Unveiling the Differential Antioxidant Activity of Maslinic Acid in Murine Melanoma Cells and in Rat Embryonic Healthy Cells Following Treatment with Hydrogen Peroxide

Maslinic acid (MA) is a natural triterpene from Olea europaea L. with multiple biological properties. The aim of the present study was to examine MA’s effect on cell viability (by the MTT assay), reactive oxygen species (ROS levels, by flow cytometry) and key antioxidant enzyme activities (by spectrophotometry) in murine skin melanoma (B16F10) cells compared to those on healthy cells (A10). MA induced cytotoxic effects in cancer cells (IC50 42 M), whereas no effect was found in A10 cells treated withMA(up to 210 M). In order to produce a stress situation in cells, 0.15mMH2O2 was added. Under stressful conditions, MA protected both cell lines against oxidative damage, decreasing intracellular ROS, which were higher in B16F10 than in A10 cells. The treatment with H2O2 and without MA produced different responses in antioxidant enzyme activities depending on the cell line. In A10 cells, all the enzymes were up-regulated, but in B16F10 cells, only superoxide dismutase, glutathione S-transferase and glutathione peroxidase increased their activities. MA restored the enzyme activities to levels similar to those in the control group in both cell lines, highlighting that in A10 cells, the highest MA doses induced values lower than control. Overall, these findings demonstrate the great antioxidant capacity of MA.General Secretariat of Universities, Research and Technology of the Ministry of Economy, Innovation, Science and Employment Government of the Junta de Andalucia (Spain) BIO-157program FEDER-INNTERCONECTA from the Spanish Government C-3650-0

Nutraceutical Role of Polyphenols and Triterpenes Present in the Extracts of Fruits and Leaves of Olea europaea as Antioxidants, Anti-Infectives and Anticancer Agents on Healthy Growth

This research has been funded by the Junta de Andalucia (Andalusian Research Plan, Junta de Andalucia, Spain) by the grant from the research group BIO-157 "Drugs, Environmental Toxics and Cellular Metabolism".There is currently a worldwide consensus and recognition of the undoubted health benefits of the so-called Mediterranean diet, with its intake being associated with a lower risk of mortality. The most important characteristics of this type of diet are based on the consumption of significant amounts of fruit, vegetables, legumes, and nuts, which provide, in addition to some active ingredients, fiber and a proportion of vegetable protein, together with extra virgin olive oil (EVOO) as the main sources of vegetable fat. Fish and meat from poultry and other small farm animals are the main sources of protein. One of the main components, as already mentioned, is EVOO, which is rich in monounsaturated fatty acids and to a lesser extent in polyunsaturated fatty acids. The intake of this type of nutrient also provides an important set of phytochemicals whose health potential is widely spread and agreed upon. These phytochemicals include significant amounts of anthocyanins, stilbenes, flavonoids, phenolic acids, and terpenes of varying complexities. Therefore, the inclusion in the diet of this type of molecules, with a proven healthy effect, provides an unquestionable preventive and/or curative activity on an important group of pathologies related to cardiovascular, infectious, and cancerous diseases, as well as those related to the metabolic syndrome. The aim of this review is therefore to shed light on the nutraceutical role of two of the main phytochemicals present in Olea europaea fruit and leaf extracts, polyphenols, and triterpenes, on healthy animal growth. Their immunomodulatory, anti-infective, antioxidant, anti-aging, and anti-carcinogenic capabilities show them to be potential nutraceuticals, providing healthy growth.Junta de Andalucia BIO-15

The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism

[Background] Maslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism. [Methods] We used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison. [Results] HT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling. [Conclusion] All these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings.This study was supported by grants BIO157 from the Andalucian regional government; SAF2008-00164 and ISCIII-RTICC (RD06/0020/0046) grants from the Spanish national government and & European Regional Development Fund (ERDF) "Una manera de hacer Europa" and by AGAUR-Generalitat de Catalunya grant 2009SGR1308, 2009 CTP 00026 and Icrea Academia award 2010 granted to M. Cascante)

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.This study was supported by grants Group BIO 157 from the Technology and Innovation Council of the Andalucian regional government and AGL2006-12210-C03-02/ALI, SAF2005-01627, ISCIII-RTICC (RD06/0020/0046) from the Spanish government and European Union FEDER funds

