“Mecanismo de acción de lípidos antitumorales sintéticos en Saccharomyces cerevisiae

[ES] Los lípidos antitumorales (ATLs) son una familia de compuestos sintéticos, de relevancia médica por su citotoxicidad específica frente a determinados protozoos y células cancerosas. Se han propuesto diversos modos de acción, pues los ATLs interactúan con los lípidos biológicos, modulando la estructura de las membranas celulares y la homeostasis lipídica y provocando efectos pleiotrópicos. Se han estudiado además varios ATLs en distintos tipos celulares. Es por tanto necesario dilucidar qué mecanismos biológicos median la citotoxicidad, cómo interactúan y qué relevancia presenta cada mecanismo según el compuesto concreto en cada tipo celular. Mientras que los estudios previos sobre el modo de acción de los ATLs han sido mayoritariamente realizados en células de mamífero, en esta tesis doctoral se utilizó la levadura de gemación Saccharomyces cerevisiae. Este organismo ha sido empleado como modelo para el estudio de fármacos, pues combina la similitud con las células de mamífero ¿misma arquitectura celular eucariota, homólogos en alrededor de un 30% de sus genes¿ con una mayor facilidad y rapidez de cultivo, manejo y manipulación genética. El trabajo aquí planteado se basa en screenings de colecciones -bien de knock-outs, bien de células con una dosis génica aumentada- que permitan aislar cepas resistentes o sensibles a los fármacos estudiados. Posteriormente se identifican procesos, mecanismos y estructuras celulares implicados en la toxicidad a los fármacos. Experimentos de biología celular adicionales permiten ampliar y completar dicha información. Específicamente se analizaron la incorporación y translocación de ATLs, el efecto de los mismos sobre la translocación de la ATPasa Pma1 y el papel de la función mitocondrial en la toxicidad por ATLs. Los resultados esperados de este trabajo, además de novedosos, tienen un amplio enfoque dada la metodología empleada. Esto permite plantear un modelo del mecanismo de acción de los ATLs que explique los fenotipos observados. Además de la información relevante al mismo organismo modelo empleado, el estudio paralelo de dichos fenómenos en otros modelos biológicos de interés sanitario abre potenciales nuevas vías de estudio del mecanismo de los ATLs.[EN] The work here raised bases in screenings of collections - good of knockouts, good of cells with a dose génica increased - that allow to isolate vine-stocks resistant or sensitive to the studied medicaments. Later processes, mechanisms and cellular structures are identified implied in the toxicity to the medicaments. Additional experiments of cellular biology allow to extend and to complete the above mentioned information. Specifically the incorporation was analyzed and translocación of ATLs, the effect of the same ones on the translocación of the ATPasa Pma1 and the paper of the function mitocondrial in the toxicity for ATLs. The results expected from this work, besides new, have a wide approach given the used methodology. This there allows to raise a model of the mechanism of action of the ATLs who explains the observed phenotypes. Besides the relevant information to the same organism used model, the parallel study of the above mentioned phenomena in other biological models of sanitary interest opens potential new routes of study of the mechanism of the ATLs

Mecanismos de acción de lípidos antitumorales sintéticos en Saccharomyces cerevisiae

