M. Emília Juan

Resveratrol Induces Apoptosis through ROS-Dependent Mitochondria Pathway in HT-29 Human Colorectal Carcinoma Cells

trans-Resveratrol is a polyphenol found in blueberries, grapes, and wine with cancer chemopreventive properties. The low bioavailability of this compound enhances its concentration in the luminal content and becomes a potential chemopreventive agent against colon cancer. In the present study, the antiproliferative and pro-apoptotic effects on the human colorectal carcinoma HT-29 cells as well as the mechanisms underlying these effects were examined. Proliferation, cytotoxicity, and apoptosis were measured by fluorescence-based techniques. Studies of dose-dependent effects of trans-resveratrol showed antiproliferative activity with an EC 50 value of 78.9 +/- 5.4 microM. Caspase-3 was activated in a dose-dependent manner after incubation for 24 h giving an EC 50 value of 276.1 +/- 1.7 microM. Apoptosis was also confirmed with microscopic observation of changes in membrane permeability and detection of DNA fragmentation. The activity of trans-resveratrol on the mitochondria apoptosis pathway was evidenced by the production of superoxide anions in the mitochondria of cells undergoing apoptosis. In conclusion, trans-resveratrol inhibits cell proliferation without cytotoxicity and induces apoptosis in HT-29. Results of the present study provide evidence demonstrating the antitumor effect of trans-resveratrol via a ROS-dependent apoptosis pathway in colorectal carcinoma.

Quantification of trans-resveratrol and its metabolites in rat plasma and tissues by HPLC.

trans-Resveratrol, a polyphenol from grapes, is being recognized as a bioactive agent with potential beneficial effects on health. However, little is known about its distribution in the organism mainly because of the lack of accurate and precise detection methods. Consequently the aim of the present study was to develop a methodology of extraction and quantification of trans-resveratrol and its metabolites in plasma, brain, testis, liver, lungs and kidney by HPLC. To this end, the time of homogenization and liquid extraction were adapted to the different tissues. The methods were validated using homogenized tissues spiked with pure trans-resveratrol. The precision (% R.S.D.) ranged from 3.7% in testis to 13.2% in lungs. Recoveries were 98.5+/-3.2% (liver), 100.1+/-1.8% (kidney), 96.5+/-7.6% (lungs), 99.0+/-0.7% (brain) and 103+/-2.7% (testicle). The limits of detection ranged from 5.5 nM in testis to 11.2 nM in kidney. After validation, the methods were applied to the assessment of the bioavailability and distribution of trans-resveratrol in rats after the intravenous administration of 15 mg/kg. At 90 min, trans-resveratrol and its glucuronide and sulfate conjugates were widely distributed in all the tissues studied. The highest concentrations (nmol/g tissue) were found in kidney (resveratrol: 1.45+/-0.35; glucuronide: 2.91+/-0.19; sulfate: not detected), and the lowest in brain (resveratrol: 0.17+/-0.04; glucuronide: not detected; sulfate: 0.04+/-0.01). In conclusion, accurate and reproducible methods have been described to identify target tissues of resveratrol as a first step to understand its mechanisms of action in vivo.

Antiproliferative and apoptosis-inducing effects of maslinic and oleanolic acids, two pentacyclic triterpenes from olives, on HT-29 colon cancer cells.

We have previously reported the anticarcinogenic effects of an olive fruit extract composed of pentacyclic triterpenes, the main components of which are maslinic acid (73.25%) and oleanolic acid (25.75%). Here we examined the effects of the individual components on proliferation, necrosis and apoptosis rates by fluorescence-based techniques in human HT-29 colon cancer cells. Oleanolic acid showed moderate antiproliferative activity, with an ec50 of 160.6 (se 10.6) micromol/l, and moderate cytotoxicity at high concentrations ( > or = 250 micromol/l). On the other hand, maslinic acid inhibited cell growth with an ec50 of 101.2 (se 7.8) micromol/l, without necrotic effects. Oleanolic acid, which lacks a hydroxyl group at the carbon 2 position, failed to activate caspase-3 as a prime apoptosis protease. In contrast, maslinic acid increased caspase-3-like activity at 10, 25 and 50 micromol/l by 3-, 3.5- and 5-fold over control cells, respectively. The detection of ROS in the mitochondria, which serve as pro-apoptotic signal, evidenced the different bioactivity of the two triterpenes. Confocal microscopy analysis revealed that maslinic acid generated superoxide anions while oleanolic acid-treated cells did not differ from the control. Completion of apoptosis by maslinic acid was confirmed microscopically by the increase in plasma membrane permeability, and detection of DNA fragmentation. In conclusion, the anticancer activity observed for olive fruit extracts seems to originate from maslinic acid but not from oleanolic acid. Maslinic acid therefore is a promising new compound for the chemoprevention of colon cancers.

