Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells.

FTIR, (1)H NMR and EPR spectroscopy studies on the interaction of flavone apigenin with dipalmitoylphosphatidylcholine liposomes.

Apigenin (5,7,4'-trihydroxyflavone) is a cancer chemopreventive agent and a member of the family of plant flavonoids. Apigenin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated by means of FTIR spectroscopy, (1)H NMR and EPR techniques. Fluorescent microscopy and electron microscopy were applied to study the apigenin effects on colon myofibroblasts and human skin fibroblasts. The strong rigidifying effect of apigenin with respect to polar head groups was concluded on the basis of the action of the flavone on partition coefficient of Tempo spin label between the water and lipid phases. The ordering effect was also found in hydrophobic region at the depth monitored by 5-SASL and 16-SASL spin labels. The inclusion of apigenin to the membrane restricted the motional freedom of polar head groups lowering penetration of Pr(3+) ions to the membranes. The (1)H NMR technique supported also the restriction of motional freedom of the membrane in the hydrophobic region, especially in the zone of CH(2) groups of alkyl chains. FTIR analysis showed that apigenin incorporates into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the C-O-P-O-C segment. It is also very likely that hydroxyl groups of apigenin link with polar groups of DPPC by water bridges. Electron and fluorescence microscopic observations revealed changes in the internal membrane organization of the examined cells. In conclusion, the changes of the structural and dynamic properties of membranes can be crucial for processes involving tumor suppression signal transduction pathways and cell cycle regulation.

Localization and interaction of genistein with model membranes formed with dipalmitoylphosphatidylcholine (DPPC).

The effect of genistein on the liposomes formed with dipalmitoylphosphatidylcholine was studied with the application of Fourier-transform infrared spectroscopy, nuclear magnetic resonance (1H NMR) and electron paramagnetic resonance techniques. Membranous structures organization of human skin fibroblasts and colon myofibroblasts was also examined using fluorescence and electron microscopy. The strongest rigidifying effect of genistein with respect to polar head groups was concluded on the basis of the effect of the flavonoid on the shape of NMR lines attributed to -N+(CH3)3 groups. The rigidifying effect of genistein with respect to the hydrophobic core of lipid membranes was also concluded from the genistein-dependent broadening of the NMR lines assigned to -CH2 groups and terminal -CH3 groups of alkyl chains. EPR data supported ordering effect of genistein of the hydrophobic core in the liquid-crystalline phase (Lalpha). The analysis of the FTIR spectra of the two-component liposomes showed that genistein incorporates into DPPC membranes via hydrogen bonding between the lipid polar head groups in the C-O-P-O-C segment and its hydroxyl groups. Both fluorescence microscopy and ultrastructural observation revealed changes in membranous structures organization as aftermath of genistein treatment. In conclusion, genistein localized within membranes changes the properties of membrane that can be followed by the changes inside cells being crucial for pharmacological activity of genistein used in cancer or other disease treatment.

The study of the quercetin action on human erythrocyte membranes.

Quercetin is a naturally occurring flavonoid that exerts multiple pharmacological effects. In our previous study, we showed that quercetin greatly affects the lipid membrane. In this report, a study of quercetin on human erythrocyte membrane has been performed to determine the influence of this flavonoid on the fluidity and the conformational changes of membrane proteins. An additional aim of the study was to find how quercetin presence affects the resistance of membrane to haemolytic agents. The results showed that incorporation of quercetin into the erythrocyte membranes caused the changes of the partition coefficient of the Tempo spin label between the water and polar head group phases. In the studies, the W/S ratio has been used as a monitor of changes in protein conformation and in the environment within the membrane. It was observed that quercetin caused an increase in protein-protein interactions in human erythrocyte membranes. Haemolytic action of quercetin in the dark was also investigated. This compound showed protective effect against hypotonic haemolysis. However, in the heat-induced haemolysis quercetin caused acceleration of haemolysis. Dark reaction of erythrocyte with quercetin resulted in a shrinkage of the cells and alteration of their shapes. From the results we have concluded that modification of erythrocyte membrane by quercetin proceeds via reaction with membrane lipids and proteins.