2D-DIGE ANALYSIS OF BUTYRATE TREATED HCC CELL LINE, HEPG2 VINCENT, LAU SIANG LIN B.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE MASTERS OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS I would like to express sincere gratitude to my principal investigator, Associate Professor Maxey Chung Ching Ming, for his guidance and support throughout the span of this project. He has given me opportunities and exposure to the different facets of scientific research. In addition, acknowledgement specifically goes to Dr. Sandra Tan and Dr. Tan Hwee Tong for their insights and assistance on the direction of the project as well as advises during times of experimental troubleshooting. I owe a large part of the rich experiences in my post-graduate studies to the members of my laboratory especially to research assistants Cynthia Liang Mui Yee, Lim Teck Kwang and Tan Gek San. Last but not least, I thank my fellow post-graduate students Hendrick Loei, Lin Qifeng and Zubaidah Ramdzan for their constant encouragement and camaraderie. TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS ABSTRACT . INDEX OF TABLES . INDEX OF FIGURES . INDEX OF ABBREVIATIONS . 12 1. LITERATURE REVIEW . 15 1.1 Hepatocellular carcinoma . 15 1.1.1 Etiological factors 15 1.1.2 Disease pathology 16 1.1.3 Disease diagnosis and classification 19 1.1.4 Current treatments of HCC 21 1.1.5 Molecular pathogenesis 23 1.2 Butyrate 25 1.2.1 Butyrate and cancer 26 1.2.2 Molecular effects of butyrate . 27 1.4 Proteomics and current platforms . 32 1.4.1 Tools in expressional proteomics . 34 1.4.2 2D-DIGE 35 1.4.3 Sample prefractionation with heparin affinity chromatography 36 2. OBJECTIVES OF THE STUDY . 39 3. MATERIALS AND METHODS 41 3.1 Materials 41 3.1.1 Cell lines . 41 3.1.2 Instruments and Equipment 42 3.1.4 General Chemicals and Reagents . 43 3.1.5 Western Blot antibodies and reagents 45 3.1.6 Softwares and Databases 46 3.2 Methods 48 3.2.1 Cell line extract preparation . 48 3.2.2 Heparin Affinity Liquid Chromatography (HPLC) . 50 3.2.3 Sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS PAGE) 52 3.2.4 2-Dimensional gel electrophoresis (2-DE) . 53 3.2.5 2-Dimension Difference Gel Electrophoresis (2D-DIGE) . 54 3.2.6 Decyder image analysis 57 3.2.7 In-gel tryptic digestion for Mass spectrometry analysis 58 3.2.8 Matrix-assisted laser desorption/ionization tandem Mass spectrometry 59 3.2.9 Bioinformatics annotation tools . 60 3.2.10 Immunobloting . 60 3.2.11 MTT cell viability assay . 62 3.2.12 Transwell migration assay 63 3.2.13 Basement membrane matrix invasion assay . 63 3.2.14 siRNA knockdown of GSN 64 4. RESULTS . 65 4.1 HCC cell lines exhibit butyrate induced cell-growth inhibition . 65 4.3 Butyrate increases expression of p21 and p53, and decreases c-Myc levels 69 4.4 2D-DIGE analysis after heparin affinity chromatography . 69 4.5 Differential protein expression in HepG2 cells after butyrate stimulation 75 4.5.1 Localization and biological functions of deregulated proteome 81 4.5.2 Butyrate-affected pathways and cellular processes 81 4.6 Immunoblotting validation of selected regulated proteins 85 4.7 Over-expression of Gelsolin (GSN) by butyrate . 86 4.8 Functional study on GSN 87 5. DISCUSSION 91 5.1 Butyrate inhibits growth but promotes migration in HCC lines 92 5.2 Using HepG2 as a cell model for proteomic analysis of drug treatment 93 5.3 Identification of novel proteins involved in butyrate . 94 5.4 Proteins differentially-expressed in HepG2 after butyrate treatment . 96 5.4.1 Changes in Metabolic Program 97 5.4.2 Tumor suppressing effects of butyrate in HepG2 cells 102 5.5 Butyrate-inhibited metastasis . 106 5.5.1 Proteins known in metastasis . 108 5.5.2 Increased expression of gelsolin after butyrate stimulation . 110 6. CONCLUSION 112 7. REFERENCE 114 ABSTRACT Hepatocellular carcinoma (HCC) is one of the top causes of cancer deaths in Asia Pacific, and its incidence is predicted to increase in the next decade. The lethality of this disease can be seen from the high mortality rates and poor prognosis with a 5-year survival rate of only 5%. For patients with extrahepatic metastasis, the life expectancy decreases to a mere months. This is due to lack of reliable biomarkers, poor understanding of HCC tumorigenesis and limited clinical therapies. Butyrate, a physiological saccharolytic fermentative by-product of the colonic microflora, is an attractive chemotherapeutic agent with curative potentials due to its cancer-specific cytostatic and apoptotic effects. As the liver is the second site of butyrate delivery via hepatic portal transport and butyrate is well tolerated by hepatocytes, butyrate treatment could be an avenue to lengthen the lifespan of HCC patients awaiting liver transplants. Despite treatment feasibility, butyrate’s mode of actions against neoplasm, such as cell arrest induction