Exploring mesenchymal stem cell derived exosome and tocotrienol (t3) as therapeutic agents in drug induced liver injury (DILI)

EXPLORING MESENCHYMAL STEM CELL-DERIVED EXOSOMES AND TOCOTRIENOL (T3) AS THERAPEUTIC AGENTS IN DRUG-INDUCED LIVER INJURY (DILI) TAN CHEAU YIH (B Eng (Hons.), UTM) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2014 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Tan Cheau Yih August 2014 i Acknowledgement I have never thought of completing another years of graduate programme after years of master degree This dissertation marks another important journey in my life and the completion of it would not be possible without the support of several people I would like to express my sincere gratitude to all of them Firstly, I would like to thank my PhD supervisor, Dr Ho Han Kiat for his advice and sharing which encouraged me to move on to this journey of research Thank you for giving me the opportunity to join the wonderful lab, the freedom to shape my research works and thoughts, the motivations and supports throughout my candidature I truly appreciate all the help and had enjoyed my years journey working with you I am also grateful to my cosupervisor, A/Prof Dan Yock Young for his precious advice and help given throughout my PhD pursuit The enthusiasm, passion and energy he has for research was contagious and motivational to me Every meeting with him is a recharging time to me, especially during hiccup periods in mice work Thank you to my thesis committees, Prof Paul Ho Chi Lui and A/Prof Theresa Tan May Chin who have guided me through all these years Thank you for the continuous supports, valuable suggestions and recommendations given I would also like to extend my deepest appreciation to our collaborators, Dr Lim Sai Kiang from Institute of Medical Biology, for the contribution of MSC-derived exosomes and Dr Fong Chee Wai from Davos Life Science Singapore, for the contributions of tocotrienol analogs This project would not be possible without their generous supports and professional advices I am also ii grateful to Ruenn Chai for his helps and inputs on the MSC-derived exosomes and also Judy Saw for her assistance in tocotrienols uptake assay The Laboratory of Liver Cancer and Drug-Induced Liver Diseases Research Group members, Lee Cheng, Yi Yun, Yun Shan, Chun Yan, Angie, Duan Yan, Sheela and Winnie, thank you for the help, support and great companies I am also grateful to the A/Prof Dan’s lab members, expecially to Jaymie and Brian for the help and encouragement given to me to overcome mice phobia Mandy, Charmaine, Pan Jing, Luqi, Li Jian, Sudheer, Hua Pey, Yi Ling, Xiu Ping, Hui Ting and Qiu Yi, thank you for the friendship and joy that we have shared together My biggest gratitude goes to Sing Teang, thank you for being there all the time with me, through ups and downs, thank you for the all the supports and companion It is a great pleasure