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-β-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.
Alkylphospholipid (APL) analogs are a new class of antitumoral agents that do not target DNA but insert themselves into the plasma membrane and subsequently trigger a broad range of biological effects, which ultimately lead to cell death (Soto and Soto, 2006; Rakotomanga et al., 2007). An important characteristic of APLs is their amphiphilic properties, enabling them to interact with cell membranes and affect cell metabolism at different levels (reviewed by Jiménez-López et al., 2010). Within this context, studies carried out in our laboratory have demonstrated that, after long-term treatment, the APL hexadecyl 2-(trimethylazaniumyl)ethyl phosphate (hexadecylphosphocholine; HePC), also known as miltefosine, alters phosphatidylcholine metabolism (Jiménez-López et al., 2004) and intracellular cholesterol trafficking and metabolism, all of which lead to an increased uptake, synthesis, and accumulation of cholesterol in the cell (Jiménez-López et al., 2006; Carrasco et al., 2008; Marco et al., 2009). Thus, we extended our studies to analyze the effects of a variety of APLs, such as [1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine] (edelfosine), [(Z)-docos-13-enyl] 2-(trimethylazaniumyl)ethyl phosphate (erucylphosphocholine; ErPC), and 1,1-dimethylpiperidin-1-ium-4-yl) octadecyl phosphate (perifosine) upon intracellular cholesterol homeostasis and found that all of them impede the esterification of plasma-membrane cholesterol via acyl-CoA/cholesterol acyltransferase activity (Carrasco et al., 2010). This effect is caused by the disruption of cholesterol movement from the plasma membrane to the endoplasmic reticulum (ER), where it is esterified, which in turn induces a significant cholesterogenic response in HepG2 cells, involving increased gene expression and higher levels of several proteins involved in the pathway for the biosynthesis and receptor-mediated uptake of cholesterol.
Cell-cholesterol levels are the result of a balance among uptake, efflux, and endogenous synthesis. Many aspects of cholesterol metabolism are well known, including its uptake, its synthesis in the ER, and its regulation via sterol regulatory element-binding protein. Nevertheless, although widely investigated, some dispute remains on how cholesterol molecules move from the plasma membrane to extracellular acceptors. The generally recognized mechanism is now thought to involve the movement of cholesterol molecules from the cell membrane through the aqueous phase to the acceptor particle, that is to say, by an aqueous diffusion mechanism. Other mechanisms have also been proposed, but with the inclusion of collision or receptor mediation (Fielding and Fielding, 2001).
The concentration of cholesterol in the membrane is particularly high in lipid rafts, which have been reported to act as molecular platforms and play a significant role in many signaling cascades (Simons and Toomre, 2000) and in the activation of immune responses (Langlet et al., 2000). Recent studies with cultured cells have suggested that APLs may act on cell signal transduction by affecting the protein composition of these rafts. Thus, treatment of human leukemia cells with edelfosine results in the translocation of Fas into membrane rafts, and this apparently triggers apoptosis (Mollinedo et al., 2010). Another raft-dependent pathway shown to be an important target of APL is the phosphatidylinositol 3-kinase/Akt pathway, which is involved in cell growth, proliferation, and survival (Engel et al., 2008).
In the present work, we have extended our studies to analyze the effects of clinically relevant APLs upon cholesterol efflux in the human hepatoblastoma cell line HepG2, which is commonly used for lipid metabolism studies because it retains many liver-specific functions. Our objective was to analyze the initial effects of APLs upon cholesterol efflux into the medium, one of the most important processes contributing to the control of cell cholesterol levels in the hope that this would provide us with information about the early mechanism of APL activity on cholesterol homeostasis. We found that all of the APLs assayed, HePC, edelfosine, ErPC, and perifosine, stimulated the efflux of cholesterol from cells into the medium but that this could be mitigated by the addition of exogenous cholesterol. The cholesterol release provoked a depletion of cholesterol levels in the plasma membrane that can be related to the activation state of Akt.
Materials and Methods
Chemicals and Reagents.
