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Characterization of the Influence of the Galactoside-Binding Protein

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Characterization of the Influence of the Galactoside-Binding Protein THE UNIVERSITY OF LIVERPOOL Characterization of the influence of the galactoside- binding protein Peanut Agglutinin on endothelial secretion of cytokines in cancer metastasis Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Weikun Wang November 2019 ACKNOWLEDGEMENTS I am grateful and indebted to my supervisor Professor Lugang Yu for his guidance and support throughout my research and for his constructive criticism and excellent advice during the preparation of this thesis. I am also very grateful to my secondary supervisor, Professor Jonathan Rhodes for his kindly advice and help throughout this period. I would like to thank colleagues from our research group, in particular, Dr. Chen Chen, Dr. Qicheng Zhao and Dr. Carrie Duckworth for their technical support and advices. I am grateful to my father Dr. Yimin Wang, my mother Mrs. Ling Xiong and my husband Mr. Yun Lin. This work would not have been possible without their financial and spiritual support. 1 AUTHOR’S DECLARATION All techniques and experiments performed and described in this thesis were undertaken by me as a student working towards the degree of Doctor of Medicine at the University of Liverpool. Neither this thesis, nor part of it has been submitted in support of an application for another degree or qualification at this or any other university or institute of learning. Weikun Wang 2 ABSTRACT Peanut agglutinin (PNA) is a dietary lectin which accounts for 0.15% of the total weight of peanut Arachis hypogaea. PNA is a galactoside-binding protein which binds highly specifically to the tumour-associated Thomsen-Friedenreich blood group antigen. Previous studies demonstrate that PNA rapidly enters the blood circulation after peanut ingestion. PNA interacts with oncofetal TF disaccharide on the transmembrane mucin protein MUC1, causing polarization of MUC1 and thus reveals functional cell surface adhesion molecules. Therefore, circulating PNA has potential effects by inducing cancer cell homotypic and heterotypic adhesion, cell survival and migration, thus potentially promoting cancer metastasis and resistance to anoikis. In addition, earlier studies from our group have shown that a human galactoside-binding galectin-3 also interacts with MUC1-TF potentially inducing cancer metastasis from vascular endothelium. It is also reported that circulating galectin-3 enhances cytokine secretion from endothelial cells, and this interaction is suggested to be responsible for the metastasis-promoting effect of galectin-3. This thesis further explores the PNA effect on endothelial secretion of metastasis-associated cytokines. The study presented here reports that PNA at a concentration similar to that found in the sera of people after eating 200g peanuts causes increase of IL-6 and MCP-1 secretion from both micro-vascular and macro-vascular endothelial cells. The 3 PNA-induced cytokine secretion was found to enhance endothelial expression of several cell surface adhesion molecules, leading to increased cancer cell- endothelial cell adhesion. The increased cytokine secretion is also shown to promote endothelial cell adhesion, proliferation and angiogenesis. The molecular mechanism of the PNA-induced cytokine secretion is also studied, and two cell surface adhesion molecules MCAM and PECAM were identified as major functional receptors of PNA. Moreover, siRNA-mediated suppression of MCAM and PECAM expression completely abolished the PNA-induced cytokine secretion from endothelial cells. This study also investigated the cell signaling involved in the potential pro-metastasis effect of PNA. The results from this study suggested that the actions of dietary PNA in cancer patients might have impacts on cancer growth and metastasis. 4 ABBREVIATIONS Akt Protein Kinase B (PKB) ASF Asialofetuin ATP Adenosine triphosphate BCL2 B-cell lymphoma 2 BRCA1/2 Breast Cancer genes 1 or 2 bFGF Basic fibroblast growth factor BSA Bovine serum albumin CAMs Cell adhesion molecules CCL2 Chemokine (C-C motif) ligand 2 (MCP-1) CCR C-C motif receptor CD146 Melanoma cell adhesion molecule (MCAM) CD23 Fc epsilon RII (FcεRII) CD31 Platelet endothelial cell adhesion molecule (PECAM) CD44 Homing cell adhesion molecule (HCAM) CM Conditioned Medium CRC Colorectal Cancer CRDs Carbohydrate recognition-binding domains CTL C-type lectins DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid ECL Enhanced chemiluminescence ECM Extracellular matrix components EDTA Ethylenediaminetetraacetic acid 5 EGF Epithelial growth factor EGM Endothelial growth media ELISA Enzyme-linked immunosorbent assay FCS Fetal calf serum Gal Galactose Glc Glucose G-CSF Granulocyte colony-stimulating factor GlcNAc