Antitumoral alkylphospholipids alter cell lipid metabolism.

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.

Lipid efflux mediated by alkylphospholipids in HepG2 cells.

Antitumoural alkylphospholipid (APL) analogues alter cholesterol homoeostasis in HepG2 cells by interfering with cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and at the same time stimulating the release of considerable quantities of membrane cholesterol. The capacity of APLs to stimulate cholesterol efflux is suppressed when cells are incubated simultaneously with APLs and serum whilst the inhibition of cholesterol transport to the ER (measured in terms of the synthesis of esterified cholesterol) persists, indicating that both effects are independent of each other. Interestingly, our results suggest that both raft and non-raft membrane domains contribute to the cholesterol released to APLs. In addition, a marked efflux of choline-bearing phospholipids (phosphatidylcholine (PC) and sphingomyelins (SM)) was found to be related to this release of cholesterol. Finally, we observed that APL micelles composed of cholesterol might act as donor/acceptor cholesterol systems. Thus, the findings of this study clearly demonstrate that antitumoural APLs act as extracellular acceptors, stimulating cholesterol and phospholipid efflux, although they may also play a role as cholesterol donors.

Design, synthesis, theoretical calculations and biological evaluation of new non-symmetrical choline kinase inhibitors.

Inhibition of Choline Kinase (ChoK) has been reported as a therapeutical target in the treatment of some kinds of tumor. In this paper, the design and synthesis of new non-symmetrical monocationic ChoK inhibitors is described, bearing a cationic head and an adenine moiety connected by linkers of different lengths. Docking studies indicate that the cationic head of these compounds could be inserted into the choline binding site of the enzyme, while the adenine moiety could be stabilized into the ATP binding site. Docking studies also support the difference of activity of the synthesized compounds, which depends on both the substituent at position 4 of the cationic head and the linker length, being dimethylamine and 1,4-diphenylbutane respectively, the most appropriate ones. Compounds 14 (IC(50) = 10.70 ± 0.40 µM) and 17 (IC(50) = 6.21 ± 0.97 µM) are the most potent ChoK inhibitors and suitable for further modification with a view to obtain more potent antitumor compounds.

Antitumoral alkylphospholipids induce cholesterol efflux from the plasma membrane in HepG2 cells.

Alkylphospholipid (APL) analogs are promising candidates in the search for treatments of cancer. Previous studies conducted in our laboratory indicate that, after prolonged treatment, they alter cholesterol homeostasis in HepG2 cells. Here we describe the effects that different APLs exert upon this cell line after a 1-h exposure in a serum-free medium, including 1) a rapid, significant increase in cholesterol efflux into the extracellular medium, which consequently provoked a depletion of cholesterol in the plasma membrane (further assays conducted in an attempt to return to control cholesterol levels were only partially successful); 2) use of methyl-ß-cyclodextrin, which indicated that APLs acted in a way similar to this agent that is used frequently to modulate membrane cholesterol levels; 3) the phosphorylation of Akt that showed that this critical regulator for cell survival was modulated by changes in cholesterol levels induced in the plasma membrane by APLs; and 4) membrane cholesterol depletion that is not related to the impairment of cholesterol traffic produced by APLs. Thus, we have found that antitumoral APLs efficiently deplete membrane cholesterol, which may be one important factor in determining the early biological actions of APLs.

Disruption of cellular cholesterol transport and homeostasis as a novel mechanism of action of membrane-targeted alkylphospholipid analogues.

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.

Alterations in the homeostasis of phospholipids and cholesterol by antitumor alkylphospholipids.

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.

Hexadecylphosphocholine alters nonvesicular cholesterol traffic from the plasma membrane to the endoplasmic reticulum and inhibits the synthesis of sphingomyelin in HepG2 cells.

The synthetic lipid analogue, hexadecylphosphocholine is an antitumoral and antileishmanial agent that acts on cell membranes and can induce apoptosis. We have previously investigated the effect of hexadecylphosphocholine on the biosynthesis and intracellular transport of cholesterol in the human hepatoma HepG2 cell line. Here we show that the traffic of endocytosed-cholesterol from LDL to the plasma membrane and the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane were unaffected by alkylphosphocholine exposure. On the contrary, cholesterol traffic from the plasma membrane to the endoplasmic reticulum was drastically interrupted after 1 h of cell exposition to HePC and, consequently, the intracellular esterification of cholesterol was substantially decreased. Our results also demonstrate that this alkylphosphocholine exclusively affected the nonvesicular, energy-independent cholesterol traffic, without altering the vesicular transport. In addition, hydrolysis of plasma membrane sphingomyelin by exogenously added sphingomyelinase resulted in enhanced plasma-membrane cholesterol esterification, but sphingomyelinase treatment did not prevent the inhibition in cholesteryl ester formation caused by hexadecylphosphocholine. We also found that sphingomyelin synthesis was significantly inhibited in HepG2 cells after exposure to hexadecylphosphocholine. Since sphingomyelin and cholesterol are major lipid constituents of membrane raft microdomains, these results suggest that hexadecylphosphocholine could disturb membrane raft integrity and thence its functionality.

