Tong, Kit Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 91-104). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.x / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.2 / Chapter 1.1.1 --- Tumor development --- p.2 / Chapter 1.1.2 --- Cell cycle --- p.4 / Chapter 1.1.3 --- Apoptosis --- p.9 / Chapter 1.1.3.1 --- The extrinsic pathway --- p.14 / Chapter 1.1.3.2 --- The intrinsic pathway --- p.16 / Chapter 1.1.3.3 --- The Bcl-2 family proteins --- p.17 / Chapter 1.1.3.4 --- Execution of apoptosis --- p.20 / Chapter 1.1.4 --- Melanoma --- p.22 / Chapter 1.2 --- Polyunsaturated fatty acids (PUFAs) --- p.24 / Chapter 1.2.1 --- "Chemistry, classification, metabolic conversion and sources …" --- p.24 / Chapter 1.2.2 --- Epidemiology studies --- p.27 / Chapter 1.2.3 --- Docosahexaenoic acid (DHA) --- p.28 / Chapter 1.2.3.1 --- Sources --- p.28 / Chapter 1.2.3.2 --- DHA and cancer --- p.29 / Chapter 1.3 --- Objectives --- p.33 / Chapter Chapter 2 --- Materials and Methods --- p.34 / Chapter 2.1 --- In vitro studies of DHA on growth and survival of human cancer cells --- p.34 / Chapter 2.1.1 --- Cell cultures --- p.34 / Chapter 2.1.2 --- Studies of growth inhibition of DHA on human cancer cells --- p.35 / Chapter 2.1.2.1 --- MTT assay --- p.35 / Chapter 2.1.2.2 --- Chemiluminescent-bromodeoxyuridine (Chemi-BrdU) immunoassay --- p.36 / Chapter 2.1.3 --- Studies of growth inhibitory mechanism of DHA on A375 cells. --- p.38 / Chapter 2.1.3.1 --- DNA -flow cytometry analysis --- p.38 / Chapter 2.1.3.2 --- Western blot analysis --- p.39 / Chapter 2.1.3.3 --- Caspase inhibitor studies --- p.42 / Chapter 2.1.3.4 --- Mitochondrial membrane potential analysis --- p.42 / Chapter 2.2 --- In vivo study of the anticancer effect of DHA on A375 cells --- p.44 / Chapter 2.2.1 --- Animals --- p.44 / Chapter 2.2.2 --- Cell inoculation and treatments --- p.44 / Chapter 2.2.3 --- Western blot analysis --- p.45 / Chapter 2.3 --- Statistical analysis --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- In vitro studies of DHA on growth and survival of human canccr cells --- p.47 / Chapter 3.1.1 --- DHA reduced proliferation and survival of human cancer cells --- p.47 / Chapter 3.1.2 --- DHA modulated cell cycle of A375 cells --- p.52 / Chapter 3.1.3 --- DHA induced apoptosis in A375 cells --- p.55 / Chapter 3.1.4 --- Caspase activations were involved in the DHA-induced apoptosis in A375 cells --- p.59 / Chapter 3.1.5 --- "Caspase 3´ة 6, 8 and 9 were activated in DHA-induced apoptosis of A375 cells" --- p.62 / Chapter 3.1.6 --- DHA dissipated mitochondrial membrane potential in A375 cells --- p.66 / Chapter 3.1.7 --- DHA triggered the mitochondrial pathway of apoptosis --- p.68 / Chapter 3.1.8 --- DHA triggered the death receptor pathway of apoptosis --- p.71 / Chapter 3.2 --- In vivo study of the anticancer effect of DHA on A375 cells --- p.74 / Chapter 3.2.1 --- Effect of DHA on the growth ofA375 xenograft in athymic Bαlb/c mice --- p.74 / Chapter 3.2.2 --- DR4 and TRAIL were upregulated by DHA treatment in A375 solid tumor --- p.77 / Chapter Chapter 4 --- Discussion --- p.79 / References --- p.91

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Introduction: The ability of different polyunsaturated fatty acids (PUFAs), especially n-3 PUFAs, to prevent the development of cancer has been under intense investigation the past three decades. Numerous studies have shown that these fatty acids can kill cancer cells in vitro as well as in vivo whilst normal cells remain unaffected. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood. This study investigated the signalling pathways modulated by docosahexaenoic acid (DHA) in an adenocarcinoma cell line, in order to shed some light on these unknown mechanisms. Materials & Methods: NCM460 (normal colon epithelial) and CaCo2 (colon adenocarcinoma) cells were cultured and treated with low doses of palmitic acid (PMA), oleic acid (OA), arachidonic acid (AA), and DHA. The effects of these fatty acids on the proliferation of the cells were measured with the MTT assay. The composition of membrane phospholipids of CaCo2 cells was determined after 48h supplementation with different fatty acids by gas chromatography. Also, CaCo2 cells were treated with DHA (10 μM) only and proteins were harvested at fixed time points ranging from 2 minutes to 48 hours. The protein inhibitors wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB 203580 (p38 inhibitor) and also RNA interference (RNAi) of the p38 MAPK protein were used to investigate cross-talk between signalling pathways. ERK, p38 MAP kinase, Akt, and p53 were then analysed by Western blotting using phospho-specific and