MITOCHONDRIAL PROTEOMICS OF COLORECTAL CANCER CELLS: STUDY OF THE EFFECTS OF BUTYRATE & SUBCELLULAR EXPRESSION OF MORTALIN KUAH LI FANG (B.Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS This project would not have been possible without the patient guidance and constant encouragement of my supervisors, Associate Professor Maxey Chung and Dr Sandra Tan I am truly grateful to Prof Chung, my project supervisor and Life Sciences coordinator, for giving me the chance and courage to pursue my Masters degree in his lab My deepest thanks also go to Sandra, who has been my mentor and dear sister in Christ, for her invaluable advice and inspiring example of perseverance and faith, in research work and life I would also like to thank Associate Professor Hooi Shing Chuan who has kindly provided the cell lines used in this project and whose lab, in Department of Physiology, first started this study of colon cancer cells and butyrate treatment I have been truly blessed for the many opportunities of growth and training in our lab team—the “MaxProteomics team” For this, I am deeply grateful to Dr Lin Qinsong, Gek San, Cynthia, Teck Kwang, and Jason—the dedicated researchers whose expertise and kind support have contributed greatly to my project and nurtured me in research life I would also like to acknowledge my fellow lab-mates and friends in MaxProteomics: THT, for his faithful teaching of techniques and help; Aida, for the amazing brainstorming and sharing sessions that never failed to motivate me; Jiayi, for the selfless sharing of knowledge and experiences; Xuxiao, for showing me how to smile through failures and disappointments; as well as the rest of my team-mates—Jack, Vincent, Hong Qing, Hendrick and Wenchun—who have added much fun and joy to the whole learning journey My heartfelt appreciation also goes to our “computer doctors”, Tiefei and Eric, for the much-needed help and counsel in IT and bioinformatics I am also thankful for the well-equipped facilities in Protein Proteomics Centre, faithfully maintained by its friendly staff I would also like to give thanks to my family, especially my parents, for their unfailing support and faith in me through the years and through the writing of this thesis Their unquestioning love, understanding and sacrifices have given me much strength and meaning in work and life Finally, I give all credit for the completion of this thesis and its potential future contribution to cancer treatment, to God, who is truly my Ever-present Help in times of need and the Sole Provider of my competence and work I would like to thank Him for the many blessings in my life, including the warm acceptance and prayer support from my spiritual family in Saint James Church His grace is indeed sufficient and His wisdom, infinite, as demonstrated by the gift of Science and research i CONTENTS ACKNOWLEDGEMENTS ……………………………………………………… i TABLE OF CONTENTS ….……………………………………………………… ii SUMMARY ……………… ……………………………………………………… viii LIST OF TABLES ………………………………………………………………… x LIST OF FIGURES ………………………………………………………………… xi ABBREVIATIONS AND SYMBOLS USED …………………………… xiii INTRODUCTION 1.1 COLORECTAL CANCER (CRC) 1.1.1 Epidemiology 1.1.2 Limits of current CRC diagnosis methods 1.1.3 Limits of current CRC treatment 1.1.4 Grounds for modern cancer research 1.1.5 Early cellular events in CRC carcinogenesis 1.1.6 Genetic models of CRC carcinogenesis 1.1.6.1 Chromosomal instability (CIN) pathway 1.1.6.2 Microsatellite instability (MIN) pathway 1.1.7 Epigenetic pathway of CRC carcinogenesis via methylation 10 1.1.8 Risk factors of CRC 11 1.1.8.1 Genetic risk factors 11 1.1.8.2 Environmental risk factors 12 1.1.8.3 Chemopreventive role of dietary fibre 12 1.1.8.4 Dietary fibre protects