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 “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). 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.2010.05.08

Atomic Force Microscopy study of lipid rafts in MDA-MB-231 human breast cancer cells after DHA incorporation / P.A. Corsetto, A. Cremona, F. Orsini, P. Arosio, I.E. Jovenitti, S. Zava, G. Montorfano, A.M Rizzo. ((Intervento presentato al convegno Convegno annuale della sezione ligure-lombardo-piemontese della società italiana di biochimica e biologia molecolare tenutosi a Novara nel 2011.

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

P.A. Corsetto;A. Cremona;F. Orsini;P. Arosio;I.E. Jovenitti;S. Zava;G. Montorfano;A. M Rizzo
2011-05-20

Abstract

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 “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). 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.2010.05.08
Omega-3 ; Cancer ; Lipid Rafts
Settore BIO/10 - Biochimica
http://www3.med.unipmn.it/lf/Abstracts_mag2011_sm.pdf
Atomic Force Microscopy study of lipid rafts in MDA-MB-231 human breast cancer cells after DHA incorporation / P.A. Corsetto, A. Cremona, F. Orsini, P. Arosio, I.E. Jovenitti, S. Zava, G. Montorfano, A.M Rizzo. ((Intervento presentato al convegno Convegno annuale della sezione ligure-lombardo-piemontese della società italiana di biochimica e biologia molecolare tenutosi a Novara nel 2011.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/239118
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