N-acetylglucosamine GM-CSF Granulocyte-macrophage colony-stimulating factor HCAM Homing cell adhesion molecule hEGF Human Epidermal Growth Factor HMVEC-Ls Human micro vascular lung endothelial cells HUVEC Human umbilical vein endothelial cells ICAM-1 Intercellular adhesion molecule-1 Ig-CAM Immunoglobulin-like Cell Adhesion Molecules IgSF Immunoglobulin superfamily IKK IκB kinase IL-6 Interleukin 6 JAK Janus kinase KSHV Kaposi’s sarcoma-associated herpesvirus MAPK Mitogen-activated protein kinases MCAM Melanoma Cell Adhesion Molecule MCP-1 Chemokine (C-C motif) ligand 2 (CCL2) MEK Mitogen-activated protein kinase MHC Major Histocompatibility Complex MIP Macrophage Inflammatory Proteins MMP Matrix metalloproteinase 6 MMR Mismatch repair MUC1 Membrane-associated sialomucin episialin NAC N-acetylcysteine NF-Kappa B Nuclear factor Kappa B NIH National Institute of Health NK Natural killer cell PAGE Polyacrylamide gel electrophoresis PBMC peripheral blood mononuclear cells PECAM Platelet endothelial cell adhesion molecule PNA Peanut agglutinin Poly-HEMA Poly-2-hydroxyethyl methacrylate RGD Arginylglycylaspartic acid (Arg-Gly-Asp) SDS Sodium dodecyl sulphate SiRNA Small interfering RNA STAT Signal Transducers and Activators of Transcription TEMED Tetramethylethylenediamine TF Thomsen-Friedenreich oncofetal carbohydrate antigen (N-acetyl-D-galectosamine linked to protein) TNF-α Tumour necrosis factor-alpha TNM The TNM Classification of Malignant tumour (, Lymph Nodes, Metastasis) cTNM Clinical TNM pTNM Pathological testing UICC Union International Cancer Control UV Ultraviolet light VEGF Vascular endothelial growth factor VCAM-1 Vascular cell adhesion protein 1 WHO World Health Organization 7 TABLE OF CONTENT ACKNOWLEDGEMENTS ................................................................................................................ 1 AUTHOR’S DECLARATION ............................................................................................................ 2 ABSTRACT ....................................................................................................................................... 3 ABBREVIATIONS ............................................................................................................................ 5 TABLE OF CONTENT ........................................................................................................................... 8 CHAPTER 1 Introduction ........................................................................................................... 14 1.1 Cancer ................................................................................................................ 14 1.1.1 General ................................................................................................................. 14 1.1.2 Biology .................................................................................................................. 17 1.2 Cancer Metastasis .............................................................................................. 20 1.2.1 General ................................................................................................................. 20 1.2.2 Inflammation and Cancer metastasis ............................................................. 23 1.2.3 The endothelium and cancer metastasis ....................................................... 29 1.3 Lectins and Cancer ............................................................................................ 32 1.3.1 General and structure ....................................................................................... 32 1.3.2 Glycosylation and Cancer .................................................................................. 36 1.3.3 Galectin-3 ............................................................................................................. 40 1.3.4 Galectin-3-MUC1 interaction in cancer metastasis...................................... 41 1.3.5 Galectin-3 induces endothelial cytokine secretion ..................................... 43 1.4 Peanut Agglutinin .............................................................................................. 45 1.4.1 General and structure ....................................................................................... 45 1.4.2 PNA binds to the Thomsen-Friedenreich antigen ........................................ 47 1.4.3 PNA induces cancer cell proliferation ............................................................ 48 1.4.4 PNA as biomarker in cancer ............................................................................. 49 1.5 Diet and Cancer ................................................................................................. 51 1.5.1 Epidemiology .....................................................................................................
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