THE USE OF ATOMIC FORCE MICROSCOPY TOGETHER WITH CLASSICAL BIOCHEMICAL TECHNIQUES TO STUDY ALTERATIONS OF MEMBRANE MICRODOMAINS INDUCED BY DHA IN MDA-MB-231 BREAST CANCER CELLS
Creators: name: CREMONA, ANDREA
Publisher: name: Universit� degli Studi di Milano
Year: 2014

description: Today, the Western diet is substantially enriched in n-6 PUFAs (shifting the balance from the optimal n-6:n-3 ratio of 1?5:1 to approximately 20:1) due to the increased consumption of food items rich in n-6 and simultaneous reduction of fish intake. Diet enriched in n-6 PUFAs determines an unbalance between bioactive lipid mediators that are involved in regulating inflammation. These bioactive products can contribute to the development of numerous chronic diseases, including cancer. Many studies have demonstrated that genetic factors contribute only for 5% to the onset of cancer, while 95% is due to environmental conditions factors, including lifestyle-related factors (tobacco, alcohol and physical activity), external stimuli (radiation and infections) and diet that represents about 30?35% of the risk factors.
There are in vitro and in vivo evidences that the DHA and EPA administration is able to reduce cancer proliferation increase apoptosis as well as inhibit angiogenesis and metastasis, and to influence cancer differentiation. Several molecular mechanisms have been proposed for the anticancer activity of n-3 PUFAs, including inhibition of cell proliferation, anti-angiogenic action and enhancement of apoptosis. The multiple actions of n-3 PUFAs appear to involve multiple mechanisms that connect the cell membrane, the cytosol, and the nucleus. N-3 PUFAs may act also modulating cell membrane FA composition. 
The plasma membrane is involved in many aspects of cell biology, including proliferation, differentiation and apoptosis. Phospholipids (PLs) fatty acid composition within cell membrane play a key role in maintaining its fluidity, structure and function. Thus, changes in membrane PLs fatty acid composition can influence the function of cells. In particular, n-3 PUFAs incorporated into membrane PLs can potentially affect a variety of plasma membrane physical properties including membrane thickness, fluidity and elasticity, structure and function in particular of membrane microdomains (lipid rafts); moreovere they can cause alteration of cell signaling pathways that lead to altered transcription factor activity and changes in gene expression. We have observed that DHA is incorporated in breast cancer cell membrane with different specificity for the PLs moiety. Moreover, we have demonstrated that the treatment with DHA determines a reduction of cell proliferation, inhibition of EGFR activity and induction of apoptotic process, suggesting that these effects might be the consequences of cell membrane alterations induced by FAs.
The main purpose of my research project was to analyze the effects of PUFA administration on membrane microdomain structure and function in breast cancer cells by a combination of biophysical and biochemical techniques. In my study I have used Atomic Force Microscopy (AFM) to perform a morpho-dimensional characterization of lipid rafts as Detergent-Resistant Membrane (DRM) isolated from MDA-MB-231 breast cells (estrogen receptor negative and over-expressing Epidermal growth factor receptor (EGFR)). 
AFM providing nanometer spatial resolution and operating in physiological-like conditions without fixation, staining, or labeling, appears to be a useful tool to visualize and quantitatively characterize the topology of biological membranes as well as of their protein content.
AFM imaging of DRM fractions showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of microdomains with lateral dimensions in the order of a few hundreds of nanometers. Moreover, AFM-immunolabeling using specific antibodies suggested the presence, in these microdomains, of a characteristic marker of lipid rafts, the protein flotillin-1. In addition, my results suggested that AFM could be an useful tool to study the phase coexistence of a Liquid-disordered (Ld) and Liquid-ordered (Lo) domain in purified membrane microdomains. 
Finally, I have investigated the role of lipid changes induced by DHA in membrane microdomain structure and function by biochemical tecniques. The data showed that the incubation with DHA determines its incorporation in all PLs, with different specificity for PI, PS and PC, and a reduction of sphingomyelin (SM) and cholesterol (Chol) content in membrane microdomains. Moreover, I have demonstrated that DHA can exclude key proteins, such as EGFR and Ras, from membrane microdomains, modifying their downstream signaling, such as Erk1/2 and Akt.