to have all of you around! My appreciation also goes to all the supporting staffs in Pharmacy department for their kind assistance: Johannes, Sek Eng, Sukaman, Kelly, Timothy, Liza, Napsiah, Ying Ying, Jenny and Mrs Teo Special thanks to NUS department of Pharmacy for enrolling me as a postgraduate student and NUS (President Graduate Fellowship) for the financial support Last but not least, I would like to dedicate this dissertation to my beloved family members and husband for their love and encouragement which gave me the strength to surmount all challenges iii Table of Contents Declaration i Acknowledgement ii Summary ix List of Publication xii List of Tables xiii List of Figures xiv List of Supplementary Tables xviii List of Abbreviations xix Chapter Introduction 1.1 Liver injury and its pattern 1.2 Clinical outcomes of drug-induced liver injury 1.3 Mechanism of liver injury 1.3.1 General drug-induced liver injury mechanism 1.3.2 APAP-induced liver injury mechanism 1.3.2.1 NAPQI formation 1.3.2.2 NAPQI and protein binding 1.3.2.3 Mitochondrial superoxide and peroxynitrite formation .10 1.3.2.4 Amplification of mitochondrial oxidant stress 12 1.3.2.5 Apoptosis and necrosis .13 1.3.3 Conclusion 16 1.4 Cellular responses to injury 17 1.4.1 Anti-oxidative responses .18 1.4.2 Anti-apoptosis 21 1.4.3 Liver inflammation 22 1.4.4 Liver regeneration 24 1.4.5 Conclusion 27 1.5 Current management in DILI 28 1.5.1 Protection 28 1.5.2 Treatment and limitations .29 1.6 Proposed strategies in managing DILI 30 iv 1.6.1 Potential protective agent: alpha-tocotrienol (-T3) 31 1.6.2 Potential regenerative agent: mesenchymal stem cells (MSC) derived exosomes 33 1.7 Aims and objectives 36 Chapter Materials and Methods 39 Materials 40 2.1.1 Preparation and quantification of MSC-derived exosomes 41 2.1.2 Preparation and quantification of Vitamin E derived -TP and T3 42 2.2 In vivo studies 43 2.2.1 Animal and diets 43 2.2.2 In vivo CCl4 induced liver injury model optimization 43 2.2.3 In vivo exosomes route of administration optimization 44 2.2.4 CCl4 induced acute liver injury induction with exosomes treatment 44 2.2.5 Measurement of serum ALT and aspartate aminotransferase (AST) release 45 2.2.6 Histologic examination 45 2.2.7 Immunohistochemistry (IHC) of PCNA .46 2.3 In vitro studies 47 2.3.1 Cell lines and culture conditions 47 2.3.2 In vitro cytotoxicity test of exosomes and Vitamin E analogs (-TP and T3 isomers) .48 2.3.3 In vitro cellular uptake of Vitamin E analogs (-TP and T3 isomers) 49 2.3.4 In vitro treatment of APAP-induced liver injury model 49 2.3.5 In vitro treatment of hydrogen peroxide (H2O2)-induced liver injury model 50 2.3.6 Cell viability assay 51 2.3.7 Isolation of total mRNA from TAMH cells .51 2.3.8 Reverse transcription and qRT-PCR 51 2.3.9 Western blots 53 2.3.10 Determination of GSH content 54 v 2.3.11 Determination of intracellular reactive oxygen species (ROS) 54 2.3.12 Determination of intracellular lipid peroxidation (LPO) 55 2.3.13 Determination of membrane potential transition (MPT) 55 2.3.14 Caspase-3 activity assay 56 2.3.15 Combination therapy 56 2.3.16 Statistical analysis 56 Chapter Results on exosomes 57 3.1 Introduction 58 3.2 Influence of exosomes against CCl4-induced liver injury in vivo model 58 3.2.1 Development of CCl4-induced hepatic injury in a mouse model 59 3.2.2 Screening of exosomes toxicity and the optimum route of exosomes administration in a mouse model .61 3.2.3 Effect of exosomes against CCl4 on biochemical indices of injury 63 3.2.4 Effect of exosomes against CCl4 on histopathological patterns of liver injury .65 3.2.5 Effect of exosomes against CCl4 on protein expression in liver regeneration 67 3.2.6 Effect of exosomes on PCNA immunohistochemical staining (IHC) 69 3.2.7 Summary 71 3.3 Influence of exosomes against APAP- and H2O2-induced liver injury in vitro model 72 3.3.1 Exosomes characterisation 72 3.3.2 Effect of exosomes on cell viability in APAP and H2O2induced toxicity 75 3.3.3 Effect of exosomes on gene regulation during priming phase of liver regeneration 78 3.3.4 Effect of exosomes on the induction of transcription factors during the G1 phase of cell cycle .81 3.3.5 Effect of exosomes on cell proliferation markers during G1 and S phase of cell cycle .83 vi 3.3.6 Effect of exosomes on caspase activity and apoptotic gene Bcl-xL .85 3.3.7 Effect of exosomes on anti-oxidant gene activity 87 3.3.8 Summary 89 3.4 Discussion 91 3.5 Conclusion 101 Chapter Results on Tocotrienol (T3) 102 4.1 Introduction 103 4.2 Characterization of tocotrienol analogs in TAMH cells 104 4.2.1 Cytotoxicity of T3 analogs in TAMH cells 105 4.2.2 Cellular uptake of different concentration of T3 analogs in TAMH cells 107 4.2.3 Summary of Vitamin E analogs characterization 110 4.3 Influence of T3 analogs against APAP- and H2O2-induced in liver injury in vitro……… 112 4.3.1 Effect of T3 analogs on APAP and H2O2-induced on cell death in TAMH cells 112 4.3.2 Effect of lower dosage of -T3 and -T3 against APAP and H2O2-induced injury on cell viability in TAMH cells 118 4.3.3 Effect of -TP and -T3 on GSH activity 122 4.3.4 Effect of -TP and -T3 on intracellular ROS 124 4.3.5 Effect of -TP and -T3 on lipid peroxidation (LPO) 126 4.3.6 Effect of -TP and -T3 on antioxidant genes activity 128 4.3.7 Effect of -TP and -T3 on mitochondrial membrane permeability transition (MPT) 131 4.3.8 Effect of -TP and -T3 on Bcl-xL anti-apoptotic gene 133 4.3.9 Effect of -TP and -T3 on caspase activity 135 4.3.10 Effect of -TP and -T3 on gene regulation in ROS induced inflammation 137 4.3.11 Effect of -TP and -T3 on protein expressions in liver regeneration 139 4.3.12 Summary 141 4.4 Discussion 143 4.5 Conclusion 155 vii Chapter Results on combination therapy of exosomes and -T3 157 5.1 Introduction 158 5.2 Effect of exosomes and -T3 against