Memoria presentada por Álvaro Cuesta Marbán para optar al grado de Doctor por la Universidad de Salamanca y realizada en Instituto de Biología Molecular y Celular del Cancer de Salamanca del CSIC.Presentaciones del trabajo en congresos: 1. 8th International Meeting on Yeast Apoptosis (2-6 mayo 2011, University of Kent, Canterbury, UK) con presentación de la ponencia Genome‐wide screenings and subcellular localization analyses uncover major events in the mechanism of action antitumor lipids in Saccharomyces cerevisiae. 2. Reunión del grupo de Control del Crecimiento, la Proliferación y la Supervivencia Celular perteneciente a la Red Temática de investigación Cooperativa en Cáncer (11-12 abril 2011), con presentación de la ponencia Mecanismos de acción de lípidos antitumorales en Saccharomyces cerevisiae por medio de screenings químico-genómicos. 3. XXXIII Congreso de la Sociedad española de bioquímica y biología molecular (14-17 septiembre 2010, Córdoba, España), con presentación del poster Mecanismo de acción de éter-lípidos antitumorales en Saccharomyces cerevisiae por medio de screenings químico-genómicos 4. Encuentro Syracuse Biomaterials Institute: 2nd Annual Offsite Meeting and Poster Session, Universidad de Syracuse, New York, con presentación del poster Identification of Potential Pharmacological Targets of Anti-tumor Lipids through Genomic Screening Congreso 2009 Northeast Regional Yeast Meeting, Universidad de Cornell, Ithaca (New York)Los lípidos antitumorales (ATLs) son una familia de compuestos sintéticos, de relevancia médica por su citotoxicidad específica frente a determinados protozoos y células cancerosas. Se han propuesto diversos modos de acción, pues los ATLs interactúan con los lípidos biológicos, modulando la estructura de las membranas celulares y la homeostasis lipídica y provocando efectos pleiotrópicos. Se han estudiado además varios ATLs en distintos tipos celulares. Es por tanto necesario dilucidar qué mecanismos biológicos median la citotoxicidad, cómo interactúan y qué relevancia presenta cada mecanismo según el compuesto concreto en cada tipo celular. Mientras que los estudios previos sobre el modo de acción de los ATLs han sido mayoritariamente realizados en células de mamífero, en esta tesis doctoral se utilizó la levadura de gemación Saccharomyces cerevisiae. Este organismo ha sido empleado como modelo para el estudio de fármacos, pues combina la similitud con las células de mamífero (misma arquitectura celular eucariota, homólogos en alrededor de un 30% de sus genes) con una mayor facilidad y rapidez de cultivo, manejo y manipulación genética. El trabajo aquí planteado se basa en screenings de colecciones -bien de knock-outs, bien de células con una dosis génica aumentada- que permitan aislar cepas resistentes o sensibles a los fármacos estudiados. Posteriormente se identifican procesos, mecanismos y estructuras celulares implicados en la toxicidad a los fármacos. Experimentos de biología celular adicionales permiten ampliar y completar dicha información. Específicamente se analizaron la incorporación y translocación de ATLs, el efecto de los mismos sobre la translocación de la ATPasa Pma1 y el papel de la función mitocondrial en la toxicidad por ATLs. Los resultados esperados de este trabajo, además de novedosos, tienen un amplio enfoque dada la metodología empleada. Esto permite plantear un modelo del mecanismo de acción de los ATLs que explique los fenotipos observados. Además de la información relevante al mismo organismo modelo empleado, el estudio paralelo de dichos fenómenos en otros modelos biológicos de interés sanitario abre potenciales nuevas vías de estudio del mecanismo de los ATLs.Peer reviewe

Alteration of plasma membrane organization by an anticancer lysophosphatidylcholine analogue induces intracellular acidification and internalization of plasma membrane transporters in yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H+-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by a Natural Sciences and Engineering Research Council of Canada discovery grant, a seed grant from the University of Calgary, a Natural Sciences and Engineering Research Council of Canada University Faculty award (to V. Z.), Canadian Institutes of Health Research Grant 14124 (to C. R. M.), Spanish Ministerio de Economia y Competitividad Grants SAF2008-02251 and SAF2011-30518, Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, co-funded by the Fondo Europeo de Desarrollo Regional of the European Union Grants RD06/0020/1037 and RD12/0036/0065, European Community's Seventh Framework Programme FP7-2007-2013 Grant HEALTH-F2-2011-256986, (PANACREAS), and Junta de Castilla y León Grants CSI052A11-2 and CSI221A12-2 (to F. M.).Peer Reviewe

Human initiator caspases trigger apoptotic and autophagic phenotypes in Saccharomyces cerevisiae

Caspases are a family of proteases that participate in the progression and execution of the apoptotic program. However, regulation of the caspase activation and their substrates has not yet been fully elucidated. Here we explore the effect of the ectopic expression of the human initiator caspases-8 and -10 in Saccharomyces cerevisiae. Our results showed that the expression of human CASP10 and CASP8 triggers certain apoptotic markers such as a massive production of reactive oxygen species (ROS), chromatin condensation and phosphatidylserine externalization, finally leading to cell death. In response to hydroxyurea (HU), yeast cells expressing caspase-10 did not reduce the replication of DNA and escaped to the intra-S checkpoint of the cell cycle. In addition, caspase-10 expression induced yeast vacuolization and a vacuole-associated phenotype resembling autophagy. Other intracellular alterations such as disorganization of the actin cytoskeleton, cell wall damage, and aberrations within the endoplasmic reticulum lumen were also associated with caspase-10 expression. Furthermore, caspase-induced cell death was completely dependent on the proteolytic activation of the enzyme but, in contrast, was not dependent on either of the endogenous yeast apoptotic proteins Aif1 and Mca1 or the mitochondria. © 2008 Elsevier B.V.This work was supported by grants from Ministerio de Ciencia e Innovación of Spain (AGL2005-07245-C03-03 to J. L. R., and SAF2008-02251 and RD06/0020/1037 - Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III to F. M.), Junta de Castilla y León (SA008B08) to A. J. Fundación “la Caixa” (BM05-30-0) and NIH grant GM621284 to A. M. N.. P. L. S. and A. C. M. are recipients of FPU predoctoral fellowships from the Spanish Ministerio de Educación y Ciencia.Peer Reviewe