against CRC via SCFAs production 13 1.2 BUTYRATE AND CRC 14 1.2.1 Butyrate as potential chemotherapeutic agent against CRC 14 1.2.2 Role of SCFA butyrate in colonic health and cancer 14 1.2.2.1 Essential nutrient of the colon 14 ii 1.2.2.2 Modulator of colonic cell growth and maturation 15 1.2.2.3 Modulator of the gut inflammation and immunity 16 1.2.2.4 Anti-cancer effects of butyrate 17 1.2.3 Molecular mechanisms for butyrate anti-neoplastc effects 17 1.2.3.1 Histone acetylation and deacetylation in gene expression 17 1.2.3.2 Role of HDAC in carcinogenesis 19 1.2.3.3 Butyrate inhibition of HDAC 19 1.2.3.4 Effects of butyrate mediated through histone hyperacetylation 23 1.2.3.5 Effects of butyrate through non-histone substrates of HDACs 24 1.3 BUTYRATE EFFECTS THROUGH THE MITOCHONDRIA IN CANCER 26 1.3.1 The mitochondrial organelle as a key player in cell life and death 26 1.3.2 Mitochondria in cancer 26 1.3.2.1 Energy metabolism in cancer cells 26 1.3.2.2 Apoptosis resistance in cancer cells 27 1.3.2.3 Other characteristics of cancer cell mitochondria 29 1.3.3 Butyrate effects through the cancer cell mitochondria 32 1.3.3.1 Alteration of the metabolic profile of cancer cell 32 1.3.3.2 Potentiation of intrinsic apoptosis via MOMP 33 1.4 THE PROTEOMICS APPROACH 1.4.1 Why proteomics? 34 34 1.4.1.1 The dynamic proteome 35 1.4.1.2 Proteomics as a discovery tool 36 1.4.2 Technologies in proteomics 36 1.4.2.1 Two-dimensional gel electrophoresis / mass spectrometry approach (2-DE/MS) 37 1.4.2.2 Two-dimensional difference gel electrophoresis (2D-DIGE) 38 1.4.3 Subproteomics—when less is more 1.4.3.1 Mitochondrial proteomics 38 39 iii 1.4.3.2 Study of the effects of butyrate on the mitochondrial proteome 1.5 PROJECT AIMS MATERIALS AND METHODS 40 41 42 2.1 CELL LINES AND CELL CULTURE 42 2.2 SAMPLE PREPARATION 42 2.2.1 Butyrate treatment and harvesting of cells 42 2.2.2 Whole cell lysates 43 2.2.3 Subcellular fractions 43 2.2.3.1 Mitochondrial enrichment 44 2.2.3.2 Cytosol enrichment 44 2.2.3.3 Nuclear enrichment 44 2.3 PROTEIN ASSAY 45 2.4 2-D DIGE 45 2.4.1 CyDye labeling 45 2.4.2 IEF 46 2.4.3 2-DE 46 2.4.4 Fluorescence scanning and protein visualisation 47 2.4.5 DeCyder image analysis 47 2.4.6 Vorum Silver Staining 48 2.5 MASS SPECTROMETRY (MS) 49 2.5.1 Enzymatic digestion of protein spots 49 2.5.2 MS and database searching 50 2.6 SDS-PAGE / WESTERN BLOT 51 2.6.1 1-D SDS-PAGE 51 2.6.2 1-D western immunodetection 52 2.6.3 2-D western immunodetection 53 2.6.4 Visualisation of immunoreactive proteins 54 iv 2.6.5 MS/MS confirmation of mortalin isoforms 2.7 BIOINFORMATIC PREDICTIONS 54 56 2.7.1 Phosphorylation sites prediction via NetPhos 56 2.7.2 Phosphorylation sites prediction via Scansite 56 57 RESULTS 3.1 ENRICHMENT OF HCT 116 MITOCHONDRIAL PROTEOME 57 3.1.1 Enrichment of mitochondrial marker upon subcellular fractionation 57 3.1.2 2-DE profile of HCT 116 mitochondrial proteome 59 3.2 MITOCHONDRIAL PROTEOMICS OF BUTYRATE-TREATED HCT 116 CELLS 67 3.2.1 Batch-to-batch reproducibility among mitochondrial fractions 67 3.2.2 2-D DIGE analysis of HCT 116 mitochondrial proteome changes upon butyrate treatment 68 Proteome alterations in the HCT 116 mitochondria upon butyrate treatment 70 3.3 BUTYRATE REGULATION OF MORTALIN EXPRESSION IN CRC CELLS 77 3.2.3 3.3.1 Characterisation of mortalin isoforms in HCT 116 whole cell and mitochondrial fractions 78 3.3.1.1 Mortalin isoforms detected at HCT 116 whole cell level 78 3.3.1.2 Mortalin isoforms detected in HCT 116 mitochondrial fraction 80 3.3.2 Butyrate-induced changes in mortalin expression in HCT 116 83 3.3.2.1 Butyrate regulation of mortalin isoforms at whole cell level 83 3.3.2.2 Butyrate regulation of HCT 116 mitochondrial