Fetal bovine serum (FBS) was from The Cell Culture Company (Pasching, Austria). Minimal essential medium (MEM), cholesterol, water-soluble cholesterol, methyl-β-cyclodextrin (MβCD), and thin layer chromatography plates were from Sigma-Aldrich (Madrid, Spain). X-ray film was from Konica Minolta (Tokyo, Japan). [7(n)3_3H]cholesterol was from PerkinElmer Life and Analytical Sciences (Waltham, MA). HePC was from Cayman Chemical (Ann Arbor, MI), edelfosine was from Calbiochem (Nottingham, UK), ErPC was from Alexis Biochemicals (Exeter, UK), and perifosine was from Selleck Chemicals (London, ON, Canada). Polyclonal anti-human phospho-Akt (Ser473) antibody and horseradish peroxidase (HRP)-linked secondary IgG were from Cell Signaling Technology (Danvers, MA). Polyclonal anti-human Akt1/2/3 primary antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The enhanced chemiluminescence (ECL) detection system was from Millipore (Billerica, MA).
The human hepatoblastoma HepG2 cell line was from The European Collection of Animal Cell Cultures (Salisbury, UK). Cells were cultured in MEM containing 10% heat-inactivated FBS supplemented with 2 mM l-glutamine, 1% nonessential amino acids, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were grown in a humid atmosphere with 5% CO2 at 37°C and subcultured at a ratio of 1:10 once a week. Cells were plated on tissue-culture dishes (Nalge Nunc International, Rochester, NY) at a density of 5 × 104 cells/cm2 and kept in culture medium before use in experimental assays at approximately 70% confluence.
Cholesterol Efflux Assays.
HepG2 cells were cultured in MEM/10% FBS for 24 h. The medium was removed, and the cells were washed twice with PBS. To label the plasma membrane, radioactive [7(n)3-3H]cholesterol (2 μCi/ml) was added in a serum-free medium for a period of 60 min. To remove any unincorporated radioactivity, the cells were washed three times with PBS containing 0.5% BSA and twice with PBS (both prewarmed to 37°C). The different treatments were added in serum-free medium, and aliquots were collected at various intervals from 0 to 45 min. Radiometric measurements of medium aliquots were made by liquid scintillation using a Beckman 6000-TA counter (Beckman Coulter, Madrid, Spain) and expressed in terms of percentage as radioactive cholesterol in medium per total radioactive cholesterol. Background (control) values were subtracted from treatment values.
Trafficking of Cholesterol from the Plasma Membrane to the Endoplasmic Reticulum.
An efficient way of measuring cholesterol transport from the plasma membrane to the ER is to determine the degree of esterification of radiolabeled cholesterol previously incorporated into the plasma membrane (Lange and Steck, 1997; Marco et al., 2009). Thus, HepG2 cells were incubated with 2 μCi/ml [7(n)3-3H]cholesterol for 60 min at 37°C and then with the different APLs in the presence or absence of 30 μg/ml cholesterol. After 45 min of exposure, the medium was removed, and the lipids were extracted from the cells following the procedure of Bligh and Dyer (1959). Cholesterol and cholesteryl esters were separated by thin layer chromatography using a mixture of hexane/diethyl ether/acetic acid (70:30:2) as solvent. Radiometric measurements of scraped lipid spots, rendered visible by exposure to iodine vapor, were made by liquid scintillation. The fraction of esterified plasma-membrane cholesterol is expressed in terms of the percentage of esterification of the total labeled cholesterol.
Study of Cholesterol Replenishment.
HepG2 cells were cultured in MEM/10% FBS for 24 h. The medium was removed, and cells were washed twice with PBS before being treated with 50 μM edelfosine or 5 mM MβCD (as positive control) or with no additions (control) for 60 min. To quantify cholesterol, lipids were extracted from half of the replicates, as described above. The other half of the samples was washed extensively with PBS to remove edelfosine or MβCD, and 1 mM water-soluble cholesterol in MEM was added. After 90 min, the cells were visualized under an inverted microscope, and lipid extraction and chromatography were carried out. Cholesterol levels were determined with the use of an enzymatic colorimetric kit from Lab Kit (Madrid, Spain).
