**4.1 Influence of Perifosine (OPP) on cell membrane fluidity as studied by EPR**

To get the information about the influence of a biologically active substance on cell membrane by EPR it is necessary to introduce a lipophilic paramagnetic probe into the membrane bilayer. This so called spin probe serves as a marker, which reflects the motion of the alkyl chains in the vicinity of the nitroxide group of the spin probe and in this way gives information about its surrounding. Motional characteristics that determine membrane fluidity are reflected in the EPR spectra line-shape. Main parameters obtained directly from the line-shape of the EPR spectra are order parameter (S) and correlation time (τc). Order parameter describes the orientational order of the phospholipids alkyl chains with S = 1 for perfectly ordered chains and S = 0 for isotropic alignment, and rotational correlation time (τc) describes the dynamics of the spin probe motion; more fluid membranes are characterized by a small τc. The changes in the EPR spectra line-shape give direct information about the external influences (temperature, interactions, damages) on cell membrane fluidity. More exact information about the membrane alterations can be obtained by computer simulation of the EPR spectra taking into account that the membrane is heterogeneous, composed of several coexisting domains with different fluidity characteristics. Therefore the EPR spectrum is composed of several spectral components reflecting different modes of restricted rotational motion of the spin probe molecules in different membrane environments (Pabst et al., 2007; Stopar et al., 2006; Strancar et al., 2003; Strancar et al., 2005).

In order to see how Perifosine (OPP) influences the plasma membrane fluidity of ER+ MCF7 and ER- MT-3 breast cancer cells, the cells were labeled with the spin probe 5P. This is a spin labeled OPP (5P), containing the nitroxide group at the 5th C atom (counting from the polar head group), (Mravljak et al., 2005). Structural formula of 5P is shown in Fig. 3.

Merida, 2007; van Dijk et al., 1997; Yang & Kazanietz, 2003). Alkylphospholipids also inhibit DAG formation by phospholipase C (Maly et al., 1995; Ruiter et al., 2001; Strassheim et al., 2000). Alkylphospholipids also activate the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) pathway responsible for apoptosis in tumor cells (Gajate et al., 1998; Nieto-Miguel et al., 2007; Nieto-Miguel et al., 2008; Nieto-Miguel et al., 2006; Ruiter et al., 2001; Ruiter et al., 1999). Apoptosis can be triggered by imbalance between apoptotic and survival signals (Ruiter et al., 2001; Ruiter et al., 1999), which can be influenced by alkylphospholipids, since they have an influence on cross-talk between several membrane

In this chapter the investigation of the interaction of Perifosine (octadecyl(1,1-di-methyl-4 piperidinium-4-yl)phosphate – OPP), with ER positive (ER+) and ER negative (ER-) breast cancer cell lines is emphasized. Perifosine was chosen since it is one of the most cancerostatically active lipids, with strong antitumor effect on xenotransplanted human breast cancer. We summarize results obtained mostly by the electron paramagnetic resonance (EPR) method in order to measure the influence of Perifosine on cell membrane fluidity and to measure transport of free and liposome incorporated Perifosine into breast

**4.1 Influence of Perifosine (OPP) on cell membrane fluidity as studied by EPR** 

To get the information about the influence of a biologically active substance on cell membrane by EPR it is necessary to introduce a lipophilic paramagnetic probe into the membrane bilayer. This so called spin probe serves as a marker, which reflects the motion of the alkyl chains in the vicinity of the nitroxide group of the spin probe and in this way gives information about its surrounding. Motional characteristics that determine membrane fluidity are reflected in the EPR spectra line-shape. Main parameters obtained directly from the line-shape of the EPR spectra are order parameter (S) and correlation time (τc). Order parameter describes the orientational order of the phospholipids alkyl chains with S = 1 for perfectly ordered chains and S = 0 for isotropic alignment, and rotational correlation time (τc) describes the dynamics of the spin probe motion; more fluid membranes are characterized by a small τc. The changes in the EPR spectra line-shape give direct information about the external influences (temperature, interactions, damages) on cell membrane fluidity. More exact information about the membrane alterations can be obtained by computer simulation of the EPR spectra taking into account that the membrane is heterogeneous, composed of several coexisting domains with different fluidity characteristics. Therefore the EPR spectrum is composed of several spectral components reflecting different modes of restricted rotational motion of the spin probe molecules in different membrane environments (Pabst et al., 2007; Stopar et al., 2006; Strancar et al., 2003;

In order to see how Perifosine (OPP) influences the plasma membrane fluidity of ER+ MCF7 and ER- MT-3 breast cancer cells, the cells were labeled with the spin probe 5P. This is a spin labeled OPP (5P), containing the nitroxide group at the 5th C atom (counting from the polar

head group), (Mravljak et al., 2005). Structural formula of 5P is shown in Fig. 3.

**4. Interaction of free Perifosine (OPP) with breast cancer cells** 

dependent signaling pathways.

cancer cells.

Strancar et al., 2005).

It enters the plasma membrane easily but due to its charge, it only slowly crosses from outer to inner side of cell membrane, therefore it is suitable spin probe for detecting changes in the properties of the outer layer of plasma membrane. For spin labeling of cell membranes, MCF7 and MT-3 cells were mixed with 5P (2 M) as a spin probe and with different amounts of Perifosine (OPP) to achieve final concentrations of OPP in extracellular medium: 0 µM, 25 µM, 50 µM or 150 µM.

Fig. 3. Structural formulas of spin probes. MeFASL(10,3) – 5-doxylpalmitoyl methylester; HFASL(10,3) – 5-doxyl-palmitic acid, ASL - spin labeled tempocholine; 5P – spin labeled OPP, containing the doxyl group at the 5th C atom (counting from the polar head group).

From the spectra the order parameter was calculated. In the absence of OPP, S = 0.66 for MT-3 and S = 0.68 for MCF7 cells were obtained. After addition of 150 µM OPP, the order parameter decreased to S = 0.57 for MT-3 and to 0.60 for MCF7 cells. At lower concentrations of Perifosine no significant differences in order parameter were observed. This result indicates that OPP increases membrane fluidity of both cell lines at concentrations higher than 50 µM. The influence of OPP is less pronounced for MCF7 as for MT-3 cells. This indicates that OPP either doesn't incorporate into the alkylphospholipid resistant, ER+ MCF7 cell membranes as well as into alkylphospholipid sensitive, ER- MT-3 cells, or it doesn't concentrate in plasma membrane of MCF7 cells at such high concentrations as it does in MT-3 cells.