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine

[Background]:Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.[Methodology/Principal Findings]:Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.[Conclusions/Significance]:The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis.This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (SAF2014-59716-R and BIO2014-56930-P), Instituto de Salud Carlos III (RD12/0036/0065 from Red TemaÂtica de Investigación Cooperativa en Cáncer, cofunded by the EU's European Regional Development Fund ± FEDER),European Community's Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS), and Spain-UK International Joint Project grant from The Royal Society-CSIC (2004GB0032). CG was supported by the RamoÂn y Cajal Program from the Spanish Ministerio de Ciencia e Innovación.AÂCM was recipient of Formación de Profesorado Universitario predoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación.Peer reviewe

Genome-wide screenings and subcellular localization analyses uncover major events in the mechanism of action of antitumor lipids in Saccharomyces cerevisiae

Trabajo presentado al "8th International Meeting on Yeast apoptosis" celebrado en Kent del 2 al 6 de mayo de 2011.-- et al.Peer Reviewe

Drug uptake, lipid rafts, and vesicle trafficking modulate resistance to an anticancer lysophosphatidylcholine analogue in yeast

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosineresistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Spanish Ministerio de Economia y Competitividad Grants SAF2005-04293, SAF2008-02251, and SAF2011-30518, Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, co-funded by the Fondo Europeo de Desarrollo Regional of the European Union Grants RD06/0020/1037 and RD12/0036/0065, European Community's Seventh Framework Programme FP7-2007-2013 Grant HEALTH-F2-2011-256986 (PANACREAS), Fundación “la Caixa” Grant BM05-30-0, Junta de Castilla y León Grants CSI052A11-2 and CSI221A12-2 (to F. M.), Spanish Ministerio de Economia y Competitividad Grants BIO2008-00194 and BIO2011-23901, Junta de Castilla y León Grant GR147 (to J. L. R.), Fondo de Investigación Sanitaria and European Commission (FIS-FEDER) Grants 06/0813 and PS09/01915, Junta de Castilla y León Biomedicine Project 2010-2011 (to C. G.), Spanish Ministerio de Economia y Competitividad Grant CTQ2010-16457 (to A. U. A.), Canadian Institutes of Health Research Grant 14124 (to C. R. M.), a Natural Sciences and Engineering Research Council of Canada discovery grant, a seed grant from the University of Calgary, and an Natural Sciences and Engineering Research Council of Canada University faculty award (to V. Z.).Peer Reviewe

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine.

Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis

Web-Based learning in dental traumatology: diagnosis in young permanent teeth.

Los objetivos alcanzados en este proyecto se pueden resumir en el desarrollo de una herramienta informática destinada al alumno de Odontología para aumentar sus conocimientos y sus capacidades en el ámbito del diagnóstico odontológico en Odontopediatría, en concreto en el ámbito de la traumatología en dientes permanentes jóvenes y desarrollar su aprendizaje autónomo en este proceso. Esta herramienta permite: -entrenar al alumno en el reconocimiento de diferentes lesiones clínicas de tejidos dentales derivadas de traumatismos orales a través del análisis de fotografías de casos reales. -entrenar al alumno en el reconocimiento de las lesiones clínicas del ligamento periodontal derivadas de traumatismos orales en fotografías de casos reales. - entrenar al alumno en el reconocimiento de las lesiones de los tejidos duros y del ligamento periodontal derivadas de traumatismos en radiografías de los mismos casos mostrados en las fotografías. .-hacer practicar al alumno la relación entre el diagnóstico clínico y el diagnóstico radiográfico en dientes permanentes traumatizados. - hacer practicar el método de clasificación de lesiones traumáticas en dentición permanente. -recordar y ampliar los conocimientos del alumno en el ámbito de la traumatología a través de resúmenes prácticos.Depto. de Especialidades Clínicas OdontológicasFac. de OdontologíaFALSEsubmitte