Multidrug Resistance Proteins Restrain the Intestinal Absorption of trans-Resveratrol in Rats

trans-Resveratrol, a natural antioxidant, has been described as a nutraceutic compound with important beneficial effects on health, but its low oral bioavailability hinders its therapeutic activity. Here, we studied the mechanisms of apical transport of trans-resveratrol in enterocytes and the role of ATP-binding cassette (ABC) transporters in the secretion of resveratrol glucuronide and sulfate resulting from the rapid intracellular metabolism. An intestinal perfusion method with recirculation in vivo was used in rats. Jejunal loops were perfused with increasing concentrations of trans-resveratrol and results showed that its uptake occurs by simple diffusion without the participation of a mediated transport. The apparent diffusion constant was 8.1 +/- 0.3 microL/(5 min.mg dry weight). The glycoprotein-P (Pgp, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) located in the apical membrane of enterocytes were investigated using specific inhibitors. The Pgp inhibitors verapamil (5 micromol/L) and cyclosporin A (5 micromol/L) did not affect the efflux of trans-resveratrol and its conjugates. The MRP2 inhibitors probenecid (2 mmol/L) and MK571 (10 micromol/L) reduced the efflux of glucuronide by 61 and 55%, respectively, and of sulfate by 43 and 28%, respectively. The BCRP inhibitor Ko143 (0.5 micromol/L) decreased the secretion of glucuronide by 64% and of sulfate by 46%. Our experiments identify MRP2 and BCRP as the 2 apical transporters involved in the efflux of resveratrol conjugates.

The bioavailability and distribution of trans-resveratrol are constrained by ABC transporters

The ABC proteins are a family of membrane transporters that mediates the extrusion from cells of a wide variety of structurally unrelated substrates. The current review focuses on the role of these efflux pumps located in the intestine on the low oral bioavailability of trans-resveratrol. The enterocytes hold in the apical membrane three transporters, namely, P-glycoprotein (P-gp), multidrug resistance associated protein 2 (MRP2) and breast cancer resistance protein (BCRP), whereas the basolateral membrane contains multidrug resistance associated protein 3 (MRP3). The use of different specific inhibitors of these transporters as well as knockout mice enabled us to conclude that MRP2 and BCRP are involved in the extrusion of trans-resveratrol glucuronide and sulfate to the intestinal lumen without the participation of P-gp. The role of these transporters as a bottleneck in the absorption of trans-resveratrol cannot be undervalued affecting not only the bioavailability of its glucuronide and sulfate but also their distribution in the different organs.

D-Fagomine lowers postprandial blood glucose and modulates bacterial adhesion.

D-Fagomine is an iminosugar originally isolated from seeds of buckwheat (Fagopyrum sculentum Moench), present in the human diet and now available as a pure crystalline product. We tested D-fagomine for activities connected to a reduction in the risk of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. The activities were: intestinal sucrase inhibition in vitro (rat mucosa and everted intestine sleeves), modulation of postprandial blood glucose in rats, bacterial agglutination and bacterial adhesion to pig intestinal mucosa. When ingested together with sucrose or starch, D-fagomine lowered blood glucose in a dose-dependent manner without stimulating insulin secretion. D-Fagomine reduced the area under the curve (0-120 min) by 20 % (P < 0·01) and shifted the time to maximum blood glucose concentration (Tmax) by 15 min at doses of 1-2 mg/kg body weight when administered together with 1 g sucrose/kg body weight. Moreover, D-fagomine (0·14 mm) agglutinated 60 % of Enterobacteriaceae (Escherichia coli, Salmonella enterica serovar Typhimurium) populations (P < 0·01), while it did not show this effect on Bifidobacterium spp. or Lactobacillus spp. At the same concentration, d-fagomine significantly (P < 0·001) inhibited the adhesion of Enterobacteriaceae (95-99 % cells in the supernatant) and promoted the adhesion of Lactobacillus acidophilus (56 % cells in the supernatant) to intestinal mucosa. D-Fagomine did not show any effect on bacterial cell viability. Based on all this evidence, D-fagomine may be used as a dietary ingredient or functional food component to reduce the health risks associated with an excessive intake of fast-digestible carbohydrates, or an excess of potentially pathogenic bacteria.

