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Corylus Avellana: a New Biotechnological Source of Anticancer Agents

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Corylus Avellana: a New Biotechnological Source of Anticancer Agents Corylus avellana: a new biotechnological source of anticancer agents Ana Gallego Palacios TESI DOCTORAL UPF / 2015 Thesis Directors: Dr. Elisabet Moyano Claramunt, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra. Dr. Mercedes Bonfill Baldrich, Departament de Productes Naturals, Biologia Vegetal i Edafologia, Universitat de Barcelona. DEPARTAMENT DE CIENCIES EXPERIMENTALS I DE LA SALUT ii “Mira a la derecha y a la izquierda del tiempo y que tu corazón aprenda a estar tranquilo” Federico García Lorca iii Agraïments/Agradecimientos/Acknowledgement En primer lugar quería agradecer a los organismos que han apoyado económicamente el trabajo plasmado en esta tesis, al Ministerio de Educación y Ciencia de España (BIO2011-29856-C02-1) y a Generalitat de Catalunya (2014SGR215). También a la Universidad Pompeu Fabra por proporcionarme la beca pre-doctoral que me ha permitido realizar este doctorado y la Universidad de Barcelona donde he realizado todo el trabajo experimental. Hay muchas personas que han contribuido, tanto directa como indirectamente, en este proyecto y a las cuales quiero agradecer. En primer lloc volia agrair a les meves directores de tesi, la Dr. Elisabeth Moyano i la Dr. Mercedes Bonfill per l’ajuda, la dedicació i el suport que m’han donat durant tots aquest anys. Eli he pogut aprendre molt de tu, no només a nivell científic sinó també a nivell personal. Gracies pel teu temps, pel teu positivisme i per les teves reflexions. A la Mercè perquè sempre has tingut la porta del teu despatx oberta per qualsevol dubte i perquè sempre m’has donat la llibertat i la independència necessària per millorar. Al Dr. Javier Palazón y a la Dr. Rosa Cusidó porque aunque oficialmente no son los directores de esta tesis han contribuido enormemente a ella, aconsejándome y ayudándome en todo momento. Especialmente al Dr. Palazón por valorarme y por todas las oportunidades que me ha ofrecido a nivel formativo, ya sea asistiendo a congresos o mediante estancias en diversas universidades. Todo ello, junto con el conocimiento que todos ellos me han aportado, me ha permitido aprender muchísimo y madurar v como científica. También, gracias a todos ellos por el buen ambiente de trabajo que han creado durante todo este tiempo. During these years I had the chance work and collaborate with some departments around Europe and I would like to acknowledge to all of them. They kindly collaborate with us, and they help me so much to improved my knowledge: to Dr. Paul Christou and Dr Teresa Capell from the University of Lleida, to Dr. Yamal Ouazzani and Dr. Emilie Adelin from the CNRS (Centre national de la recherche scientifique, Gif-sur Yvette, France) to Dr Regine Eibl and Nicole Imseng from the University of Zurich and to Dr Isidoro Mentón and Dr. M Isabel V. Baanante from the University of Barcelona. All of them and all the students from their groups were really friendly and professionals and it was a pleasure for me works with them and learns from their experience. También quería agradecerle al Dr. Manuel Pastor su paciencia y ayuda con el diseño del experimento fraccional factorial y con los análisis estadísticos. A los servicios científicotécnicos de la UB, especialmente al Dr Isidre Casals, a la Dr. Olga Jáuregui, al Dr. Albert Adeva y al Dr. David Bellido por su apoyo. Me gustaría recalcar la ayuda de Olga, de la que he podido aprender mucho y la cual me ha enseñado la importancia de trabajar con seriedad y rigor, siendo siempre lo más crítica posible con todo aquello obtenido. Durante estos años he podido trabajar con muchas personas de todo el mundo, creándose un ámbito multicultural que me ha permitido aprender muchas culturas y abrir mi mente. Con muchos de ellos he podido compartir grandes momentos y experiencias como con Miriam, Karla, Rafa, Liliana, Diego, Raúl. Gracias por todo y por vi todo lo que vendrá. También he tenido la oportunidad de trabajar con muchas personas que han hecho estancias en nuestro laboratorio. Algunos de ellos han colaborado activamente con esta tesis, como Kristina, Lucie, Victor, Xavi, David, Paula y Gemma. Con todos ellos tuve la posibilidad de trabajar mano a mano, formando un equipo y aprendiendo el uno del otro continuamente. Fuisteis de gran apoyo en los momentos difíciles. También hubieron otras personas que aunque no trabajaron directamente conmigo, amenizaron el trabajo y con los que también compartí grandes momentos: Oscar, Jannet, Dulce, Alejandra, Morteza, Ana Belen, Maryam, Esther, Mohamad, Nadia, Suellen, Marta, Maria, Ana Luisa, Farnoosh, Marta, Fran, Adrian, Andrea, Said, Arif, Burhan, Sara, Hajar, Virginie, Ozçag, Aida, Ali, David, Carmen…A Yaiza, a la que conocí en el laboratorio y se convirtió en una de mis mejores amigas y en la mejor compañera de piso que alguien puede tener. Por nuestras noches de terapia, nuestras cervezas de consuelo, por nuestra peculiar visión del mundo y por el gran apoyo