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 dosagesMAinduced cellular damage by apoptosis as seen in the results obtained.This study has been supported, in part, by funds of the consolidated Research Group BIO-157, from the General Secretariat of Universities, Research and Technology of the Ministry of Economy, Innovation, Science and Employment Government of the Junta de Andaluc´ıa (Spain), and by the Research Contract no. C-3650-00 under the program FEDER-INNTERCONECTA from the Spanish Government and European Union FEDER funds. Amalia P´erez-Jim´enez is a recipient of a postdoctoral research fellowship Torres- Quevedo no. PTQ 12-05739

Involvement of the PI3K/AKT Intracellular Signaling Pathway in the AntiCancer Activity of Hydroxytyrosol, a Polyphenol from Olea europaea, in Hematological Cells and Implication of HSP60 Levels in Its Anti-Inflammatory Activity

Hydroxytyrosol (HT), the main representative of polyphenols of olive oil, has been described as one of the most powerful natural antioxidants, also showing anti-inflammatory, antimicrobial, cardioprotective and anticancer activity in different type of cancers, but has been little studied in hematological neoplasms. The objective of this work was to evaluate the anticancer potential of HT in acute human leukemia T cells (Jurkat and HL60) and the anti-inflammatory potential in murine macrophages (Raw264.7). For this, cytotoxicity tests were performed for HT, showing IC50 values, at 24 h, for Jurkat, HL60 and Raw264.7 cells, of 27.3 mu g center dot mL(-1), 109.8 mu g center dot mL(-1) and 45.7 mu g center dot mL(-1), respectively. At the same time, HT caused cell arrest in G(0)/G(1) phase in both Jurkat and HL60 cells by increasing G(0)/G(1) phase and significantly decreasing S phase. Apoptosis and cell cycle assays revealed an antiproliferative effect of HT, decreasing the percentage of dividing cells and increasing apoptosis. Furthermore, HT inhibited the PI3K signaling pathway and, consequently, the MAPK pathway was activated. Inflammation tests revealed that HT acts as an anti-inflammatory agent, reducing NO levels in Raw264.7 cells previously stimulated by lipopolysaccharide (LPS). These processes were confirmed by the changes in the expression of the main markers of inflammation and cancer. In conclusion, HT has an anticancer and anti-inflammatory effect in the cell lines studied, which were Raw264.7, Jurkat, and HL60, and could be used as a natural drug in the treatment of liquid cancers, leukemias, myelomas and lymphomas.General Secretariat of Universities, Research and Technology of the Ministry of Economy, Innovation, Science and Employment of the Government of the Junta de Andalucia, Spain BIO-15

Efficient In Vitro and In Vivo Anti‐Inflammatory Activity of a Diamine‐PEGylated Oleanolic Acid Derivative

Recent evidence has shown that inflammation can contribute to all tumorigenic states. We have investigated the anti‐inflammatory effects of a diamine‐PEGylated derivative of oleanolic acid (OADP), in vitro and in vivo with inflammation models. In addition, we have determined the sub‐cytotoxic concentrations for anti‐inflammatory assays of OADP in RAW 264.7 cells. The in‐ flammatory process began with incubation with lipopolysaccharide (LPS). Nitric oxide production levels were also determined, exceeding 75% inhibition of NO for a concentration of 1 μg/mL of OADP. Cell‐cycle analysis showed a reversal of the arrest in the G0/G1 phase in LPS‐stimulated RAW 264.7 cells. Furthermore, through Western blot analysis, we have determined the probable molecular mechanism activated by OADP; the inhibition of the expression of cytokines such as TNF‐α, IL‐1β, iNOS, and COX‐2; and the blocking of p‐IκBα production in LPS‐stimulated RAW 264.7 cells. Finally, we have analyzed the anti‐inflammatory action of OADP in a mouse acute ear edema, in male BL/6J mice treated with OADP and tetradecanoyl phorbol acetate (TPA). Treatment with OADP induced greater suppression of edema and decreased the ear thickness 14% more than diclofenac. The development of new derivatives such as OADP with powerful anti‐inflammatory effects could represent an effective therapeutic strategy against inflammation and tumorigenic processes

