The modulation of various signal transduction pathways in colorectal carcinoma cells by docosahexaenoic acid

[摘要] ENGLISH ABSTRACT: Introduction: The ability of different polyunsaturated fatty acids (PUFAs),especially n-3 PUFAs, to prevent the development of cancer has been underintense investigation the past three decades. Numerous studies have shownthat these fatty acids can kill cancer cells in vitro as well as in vivo whilst normalcells remain unaffected. Unfortunately, the cellular and molecular mechanismsresponsible for this phenomenon are still poorly understood. This studyinvestigated the signalling pathways modulated by docosahexaenoic acid(DHA) in an adenocarcinoma cell line, in order to shed some light on theseunknown mechanisms.Materials & Methods: NCM460 (normal colon epithelial) and CaCo2 (colonadenocarcinoma) cells were cultured and treated with low doses of palmitic acid(PMA), oleic acid (OA), arachidonic acid (AA), and DHA. The effects of thesefatty acids on the proliferation of the cells were measured with the MTT assay.The composition of membrane phospholipids of CaCo2 cells was determinedafter 48h supplementation with different fatty acids by gas chromatography.Also, CaCo2 cells were treated with DHA (10 μM) only and proteins wereharvested at fixed time points ranging from 2 minutes to 48 hours. The proteininhibitors wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB203580 (p38 inhibitor) and also RNA interference (RNAi) of the p38 MAPKprotein were used to investigate cross-talk between signalling pathways. ERK,p38 MAP kinase, Akt, and p53 were then analysed by Western blotting usingphospho-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 weretoxic to normal cells. In addition, DHA was shown to be most effective to killCaCo2 cells whilst protecting NCM460 cells and a subsequent dose response experiment revealed that lower concentrations are most suitable for thispurpose. DHA was also shown to be readily incorporated into phospholipids,along with AA. This is associated with increased membrane fluidity, whichcould affect the localisation, and downstream effects, of various signallingproteins within the membrane. Western blot analysis revealed a rapid increasein activity in most proteins under investigation, especially ERK and Akt(Ser473). Long-term DHA supplementation suppressed the full activation ofAkt. This down regulation of survival signalling could lead to cell death inCaCo2 cells. In addition, it was shown that after 48h, DHA induced thecleavage of caspase-3 and PARP, which is indicative of apoptosis. RNAiexperiments suggested a possible role for p38 MAPK in the phosphorylation ofp53 at Ser15, a site which is associated with DNA damage.Conclusion: DHA exerts its effects by means of cellular signal transductionpathways, particularly by suppression of the important survival-related kinase,Akt. This could have implications for future therapeutic interventions in cancerpatients, as fatty acids are safe to use and do not interfere with the functionalityof normal tissue.

[发布机构] Stellenbosch University