Hydroxytyrosol targets extracellular matrix remodeling by endothelial cells and inhibits both ex vivo and in vivo angiogenesis

This is the preprint version of our manuscript, corresponding to the article that has been 
published in final form at FOOD CHEMISTRY with DOI: 10.1016/j.foodchem.2016.10.111The health benefits of olive oil are attributed to their bioactive compounds, such as hydroxytyrosol. Previously, we demonstrated that hydroxytyrosol inhibits angiogenesis in vitro. The present study aimed to: i) get further insight into the effects of hydroxytyrosol on extracellular matrix remodeling; and ii) test whether hydroxytyrosol is able to inhibit angiogenesis ex vivo and in vivo. Hydroxytyrosol induced a shift toward inhibition of proteolysis in endothelial cells, with decreased expression of extracellular matrix remodeling-enzyme coding genes and increased levels of some of their inhibitors. Furthermore, this work demonstrated that hydroxytyrosol, at concentrations within the range of its content in virgin olive oil that can be absorbed from moderate and sustained virgin olive oil consumption, is a strong inhibitor of angiogenesis ex vivo and in vivo. These results suggest the need for translational studies to evaluate the potential use of hydroxytyrosol for angio-prevention and angiogenesis inhibition in clinical setting.This work was supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER). The “CIBER de Enfermedades Raras” is an initiative from the ISCIII (Spain). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript

Inquiry based learning: why buying a car with a tree included? Enhancing science and mathematic learning

International reports reveal a deficient situation in relation to science and mathematics learning, which can be considered as an obstacle for the education of literate and informed citizens and the qualification and the preparation of future scientists and engineers. This situation may be partly attributed to the way science and mathematics are taught at school. Research on effective teaching approaches shows that inquiry based learning (IBL) improves students’ engagement and motivation for science and mathematics learning and promotes the development of process skills, critical thinking and conceptual understanding of some science and mathematics topics. The present work describes the design and implementation of an instructional approach for enhancing science and mathematics learning through IBL. The instructional approach is based on the design of an interdisciplinary task which starts by challenging students through the analysis of an advertisement. The initial scenario engages students in an investigation process to look for evidence and understanding while acquiring meaningful learning of key science topics and mathematical tools. The task also takes advantage of current technological resources to facilitate and support the overall inquiry process. (Orig.

Interplay between glucose and palmitate uptake in breast carcinoma in vitro

One of the most studied tumor cells lines in vitro is the breast carcinoma MDA-MB-231 cell line. Several studies have proved its glycolytic profile, namely known as the Warburg effect. Glutamine oxidation is also important for its metabolism. Nevertheless, the use of fatty acids for obtaining energy in these cells is still rising. Palmitic acid is the most common saturated fatty acid, containing sixteen carbons in its structure. However, the use of palmitate for metabolic studies in MDA-MB-231 is not very extended due to its pro-apoptotic effect in this cell line after certain time exposure. Nonetheless, in this work we used palmitate as a metabolic fuel for just 30 minutes in order to see the almost immediate response of the cells to its presence, after a 30 minutes fast period. Our results show that MDA-MB-231 cells are not able of oxidizing palmitate nor producing lactate from it. Simultaneous presence of palmitate with glucose or with glutamine does not affect glucose nor glutamine uptake in these cells. However, we observed that even low concentrations of glucose increase palmitate uptake in MDA-MB-231 after a 30 minutes incubation. Treatment with 5 mM 2-deoxyglucose also for 30 minutes counters this rise, since 2-deoxyglucose diminishes palmitate uptake. Increasing glucose concentration to the same dosis of 2-deoxyglucose leads to a prevalence of the glucose effect on palmitate uptake. The exact role of glucose and glucose derivatives should be further studied in order to know more about palmitate metabolism in this cell line.Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). This communication has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

Identification and characterization of new anti-angiogenic compounds from natural sources

The inhibition of angiogenesis has attracted broad attention in the field of pharmacological research, not only for cancer, but for other angiogenesis dependent diseases including ophthalmic, cutaneous and inflammatory diseases, as well as a number of rare diseases. Our research group has characterized multiple new natural bioactive compounds with multitargeted antiangiogenic effects by employing a well-established set of in vitro, in vivo and ex vivo preclinical models of angiogenesis. Most of them have been isolated from plants and terrestrial microorganisms, mainly due to their higher availability and because their therapeutic effects had been previously known in folk traditional medicines. In vitro primary screening includes cell differentiation and toxicity and proliferation assays. Secondary screening involves several experiments to evaluate effects on adhesion, migration, invasion, apoptosis or cell cycle analysis, among others. Additionally, we perform a further molecular characterization analyzing possible signaling pathways that are affected to elucidate their mechanism of action. The characterization is completed with the ex vivo aortic ring assay, and in vivo assays, as CAM and zebrafish assays, to ensure the anti-angiogenic ability. As a fruit of the mentioned screening, a number of compounds with remarkable anti-angiogenic activity have been identified and characterized.Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain)]. This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

Lactate Oxidation in Endothelial Cells: A Feature of All Endothelial Cells?