mortalin isoforms 85 3.3.2.3 Detection of serine and tyrosine phosphorylation in butyrateregulated mitochondrial mortalin isoforms 85 3.3.2.4 Prediction of phosphorylation sites in mortalin 88 3.3.2.5 Butyrate effects on mortalin level in HCT 116 subcellular fractions 91 3.3.2.6 Regulation of mortalin isoforms in HCT 116 nuclear fraction 93 v 3.3.2.7 Regulation of mortalin isoforms in HCT 116 cytosolic fraction 3.3.3 93 Butyrate-induced changes in mortalin expression in HT-29 95 3.3.3.1 Butyrate regulation on mortalin level in HT-29 subcellular fractions 95 3.3.3.2 Butyrate-induced changes at HT-29 whole cell level 98 3.3.3.3 Butyrate-induced changes in HT-29 mitochondrial fraction 98 3.3.3.4 Butyrate-induced changes in HT-29 nuclear fraction 99 3.3.3.5 Butyrate-induced changes in HT-29 cytosolic fraction 99 3.3.4 Differential regulation of mortalin isoforms in HCT 116 and HT-29 upon butyrate treatment DISCUSSION 4.1 HCT 116 MITOCHONDRIAL PROTEOME 101 103 103 4.1.1 Enrichment of mitochondrial proteome 103 4.1.2 2-DE/MS/MS survey of HCT 116 mitochondrial proteome 105 4.2 MITOCHONDRIAL PROTEOMICS OF BUTYRATE-TREATED HCT 116 4.2.1 4.2.2 106 2-D DIGE analysis of HCT 116 mitochondrial proteome changes upon butyrate treatment 106 Butyrate-regulated mitochondrial proteins in HCT 116 108 4.2.2.1 Oxidative metabolic enzymes 108 4.2.2.2 Redox enzymes 109 4.2.2.3 Respiratory chain complex subunits 111 4.2.2.4 Players of apoptosis and cell death 111 4.2.2.5 Members of the mitochondrial translation system 112 4.2.2.6 Regulators of mitochondrial protein import and folding 112 4.3 BUTYRATE REGULATION OF MORTALIN EXPRESSION IN CRC CELLS 4.3.1 Characterisation of mortalin isoforms with differential subcellular expression in HCT 116 4.3.1.1 Mortalin was present most abundantly in HCT 116 mitochondrial fractions 114 115 116 vi 4.3.1.2 Different mortalin isoforms were found in different subcellular fractions in HCT 116 cells 117 4.3.1.3 pSer and pTyr residues were detected mainly in mortalin isoforms a, b, and c 120 4.3.1.4 Mortalin was found more abundantly in HCT 116 nuclear fraction compared to the cytosolic fraction 121 4.3.1.5 Two mortalin isoforms, d and e, were preferentially present in HCT 116 mitochondrial and nuclear fractions 122 4.3.2 4.3.3 Butyrate effects on the mortalin level in HCT 116 subcellular fractions 124 Differential regulation of HCT 116 mortalin isoforms by butyrate 124 4.3.3.1 HCT 116 mortalin isoforms were regulated by butyrate only at specific subcellular levels 125 4.3.3.2 Re-distribution of mortalin isoforms d and e was detectable upon prolonged butyrate treatment 126 4.3.3.3 Mortalin isoform d was specifically associated with butyrateinduced cell death 127 4.3.3.4 Butyrate preferentially down-regulated mortalin isoform d in HCT 116 mitochondrial fraction 128 4.3.4 Comparison of butyrate regulation of mortalin HCT 116 and HT-29 131 4.3.4.1 Extensive down-regulation of subcellular mortalin isoforms was associated with resistance to butyrate-induced apoptosis 131 4.3.4.2 HT-29 expressed only trace levels of mortalin isoforms d and e 133 CONCLUSION AND FUTURE DIRECTIONS 135 BIBLIOGRAPHY 138 LIST OF CONFERENCES FOR POSTER PRESENTATIONS 156 vii SUMMARY Colorectal cancer (CRC) is currently the most prevalent malignancy in developed countries, accounting for over half a million deaths annually The high mortality of CRC is largely due to late diagnosis and the lack of effective adjunctive treatment and preventive chemotherapeutics Butyrate, a short-chain fatty acid produced during colonic bacteria fermentation of dietary fibre, has been shown to protect against CRC by inducing growth arrest, differentiation and apoptosis of cancer cells In addition to regulating gene