected to play various roles in the human body. The local influence of exogenous opioid peptides on gastrointestinal functions has been widely reported. After passing the gut barrier, β-casomorphins may affect the functions of immunological system, as well as dopaminergic, serotoninergic and GABA-ergic systems in brain, regulate the opioid receptor development and elicit behavioral effects. However, possibilities and mechanisms of the intestinal transport of β-casomorphins in human body in vivo have not been reported so far. In our research, the transepithelial transport of μ-opioid receptor agonists – human β-casomorphin-5 and 7(BCM5, BCM7) and antagonist – lactoferroxin A (LCF A) have been investigated using Caco-2 monolayer. In order to determine the pathway of investigated peptide transport across Caco-2 monolayer, two directions of the transport (apical to basolateral and basolateral to apical) have been studied. All investigated peptides were transported across the human intestinal cell line Caco-2 and the curves of cumulative amount of transported peptides in time were linear in each case. In addition, the hydrolysis of β-casomorphins during 60 min of experiment by dipeptidyl peptidase IV was observed. The data suggest the possibility of transport of opioid peptides derived from food across human intestinal mucosa.     

Activity and location of olive oil phenolic antioxidants in liposomes

The antioxidant activity of hydroxytyrosol, hydroxytyrosol acetate, oleuropein, 3,4-dihydroxyphenylelenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylelenolic acid dialdehyde (3,4-DHPEA-EDA) towards oxidation initiated by 2,2'-azobis(2-amidinopropane) hydrochloride in a soybean phospholipid liposome system was studied. The antioxidant activity of these olive oil phenols was similar and the duration of the lag phase was almost twice that of alpha-tocopherol. Trolox, a water-soluble analogue of alpha-tocopherol, showed the worst antioxidant activity. However, oxidation before the end of the lag phase was inhibited less effectively by the olive oil phenols than by alpha-tocopherol and Trolox. Synergistic effects (11-20% increase in lag phase) were observed in the antioxidant activity of combinations of alpha-tocopherol with olive oil phenols both with and without ascorbic acid. Fluorescence anisotropy of probes and fluorescence quenching studies showed that the olive oil phenols did not penetrate into the membrane, but their effectiveness as antioxidants showed they were associated with the surface of the phospholipid bilayer.     

Pre-treatment of Caco-2 cells with zinc during the differentiation phase alters the kinetics of zinc uptake and transport(2)

The Caco-2 cell model was used to study the efficiency of absorption and endogenous excretion of zinc (Zn) regulated by dietary Zn concentration. Cells were seeded onto high pore-density membranes and maintained in medium supplemented with 10% FBS. After confluence, cells were treated with 5 or 25 μmol Zn/L for 7 d, and Zn uptake and transport were measured in both apical (AP) and basolateral (BL) directions by using ⁶⁵Zn. Similar cells were labeled with ⁶⁵Zn and the release of Zn to the AP and BL sides was measured. The AP uptake of Zn in cells exposed to 25 μmol Zn/L was slower (p < 0.05) than that in cells exposed to 5 μmol Zn/L. The AP to BL transport rate in the 25 μmol Zn/L group was only 40% (p < 0.05) of that in the 5 μM group. In contrast, the rate of BL Zn uptake was 4-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). The BL to AP transport rate was 2-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Basolateral uptake was 6 to 25 times greater (p < 0.05) than AP uptake for cells treated with 5 and 25 μmol Zn/L, respectively. The rate of Zn release was enhanced about 4-fold (p < 0.05) by 25 μmol Zn/L treatment. Release to the BL side was 10 times greater than to the AP side. Zn-induced metallothionein (MT), thought to down-regulate AP to BL Zn transport, was 4-fold higher (p < 0.001) in the 25 μmol Zn/L group than in the 5 μM group, but the rate of BL Zn release was higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Induced changes in transport rates by media Zn concentrations could involve the up- and/or down-regulation of Zn influx and efflux proteins such as the ZIP and ZnT families of Zn transporters.     

