Olga Wesołowska

Interaction of the chemopreventive agent resveratrol and its metabolite, piceatannol, with model membranes.

Resveratrol and piceatannol are plant-derived polyphenols possessing extremely wide range of biological activities such as cancer chemopreventive, cardio- and neuroprotective, antioxidant, anti-inflammatory, anticancer and lifespan extending properties. Despite great interest in these stilbenes, their interactions with lipid bilayers have not been extensively studied. In the present work, the interaction of both resveratrol and piceatannol with model membranes composed of phosphatidylcholine (DMPC and DPPC) was investigated by means of fluorescence spectroscopy, differential scanning calorimetry (DSC) and electron spin resonance spectroscopy (ESR). Generalized polarization of two fluorescent probes Laurdan and Prodan measured in pure lipid and lipid:stilbene mixtures revealed that resveratrol and piceatannol changed bilayer properties in both gel-like and liquid crystalline phase and interacted with lipid headgroup region of the membrane. These findings were corroborated by DSC experiments in which the stilbene-induced decrease of lipid melting temperature and transition cooperativity were recorded. Resveratrol and piceatannol restricted also the ESR-measured mobility of spin probes GluSIN18, 5DSA and 16DSA with nitroxide group localized at different depths. Since the most pronounced effect was exerted on the spin probe located near membrane surface, we concluded that also ESR results pointed to the preferential interaction of resveratrol and piceatannol with headgroup region of lipid bilayer.

Trifluoperazine induces domain formation in zwitterionic phosphatidylcholine but not in charged phosphatidylglycerol bilayers.

The interaction of trifluoperazine with the zwitterionic lipids dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine and with anionic dimyristoylphosphatidylglycerol was studied by means of microcalorimetry and fluorescence spectroscopy. Intercalation of drug molecules into the lipid bilayers was confirmed by the observed differential scanning calorimetry peak broadening and the decrease in chain-melting temperatures. For trifluoperazine:lipid mole ratios higher than 0.4 and 0.6 (for dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, respectively) the deconvolution of transition profiles into two Gaussian components was possible, which suggests phase separation in the studied mixtures. Deconvolution of the thermograms was not possible for any of the drug:dimyristoylphosphatidylglycerol mole ratios studied. To confirm the existence of phase separation in trifluoperazine-phosphatidylcholine mixtures fluorescence spectroscopy experiments were performed using Laurdan as a probe. From the generalised polarisation versus excitation wavelength dependences, recorded at different temperatures, we conclude that a phase separation occurs in the gel state of the studied trifluoperazine-phosphatidylcholine mixtures. We attribute the existence of domains in the bilayer to the dissimilar interactions of two protonation forms of trifluoperazine with phosphatidylcholine molecules. Structural defects present at domain boundaries could be related to the trifluoperazine induced increase of membrane permeability and fluidity. This may partially explain the mechanism of multidrug resistance modulation by trifluoperazine.

8-Prenylnaringenin is an inhibitor of multidrug resistance-associated transporters, P-glycoprotein and MRP1

Flavonoids with hydrophobic e.g. prenyl substituents might constitute the promising candidates for multidrug resistance (MDR) reversal agents. The interaction of 8-prenylnaringenin (8-isopentenylnaringenin), a potent phytoestrogen isolated from common hop (Humulus lupulus), with two multidrug resistance-associated ABC transporters of cancer cells, P-glycoprotein and MRP1, has been studied for the first time. Functional test based on the transport of fluorescent substrate BCECF revealed that the flavonoid strongly inhibited MRP1 transport activity in human erythrocytes (IC(50)=5.76+/-1.80muM). Expression of MDR-related transporters in drug-sensitive (LoVo) and doxorubicin-resistant (LoVo/Dx) human colon adenocarcinoma cell lines was characterized by RT-PCR and immunochemical methods and elevated expression of P-glycoprotein in resistant cells was found to be the main difference between these two cell lines. By means of flow cytometry it was shown that 8-prenylnaringenin significantly increased the accumulation of rhodamine 123 in LoVo/Dx cells. Doxorubicin accumulation in both LoVo and LoVo/Dx cells observed by confocal microscopy was also altered in the presence of 8-prenylnaringenin. However, the presence of the studied compound did not increase doxorubicin cytotoxicity to LoVo/Dx cells. It was concluded that 8-prenylnaringenin was not able to modulate MDR in human adenocarcinoma cell line in spite of the ability to inhibit both P-glycoprotein and MRP1 activities. To our best knowledge, this is the first report of 8-prenylnaringenin interaction with clinically important ABC transporters.