expression through its histone deacetylase inhibitor (HDACi) activities, butyrate has also been shown to directly affect the energy metabolism and apoptotic resistance in cancer cells The mitochondrion has been shown to be a crucial player in the anti-neoplastic effects of butyrate, but the precise molecular pathways remain unclear To determine the direct biochemical effects of butyrate on the mitochondria of colon cancer cells, we used a sub-proteomics approach to study the differential expression of mitochondrial proteins in HCT-116 human CRC cells treated with 5mM butyrate for 24 hours To accomplish this, mitochondrial fractions of the untreated and butyratetreated samples were compared by 2-dimensional difference gel electrophoresis (2-D DIGE) Subsequent tandem mass spectrometry (MS/MS) analysis identified 28 butyrate-regulated proteins, out of which 18 were found to have mitochondrial functions based on published literature These proteins include metabolic and redox enzymes; regulators of protein import, folding and assembly in the organelle; components of the mitochondrial translational apparatus; subunits of various respiratory complexes; as well as translocated proteins involved in apoptosis and cell death This supports the association of the protective effects of butyrate with its biochemical effects on the mitochondria of cancer cells viii In particular, one of the mitochondrial proteins found to be significantly downregulated by butyrate, was the mitochondrial heat shock protein 70 (mthsp70), which is also known as mortalin This protein was recently reported to be overexpressed in colorectal adenocarcinomas and positively correlated with poor prognosis in CRC The down-regulation of mortalin by the anti-cancer agent, butyrate, could be an important mechanism of chemoprotection by the latter Using immunoassays, we characterised mortalin isoforms (a, b, c, d, and e) and their specific regulation by butyrate, in the whole cell lysate and various subcellular fractions of HCT 116 cells Most interestingly, two mortalin isoforms, d and e, were found to be significantly suppressed by butyrate in both the mitochondrial and nuclear fractions, but up-regulated in the whole cell lysate upon prolonged butyrate treatment In addition, isoform d was found to be present specifically in the floating, apoptotic cell population of 72-hr butyrate-treated HCT 116 cells, further indicating the association between this mortalin isoform with butyrate-induced apoptosis Furthermore, we also showed that these two mortalin isoforms were expressed in much lower levels in the subcellular fractions of HT-29 cells, a relatively more butyrate-resistant 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Subcellular distribution of mortalin and its regulation by 24-hr butyrate treatment in HCT 116 cells 92 3.16 Expression of mortalin in HCT 116 nuclear and cytosolic fractions upon 24-hr butyrate treatment 94 3.17 Subcellular distribution of mortalin and its regulation by 24-hr butyrate treatment in HT-29 cells 97 3.18 Expression of mortalin in HT-29 whole-cell and subcellular fractions upon 24-hr butyrate. .. Enrichment of mortalin isoforms and presence of additional isoforms in HCT 116 mitochondrial fraction, relative to whole cell lysate 82 3.12 Butyrate effects on the 2-DE profile of mortalin isoforms in HCT 116 whole cell lysates 84 xi 3.13 Butyrate effects on the 2-DE profile of mortalin isoforms in HCT 116 mitochondrial fractions 87 3.14 In silico prediction of phosphorylation sites in mortalin protein sequence... butyrate- treated mitochondrial fractions of HCT 116 cells 69 3.7 Butyrate regulation of well-characterised mitochondrial proteins in HCT 116 mitochondrial fraction 71 