NATURAL BIOACTIVE COMPOUNDS AS INHIBITORS OF CANCER TARGETS WILSON MALDONADO ROJAS MSc. NATURAL BIOACTIVE COMPOUNDS AS INHIBITORS OF CANCER TARGETS WILSON MALDONADO ROJAS MSc. UNIVERSITY OF CARTAGENA SCHOOL OF PHARMACEUTICAL SCIENCES Ph.D. PROGRAM IN ENVIRONMENTAL TOXICOLOGY CARTAGENA DE INDIAS 2016 NATURAL BIOACTIVE COMPOUNDS AS INHIBITORS OF CANCER TARGETS WILSON MALDONADO ROJAS MSc. This work is a document submitted as a requirement for obtain Ph.D. In Environmental Toxicology Prof. JESÚS OLIVERO VERBEL. Ph.D. Director Environmental and Computational Chemistry Group University of Cartagena UNIVERSITY OF CARTAGENA SCHOOL OF PHARMACEUTICAL SCIENCES Ph.D. PROGRAM IN ENVIRONMENTAL TOXICOLOGY CARTAGENA DE INDIAS 2016 CAUTIONARY NOTE The University is not responsible for the items issued by students in their thesis work. Only ensure that nothing contrary is published to dogma and Catholic morals and because the theses do not contain personal attacks against someone, rather look in them the desire to seek truth and justice. "Article 23 of Resolution 13, July 1946 . ACCEPTANCE NOTE __________________________ __________________________ __________________________ __________________________ Jury President __________________________ Jury __________________________ Jury __________________________ Jury Cartagena de Indias, 04th August 2016 DEDICATION To God who created the universe, manager and participant in each of my plans and dreams, by which it was possible to reach this achievement... To my mother Emilse Rojas Blanco and my father Antonio Maldonado Rodriguez for being my motivation to excel at all times, teaching me the real value of things, the perseverance to achieve the proposed goals, and even when they are not with me right now, I remember them at all times and dedicatethis triumph from the bottom of my heart... To Liliana Amaya, mother of my three beautiful daughters Carolina, Paula Andrea, and my little Valeria, who are my main motivation, my most precious treasures, and constant source of warmth, tenderness and love... To my brothers Oscar, Antonio, Luis Alfonso and Luz Marina, and my whole family for the unconditional support given in all these years of hard work and dedication despite the adversities... To All my friends and colleagues for their help... This is for you all. ACKNOWLEDGMENTS Firstly, I would like to express my gratitude to God for giving me the strength and the ability to move forward in every step of my personal training, for his immense compassion and incomparable love, to him be glory and honor forever. Besides God, I would also like to thank my family for their support, motivation, confidence and time, many thanks indeed have been each of you builders of this achievement. My sincere thanks to Professor Jesus Olivero Verbel, my advisor and friend, for his teachings, advices, patience, unconditional support in good and bad times , and the opportunity provided to know and let me be part of his workgroup, and to grow professionally and personally taught by knowledge and high regard, thank you for everything. To all members of the Environmental and Computational Chemistry Group, an excellent group of people and professionals thanks to all without exception, for its immense collaboration. To undergraduate students María Isabel Olivero, Juan Carlos Salinas, Jessica Cerón, Joyce Ramírez, young researchers Karen Rivera, Nadia Coronado, Juan David Sierra, Alejandra Manjarres, Angela Bertel Sevilla and Ricardo Hernandez. To all my professors from the Ph.D program in Environmental Toxicology, especially Prof. Boris Johnson Restrepo, for all his knowledge and advice during the project, and Prof. Yovanni Marrero Ponce, for his valuable support throughout my training in the DTA, the confidence, advices and knowledge transmitted. To all personnel at the School of Pharmaceutical Sciences, administratives and all staff in general. To the University of Cartagena, alma mater, thank you very much for giving me the opportunity to continue my training process. I also want to thank Professor Enrique Melendez, an extraordinary scientist and a great person, for giving me the opportunity to work with his research group at the University of Puerto Rico, Mayaguez, as well as, his team of researchers, Xiomara Narvaez, José Carmona and Moralba Dominguez, for their great disposition and patience to train me, and give me part of their knowledge to achieve some of the results presented in this thesis. I would also like to thank people from other research groups who helped me with the activities of my project in my international internship in Puerto Rico, some of them are Carlos Ortega, Leonardo Pacheco, Jorge Guette, Jose Luis Ruiz and Andres Roman. And to all those who supported and believed in me, thanks a lot. Finally, I wish to thank to the University of Cartagena, Cartagena (Colombia); as well as as the program for supporting research groups, third call for mobility of students from research groups at the University of Cartagena, 2014 sponsored by the Vice-Rectory for Research of the University of Cartagena (2013–2016), CENIVAM (RC-0572-7-2012) and the National Program for Doctoral Studies in Colombia (Colciencias, 528-2011). Natural Bioactive Compounds as Inhibitors of Cancer Targets. Ph.D. In Environmental Toxicology CONTENTS ABSTRACT ....................................................................................................................... 