Fernando J. Reyes-Zurita

Maslinic acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29 human colon-cancer cells via the mitochondrial apoptotic pathway

We have investigated the mechanisms of maslinic acid with regard to its inhibitory effects on the growth of HT29 colon-cancer cells. High concentrations of maslinic acid are present in the protective wax-like coating of olives. Our results show that treatment with maslinic acid results in a significant inhibition of cell proliferation in a dose-dependent manner and causes apoptotic death in colon-cancer cells. We found that it inhibits considerably the expression of Bcl-2 whilst increasing that of Bax; it also stimulates the release of mitochondrial cytochrome-c and activates caspase-9 and caspase-3. All these results point clearly to the activation of the mitochondrial apoptotic pathway in response to the treatment of HT29 colon-cancer cells with maslinic acid. Our results suggest that maslinic acid has the potential to provide significant natural defence against colon-cancer.

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein‐turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2‐DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish‐farm conditions and fed with a 100 mg/kg MA‐enriched diet (MA100). After the comparison of the protein profiles from MA100 fed fish and from control, 49 protein spots were found to be altered in abundance (≥2‐fold). Analysis by MALDI‐TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S‐adenosyl methionine‐dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6‐phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4‐hydroxyphenylpyruvic dioxygenase, methylmalonate‐semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat‐shock protein, 58 kDa glucose‐regulated protein, cytokeratin E7, type‐II keratin, intermediate filament proteins, 17‐β‐hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6‐phosphate dehydrogenase, elongation factor 2, 60 kDa heat‐shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein‐expression levels of these proteins, we proposed a cellular‐signalling pathway to explain the hepatic‐cell response to the intake of a diet containing MA.

Witch hazel (Hamamelis virginiana) fractions and the importance of gallate moieties--electron transfer capacities in their antitumoral properties.

Witch hazel (Hamamelis virginia) extracts are used in traditional medicine. They are particularly rich in gallate esters included in proanthocyanidins, hydrolyzable tannins (galloylated sugars), and methyl gallate. This study examines the response of human colon cancer cells to treatment with fractions obtained from a witch hazel polyphenolic extract. The results are compared with those obtained previously with homologous fractions from grape (less galloylated) and pine (nongalloylated). Witch hazel fractions were the most efficient in inhibiting cell proliferation in HT29 and HCT116 human colon cancer cell lines, which clearly shows that the more galloylated the fractions, the more effective they were at inhibiting proliferation of colon cancer cells. Witch hazel fractions were, in addition, more potent in arresting the cell cycle at the S phase and inducing apoptosis; they also induced a significant percentage of necrosis. Interestingly, the apoptosis and cell cycle arrest effects induced were proportional to their galloylation. Moreover, witch hazel fractions with a high degree of galloylation were also the most effective as scavengers of both hydroxyl and superoxide radicals and in protecting against DNA damage triggered by the hydroxyl radical system. These findings provide a better understanding of the structure-bioactivity relationships of polyphenolics, which should be of assistance in choosing an appropriate source and preparing a rational design for formulations of plant polyphenols in nutritional supplements.

Semi-synthesis of acylated triterpenes from olive-oil industry wastes for the development of anticancer and anti-HIV agents

A broad set of potential bioactive conjugate compounds has been semi-synthesized through solution- and solid-phase organic procedures, coupling two natural pentacyclic triterpene acids, oleanolic (OA) and maslinic acids (MA), at the hydroxyl groups of the A-ring of the triterpene skeleton, with 10 different acyl groups. These acyl OA and MA derivatives have been tested for their anti-proliferative (against the b16f10 murine melanoma cancer cells) and antiviral (as inhibitors of the HIV-1-protease) effects. Several derivatives have shown high levels of early and total apoptosis (up to 90%). Most of the compounds that exhibited anti-proliferative effects also generated ROS, probably involving the activation of an intrinsic apoptotic route. The only four compounds that did not cause the release of ROS could be related to the participation of a probable extrinsic activation of the apoptosis mechanism. A great number of these acyl OA and MA derivatives have proved to be potent inhibitors of the HIV-1-protease, the most active inhibitors having IC50 values between 0.31 and 15.6 μM, these values being between 4 and 186 times lower than their non-acylated precursors. The potent activities exhibited in the apoptosis-activation processes and in the inhibition of the HIV-1-protease by some OA and MA acylated derivatives imply that these compounds could be used as new, safe, and effective anticancer and/or antiviral drugs.

Solid-Phase Library Synthesis of Bi-Functional Derivatives of Oleanolic and Maslinic Acids and Their Cytotoxicity on Three Cancer Cell Lines

A wide set of 264 compounds has been semisynthesized with high yields and purities. These compounds have been obtained through easy synthetic processes based on a solid-phase combinatorial methodology. All the members of this library have one central core of a natural pentacyclic triterpene (oleanolic or maslinic acid) and differ by 6 amino acids, coupled with the carboxyl group at C-28 of the triterpenoid skeleton, and by 10 different acyl groups attached to the hydroxyl groups of the A-ring of these molecules. According to the literature on the outstanding and promising pharmacological activities of other similar terpene derivatives, some of these compounds have been tested for their cytotoxic effects on the proliferation of three cancer cell lines: B16-F10, HT29, and Hep G2. In general, we have found that around 70% of the compounds tested show cytotoxicity in all three of the cell lines selected; around 60% of the cytotoxic compounds are more effective than their corresponding precursors, that is, oleanolic (OA) or maslinic (MA) acids; and nearly 50% of the cytotoxic derivatives have IC50 values between 2- to 320-fold lower than their corresponding precursor (OA or MA).