APAP- and H2O2-induced liver injury in cell viability 159 5.3 Discussion on the combination therapy of exosomes and -T3 161 Chapter Conclusion and future prospectives 162 6.1 Recapitualtion of overall hypothesis and study aims 163 6.2 Conclusion of MSC-derived exosomes 164 6.3 Conclusion on T3 167 6.4 Conclusion of exosomes and -T3 combination therapy 170 6.5 Overall conclusion 171 6.6 Recommendations for future work 173 References 176 Appendices 194 viii Summary Drug-induced acute liver injury (DILI) is a major clinical problem arising from both diseases and therapeutic misadventures This issue not only translates into significant morbidities and mortalities worldwide, but also causes the repercussions of drug removal from market and socio-economic burden Management of DILI is often limited to cessation of drug use and supportive therapy, as there are no therapeutically proven natural hepatoprotective agents Current treatment using N-acetylcysteine (NAC) has narrow therapeutic window and only effective when administered at a very early stage of the injury To address this unmet need, we are interested in exploring two potential natural hepatoprotective or hepatoregenerative agents, exosomes and alpha-tocotrienol (-T3) in the acute liver injury model Hitherto mesenchymal stem cell (MSC) and the conditioned medium (MSCCM) was shown to be effective in treating various organ failure, including liver Later, MSC-CM derived exosomes was identified to play a vital functional role in tissue repair Nevertheless, the hepatoprotective or hepatoregenerative effect of exosomes has never been demonstrated On the other hand, Vitamin E has been well-known for its antioxidant property with -tocopherol (-TP) being the most active form However, recent research showed that tocotrienols (or T3, another subtype of Vitamin E) analogs exert better functions in health and disease distinct from -TP, especially -T3 in overcoming neuronal injury and ischemic perfusion injury However, the hepatoprotective effect of specific analogs of T3 has yet to be identified Therefore, our overarching aim was to determine if MSC-CM derived ix 128 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corticosteroids Postgrad Med J 1976, 52:631-633 Paumgartner G, Beuers U: Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited Hepatology 2002, 36:525-531 Heinrich PC, Castell JV, Andus T: Interleukin-6 and the acute phase response Biochem J 1990, 265:621-636 193 APPENDICES 194 APPENDICES I Supplementary table Acute phase plasma proteins in human and rat [242] 195 APPENDICES II Supplementary table Proteomic profile of independently prepared exosomes as determined by LC MS/MS and antibody arrays [187] A2M CD81 FAM29A IGFBP3 LYAR PSMB4 SLC7A10 ABI3BP CD82 FAM3B IGFBP4 LYZ PSMB5 SLC7A5 ACAA2 CD9 FAM64A IGFBP6 MADH4 PSMB6 SMARCA4 ACAT2 CDC2L5 FAM71F1 IGFBP7 MAMDC2 PSMB7 SMC1A ACLY CDC42 FAP IGHA1 MAP1A PSMB8 SORT1 