Influence of silybin on biophysical properties of phospholipid bilayers

AbstractAim:Silybin (silibinin) is major biologically active flavonolignan extracted from milk thistle (Sylibum marianum). Its biological activities include hepato-protection, anticancer properties, and antioxidant- and membrane-stabilizing functions. Although membranes are postulated to be one of the cellular targets for silybin, little is known about its interaction with phospholipid bilayers.Methods:In the present work, the interactions of silybin with phosphatidylcholine bilayers were studied in detail using fluorescence spectroscopy, microcalorimetry and electron spin resonance techniques.Results:The results showed that silybin interacted with the surface of lipid bilayers. It affected the generalized polarization of the fluorescent probe Prodan, while not influencing the more deeply located Laurdan. Silybin lowered the main phospholipid phase transition temperature as judged by microcalorimetry, and caused the immobilization of spin probe Tempo-palmitate located on the surface of membranes. The mobility of spin probes 5- and 16-doxyl stearic acid was not affected by silybin. Silybin-induced quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence indicated that some flavonoid molecules partitioned into the hydrophobic region of membranes, which did not change significantly the biophysical properties of the deeper membrane regions.Conclusion:Such a behavior of silybin in membranes is in accordance with its postulated biological functions and neglectable side effects of therapies using silybin.

Interactions of Phenothiazines with Lipid Bilayer and their Role in Multidrug Resistance Reversal

The mechanism of multidrug resistance (MDR) reversal is not fully understood yet. Interaction of MDR modifiers with lipid bilayer of cell membranes and alterations of fluidity or other biophysical properties of plasma membrane might be an important factor in mechanism of MDR modulation and reversal. In this review we focus on phenothiazines which belong to the group of drugs known to modify MDR in different types of cells, from cancer cells up to various kinds of microorganisms. First, the aggregation properties of phenothiazines and their interactions with lipid bilayers are described. The localization of phenothazine derivative molecules in bilayers and alteration of membrane properties are discussed. Apart from the influence on model bilayers also the interactions of phenothiazines with cellular membranes (especially of erythrocytes) are reviewed. In subsequent sections the anti-MDR activity of phenothiazine derivatives observed in microorganisms and in cancer cells is described. The possible molecular mechanisms involved in MDR reversal by these compounds are presented. The direct interactions of phenothiazines with multidrug transporters and other effects of these modulators on plasma membranes are discussed. Finally, the structural features of phenothiazine derivatives essential for their optimal MDR reversal activity are described.

New phenothiazine-type multidrug resistance modifiers: anti-MDR activity versus membrane perturbing potency ☆

The phenothiazine multidrug resistance (MDR) modulators are chemically diversified but share the common feature to be hydrophobic cationic molecules. Molecular mechanisms of their action may involve interactions with either P-glycoprotein or membrane lipid matrix. In the present work we study the anti-MDR and biophysical membrane effects of new phenothiazine derivatives differing in the type of group substituting phenothiazine ring at position 2 (H-, Cl-, CF(3)-) and in the side chain group (NHCO(2)CH(3) or NHSO(2)CH(3)). Within each phenothiazine subset we found that anti-MDR activity (determined by P-glycoprotein inhibition assessed by flow cytometry) correlates with the theoretically calculated hydrophobicity value (logP) and experimental parameters (determined by calorimetry and fluorescence spectroscopy) of lipid bilayers. It is concluded that the biological and biophysical activity of phenothiazine derivatives depends more on the type of ring substitution than on the nature of the side chain group.

The alterations of lipid bilayer fluidity induced by newly synthesized phenothiazine derivative

Using fluorescence spectroscopy, calorimetry and ESR the interactions of the phenothiazine derivative 2-trifluoromethyl-10-(4-[methylsulfonylamid]buthyl)-phenothiazine (FPhMS) with lipids were studied. Calorimetry showed biphasic effect of FPhMS on main phase transition of DPPC. At molar ratios up to 0.06 drug induced decrease of transition temperature and enthalpy, while at higher concentrations it caused subsequent increase of these parameters. For all concentrations studied we observed gradual broadening of transition peaks. Fluorescence polarization revealed that in FPhMS/lipid mixtures, order in bilayers is decreased in the gel state and increased in the liquid crystalline state. ESR experiment showed that at molar ratio of 0.06, FPhMS reduces the mobility of spin probes located in both polar and hydrophobic regions. Comparing observed effects with those reported for cholesterol/lipid mixtures, we conclude that at higher concentrations FPhMS presumably induces a new mode of bilayer packing. This structure is less co-operative than an unperturbed bilayer, but locally the mobility of lipid molecules is decreased.

Polyphenols counteract tumor cell chemoresistance conferred by multidrug resistance proteins.

One of the main reasons of cancer resistance to chemotherapeutic treatment is the presence of different ABC multidrug transporters in plasma membranes. The transporters extrude wide spectrum of anticancer agents out of cancer cells at the expense of energy derived from ATP-hydrolysis. Plant-origin polyphenolic compounds, mainly flavonoids and stilbenes or their synthetic derivatives, can modulate the main ABC transporters responsible for cancer drug resistance, including P-glycoprotein, MRP1 and BCRP. The recent studies on different resistant cancer cell lines enabled the discovery of a number of polyphenolic compounds able to reverse drug resistance in vitro and these compounds could be promising candidates for further clinical trials. The review summarizes the recent advances in the field of polyphenols interaction with ATP-binding cassette multidrug transporters. The mechanism of flavonoids interactions with the multidrug transporters and the structure-activity relationship are also discussed.

Interaction of prenylated chalcones and flavanones from common hop with phosphatidylcholine model membranes.

Common hop (Humulus lupulus) constitutes a source of numerous prenylated chalcones such as xanthohumol (XH) and flavanones such as 8-prenylnaringenin (8-PN) and isoxanthohumol (IXH). Range of their biological activities includes estrogenic, anti-inflammatory, anti-infective, anti-cancer, and antioxidant activities. The aim of the present work was to characterize the influence of prenylated polyphenols on model 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes by means of differential scanning calorimetry (DSC), fluorescence and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies. All studied compounds intercalated into DPPC bilayers and decreased its melting temperature as recorded by DSC, Laurdan and Prodan fluorescence, and ATR-FTIR. Polyphenols interacted mainly with glycerol backbone and acyl chain region of membrane. Magnitude of the induced effect correlated both with lipophilicity and molecular shape of the studied compounds. Elbow-shaped 8-PN and IXH were locked at polar-apolar region with their prenyl chains penetrating into hydrophobic part of the bilayer, while relatively planar XH molecule adopted linear shape that resulted in its deeper insertion into hydrophobic region. Additionally, by means of DSC and Laurdan fluorescence IXH was demonstrated to induce lateral phase separation in DPPC bilayers in gel-like state. It was assumed that IXH-rich and IXH-poor microdomains appeared within membrane. Present work constitutes the first experimental report describing interactions of prenylated hop polyphenols with phospholipid model membranes.

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids.

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.