There has been considerable evidence recently demonstrating the anti-tumour effects of flavonols. Quercetin, an ubiquitous bioactive flavonol, inhibits cells proliferation, induces cell cycle arrest and apoptosis in different cancer cell types. The precise molecular mechanism of quercetin-induced apoptosis in human breast cancer cells is unclear. The purpose of this study was to investigate effects of quercetin on cell viability and to determine its underlying mechanism in human breast cancer MDA-MB-231 cells. Quercetin decreased the percentage of viable cells in a dose- and time-dependent manner, which was associated with cell cycle arrest and apoptosis. Quercetin did not increase reactive oxygen species generation but increased cytosolic Ca2+ levels and reduced the mitochondrial membrane potential (ΔΨm). Quercetin treatment promoted activation of caspase-3, -8 and -9 in MDA-MB-231 cells. Caspase inhibitors prevented the quercetin-induced loss of cell viability. Quercetin increased abundance of the pro-apoptotic protein Bax and decreased the levels of anti-apoptotic protein Bcl-2. Confocal laser microscope examination indicated that quercetin promoted apoptosis-inducing factor (AIF) release from mitochondria and stimulated translocation to the nucleus. Taken together, these findings suggest that quercetin results in human breast cancer MDA-MB-231 cell death through mitochondrial- and caspase-3-dependent pathways.

Direct effects of ethanol on the interaction of cytosolic lipid transfer proteins with ligands are not known. In this study, recombinant liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2) were used in conjunction with a series of fluorescent fatty acid probe molecules to compare the relative dielectric properties of the ligand binding sites and to examine the effects of ethanol in vitro on ligand interaction with these proteins. L-FABP and SCP-2 exhibited broad but distinct ligand specificities. Although NBD-stearic acid bound with high affinity to both proteins, emission spectra showed that the relative dielectric constant of the ligand binding site in SCP-2 was significantly lower than in L-FABP, 2 vs 24. Furthermore, affinities of L-FABP for NBD-fatty acid probes were NBD-stearic acid > NBD-lauric acid >>> NBD-hexanoic acid, NBD-acetic acid. In contrast, SCP-2 bound only NBD-stearic acid with a Kd of 0.23 microM and Bmax of 0.98 mol/mol. This observation of SCP-2 specifically binding the fluorescent NBD-stearic acid was confirmed with RdB-stearic acid and the naturally fluorescent cis-parinaric acid, both of which had similar affinities and stoichiometries. Ethanol in vitro had no effect on L-FABP-NBD-stearic acid binding. However, ethanol at physiological concentrations (25 mM) dramatically inhibited NBD-stearic acid binding to SCP-2. In conclusion, the data show that both L-FABP and SCP-2 specifically bind fluorescent fatty acids. However, the ligand binding sites of L-FABP and SCP-2 differed dramatically in their dielectric properties and their sensitivity to ethanol.

Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3-4-, 14-15-, and 24-25-month-old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)-quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol-to-phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins. Key Words: Fluidity-Cholesterol-Brain-Synaptic plasma membrane-Lipid asymmetry-Aging-Mice.