que nos hemos dado mutuamente durante todos estos años. A los de arriba: Nati, Laura, Jean, Ying, Carmellina, Patricia, Olivia, Carolina, Lucia, Gabriela, Javier, Luciana, y a los de al lado: Kosta, Miren, Joan, Xavi, Marta, Ambra…porque siempre hemos sido una gran familia y hemos compartido muchas comidas, salidas y grandes charlas. A mis amigos de toda la vida Juana y Jordi, y a todo el grupo de querer es poder: Calero, Xavi, Javi, Laura, Joan, Adrià, Sandra, David, Iván y Diego. Siempre me habéis apoyado y animado, me habéis mantenido con los pies en la tierra alejándome de mi vii pequeño mundo y haciéndome ver que siempre hay otro punto de vista en la vida. Gracias por todo lo que hemos compartido, y por todo aquello que vendrá. A Oscar porque es difícil que la vida nos junte, pero por mucho tiempo que pasemos sin vernos siempre tendremos una gran amistad. A les unifriends, perquè des del principi hem estat juntes i encara ho estem, tot i que algunes hagin marxat ben lluny. Pels nostres sopars japonesos, les nostres converses i el suport que sempre ens hem donat: Mireia, Monica, Miriam, Laura, Anna, Angela i Cris. Y por último, uno de los pilares más importantes de mi vida, a mi familia. Porque siempre habéis estado allí, en los momentos más felices y en los momentos más tristes. Siempre me habéis apoyado y aconsejado en todo. A mi padre porque siempre me has ayudado, apoyado y comprendido (aunque no siempre ha sido fácil), queriendo lo mejor para mí. A mi madre porque me ha guiado y ha hecho que hoy sea lo que soy, porque me enseñó a ser crítica y a luchar. A Raquel, Sergio y Eloy y a David, Mónica y Lucas porque siempre estáis ahí, porque pese a ser la freak de la familia, siempre me habéis apoyado y comprendido. Siempre contaremos los unos con los otros. También, a Laura por nuestras comidas de primas, por tu gran apoyo y comprensión y por nuestra complicidad. Siempre estaremos allí, la una para la otra. A todos vosotros, y a muchos más, muchas gracias por haber contribuido a este periodo de mi vida, sin vosotros nada hubiese sido igual. viii ix x Abstract The difficulty of increasing taxane production and reducing its costs has prompted a search for new biotechnological sources. The unexpected discovery of taxanes in Corylus avellana has generated considerable interest in studying this plant and its derived cell cultures. We therefore focused this work on the study of cell suspension cultures of C. avellana as a new biotechnological approach to the production of taxol and related taxanes. With this goal, we optimized a scale-up process, the type of elicitor and moment of elicitation, cell culture growth, and an analytical technique to detect the compounds of interest. Also, C. avellana tree extracts were analyzed, leading to the identification of compounds with antiproliferative activity against cancer cell lines. These results provide new insights into cell suspension cultures of C. avellana and how to increase taxane production. xi xii Resum La dificultat d’incrementar la producció de taxans i de reduir els seus costos ha promogut la cerca de noves fonts biotecnològiques. El fet de trobar taxol i taxans a Corylus avellana ha incrementat l’ interès en el seu estudi. Per aquesta raó, hem focalitzat aquest treball en trobar noves estratègies per la producció de taxans en suspensions cel·lulars de C. avellana. Amb aquest objectiu, hem optimitzat el procés d’escalat, l’elicitació i el creixement dels cultius, així com la tècnica analítica per identificar i quantificar els compostos d’interès. A més, hem estudiat diversos extractes de l’arbre, fet que ens ha permès trobar altres compostos amb activitat antiproliferativa en línies cel·lulars cancerígenes. Tots els resultats proporcionen noves eines pel cultiu de suspensions cel·lulars de C. avellana i per a l’increment de la producció de taxans. xiii xiv Index Pages Acknowledgments.......................................................... v-viii Abstract.......................................................................... xi Resum............................................................................. xiii Abbreviations…………………………………………. 19, 20 Justification and aims…………………………………. 23, 24 1. Introduction........................................................ 27-58 1.1 Corylus avellana L………………………... 29-33 1.1.1 Botanical description…………….... 29-31 1.1.2 Nutritional profile............................ 31, 32 1.1.3 Phytochemical profile...................... 32, 33 1.2 Taxol and related taxanes............................. 33-40 1.2.1 Mechanism of action........................ 36,37 1.2.2 Difficulties in taxol production........ 37-40 1.3 Empirical approach to increase high-value secondary metabolites in plant culture......... 40-51 1.3.1 Significance of the medium composition in cell growth............... 42-44 1.3.2 Significance of the elicitation in secondary metabolite production..... 44-49 1.3.3 Scale-up............................................ 49-51 1.4 Improving taxane analytical detection......... 51-55 1.5 Drug discovery from medicinal plants........
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