ACSL1 CDH13 FASN IGHA2 MAP2K6 PSMB9 SPACA1 ACTA1 CDIPT FAT IGHG1 MAP3K1 PSMC5 SPARC ACTA2 CDK5R2 FAT2 IGHG2 MARCKS PSMD11 SPOCK1 ACTB CEACAM8 FAT4 IGHG4 ACTG2 CFB FBLN1 IGHM MAT1A PSMD6 SPTAN1 ACTN1 CFI FBN1 IGJ MBD3 PSMD7 SPTBN1 ACTN2 CFL1 FBN2 IGKC MCC PTGFRN SPTBN4 ACTN3 CFL2 FBXW8 IGKV1-5 MCM10 PTK7 SRGN ACTN4 CFTR FEN1 IGL@ MDH1 PTPRK SRI ACTR1A CHMP2A FER1L3 IGLV4-3 MDH2 PTRF SRPX2 ACTR2 CHST12 FGA IGSF8 ME1 ACTR3 CITED1 FGB IL10 MECP2 PTX3 STAT1 ADAM10 CLASP2 FGF16 IL11 MFAP4 PXDN STC1 ADAM9 CLDN1 FGF18 IL13 MFGE8 PZP STC2 FGF19 IL15RA MFSD2 QPCTL STOM ADAMTS12 CLEC11A MARCKSL1 PSMD14 SPRY4 PTTG1IP ST6GALNAC6 AEBP1 CLIC1 FGFRL1 IL17B MIF QSOX1 STOML3 AFM CLIC6 FGG IL17R MMP1 RAB10 STX12 AGRN CLPX FLG2 IL19 MMP10 RAB11B STX2 AHCY CLSTN1 FLJ13197 IL1F9 MMP2 RAB14 SURF4 AHNAK2 CLTA FLJ22184 IL1RAP MMP3 RAB15 SVEP1 AHSG CLTC FLJ32784 IL1RAPL1 MOS RAB1A SYT1 AKR1B1 CLTCL1 FLNA IL1RL2 MPO RAB1B SYT9 AKR7A2 CLU FLNB IL22RA1 MPZL1 RAB2A TAAR2 ALB CMIP FLNC IL23A MRC2 RAB33B TAGLN ALCAM CNGB1 FLOT1 IL3 MSN RAB35 TALDO1 ALDH2 COL12A1 FLOT2 IL5 MXRA5 RAB39B TAS2R60 ALDH3A2 COL14A1 FLT1 IL6ST MYADM RAB5A TCN1 ALDH6A1 COL18A1 FN1 IL7 MYCBPAP RAB5B TF ALDH7A1 COL1A1 FREM3 IL8 MYH14 RAB5C TFG ALDH9A1 COL1A2 FST INHBA MYH9 RAB6A TFRC 196 ALDOA COL2A1 FTL INHBB MYL6B RAB7A TGFB1 ALDOB COL3A1 FUCA2 INSR MYO1C RAB8A TGFB2 ALDOC COL4A1 GALNT5 IQGAP1 NBL1 RAB8B TGFBI ALOX12P2 COL4A2 GANAB ITGA11 NEFH RAC1 TGM2 ANG COL4A3 GAPDH ITGA2 NEK10 RAC2 TGOLN2 ANGPTL2 COL5A1 GAPDHS ITGA3 NID1 RAD21 THBS1 ANPEP COL5A2 GARS ITGA4 NLRP8 RALA THBS2 ANXA1 COL6A1 GAS6 ITGA5 NME1 RAN THY1 ANXA11 COL6A2 GDF1 ITGAL NOMO1 RAP1A TIMP1 ANXA2 COL6A3 GDF11 ITGAV NRAS RAP1B TIMP2 ANXA2P1 COL7A1 GDF3 ITGB1 NRG2 RAP1B TIMP3 ANXA3 COMP GDF5 ITGB5 NRLN1 RAP2C TKT ANXA4 COPB1 GDF8 ITIH2 NRP1 RARRES1 TLN1 ANXA5 COPS3 GDF9 ITIH4 NT5E RASA1 TMBIM1 ANXA6 COPS4 GDI1 ITPR2 NTF5 RASA4 TMED10 ANXA7 COPS8 GDI2 JUP NUSAP1 RB1CC1 TMED9 AP1S1 CPS1 GFRA3 KIAA0146 OBFC1 APEH CREG1 GLDC KIAA0256 APOA1 CRIPT GLUD1 KIAA0467 APOE CRTAP GNA13 KIAA1881 OPRM1 APP CSF1 GNAI2 KPNB1 OSM ARF1 CSF2 GNAL KRT1 OTC ARF4 CSF3 GNAS KRT10 ARF5 CSPG4 GNAT3 KRT13 OXTR RNF40 TNFRSF1A ARHGAP18 CST4 GNB1 KRT14 P4HB RPL10A TNFSF18 ARHGAP23 CTA-221G9.4 GNB2 KRT15 PAICS RPL12 TNFSF5 ARHGDIA CTBP2 GNB4 KRT16 PAN3 RPL15 TPBG ARHGEF1 CTNNA1 GNG12 KRT17 PAPPA RPL18 TPI1 ARL6IP5 CTNNA2 GNPDA1 KRT18 PARP10 RPL23 TRAP1 ARMS2 CTNNB1 GOT2 KRT19 PARP16 RPL29 TREM1 ARPC3 CTNND1 GPC1 KRT2 PARVG RPL35A TREML2P ARPC4 CTSG GPC5 KRT27 PC RPLP0 TRIM40 ARPC5 CXCL16 GPI KRT28 PCOLCE RPS10 TRIM41 ASH1L CXCL2 GPR112 KRT3 PDCD6 RPS16 TSN ASL CXorf39 GREM1 KRT4 PDCD6IP RPS18 TSNAX ATP1A1 CYBRD1 GRM2 KRT5 PDGFA RPS2 TSPAN14 ATP1B3 DBF4B GRM3 KRT6A PDGFC RPS24 TSPAN4 ATP2B1 DCD GRM7 KRT6B PDGFRB RPS27A TSPAN6 ATP2B4 DCHS2 GSN KRT6C PDIA3 RPS3 TSPAN9 ATP5A1 DCLK2 GSTM1 KRT7 PEBP1 RPS4X TSTA3 ATP5B DCN GSTM2 KRT72 PFAS RPS5 TTLL3 197 RCOR2 TMEM16B ODZ3 RDH5 TMEM2 OFD1 RFTN1 TMEM47 