Short-chain fatty acid modulation of apoptosis in gastric and colon cancer cells.

IV DECLARATION . VI ACKNOWLEDGEMENTS . VII CHAPTER 1: SHORT-CHAIN FATTY ACID MODULATION OF APOPTOSIS IN GASTRIC AND COLON CANCER. 1 1.1: INTRODUCTION . 1 1.2: APOPTOSIS . 2 1.3: ROLE OF THE OXIDATIVE PENTOSE PATHWAY, GLUTATHIONE AND REACTIVE OXYGEN SPECIES IN APOPTOSIS . 3 1.4: SHORT-CHAIN FATTY ACIDS . 9 1.5: SCFAS AND THE GSH DEFENCE SYSTEM . 11 1.6: CANCER, GLUCOSE METABOLISM AND SCFAS. 12 1.7: SUMMARY. 14 CHAPTER 2: A NOVEL TECHNIQUE FOR THE RAPID MEASUREMENT OF DGLUCOSE METABOLISM AND OXIDATIVE PENTOSE PATHWAY ACTIVITY IN THE KATO III GASTRIC CANCER CELL LINE. 16 2.1: INTRODUCTION . 16 2.2: MATERIALS AND METHODS. 19 2.2.1: Materials. 19 2.2.2: Kato III cell culture . 19 2.2.3: Study protocols . 19 2.2.3.1: Experimental set up of technique . 19 2.2.3.2: D-glucose oxidation and OPP activity following serum deprivation . 20 2.2.3.3: D-glucose oxidation and OPP activity following incubation with DHEA . 20 2.2.3.4: D-glucose oxidation and OPP activity following incubation with IGF-I . 20 2.2.4: Technique for assessment of 1-C-D-glucose oxidation . 21 2.2. 5: Assay for G6PDH activity . 22 2.2. 5.1: Preparation of cytosol . 22 2.2. 5.2: G6PDH assay. 22 2.2. 6: Statistics. 23 2.3: RESULTS . 24 2.3.1: Initial validation of technique . 24 2.3.2: Effects of serum deprivation. 24 2.3.2.1: Kato III cell viability . 24 2.3.2.2: G6PDH activity . 29 2.3.2.3: 1-C-D-glucose oxidation. 29 2.3.3: Effects of DHEA treatment . 29 2.3.3.1: Kato III cell viability . 29 2.3.3.2: G6PDH activity . 32 2.3.3.3: 1-C-D-glucose oxidation. 32 2.3.4: Effects of IGF-I treatment on Kato III cells . 32 2.3.4.1: Kato III cell viability . 32 2.3.4.2: G6PDH activity . 33 2.3.4.3: 1-C-D-glucose oxidation. 33 2.4: DISCUSSION . 39 CHAPTER 3: BUTYRATE AND PROPIONATE MODULATE APOPTOSIS AND CELL CYCLE IN KATO III GASTRIC CARCINOMA CELLS. 43 3.1: INTRODUCTION . 43 3.2: MATERIALS AND METHODS . 44 3.2.1: Materials. 44 3.2.2: Kato III cell culture . 44 3.2.3: Experimental design . 44 3.2.4: Assays . 45 3.2.4.1: Cell viability measurements . 45 3.2.4.2: Cell cycle analysis . 46 3.2. 5: Statistics. 46 3.3: RESULTS . 48 3.3.1: Cell viability . 48 3.3.1.1: Butyrate decreased viability and induced apoptosis and necrosis in Kato III cells . 48 3.3.1.2: Propionate decreased cell viability and induced apoptosis in Kato III cells . 50 3.3.1.3: Butyrate/propionate combination treatment decreased cell viability and induced apoptosis in Kato III cells . 52 3.3.1.4: Butyrate/propionate combined treatment decreased viability greater than either SCFA alone. 54 3.3.2: Cell cycle . 61 3.3.2.1: Butyrate induced S and G2-M phase arrest in Kato III cells . 61 3.3.2.2: Propionate induced G2-M phase arrest in Kato III cells . 61 3.4: DISCUSSION . 65 CHAPTER 4: SHORT-CHAIN FATTY ACID MODULATION OF INTRACELLULAR PROTECTION AND D-GLUCOSE METABOLISM IN KATO III HUMAN GASTRIC CARCINOMA CELLS. 68 4.1: INTRODUCTION . 68 4.2: MATERIALS AND METHODS . 70 4.2.1: Materials. 70 4.2.2: Kato III cell culture . 70 4.2.3: Experimental design . 70 4.2.4: Assays . 71 4.2.4.1: G6PDH activity . 71 4.2.4.2: GSH availability . 71 4.2.4.3: 1-C-glucose oxidation . 72 4.2. 5: Statistics. 72 4.3: RESULTS . 74 4.3.1: Oxidative pentose pathway activity . 74 4.3.1.1: Butyrate increased OPP activity in Kato III cells . 74 4.3.1.2: Propionate increased OPP activity in Kato III cells . 74 4.3.2.3: Butyrate/