Hexadecylphosphocholine interferes with the intracellular transport of cholesterol in HepG2 cells.

We have shown, in a previous publication, that nontoxic concentrations of hexadecylphosphocholine exert an antiproliferative effect on HepG2 cells. Hexadecylphosphocholine also interferes with the biosynthesis of cholesterol and phosphatidylcholine. We have now extended our studies to try to establish the molecular mechanism by which hexadecylphosphocholine disrupts cholesterol homeostasis. Using radiolabelled substrates we determined the effect of hexadecylphosphocholine on cholesterol synthesis, the destiny of cholesterol from low-density lipoprotein and the transport of cholesterol between the plasma membrane and the endoplasmic reticulum. Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by western blotting and RT-PCR, respectively. HepG2 cells exposed to hexadecylphosphocholine showed an increase in cholesterol biosynthesis when acetate, but not mevalonate, was used as a substrate. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) and low-density lipoprotein receptor, as well as the corresponding mRNA expression, increased after 24 h of treatment with hexadecylphosphocholine. Cholesteryl linoleate in low-density lipoprotein uptake and further hydrolysis of these esters increased but the cholesterol esterification was reduced after 6 h of treatment with alkylphosphocholine. Cholesterol transport from the plasma membrane to the endoplasmic reticulum was impaired by hexadecylphosphocholine. In conclusion, hexadecylphosphocholine interfered with the transport of cholesterol from the cell surface to the endoplasmic reticulum, leading to a depletion of cholesterol in the endoplasmic reticulum and a deregulation of cholesterol biosynthesis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine synthesis produces an alteration in the phosphatidylcholine/cholesterol ratio that may well be responsible for the antiproliferative activity exhibited by hexadecylphosphocholine in HepG2 cells.

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes.

We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA.

Hexadecylphosphocholine disrupts cholesterol homeostasis and induces the accumulation of free cholesterol in HepG2 tumour cells.

Hexadecylphosphocholine (HePC) is a synthetic lipid belonging to the alkylphosphocholines (APC), a new group of antiproliferative agents that are proving to be promising candidates in anticancer therapy. We reported in a previous study that HePC interferes with phosphatidylcholine (PC) synthesis in HepG2 cells via both CDP-choline and phosphatidylethanolamine (PE) methylation. We have subsequently extended our studies to show that HePC interferes with sphingolipid metabolism by hindering the formation of sphingomyelin (SM), an effect accompanied by a substantial increase in the incorporation of the exogenous lipogenic precursors into ceramides. Interestingly, we demonstrate for the first time that HePC strongly inhibits the esterification of free cholesterol (FC) by acting at the level of acyl CoA:cholesterol acyltransferase (ACAT) (EC 2.3.1.26) activity. This effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to a rise in the levels of FC in cells. We are left in no doubt that the imbalance in the metabolism of membrane-lipid components vital to cell survival may well be responsible for the observed DNA fragmentation and activation of caspase-3, an enzyme involved in the cell apoptosis found in this study.

Ethanol specifically alters the synthesis, acylation and transbilayer movement of aminophospholipids in rat-liver microsomes.

By experimenting with the aminoalcohols [3-3H]serine and [2-14C]ethanolamine we have been able to relate the effects of ethanol upon the biosynthesis of radioactive aminophospholipids (APL) in rat-liver microsomes and their distribution within the bilayer. The translocation of newly synthesized molecules of aminophospholipids labeled with different fatty acids was also investigated. The synthesis of phosphatidylserine (PS) and phosphatidylethanolamine (PE) by base-exchange reaction (BES) was inhibited in membranes exposed to ethanol in direct response to its concentration. In addition, 100 mM ethanol specifically inhibited the transport of newly synthesized PS to the inner leaflet, resulting in similar levels of PS in both leaflets of the bilayer. The inhibition of PE synthesis by ethanol caused a decrease in its distribution in both inner and outer leaflets. An in vitro study of the incorporation of radioactive palmitate and oleate into the PS and PE of microsomes incubated with ethanol showed a decrease in the radioactivity levels of PE, suggesting that ethanol was specifically inhibiting the corresponding acyltransferase. It specifically altered the transbilayer movement of newly acylated phospholipids, modifying the distribution of palmitoyl- and oleoyl-acylated PS and PE in both leaflets. These results demonstrate for the first time that ethanol interferes with both the synthesis and intramembrane transport of aminophospholipids in endoplasmic reticulum (ER) membranes. Bearing in mind that if a membrane is to function properly its structure must be in optimum condition; it is evident that the observed processes may be responsible to some degree for the pathophysiological effects of alcohol upon cells.

Hexadecylphosphocholine inhibits phosphatidylcholine synthesis via both the methylation of phosphatidylethanolamine and CDP-choline pathways in HepG2 cells.

We reported in a recent publication that hexadecylphosphocholine (HePC), a lysophospholipid analogue, reduces cell proliferation in HepG2 cells and at the same time inhibits the biosynthesis of phosphatidylcholine (PC) via CDP-choline by acting upon CTP:phosphocholine cytidylyltransferase (CT). We describe here the results of our study into the influence of HePC on other biosynthetic pathways of glycerolipids. HePC clearly decreased the incorporation of the exogenous precursor [1,2,3-3H]glycerol into PC and phosphatidylserine (PS) whilst increasing that of the neutral lipids diacylglycerol (DAG) and triacylglycerol (TAG). Interestingly, the uptake of L-[3-3H]serine into PS and other phospholipids remained unchanged by HePC and neither was the activity of either PS synthase or PS decarboxylase altered, demonstrating that the biosynthesis of PS is unaffected by HePC. We also analyzed the water-soluble intermediates and final product of the CDP-ethanolamine pathway and found that HePC caused an increase in the incorporation of [1,2-14C]ethanolamine into CDP-ethanolamine and phosphatidylethanolamine (PE) and a decrease in ethanolamine phosphate, which might be interpreted in terms of a stimulation of CTP:phosphoethanolamine cytidylyltransferase activity. Since PE can be methylated to give PC, we studied this process further and observed that HePC decreased the synthesis of PC from PE by inhibiting the PE N-methyltransferase activity. These results constitute the first experimental evidence that the inhibition of the synthesis of PC via CDP-choline by HePC is not counterbalanced by any increase in its formation via methylation. On the contrary, in the presence of HePC both pathways seem to contribute jointly to a decrease in the overall synthesis of PC in HepG2 cells.

Hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis and the proliferation of HepG2 cells.

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.

Resistance of HepG2 cells against the adverse effects of ethanol related to neutral lipid and phospholipid metabolism.

The influence of both short- and long-term ethanol exposure on the lipid metabolism was determined in the human hepatoma cell line HepG2. Ethanol did not cause any cytotoxicity or lipid peroxidation even after 7 days of 100 mM ethanol treatment of HepG2 cells. Incubation of cells in the presence of [1-(14)C]ethanol demonstrated that these cells actively metabolize ethanol to acetyl CoA, incorporating the radioactive label into neutral lipids and phospholipids. [1,2,3-(3)H]glycerol was efficiently used in phospholipid and neutral lipid biosynthesis, showing higher radioactivity in phosphatidylcholine, phosphatidylethanolamine and triacylglycerols. Exposure of HepG2 cells to 100 mM ethanol for 24 hr did not significantly modify the incorporation of glycerol into newly synthesized phospholipids and neutral lipids, nor was lipid degradation affected by the presence of ethanol. When the alcohol treatment was prolonged for 7 days, incorporation of [1,2,3-(3)H]glycerol into triacylglycerols and diacylglycerols showed a slight increase concomitantly with decreased radioactivity in the major phospholipids, phosphatidylcholine and phosphatidylethanolamine. In addition, these changes were associated with a greater release of radiolabeled triacylglycerols into the culture medium. These results indicate that ethanol does not cause in HepG2 cells the marked lipogenic stimulation widely shown in hepatocytes, and demonstrate that HepG2 cells strongly resist the adverse effects of ethanol. Since these cells lack the isoenzymatic form of cytochrome P(450) mainly involved in the ethanol metabolism (namely cytochrome P(450)2E1) and also are devoid of alcohol dehydrogenase activity, we propose that the toxic actions of ethanol on liver must be linked to the activity of one or both of these systems.