and metastasis abrogation, remains oblique in liver cancer cells. In this study, we employed a novel proteomics workflow using heparin affinity chromatography and 2-Dimension Difference Gel Electrophoresis (2D-DIGE) to identify butyrate-induced changes in proteins in the HepG2 liver cancer cell line. We focused on cytoskeletal-related proteins in cell migration and invasion pathways which are enriched via heparin interaction. Our results revealed that the three HCC cell lines HepG2, HCC-M and Hep3B exhibited reduced cell viability significantly after butyrate treatment. The treatment however resulted in significant increased cell motility in all the cell lines. From this proteomics screening of butyrate-treated HepG2 cells, a total of 52 proteins’ expressions were detected to be dysregulated. These butyrate-induced proteins are mainly involved in glucose catabolism, urea cycle and nucleic acid synthesis. This study also yielded a list of cancer-associated proteins that were regulated after treatment thus suggesting the reversal of the malignant phenotype of HCC cells by butyrate. A group of proteins involved in cell migration and cytoskeletal reorganization was identified in this study which included LRRC15, NME1, and GSN. These proteins may help us to understand the mechanisms behind the changes in cell mobility after butyrate treatment. Gelsolin (GSN) is one of the targets up-regulated by butyrate treatment in HepG2 cells indicating its possible involvement in butyrate’s effect on cell motility or invasion. HepG2 with siRNA-mediated GSN knockdown resulted in increased basement membrane invasion. This suggested that up-regulation of GSN by butyrate is crucial for butyrate-mediated suppression of metastasis. INDEX OF TABLES Table No. Description Page No. Table 3.1: Labeling strategy for 2D-DIGE of heparin-bound and unbound 56 protein fractions. control-treated paired biological replicate were analyzed. Table 3.2: Concentration of primary and secondary antibodies used for western 62 blot Table 4.1: 76 Protein spots detected to be regulated with more than 1.5-fold from 2D-DIGE of heparin-bound and unbound samples. Spot numbers are numbered by DeCyderTM. The ‘Appearance’ column depicts the number of replicate gels that each individual spot was detected in. Table 4.2: Regulated protein spots identified by MALDI TOF/TOF mass 78 spectrometry after in-gel tryptic digestion, with total ion score C.I.% and best ion score C.I.%. Table 5.1: Cellular movement-related proteins identified in butyrate-treated 108 HepG2 dataset as categorized by IPA. INDEX OF FIGURES Figure No. Description Page No. Figure 4.1: Cell viability of (A) HepG2, (B) HCC-M, and (C) Hep3B HCC cell 66 lines were quantified using MTT assay. Cells were treated with mM butyrate treatment for 120 hrs at 12-, 24-, 48-, 72-, 96-, and 120-hr time-points. Respective non-treated controls were done in parallel. Figure 4.2: Migration potential of HepG2, HCC-M and Hep3B cell lines before 68 and after 22 hrs of mM butyrate treatment was assessed via boyden transwell assay. HCT116 colon cancer cell line was performed as an experimental control. The number of migrated cells treated with butyrate was expressed as a ratio of migrated control cells. Figure 4.3: 1-D western blot of p21, c-Myc, and p53 on mM, 24 hr butyrate- 69 treated (Trt) and control (Crtl) HepG2 whole cell extracts with GAPDH as loading control. Figure 4.4: 71 HPLC elution profile, at 280 nM absorbance, of mg (A) control and (B) butyrate-treated HepG2 cell lysates with protein fractions collected in a two-step program: (I) The larger peak contains unbound proteins and (II) a smaller peak contains heparin-bound proteins eluted at 0.8 M NaCl. Figure 4.5: 72 Representative images of 2D-DIGE analysis of (A) unbound and (B) heparin-bound protein fractions. 120 µg of proteins from Cydyelabeled internal standard, control and treatment samples were combined and electrophoresed on the same IPG strip of pH 3–10 nonlinear. Gel images were acquired with Typhoon fluorescent scanner. Figure 4.6: Representative silver-stained gel images of (A) unbound and (B) 73 heparin-bound protein fraction proteins. Figure 4.7: Distribution of identified butyrate-induced differentially expressed 82 proteins categorized by (A) cellular localization and (B) biological functions based on GO consortium. Figure 4.8: 83 Top biological functions and canonical pathways identified from butyrate-regulated proteins using IPA. (A) Top molecular and cellular functions (B) Selected study-relevant canonical pathways. All function and pathway analysis represented here are significant at p < 0.05. Figure 4.9: Top network ‘cancer’ from IPA. Schematic illustration of the 84 interaction behavior of differentially expressed proteins. Arrows described the direct associations between the proteins. Solid lines and dotted lines represent direct and indirect interactions. 