Katarzyna Hąc-Wydro

Cholesterol and phytosterols effect on sphingomyelin/phosphatidylcholine model membranes—Thermodynamic analysis of the interactions in ternary monolayers

In this work thermodynamic analysis of the interactions between lipids in ternary sphingomyelin/DPPC/sterol Langmuir films were performed to compare the effect of cholesterol, beta-sitosterol and stigmasterol on a model membrane. The condensing effect of the respective sterols and the interactions between molecules in ternary mixtures were analyzed on the basis of the excess area per molecule and the excess free energy of mixing values. The stability of the mixed monolayers was verified with the free energy of mixing values. The conclusions on the ordering effect of sterols were drawn from the analysis of the compression modulus values. It was found that the stoichiometry of the mixed films of the highest thermodynamic stability and of the strongest interactions is the same for all the sterols investigated. The results obtained prove that the mammalian sterol induces the strongest contraction of the area and reveals the strongest stabilizing and ordering effect among the investigated sterol. Stigmasterol was found to condense a model membrane in a weaker extent as compared to beta-sitosterol, however, the differences in ordering properties of both phytosterols are less pronounced. The magnitude of the influence of the investigated sterols on a model membrane was thoroughly discussed from the point of view of the structure of their side chain, which determines the geometry of a sterol molecule.

Effect of saturation degree on the interactions between fatty acids and phosphatidylcholines in binary and ternary Langmuir monolayers

Fatty acids molecules have strong influence on membrane properties. In this work the interactions between fatty acids and phosphatidylcholines were studied in mixed binary and ternary Langmuir monolayers. The compounds investigated, both fatty acids and phospholipids were of the same hydrocarbon chain length and differed in their saturation degree (stearic acid (C18:0)), oleic acid (C18:1)), linoleic acid (C18:2) and distearoylphosphatidylcholine-DSPC and dioleoylphosphatidylcholine-DOPC). It was found that the investigated fatty acids interact more strongly with saturated phospholipid (DSPC) and more strongly affect the molecular organization of DSPC films as compared to DOPC monolayer. The saturated fatty acid (stearic acid) makes the phospholipid monolayers more rigid, while unsaturated fatty acids decrease the condensation of phospholipid films. The differences in the effect of the respective fatty acids on phosphatidylcholines monolayers were thoroughly analyzed from the point of view of geometry of the lipids molecules, which is determined by the saturation of the hydrocarbon chain.

Edelfosine disturbs the sphingomyelin–cholesterol model membrane system in a cholesterol-dependent way – The Langmuir monolayer study

Synthetic alkyl-lysophospholipids, represented by edelfosine (ED), reveal strong anticancer activity and therefore are promising drugs used in anticancer therapy. Primary target for edelfosine is cellular membrane, which is in contrast to traditional cytostatics affecting DNA. The mechanism of antitumor activity of edelfosine was hypothesized to be related to its accumulation in membrane rafts. Inspired by these findings, we have performed the Langmuir monolayer studies on the influence of edelfosine on systems composed of sphingomyelin (SM) and cholesterol (Chol), being the principal components of membrane rafts. Sphingomyelin-cholesterol proportion in monolayers was varied to reflect the composition of solely membrane rafts (SM/Chol=2:1) and contain excess of cholesterol (SM/Chol=1:1 and 1:2). Into these systems, edelfosine was added in various concentrations. The analysis of surface pressure-area isotherms, complemented with films visualization with Brewster angle microscopy (BAM) allowed us to compare the effect of edelfosine on condensation and ordering of SM/Chol monolayers. The results evidenced that the influence of ED on the interactions in model membranes and its fluidizing effect is highly cholesterol-dependent. The strongest decrease of monolayer ordering was observed for model raft system, while the excess of cholesterol present in the remaining mixtures was found to weaken the fluidizing effect of the drug.

