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

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

Caracterización de los efectos antioxidantes y proliferativos del ácido maslínico en diferentes líneas tumorales

Tesis Univ. Granada. Programa Oficial de Doctorado en: Bioquímica y Biología MolecularLa realización de esta tesis doctoral ha sido posible gracias al disfrute de una beca de formación para personal de investigación dentro del contrato de investigación nº 3341-00, adscrito al Proyecto de Investigación PPT-010000-2009-017 de la Fundación Parque Tecnológico Ciencias de la Salud, Empresa Biomaslinic, S.L. La investigación desarrollada ha sido financiada por el Plan Andaluz de Investigación (PAI) dentro de la subvención concedida al grupo de investigación BIO-157

The Efficacy of Intratissue Percutaneous Electrolysis (EPI^®) and Nutritional Factors for the Treatment of Induced Tendinopathy in Wistar Rats: Hepatic Intermediary Metabolism Effects

Achilles tendinopathy (TP) is characterized as the third most common disease of the musculoskeletal system, and occurs in three phases. There is currently no evidence of effective treatment for this medical condition. In this study, the modulatory effects of the minimally invasive technique intratissue percutaneous electrolysis (EPI) and combinations of EPI with four nutritional factors included in the diet, hydroxytyrosol (HT), maslinic acid (MA), glycine, and aspartate (AA), on hepatic intermediary metabolism was examined in Wistar rats with induced tendinopathy at various stages of TP. Results obtained showed that induced tendinopathy produced alterations in the liver intermediary metabolisms of the rats. Regarding carbohydrate metabolism, a reduction in the activity of pro-inflammatory enzymes in the later stages of TP was observed following treatment with EPI alone. Among the combined treatments using nutritional factors with EPI, HT+EPI and AA+EPI had the greatest effect on reducing inflammation in the late stages of TP. In terms of lipid metabolism, the HT+EPI and AA+EPI groups showed a decrease in lipogenesis. In protein metabolism, the HT+EPI group more effectively reduced the inflammatory effects of induced TP. Treatment with EPI combined with nutritional factors might help regulate intermediary metabolism in TP disease and reduce the inflammation process

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

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

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

Altres ajuts: Secretaría General de Universidades, Investigación y Tecnología de la Consejería de Economía, Innovación, Ciencia y Empleo de la Junta de Andalucía.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 µg·mL, 109.8 µg·mL and 45.7 µg·mL, respectively. At the same time, HT caused cell arrest in G0/G1 phase in both Jurkat and HL60 cells by increasing G0/G1 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

The oleanolic acid derivative, 3-O-succinyl-28-O-benzyl oleanolate, induces apoptosis in B16–F10 melanoma cells via the mitochondrial apoptotic pathway

Oleanolic acid (1) is a pentacyclic triterpene present in olive pomace, which is known to induce apoptosis and to have anti-tumor properties; however, high concentrations of this product are necessary to produce cytotoxic effects. The 3-O-succinyl-28-O-benzyl oleanolate derivative (4) presents greater cytotoxicity and apoptosis effects than its natural precursor, oleanolic acid, or its benzyl derivative (2). This study examines the response of B16–F10 melanoma cells to treatment with compound 4, in comparison to 1 and 3. Our studies show that treatment with 4 results in a significant inhibition of cell proliferation in a dose-dependent manner and causes apoptotic cell death. At concentrations inhibiting cell growth by 50% and 80%, compound 4 induces strong G0/G1 cell-cycle arrest, around 72–95% apoptosis, and mitochondrial disturbances confirmed by FACS analysis, which probably involve the activation of the intrinsic apoptotic route. Morphological changes including cell shrinkage, chromatin condensation, and loss of nuclear architecture were also observed. In this report, we demonstrated for the first time that in melanoma cancer cells, compound 4 exerts a significant anti-proliferation effect by inducing the apoptotic process with mitochondrial depolarization. These findings support the role of compound 4 as a new, potential therapeutic tool against aberrant cell proliferation in melanoma.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