Department of Physiological Sciences

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Browsing Department of Physiological Sciences by Subject "Adenocarcinoma"

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    The modulation of various signal transduction pathways in colorectal carcinoma cells by docosahexaenoic acid
    (Stellenbosch : University of Stellenbosch, 2006-12) Du Toit, Joe-Lin; Engelbrecht, A. M.; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
    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.