Incorporation of Pentacyclic Triterpenes into Mitochondrial Membrane

Article pubs.acs.org/JPCB

Incorporation of Pentacyclic Triterpenes into Mitochondrial MembraneStudies on the Interactions in Model 2D Lipid Systems Michał Flasiński,* Katarzyna Hąc-Wydro, and Marcin Broniatowski Department of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 3, 30-387 Kraków, Poland S Supporting Information *

ABSTRACT: Three representatives of naturally occurring pentacyclic triterpenes (PTs) were subjected to comprehensive studies aimed at the analysis of their interactions with phospholipids found naturally in mitochondrial membrane. To reach this goal, the selected compoundsα-amyrin (AMalf), betulinic acid (BAc), and ursolic acid (Urs)were incorporated into two-component and multicomponent Langmuir monolayers acting as a model of mitochondrial membrane. As the lipids characteristic for mitochondria, phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), and cardiolipin (BHCL) were chosen. Our studies were motivated by the fact that, according to the literature, the anticancer activity of PTs is correlated with their ability to incorporate into mitochondrial membrane and modify its properties. The undertaken studies were based on the surface pressure (π)−molecular area (A) isotherm registration complemented with the thermodynamic analysis and BAM visualization. It was found that all three terpenes with the exception of high betulinic acid proportion (30 and 50%) interact beneficially with POPC in two-component monolayers, while incorporation of BAc and Urs into POPE film is energetically unfavorable. As far as the model mitochondrial membrane composed of POPC/POPE/BHCL is concerned, the largest destructive influence (high positive values of ΔGExc and decrease of the model monolayer condensation) was found in the case of terpene acids, while the effect of α-amyrin was energetically favorable. We postulated that the origin of the observed findings is connected with the specific interactions between bolaamphlilic terpene acids and POPE, known from its propensity to form intermolecular hydrogen bonds.

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INTRODUCTION Naturally occurring pentacyclic triterpenes (PTs) prove their vast pharmaceutical potential in the fields of anticancer,1−4 antiinflammatory,5,6 and antimicrobial therapy7−9 as well as in Alzheimer treatment.10 PTs being the secondary plant metabolites are mainly stored in leaves, stem bark, fruit peels, and resins.11 In some plants, pentacyclic triterpenes occur together, whereas in other ones characteristic terpene can be predominant. These compounds constitute a large family of isoprenoid-based molecules synthesized in living plant organisms in the process of squalene cyclization.12,13 One of the commonly applied classifications divides them into three main groupslupanes, oleananes, and ursaneswhich differ in regard to the size of cycyloalkane ring E, the kind of its substituents, as well as the presence of a double bond in the C ring (Scheme 1). Over the last several years, there has been a growing body of evidence proving a wide spectrum of biological activity displayed by PTs. Naturally, one of the key properties that draws scientists’ special attention is the capability of terpenes to induce apoptosis in a variety of cancer cells including those known as multidrug resistant.14,15 According to the literature reports, anticancer activity of PTs and especially terpene acids is associated with their direct influence on cancer cell © 2014 American Chemical Society

mitochondria. Biological studies revealed that betulinic acid being probably the most active in this case acts selectively and concentration dependently toward tumor cells, sparing normal ones.16 It was proposed that, because of similarity in the chemical structure to molecules of sterols, terpene acids are able to incorporate into lipid membranes and as a result of this to modify their physicochemical properties.4,17,18 Such a behavior was observed for betulinic acid and its analogues which are capable of insertion into erythrocyte membranes, leading to the alteration of their morphology.18 In the mentioned study, the observed changes in the shape of normal red blood cells to stomatocytes or echinocytes were recognized to be responsible for the resistance to parasite infection. Similarly to cholesterol, terpene molecules when incorporated into lipid bilayers affect the fluidity and permeability of the membrane which may be responsible for morphological changes. Unfortunately, as far as the terpene molecules are concerned, there is lack of knowledge correlating their specific chemical structures with the properties at the level of cellular membrane. This fact should be of special interest bearing in Received: August 29, 2014 Revised: October 17, 2014 Published: October 21, 2014 12927

dx.doi.org/10.1021/jp508743j | J. Phys. Chem. B 2014, 118, 12927−12937

The Journal of Physical Chemistry B

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Scheme 1. Chemical Structures of the Investigated PTs

