Università degli Studi di Milano SCUOLA DI DOTTORATO IN SCIENZE BIOCHIMICHE, NUTRIZIONALI E METABOLICHE Dipartimento di Biotecnologie Mediche e Medicina Traslazionale DOTTORATO DI RICERCA IN BIOCHIMICA XXVI CICLO-BIO/10 The natural flavonoid Luteolin induces apoptosis in colon cancer cells by dysregulating the sphingolipid rheostat Loubna ABDEL HADI Matricola n.R09265 Docente guida: Prof. Laura RIBONI Coordinatore del Dottorato: Prof. Francesco BONOMI To the soul of children who were victims in conflicts zones all over the world and most especially Syrian children who are being abused and killed since 3 years and up to this moment 2 INDEX ABBREVIATION .............................................................................................................................................................. 4 SUMMARY........................................................................................................................................................................ 5 INTRODUCTION .............................................................................................................................................................. 9 1. Colorectal Cancer ....................................................................................................................................................... 9 1.1. Colorectal cancer features .................................................................................................................................. 9 1.2. Diet in colon cancer as a double-edge sword ................................................................................................... 23 1.3. Caco-2 cells as tumour and healthy models ..................................................................................................... 30 2. Flavonoids and Luteolin ........................................................................................................................................... 32 2.1 Flavonoids........................................................................................................................................................ 32 2.2. Luteolin ............................................................................................................................................................ 35 3. Sphingolipids ............................................................................................................................................................ 47 3.1. Overview on Sphingolipids.............................................................................................................................. 47 3.2. Sphingolipids and colon tumorigenesis ........................................................................................................... 67 AIM OF THE WORK ...................................................................................................................................................... 80 MATERIALS AND METHODS ...................................................................................................................................... 82 Materials ....................................................................................................................................................................... 82 Methods ........................................................................................................................................................................ 84 4.1. Cell culture....................................................................................................................................................... 84 4.2. Enzyme assays ................................................................................................................................................. 85 4.3. Phospholipid content and composition ............................................................................................................ 87 4.4. Cell treatments ................................................................................................................................................. 88 4.5. Determination of cell viability: MTT assay ..................................................................................................... 89 4.6. Fluorescence studies ........................................................................................................................................ 89 4.7. Phospho-Akt immunoblotting .......................................................................................................................... 90 4.8. Pulse/chase experiments with radiolabeled sphingosine .................................................................................. 91 4.9. Protein assays ................................................................................................................................................... 94 4.10. Statistical analysis ............................................................................................................................................ 94 RESULTS ......................................................................................................................................................................... 95 5.1. Characterization of tumoural and differentiated CaCo-2 cells ......................................................................... 95 5.2. Effect of LU on tumoural and differentiated Caco-2 cell viability ................................................................ 103 5.3. Role of Cer as mediator of LU toxicity in tumoural Caco-2 cells ................................................................. 104 5.4. Effect of LU on Sph metabolism in tumoural Caco-2 cells ........................................................................... 107 5.5. Accumulation of Cer in the endoplasmic reticulum in LU-treated cells: possible impairment of Cer trafficking ............................................................................................................................................................... 109 5.6. Effect of Luteolin on sphingolipids rheostat in tumoural Caco-2 cells .......................................................... 113 5.7. Effect of LU on Sphingosine kinases activity in tumoural Caco-2 cells ........................................................ 114 5.8. S1P acts as a Cer antagonist in favouring tumoural Caco-2 cell survival against LU toxicity ...................... 115 DISCUSSION ................................................................................................................................................................. 118 REFERENCES ............................................................................................................................................................... 125 ACKNOWLEDGMENTS .............................................................................................................................................. 145 3 ABBREVIATION ACF Aberrant crypt foci ALP Alkaline phosphatase Alk-SMase Alkaline Sphingomyelinase A-SMase Acidic Sphingomyelinase BFA Brefeldin A BODIPY-C5-Cer N-(4,4-difluoro-5,7-dimethyl-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosine) CDase Ceramidase Cer Ceramide CRC Colorectal cancer C2-Cer N-acetyl-D-erythro-sphingosine, C6-Cer N-hexanoyl-D-erythro-sphingosine D609 O-Tricyclo[5.2.1.02,6] dec-9-yl dithiocarbonate potassium salt ER Endoplasmic reticulum EMEM Minimum essential medium eagle FCS Fetal Calf serum GalCer Galactosylceramide GlcCer Glucosylceramide GSLs Glycosphingolipids Gb3 Globotriaosylceramide LacCer Lactosylceramide LU Luteolin LY LY294002 MTT 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide N-SMase Neutral Sphingomyelinase PBS Phosphate-buffered saline PlsEtn Phosphoethanolamine plasmalogen PM Plasma membrane PNPP p-Nitrophenyl-phosphate PNP p-Nitrophenol PI3K Phosphatidylinositol-3-OH kinase SLs Sphingolipids Sph Sphingosine SM Sphingomyelin SMase Sphingomyelinase SPHKI Sphingosine kinase I SPHKII Sphingosine kinase II S1P Sphingosine-1-phoshate 4 SUMMARY Colorectal cancer is one of the most common malignancies and a leading cause of cancer death in the world. More powerful and safer therapeutic approaches are urgently needed to reduce mortality and garner better curative effects. In this regard, dietary supplements capable of preventing carcinogenesis and inhibiting the growth of colon carcinoma cells have generated intense interest. Luteolin (LU), a common dietary flavonoids, has emerged as a powerful anticancer agents able to sensitize different cancer cells, including colon cancer ones, to therapeutic-induced cytotoxicity. However, the molecular mechanisms underlying LU effects in colon cancer are largely unknown. Sphingolipids have critical functions as signaling molecules. In particular, Ceramide (Cer) and Sphingosine-1-phosphate (S1P) are involved as key antagonist mediators in regulating crucial cell responses such as proliferation and apoptosis. Cer can act as a second messenger, and, by activating a variety of signaling pathways, is able to promote growth arrest, apoptosis, or cell differentiation. To the opposite, the sphingoid mediator S1P can act as a potent mitogen and survival factor for a variety of cell types. These two lipids together form the “sphingolipid rheostat” regulating the balance between cell growth and cell death. The objective of our study was to investigate the effects and the molecular mechanisms of LU in colon