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Proteomics. 8:5086-96. 129 [...]... transcriptional activities of genes affected by butyrate treatment Changes in gene expression are ascribed to direct effects of HDACi on gene promoters as well as assorted downstream secondary effects Promoters of butyrateresponsive genes consist of the butyrate response elements,’ and one of the direct action of butyrate is mediated through transcription factors Sp1 and Sp3, such as the promoter of p21/WAF-1 For... cell surface, butyrate can modulate TNFR1, -R2, FADD and Fas-R/CD95, clearly showing its stimulation and restoration of susceptibility of tumor cells to death ligand-induced cell death The ease to commit malignant cells to apoptosize is furthered by butyrate- mediated elimination of antiapoptotic proteins such as XIAP Hitherto, one of the key characteristics of cancer cells is its loss of differentiated... treatment The immense advantages of proteomics offered by 2DE have not been utilized in this field In this study, we employed 2D- DIGE to uncover proteome changes that occur in the hepatoma cell line HepG2 In a cellular milieu, butyrate has been known to inhibit HDACs, resulting in a change of global acetylation profile Besides histones, the acetylation status of a variety of HDAC-regulated proteins is... human saliva Most of the absorbed butyrate is catabolized by the colonic epithelium, with remaining amounts of butyrate 25 transported in the portal blood The liver absorbs the rest of the micronutrient with little left for venous systemic circulation In addition of being an energy alternative for the epithelial cells, a plethora of colonic health-related processes are also influenced by butyrate These... stem cells to differentiation Butyrate s short systemic half-life has been overcome by 26 stabilization using carrier groups such as arginine butyrate rendering it an effective yet safe chemotherapeutic alternative The exploitation of butyrate however is done without a complete understanding of its molecular mechanisms Our previous work had focused on the actions of butyrate on colorectal cancer cells. .. small molecule in the liver even in cases of decompensation 1.2.2 Molecular effects of butyrate One of the main mechanism of actions through which butyrate acts is via inhibition of HDACs in the cell, resulting in the hyperacetylation of histones, chromatin relaxation and targeted changes in gene expression It is crucial to note that though similar alterations of gene expression can be observed in distinct... labelling methods, shotgun proteomics and array technologies 2-DE and 2D- DIGE of butyrate- treated HCC cells followed by tandem MS identification of differentially expressed protein spots are carried out in this study Stable-isotope labelling involves labelling of sample proteins prior to separation via liquid chromatography before tandem MS analysis Common labelling techniques in proteomics include cleavable... GSN (GSN-KD) and HCT116 as reference (B) MatrigelTM invasion assay for No siRNA, NTC and GSN-KD All migrated treated cells are expressed as a ratio of corresponding control cells without butyrate treatment 11 INDEX OF ABBREVIATIONS 1-D One-dimensional 2-DE Two-dimensional electrophoresis 2D- DIGE Two-dimensional difference gel electrophoresis Acc No Accession number ACN Acetonitrile AFB1 Aflatoxin B1... is chronic interstitial inflammation of the liver, demarcated by the replacement of normal tissue with fibrous tissue and loss of functional liver cells (Farazi, 2006) The detriment process often entails repetitive cycles of liver damage and tissue repair, preceding fibrosis, where persistent injuries are permanently filled by tough scar tissues instead of liver cells It is an irreversible process leading... cell viability assay measured at 550 nm absorbance of GSN- 89 knockdown HepG2 cells and respective non-targeting controls after 5 mM butyrate treatment at 24-, 48-, and 72-hr timepoints Figure 4.15: 90 Motility functional assay of GSN-knowndown cells (A) Transwell migration assay for HepG2 cells without siRNA treatment (No siRNA), treated with non-targeting siRNA (NTC), siRNA against GSN (GSN-KD) and . inflammation of the liver, demarcated by the replacement of normal tissue with fibrous tissue and loss of functional liver cells (Farazi, 2006). The detriment process often entails repetitive cycles of. increased cell motility in all the 3 cell lines. From this proteomics screening of butyrate-treated HepG2 cells, a total of 52 proteins’ expressions were detected to be dysregulated. These butyrate-induced. also yielded a list of cancer-associated proteins that were regulated after treatment thus suggesting the reversal of the malignant phenotype of HCC cells by butyrate. A group of proteins involved