3.8 Other butyrate- regulated proteins in HCT 116 mitochondrial fraction 72 3.9 2-DE profile of mortalin isoforms in HCT 116 whole cell lysate 79 3.10 2-DE profile of mortalin isoforms in HCT 116 mitochondrial fraction 81 3.11 Enrichment of mortalin. .. route of administration of exisiting and new therapeutic agents would be tailor-made to enhance the recovery and quality of life of the individual Such ideal forms of cancer therapy rely heavily on understanding the molecular mechanisms involved in carcinogenesis and the mechanism(s) of action of effective chemotherapeutic agents 1.1.5 Early cellular events in CRC carcinogenesis The intestinal epithelium... Scansite and recent literature 90 3.5 Summary of the subcellular expression and butyrate regulation of mortalin isoforms in HCT 116 and HT-29 102 x LIST OF FIGURES Figure PAGE 1.1 Global and local rates of incidence and mortality of CRC 2 1.2 Molecular changes underlying the CRC carcinogenesis through ACS 8 1.3 Butyrate ion and its role as a SCFA in healthy colonic homeostasis 16 1.4 Regulation of gene... through the activities of HATs and HDACs 18 1.5 The HDAC family in humans 21 1.6 HDACi effects of butyrate on histone and non-histone proteins 22 1.7 Mechanisms of MOMP proposed by current literature 29 3.1 1-DE profile of whole cell lysates and mitochondrial fractions; and immunodetection of mitochondrial marker enrichment 58 3.2 DIGE comparison of the proteomes of HCT 116 crude whole cell lysate and. .. driven the search of new technologies and treatment regimens to enable earlier diagnosis and higher survival rate of CRC patients The current challenges in CRC cancer research can be summarised as follows:(i) The search for early biomarkers, which are also more sensitive and specific, for the rapid diagnosis and subsequent prognosis of the disease (ii) The discovery and optimisation of new chemotherapeutic... INTRODUCTION 1.1 COLORECTAL CANCER (CRC) 1.1.1 Epidemiology Colorectal cancer (CRC) forms in the tissues of the colon and rectum, and is currently the third most common cancer worldwide, ranking after lung and breast cancers (Kamangar et al., 2006) This malignancy alone accounts for over a million new cancer cases and over 500,000 cancer deaths annually (Figure 1.1) Its incidence concentrates in the developed... date, many hypotheses have been made for the beneficial effects of dietary fibre in preventing CRC These include the stool bulking properties of roughage, promotion of bowel movement, reduction of colonic pH, alteration of bile acid metabolism, and the increase in the production of short chain fatty acids (SCFAs) These can minimise mucosal damage by carcinogens and toxic by-products of colonic bacterial... propionate, and butyrate — are metabolized via mitochondrial β-oxidation as the primary and preferred energy source of healthy colonocytes (Heerdt and Augenlicht, 1991) They are known to decrease colonic and faecal pH, and are important for the establishment and maintenance of the colonic mucosal homeostasis in both humans and rodents (Harig et al., 1989; Tappenden et al., 1997) 13 Introduction 1.2 BUTYRATE AND ... death This supports the association of the protective effects of butyrate with its biochemical effects on the mitochondria of cancer cells viii In particular, one of the mitochondrial proteins... Enrichment of mortalin isoforms and presence of additional isoforms in HCT 116 mitochondrial fraction, relative to whole cell lysate 82 3.12 Butyrate effects on the 2-DE profile of mortalin isoforms... Scansite and recent literature 90 3.5 Summary of the subcellular expression and butyrate regulation of mortalin isoforms in HCT 116 and HT-29 102 x LIST OF FIGURES Figure PAGE 1.1 Global and local