Erythrodiol, a natural triterpenoid from olives, has antiproliferative and apoptotic activity in HT-29 human adenocarcinoma cells

Erythrodiol is the precursor of pentacyclic triterpenic acids present in Olea Europaea. Although olive oil and some of its constituents are reported to have anticarcinogenic activities, erythrodiol has not been assessed in its cell biological functions in detail. We therefore determined its effects on cell growth and apoptosis in human colorectal carcinoma HT-29 cells. Proliferation, cytotoxicity, and apoptosis were measured by fluorescence-based techniques. Erythrodiol inhibited cell growth with an EC50 value of 48.8 +/- 3.7 microM without any cytotoxic effects in a concentration range up to 100 microM. However, exposure of cells for 24 h to 50, 100, and 150 microM erythrodiol increased caspase-3-like activity by 3.2-, 4.8-, and 5.2-fold over that in control cells. We here demonstrate for the first time that, in colon adenocarcinoma cells, erythrodiol exerts antiproliferative and proapoptotic activity.

Assessment of the safety of maslinic acid, a bioactive compound from Olea europaea L.

SCOPE Maslinic acid, the main pentacyclic triterpene of the cuticle of Olea europaea L. fruit, has multiple beneficial effects on health, most notably antitumor and hypoglycemic properties. Notwithstanding the biological activities, there is a lack of knowledge about its safety. Therefore, the purpose of this study was to evaluate whether high doses of maslinic acid have harmful effects on Swiss CD-1 male mice. METHODS AND RESULTS The single oral administration of the pentacyclic triterpene at 1000 mg/kg to mice did not produce any signs of morbidity or mortality. The repeated daily oral administration of 50 mg/kg of maslinic acid for 28 days did not induce any sign of toxicity during the experimental period. Body weight did not differ between mice that received the triterpene and the control group. Similarly, hematological and biochemical variables were not affected by the treatment. Histopathologic examination of the organs revealed that there were no differences between the control and the treated mice. CONCLUSION Taken together the results obtained from the acute and the repeated intake of maslinic acid indicate that the compound does not exert any adverse effects on the variables tested in mice, thus suggesting a sufficient margin of safety for its putative use as a nutraceutical.

Determination of dihydroresveratrol in rat plasma by HPLC.

Dihydroresveratrol is a metabolite of trans-resveratrol formed in the intestine by the hydrogenation of the double bond by microflora. The aim of the present study was to validate a method to measure dihydroresveratrol in rat plasma and then to quantify its plasmatic concentration after the oral administration of 60 mg/kg to Sprague-Dawley rats. Dihydroresveratrol was extracted from acidified plasma with a C18 cartridge, eluted with methanol, and concentrated prior to HPLC analysis with diode-array detection (HPLC-DAD) at 276 nm. The method was validated by spiking blank plasma samples with pure dihydroresveratrol, obtaining a linear correlation and good interday and intraday precisions, expressed as coefficient of variation (<7%). The average recovery was 96.7% and the limit of detection was 275 nM. The oral administration of dihydroresveratrol to rats and its subsequent detection, along with dihydroresveratrol glucuronide and sulfate, provides evidence of its absorption and metabolism.

Determination of maslinic acid, a pentacyclic triterpene from olives, in rat plasma by high-performance liquid chromatography.

Maslinic acid, a pentacyclic triterpene from olives, has been reported to exert beneficial effects on health, including anticarcinogenic activity. Despite its importance, little is known about its bioavailability in both humans and animals. A fundamental step for this evaluation consisted of measuring this compound in blood. Therefore, a simple high-performance liquid chromatography (HPLC) method with diode array detection has been developed. Maslinic acid contained in plasma was extracted twice using ethyl acetate. After centrifugation, the organic fraction was evaporated to dryness and the residue was reconstituted with methanol/water (75:25, v/v) and analyzed by HPLC. The method was validated by obtaining a linear correlation (r(2) = 0.999) and an average recovery of 99%. Precision expressed as the coefficient of variation ranged from 1.23 to 9.06%. The oral administration of maslinic acid (50 mg/kg) to rats and its subsequent detection in plasma showed that the method is suitable for absorption, distribution, and metabolism studies.