RGN TMEM51 RHOC TNC RMND5A TNFRSF11B OXNAD1 RNF123 TNFRSF12A ATP8B3 DCTN1 GSTM5 KRT73 PFKFB3 RPSA TTN ATRN DECR1 GSTO1 KRT74 PFN1 RRAS2 TTYH3 ATXN1 DEFA1 GSTP1 KRT76 PFN2 RTN4 TUBA1A AXL DIP2B GTPBP2 KRT77 PGAM2 RUVBL1 TUBA1B BASP1 DIRAS2 GYLTL1B KRT78 PGD S100A11 TUBA1C BDNF DKFZp686D0972 GZMA KRT79 PGK1 S100A13 TUBB BGN DKK1 H2AFV KRT8 PGLYRP2 S100A8 TUBB2A BHMT2 DKK3 H2AFX KRT80 PIGR S100P TUBB2C BRMS1 DMBT1 HBB KRT84 PIP SAA4 TUBB3 BSG DNASE1L1 HBE1 KRT9 PKM2 SASS6 TUBB6 C11orf59 DNPEP HDAC5 LACRT PLAB SCAMP3 UBA52 C1orf78 DPYS HERC5 LAMA4 PLAU SCGB2A1 UBB C1R DPYSL2 HGF LAMB1 PLEC1 SCYE1 UBE1 C1S DSP HGFR LAMC1 PLEKHG3 SDC1 UBE2N C20orf114 DULLARD HISPPD2A LAMP1 PLOD1 SDC2 UGP2 C3 ECM1 HIST1H2AE LAMP2 PLOD2 SDC4 UNC13B C5orf24 ED1 HIST1H2BA LAP3 PLOD3 SDCBP UNC45A C9orf19 EDG2 HIST1H2BL LCN1 PLP2 SEC14L4 VAMP3 C9orf91 EDIL3 HIST1H4H LCN2 PLSCR3 SEMA5A VANGL1 CACNA2D1 EEA1 HIST2H2BE LDHA PLTP Sep-02 VASN CACNA2D4 EEF1A1 HLAA LDHAL6B PLUNC Sep-07 VAT1 CALR EEF1G HMGCS2 LDHB PNO1 SERINC5 VCAN CAND1 EEF2 HNRNPA1 LEPRE1 PODN SERPINA1 VCL CAP1 EFEMP2 HP LGALS1 POLN SERPINB3 VCP CAPNS1 EHD1 HPX LGALS3 POSTN SERPINE1 VEGFC CAPZA1 EHD2 HRSP12 LGALS3BP POTE2 SERPINE2 VIL1 CASP14 EHD4 HSP90AA1 LGALS8 PPIA SERPINF1 VIL2 CAT EIF4A1 HSP90AB1 LGR6 PPIB SFN VIM CAV1 EMILIN1 HSP90B1 LIF PPME1 SFRP1 VTI1A CCDC129 ENG HSPA1A LMNA PPP1CC SFRP4 VTN CCDC64B ENO1 HSPA1L LOC124220 PRDM16 SHANK3 WDR49 CCL2 ENO2 HSPA5 LOC283523 PRDX1 SLAIN1 WDR52 CCL20 ENO3 HSPA6 LOC284297 PRDX6 SLC16A1 WNT5A CCL28 ENTPD4 HSPA8 LOC388344 PRNP SLC16A3 YBX1 CCL7 ENTPD4;LOXL2 HSPB1 LOC389827 PRR4 SLC1A4 YWHAB CCR4 EPB41L3 HSPD1 LOC442497 PRSS23 SLC1A5 YWHAE CCR5 EPHA2 HSPG2 LOC653269 PSMA1 SLC22A2 YWHAG CCT5 EPO HTRA1 LOC727942 PSMA2 SLC25A10 YWHAQ 198 CCT6A ESM1 HYI LOC728320 PSMA3 SLC25A13 YWHAZ CD109 ETFB ICAM1 LOC728378 PSMA4 SLC2A1 ZBTB4 CD151 F2R ICAM5 LOC730013 PSMA5 SLC2A3 ZNF134 CD248 F3 IDH3B LRP1 PSMA6 SLC38A2 ZNF503 CD276 F8 IFITM2 LRP6 PSMA7 SLC38A3 ZNF614 CD44 FADD IFNG LRRFIP2 PSMB1 SLC39A14 CD47 FAH IFRD1 LTBP1 PSMB10 SLC3A2 CD59 FAM108A1 IFT140 LTBP2 PSMB2 SLC44A1 CD63 FAM129B IGF2R LTF PSMB3 SLC44A2 Regular font: identified by LC MS/MS Underline: identified by antibody arrays Bold and Underline: identified by both LC MS/MS and antibody arrays Bold: identified by LC MS/MS and was found to be present in at least 50% of exosomes characterized 199 ... protection pathways in APAP- and H2O2 -induced liver injury in TAMH cells 156 Figure 44 Combination therapy of exosomes and -T3 in APAP- and H2O 2induced liver injury in TAMH cells ... virally -induced disease predominated in early 90s but has substantially declined in the past few years, with most acute injury cases now arising from drug induced- liver injury (DILI) [7] In the... Chapter Introduction 1.1 Liver injury and its pattern 1.2 Clinical outcomes of drug- induced liver injury 1.3 Mechanism of liver injury 1.3.1 General drug- induced liver