**3.2 Inhibition of phosphatidylcholine biosinthesis**

Inhibition of phosphatidylcholine (PC) biosynthesis is a major alkylphospholipid target (Fig. 2). Inhibition of the biosynthesis of PC causes stress on cells sufficient to trigger apoptosis. In the endoplasmic reticulum, alkylphospholipids inhibit CTP (phosphocholine cytidyltransferase, CT), which chatalyses the rate-limiting step of the *de novo* PC synthesis. Alkylphospholipids inhibit CT in all exponentially growing tumor and normal cells, including leukemic and endothelial cells (Zerp et al., 2008). Synthesis of PC is essential for cell proliferation and is upregulated in tumor cells. PC is not only the most abundant membrane lipid and crucial for new membrane formation, but also the precursor for the second messengers diacylglycerol (DAG) and phosphatidic acid (PA) and for

Anti-cancer mechanisms of alkylphospholipids have been described and extensively discussed in some recent reviews (Danker et al., 2010; Gajate & Mollinedo, 2002; Gills & Dennis, 2009; van Blitterswijk & Verheij, 2008). Early interest focussed on immune stimulating activity of alkylphospholipids. It could be demonstrated that Miltefosine and other lipids of this class are able to activate T-cells and macrophages to express and release chemokines like GM-CSF (Vehmeyer et al., 1992), IFgamma (Hochhuth et al., 1992) and/or nitric oxide (NO) (Zeisig et al., 1995). This effect could be improved if the alkylphospholipids were used in liposomal form. Because of their amphiphilic structure, alkylphospholipids are able to form lamellar bilayers, if combined with lipids of opposite molecular shape. Liposomes were taken up by macrophages much better than the free, micellar lipids and induced, after cellular uptake, the release of IF gamma and NO (Eue et al., 1995). But their potency as immune stimulator was limited and not sufficient enough amount of chemokines was released for a complete inhibition of tumor cell proliferation.

Due to their amphiphilic nature alkylphospholipids are easily incorporated into cell membranes in substantial amounts and then spread among intracellular membrane compartments, where they accumulate and interfere with a wide variety of key enzymes (Unger et al., 1992; van Blitterswijk et al., 1987). At lower, clinically relevant concentrations alkylphospholipids interfere with phospholipid turnover and lipid-based signal transduction pathways. In mouse S49 lymphoma cells alkylphospholipids accumulate in detergent-resistant, sphingolipid- and cholesterol-enriched lipid raft domains and are rapidly internalized by clathrin-independent, raft-mediated endocytosis (van der Luit et al., 2007). Alkylphospholipid uptake in KB carcinoma cells, however, appears to be raftindependent and mediated by a yet unidentified ATP-dependent lipid transporter (Vink et al., 2007). In leukemic cells treatment with alkylphospholipids induces the formation of membrane raft aggregates containing Fas/CD95 death receptor and the adaptor molecule Fas-associated death domain-containing protein (FADD), which are critical in the triggering of apoptosis (Gajate et al., 2009). Miltefosine and other alkylphospholipids also alter intracellular cholesterol traffic and metabolism leading to an increased uptake, synthesis and accumulation of cholesterol in the cell (Carrasco et al., 2008; Jimenez-Lopez et al., 2006; Marco et al., 2009). As cholesterol and sphingomyelin content are critical for the integrity and functionality of membrane lipid rafts, the disturbance of the cholesterol/sphingomyelin

ratio could alter signaling pathways associated with these membrane domains.

Inhibition of phosphatidylcholine (PC) biosynthesis is a major alkylphospholipid target (Fig. 2). Inhibition of the biosynthesis of PC causes stress on cells sufficient to trigger apoptosis. In the endoplasmic reticulum, alkylphospholipids inhibit CTP (phosphocholine cytidyltransferase, CT), which chatalyses the rate-limiting step of the *de novo* PC synthesis. Alkylphospholipids inhibit CT in all exponentially growing tumor and normal cells, including leukemic and endothelial cells (Zerp et al., 2008). Synthesis of PC is essential for cell proliferation and is upregulated in tumor cells. PC is not only the most abundant membrane lipid and crucial for new membrane formation, but also the precursor for the second messengers diacylglycerol (DAG) and phosphatidic acid (PA) and for

**3. Mode of action of APL** 

**3.1 Uptake and absorption of alkylphospholipids** 

**3.2 Inhibition of phosphatidylcholine biosinthesis** 

sphingomyelin (SM) in membrane lipid rafts. Inhibition of PC biosynthesis blocks the downstream sphingomyeline synthase (SMS) that catalyzes synthesis of sphingomyelin and diacylglycerol in the trans-Golgi (van Blitterswijk et al., 2003). Possible consequence is the accumulation of the ceramide, which is a second SMS substrate and can trigger apoptosis (Wieder et al., 1998). Another consequence of the PC shortage is the oxidative stress with reactive oxygen species (ROS) formation (Smets et al., 1999; Vrablic et al., 2001; Wagner et al., 1993).

Fig. 2. Alkylphospholipid targets in lipid metabolism and signalling pathways summarized after van Blitterswijk et al. (van Blitterswijk & Verheij, 2008).