Immunoblot Analysis of Akt Activation.
HepG2 cells growing in log phase were deprived of serum overnight and incubated with MEM in the absence (only vehicle) or presence of 20 μM edelfosine, 20 μM perifosine, or 5 mM MβCD for 30 min. To stimulate Akt, the cells were incubated with 100 nM human insulin-like growth factor (IGF)-1 for 15 min, and the phosphorylation state of Akt was measured. In addition, the effect of cholesterol replenishment on basal Akt activation was analyzed after washing three times with PBS supplemented with 0.5% fatty acid-free BSA and further incubation with 1 mM water-soluble cholesterol for 45 min. After treatment, the cells were washed twice, scraped into ice-cold PBS, pH 7.4, and centrifuged at 100g for 10 min at 4°C. Cell pellets were suspended in 0.1 ml of ice-cold lysis buffer consisting of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and a protease inhibitor cocktail from Sigma-Aldrich and incubated on ice for 30 min with occasional shaking. Cell lysates were centrifuged at 10,000g for 15 min at 4°C, and the supernatants were stored at −80°C until use; an aliquot was taken to determine protein concentration. Equal amounts of lysate protein (60 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Prestained broad-range protein-molecule mass markers were used during electrophoresis. Membranes were blocked in PBS containing 5% nonfat dry milk and 0.1% Tween 20 and then incubated with phospho-Akt primary antibody (1:1000) in blocking solution at 4°C with gentle shaking overnight. After several washes in PBS containing 0.1% Tween 20, the membranes were incubated with HRP-conjugated IgG (1:5000) as secondary antibody for 60 min. Immunoreactive proteins were detected by autoradiography using a chemiluminescent HRP substrate and exposure to X-ray film. After incubation with an antibody-stripping solution consisting of 60 mM Tris-HCl, pH 6.8, 100 mM β-mercaptoethanol, and 2% SDS for 30 min at 60°C, blots were incubated with total-Akt primary antibody (1:1000). Densitometric analysis was carried out using the ImageJ gel-digitizing software (http://rsbweb.nih.gov/ij/).
Results are expressed as mean ± S.E.M. One-way analysis of variance with post hoc comparisons by Scheffé's test was carried out (SPSS 13.0; SPSS, Inc., Chicago, IL). P values <0.05 were considered to be statistically significant.
Cell-protein content was determined in the cell homogenates by the Bradford (1976) method using BSA as standard. Cell morphology was observed under an inverted microscope.
Effects of Hexadecylphosphocholine on Cholesterol Efflux.
We recently reported that long-term exposure to HePC causes deregulation in the homeostasis of cholesterol in HepG2 cells, both in terms of synthesis and intake, but we had not yet explored the possible effects of HePC on cholesterol efflux. Therefore, in this work, we have extended our studies to analyze further the possible manner in which HePC might influence cholesterol efflux. To this end, we treated cells with [7(n)3-3H]cholesterol for 60 min to label the plasma membrane and then exposed them to different quantities of HePC, determining the recovery of the [7(n)3-3H]cholesterol in the medium after a 45-min incubation. Because serum acts as an extracellular cholesterol acceptor, the studies on cholesterol efflux were routinely conducted in a serum-free medium. As can be seen in Fig. 1, HePC significantly increases cholesterol efflux from the cells in a dose-dependent manner. Thus, in the presence of 50 μM HePC, HepG2 cells released up to 40% of total radiolabeled plasma-membrane cholesterol. Bearing these results in mind, we determined the time course of cholesterol efflux in cells exposed to 50 μM HePC (the most efficient concentration assayed) and compared it with cholesterol efflux in cells exposed to MβCD, a well known cholesterol-depleting agent. We then labeled the cholesterol in plasma membrane for 60 min and followed the kinetics of cholesterol efflux both in the presence of HePC or MβCD and in their absence as controls. As has been shown in other cell types (Zidovetzki and Levitan, 2007), when HepG2 cells were exposed to 5 mM MβCD, we observed the existence of two distinct plasma membrane cholesterol pools, a fast pool removed with a half-time (t1/2) of 85 s and a slow pool with a t1/2 of 10 min. Although at 30 min, 5 mM MβCD was significantly more efficient in releasing cholesterol than 50 μM HePC (*, P < 0.001), the efflux kinetics were similar (Fig. 2). It is noteworthy that the cholesterol efflux induced at 30 min by 50 μM HePC was significantly higher (*, P < 0.001) than that observed in cells treated with the same concentration of MβCD.
