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Aim: To test whether membrane fluidity and its changes are important for the sensitivity of cells to the action of perifosine (OPP), a new anticancer drug targeting cell membrane and not DNA., Method: Influence of OPP on the membrane structure of OPP-resistant MCF7, and OPP-sensitive MT3 breast cancer cell lines, as well as of mouse fibroblasts (L929) cell lines, and model cells (liposomes) was investigated by electron paramagnetic resonance, using spin labeled derivative of OPP (P5) and 5-doxylpalmitoyl methylester (MeFASL(10,3)) as spin probes., Results: OPP increased membrane fluidity of all cell lines at concentrations higher than 50 μM (on the level of P≤0.05, t test). In cells, the differences were observed only by P5 and not by MeFASL(10,3). Average order parameter Seff decreased for about 12% in MCF7 and L929 and only for 8% in OPP-sensitive MT3 cells, showing that there was no correlation between membrane fluidity changes and sensitivity of cells to OPP. The only correlation we found was between OPP sensitivity and the cell growth rate. In liposomes, both spin probes were sensitive to the action of OPP. Seff decreased with increasing concentration of OPP. For MeFASL(10,3) a significant decrease was observed at 4 mol% OPP, while for P5 it was observed at 8 mol%., Conclusion: Influence of OPP on plasma membrane fluidity of breast cancer cells is not the determining factor in the sensitivity of cells to OPP.

The effects of tricylic antidepressant clomipramine (CLO) on the membrane properties of saturated dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as well as on unsaturated egg yolk phosphatidylcholine liposomes were investigated by the electron paramagnetic resonance spin-labeling technique, in combination with the simulation of the spectra, taking into account that the membrane is heterogeneous and composed of the regions with different fluidity characteristics. Different spin labels, monitoring membrane properties in the upper and inner parts of the membrane, were used. In general, two spectral components, having different motional characteristics, were detected in all liposomes investigated. In liposomes with saturated chains, CLO decreased the phase-transition temperature, disordered the membrane, and increased polarity in the upper part of the membrane. However, less impact was observed in liposomes with unsaturated chains. In dipalmitoyl phosphatidylcholine liposomes, it also induced molecular rearrangements near the pretransition temperature. The presence of 30 mol% cholesterol increased the fluidizing effect of CLO and modified the lateral diffusion of nitroxide in the inner part of the membrane. A unique anomalous increase in diffusion of nitroxide, dependent on CLO concentration, was detected in the temperature region where the phosphatidylcholine membrane without cholesterol experiences the phase transitions. Since the changes in the central part of the membrane were even more pronounced than in the upper part of the membrane, it could be concluded that CLO incorporates into the membrane with its hydrophobic ring parallel to the phospholipid chains.

Standard methods of characterization of electron paramagnetic resonance (EPR) spectra of spin-labeled biomembranes limit the resolution of lateral heterogeneity to only two or three domain types. This disables examination of the structure-function relationship in complex membranes, which might be composed of a larger number of different domain types. To enable exploration of this kind, a new approach based on analysis of EPR spectra with multi-run, hybrid evolutionary optimization is proposed here. From the multiple runs a quasi-continuous distribution of membrane spectral parameters (order parameter, proportion of spectral component, polarity correction factor, rotational correlation time and broadening constant) can be constructed and presented by a new presentation technique CODE (colored distribution of E PR spectral parameters). Through this the concept of a "soft" picture of membrane heterogeneity is introduced, in contrast to the standard "discrete" domain picture. The "soft" characterization method, established on synthetic spectra, was used to examine the lateral heterogeneity of liposome membranes as well as of membranes of neutrophils from healthy and asthmatic horses. In liposome membranes the determined number of domain types was the same as already established by standard procedures of EPR spectra line-shape interpretation. In membranes of neutrophils a quasi-continuous distribution of membrane domain properties was detected by the new method.