Effect of edelfosine on tumor and normal cells model membranes—A comparative study

This work is aimed at comparing the effect of edelfosine (Ed) on normal and tumor cell membrane imitated by the Langmuir monolayers prepared from cholesterol and phosphatidylcholines. To keep the differences in the fluidity of these membranes, model membrane of normal cell has been composed from saturated phospholipid (DPPC) and contained a higher proportion of cholesterol (cholesterol:DPPC=0.67) than tumor cell model membranes prepared from unsaturated phosphatidylcholine (cholesterol:POPC=0.25). The results proved that the incorporation of edelfosine modifies the organization and interactions between molecules in both model systems. The interactions in cholesterol/DPPC/edelfosine monolayers are stronger than in cholesterol/DPPC film. On the other hand, the interactions in cholesterol/POPC/edelfosine system are weaker than in cholesterol/POPC monolayer, thus the incorporation of edelfosine is, from thermodynamical point of view, unfavorable for binary cholesterol/POPC monolayer. Edelfosine has been found to increase the fluidity of model membranes, however, at a lower concentration (up to 5% of edelfosine in model system) this compound affects only the condensation of tumor cell model membrane, while practically does not modify the organization of normal model system. It has been suggested that edelfosine inserts into tumor cellular membranes more easily than into normal cell membrane and cholesterol/edelfosine interactions determine the interactions between molecules in the investigated mixed monolayers.

The replacement of cholesterol by phytosterols and the increase of total sterol content in model erythrocyte membranes.

The activity of phytosterols on human organism includes the ability of these compounds to incorporate into membranes. In the consequence the plant sterols are able to increase total sterol concentration in membrane or/and to replace cholesterol molecules. The aim of this work was to compare the influence of both these effects on the properties of model erythrocyte membranes. Moreover, the interactions between the plant sterols (beta-sitosterol and stigmasterol) and saturated-monounsaturated phosphatidylcholine were investigated and the condensing and ordering potency of these phytocompounds on membrane phospholipids were thoroughly analyzed. It was found that the addition of the plant sterols into model membrane modifies the condensation, ordering and interactions in the system. Moreover, the replacement of mammalian sterol by phytosterol more strongly influences the model system than even a 10% increase of total sterol concentration induced by the incorporation of the plant sterol, at constant content of cholesterol. The investigated plant sterols at their lower concentration in the mixed system are of similar effect on its properties. At higher content stigmasterol was found to modify the properties of model membrane more strongly than beta-sitosterol.

The influence of plant stanol (β-sitostanol) on inner leaflet of human erythrocytes membrane modeled with the Langmuir monolayer technique

The presence of dietary phytocompounds - plant sterols and stanols - in human plasma and membranes raises the question on their influence on membrane properties. A good way to get an insight into interactions of these biomolecules with membranes at molecular level is to perform experiments on artificial systems, e.g. Langmuir monolayers, composed of membrane lipids. In this paper the influence of plant stanol - β-sitostanol - on monolayers imitating the inner leaflet of human membrane, composed of phosphatidylethanolamine (POPE)/phosphatidylserine (POPS)/cholesterol (Chol) was studied. Two effects have been investigated and compared, namely the systematic increase of plant stanol (β-sitostanol) content in the mixed film as well as the replacement of cholesterol by β-sitostanol in POPE/POPS/Chol monolayer. The analysis of the collected data evidenced that the plant stanol strongly influences the condensation and interactions in POPE/POPS/Chol film. BAM images taken for the studied monolayers demonstrated that the presence of β-sitostanol in the mixture leads to the formation of 3D crystallites within the film, which seems to result from a limited solubility of plant stanol in phospholipids environment. All these effects have got intensified upon systematic elimination of cholesterol and its replacement by plant stanol in the mixed monolayer. The obtained results evidence that the presence of plant stanol may significantly alter organization of the inner leaflet of human membrane.

The relationship between the concentration of ganglioside GM1 and antitumor activity of edelfosine--the Langmuir monolayer study.

One of the characteristic features of tumor membranes is altered concentration of gangliosides level and their over-expression in tumor progression. This fact should be considered when the mechanism of activity and selectivity of edelfosine (ED)--a membrane-active anticancer drug--is investigated. Strong affinity of this drug to ganglioside GM1, found in binary mixed monolayers, encouraged us for a deeper investigations on more complex model systems. In this work we have studied the influence of edelfosine on the interactions between molecules in model sphingomyelin/cholesterol monolayer, mimicking a tumor membrane, containing ganglioside in increasing proportion that is characteristic of cancer progression. It was found that edelfosine in very low concentration (1%) practically does not influence on model membrane system, independently of the proportion of ganglioside. On the other hand, at the increased ED concentration (10%), the interactions between the lipids in the mixed system become progressively stronger upon the increase of ganglioside concentration as compared to those in the model tumor membrane. The results of our investigations show that the affinity of edelfosine towards tumor cells may be correlated with over-expression of gangliosides in cancer cells.