Effect of different olive oil-derived antioxidants (hydroxytyrosol and 3,4-dihydroxyphenylglycol) on the quality of frozen-thawed ram sperm

The aim of the present study was to evaluate the effect of the addition of different concentrations of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during the freezing-thawing process. Sperm was collected, pooled and diluted with commercial extenders and then divided into aliquots supplemented with different concentrations (10 μg/ml, 30 μg/ml, 50 μg/ml and 70 μg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. A control group, without antioxidant, was also prepared. Sperm motility, viability, acrosome integrity, mitochondrial membrane potential and lipid peroxidation (LPO) were assessed. The results showed that frozen-thawed ram spermatozoa exhibited lower values for motility, membrane integrity, acrosome and mitochondrial membrane potential than fresh samples (P ≤ 0.01). However, when antioxidants were added, thawed spermatozoa exhibited relatively low LPO, recording values similar to fresh spermatozoa; by contrast, the control group of frozen-thawed spermatozoa without antioxidants exhibited significantly higher LPO (P ≤ 0.01). The addition of a HT+DHPG mixture (MIX) had a negative impact on sperm membrane and acrosome integrity, suggesting that a pure antioxidant supplementation has the potential to offer superior results. In conclusion, HT and DHPG exhibited a positive effect on the frozen-thawed spermatozoa inasmuch as they reduced the LPO. These olive oil-derived antioxidants have the potential to improve frozen-thawed sperm quality, although further studies should be carried out to analyse the antioxidant effect at different times after thawing.     

蒙脱石对细菌黏附Caco-2细胞的影响

采用Caco-2细胞培养模型,观察两歧双歧杆菌、嗜酸乳杆菌、嗜水气单胞菌、副溶血弧菌、大肠杆菌、鼠伤寒沙门菌的黏附率,并在培养液中加入蒙脱石,计算蒙脱石对细菌黏附的阻断率,探讨蒙脱石对上述细菌黏附作用的影响。结果表明:所试菌与Caco-2细胞均有不同程度的黏附作用;蒙脱石对细菌黏附Caco-2细胞均有不同程度的阻断作用,对病原菌黏附Caco-2细胞的阻断作用要明显大于其对益生菌的阻断效果,其中对大肠杆菌、鼠伤寒沙门菌、嗜水气单胞菌、副溶血弧菌黏附的阻断率分别为54.22%、48.41%、60.53%、50.64%,而对两歧双歧杆菌、嗜酸乳杆菌黏附的阻断率分别为25.64%和21.49%。结果提示蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位。     

Determination of thalidomide in transport buffer for Caco-2 cell monolayers by high-performance liquid chromatography with ultraviolet detection

We report simple validated HPLC methods for the determination of thalidomide in the transport buffer for the human colonic cell line (Caco-2) cell monolayers. An aliquot of 50 microl of the mixture was injected onto a Spherex C(18) column (150 x 4.6 mm; 5 microm) at a flow-rate of 0.5 ml/min of mobile phase consisting of acetonitrile-10 mM ammonium acetate buffer (24:76, v/v, pH 5.5), and thalidomide was detected by ultraviolet detector at a wavelength of 220 nm. Calibration curves for thalidomide were constructed at the concentration range of 0.025-1.0 and 1.0-50 microM in transport buffer. The validated methods were used to determine the transport of thalidomide by Caco-2 monolayers. The transport across the monolayers from the apical (A) to basolateral (B) side was similar to that from B to A side. The apparent permeability coefficient (P(app)) values of thalidomide at 10-300 microM from the A to B and from B to A side was 2-6 x 10(-5) cm/s, with a marked decrease in P(app) values from A to B side at increased thalidomide concentration. The A to B transport appears to be dependent on temperature and sodium ion. Sodium azide, 2,4-dinitrophenol (both ATP inhibitors), 5-fluorouracil, cytidine and glutamic acid significantly inhibited the transport of thalidomide. These results indicate that the transport of thalidomide by Caco-2 monolayers was rapid, which might involve an energy-dependent mechanism.