Resumen de la comunicaciónMetabolism of endothelial cells is a topic that has gained an increasing interest in the last years. This is due to their role in the angiogenic process, which is pathologically upregulated in several diseases, such as retinopathies, diabetes and cancer. Glycolysis, among other metabolic routes, has been found to be essential for triggering the angiogenic switch. Additionally, it has been seen that endothelial cells are able to take up lactate from the extracellular media, for example in the case of the tumor microenvironment, where cancer cells would have secreted high amounts of this metabolite. Endothelial cells would oxidize this lactate for obtaining energy, but lactate can also act as a signaling molecule for the angiogenic process. However, experiments to determine the molecular fate of lactate have been performed using only macrovascular endothelial cells. The aim of the present work is to prove whether microvascular endothelial cells are also able to take up and oxidize lactate. For this purpose, fluorimetry, isotopic labeling and Seahorse experiments were used to study the metabolism of a human microvascular endothelial cell line (HMEC). The expression levels of transcripts and proteins of different enzymes and transporters related to lactate metabolism were estimated by qPCR and Western blotting. The results obtained indicate that these cells rely on glycolysis for their metabolism, while the oxidation of glucose and glutamine seems to be considerably low. On the other hand, no lactate oxidation could be detected. We then checked the mRNA expression of the two isoenzymes of lactate dehydrogenase (LDH) and the two main lactate transporters, MCT1 and MCT4, and found that levels of LDH-B and MCT1 were undetectable. We failed to measure any MCT1 mRNA or protein expression either in normoxia or hypoxia. Hence, we can conclude that at least this microvascular endothelial cell line cannot use extracellular lactate as a metabolic fuel.Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

Fasentin, a glucose uptake inhibitor, is also able to inhibit angiogenesis

Es comunicación a congreso en formato pósterThe role of glucose on endothelial cell (EC) metabolism and angiogenesis has been an emerging issue in the last few years. Some inhibitors of glucose metabolism, such as 2-deoxyglucose, have been shown to have anti-angiogenic effects. Fasentin is a poor-studied inhibitor of glucose uptake which modulates GLUT-1 and GLUT-4 transporters in cancer cells. We wanted to test its possible effect on EC glucose uptake, showing a light decrease in HMEC at 100 µM. Lower doses did not affect this characteristic of glucose metabolism. In line with this fact, fasentin at 100 µM totally inhibited tube formation on Matrigel in these cells. This anti-angiogenic effect is not likely to be helped by a pro-apoptotic effect of fasentin but, as proved with additional assays, it could be due to a decrease on the signaling for extracellular matrix degradation. More research would be necessary in order to elucidate its fine regulation on angiogenesis and metabolism.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. [Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"]

Novel application assigned to toluquinol: inhibition of lymphangiogenesis by interfering with VEGF-C/VEGFR-3 signalling pathway