Studies on β-sitosterol and ceramide-induced alterations in the properties of cholesterol/sphingomyelin/ganglioside monolayers.

Phytosterol-β-sitosterol promotes apoptosis in various cancer cells and inhibits their growth. Supplementation of cancer cells with this compound causes modifications in membrane composition, namely, substitution of cholesterol (Chol), decrease of sphingomyelin (SM) content and increase of ceramide (Cer) level. The aim of this work was to investigate the influence of partial replacement of cholesterol by plant sterol, substitution of sphingomyelin by ceramide and both these factors simultaneously on the properties of the monolayers composed of major lipids identified in breast cancer membranes, namely Chol/SM/GM3 mixtures. Brewster Angle Microcopy experiments and the analysis of the isotherms recorded during films compression and resulting parameters evidenced that β-sitosterol weakens the interactions between molecules, decreases films stability and condensation. The influence of ceramide on sterol/SM/GM3 films was reflected in strong modifications of their texture, however, the morphology of monolayer was determined by the structure of sterol present in the system. It was also found, that simultaneous replacement of 50mol% of Chol and SM by phytosterol and Cer, respectively, induces lipids segregation, which is manifested in large diversity of phases observed in BAM images. To facilitate the analysis of the data collected for multicomponent monolayers, the properties of selected sterol/GM3, sterol/Cer, SM/GM3, Cer/GM3 binary films were also investigated. The obtained results evidenced that the studied herein modifications in the composition of Chol/SM/GM3 monolayer, reflecting compositional alterations induced by phytosterol in cancer membranes, strongly affect the organization of model system, therefore they should be considered in the studies on anticancer mechanism of β-sitosterol.

Langmuir monolayers studies on the relationship between the content of cholesterol in model erythrocyte membranes and the influence of β-sitosterol

Plant sterols, which are well known dietary taken cholesterol lowering agents, can incorporate into erythrocyte membranes. However, the influence of these compounds on membrane properties seems to be unclear. Since the composition of erythrocytes undergoes changes, e.g., in pathological cases, in this work the relationship between the proportion of lipids in model membranes (Langmuir monolayers) and the effect of β-sitosterol was analyzed. The investigations were carried out for cholesterol/phosphatidylcholine (POPC)/sphingomyelin films differing in the proportion of cholesterol and containing the plant sterol in various concentrations. Additionally, the experiments based on systematic replacement of cholesterol in model membranes were performed. Based on the analysis of mean area and the excess area per molecule, compressional modulus and the excess free energy of mixing values as well as BAM images it was evidenced that the effect of the plant sterol depends on the composition of model membrane. However, it was also found that, in the investigated range of monolayer composition, the increase of total sterol content in the mixed film caused by the presence of β-sitosterol practically does not influence the molecular packing and interactions in the investigated monolayers. On the other hand, the replacement of cholesterol by β-sitosterol causes a decrease of condensation and weakens the interactions between molecules in the mixed film. These effects are the strongest for the systems of the highest content of cholesterol. The most important finding is that the variations in the composition of membranes, widely occurring in natural systems, may influence the effect of phytosterols.

Searching for the role of membrane sphingolipids in selectivity of antitumor ether lipid-edelfosine.

Edelfosine is a synthetic antitumor lipid of high selectivity. Its activity on membrane level inspired the investigations on edelfosine-lipid interactions to verify, which of the membrane components may be responsible for the selectivity of this drug. Because of overexpression of gangliosides in tumor progression and the ability of edelfosine to insert into membrane rafts, we have chosen two sphingolipids, i.e. sphingomyelin and ganglioside to investigate in mixtures with edelfosine. It was found that edelfosine shows strong affinity to ganglioside in contrast to sphingomyelin. Differences in the interactions of edelfosine with sphingolipids were analyzed from the point of view of the structure and shape of the interacting molecules. The comparison of the results with those previously reported for edelfosine mixed with other membrane components, allowed us to suggest that gangliosides may be considered as target molecules attracting edelfosine to tumor cells.