Alkylphospholipids (APLs) represent a new class of drugs which do not interact directly with DNA but act on the cell membrane where they accumulate and interfere with lipid metabolism and signalling pathways. This review summarizes the mode of action at the molecular level of these compounds. In this sense, a diversity of mechanisms has been suggested to explain the actions of clinically-relevant APLs, in particular, in cancer treatment. One consistently reported finding is that APLs reduce the biosynthesis of phosphatidylcholine (PC) by inhibiting the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CT). APLs also alter intracellular cholesterol traffic and metabolism in human tumour-cell lines, leading to an accumulation of cholesterol inside the cell. An increase in cholesterol biosynthesis associated with a decrease in the synthesis of choline-containing phospholipids and cholesterol esterification leads to a change in the free-cholesterol:PC ratio in cells exposed to APLs. Akt phosphorylation status after APL exposure shows that this critical regulator for cell survival is modulated by changes in cholesterol levels induced in the plasma membrane by these lipid analogues. Furthermore, APLs produce cell ultrastructural alterations with an abundant autophagic vesicles and autolysosomes in treated cells, indicating an interference of autophagy process after APL exposure. Thus, antitumoural APLs interfere with the proliferation of tumour cells via a complex mechanism involving phospholipid and cholesterol metabolism, interfere with lipid-dependent survival-signalling pathways and autophagy. Although APLs also exert antiparasitic, antibacterial, and antifungal effects, in this review we provide a summary of the antileishmanial activity of these lipid analogues. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

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The alkylphospholipid analog miltefosine (hexadecylphosphocholine) is a membrane-directed antitumoral and antileishmanial drug belonging to the alkylphosphocholines, a group of synthetic antiproliferative agents that are promising candidates in anticancer therapy. 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 miltefosine and other alkylphospholipids on the human hepatoma HepG2 cell line, with a special emphasis on lipid metabolism. Results obtained in our laboratory indicate that miltefosine displays cytostatic activity and causes apoptosis in HepG2 cells. Likewise, treatment with miltefosine produces an interference with the biosynthesis of phosphatidylcholine via both CDP-choline and phosphatidylethanolamine methylation. With regard to sphingolipid metabolism, miltefosine hinders the formation of sphingomyelin, which promotes intracellular accumulation of ceramide. We have demonstrated for the first time that treatment with miltefosine strongly impedes the esterification of cholesterol and that this effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to higher levels of cholesterol in the cells. Indeed, miltefosine early impairs cholesterol transport from the plasma membrane to the endoplasmic reticulum, causing a deregulation of cholesterol homeostasis. Similar to miltefosine, other clinically-relevant synthetic alkylphospholipids such as edelfosine, erucylphosphocholine and perifosine show growth inhibitory effects on HepG2 cells. All the tested alkylphospholipids also inhibit the arrival of plasma-membrane cholesterol to the endoplasmic reticulum, which induces a significant cholesterogenic response in these cells, involving an increased gene expression and higher levels of several proteins related to the pathway of biosynthesis as well as the receptor-mediated uptake of cholesterol. Thus, membrane-targeted alkylphospholipids exhibit a common mechanism of action through disruption of cholesterol homeostasis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine and sphingomyelin biosyntheses certainly alter the ratio of choline-bearing phospholipids to cholesterol, which is critical for the integrity and functionality of specific membrane microdomains such as lipid rafts. Alkylphospholipid-induced alterations in lipid homeostasis with probable disturbance of the native membrane structure could well affect signaling processes vital to cell survival and growth.

Background and purpose:  Alkylphospholipid (APL) analogues constitute a new class of synthetic anti‐tumour agents that act directly on cell membranes. We have previously demonstrated that hexadecylphosphocholine (HePC) alters intracellular cholesterol traffic and metabolism in HepG2 cells. We now extended our studies to analyse the effects of other clinically relevant APLs, such as edelfosine, erucylphosphocholine and perifosine on intracellular cholesterol homeostasis.Experimental approach:  Using radiolabelled substrates we determined the effect of APLs on cholesterol metabolism and cholesterol traffic from the plasma membrane to the endoplasmic reticulum (ER). Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by Western blot and RT‐PCR respectively. Membrane raft and non‐raft fractions were isolated from HepG2 cells by a detergent‐free method.Key results:  All APLs inhibited the transport of cholesterol from the plasma membrane to the ER, which induced a significant cholesterogenic response in HepG2 cells. This response involved an increased gene expression and higher levels of several proteins related to the biosynthesis and the receptor‐mediated uptake of cholesterol. Cell exposure to the APL‐representative HePC enhanced the content of cholesterol mainly in the membrane raft fractions, compared with the untreated cells.Conclusions and implications:  Membrane‐targeted APLs exhibited a novel and common mechanism of action, through disruption of cholesterol homeostasis, which in turn affected specific lipid microdomains of cellular membranes.

Hexadecylphosphocholine (HePC) is a synthetic lipid representative of a new group of antiproliferative agents, alkylphosphocholines (APC), which are promising candidates in anticancer therapy. Thus we have studied the action of HePC on the human hepatoblastoma cell line HepG2, which is frequently used as a model for studies into hepatic lipid metabolism. Non‐toxic, micromolar concentrations of HePC exerted an antiproliferative effect on this hepatoma cell line. The incorporation into phosphatidylcholine (PC) of the exogenous precursor [methyl‐¹⁴C]choline was substantially reduced by HePC. This effect was not due to any alteration in choline uptake by the cells, the degradation rate of PC or the release of PC into the culture medium. As anaccumulation of soluble choline derivatives points to CTP:phosphocholine cytidylyltransferase (CT) as the target of HePC activity we examined its effects on the different enzymes involved in the biosynthesis of PC via CDP–choline. Treatment with HePC altered neither the activity of choline kinase (CK) nor that of diacylglycerol cholinephosphotransferase (CPT), but it did inhibit CT activity in HepG2 cells. In vitro HePC also inhibited the activity of cytosolic but not membrane‐bound CT. Taken together our results suggest that HePC interferes specifically with the biosynthesis of PC in HepG2 cells by depressing CT translocation to the membrane, which may well impair their proliferation.