15 RESUMEN ........................................................................................................................ 17 CHAPTER 1. THESIS OVERVIEW ................................................................................... 22 1.1 RATIONALE ........................................................................................................... 22 1.2. HYPOTHESIS ....................................................................................................... 25 1.3. PERTINENCE ....................................................................................................... 25 1.4. OBJECTIVES AND THESIS STRUCTURE .......................................................... 27 1.5. REFERENCES ...................................................................................................... 28 CHAPTER 2. INTRODUCTION TO THE CANCER .......................................................... 35 2.1. CANCER OVERVIEW ........................................................................................... 35 2.2. DNA METHYLATION IN SKIN CANCER .............................................................. 37 2.3. MOLECULAR PROTEIN-LIGAND DOCKING ....................................................... 38 2.4. NATURALLY OCCURRING COMPOUNDS AND THEIR APPLICATION IN MEDICINE .......................................................................................................................... 39 2.5. MOLECULAR BIOLOGY ASSAYS (PCR/RFLP) .................................................. 40 2.6. CELL VIABILITY ASSAYS .................................................................................... 41 2.7. SPECTROSCOPIC METHODS USED TO EVALUATE PROTEIN-LIGAND INTERACTIONS ................................................................................................................. 41 2.8. REFERENCES ...................................................................................................... 42 CHAPTER 3. POTENTIAL INTERACTION OF NATURAL DIETARY BIOACTIVE COMPOUNDS WITH COX-2 .................................................................................................. 49 3.1. ABSTRACT ........................................................................................................... 49 3.2. INTRODUCTION ................................................................................................... 50 3.3. MATERIALS AND METHODS .............................................................................. 50 3.4. RESULTS AND DISCUSSION .............................................................................. 54 3.5. CONCLUSIONS .................................................................................................... 67 3.6. REFERENCES ...................................................................................................... 67 CHAPTER 4. FOOD-RELATED COMPOUNDS THAT MODULATE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE MAY ACT AS ITS INHIBITORS ............................. 77 4.1. ABSTRACT ........................................................................................................... 77 4.2. INTRODUCTION ................................................................................................... 78 9 Natural Bioactive Compounds As Inhibitors Of Cancer Targets. Ph.D. In Environmental Toxicology 4.3. MATERIALS AND METHODS .............................................................................. 79 4.4. RESULTS AND DISCUSSION .............................................................................. 82 4.5. CONCLUSIONS .................................................................................................... 90 4.6. REFERENCES ...................................................................................................... 91 CHAPTER 5. COMPUTATIONAL FISHING OF NEW DNA METHYLTRANSFERASE INHIBITORS FROM NATURAL PRODUCTS ......................................................................... 99 5.1. ABSTRACT ........................................................................................................... 99 5.2. INTRODUCTION ................................................................................................