Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN ONCOLOGIA, EMATOLOGIA E PATOLOGIA Ciclo XXXI Settore Concorsuale di afferenza: 06/A2 Settore Scientifico disciplinare: MED/04 MEMBRANE FATTY ACID REMODELING ALONG CYTOTOXIC EVENTS INDUCED BY A NOVEL COPPER-BASED ANTITUMORAL AGENT AND TUMOR PROGRESSION IN MICE MODELS Presentata da: Maria Louka Coordinatore Dottorato Supervisore Prof. Pier-Luigi Lollini Prof. Andrea Bolognesi Esame finale anno 2019 To my family and Bastien, ‘The route marks an address. And a route is much more than a result.’ Jorge Bucay - Let Me Tell You a Story Acknowledgements First and foremost, I would like to express my gratitude to Dr. C. Ferreri and Dr. C. Chatgilialoglu for giving me the opportunity to perform this 3-year training within Lipinutragen and take part in the European research network 'ClickGene'. This was a particularly rich experience and their support and encouragement guided me through it. I would like to thank Prof. A. Bolognesi for offering me a Ph.D candidate position at the University of Bologna and giving me access to his laboratory. The thesis supervision was accompanied by his insightful comments and suggestions. Special thanks to the members of his group Dr. L. Polito and Dr. M. Bortolloti for their invaluable guidance and help in experiments execution. I am also grateful to Dr. V. Sunda, Dr. A. Sansone and G. Menounou for sharing their knowledge and being always willing to lend a hand. Thanks to all the members of Lipinutragen laboratory and Bio Free Radicals’ group at ISOF, CNR that created a nice working environment and supported me, whenever needed. Furthermore, I appreciate my collaboration with Prof. E. Efthimiadou and Dr. M. Krokidis, who hosted me in their lab during my secondment at the NCSR Demokritos and contributed to my research activities. A special mention to G. Toniolo and N. Fantoni for producing and providing me materials within the frame of collaborative projects. I gratefully acknowledge the Marie Skłodowska-Curie action for providing my fellowship. This project has indeed received funding from the European Union's Horizon 2020 framework programme for research and innovation under grant agreement No 642023. Finally, I would also like to address thanks to all my friends who contributed to my every day’s happiness and personal balance. Last but not least, I am grateful towards my family and Bastien for their unconditional love and support throughout this ‘journey’. Table of contents Table of contents Abstract ........................................................................................ IV 1. Introduction .................................................................................1 1.1 Fatty acid-based membrane lipidomics .............................................. 1 1.1.1 Plasma membrane ............................................................................................... 1 1.1.2 Membrane fatty acids .......................................................................................... 5 1.1.3 Membrane role in cell proliferation and survival related signaling .................. 18 1.2 Membrane lipidome in cancer .......................................................... 20 1.2.1 Altered fatty acid synthesis in cancer ................................................................ 21 1.2.2 Fatty acid composition of tumor patients’ erythrocytes .................................... 25 1.2.3 Antineoplastic agents affecting membrane’s structure and properties .............. 27 1.2.4 Fatty acid supplementation in cancer prevention and co-adjuvant treatment ... 28 2. Materials and methods .............................................................32 2.1 Materials .............................................................................................. 32 2.1.1 Reagents ............................................................................................................ 32 2.1.2 Instruments ........................................................................................................ 33 2.2 Methods ............................................................................................... 33 2.2.1 Cell cultures ....................................................................................................... 33 2.2.2 Cell viability assay ............................................................................................ 33 2.2.3 Evaluation of apoptosis ..................................................................................... 34 2.2.4 Microsomes isolation ........................................................................................ 34 2.2.5 Protein concentration ......................................................................................... 36 2.2.6 Microsomes activity .......................................................................................... 36 2.2.7 SCD1 activity assay .......................................................................................... 37 2.2.8 Phospholipid Extraction .................................................................................... 37 2.2.9 Thin layer chromatography ............................................................................... 38 2.2.10 Gas chromatographic fatty acid analysis ......................................................... 38 2.2.11 Animal studies ................................................................................................. 42 2.2.12 Xenografts model construction ....................................................................... 42 Table of contents 2.2.13 Statistical analysis ........................................................................................... 43 3. The copper complex: Cu-TPMA-Phen ...................................44 3.1 Background and Objectives .............................................................. 44 3.2 Molecular structure ............................................................................ 46 3.3 Effects on cell viability ....................................................................... 47 3.3.1 Cytotoxicity ....................................................................................................... 47 3.3.2 Evaluation of cell death ..................................................................................... 49 3.3.3 Caspase activation ............................................................................................. 51 3.3.4 Protection by inhibitors/scavengers .................................................................. 53 3.4 Effects on membrane lipidome .......................................................... 56 3.4.1 Membrane fatty acid analysis of copper treated cells ....................................... 56 3.4.2 Time-course lipidomics ..................................................................................... 60 3.4.3 Membrane fatty acid analysis after inhibitors/scavengers pre-treatment .......... 62 3.5 Cell-free SCD1 activity assay ............................................................ 64 3.6 Fatty acids supplementation .............................................................. 65 3.7 Drug encapsulation in pH-sensitive polymeric nanoparticles........ 67 3.7.1 Cytotoxicity of encapsulated Cu-TPMA-Phen ................................................. 67 3.7.2 Membrane lipidomics of encapsulated Cu-TPMA-Phen .................................. 69 3.8 Conclusions ......................................................................................... 71 4. Membrane remodeling in tumor-bearing mice models ........73 4.1 Background and Objectives .............................................................. 73 4.2 Erythrocyte membrane fatty acid analysis ...................................... 75 4.2.1 Healthy Swiss vs control SCID mice ................................................................ 75 4.2.2 Tumor-bearing mice at different stages............................................................. 78 4.2.3 Tumor-bearing SCID mice treated with iron nanoparticles and bleomycin ..... 85 4.2.4 Healthy Swiss mice treated with iron nanoparticles and bleomycin ................. 88 4.3 Conclusions ......................................................................................... 92 7. Abbreviations ............................................................................94 Table of contents 8. References ..................................................................................96 Abstract Abstract Lipid metabolic reprogramming is an established hallmark of cancer development, which among others includes a distinct fatty acid biosynthesis and metabolism due to the enhanced cellular growth and proliferation of cancer cells. Cell membranes are built by phospholipids, which are fatty acid-containing lipid species. Alterations to the fatty acid content may influence the membrane properties, such as fluidity, permeability and membrane lipid-related signaling, thus favoring tumor growth, progression and metastasis. The membrane remodeling is highly affected by the intracellular lipid pool, which in turn depends on both the endogenously produced fatty acids and their dietary intake. Key enzymes that are involved in lipid biosynthesis, show increased activity along tumor development and progression. Some chemotherapeutics have also been reported to influence the characteristics of the plasma membrane, while the final outcome of chemical exposure depends on membrane-related signaling (e.g. lipid rafts