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EXPERIMENTAL SECTION Materials. Pentacyclic triterpenes (PTs) applied in our studiesα-amyrin (AMalf), betulinic acid (BAc), and ursolic acid (Urs)of the highest available purity (99%) were supplied by Sigma-Aldrich. Lipids applied to obtain the model mitochondrial membranesynthetic 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE), and cardiolipin extracted from bovine heart (BHCL)were products of the highest purity available (≥99%) from Avanti Polar Lipids, Inc. Cholesterol (≥99%) was purchased from Sigma-Aldrich. In routine experiments on one-component monolayers, 100−200 μL of 0.25 mg/mL lipid solution in chloroform/methanol 9/1 (v/v) mixture was deposited onto a water subphase with a Hamilton microsyringe, precise to 1.0 μL. In order to obtain two-component and multicomponent Langmuir films, mixed solutions of the declared compositions were prepared from the respective stock solutions. In each experiment, the monolayer was left after spreading for 10 min before the compression was initialized with a barrier speed of 20 cm2/min. Chloroform of spectroscopic purity (99.9% stabilized by ethanol) as well as methanol (99%) were provided by Sigma-Aldrich. As a subphase, ultrapure water of resistivity ≥18.2 MΩ·cm obtained from a Milli-Q system was applied. Methods. The experiments were performed with the NIMA (UK) Langmuir trough (total area = 300 cm2) placed on an antivibration table. Surface pressure was measured with an accuracy of ±0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The constant temperature during experiments (20 °C) was controlled thermostatically with the circulating water system. Brewster angle microscopy experiments were performed with an UltraBAM instrument (Accurion GmbH, Gö ttingen, Germany) equipped with a 50 mW laser emitting light of p polarization at a wavelength of 658 nm, a 10× magnification objective, a polarizer, an analyzer, and a CCD camera. The spatial resolution of the BAM was 2 μm. Composition of the Model Systems. In preliminary experiments aimed at analysis of the miscibility and interactions between three representatives of pentacyclic triterpenes AMalf, BAc, and Ursand main lipids occurring in mammalian mitochondrial membranePOPC and POPEtwo-component Langmuir monolayers of three PT proportions (10, 30, and 50%) were investigated. For experiments on the model mitochondrial membrane, three-component lipid mixtures of POPC/POPE/BHCL were prepared. The ratio of these lipids was according to the literature set as POPE/POPC = 0.75 and POPE/BHCL = 2.0,27 which corresponds to a composition of 47.05 mol % of POPC, 35.30 mol % of POPE, and 17.65 mol %

mind that PTs in contrast to cholesterol possess in molecules additional functional groups that shape their properties and may be responsible for the different membrane activity. The significance of the substituents’ reactivity in the context of PT behavior in a membrane environment was highlighted by the other authors.18 In our investigations, we applied the Langmuir monolayer technique in order to construct the most efficient model environment for the studies aimed at elucidating the interactions between pentacyclic triterpenes and lipids occurring in high proportion in mitochondrial membrane. It should be highlighted that such an approach is extensively applied because of numerous advantages, for example, uncomplicated experimental conditions maintenance, strict control of the monolayer composition, as well as effortless manipulation of the surface density by monolayer compression/expansion. Experiments were based on the surface pressure (π)−mean molecular area (A) isotherms registration for two-component monolayers composed of PTs and two main phospholipids found in mitochondrial membrane, i.e., POPC and POPE as well as for multicomponent model membrane imitating lipid composition of the inner mitochondrial membrane. On the basis of the registered isotherms, the compression modulus Cs−1 values were calculated for the studied monolayers to discuss their condensation. Moreover, the miscibility and interactions between components were estimated on the basis of the thermodynamic analysis carried out with the calculation of the excess free energy of mixing (ΔGExc). Additionally, the morphology of the studied surface films was visualized with Brewster angle microscopy (BAM) in order to verify miscibility and to observe phase separation. For our studies, we selected three representatives of the pentacyclic triterpene family, namely, two terpene acidsbetulinic acid (BAc) and ursolic acid (Urs)as well as an ursane-type compoundα-amyrin (AMalf). The choice of these terpenes was preceded by the preliminary studies presented in our previous articles.19,20 Betulinic acid was chosen because of its large anticancer potential linked to the ability of mitochondrial membrane permeabilization.21,22 In contrast, ursolic acid is rather known from its antimicrobial and anti-inflammatory activity.23,24 Moreover, our studies proved that this terpene reveals the ability to disintegrate cardiolipin rich domains, which is of importance taking into account that these negatively charged lipids are characteristic components of mitochondrial membrane.25,26 It is also important that Urs, on the contrary to BAc, does not discriminate over cardiolipins differing in chemical structures of hydrophobic fragments.19 The last among the terpenes of choice, α-amyrin, in preliminary experiments revealed strong energetically unfavorable interactions with cardiolipins; therefore, its influence on mitochondria mimicking membrane should be elucidated. 12928



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Figure 1. Surface pressure (π) vs mean molecular area (A) isotherms registered for monolayers of the investigated representatives of pentacyclic triterpenes: α-amyrin (AMalf), ursolic acid (Urs), and betulinic acid (BAc) as well as membrane lipids: cardiolipin (BHCl), cholesterol (Chol), POPC, and POPE.