Alkylphospholipid-Dependent Efflux Modulation by Cholesterol Coaddition.
The results shown in Figs. 1 and 2 prompted us to see whether the action of HePC on the efflux of cholesterol could be affected by the presence of exogenous cholesterol. Thus, we assayed the rate of plasma-membrane [7(n)3-3H]cholesterol efflux in the presence of 50 μM HePC and different quantities of cholesterol and found that the addition of 15 μg/ml cholesterol clearly reduced the quantity of cholesterol released into the medium without affecting the efflux kinetics (Fig. 3). It is noteworthy that the levels of cholesterol higher than 30 μg/ml abrogated the efflux of cholesterol induced by HePC.
Cholesterol Efflux Induced by Other Alkylphospholipid Analogs.
Because we recently demonstrated that other APLs, such as edelfosine, ErPC, and perifosine, disrupt cholesterol homeostasis in HepG2 cells via a mechanism similar to that of HePC (Carrasco et al., 2010), we went on to study the influence of these APLs on cholesterol efflux both in the presence and absence of exogenous cholesterol. The high capacity of the APLs to stimulate cholesterol efflux is shown in Table 1. As can be seen, three of the APLs assayed induced a concentration-dependent release of cholesterol, with edelfosine clearly being the most efficient since nearly 50% of plasma-membrane cholesterol from the HepG2 cells found in the medium after 30 min of incubation. Cholesterol efflux in the presence of the four APLs followed the pattern: edelfosine > HePC > perifosine > ErPC. It is noteworthy that ErPC exhibited a clear difference in its ability to release cholesterol from the cell given that all of the concentrations of this APL assayed resulted in effluxes of similar quantities of cholesterol into the medium. What was noticeable, however, was that all of the APLs analyzed exhibited similar efflux kinetics to that observed with HePC (data not shown). As far as the coaddition of cholesterol is concerned, in just the same way as we had observed previously with HePC, we found that exogenous cholesterol reduced the efficacy of edelfosine and perifosine to stimulate cholesterol efflux, whereas it had little effect upon ErPC.
Cholesterol Coaddition Does Not Impede the Interruption of Cholesterol Traffic Induced by APLs.
We also studied the manner in which APLs interfered in the traffic of cholesterol from the plasma membrane to the ER under conditions of enhanced cholesterol efflux (i.e., after exposure to only APL) and after exposure with APL plus cholesterol, where cholesterol efflux is not modified by the APL. Considering that the best experimental procedure to study any interference in the traffic of cholesterol from the plasma membrane to the ER is to analyze the rate of synthesis of esterified cholesterol from plasma-membrane cholesterol, we labeled the cholesterol in the plasma membrane and then exposed the HepG2 cells to the different APLs, both in the presence and absence of 30 μg/ml exogenous cholesterol, and determined the radioactivity appearing in the esterified cholesterol. The data in Fig. 4 show clearly that, as we have reported in a previous publication (Carrasco et al., 2010), all of the APLs assayed impeded the arrival of plasma-membrane cholesterol to the ER and thus its subsequent esterification but also that this interference was unrelated to the release of plasma-membrane cholesterol because it was observed both in the presence and absence of exogenous cholesterol.
Effects of Alkylphospholipids on Cell Morphology.
Cells exposed to the different APLs for 30 min became abnormally rounded in shape. A similar morphological change has been reported by other authors in cells depleted of cholesterol by exposure to MβCD (Park et al., 2009). Thus, in our study, we further examined whether the presence of cholesterol might prevent the changes in cell morphology induced by APLs. As illustrated in Fig. 5, HePC and edelfosine treatments clearly brought about changes in cell morphology, from flattened to rounded within 30 min. It is interesting to note that according to the cholesterol studies reported above (Table 1), this change was broadly mitigated by the coaddition of cholesterol. We observed the same effects with the other APLs (data not shown).
