Chemical-Physical Changes in Cell Membrane Microdomains of Breast Cancer Cells After Omega-3 PUFA Incorporation

Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and progression of breast cancer. However, no clear data are present in literature that could demonstrate how n\ua0-\ua03 PUFA can interfere with breast cancer growth. It is suggested that these fatty acids might change the structure of cell membrane, especially of lipid rafts. During this study we treated MCF-7 and MDA-MB-231 cells with AA, EPA, and DHA to assess if they are incorporated in lipid raft phospholipids and are able to change chemical and physical properties of these structures. Our data demonstrate that PUFA and their metabolites are inserted with different yield in cell membrane microdomains and are able to alter fatty acid composition without decreasing the total percentage of saturated fatty acids that characterize these structures. In particular in MDA-MB-231 cells, that displays the highest content of Chol and saturated fatty acids, we observed the lowest incorporation of DHA, probably for sterical reasons; nevertheless DHA was able to decrease Chol and SM content. Moreover, PUFA are incorporated in breast cancer lipid rafts with different specificity for the phospholipid moiety, in particular PUFA are incorporated in PI, PS, and PC phospholipids that may be relevant to the formation of PUFA metabolites (prostaglandins, prostacyclins, leukotrienes, resolvines, and protectines) of phospholipids deriving second messengers and signal transduction activation. The bio-physical changes after n\ua0-\ua03 PUFA incubation have also been highlighted by atomic force microscopy. In particular, for both cell lines the DHA treatment produced a decrease of the lipid rafts in the order of about 20-30\ua0%. It is worth noticing that after DHA incorporation lipid rafts exhibit two different height ranges. In fact, some lipid rafts have a higher height of 6-6.5\ua0nm. In conclusion n\ua0-\ua03 PUFA are able to modify lipid raft biochemical and biophysical features leading to decrease of breast cancer cell proliferation probably through different mechanisms related to acyl chain length and unsaturation. While EPA may contribute to cell apoptosis mainly through decrease of AA concentration in lipid raft phospholipids, DHA may change the biophysical properties of lipid rafts decreasing the content of cholesterol and probably the distribution of key proteins

New insights into the genetic etiology of Alzheimer's disease and related dementias.

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele

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 \u201crafts\u201d. 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)

Atomic force microscopy imaging of lipid rafts of human breast cancer cells

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-

Chemical-physical changes in breast cancer lipid rafts after omega-3 PUFA incorporation

Recent studies suggest that \u3c9-3 PUFA can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. \u3c9-3 PUFA could alter cancer growth influencing cell replication, cell cycle, and cell death. The mechanisms of PUFA action are still unclear. The aim of this study is to demonstrate that \u3c9-3 PUFA reduce breast adenocarcinoma growth through their incorporation in cell membrane, especially in lipid rafts. We have observed with viability tests, cell morphology analyses and apoptotic markers levels, that EPA and DHA induce apoptosis. We have demonstrated that AA, EPA and DHA are incorporated in breast cancer membrane and isolated lipid rafts with different specificity for the phospholipids moiety. Only the treatment with DHA induces a reduction of cholesterol content in lipid rafts, indicating a possible change in rafts organization. Physical changes in the breast cancer lipid rafts were also assessed with atomic force microscopy (AFM) after \u3c9-3 treatment. The reduction of saturation degree and cholesterol content might induce membrane structural instability causing proteins (i.e. EGFR) displacement. In fact we have observed that in MDA-MB-231 cells, DHA reduces the EGFR content, but not EPA. Both \u3c9-3 PUFA reduce the activation of EGFR. In conclusion, \u3c9-3 PUFA alter lipid raft stability and might modify cellular signalling in breast cancer cells

Atomic Force Microscopy study of lipid rafts in MDA-MB-231 human breast cancer cells after DHA incorporation

INTRODUCTION: 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 by microdomains of saturated lipids that segregate together to form lipid \u201crafts\u201d. 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). Here we report a biophysical approach to investigate lipid rafts fatty acid and protein composition in MDA-MB-231 treated with DHA, applying spectroscopic and imaging 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). RESULTS: Biochemical analyses show that DHA increases the unsaturated state of phospholipids in lipid rafts of breast cancer cells, therefore, alters their physical-chemical properties. Many acylated proteins directly interact with membrane lipid bilayers by their saturated acyl moieties. Then we suggest that altered lipid composition of microdomains might determine the displacement of proteins from lipid rafts in n-3 PUFA-treated cells with alteration of signal transduction, with particular regards to EGFR. In addition, morpho-dimensional changes in lipid rafts are visualized and 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. (2004) Clin. Cancer Res. 10, 1241-1249 2. Foster LJ et al. (2003) Proc. Natl. Acad. Sci USA 100, 5813-5818 3. Corsetto PA et al. (2010) Chem. Phys. Lip. S28, doi: 10.1016/j.chemphyslip.2010.05.0