Maslinic Acid-Enriched Diet Decreases Intestinal Tumorigenesis in ApcMin/+ Mice through Transcriptomic and Metabolomic Reprogramming

Chemoprevention is a pragmatic approach to reduce the risk of colorectal cancer, one of the leading causes of cancer-related death in western countries. In this regard, maslinic acid (MA), a pentacyclic triterpene extracted from wax-like coatings of olives, is known to inhibit proliferation and induce apoptosis in colon cancer cell lines without affecting normal intestinal cells. The present study evaluated the chemopreventive efficacy and associated mechanisms of maslinic acid treatment on spontaneous intestinal tumorigenesis in ApcMin/+ mice. Twenty-two mice were randomized into 2 groups: control group and MA group, fed with a maslinic acid–supplemented diet for six weeks. MA treatment reduced total intestinal polyp formation by 45% (P<0.01). Putative molecular mechanisms associated with suppressing intestinal polyposis in ApcMin/+ mice were investigated by comparing microarray expression profiles of MA-treated and control mice and by analyzing the serum metabolic profile using NMR techniques. The different expression phenotype induced by MA suggested that it exerts its chemopreventive action mainly by inhibiting cell-survival signaling and inflammation. These changes eventually induce G1-phase cell cycle arrest and apoptosis. Moreover, the metabolic changes induced by MA treatment were associated with a protective profile against intestinal tumorigenesis. These results show the efficacy and underlying mechanisms of MA against intestinal tumor development in the ApcMin/+ mice model, suggesting its chemopreventive potential against colorectal cancer.

Antitumour activity on extrinsic apoptotic targets of the triterpenoid maslinic acid in p53-deficient Caco-2 adenocarcinoma cells

We report that a novel triterpenoid, (2a,3b)-2,3-dihydroxyolean-12-en-28-oic acid (maslinic acid), isolated from olive pomace from Olea europaea, triggers primarily the extrinsic and later the intrinsic apoptotic pathways in Caco-2 human colon-cancer cells. Apoptosis induced by maslinic acid was confirmed by FACS analysis using annexine-V FICT staining. This induction of apoptosis was correlated with the early activation of caspase-8 and caspase-3, the activation of caspase-8 was also correlated with higher levels of Bid cleavage and decreased Bcl-2, but with no change in Bax expression. Maslinic acid also induced a sustained activation of c-Jun N-terminal kinase (JNK). Incubation with maslinic acid also resulted in the later activation of caspase-9, which, together with the lack of any Bax activation, suggests that the mitochondrial pathway is not required for apoptosis induced by maslinic acid in this cell line. In this study we found that the mechanism of apoptotic activation in p53-deficient Caco-2 cells differs significantly from that found in HT-29 cells. Natural agents able to activate both the extrinsic and intrinsic apoptotic pathways by avoiding the mitochondrial resistance mechanisms may be useful for treatment against colon cancer regardless of its aetiology.

Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells.

Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin.

A New HPLC-MS Method for Measuring Maslinic Acid and Oleanolic Acid in HT29 and HepG2 Human Cancer Cells

Maslinic acid (MA) and oleanolic acid (OA), the main triterpenic acids present in olive, have important properties for health and disease prevention. MA selectively inhibits cell proliferation of the HT29 human colon-cancer cell line by inducing selective apoptosis. For measuring the MA and OA concentration inside the cell and in the culture medium, a new HPLC-MS procedure has been developed. With this method, a determination of the amount of MA and OA incorporated into HT29 and HepG2 human cancer-cell lines incubated with different concentrations of MA corresponding to 50% growth inhibitory concentration (IC50), IC50/2, IC50/4, and IC50/8 has been made. The results demonstrate that this method is appropriate for determining the MA and OA concentration in different types of cultured cells and reveals the specific dynamics of entry of MA into HT29 and HepG2 cells.

Maslinic Acid, a Triterpene from Olive, Affects the Antioxidant and Mitochondrial Status of B16F10 Melanoma Cells Grown under Stressful Conditions

Maslinic acid (MA) is a natural compound whose structure corresponds to a pentacyclic triterpene. It is abundant in the cuticular lipid layer of olives. MA has many biological and therapeutic properties related to health, including antitumor, anti-inflammatory, antimicrobial, antiparasitic, antihypertensive, and antioxidant activities. However, no studies have been performed to understand the molecular mechanism induced by this compound in melanoma cancer. The objective of this study was to examine the effect of MA in melanoma (B16F10) cells grown in the presence or absence of fetal bovine serum (FBS). We performed cell proliferation measurements, and the reactive oxygen species (ROS) measurements using dihydrorhodamine 123 (DHR 123) and activities of catalase, glucose 6-phosphate dehydrogenase, glutathione S-transferase, and superoxide dismutase. These changes were corroborated by expression assays. FBS absence reduced cell viability decreasing IC50 values of MA. The DHR 123 data showed an increase in the ROS level in the absence of FBS. Furthermore, MA had an antioxidant effect at lower assayed levels measured as DHR and antioxidant defense. However, at higher dosages MA induced cellular damage by apoptosis as seen in the results obtained.