Том 14, № 4 (2014)

The Langmuir monolayer technique is one of the methods used to build models of cellular membranes and enables to investigate the interactions of membrane components with other biomolecules. This method has been applied to study the effect of edelfosine - a synthetic alkyl-lysophospholipid analog - on model lipid membranes in order to get insight into its mode of action and selectivity. Edelfosine is mainly known for its anticancer properties, although it is also applied in the treatment of other diseases, like autoimmune, anti-HIV and antiparasitic. In this review we focus on its antitumor activity (although some other aspects of its therapeutic effects are also indicated) and summarize the results obtained so far with use of the monolayer technique. The application of this method evidenced for a key role of cholesterol and membrane rafts in the mechanism of anticancer activity of edelfosine. As regards the selectivity of this drug, the obtained results proved that the difference in fluidity of tumor versus normal cell membrane is important but probably not the only factor determining an easier incorporation of edelfosine into cancer cells. Further studies show that edelfosine is of strong affinity to gangliosides, which may be considered as molecules targeting edelfosine into cancer cell membrane.

A fluorescent analog of ET-18-OCH3, 1-O-(7'-N,N-dimethylamino-3'-pentadecanoyl-1'-naphthyl)-2-O-methyl-sn-glycerophosphocholine (1), was synthesized and its bioactivity was screened against 12 human cancer cell lines. The bioactivity of 1 was found to differ markedly from that of ET-18-OCH3. Growth of two prostate cell lines (PC3 and DU145) and a glioma cell line (U251) was significantly affected by 1, with IC50 values of 2, 6, and 12 µM, respectively. Compound 1 was cytotoxic to PC3 cells by caspasedependent apoptosis. The subcellular distribution of 1 differed from that reported for a phenyl-polyene analog of ET-18-OCH3; 1 was found to be localized in the endoplasmic reticulum, mitochondria, and lysosomes but not in the plasma membrane or nucleus of PC3 cells. However, no differences in accumulation of 1 were found between PC3 and cells that were not affected by the compound, implying that the selective PC3 cytotoxicity is a consequence of specific molecular components of PC3 cells.

Alkylphospholipid (APL) analogues are promising candidates in the search for treatments for cancer. In contrast to standard chemotherapeutic drugs, these lipophilic agents target the cell membrane without interacting directly with DNA. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of clinically-relevant APLs, such as hexadecylphosphocholine (HePC), edelfosine, erucylphosphocholine (ErPC) and perifosine on the human hepatoma HepG2 cell line, which is commonly used for lipid metabolism studies with a special emphasis on cholesterol metabolism. One consistent finding is that HePC and other APLs cause a reduction in the biosynthesis of phosphatidylcholine (PC) by inhibiting the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CT). Our research group has been at the forefront in demonstrating that exposure to APLs affects cholesterol homeostasis in mammalian cells. Treatment with HePC, for example, causes a marked enhancement in cholesterol synthesis, which has been related to an impairment in the arrival of cholesterol at the endoplasmic reticulum (ER). In a similar way to HePC, edelfosine, ErPC and perifosine increase the de novo synthesis and uptake of cholesterol and also inhibit the arrival of plasma-membrane cholesterol at the ER, which induces a significant cholesterogenic response in these cells, involving an increase in gene expression and higher levels of several proteins related to the biosynthetic pathway and receptor-mediated uptake of cholesterol. It is generally accepted nowadays that the maintenance of a tightly controlled free-cholesterol/PC ratio is crucial to optimum cell behaviour and that alterations to this ratio may lead to necrosis and/or apoptosis. Our results have considerable bearing on this idea because an increase in cholesterol biosynthesis associated with a decrease in the synthesis of choline-containing phospholipids and cholesterol esterification leads to a modification in the free-cholesterol/PC ratio in cells exposed to APLs. It is well accepted that cholesterol is critical for the formation of lipid rafts and therefore drugs that alter cell cholesterol content should modify the properties of these membrane domains and consequently the signal-transduction pathways, which depends upon lipid-raft integrity. Results on the whole show that APLs share a common active mechanism consisting of disrupting PC and sphingomyelin (SM) biosyntheses and cholesterol homeostasis, all of which leads to a disturbance in the native membrane structure, thus affecting signaling processes vital to cell survival and growth.

Integrin-dependent adhesion of tumor cells to extracellular matrix proteins provides anchorage-dependent protection from cell death. In the present investigation we aimed to understand whether and how the paradigmatic membrane-targeted synthetic phospholipid analog erufosine is relevant for tumor cell adhesion to extracellular matrix proteins, cell survival and migration. The antineoplastic action of erufosine was analyzed with glioblastoma and prostate cancer cells adhering to fibronectin or collagen I using proliferation, adhesion and migration assays. The composition of adhesion contacts containing activated β1 integrins was studied using immunofluorescence. The importance of β1 integrins for the observed effects was analyzed in fibroblasts proficient or deficient in β1 integrin expression. Adhesion to collagen I and fibronectin increased the death threshold in serum-deprived tumor cells. Moreover, β1 integrin-deficient cells were more sensitive to erufosine-treatment compared to β1 integrin proficient cells suggesting a role of β1 integrins for matrix-mediated death resistance. Most importantly, erufosine disturbed the maturation of the cell adhesion complexes containing paxillin, activated β1 integrins and phosphorylated FAK, leading to a reduction of survival signals and inhibition of tumor cell adhesion and migration. These findings suggest that membrane-targeted synthetic phospholipids analogs may be of value for counteracting matrix-mediated treatment resistance in combined treatment approaches with radiotherapy or chemotherapy.

Synthetic alkylphospholipids (APLs), exhibit similarity to the platelet-activating factor (PAF). These compounds have antiproliferative effects on tumour cells and can therefore be regarded as a new class of drugs. Unlike classic cytostatic agents, synthetic alkylphospholipids do not interfere with the DNA or the mitotic spindle apparatus. Instead, due to their aliphatic character, alkylphospholipids accumulate in cell membranes, where they have an impact on lipid metabolism and lipid-dependent signalling pathways which leads to inhibition of proliferation and induction of apoptosis in malignant cells. Normal cells remain unaffected by these compounds. Glycosidated phospholipids, are a novel class of alkylphospholipids, in which carbohydrates or carbohydrate-related molecules are introduced in the chemical lead of PAF. These hybrid alkylphospholipids also exhibit anti-proliferative capacity. Furthermore, members of this subfamily also modulate cell adhesion, differentiation, apoptosis and migration of tumour cells. Among the members of this group, Inositol-C2-platelet-activating factor (Ino-C2-PAF) is the most effective compound developed so far. Recently, we also showed that Ino-C2-PAF exhibited the strongest impact on the gene expression levels of immortalised keratinocytes in comparison to edelfosine and another glycosidated alkylphospholipid, Glucose-platelet-activating factor (Glc-PAF). Furthermore, Ino-C2-PAF reduced the expression of genes encoding proteins associated with inflammation and the innate and acquired immune responses.

Perifosine treatment exhibits a complex molecular response including the inhibition of Akt or the induction of apoptosis via clustering of death receptors in lipid rafts. However, the molecular response can vary between different tumor entities and the contribution of each target pathway to the activity of Perifosine might be distinct depending on the tumor entity or the agent combined with Perifosine. In this review we discuss the current view on the mechanism of action of perifosine in cancer and the contribution of the molecular targets of Perifosine to its activity.