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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17350 |
| Date | 12 1900 |
| Creators | Du Toit, Joe-Lin |
| Contributors | Engelbrecht, A.M., University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xx, 121 leaves : ill. |
| Rights | University of Stellenbosch |
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