**3.3 Influence of alkylphospholipids on major signaling pathways**

Alkylphospholipids interfere with phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB)/Akt survival pathway, which is important for proliferation, differentiation, survival and intracellular trafficking (Fig. 2). They inhibit phosphorylation and recruitment of PKB/Akt to the membrane, which is essential for its activation (Elrod et al., 2007; Kondapaka et al., 2003; Rahmani et al., 2005; Tazzari et al., 2008) probably by decreased production of PIP3 (Gills & Dennis, 2009; van Blitterswijk & Verheij, 2008).

Alkylphospholipids inhibit PC hydrolysis to phosphatidic acid (PA) by phospholipase D and further to diacylgycerol (DAG) (Kiss & Crilly, 1997; Lucas et al., 2001). PA and DAG are second messengers, essential for the mitogen-activated protein kinase (MAPK) pathways, which regulate mitosis, metabolism, survival, apoptosis and differentiation (Chen et al., 2001; Kyriakis & Avruch, 2001; Pearson et al., 2001). PA is also involved in the activation of protein kinase C-ζ (Limatola et al., 1994), mTOR (Fang et al., 2001) and c-Raf (Rizzo et al., 2000). DAG activates proteins with the C1 domain, such as protein kinases C and D, Ras guanine-releasing protein (RasGRP) and indirectly MAPK/ERK pathway (Carrasco &

Interaction of Alkylphospholipid Formulations with Breast Cancer

0 µM, 25 µM, 50 µM or 150 µM.

concentrations as it does in MT-3 cells.

**4.2 Transport of Perifosine (OPP) into the cell** 

Cells in the Context of Anticancer Drug Development 367

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:

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

To get information about the transport of Perifosine (OPP) into breast cancer cells by EPR, several spin labeled OPPs were synthesized in our group (Mravljak et al., 2005), which have an EPR detectable nitroxide group at various positions along the alkyl chain. We have chosen spin labeled OPP (5P) (structural formula in Fig. 3) with the lowest critical micellar concentration (CMC) of all synthesized spin labeled OPPs (Mravljak et al., 2005). Its CMC is around 10 μM, while 14P (spin labeled OPP, containing the nitroxide group at the 14th C atom) exhibited the CMC of around 200 μM (Mravljak et al., 2005). Edelfosine and OPP disperse in water in the form of micelles, due to their inverted-cone shape (Busto et al., 2007), displaying a CMC at 2.5 - 3 μM (Rakotomanga et al., 2004). It appears that addition of the doxyl group to OPP distorts the inverted-cone shape of molecule to greater extent when it is placed further away from the polar head group, which results in increased CMC. Since the CMC of 5P is similar to CMC of other similar alkylphospholipids one can assume that

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 dependent signaling pathways.