Innovamos, jugamos y aprendemos combinando conocimientos de diferentes áreas

El objetivo principal de este proyecto de innovación docente es elaborar nuevas herramientas docentes para la enseñanza de la Bioquímica y la Fisiología, así como facilitar su aprendizaje y la relación de los conocimientos adquiridos entre ambas asignaturas en diferentes Grados tanto del Campus de Melilla como del Campus de Granada. Para ello nos planteamos los siguientes objetivos específicos: 1. Identificar una historia para hilar el contenido de la asignatura de Bioquímica. 2. Crear ligas o torneos para retar a los alumnos en su aprendizaje con estos juegos. 3. Relacionar contenidos entre las asignaturas de Bioquímica y Fisiología. 4. Evaluar el material didáctico al final del semestre mediante cuestionarios para comprobar su evolución en el aprendizaje de los conocimientos adquiridos. Por otro lado, también pretendemos que los alumnos identifiquen los conocimientos y competencias adquiridos y la utilidad y aplicación de esas tareas y habilidades en el ámbito empresarial, como un primer paso para el autoconocimiento profesional y la búsqueda de empleo. La dinámica de juego empleada será la realización de un Escape Room Educativo Virtual. Para alcanzar el primer objetivo en clase, identificaremos una historia o juego de interés entre los alumnos de primero. Si existen varias, se elegirá la que sea propuesta por el mayor número de alumnos. Tras la presentación de la historia, crearemos una liga o torneo para retar a los alumnos entre ellos. En el caso de que haya algún alumno/a con necesidades especiales, se nombrar un alumno colaborador, el cual le facilitará su participación durante la realización del juego. Además, el contenido será evaluado de modo que todos los alumnos tengan acceso a él, esto es, aumento del tamaño de letra, grabación de voz en off en caso necesario, aumento en el tiempo de realización de la actividad, etc. Algunas de las preguntas introducidas en el juego permitirán la relación de contenidos entre las asignaturas de Bioquímica y Fisiología y su aplicabilidad en su futuro profesional. Por último, al finalizar la actividad se les pasará a los alumnos un cuestionario para evaluar su satisfacción con este proyecto de innovación docente. Dicho proyecto docente ha sido aplicado al final del semestre en los Grados de Enfermería, Fisioterapia y el Doble Grado en Educación Primaria y Ciencias de la Actividad Física y del Deporte del Campus de Melilla y en los Grado de Fisioterapia y el Grado en Ciencias de la Actividad Física y del Deporte del Campus de Granada en la Universidad de Granada.The main objective of this teaching innovation project is to develop new teaching tools for the teaching of Biochemistry and Physiology, as well as to facilitate their learning and the relationship of the knowledge acquired between both subjects in different Degrees in both Melilla and Granada Campus at the University of Granada. To this end, we set ourselves the following specific objectives: 1. Identify a story to string together the content of the Biochemistry subject. 2. Create leagues or tournaments to resume students in their learning with these games. 3. Relate content between the subjects of Biochemistry and Physiology. 4. Evaluate the didactic material at the end of the semester by means of questionnaires to verify its evolution in the learning of the acquired knowledge. On the other hand, we also intend that students identify the knowledge and skills acquired and the usefulness and application of these tasks and skills in the business field, as a first step for professional self-knowledge and job search. The game dynamics used will be the realization of a Virtual Educational Escape Room. To achieve the first objective in class, we will identify a story or game of interest among the first graders. If there are several, the one proposed by the largest number of students will be chosen. After the presentation of the story, we will create a league or tournament to take the students back to each other. In the event that there is a student with special needs, a collaborating student will be appointed, who will facilitate their participation during the game. In addition, the content will be evaluated so that all students have access to it, that is, increased font size, voiceover recording if necessary, increased time to complete the activity, etc. Some of the questions presented in the game will allow the content relationship between the subjects of Biochemistry and Physiology and its applicability in their professional future. Finally, at the end of the activity, students will be given a questionnaire to assess their satisfaction with this teaching innovation project. This teaching project has been applied at the end of the semester in the Degrees of Nursing, Physiotherapy and the Double Degree in Primary Education and Physical Activity and Sports Sciences of the Melilla Campus and in the Degrees of Physiotherapy and the Degree in Sciences of the Physical Activity and Sports of the Granada Campus at the University of Granada

Evaluación nutricional y bioquímica del ácido maslínico, triterpeno natural, sobre el crecimiento de la dorada (Sparus aurata). Caracterización cinética y proteómica

Tesis Univ. Granada. Departamento de Bioquímica y Biología Molecular I. Leída el 24 de julio de 200

Are Ancestral Medical Practices the Future Solution to Today’s Medical Problems?

Our cells and organs are threatened and, in most cases, constantly subjected to the aggression of numerous situations, both endogenous, characterized by unfavorable genetics, and exogenous, by deficient or inadequate nutrition, and even by a hostile environment; in most cases, they ultimately cause a cascade of degenerative and cardiovascular diseases, cancer, and infections, as well as those related to the metabolic syndrome, all of which eventually generate irreversible damage to the organism and, consequently, a significant deterioration in its survival [...