total antibodies. The cleavage of the apoptotic proteins, caspase-3 and PARP were also analysed. Results and discussion: MTT assays revealed that none of the fatty acids were toxic to normal cells. In addition, DHA was shown to be most effective to kill CaCo2 cells whilst protecting NCM460 cells and a subsequent dose response experiment revealed that lower concentrations are most suitable for this purpose. DHA was also shown to be readily incorporated into phospholipids, along with AA. This is associated with increased membrane fluidity, which could affect the localisation, and downstream effects, of various signalling proteins within the membrane. Western blot analysis revealed a rapid increase in activity in most proteins under investigation, especially ERK and Akt (Ser473). Long-term DHA supplementation suppressed the full activation of Akt. This down regulation of survival signalling could lead to cell death in CaCo2 cells. In addition, it was shown that after 48h, DHA induced the cleavage of caspase-3 and PARP, which is indicative of apoptosis. RNAi experiments suggested a possible role for p38 MAPK in the phosphorylation of p53 at Ser15, a site which is associated with DNA damage. Conclusion: DHA exerts its effects by means of cellular signal transduction pathways, particularly by suppression of the important survival-related kinase, Akt. This could have implications for future therapeutic interventions in cancer patients, as fatty acids are safe to use and do not interfere with the functionality of normal tissue. / AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van verskillende poli-onversadigde vetsure (POVSe), veral n-3 POVSe, om die ontstaan van kanker te voorkom, is intens nagevors die afgelope drie dekades. Menigte studies het aangevoer dat hierdie vetsure kankerselle in vitro asook in vivo kan doodmaak, terwyl normale selle nie daardeur beïnvloed word nie. Ongelukkig word die sellulêre and molekulêre meganismes onderliggend tot hierdie verskynsel nie goed begryp nie. Hierdie studie het verskeie seintransduksie-paaie wat deur dokosaheksaenoësuur (DHS) in ‘n adenokarsinoom sellyn gemoduleer word, ondersoek. Materiale & Metodes: NCM460 (normale kolonepiteel) en CaCo2 (kolon adenokarsinoom) selle is onderhou in ‘n selkultuur-laboratorium en behandel met lae dosisse palmitiensuur (PMS), oleïensuur (OS), aragidoonsuur (AS), en DHS. Die invloed van hierdie vetsure op die proliferasie van die selle is d.m.v. die MTT toets bepaal. The samestelling van membraan-fosfolipiede van CaCo2 selle is na 48h behandeling met die verskillende vetsure bepaal deur middel van gaschromatografie. Die CaCo2 selle is ook met DHA (10 μM) alleenlik behandel en teen vaste tydpunte wat wissel van 2 minute tot 48h, waarna proteïene geëkstraeer is. Die proteïen-inhibitore wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor), en SB 203580 (p38 inhibitor) asook RNAinterferensie (RNAi) teen die p38 MAPK proteïen is ingespan om oorvleueling tussen seintransduksie–weë te ondersoek. ERK, p38 MAPK, Akt, en p53 is geanaliseer deur middel van die Western–klad metode met fosfo–spesifieke en totale antiliggame. Die kliewing van die apoptotiese proteïene caspase-3 en PARP is ook bepaal. Resultate en bespreking: MTT toetse het ontul dat geen vetsure toksies was vir die normale selle nie. Daar is ook gevind dat DHS die mees effektiewe vetsuur was om CaCo2 selle te dood, terwyl NCM460 selle beskerm word. Gevolglik het ‘n dosis-respons eksperiment getoon dat laer konsentrasies die beste geskik is vir hierdie doel. Daar is ook gevind dat DHA maklik in fosfolipiede geïnkorporeer word, tesame met AS. Dit word geassosieer met verhoogde membraan-vloeibaarheid, wat die ligging, en ook stroom-af werking, van verskeie seintransduksie proteïene in die membraan, kan beïnvloed. Westernklad analises het ‘n vinnige verhoging in die aktiwiteite van die meeste proteïene onder die soeklig, getoon, veral ERK en Akt (Ser473). Langdurige DHS behandeling het die maksimale aktiwiteit van Akt onderdruk. Hierdie afname van oorlewing-gerigte seine kan lei tot seldood in CaCo2 selle. Daar is boonop geving dat DHS die kliewing van caspase-3 en PARP geïnduseer het na 48, wat dui op apoptose. Uit die RNAi eksperiment kon daar ook ‘n moontlike rol vir p38 MAPK in die fosforilering van p53 by Ser15, wat geassosieer word met DNS-skade, getoon word. Gevolgtrekking: DHS beoefen sy effekte deur middel van seintransduksie paaie, veral deur die oorlewing-geassosieerde kinase, Akt, te onderdruk. Dit kan implikasies hê vir toekomende terapeutiese ingrypings in kankerpasiënte, aangesien vetsure veilig is om te gebruik en nie skadelik is vir normale weefsel nie.