pentacyclic triterpenes (Figure 1, left panel). The isotherm registered for the monolayer of AMalf possesses the most steep course, which indicates solid character of the studied surface film. Moreover, the surface pressure starts to rise at relatively the smallest molecular area (60 Å2/molecule). The monolayer collapses at 36 mN/m, which corresponds to 40 Å2/molecule. On the other hand, in the case of Urs and BAc monolayers, which can be characterized as liquid condensed (LC), π starts to increase at a notably larger mean area per molecule, i.e., ∼80 and 65 Å2, respectively. The other difference is connected with the surface pressure characteristic for the collapse of triterpene monolayers. It can be seen that this parameter decreases in the following order: AMalf > BAc > Urs. It should be however stressed here that these monolayers are very stiff which may often lead to deformation of the Wilhelmy plate during compression. This fact can result in lower reproducibility of the isotherms in the region of higher surface pressure, close to the monolayer collapse. In Figure 1 (right panel), π−A isotherms registered for monolayers of the investigated membrane lipids were gathered. In short, the virtually perpendicular isotherm of the cholesterol monolayer reveals the solid state of the surface film in the whole range of surface pressures up to the collapse of the film at 43 mN/m. One molecule of this sterol occupies in the monolayer an area of approximately 38 Å2. On the other hand, in the case of the isotherm for the cardiolipin monolayer, an increase of the surface pressure can be noticed at ca. 230 Å2/ molecule which is connected with a relatively very large crosssectional area of a BHCl molecule having four hydrophobic linoleoyl chains. The slope of the isotherm is rather mild, whereas the collapse of the monolayer can be observed at π = 45 mN/m, which corresponds to a molecular area of 105 Å2. The isotherms for two phospholipids, POPC and POPE, lay between those described above. The surface pressure starts to rise at relatively similar molecular areas of 96 and 101 Å2 for monolayers of POPE and POPC, respectively. The most important difference between both curves is connected with the presence of LE to LC phase transition seen in the course of the POPE isotherm at ∼34 mN/m.30

of BHCL. Into this mixture, the investigated triterpenes in proportion of 5, 15, and 30% were incorporated. The Analysis of the Isotherms. One of the parameters analyzed in this study was the compression modulus, expressed as Cs−1 = −A(dπ/dA).28 This parameter was calculated directly from the surface pressure (π)−mean area (A) isotherm and presented graphically as the Cs−1 (max) vs monolayer composition. Interactions between components in the mixed Langmuir monolayers were analyzed quantitatively on the basis of the excess free energy of mixing (ΔGExc), defined as ΔGExc = NA∫ π0 A12 − (A1X1 + A2X2) dπ, where A12 is the mean molecular area for a given composition of a binary film at a given surface pressure, A1 and A2 are the mean molecular areas for pure monolayers of components 1 and 2, respectively, taken at the same surface pressure, while X1 and X2 indicate molar fractions of the components in the mixture and NA is Avogadro’s number.29 In the thermodynamic analysis performed for a fourcomponent system, we were interested predominantly in the effects caused by the addition of a small (potentially pharmacological) proportion of PTs; therefore, in the calculations of ΔGExc, the model membrane of POPC/ POPE/BHCL of the fixed molar ratio was treated as one component.

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RESULTS AND DISCUSSION One-Component Monolayers and Binary Systems. In the initial step of our studies, interactions between three representatives of pentacyclic triterpenesAMalf, BAc, and Ursand main phospholipids occurring in mitochondrial membranes, i.e., POPC and POPE, were analyzed. For the clarity of data presentation, the surface pressure (π)−molecular area (A) isotherms registered for one-component monolayers of each lipid investigated in our contribution were presented in Figure 1. Additionally, in the Supporting Information, BAM images registered for one-component monolayers of BAc and Urs can be found. As can be seen in Figure 1, the obtained compression curves differ significantly in the case of all three investigated 12929



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Figure 2. Surface pressure (π) vs mean molecular area (A) isotherms registered for two-component monolayers of the investigated triterpenes and phospholipids POPC and POPE.

Figure 3. Maximal values of the compression modulus vs terpene proportion in the mixed two-component POPC/Terp and POPE/Terp systems.

according to applied triterpene, a characteristic kink in the course of the isotherm for mixed monolayers appears. This effect can be observed for AMalf/POPC 50%/50% and BAc/ POPC 30%/70% systems; however, it is not visible in the monolayer of Urs/POPC. This finding may suggest a phase separation occurring at higher surface pressures at which the one-component monolayer of terpene collapses. A similar effect takes place also for the mixed films with POPE; however, the

In the next step of our studies, surface pressure−area isotherms for two-component monolayers were registered and the obtained results were presented in Figure 2. It can be seen that, in the case of mixed monolayers with AMalf and BAc, isotherms for two-component films lay between curves characteristic for one-component monolayers. For mixtures with POPC, one can find that the collapse pressures change with the monolayer composition and, 12930



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Figure 4. Excess free energy of mixing (ΔGExc) calculated at the surface pressure of 10 (a, c) and 25 mN/m (b, d) for the investigated twocomponent monolayers containing POPC (top row) and POPE (bottom row) as well as terpenes in the proportion of 10, 30, and 50%.

situation at high π is additionally more complex because of the phase transition present in the monolayer of pure POPE. Interestingly, this phenomenon is also observed in the isotherms of mixed systems at very similar surface pressure (∼34 mN/m). Additional information regarding characteristics of the mixed monolayers, especially at the lower surface pressure, were obtained on the basis of parameters calculated directly from the isotherms. These results were presented and discussed below. On the basis of π−A isotherms for mixed POPC/Terp and POPE/Terp systems, the compression modulus was calculated and presented as a function of terpene content in Figure 3. Calculation of the compression moduli enables us to discuss the condensation of the surface film which provides not only the information on the Langmuir film state but also the mechanistic properties of a membrane mimicking system. As can be seen in Figure 3, monolayers of both phospholipids achieve a liquid condensed (LC) state, since the CS−1max values seen in the plots equal 135 and 242 mN/m for monolayers of POPC and POPE, respectively. On the other hand, as far as the compression moduli of terpene monolayers are concerned, CS−1max values reach 445, 110, and 110 mN/m for surface films of AMalf, BAc, and Urs, respectively, which is in accordance with previous studies.19,20 Interestingly, these differences in the condensation of pure terpene monolayers do not translate into the effects observed in the mixed films. In the case of POPC monolayer, it can be seen that the highest increase of the condensation can be observed in the presence of ursolic acid, whereas the effect is the weakest when BAc was applied. It is also worth mentioning that incorporation of only 10% of terpenes into the POPC monolayer causes small diminishing of its condensation, while, for a higher amount, the