Study of Cholesterol Replenishment in Alkylphospholipid-Treated Cells.
We have demonstrated in this study that exposure of HepG2 cells to APLs stimulates cholesterol efflux from the cell surface and also causes the cells to become round in shape. It has also been reported recently that cholesterol depletion produced by either MβCD or statins induces cell death concomitantly with a decrease in levels of cell-surface lipid rafts (Li et al., 2006), which are restored by cholesterol replenishment. Thus, we went on to examine whether stimulation of the efflux of cholesterol induced by APLs was able to deplete cholesterol levels in the HepG2 cells and, if this were so, whether subsequent incubation with exogenous cholesterol could replenish these cholesterol levels and restore normal cell morphology. To this end, we chose edelfosine as the representative APL. After a 60-min exposure to edelfosine or MβCD (as positive control) and further washings with PBS, we added 1 mM water-soluble cholesterol. In accordance with previous reports by several authors working with other cell lines (Haynes et al., 2000; Rouquette-Jazdanian et al., 2006), we found that approximately 60% cellular cholesterol was depleted by MβCD from the HepG2 cells (Fig. 6). It is noteworthy that 50 μM edelfosine also stimulated the efflux of similar quantities of cholesterol after the same time. Apart from this, both cholesterol levels and the morphology of the MβCD-treated cells returned to the control situation after cholesterol replenishment, whereas edelfosine-treated cells showed no improvement in morphology or a complete return to control cholesterol levels.
Comparative Analysis of the Effects of Methyl-β-cyclodextrin and Alkylphospholipids on the Activation Status of Akt.
To study the possible role of APLs on Akt activity, we conducted immunoblotting assays to analyze the effect caused by edelfosine and perifosine on the level of phosphorylated active Akt after stimulation with IGF-1. Exposure of the cells to edelfosine, perifosine, or 5 mM MβCD for 30 min produced a significant decrease in the phospho-Akt/Akt ratio in the assayed cell lysates, as a result of reduced phospho-Akt, after IGF-1 stimulation (Fig. 7A). Figure 7B shows that cholesterol replenishment restores the state of phosphorylation of Akt in MβCD-treated cells but not in the APL-exposed cells.
Our laboratory has reported that HePC alters cholesterol homeostasis in HepG2 cells by impairing cholesterol trafficking from the plasma membrane to the ER (Marco et al., 2009). More recently, we have extended our studies and demonstrated that other APLs, such as edelfosine, perifosine, and ErPC, produce similar effects on cholesterol homeostasis, demonstrating that all of the APLs assayed exhibited a common mechanism of action (Carrasco et al., 2010). In this work, we analyze the early action mechanism of these APLs, which may trigger the alterations in cholesterol homeostasis produced by these compounds. Because intracellular cholesterol metabolism is controlled by influx, synthesis, and efflux, we investigated the possible effect of APLs on the release of cellular cholesterol into a serum-free medium (i.e., in the absence of any physiological or synthetic acceptors). Our data demonstrate that exposure to HePC (as well as edelfosine, perifosine, and ErPC) results in a significant efflux of cholesterol from the plasma membrane into the extracellular medium in HepG2 cells. This effect has not been described until now and is of great interest because in vitro biophysical and biochemical studies into APL activity are frequently carried out in a serum-free medium where cholesterol is released by APLs. This should be born in mind when interpreting some of the actions of APLs.
