........................................................................................................................................ 39 Introduction .................................................................................................................................. 40 Materials and Methods ................................................................................................................. 43 Results .......................................................................................................................................... 50 GGA treatment upregulates PUMA gene expression in HuH-7 cells .................................. 50 Mutant p53 is involved in GGA-induced cell death of HuH-7 cells ................................... 50 Mutant p53 relocates from the cytoplasm to the nucleus by GGA treatment ....................... 51 GGA-induced changes in native forms of p53 in the cytoplasmic space ............................. 52 p53 is released from putative huge complexes with GGA treatment .................................. 53 Nuclear translocation of mutant p53 upregulates PUMA mRNA levels in HuH-7 cells .......... 54 Discussion ..................................................................................................................................... 55 Figures .......................................................................................................................................... 59 Chapter IV: GGA induces unfolded protein response in HuH-7 cells ...................................... 68 Abstract ......................................................................................................................................... 69 Introduction .................................................................................................................................. 70 Materials and Methods ................................................................................................................. 72 Results .......................................................................................................................................... 77 Rapid induction of XBP1 mRNA splicing by GGA treatment in HuH-7 cells ..................... 77 Structure specificity of diterpenoid-induced splicing of XBP1 mRNA in HuH-7 cells ......... 78 Nuclear accumulation of XBP1s after GGA treatment ..................................................... 79 Specificity of diterpenoid-induced cell death in HuH-7 cells ............................................ 79 GGA-induced XBP1 splicing shared a similarity with lipid-induced UPR .......................... 80 Rescue from GGA-induced cell death by oleate co-treatment ........................................... 81 Inhibition of GGA-induced UPR attenuates GGA-induced accumulation of LC3-II ............ 81 Discussion ..................................................................................................................................... 83 Figures .......................................................................................................................................... 87 Chapter V: General Discussion .................................................................................................. 101 How GGA affects p53 in HuH-7 cells? ...................................................................................... 102 GGA-induced UPR is an upstream signal of GGA-induced autophagy .................................... 104 Conclusions ................................................................................................................................ 105 Figure .......................................................................................................................................... 106 Acknowledgements ....................................................................................................................... 107 References ..................................................................................................................................... 108 List of abbreviations ACSL3; acyl-CoA synthetase long-chain family member 3 ATF6; activating transcription factor 6 ATRA; all-trans retinoic acid Atg8; autophagy-related gene-8 BBC3; BCL2-binding component 3 BECN1; beclin 1 BH-3; bcl-2 homology domain 3 BiP; binding immunoglobulin protein BMH; bis(maleimido) hexane BN-PAGE; blue native-gradient PAGE BSA; bovine serum albumin cDNA; complementary DNA COX; cytochrome c oxidase complex DIC; differential interference contrast DDIT3; DNA-damage-inducible transcript 3 m ; mitochondrial inner membrane potential D-MEM; Dulbecco’s modified Eagle’s medium DRAM; damage-regulated autophagy modulator eIF2; eukaryotic initiation factor 2 alpha ER; endoplasmic reticulum FA; farnesoic acid FBS; fetal bovine serum F1,6-DP ; D-fructose 1,6-bisphosphate F2,6-DP; D-fructose 2,6-bisphosphatase F6-P; D-fructose 6-phosphate GFP; green fluorescent protein GGA; geranylgeranoic acid GGOH; geranylgeraniol GRP78; glucose-regulated protein 78 G6-P; glucose 6-phosphate HGNC; HUGO Nomenclature Committee HRP; horseradish peroxidase HSPs; heat-shock proteins IRE1; inositol requiring 1 KEGG: Kyoto Encyclopedia of Genes and Genomes LC3; microtubule-associated protein 1 light chain 3 MCP; multi-channel plate MD; mega dalton MPCs; multiple protein complexes m/z; mass-to-charge ratio DH; -nicotinamide adenine dinucleotide, reduced NetCDF; network common data file ODC; ornithine decarboxylase OPLS-DA; orthogonal partial least squares-discriminant analysis PAGE; polyacrylamide gel electrophoresis PARC; p53-associated, parkin-like cytoplasmic protein PBS(-); phosphate buffered saline, Ca-free PBS-T; PBS(-) containing 0.1% polyoxyethylene sorbitan monolaurate PCA; principal component analysis PDIA4; protein disulfide isomerase family A, member 4 PEP; phosphoenolpyruvic acid PERK; protein kinase RNA (PKR)-like ER kinase PKR; protein kinase RNA PUMA; p53 upregulated modulator of apoptosis PVDF; Polyvinylidene difluoride Q-Tof/MS; quadrupole time-of-flight type mass spectrometry RIPA; radioimmuno precipitation assay RNase; endoribonuclease ROS; reactive oxygen species RT-qPCR; reverse-transcription real-time polymerase chain reaction SAGE; serial analysis of gene expression SCD1; stearoyl-CoA desaturase 1 SCO2; Synthesis of cytochrome c oxidase 2 SDS; sodium dodecylsulfate SSAT; spermidine/spermine-N 1 -acetyltransferase TE; 10 mM Tris-HCl, pH 7.4 and 1 mM EDTA buffer TIGAR; TP53-induced glycolysis and apoptosis regulator TP53; Tumor protein 53 UPLC; ultra-performance liquid chromatography UPR; unfolded protein response VIP; variable importance in the projection XBP1: X-box binding protein 1 WT; wild type 9CRA; 9-cis retinoic acid