trend is more complex. For 30% terpene, ursolic acid causes an increase of the monolayer condensation, whereas at the highest proportion of PT (50%) also for amyrin CS−1max is higher as compared to that value characteristic for pure POPC. The situation is different in the case of the phosphatidylethanolamine monolayer (Figure 3, right panel). One can find that, regardless of the applied terpene and its concentration in the binary system, the surface film of POPE always loses its condensation. Moreover, as can be seen in Figure 3, this effect is the strongest in the case of the smallest amount of triterpenes. It should be emphasized that modification of the model membrane condensation and in particular its decrease, as observed in the majority of studied systems, may implicate the activity of investigated terpenes in membranes rich in these monounsaturated phospholipids. In order to analyze the mixing and mutual interactions in the studied two-component systems, in the next step of our study, thermodynamic calculations based on the excess free energy of mixing (ΔGExc) were carried out. The results were presented in Figure 4. The results presented in Figure 4 differ significantly depending on the composition of the binary monolayers. The largest differences are connected with the class of membrane phospholipids: PC vs PE. One can find that the observed trends concerning signs of ΔGExc are similar for both surface pressures investigated in this study, i.e., 10 and 25 mN/m. Also, the mutual proportions between values of the excess free energy of mixing for the investigated terpene monolayers are similar at these surface pressures; however, at 25 mN/m, these values are in all cases notably larger. Generally, it can be seen that interactions of terpenes with the investigated lipids are thermodynamically favorable when these molecules are 12931



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Figure 5. Surface pressure (π) vs mean molecular area (A) isotherms registered for multicomponent monolayers imitating mitochondrial membrane mixed with the corresponding triterpene or cholesterol.

or plant stanol,33 where ΔGExc values are positive in the mixed monolayers with PE and negative with PC. The other problem that should be addressed here is the mutual miscibility of the binary monolayer components. To solve this problem, we need to combine information obtained from the isotherm analysis, microscopic observations, and thermodynamic considerations. In the case of PT/POPE monolayers, the situation is relatively simple, especially in the case of triterpene acid for which in two-component monolayers interactions are thermodynamically unfavorable. Moreover, in the BAM images registered for these systems (Figure S3, Supporting Information), small bright domains indicating phase separation appear at a surface pressure of ca. 20 mN/m. Finally, in the course of the π−A isotherms registered for triterpene/ POPE Langmuir films, strong alteration of curve course can be observed prior to the monolayer collapse. Such behavior is often observed for monolayers in which phase separation takes place.34 On the other hand, two collapses in the π−A isotherms registered for binary monolayers with POPC are only visible for the highest proportion of AMalf and 30 and 50% of BAc. Interestingly, in these cases, ΔGExc is positive, which may confirm reduced miscibility in the BAc/POPC system. For the remaining monolayers, i.e., AMalf/POPC and Urs/POPC, one can find that the components are miscible up to the relatively high surface pressure of ca. 30 mN/m, at which bright domains of the 3D phase appear at the air/water interface (Figure S2, Supporting Information). Multicomponent Systems. Let us now proceed to the results of the studies focused on the influence of selected pentacyclic triterpenes on the multicomponent monolayer imitating mitochondrial membrane. As indicated in the Experimental Section, this model membrane was composed of POPC, POPE, and cardiolipin characteristic of mammalian mitochondria. Into this artificial system, the investigated PTs were added in molar proportion of 5, 10, and 15%. Additionally, for comparison, a model mitochondrial membrane containing 5% cholesterol was prepared. All of the π−A isotherms registered for multicomponent monolayers were compiled in Figure 5. As can be seen in Figure 5, only in the case of AMalf in the multicomponent system, the π−A isotherms move gradually

incorporated into the monolayer of POPC, which manifests in the negative values of the excess free energy of mixing (Figure 4a and b). On the other hand, in the mixtures of POPE/PTs, the sign of ΔGExc is positive with the only exception being the monolayer containing 30% α-amyrin. This means that interactions in the mixed films are more repulsive or less attractive as compared to the situation found in the onecomponent monolayers of the respective compounds. There are also significant differences between the terpenes. Namely, in the case of the mixed monolayer with POPC, the largest negative values of ΔGExc can be found for AMalf (10%), Urs (30%), and both of these terpenes when added in the largest proportion tested (50%). It should also be pointed out that incorporation of PTs into these systems results in the increase of their stability. The obtained results show that, in contrast to AMalf and Urs, betulinic acid behaves different when included in PC film and in the case of its higher proportion, i.e., 30 and 50%, values of ΔGExc are close to zero. Thermodynamic considerations lead to considerably different conclusions when we move to the results obtained for monolayers containing POPE. At first glance, it can be seen that, in the case of ursolic acid (10 and 30%), the interactions in binary films are the most unfavorable among all the investigated systems, whereas for the highest proportion of terpene (50%), in the presence of BAc, the largest positive values of the excess function were found (Figure 4d). Interestingly, when changing the proportion of the terpene in the binary monolayer, a different trend can be observed for BAc and Urs. Namely, with the increase of betulinic acid proportion, the interactions in the surface film become thermodynamically more unfavorable, whereas the situation is opposite for the second triterpene acid. The third investigated PT, α-amyrin, behaves in the mixed monolayer with POPE very different as compared to BAc and Urs. In this case, ΔGExc values oscillate around zero, depending on the exact proportion of AMalf in the binary film. The results presented above revealed that POPE but not POPC discriminates between triterpenes possessing different structural motifs: bolaamphiphilic molecules of terpene acids vs monoamphiphilic AMalf.31 At the same time, it is worth mentioning here that both betulinic and ursolic acid interact in binary monolayers with PC and PE in the same way as cholesterol32 12932



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and 113 mN/m for Urs and BAc, respectively. On the other hand, with the exception of betulinic acid, when a larger amount of terpenes was added (30%), the condensation of the monolayer was notably higher. The obtained results show that, among the investigated PTs, α-amyrin and Urs (excluding the proportion of 5%) reveal a weak condensing effect on the model system, whereas BAc causes disorganization of the artificial mitochondrial membrane. The above-mentioned finding concerning AMalf can be explained taking into account that this terpene reveals the largest condensation in the onecomponent monolayer (solid state), which makes it similar to the surface films of sterols whose molecules are also monoaphiphilic. To attain quantitative information regarding interactions of the investigated terpenes with the main components of the mitochondrial membrane, the excess free energy of mixing was calculated for this four-component system. The obtained results were presented in Figure 7. The values of the excess free energy of mixing (ΔGExc) are positive in the case of terpene acids within the whole range of the studied concentrations, while for α-amyrin the excess function has a negative sign. This observation concerns monolayers studied at both surface pressures (10 and 25 mN/m); however, just like in the case of two-component films, the absolute values of ΔGExc are larger at higher π. The most unfavorable interactions were found in the presence of BAc in mixed films, virtually regardless of the terpene content. On the other hand, the effect of Urs and AMalf is concentration dependentgenerally, in both cases, the larger the proportion of terpene, the more beneficial the interactions become. The trend observed in these results attained for the model membrane is similar to tendencies found in the studies on two-component monolayers containing POPE. In both of these cases, terpene acids influence lipid monolayers to a similar extentinteractions are energetically unfavorable, and absolute values of ΔGExc are very high. Furthermore, different behavior of AMalf is also characteristic for two- and four-component systems containing monounsaturated PE. In conclusion, it could be stated that, as far as the model membrane is concerned, the observed behavior may be attributed to the presence of POPE (35.5%) rather than other lipids. Taking together the results of condensation and thermodynamic analysis, it can be concluded that the strongest destructive effect toward the model mitochondrial membrane reveals betulinic acid, even if its concentration is as low as 5%.

with triterpene content toward lower molecular areas. Additionally, the course of the isotherms for monolayers containing 5% α-amyrin and cholesterol is nearly the sameboth curves overlap. The situation is different for the remaining two systems where isotherms corresponding to mixed monolayers do not lay below the curve characteristic for the surface film without terpene. In order to obtain more information concerning mutual interactions between the studied terpenes and the model membrane, additional parameters were calculated on the basis of registered compression curves. As far as the degree of condensation of the model mitochondrial membrane is concerned, it was found that the maximal value of CS−1 equals 145 mN/m, indicating that this surface film is in a liquid condensed (LC) state (Figure 6).

Figure 6. Maximal values of the compression modulus vs cholesterol/ terpene proportion in the mixed monolayer imitating mitochondrial membrane.

Addition of 5% cholesterol into this mixture does not change the condensation noticeably (i.e., only to ∼154 mN/m), which is an important finding, since the content of this sterol in the outer leaflet of the mitochondrial membrane is very small.27,35 Interestingly, a very similar result was found when, instead of cholesterol, 5% α-amyrin was added into the artificial membrane. In contrast, a significantly larger and opposite effect was observed for the investigated triterpene acids. In these cases, the surface film decreases its condensation to 125

Figure 7. Excess free energy of mixing (ΔGExc) calculated at surface pressures of 10 (a) and 25 mN/m (b) for the investigated multicomponent monolayers mimicking mitochondrial membrane containing 5, 15, and 30% terpenes. 12933



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Figure 8. BAM images for multicomponent monolayers imitating mitochondrial membrane that contains 5 and 30% BAc (top and middle rows) and 5% Urs (bottom row). The values presented in the left top corners of the photos indicate the surface pressure at which the image was recorded.

The second triterpene acid, Urs, shows qualitatively similar behavior, however, of a significantly smaller potency. Finally, as far as AMalf is concerned, it demonstrates completely different, adverse activity. To elucidate the problem of morphology in the investigated model lipid membranes, in situ visualization with a BAM microscope was performed. The collected images were gathered in Figure 8. At this point, it should be highlighted that both the one-component and mixed monolayers containing α-amyrin as well as the model POPC/POPE/ BHCL membrane were found to be homogeneous in the whole range of surface pressures; therefore, we presented here only images for mixed monolayers containing BAc and Urs. In Figure 8, BAM images taken for mixed monolayers of mitochondrial model membrane and terpene acids were presented. In both cases at 5% of PTs surface films lose their homogeneity at ca. 20−25 mN/m when in images very small bright spots appear. With the further compression, their number and size grow and at π = 30−40 mN/m they cover a large area of the images. A markedly different morphology can be observed in the case of BAc added to the model membrane in the higher proportion (30%). In this case, brighter grass-like domains can be seen at the interface from the very low surface pressure until the massive phase separation that proceeds from ca. 30 mN/m. This BAM visualization corroborates previous conclusions based on the thermodynamic analysisBAc and Urs reveal limited mixing with lipids of the model membrane, leading to the phase separation occurring especially at the higher surface pressures. On the other hand, as was mentioned above, addition of AMalf into the model mitochondrial membrane does not lead to the phase separation. This fact

together with the beneficial interactions provides strong evidence for the miscibility in the AMalf/POPC/POPE/ BHCL system. In the next step of our studies, the comparison of PTs and cholesterol influence on the model mitochondrial membrane was performed. Both terpenes and sterol were added to the artificial membrane in a small, 5% amount. This proportion was applied, since it is equal to the content of cholesterol found naturally in the membrane of mammalian mitochondria.35 The obtained results gained at a surface pressure of 25 mN/m were compared in Figure 9. It can be noticed that the smallest impact on the model membrane reveals cholesterol and α-amyrin, for which the values of ΔGExc equal 100 and −70 J/mol, respectively. For cholesterol, this result is rather expected bearing in mind that according to some literature reports this sterol at very small proportion can occur in the outer leaflet of the mitochondrial membrane. On the other hand, it could be expected that AMalf is ineffective in modulation of these membrane properties and cannot be applied pharmaceutically as a mitochondrial toxin of cancer cells. Significantly stronger interactions were found in the case of terpene acids, especially for BAc which is known to be the most effective in cancer therapy.22 It is worth mentioning that the observed positive sign of ΔGExc, indicating energetically unfavorable interactions between terpenes and components of the model membrane, means that incorporation of BAc and Urs into the mitochondrial membrane causes a destructive effect. The following questions arise here: What is the potential mechanism of mitochondrial toxicity, and why are terpene acids more potent in this activity than amyrins? The studies on literature reports concerning this issue suggest that 12934



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no longer energetically favorable. Thus, it is possible that at a particular surface pressure value the reorientation of the terpene molecules begins. However, the turnover of the terpene molecule can be destructive for the organization of the adjacent phospholipid molecules and in consequence can lead to local collapse and nucleation of 3D domains. In the case of BAc, another problem is the presence of an iso-allyl substituent at C19, which may cause additional destabilization of the reversed orientation and can be a reason for the strongest unfavorable interactions observed in the mixed monolayers containing this terpene. Apart from the above discussion, there is another fact that should be commented here. As we argued in our previous paper concerning the studies on the interactions between PTs and anionic membrane phospholipids (CLs and PGs), α-amyrin shows the highest propensity to disintegrate cardiolipin rich membranes, including mitochondria.20 On the basis of those results, AMalf was selected to the present investigations as a molecule of potentially high activity that could be applied in anticancer therapy. Now it is necessary to correlate all these results. On one hand, AMalf is able to destruct BHCL containing domains but, when incorporated into the model mitochondrial membrane that apart from cardiolipin contains a high proportion of POPC and POPE, the overall effect is different. The reason for such behavior is probably connected with the compensation of unfavorable interactions in the AMalf/BHCl system with strong beneficial interactions between amyrin and POPC. It is worth highlighting that the studies performed with the application of model membranes enable us to draw conclusions regarding interactions between lipid molecules, as long as we correlate results from the simplest two-component systems with those obtained in membrane mimicking multilipid films. The aim of the undertaken studies was to shed new light onto the influence of three representatives of pentacyclic triterpenesα-amyrin, betulinic, and ursolic acidon the model membrane of mammalian mitochondria. The motivation for such investigations comes from the fact that until now relatively little is known about the mechanisms of membranerelated activity of PTs. On the other hand, in the literature, there are reports claiming that terpene acids, like BAc and Urs, are capable of apoptosis induction in tumor cell lines.2,4 It is also accepted that the target site of this activity is mitochondrial membrane.37 Therefore, for our studies, we constructed an artificial multicomponent membrane composed of lipids in ratios characteristic for the membrane of this organelle. Additionally, in order to systematize the knowledge about terpene−phospholipid interactions, investigations on binary lipid films complemented these studies. In our research, we applied the Langmuir monolayer technique combined with Brewster angle microscopy dedicated for in situ visualization of the surface films. The major conclusions were attained on the basis of the thermodynamic analysis carried out for the surface pressure−molecular isotherms registered for mixed (model) systems. The other important parameter, i.e., condensation of the model membranes, was discussed on the basis of the calculated compression modulus. The main conclusions from the performed studies were summarized below.

Figure 9. Comparison of the excess free energy of mixing (ΔGExc) values calculated at a surface pressure of 25 mN/m for the investigated multicomponent monolayers mimicking mitochondrial membrane containing 5% cholesterol and respective terpenes.

the generation of reactive oxygen species (ROS) caused by the incorporation of PTs may be responsible for phospholipid peroxidation leading to the membrane permeation.17 However, the exact mechanism of this process has never been elucidated. The explanation based on ROS activity seems to be justified when we consider a real biological membrane in the cellular activity of a living organism, but in the simplified (model) system studied herein, this is not the case. To find the reasons of the obtained results, at the beginning, we must refer to the differences in the molecular structures of the investigated terpene acids and AMalf. Both BAc and Urs are bolaamphlilic molecules possessing in their chemical structures two polar groups which depending on experimental conditions may play the function of a polar headgroup.31,36 Our previous studies carried out with application of synchrotron radiation (GIXD technique) and BAM observation revealed that both phases (i.e., perpendicular with the OH group in contact with water and tilted with the COOH group immersed in the subphase) in the monolayer of BAc are present at the lower surface pressure.31 In contrast, α-amyrin is a molecule possessing, instead of a carboxylic group, a methyl group, which implicates only one possible orientation at the air/water interface. These facts characterize terpene molecules, but on the other hand, of the utmost importance is also the structure of membrane phospholipids. Our results show that the presence of POPE in the model membrane can be responsible for the discrimination between terpene acids and α-amyrin. Phosphatidylethanolamines are known for their propensity to form intermolecular hydrogen bonding with adjacent molecules in the monolayer as well as with water. This property is directly connected with the presence of an ethanolamine group being a relatively strong Hbond donor. As far as the molecules of terpene acids are concerned, among two polar groups, OH at the C3 carbon atom and COOH at C28, the second one is a stronger hydrogen bond acceptor. Therefore, it is possible that the interactions between the ethanolamine headgroup of POPE and the carboxylic substituents in BAc and Urs molecules enforce this tilted orientation of acid molecules at the interface. Such organization is possible at low surface pressures, but upon compression, the monolayers try to achieve higher condensation and the tilted orientation of a triterpene acid molecule is

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CONCLUSIONS Our experiments revealed that all three terpenes with exception of higher BAc proportion (30 and 50%) interact preferentially with POPC in two-component Langmuir monolayers, whereas incorporation of BAc and Urs into POPE film is energetically 12935



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Molecular Targets, Pharmacokinetics and Clinical Studies. Biochem. Pharmacol. 2013, 85, 1579−1587. (4) Gopal, D. V. R.; Narkar, A. A.; Badrinath, Y.; Mishra, K. P.; Joshi, D. S. Betulinic Acid Induces Apoptosis in Human Chronic Myelogenous Leukemia (CML) Cell Line K-562 Without Altering the Levels of Bcr-Abl. Toxicol. Lett. 2005, 155, 343−351. (5) Ikeda, Y.; Murakami, A.; Ohigashi, H. Ursolic Acid: An anti- and Pro-inflammatory. Mol. Nutr. Food Res. 2008, 52, 26−42. (6) Sami, A.; Taru, M.; Salme, K.; Jari, Y.-K. Eur. Pharmacological Properties of the Ubiquitous Natural Product Betulin. J. Pharm. Sci. 2006, 29, 1−13. (7) Baltina, L. A.; Flekhter, O. B.; Nigmatullina, L. R.; Boreko, E. I.; Pavlova, N. I.; Nikolaeva, S. N.; Savinova, O. V.; Tolstikov, G. A. Lupane Triterpenes and Derivatives with Antiviral Activity. Bioorg. Med. Chem. Lett. 2013, 13, 3549−3552. (8) Dominguez-Carmona, D. B.; Escalante-Erosa, F.; Garcia-Sosa, K.; Ruiz-Pinell, G.; Gutierrez-Yapu, D.; Chan-Bacab, M. J.; GiménezTurba, A.; Peña-Rodriguez, L. M. Antiprotozoal Activity of Betulinic Acid Derivatives. Phytomedicine 2010, 17, 379−382. (9) Pavlova, N. I.; Savinovaa, O. V.; Nikolaevaa, S. N.; Borekoa, E. I.; Flekhterb, O. B. Antiviral Activity of Betulin, Betulinic and Betulonic Acids Against Some Enveloped and Non-enveloped Viruses. Fitoterapia 2003, 74, 489−492. (10) Yoo, K.-Y.; Park, S.-Y. Terpenoids as Potential Anti-Alzheimer’s Disease Therapeutics. Molecules 2012, 17, 3524−3538. (11) Jager, S.; Trojan, H.; Kopp, T.; Laszczyk, M.; Scheffler, A. Pentacyclic Triterpene Distribution in Various Plants − Rich Sources for a New Group of Multi-Potent Plant Extracts. Molecules 2009, 14, 2016−2031. (12) Nes, D. W.; Heftmann, E. A Comparison of Triterpenoids with Steroids as Membrane Components. J. Nat. Prod. 1981, 44, 377−400. (13) Xu, R.; Fazio, G. C.; Matsuda, S. P. T. On the Origins of Triterpenoid Skeletal Diversity. Phytochemistry 2004, 65, 261−291. (14) Selzer, E.; Pimentel, E.; Wacheck, V.; Schlegel, W.; Pehamberger, H.; Jansen, B.; Kodym, R. Effects of Betulinic Acid Alone and in Combination with Irradiation in Human Melanoma Cells. J. Invest. Dermatol. 2000, 114, 935−940. (15) Li, Y.; He, K.; Huang, Y.; Zheng, D.; Gao, C.; Cui, L.; Jin, Y.-H. Betulin Induces Mitochondrial Cytochrome c Release Associated Apoptosis in Human Cancer. Cells. Mol. Carcinog. 2010, 49, 630−640. (16) Gao, M.; Lau, P. M.; Kong, S. K. Mitochondrial Toxin Betulinic Acid Induces In Vitro Eryptosis in Human Red Blood Cells Through Membrane Permeabilization. Arch. Toxicol. 2014, 88, 755−768. (17) Samudio, I.; Konopleva, M.; Pelicano, H.; Huang, P.; Frolova, O.; Bornmann, W.; Ying, Y.; Evans, R.; Contractor, R.; Andreeff, M. A Novel Mechanism of Action of Methyl-2-cyano-3,12 Dioxoolean-1,9 Diene-28-oate: Direct Permeabilization of the Inner Mitochondrial Membrane to Inhibit Electron Transport and Induce Apoptosis. Mol. Pharmacol. 2006, 69, 1182−1193. (18) Ziegler, H. L.; Franzyk, H.; Sairafianpour, M.; Tabatabai, M.; Tehrani, M. D.; Bagherzadeh, K.; Hagerstrand, H.; Staerk, D.; Jaroszewski, J. W. Erythrocyte Membrane Modifying Agents and the Inhibition of Plasmodium Falciparum Growth: Structure−Activity Relationships for Betulinic Acid Analogues. Bioorg. Med. Chem. 2004, 12, 119−127. (19) Broniatowski, M.; Flasiński, M.; Zięba, K.; Miśkowiec, P. Interactions of Pentacyclic Triterpene Acids with Cardiolipins and Related Phosphatidylglycerols in Model Systems. Biochim. Biophys. Acta 2014, 1838, 2530−2538. (20) Broniatowski, M.; Flasiński, M.; Zięba, K.; Miśkowiec, P. Langmuir Monolayer Studies of the Interaction of Monoamphiphilic Pentacyclic Triterpenes with Anionic Mitochondrial and Bacterial Membrane Phospholipids  Searching for the Most Active Terpene. Biochim. Biophys. Acta 2014, 1838, 2460−2472. (21) Fulda, S.; Scaffidi, C.; Susin, S. A.; Krammer, P. H.; Kroemer, G.; Peter, M. E.; Debatin, K. M. Activation of Mitochondria and Release of Mitochondrial Apoptogenic Factors by Betulinic Acid. J. Biol. Chem. 1998, 273, 33942−33948.

unfavorable. This obviously results in the behavior of these PTs in a more complex model membrane system that contained POPC (47.05%), POPE (35.30%), and BHCL (17.65%). It was found that a large destructive effect (expressed here as the high positive values of ΔGExc) on the model mitochondria membrane is manifested by terpene acids, while the influence of α-amyrin is energetically favorable. Our studies show that PTs are active toward the artificial membrane even at a concentration as low as 5%, which is a good prognostic for potential pharmacological application of these compounds. We also found that incorporation of terpene acids into the investigated model membrane causes a decrease of the condensation, which distinguishes the activity of these molecules from the properties of cholesterol and α-amyrin. It is worth mentioning that verification of immiscibility in the studied systems was possible with application of the BAM technique which makes it possible to observe phase separation for the systems in which thermodynamic analysis proved energetically unfavorable interactions. Finally, we proposed that the presence of POPE in mixed monolayers is responsible for the disorganizing properties of PT acids. To elucidate this finding, we must refer to the properties of BAc and Urs in onecomponent monolayers.31 It is possible that, in the presence of POPE being a hydrogen bond donor, terpene acids organize at the air/water interface with their H bond acceptor COOH group immersed into the subphase. Such organization allowable at low surface pressure cannot be maintained during compression, which causes disruption of the monolayer and as a result phase separation. In conclusion, our studies revealed that, in contrast to AMalf, terpene acids are able to directly disorganize artificial POPC/POPE/BHCL membrane which may be the crucial step in the process of tumor mitochondria disruption caused by BAc and Urs.

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ASSOCIATED CONTENT

S Supporting Information *

Selected BAM images recorded during compression of both one- and two-component monolayers. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: fl[email protected]. Fax: +48 0-12-634-05-15. Phone: +48 0-12-664-67-97. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was financed by the National Science Centre (No. DEC-2012/05/B/ST5/00287). The research was carried out with the equipment (UltraBAM) purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Contract No. POIG.02.01.00-12-023/08).

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REFERENCES

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Incorporation of Pentacyclic Triterpenes into Mitochondrial Membrane

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