Effects of DHA incorporation in membrane lipid rafts of MDA-MB-231 human breast cancer cells

One of the major targets for breast cancer therapy is the epidermal growth factor receptor (EGFR). EGFR is a transmembrane protein with intrinsic protein tyrosine kinase activity that is activated by ligand binding, most important being EGF. EGFR over-expression contributes to increased cell proliferation and migration in breast cancer (1). Recent findings in membrane biology suggest that the plasma membrane is composed of microdomains of saturated lipids that segregate together to form lipid “rafts”. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane microdomains resistant to solubilization by nonionic detergents at low temperatures. Lipid rafts are enriched in several signaling proteins, including EGFR (2). N-3 polyunsaturated fatty acids (PUFA), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decrease proliferation and induce apoptosis in EGFR over-expressing MDA-MB-231 human breast cancer cells (3,4). Here we report a biophysical approach aimed at investigating the changes in lipid composition, induced by DHA incorporation, and their correlation with EGFR content modifications in MDA-MB-231 cell lipid rafts applying imaging and spectroscopic tools, namely AFM and FTIR microspectroscopy. Moreover, the biophysical approach is coupled to a detailed biochemical analysis by means of biochemical assays (SDS-PAGE, Western Blotting and HPLC/GC). Biochemical analyses show that DHA increases the unsaturated degree of phospholipids in lipid raft fatty acids of breast cancer cells, therefore, it alters their physical-chemical properties Many acylated proteins directly interact with membrane lipid bilayers by their saturated acyl moieties. Then we suggest that the altered lipid rafts in n-3 PUFA-treated cells with alteration of signal transduction. In addition, morpho-dimensional changes in lipid rafts are visualized and quantitatively analyzed by AFM studying purified membrane samples both before and after the DHA incorporation. AFM technique allows to obtain three-dimensional images of the surface topography of lipid microdomains at nanometer resolution in a physiological-like environment thus providing structural/functional insights that cannot be obtained with more conventional approaches. High resolution AFM imaging shows on MDA-MB-231 lipid rafts, after DHA incorporation, features in agreement, for dimensions and shape, with membrane proteins. A more accurate investigation using specific antibodies could confirm, in the next future, the nature of the observed structures and allow their identification. These preliminary results suggest that AFM could be an useful tool to characterize changes in the membrane protein content induced by DHA treatment at single protein level. (1) Muller-Todow C et al., Clin Cancer Res, 10, 1241-1249 (2004). (2) Foster LJ et al., Proc Natl Acad Sci USA, 100, 5813-5818 (2003). (3) Schley PD et al., Breast Cancer Res Treat, 92, 187-195 (2005). (4) Corsetto PA et al., Chem Phys Lip, 163, S28 (2010).