BACKGROUND AND PURPOSE Lymphangiogenesis is an important biological process associated with the pathogenesis of several diseases, including metastatic dissemination, graft rejection, lymphoedema and other inflammatory disorders. The development of new drugs that block lymphangiogenesis has become a promising therapeutic strategy. In this study, we investigated the ability of toluquinol, a 2-methyl-hydroquinone isolated from the culture broth of the marine fungus Penicillium sp. HL-85-ALS5-R004, to inhibit lymphangiogenesis in vitro, ex vivo and in vivo. EXPERIMENTAL APPROACH We used human lymphatic endothelial cells (LECs) to analyse the effect of toluquinol in 2D and 3D in vitro cultures and in the ex vivo mouse lymphatic ring assay. For in vivo approaches, the transgenic Fli1:eGFPy1 zebrafish, mouse ear sponges and cornea models were used. Western blotting and apoptosis analyses were carried out to search for drug targets. KEY RESULTS Toluquinol inhibited LEC proliferation,migration, tubulogenesis and sprouting of new lymphatic vessels. Furthermore, toluquinol induced apoptosis of LECs after 14 h of treatment in vitro, blocked the development of the thoracic duct in zebrafish and reduced the VEGF-C-induced lymphatic vessel formation and corneal neovascularization in mice. Mechanistically, we demonstrated that this drug attenuates VEGF-C-induced VEGFR-3 phosphorylation in a dose-dependentmanner and suppresses the phosphorylation of Akt and ERK1/2. CONCLUSIONS AND IMPLICATIONS Based on these findings, we propose toluquinol as a new candidate with pharmacological potential for the treatment of lymphangiogenesis-related pathologies. Notably, its ability to suppress corneal neovascularization paves the way for applications in vascular ocular pathologies.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work has been supported by personal funding by FP7-PEOPLE-2013-IEF Marie Curie Postdoctoral Fellowship (MGC). Acknowledged are the supporting grants from the Action de Recherche Concertée (ARC) (Université de Liège), the Fonds de la Recherche Scientifique-FNRS (F.R.S.-FNRS), the Foundation Against Cancer (foundation of public interest), the Centre Anticancéreux près l’Université de Liège, the Fonds Léon Fredericq (University of Liège), the Interuniversity Attraction Poles Programme-Belgian Science Policy (all from Belgium) and the Plan National Cancer (« Service Public Federal » from Belgium). Research in the lab of A.R.Q. and M.A.M. was supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER)

The noni anthraquinone damnacanthal is a multi-kinase inhibitor with potent anti-angiogenic effects

Este es el manuscrito que fue aceptado y que finalmente se publicó en Cancer Letters con el DOI: 10.1016/j.canlet.2016.10.037The natural bioactive compound damnacanthal inhibits several tyrosine kinases. Herein, we show that -in fact- damancanthal is a multi kinase inhibitor. A docking and molecular dynamics simulation approach allows getting further insight on the inhibitory effect of damnacanthal on three different kinases: vascular endothelial growth factor receptor-2, c-Met and focal adhesion kinase. Several of the kinases targeted and inhibited by damnacanthal are involved in angiogenesis. Ex vivo and in vivo experiments clearly demonstrate that, indeed, damnacanthal is a very potent inhibitor of angiogenesis. A number of in vitro assays contribute to determine the specific effects of damnacanthal on each of the steps of the angiogenic process, including inhibition of tubulogenesis, endothelial cell proliferation, survival, migration and production of extracellular matrix remodeling enzyme. Taken altogether, these results suggest that damancanthal could have potential interest for the treatment of cancer and other angiogenesisdependent diseases.Supported by grants BIO2014-56092-R (MINECO and FEDER), P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). JAGV had the financial support of Vicerrectorado de Investigación y Transferencia (University of Málaga, Spain). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript

Homocysteine treatment alters redox capacity of both endothelial and tumor cells

Homocysteine is a non-proteinogenic amino acid playing key roles in two interconnected metabolic pathways, namely, the activated methyl cycle and the linear trans-sulfuration pathway that allows the conversion of methionine to cysteine. A dysregulation of intracellular homocysteine metabolism could yield an increased export of this amino acid, leading to hyperhomocysteinemia, which has been associated with an increased risk of cardiovascular diseases. In spite of decades of experimental effort, there is no definitive consensus on what could be the molecular mechanisms whereby hyperhomocysteinemia could contribute to cardiovascular disease. The redox active nature of homocysteine has favored the idea of an induction of oxidative stress as the underlying mechanism of homocysteine toxicity. In contrast, homocysteine can also behave as an anti-oxidant. The present work is aimed to further analyze the capacity of homocysteine to modulate the redox capacity of both endothelial and tumor cells. [Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain)].Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Synthesis and biological activity of a new class of antitumor cyclopeptides based on the solomonamides

Solomonamides A (1) and B (2) are novel natural products recently isolated from the marine sponge Theonella swinhoei [1]. Preliminary structural studies revealed an unprecedented cyclic peptide type structure. Interestingly, solomonamide A exhibits anti-inflammatory activity, showing potent reduction (60%) of inflammation at a very low concentration of 100 µg/kg in animal models. However, the scarcity of these compounds from their natural sources has been a drawback for further pharmacological assays. In fact, the anti-inflammatory activity of solomonamide B was not evaluated due to the limited amounts. This difficulty to access large amounts of these compounds makes quite difficult to gain insight into their biological profiles and mechanism of action and justifies the chemical synthesis of this new class of cyclic peptides. As a consequence, the solomonamides have been the subject of several synthetic efforts [2] notably by the Reddy group who has recently reported the first total synthesis of solomonamide B based on a intramolecular Heck reaction, which led to a revision of the initially proposed structure for 2 [3].Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech