Cholesterol As a Factor Regulating the Influence of Natural (PAF And

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Biochimica et Biophysica Acta 1828 (2013) 2700–2708

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbamem

Cholesterol as a factor regulating the inﬂuence of natural (PAF and lysoPAF) vs synthetic (ED) ether lipids on model lipid membranes

Michał Flasiński ⁎,Paweł Wydro, Katarzyna Hąc-Wydro, Patrycja Dynarowicz-Łątka

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland

article info abstract

Article history: In this work we have performed a comparative study on the effect of antineoplastic ether lipid-edelfosine (ED), Received 12 May 2013 its natural analogs — Platelet Activating Factor (PAF) and its precursor (lyso-PAF), both lacking anticancer prop- Received in revised form 16 July 2013 erties, on cholesterol/phosphatidylcholine (Chol/PC) monolayers, serving as model membranes. Since all the Accepted 19 July 2013 above ether lipids are membrane active, it can be expected that their effect on membranes may differentiate Available online 29 July 2013 their biological activity. Our investigations were aimed at studying potential relationship of the effect of ED, PAF and lyso-PAF on model membranes, differing in condensation. We have modified molecular packing of Keywords: Ether lipid Chol/PC model systems either by increasing the level of sterol in the system or changing the structure of PC, Langmuir monolayer while keeping the same sterol content. Additionally, we have performed a detailed comparison of the miscibility Monolayer condensation of ED, PAF and lyso-PAF with various membrane lipids. The collected data evidenced that all the investigated Anticancer effect ether lipids influence Chol/PC films in the same way; however, in a different magnitude. Moreover, the interactions of ED, PAF and lyso-PAF with model membranes were the strongest at the highest level of sterol in the system. A thorough analysis of the obtained results has proved that the effect of the investigated ether lipids on membranes is not dependent on the condensation of the system, but it is strongly determined by the concentration of cholesterol. Since ED was found to interact with model membranes stronger than PAF and lyso-PAF, we have suggested that this fact may contribute to differences in cytotoxicity of these compounds. © 2013 Elsevier B.V. All rights reserved.

1. Introduction cancer cells sparing normal cells [1,5]. Secondly, edelfosine is not equal- ly active on various lines of the same cancer cells [9,12,13]. It can be hy- Biological membrane acts as a barrier through which various drugs pothesized that, regardless the exact mechanism of action of this reach the interior of a cell, and frequently biomembrane itself is a target compound, such a high selectivity of edelfosine may originate from dif- for various pharmaceuticals. A good example of this kind of drugs are syn- ferent ability of drug molecules to incorporate into various cellular thetic antitumor lipids covering two major groups of compounds, namely membranes. In other words, since anticancer action of edelfosine re- the alkyl ether phospholipids (known also as alkyl-lysophospholipid — quires its incorporation into the cell, the composition and properties ALPs) and the alkylphosphocholines (APCs) [1]. These molecules may regulate sensitivity of a cell toward the action of this drug. penetrate into membranes of cancer cells and cause a large diversity of As regards molecular structure of ED, it is closely related to naturally both biochemical effects (e.g. activation of Fas-death receptors, increase occurring family of ether lipids termed Platelet Activating Factor (1-O- of caspase activity, co-clustering of signaling molecules and rafts, inhibi- alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) as well as to its tion of the synthesis of phosphatidylcholines [2–5]) and disturbances in deacetylated precursor — lyso-PAF. PAF is a well known from its ability membrane organization (e.g. decrease in the stored curvature elastic en- to induce aggregation and degranulation of platelets [14],andactivityin ergy, displacement of proteins from rafts or inhibition of the activity of signaling [15] or inflammatory processes [16,17]. On the other hand, membrane-associated protein, membrane depolarization and disordering lyso-PAF is biologically inactive [18] or acts antagonistically to PAF in membrane channels [6–8]). A consequence of this action is apoptosis, [19]. Phospholipid-like structure and amphiphatic nature of PAF causes and finally cell death. that this compound, similarly to ED, may incorporate into membranes A representative of ALPs drugs is edelfosine (ED) — asinglechain affecting their organization and properties [20–23].Interestingly,very synthetic phospholipid of well documented antineoplastic activity to- similar structure of these three compounds, that is PAF, lyso-PAF wards various cell lines of leukemia [5,9,10], breast [11] or lung [12] and ED, does not reflect in their similar biological activity. Namely, cancer. It is highly important that edelfosine acts as an apoptosis- PAF is not able to induce apoptosis in cancer cells [1], while ED in promoter very selectively. First of all, this drug induces apoptosis in contrast to PAF does not cause aggregation of platelets [24,25].Tak- ing into account that all these compounds are membrane-active it ⁎ Corresponding author. Tel.: +48 12 663 20 82; fax: +48 12 634 05 15. would be very interesting to verify if one of the factors differentiat- E-mail address: fl[email protected] (M. Flasiński). ing biological activity of ED, PAF and lyso-PAF is their different effect

0005-2736/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamem.2013.07.024 M. Flasiński et al. / Biochimica et Biophysica Acta 1828 (2013) 2700–2708 2701 on membranes. The experiments in this field have widely been Milli-Q water was used. Brewster angle microscopy experiments were performed for ED to gain insight into the mechanism of anticancer effect performed with UltraBAM instrument (Accurion GmbH, Goettingen, of this molecule [26,27]. However, studies on the influence of PAF or lyso- Germany) equipped with a 50 mW laser emitting p-polarized light at a PAF on membranes are rather scarce and for artificial membranes they wavelength of 658 nm, a 10× magnification objective, polarizer, analyzer have been done only in selected binary systems (e.g. cholesterol/PAF, and a CCD camera. The spatial resolution of BAM was 2 μm. phosphatidylethanolamine/PAF or lyso-PAF or DPPC/PAF [28,29]). Unfor- tunately, this is not sufficient to draw coherent conclusions on the origin 2.3. Composition of the studied monolayers of differences in the activity of these compounds. In this work we have compared the effect of PAF, lyso-PAF and ED on As model membranes Chol/DSPC = 1:2, Chol/SOPC = 1:2, Chol/ cholesterol/phosphatidylcholine (Chol/PC) monolayers. The studied SOPC = 1:1 and Chol/SOPC = 2:1 films, differing in the packing of mixtures differ in the level of cholesterol or in the structure of PC mole- molecules, were used. Into these films ED, PAF or lyso-PAF in various cule, resulting in different film condensation. The incorporation of ether mole fractions (0.05; 0.15, 0.3) were incorporated. lipids into these monolayers allowed us to analyze the correlation be- To compare the miscibility of the ether lipids with DSPC and SOPC, tween the molecular packing and the effect induced by ED, PAF and the mixtures of the respective phosphatidylcholines with ED, PAF and lyso-PAF, as well as to verify if the foregoing correlation is determined lyso-PAF were investigated. Moreover, to be able to perform the analysis by the level of cholesterol or by molecular packing in model systems. of the interactions of the studied ether lipids with various membrane Additionally, antitumor efficacy of ED towards various leukemic cells lipids, we have completed the experiments with studies on the proper- encouraged us to undertake these experiments. Namely, it was found ties of ganglioside GM3/ether lipids films. that normal leukocytes as well as immature human promyeloblastic leukemia cells (K-562) are insensitive to apoptotic effect of ED, in con- 2.4. Data analysis trast to human promyelocytic leukemia cells (HL-60) [9,13,30].Itwas suggested that these differences may be correlated with the level of cho- For all of the monolayers studied herein the values of the excess area lesterol in membranes. This thesis is based on the fact that the incorpo- of mixing (AExc)werecalculatedatthesurfacepressureofπ =30mN/m ration of ED is faster into the cells of lower sterol level and that the from Eq. (1) [33]: concentration of cholesterol is higher in resistant to ED K-562 cells than in ED-sensitive HL-60 cells [30,31 and references therein]. Since AExc ¼ A−Aid ð1Þ one of the key role of cholesterol in membranes is to regulate their fluidity, the question raises whether the effect of ED depends on the where A are the values of the mean area per molecule derived from the presence of cholesterol, or it is determined only by the packing of isotherms at a given surface pressure, while Aid are the areas correspond- membrane. This question is additionally justified, taking into considering to ideal mixing. The Aid values for binary monolayers were calculated ation that ED-sensitive and resistant cells differ in the content of other according to Eq. (2) lipids, which also may change membranes fluidity [9,13,32]. The results id ¼ þ : ð Þ presented in this work provide new important information in this A A1X1 A2X2 2 context.

Where A1 and A2 are the mean area per molecule for the respective 2. Experimental one component ﬁlms, while X1 and X2 are the mole fractions of the respective components in binary ﬁlm [33]. In the case of ternary Chol/PC/ether 2.1. Materials lipid monolayers the areas for ideal mixing were calculated from Eq. (3):

The investigated ether lipids: C PAF (1-O-octadecyl-2-acetyl-sn- id ¼ þ ð Þ 18 A AChol=PC XChol=PC Aether lipidXether lipid 3 glycero-3-phosphocholine) and C18lyso-PAF (1-O-octadecyl-sn-glycero- 3-phosphocholine) of high purity (N99%) were purchased from Bachem where AChol/PC denotes the mean area per molecule values estimated from AG Switzerland, whereas edelfosine, ED (1-O-octadecyl-2-O-methyl- the isotherms for Chol/PC mixtures at a given surface pressure and XChol/PC rac-glycero-3-phosphocholine), of purity ≥99.1%, was provided by Biaffin is its mole fraction in Chol/PC/ether lipid film. Aether lipid and Xether lipid refer GmbH&Co KG, Germany. Double-chained phospholipids: 1,2-distearoyl- to one component ether lipid films. sn-glycero-3-phosphocholine (DSPC) and 1-stearoyl-2-oleoyl-sn- The excess free energy of mixing (ΔGExc) values for binary monolayers glycero-3-phosphocholine (SOPC) as well as ganglioside GM3 (Milk, was calculated according to Eq. (4) [33]. Bovine-Ammonium Salt) were the products of high purity (≥99%) purchased from Avanti Polar Lipids, Inc. Cholesterol (Chol) (≥99%) Zπ Zπ was supplied by Sigma. Spreading solutions of DSPC, SOPC and ΔGExc ¼ N AExc dπ ¼N A−Aid dπ ð4Þ

GM3 were prepared in chloroform:methanol 9:1 v/v mixture, 0 0 while cholesterol was dissolved in chloroform. The solvents were purchased from Aldrich (HPLC grade, ≥99.9%). The mixtures were where Aid values were calculated from Eq. (2). prepared from the respective stock solutions and deposited onto water subphase with the Hamilton micro syringe (±2.0 μL). After 3. Results spreading the ﬁlms were left for 10 min. before the compression was initiated (barrier speed of 20 cm2/min). 3.1. The effect of ED, PAF and lyso-PAF on cholesterol/PC monolayers

2.2. Methods In the first step of our studies, the surface pressure-area isotherms for Chol/PC monolayers differing in the content of ether lipids (5; 15 The experiments were performed with NIMA (UK) Langmuir trough and 30%) were recorded. In Fig. 1a–c the isotherms collected for Chol/ (total area = 300 cm2) placed on an anti-vibration table. Surface pres- DSPC/ether lipids films are shown, while in Fig. 2a–c the results for sure was measured (±0.1 mN/m) using Wilhelmy plate made of fil- Chol/SOPC/PAF mixtures are presented. The curves recorded for the ter paper (ashless Whatman Chr1) connected to an electrobalance. remaining systems (Chol/SOPC/ED and Chol/SOPC/lyso-PAF) are shown The subphase temperature (20 °C) was controlled thermostatically in Supplementary materials (Figs. S1 and S2). As it can be seen, the (±0.1 °C) by a circulating water system. For the experiments Ultrapure incorporation of PAF, lyso-PAF or ED into Chol/DSPC mixture shifts the 2702 M. Flasiński et al. / Biochimica et Biophysica Acta 1828 (2013) 2700–2708

a Chol/DSPC = 1:2 a 60 Chol/SOPC = 1:2 5% PAF 5% PAF 60 15% PAF 15% PAF 30% PAF 30% PAF PAF 40 PAF 40 , mN/m π , mN/m

π 20 20

0 0 35 70 105 140 35 70 105 140 Area, Å2/molecule Area, Å2/molecule b 60 Chol/DSPC = 1:2 b Chol/SOPC = 1:1 5% lyso-PAF 5% PAF 60 15% lyso-PAF 15% PAF 30% lyso-PAF 30% PAF lyso-PAF 40 PAF 40 , mN/m π

, mN/m 20 π 20

0 0 35 70 105 140 35 70 105 140 2 Area, Å /molecule Area, Å2/molecule c 60 c Chol/DSPC = 1:2 Chol/SOPC = 2:1 5% ED 60 15% ED 5% PAF 30% ED 15% PAF ED 40 30% PAF PAF 40 , mN/m π

, mN/m 20 π 20

0 0 35 70 105 140 35 70 105 140 2 Area, Å /molecule Area, Å2/molecule

Fig. 1. The surface pressure-area isotherms for Chol/DSPC/ether lipid monolayers. Fig. 2. The surface pressure-area isotherms for Chol/SOPC/PAF monolayers, differing in the proportion of Chol-to-SOPC. isotherms to larger molecular areas and modifies their shape, proving a cholesterol in Chol/SOPC film (2:1 proportion), this monolayer is less more liquid-like state of the film. At 30% of PAF in Chol/DSPC mixture, condensed (which reflects in larger mean molecular area at a given sur- two collapse surface pressure regions appear in the isotherm, suggesting face pressure) as compared to Chol/DSPC = 1:2 monolayer. the occurrence of a phase separation in the system. This kind of phenom- The AExc (Eq. (1)) values for binary Chol/PC mixtures in the absence enon was not observed for the remaining mixed monolayers. The effect of of ether lipid were found to be negative (data not shown), which proves ether lipids on the characteristics of the curve for Chol/SOPC monolayers miscibility and favorable packing of molecules being a consequence of is more complicated that the variation in the position and course of the their attractive interactions in the system. This is in accordance with isotherms, provoked by PAF, lyso-PAF and ED, depend on the kind of previously published data [34–36]. In the case of ternary monolayers, the investigated ether lipid, its concentration as well as the level of choles- the AExc values were calculated in respect to Chol/PC film and one com- terol in the system and the surface pressure. In the main body of this ponent ether lipid monolayer (Eqs. (1) and (3)). Such an approach article we present only the compression isotherms registered for mono- allowed us to analyze changes in this parameter caused by the respec- layers of Chol/SOPC with PAF, while the data recorded for mixtures tive ether lipids in comparison to binary Chol/PC films. containing lyso-PAF and ED are gathered in Fig. S1 and S2 in Supplemen- As it can be noticed in Fig. 3, for all the systems a very similar trend is tary materials. observed. Namely, initially (at 5% of the ether lipid) values of AExc slight- From the isotherms recorded for the investigated mixed mono- ly increase (except for Chol/SOPC = 2:1 + PAF or ED mixtures), while layers, the values of the mean area per molecule (A) were estimated with further addition of ether lipid, they systematically decrease and be- and compared with each other. As regards to binary Chol/PC films, the come negative. Moreover, the AExc values are lower for the systems of values of the mean area per molecule for the isotherms compressed to higher cholesterol level. Only in the case of Chol/DSPC/ED monolayers, π = 30 mN/m were the smallest for Chol/DSPC = 1:2 film, which indi- the AExc values increase with ED concentration. Considering the way cates that in this mixture the molecules are the most closely packed. For of calculation of AExc the obtained results should be interpreted as fol- Chol/SOPC films, A values decrease with the increase of sterol content in lows. Positive values of AExc indicate that the mean area per molecule the system. It should be pointed out that even at the highest level of for ternary mixtures is higher than those resulting from the additivity M. Flasiński et al. / Biochimica et Biophysica Acta 1828 (2013) 2700–2708 2703 a

0 0

Chol/DSPC=1:2

Chol/SOPC=1:2

/molecule -4 2 , Å

/molecule -5

Chol/PC/PAF 2 Exc , Å A Chol/DSPC = 1:2

Chol/SOPC = 1:2 Exc -8 Chol/SOPC = 1:1 A Chol/SOPC=1:1 Chol/SOPC = 2:1 01530 -10 % of PAF Chol/SOPC=2:1

b Fig. 4. The AExc values at π = 30 mN/m for the studied Chol/PC/ether lipid ﬁlms. Filled columns, open columns and patterned columns refer to ED, PAF and lyso-PAF-containing 0 ﬁlms, respectively.

The most pronounced effect of edelfosine among all the studied ether lipids was found also at its lower proportion in the mixed monolayers.

/molecule -4

2 Comparing the effect of the remaining ether lipids (PAF vs lyso-PAF) it ﬂ

, Å can be observed that their in uence depends on the level of cholesterol Chol/PC/lyso-PAF

Exc in the system. Namely, at lower content of Chol more negative values of Chol/DSPC = 1:2 A Exc fi Chol/SOPC = 1:2 A are for lyso-PAF-containing lms, for Chol/SOPC = 1:1 mixture -8 fi fi Chol/SOPC = 1:1 these values are similar for both ether lipids-containing lms, and nal- Chol/SOPC = 2:1 ly at 2:1 Chol to SOPC proportion they become more negative for PAF- containing system. Similar relation between the AExc values for PAF vs 01530lyso-PAF-containing Chol/SOPC monolayer was found at lower propor- % of lyso-PAF tion of these ether lipids in the systems. For Chol/DSPC monolayers the c AExc values change from positive for ED-containing mixtures to slightly negative for lyso-PAF containing monolayers. 0 To verify potential differences in the effect of the respective ether lipids on morphology of the monolayers, BAM images for the studied systems were taken. As regards Chol/DSPC = 1:2 mixtures, the images recorded after incorporation of a given amount of ED, PAF or lyso-PAF

/molecule -4 into the system were similar for all the studied ether lipids. Therefore 2 in Fig. 5 only the pictures for PAF-containing systems are shown. , Å Chol/PC/ED Since the morphology of Chol/DSPC mixtures was widely investigat- Exc

A Chol/DSPC = 1:2 ed with BAM technique the pictures for this system are not presented in -8 Chol/SOPC = 1:2 this work. The images published for these mixtures [34] evidenced for Chol/SOPC = 1:1 the existence of condensed domains in gaseous phase at large molecular Chol/SOPC = 2:1 areas and low surface pressures. However, at higher pressures, the film 01530was completely homogenous up to the collapse. At 5% of ether lipid con- % of ED tent in the mixture (Fig. 5), the coexistence of fluid and gaseous phase can be noticed. With film compression, the gaseous phase vanishes fi π Fig. 3. The excess area per molecule values for Chol/PC/ether lipid lms at =30mN/m. whereas condensed domains dispersed in fluid phase are being formed. With the increase of the surface pressure, these domains join together rule. This means that the incorporation of ether lipid decreases the con- and form chain-like structures observed up to the collapse. densation of Chol/PC monolayer and weakens the interactions between The incorporation of a higher amount of ether lipid into the system, molecules. On the other hand, the negative values of this parameter visibly modifies the films' texture. Namely, at 30% of ether lipid in the indicate the stabilizing effect of ether lipid on Chol/PC film and more fa- mixed film at large areas per molecules, the domains of condensed vorable interactions between molecules than in Chol/PC monolayer. phase are formed within a gaseous region. Upon compression, the num- Moreover, the higher the concentration of cholesterol in model mem- ber of these domains increases rapidly and at higher surface pressures brane, the more pronounced the above mentioned effect is. they start to merge and cover practically the entire surface. Generally, It is also interesting to note that in contrast to the systems containing BAM images recorded for monolayers of Chol/DSPC/ether lipid prove ED, the AExc values for Chol/DSPC/PAF and Chol/SOPC = 1:2 + PAF or that the single-chained compounds do not mix with the model Chol/ lyso-PAF are very similar. DSPC system, namely in the presented photos the inhomogeneity of To make the comparison of the effect of PAF, lyso-PAF and ED on the the monolayers was observed during the whole compression. This can respective model membranes more clearer, the AExc values for all the be also inferred from the positive values of AExc for mixtures containing studied systems containing 30% of particular ether lipids have been ED and PAF. compiled in Fig. 4. In the same figure (Fig. 5), BAM images for Chol/SOPC/ether lipid The figure presented above shows that the addition of ether lipids films are shown. As it was evidenced previously [34], Chol/SOPC into the investigated Chol/SOPC mixtures decreases AExc values and monolayers visualized with BAM in a wide range of surface pressures this effect is definitely the strongest in the case of incorporation of ED (π = 1 ÷ 36 mN/m) are completely homogenous. Similarly to Chol/ molecules into Chol/SOPC systems (the most negative values of AExc). DSPC/ether lipid monolayers discussed above, also in the case of 2704 M. Flasiński et al. / Biochimica et Biophysica Acta 1828 (2013) 2700–2708

Chol/DSPC(1:2) /PAF (5%)

π = 0 mN/m π = 2.5 mN/m π = 10 mN/m π = 30 mN/m π = 45 mN/m

Chol/DSPC(1:2)/PAF (30%)

π = 0 mN/m π = 1.5 mN/m π = 10 mN/m π = 30 mN/m π = 45 mN/m

Chol/SOPC(1:2)/PAF (5%)

π = 0 mN/m π = 0.01 mN/m π = 0.05mN/m π = 10 mN/m π = 30 mN/m

Chol/SOPC(1:2)/PAF (30%)

π = 0 mN/m π = 0.01 mN/m π = 0.05mN/m π = 10 mN/m π = 45 mN/m

Fig. 5. BAM images taken for Chol/PC(1:2)/PAF monolayers.

Chol/SOPC = 1:2 films after incorporation of both 5% and 30% of ether conclusions can also be drawn from the analysis of the images regis- lipids, a very similar trend in evolution of the morphology of monolayers tered for the remaining monolayers of Chol/SOPC/ether lipids (see was noticed in BAM pictures for ED, PAF and lyso-PAF-containing films. Supplementary materials S3, S4, S5). Therefore, in Fig. 5 only the images for Chol/SOPC/PAF mixed films are presented, while subtle differences between the studied systems are de- 3.2. Miscibility and interactions of ED, PAF and lyso-PAF with membrane scribed below. lipids At 5% content of PAF and lyso-PAF in the mixed films, the images are practically identical. At large molecular areas and zero surface pressure, To compare the miscibility and interactions of the studied ether the coexistence of 2D gas and fluid can be observed. With film compres- lipids with phosphatidylcholines, we have also investigated the sion, the gaseous phase vanishes and large oval domains within fluid properties of binary ether lipid/DSPC and ether lipid/SOPC films. phase can be detected in the images up to the pressure of ca. 1.5 mN/m. Based on the recorded surface pressure-area curves we have calcu- Then, the films remain homogenous up to ca. 30 mN/m, where small con- lated the AExc values at π =30mN/maswellaswehavetaken densed domains start to appear. These domains persist up to the collapse BAM images at various stages of films compression. The surface point; however, they are much smaller than those detected at lower sur- pressure-area curves for PC/ether lipid films (Xether lipid =0.3)are face pressures. In the case of Chol/SOPC (1:2)/ED (5%) mixture, the film is presented in Supplementary materials (S6), while AExc values for homogenous up to ca. 11 mN/m, however; during further compression these systems are shown in Fig. 6. the domains of the condensed phase systematically appear at the As regards to the results of SOPC/ether lipid monolayers, both the interface. negative values of the AExc as well as homogeneity of the film observed As regards to mixtures containing 30% of ether lipid, at large molec- in BAM images (data not presented) suggest that ED, PAF and lyso-PAF ular areas, the coexistence of fluid and gaseous phase can be observed in mix favorably with SOPC in monolayers. Visibly more negative values of the pictures. With the compression, the gaseous phase vanishes and AExc for SOPC/ED monolayers may suggest that this synthetic ether lipid fluid phase exists at the interface. Then (at π = 0.05; 0.08 and 1 mN/m interacts more favorably with SOPC than PAF and lyso-PAF. On the other for PAF, ED and lyso-PAF, respectively), very small domains of rather hand, the results obtained for DSPC/ether lipid films prove that the low degree of condensation, existing within a fluidmatrixuptoπ =6; mixing of molecules in these systems is unfavorable. The course of the 7.5 and 5 mN/m for PAF, ED and lyso-PAF, respectively are clearly visible. isotherms (Supplementary materials, S6), that is the presence of two Above these surface pressure regions, the monolayers are homogenous. In collapse points within the curves suggests immiscibility of films compo- the case of ED-containing system, the film remains uniform up to the col- nents. This conclusion is supported by positive values of AExc obtained lapse, while for PAF and lyso-PAF-containing mixtures at π ≈ 35 mN/m for these mixed films as well as BAM images taken for these mixtures. the condensed domains appear. The above discussed morphology of the In Fig. 7 BAM images for DSPC/ED monolayers are shown (similar mixed films containing unsaturated phospholipid (SOPC) confirms the pictures were taken for mixtures containing the remaining ether lipids miscibility of the components. It is worth mentioning that similar therefore they are not presented herein). The pictures registered at M. Flasiński et al. / Biochimica et Biophysica Acta 1828 (2013) 2700–2708 2705

4 Table 1 DSPC/ether lipid The comparison of the miscibility of various lipids with ED, PAF and lyso-PAF in Langmuir monolayers. The information concern binary ﬁlms of Xether lipid = 0.3 at the surface pressure π =30mN/m. (+) denotes positive deviations from ideality, (−) denotes negative deviations from ideality. Please see comments in the text below. 2 Deviations from ideality/miscibility

PAF lyso-PAF ED /molecule 2 SOPC/ether lipid DSPC (+)/phase separation (+)/phase separation (+)/phase separation , Å SOPC (−)/miscibility (−)/miscibility (−)/miscibility Exc 0 a a a

A DSPE (+)/phase separation (+)/phase separation (+)/phase separation SOPE (+)/miscibilitya (−)/miscibilitya (+)/phase separationa DOPE (−)/miscibilitya (−)/miscibilitya (−)/miscibilitya C18:0-SM (+)/phase separationb (+)/phase separationb (+)/phase separationb Ganglioside (−)/miscibility (−)/miscibility (−)/miscibility -2 GM3 Cholesterol (−)/miscibilityc (−)/miscibilityb (−)/miscibilityd

Exc π a Fig. 6. The A values for PC/ether lipid monolayers at Xether lipid = 30% and =30mN/m. Data taken from ref. [29]. Open columns, patterned columns and filled columns refer to ED, PAF and lyso-PAF- b Data taken from ref. [42]. containing films, respectively. c Data taken from ref. [28]. d Data taken from ref. [43]. large molecular areas evidence the formation of domains of relatively high degree of condensation, which initially is small but they increase (at 30% of ether lipid) is highly negative (ΔGExc = −760; −1100 and in size and number with film compression. For all the systems, these −1451 J/mol for PAF, lyso-PAF and ED). However, comparing the forego- condensed domains in less condensed phase are visible in pictures up ing ΔGExc for GM3/ether lipids with those published for cholesterol/ether to the surface pressure near the first collapse in the respective iso- lipids films (ΔGExc ≈−3000 J/mol) it is clear that ED, PAF and lysoPAF therms. Then, the films texture changes to be more uniform, however, interact more favorably with cholesterol as compared to ganglioside. the monolayers remain inhomogeneous up to the collapse. In Table 1 we have compiled the information on the mixing of the 4. Discussion ether lipids with different membrane lipids, i.e. studied herein as phosphatidylcholines as well as phosphatidylethanolamines — PE, Edelfosine, PAF and lyso-PAF were found to be of different – or even sphingomyelin — SM and cholesterol in binary Langmuir monolayers. opposite – biological effects on cells. The ability of these ether lipids to Apart from the foregoing compounds, important class of membrane incorporate into membranes may infer that differences in their mem- lipids considered as markers of various tumor cells are gangliosides brane activity may be the key factor responsible for their different cellu- [37,38]. These molecules are characterized by the presence of sugar lar activity. A good way to verify this hypothesis is to compare the moiety in the head group fragment, thus their structure is markedly influence of ED, PAF and lyso-PAF on model lipids membranes, which different from SM or PE molecules. Therefore, it would be interesting are highly useful and widely applied to study the effects exerted by var- to compare the interactions of ether lipids also with a ganglioside. A ious biomolecules on membranes [44,45]. Therefore in this work we good candidate is GM3, which is overexpressed in various edelfosine have performed the investigations on the effect of these three ether sensitive cells, e.g. breast, brain or lung tumor [39–41]. Unfortunately, lipids on mixed cholesterol/phosphatidylcholine monolayers. The con- the comparative monolayer studies in this field have not been densation of these systems was modified twofold — either by the level performed for PAF, lyso-PAF and ED. Therefore, to complete our analysis of cholesterol or by the structure of a phospholipid. We have also we carried out additional experiments for mixed films composed of performed a detailed comparison of miscibility and interactions of ganglioside GM3 and ether lipids (the isotherms for these systems con- these compounds with various membrane lipids in binary films. All taining 30% of ED, PAF or lyso-PAF, respectively are shown in Supple- these experiments were aimed at verifying the potential correlation be- mentary materials, S6); BAM images evidence that these monolayers tween molecular packing of model system and the influence of particu- are completely homogenous in the whole range of the surface pressure lar ether lipids on the monolayer. therefore they are not shown). The obtained results evidenced very similar trend as regards to the Data compiled in Table 1 evidence similar tendency in the mixing effect of particular ether lipids on the respective Chol/PC film. As it properties of all the studied ether lipids with respective membrane lipids. was found for Chol/SOPC films, differing in the level of cholesterol, the The results collected for ED, PAF and lyso-PAF prove that miscibility of the addition of low proportion of ether lipid (5%) destabilizes the systems, studied ether lipids with saturated PC, PE as well as synthetic C18:0 SM is which manifest in higher – as compared to Chol/PC film – values of highly unfavorable and phase separation was detected in the respective the excess area of mixing. However, further increase of ether lipid con- systems. On the other hand, as it was evidenced in our experiments all tent in Chol/SOPC monolayer strengthens the interactions between the studied ether lipids mix and interact favorably with ganglioside molecules as compared to those in the respective Chol/SOPC film. It GM3 and cholesterol. The excess free energy of mixing values (ΔGExc) was also found that thermodynamically favorable influence of all the calculated based on the isotherms presented in S5 for GM3/ether lipids studied ether lipids becomes more pronounced with the increase of

Fig. 7. BAM images for DSPC/ED monolayers at 30% of ether lipid in the mixture. 2706 M. Flasiński et al. / Biochimica et Biophysica Acta 1828 (2013) 2700–2708 cholesterol level in the mixture. Moreover, the comparison of the results The results showing that ether lipids interact favorably not only with for all the studied ether lipids allows one to conclude that the strongest lipids possessing unsaturated chains but also with ganglioside and cho- effect on Chol/SOPC films is induced by ED. Based only on this part of our lesterol could be explained on the basis of some additional consider- experiments, a general conclusion can be formulated that in the case ations regarding structural aspects. It seems like the key parameter where the condensation of the film was modified by the level of choles- here is the organization of the single-chained phospholipids' molecules terol, the influence exerted by ether lipids strengthened with sterol at the air/water interface. As it was proven by us in one of previously concentration. published articles [46], molecules of PAF and lyso-PAF are tilted from On the other hand, the results of experiments, in which the condensa- the surface normal and furthermore, they are strongly immersed into tion of model membrane was modified by the structure of PC at constant water subphase, meaning that the head-group region of these mole- level of cholesterol (Chol/DSPC = 1:2 vs Chol/SOPC = 1:2) as well as the cules are exceptionally large. Because of this reason, the incorporation studies on binary ether lipid/membrane lipid films provided additional of such molecules into periodically ordered monolayer of saturated insight into the role of monolayers packing in the effect of ether lipids. PCs or SM (typically at π = 30 mN/m only slightly tilted from surface The values of the excess area of mixing (AExc) obtained for Chol/DSPC normal) is unfavorable. The situation is completely different for the 1:2 and Chol/SOPC 1:2 in the presence of ether lipids are slightly lower monolayer of GM3, in which molecules with the bulky head-groups for Chol/SOPC system. Although for PAF and lyso-PAF-containing mix- are organized similarly to the investigated single-chained lipids. For ex- tures they are very comparable between those for Chol/SOPC and Chol/ ample, in the case of monolayer of ganglioside GM1 small fraction of the DSPC films, for ED-containing monolayer the differences are more pro- periodically organized phase was observed at the interface at high sur- nounced. Since these systems differ only in the structure of phosphatidyl- face pressure of 45 mN/m [47]. Therefore, in this case, the incorporated choline, slight strongest interactions in the Chol/SOPC/ether lipid films molecules of ether lipid do not disturb the organization of surface film. may result from more favorable mixing of ether lipids with SOPC as com- At this point the question arises: if such a justification is correct, how pared to DSPC and weaker interactions between cholesterol and SOPC as to explain the favorable interactions of single-chained lipids with cho- compared to DSPC. This part of our experiments indicates that at a given lesterol? First of all, it is well known that cholesterol interacts more fa- level of cholesterol the saturation of PC molecules is not without the influ- vorable with phospholipids having large head-group (PCs) over those ence on the effect of ether lipids on model systems. However, comparing with smaller ones (e.g. PEs) [48]. Secondly, a rigid molecule of cholester- the results obtained for all the studied systems it seems that the level of ol possesses a very small polar head (that is single hydroxyl group), an- cholesterol more strongly differentiates the effect of ether lipids on choring this lipid into aqueous subphase. Hence, the potential repulsing model membranes than the structure of PC molecule. interactions of large head regions of ether lipid molecules are signifi- To verify the relation between the condensation of Chol/PC films and cantly reduced by the incorporation of sterol molecules localized in the effect of ether lipids, we have analyzed the mean molecular areas for the region of hydrophobic chains. Chol/PC monolayers at 30 mN/m. The values of this parameter are the The obtained data allow us also to refer to the issue on high selectiv- smallest for DSPC/Chol then for Chol/SOPC = 2:1, 1:1 and the largest ity of ED towards various cells. As it was mentioned in the Introduction, for Chol/SOPC 1:2. Thus, if there is a simple correlation between conden- the cells of higher sterols level (e.g. immature human promyeloblastic sation of monolayer and the effect of ether lipids, the trend of changes in leukemia cells — K-562) are insensitive to apoptotic effect of ED. On the effect of ED, PAF and lyso-PAF should be reflected in our results in the other hand, the cells of lower sterol level e.g. human promyelocytic the same order (that is: Chol/DSPC = 1:2, Chol/SOPC = 2:1 and then leukemia cells (HL-60) are sensitive to ED [9,13,30]. It was postulated the remaining systems). However, the above relationship was not that these differences are due to a faster incorporation of ED into the found. Instead, we observed that the strongest effect induced by these cells of lower sterol level [30,31 and references therein]. Our results ether lipids is on the system of the highest sterol level. Thus, the concen- on model Chol/PC membrane evidenced that strong interactions be- tration of sterol seems to be more important from the point of view of tween ether lipids and cholesterol influence their effect on membranes. the effect of ether lipids than the monolayer condensation. It is possible that at a higher sterol level in the system these ED/sterol in- The foregoing conclusion is also supported by the results of the stud- teractions do not change the membrane organization in a degree suffi- ies on miscibility and interactions of PAF, lyso-PAF and ED on mono- cient to induce cell toxicity. However, at lower cholesterol level any layers formed by structurally different membrane lipids. As a result, all decrease in the concentration of this lipid in the membrane may lead these three ether lipids display very similar behavior in the monolayers to serious membrane disorders e.g. damage of rafts organization and with particular lipids. However, a deeper analysis of the foregoing data functioning. provides interesting conclusion of the role of monolayer packing in the Finally, we have found that natural ether lipids: PAF and lyso-PAF af- effect of ether lipids. It was found that ED, PAF and lyso-PAF tend to fect model membranes in the same way; however, in a weaker degree phase separate with saturated PCs, SM and PEs [28,29]. However, the than synthetic ED. Taking into account that anticancer activity of ED is presence of monounsaturated chain(s) in the molecules of PC and PE related to perturbations in membrane organization induced by this improves miscibility of ED, PAF and lyso-PAF with these compounds. drug, the observed weaker effect of PAF and lyso-PAF on model systems Considering only the foregoing facts it could be proposed that both may explain – to some degree – strong differences in antineoplastic ac- PAF and its lyso derivative as well as ED pack more favorably in more tivity of these compounds (e.g. the lack of apoptotic activity of PAF vs fluid films, which are formed by phospholipids having their chain(s) strong anticancer effect of ED). On the other hand, the results obtained with cis double bond. However, the results obtained for ganglioside herein do not allow us to explain strong differences in biological activity GM3/ether lipids and cholesterol/ether lipid monolayers contest this in- of PAF and lyso-PAF. As it was found, the effect of these two compounds terpretation. Both for GM3 and cholesterol-containing films highly neg- on the studied model systems does not differ drastically. The subtle dif- ative deviations from ideality were found indicating favorable mixing ferences in the results for PAF vs lyso-PAF do not authorized us to draw and interactions between molecules both at low and high content of conclusion that the differences in the activity of these two compounds ED, PAF and lyso-PAF in the mixed films. Considering GM3/ether lipids at membrane level may affect their biological properties. On the other films, these deviations are much more negative than those found for hand, it is possible that the composition of the studied herein model sys- SOPC/ether lipid and DOPE/ether lipid, although the condensation of tems was not sufficient to observe differences in the influence of PAF and GM3 film is higher as compared to the mentioned above phospholipids lyso-PAF on membrane. This issue requires further analysis from the point (A = 53.1, 57.7 and 64.0 Å2/molecule for GM3, SOPC and DOPE, respec- of view of the existence of some specific lipid, which potentially may dif- tively). This fact contradicts a thesis that the effect of ether lipids on ferentiate the membrane activity of PAF and lyso-PAF. It should be also membrane is determined only by the packing of molecules in the taken into account that biological activity of the foregoing natural ether mixed system. lipids may not be determined by their behavior in membranes. M. Flasiński et al. / Biochimica et Biophysica Acta 1828 (2013) 2700–2708 2707

5. Conclusions [11] H.K. Na, Y.J. Surh, The antitumor ether lipid edelfosine (ET-18-O-CH3) induces apoptosis in H-ras transformed human breast epithelial cells: by blocking ERK1/2 and p38 mitogenactivated protein kinases as potential targets, Asia Pac. J. Clin. Nutr. 17 (2008) The results obtained in our studies show that ED, PAF and lyso-PAF 204–207. affect model chol/PC membranes in similar manner, however, the effect [12] S.H. Shafer, C.L. Williams, Non-small andsmallcelllungcarcinomacelllines – exhibit cell type-specific sensitivity to edelfosine-induced cell death and differ- of ED is stronger than the effect of the remaining ether lipids. This in ent cell line-specific responses to edelfosine treatment, Int. J. Oncol. 23 (2003) turn – allows one to speculate that differences in the degree of perturba- 389–400. tions induced by ED vs PAF (or lyso-PAF) on model membranes may dif- [13] M.C.Chabot,R.L.Wykle,E.J.Modest,L.W.Daniel,Correlationofetherlipidcontentof ferentiate also cytotoxic properties of these compounds. Although the human leukemia cell lines and their susceptibility to 1-O-Octadecyl-2-O-methyl- rac-glycero-3-phosphocholine, Cancer Res. 49 (1989) 4441–4445. foregoing findings may explain differences in antitumor properties of [14] M.I.Gurr,J.L.Harwood,K.N.Frayn,LipidBiochemistry,5theditionBlackwellScienceLtd, ED vs PAF, the obtained data do not clarify strong differences in biolog- 2002. ical activity of PAF vs lyso-PAF. Since the effect of both these compounds [15] F. Snyder, T.-Ch Lee, R.L. Wykle, Ether-linked lipids and their bioactive species, in: D.E. Vance, J.E. Vance (Eds.), Biochemistry of Lipids, Lipoproteins and Membranes4th on model membranes studied herein was very similar, we can postulate edition, , 2002, , (chapter 9). that there is other specific lipid, which differentiates the effect of these [16] R. Göggel, S. Uhlig, The inositol trisphosphate pathway mediates platelet-activating- compounds on membranes, or – alternatively – the effect of these factor-induced pulmonary oedema, Eur. Respir. J. 25 (2005) 849–857. [17] L. Kemeny, V. Trach, A. Dobozy, Effect of locally applied WEB 2086, a platelet- ether lipids on membranes is not the factor determining their biological activating factor antagonist, on inflammatory skin conditions in mice, Arch. Dermatol. activity. However, this issue requires further analysis. Res. 288 (1996) 492–494. The most important conclusions provided in this paper concern the [18] G. Montrucchio, G. Alloatti, G. Camussi, Role of platelet-activating factor in cardio- vascular pathophysiology, Physiol. Rev. 80 (2000) 1669–1700. role of the packing of molecules in model membrane in the effect in- [19] E.J. Welch, R.P. Naikawadi, Z. Li, P. Lin, S. Ishii, T. Shimizu, C. Tirupphathi, X. Du, P.V. duced by the respective ether lipids. The analysis of the data collected Subbaiah, R.D. Ye, Opposing effects of platelet-activating factor and lyso-platelet- for monolayers of different condensation modified by the content of activating factor on neutrophil and platelet activation, Mol. Pharmacol. 75 (2009) 227–234. cholesterol or by the saturation of PC molecule, as well as the results [20] L. Mrówczyńska, H. Hagerstrand, Platelet-activating factor interaction with the for binary ether lipid/membrane lipid films surely indicate that packing human erythrocyte membrane, J. Biochem. Mol. Toxicol. 23 (2009) 345–348. of molecules in the model system is not a deciding factor regulating the [21] A. Kantar, P.L. Giorgi, G. Curatola, R. Fiorini, Effect of PAF on erythrocyte membrane fl – influence of PAF, lyso-PAF and ED on membrane. However, our results heterogeneity: a uorescence study, Agents Actions 32 (1991) 347 350. [22] A. Kantar, P.L. Giorgi, R. Fiorini, Effect of PAF on human lymphocyte membranes: indicate for a key role of cholesterol in this aspect. a fluorescence study, Agents Actions 38 (1993) C115–C117, (Spec. No.). [23] J. Chen, L. Yang, J.M. Foulks, A.S. Weyrich, G.K. Marathe, T.M. McIntyre, Intracellular Acknowledgements PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis, J. Lipid Res. 48 (2007) 2365–2376. [24] K. Danker, W. Reutter, G. Semini, Glycosidated phospholipids: uncoupling of signal- This project was financed by the National Science Centre (No DEC- ing pathways at the plasma membrane, Br. J. Pharmacol. 160 (2010) 36–47. 2011/01/B/ST4/00910). [25] I. Kudo, I.S. Nojima, H.W. Chang, R. Yanoshita, H. Hayashi, E. Kondo, H. Nomura, K. Inoue, Antitumor activity of synthetic alkylphospholipids with or without PAF activ- The research was carried out with the equipment (UltraBAM) ity, Lipids 22 (1987) 862–867. purchased thanks to the financial support of the European Regional [26] A. Więcek, P. Dynarowicz-Łątka, N. Vila-Romeu, M. Nieto-Suarez, M. Flasiński, Inter- Development Fund in the framework of the Polish Innovation Economy actions between an anticancer drug – edelfosine – and DPPC in Langmuir monolayers, Colloids Surf. A 321 (2008) 201–205. Operational Program (contract no. POIG.02.01.00-12-023/08). [27] K. Hąc-Wydro, P. Dynarowicz-Łątka, Effect of edelfosine on tumor and normal cells model membranes — a comparative studies, Colloids Surf. B 76 (2010) Appendix A. Supplementary data 366–369. [28] M. Flasiński, M. Broniatowski, P. Wydro, K. Hąc-Wydro, P. Dynarowicz-Łątka, Behav- ior of platelet activating factor in membrane-mimicking environment. Langmuir Supplementary data to this article can be found online at http://dx. monolayer study complemented with grazing incidence x-ray diffraction and Brew- doi.org/10.1016/j.bbamem.2013.07.024. ster angle microscopy, J. Phys. Chem. B 116 (2012) 10842–10855. [29] K. Hąc-Wydro, M. Flasiński, P. Wydro, P. Dynarowicz-Łątka, Towards the understanding of the behavior of single-chained ether phospholipids in model biomembranes: interac- References tions with phosphatidylethanolamines in Langmuir monolayers, Colloids Surf. B 97 (2012) 162–170. [1] C. Gajate, F. Mollinedo, Biological activities, mechanisms of action and biomedical pros- [30] L. Diomede, A. Bizzi, A. Magistrelli, E.J. Modest, M. Salmona, A. Noseda, Role of cell pect of the antitumor ether phospholipid ET-18-OCH3 (Edelfosine), a proapoptotic cholesterol in modulating antineoplastic ether lipid uptake, membrane effects and agent in tumor cells, Curr. Drug Metab. 3 (2002) 491–525. cytotoxicity, Int. J. Cancer 46 (1990) 34–346. [2] W.J. van Blitterswijk, M. Verheij, Anticancer mechanisms and clinical application of [31] L. Diomede, B. Piovani, E.J. Modest, M. Salmona, The effect of culture medium com- alkylphospholipids, Biochim. Biophys. Acta 1831 (2013) 663–674. position on ether lipid cytotoxic activity, Lipids 28 (1993) 189–192. [3] F. Mollinedo, C. Gajate, Lipid rafts, death receptors and CASMERs: new insights for [32] M. Levi, P. Wilson, S. Nguyen, E. Iorio, O. Sapora, T. Parasassi, In K562 and HL60 cells cancer therapy, Future Oncol. 6 (2010) 491–494. membrane ageing during cell growth is associated with changes in cholesterol con- [4] T. Nieto-Miguel, R.I. Fonteriz, L. Vay, C. Gajate, S. Lopez-Hernandez, F. Mollinedo, centration, Mech. Ageing Dev. 97 (1997) 109–119. Endoplasmic reticulum stress in the proapoptotic action of edelfosine in solid [33] I.S. Costin, G.T. Barnes, Two-component monolayers. II. Surface pressure-area relations tumor cells, Cancer Res. 1 (2007) 10368–10378. for the octadecanoi-docosyl sulphate system, J. Colloid Interface Sci. 51 (1975) [5] F. Mollinedo, J. de la Iglesia-Vicente, C. Gajate, A. Estella-Hermoso de Mendoza, J.A. 106–121. Villa-Pulgarin, M. de Frias, G. Roué, J. Gil, D. Colomer, M.A. Campanero, M.J. [34] P. Wydro, S. Knapczyk, M. Łapczyńska, Variations in the condensing effect of choles- Blanco-Prieto, In vitro and In vivo selective antitumor activity of edelfosine against terol on saturated versus unsaturated phosphatidylcholines at low and high sterol mantle cell lymphoma and chronic lymphocytic leukemia involving lipid rafts, concentration, Langmuir 27 (2011) 5433–5444. Clin. Cancer Res. 16 (2010) 2046–2054. [35] T.H. Chou, Ch.H. Chang, Thermodynamic behavior and relaxation processes of mixed [6] M. Dymond, G. Attard, A.D. Postle, Testing the hypothesis that amphiphilic antineo- DPPC/cholesterol monolayers at the air/water interface, Colloids Surf. B 17 (2000) plastic lipid analogues act through reduction of membrane curvature elastic stress, 71–79. J. R. Soc. Interface 5 (2008) 1371–1386. [36] K. Gong, S.S. Feng, M.L. Go, P.H. Soew, Effects of pH on the stability and compressibil- [7] A.B. Gomide, C.H. Thomé, G.A. dos Santos, G.A. Ferreira, V.M. Faça, E.M. Rego, L.J. ity of DPPC/cholesterol monolayers at the air/water interface, Colloids Surf. A 207 Greene, R.G. Stabeli, P. Ciancaglini, R. Itri, Disrupting membrane raft domains by (2002) 113–125. alkylphospholipids, Biochim. Biophys. Acta 1828 (2013) 1384–1389. [37] Y. Gu, J. Zhang, W. Mi, J. Yang, F. Han, X. Lu, W. Yu, Silencing of GM3 synthase sup- [8] M. Potier, A. Chantome, V. Joulin, A. Girault, S. Roger, P. Besson, M.-L. Jourdan, J.-Y. presses lung metastasis of murine breast cancer cells, Breast Cancer Res. 10 (2008) LeGuennec, P. Bougnoux, C. Vandier, The SK3/KCa2.3 potassium channel is a new R1, (1). cellular target for edelfosine, Br. J. Pharmacol. 162 (2011) 464–479. [38] R.G. Fish, Role of gangliosides in tumor progression: a molecular target for cancer [9] B.A. Wagner, G.R. Buettner, L.W. Oberley, C.P. Burns, Sensitivity of K562 and HL-60 therapy? Med. Hypotheses 46 (1996) 140–144. cells to edelfosine, an ether lipid drug, correlates with production of reactive oxygen [39] G. Marquina, H. Waki, L.E. Fernandez, K. Kon, A. Carr, O. Valiente, R. Perez, S. Ando, species, Cancer Res. 58 (1998) 2809–2816. Gangliosides expressed in human breast cancer, Cancer Res. 56 (1996) 5165–5171. [10] A. Estella-Hermoso de Mendoza, M.A. Campanero, J. De la Iglesia-Vicente, C. Gajate, [40] T.N. Seyfried, P. Mukherjee, Ganglioside GM3 is antiangiogenic in malignant brain F. Mollinedo, M.J. Blanco-Prieto, Anti-tumour alkyl ether lipid edelfosine: tissue dis- cancer, J. Oncol. 2010 (2010) 961243, http://dx.doi.org/10.1155/2010/961243. tribution and pharmacokinetic behaviour in healthy and tumour-bearing immune [41] R. Fuentes, R. Allman, M.D. Mason, Ganglioside expression in lung cancer cell lines, suppressed mice, Cancer Res. 15 (2009) 858–864. Lung Cancer 18 (1997) 21–33. 2708 M. Flasiński et al. / Biochimica et Biophysica Acta 1828 (2013) 2700–2708

[42] M. Flasiński, K. Hąc-Wydro, P. Wydro, M. Broniatowski, P. Dynarowicz-Łątka, Inter- [45] M. Arczewska, M. Gagoś, Molecular organization of antibiotic amphotericin B actions between single-chained ether phospholipids and sphingomyelin in mixed in dipalmitylophosphatidylcholine monolayers induced by K+ and Na+ ions: the monolayers at the air/water interface-Grazing incidence X-ray diffraction and Brewster Langmuir technique study, Biochim. Biophys. Acta 1808 (2011) 2706–2713. angle microscopy studies, Colloids Surf. B 111 (2013) 43–51. [46] M. Flasiński, M. Broniatowski, P. Wydro, P. Dynarowicz-Łątka, Comparative charac- [43] A. Więcek, P. Dynarowicz-Łątka, J. Miñones Jr., O. Conde, M. Casas, Interactions be- teristics of membrane-active single-chained ether phospholipids: PAF and Lyso-PAF tween an anticancer drug – edelfosine – and cholesterol in Langmuir monolayers, in Langmuir monolayers, J. Phys. Chem. B 116 (2012) 3155–3163. Thin Solid Films 516 (2008) 8829–8833. [47] J. Majewski, T.L. Kuhl, K. Kjaer, G.S. Smith, Packing of ganglioside-phospholipid mono- [44] M. Rodrigues, A. Santos, B.G. de la Torre, G. Radis-Baptista, D. Andreu, N.C. Santos, layers: an x-ray diffraction and reﬂectivity study, Biophys. J. 81 (2001) 2707–2715. Molecular characterization of the interactions of crotamine-derived nucleolar [48] P. Wydro, K. Hąc-Wydro, Thermodynamic description of the interactions between targeting peptide with lipid membranes, Biochim. Biophys. Acta 1818 (2012) lipids in ternary Langmuir monolayers: the study of cholesterol distribution in 2707–2717. membranes, J. Phys. Chem. B 111 (2007) 2495–2502.