Several studies have shown that APLs are surface-active molecules, with a high affinity for membranes but weak detergent activity (Busto et al., 2007). Because of their inverted-cone shape, HePC, edelfosine, and perifosine disperse in water in the form of micelles (Busto et al., 2008), displaying a critical micellar concentration at 2.5 to 3 μM (Rakotomanga et al., 2004). As mentioned under Results, ErPC exhibits a behavior different from that of other APLs on cholesterol release from the plasma membrane. This may possibly be attributed to the fact that ErPC, because of its unsaturated cis-13-docosenol derivative structure, forms lamellar structures rather than micelles (Dymond et al., 2008). In any case, as stated above, the efflux of cholesterol into the acceptor-free medium increases significantly in the presence of the different APLs assayed, and we hypothesize that, because of their high affinity for cholesterol, aggregates of APLs may act by removing cholesterol from the cell surface, as do other physiological and synthetic acceptors, such as serum, cyclodextrins, high-density lipoproteins, and phospholipid vesicles.
A comparison between the efficiency of HePC and MβCD for releasing cholesterol reveals that, in 30 min, a 100-fold lower concentration of HePC released around 30% of the cholesterol released by MβCD and that the rates of APL-induced cholesterol efflux were far in excess of those achieved with the same low concentration of MβCD. An analysis of the kinetics of cholesterol efflux in erythrocytes has demonstrated the existence of a fast kinetic pool (Steck et al., 2002), but other studies undertaken with different cell lines have reported that free cellular cholesterol exists in two kinetic pools (Zidovetzki and Levitan, 2007). Rouquette-Jazdanian et al. (2006) suggest that the fast pool was associated with the raft domains, whereas the slow pool was associated with non–raft-membrane fractions. Our time course efflux experiments also demonstrated that, in HepG2 cells, there were two distinct plasma-membrane cholesterol pools given that the kinetics of cholesterol efflux to 5 mM MβCD was biexponential. It is noteworthy that the efflux kinetics with HePC and other APLs was similar, with a fast cholesterol release lasting a few minutes followed by a slower efflux thereafter.
Of special interest in our study is the fact that, with the exception of ErPC, the coaddition of small quantities of cholesterol (from 15 to 60 μg/ml) with the APLs significantly reduced their capacity to induce cholesterol efflux from the plasma membrane. A similar reduction in the cholesterol-releasing efficiency of MβCD caused by the presence of cholesterol has been reported previously (Li et al., 2006; Park et al., 2009), and it is now accepted, in fact, that MβCD can act as a cholesterol donor-acceptor system because the ratio between the quantities of cholesterol and cyclodextrin in the complexes determines whether it will act as cholesterol acceptor or donor. At the moment, we cannot offer any unequivocal explanation for the cholesterol buffering effect on the APL-induced cholesterol efflux, but it is probable that the capability of APL micelles to accept cholesterol decreases concomitantly with an increase in the quantity of exogenous cholesterol complexed with them. On the other hand, Rakotomanga et al. (2004) have shown that HePC molecules are inserted into the monolayers of lipids as monomers until critical micellar concentration. At higher concentrations, however, HePC micelles are deployed at the interface as groups of monomers in sterol monolayers and so the presence of cholesterol might stabilize the micellar structure, thus decreasing the number of APL molecules that can deabsorb from the micelle to interact with the membrane. This could be the explanation for the well known fact that the effect of APLs is reduced by the presence of serum and other compounds (Ménez et al., 2007), because the interaction of APLs with cholesterol would decrease their effective concentration. Moreover, the reduction in the capability of APLs to release the cholesterol from cells in the presence of exogenous cholesterol may be the reason for the lower hemolytic effect found by other authors in the presence of combinations of APLs and cholesterol (Busto et al., 2008).
It is well documented that the efflux of cholesterol from the plasma membrane to an acceptor molecule in the medium results in the stimulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity and inhibition of cholesterol esterification (Field et al., 1998). Therefore, we wondered whether the possible alteration of plasma-membrane cholesterol caused by the efflux induced by APLs might be involved in the alterations produced by APLs on cholesterol homeostasis reported previously by our research group (Jiménez-López et al., 2010). In this work, we have demonstrated that the interference of APLs on cholesterol traffic from the plasma membrane to the ER is not related to cholesterol efflux, because it is to be seen both when cholesterol is released into the extracellular medium (incubation with APLs alone) and when cholesterol is not released into the medium (cholesterol coadded with APLs).
Furthermore, we went on to analyze whether the efflux of cholesterol induced by APLs altered cholesterol levels in HepG2 cells and, if so, whether the effects were reversible by cholesterol replenishment. As stated under Results and in accordance with reports by other authors using different cells lines (Li et al., 2006; Park et al., 2009), the exposure of HepG2 cells to 5 mM MβCD significantly depletes their cholesterol content and at the same time causes them to become round in shape. These changes are reversed by cholesterol replenishment. It is noteworthy that, although APLs also stimulate a depletion of cellular cholesterol and a similar morphological change, these effects are not fully reversed by the addition of exogenous cholesterol.
Several lines of study have demonstrated that depletion of cholesterol from the plasma membrane causes the disruption of lipid rafts and the consequent release of some of their constituents into non–raft-membrane domains, which renders them nonfunctional. Among the protein components whose activities have been proven to be modulated by association/dissociation to rafts, we were most interested in Akt. Other authors have reported alterations in the activity of this protein after treatment with perifosine and other APLs (Engel et al., 2008), although the mechanism involved in this effect is not yet fully understood. Because cholesterol is an essential lipid component of the rafts involved in Akt activation and, as we have demonstrated here, cholesterol is depleted by exposure to APLs, we looked to see whether changes in plasma-membrane cholesterol levels altered the activation state of Akt. We used MβCD as a positive control and found that incubation with MβCD decreased the phosphorylation of Akt and that, after replenishment with cholesterol, the levels of phosphorylated Akt returned to control values, all of which are in accordance with data reported in other cell lines after treatment with cyclodextrins (Park et al., 2009). Likewise, the depletion of cholesterol produced after exposure to APLs led to a reduction of Akt phosphorylation in HepG2 cells, suggesting that APLs may act on Akt via the depletion of plasma-membrane cholesterol. Moreover, in this case, the addition of cholesterol did not fully replenish cholesterol levels and did not restore the phosphorylation of Akt to control values. Thus, we propose that the depletion in plasma-membrane cholesterol caused by the efflux of cholesterol to the APL acceptor may be one of the underlying mechanisms for Akt inactivation reported previously by other authors.
Although further studies are needed, a common final mechanism for the APLs seems to underlie all of our observations; namely that the aqueous aggregates of APLs lead to a displacement of cholesterol from the plasma membrane, which can destabilize membrane microdomains and disturb their function in tumor cells. Thus, although APLs may act as an acceptor, as does MβCD, which binds cholesterol with high specificity, some amphiphilic compounds could insert themselves into the membrane, leading a displacement of cholesterol (Lange et al., 2009). Thus, we cannot rule out the possibility that amphipathic APLs might also act via a similar mechanism or even that both mechanisms might act cooperatively in the plasma-membrane cholesterol efflux. Thus, here we describe a new property of antitumoral APLs, those acting as efficient agents to deplete membrane cholesterol. Because these APLs are widely used as chemotherapeutic and antimicrobial drugs, the effect described in this article should be taken into account in experimental situations or clinical administration.
Participated in research design: Marco, Segovia, and Carrasco.
Conducted experiments: Ríos-Marco, Marco, Jiménez-López, and Carrasco.
Performed data analysis: Ríos-Marco and Carrasco.
Wrote or contributed to the writing of the manuscript: Ríos-Marco, Jiménez-López, Marco, Segovia, and Carrasco.
Other: Carrasco acquired funding for the research.
We thank Xiomara Gálvez for technical support and Jon Trout for revising the English text.
This work was supported by the Carlos III Institute of the Spanish Ministry of Health [Grant PI061268]. P.R.-M. holds a fellowship funded by the Spanish Ministry of Science and Innovation.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- endoplasmic reticulum
- [(Z)-docos-13-enyl] 2-(trimethylazaniumyl)ethyl phosphate (erucylphosphocholine)
- hexadecyl 2-(trimethylazaniumyl)ethyl phosphate (hexadecylphosphocholine)
- phosphate-buffered saline
- bovine serum albumin
- horseradish peroxidase
- minimal essential medium
- fetal bovine serum
- insulin-like growth factor.
- Received July 19, 2010.
- Accepted December 6, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics