Glycerophospholipid polyunsaturation modulates resveratrol action on biomimetic membranes.

The phytoalexin resveratrol has received increasing attention for its potential to prevent oxidative damages in human organism. To shed further light on molecular mechanisms of its interaction with lipid membranes we study resveratrol influence on the organisation and mechanical properties of biomimetic lipid systems composed of synthetic phosphatidylcholines with mixed aliphatic chains and different degree of unsaturation at sn-2 position (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, PDPC). High-sensitivity isothermal titration calorimetric measurements reveal stronger spontaneous resveratrol association to polyunsaturated phosphatidylcholine bilayers compared to the monounsaturated ones resulting from hydrophobic interactions, conformational changes of the interacting species and desolvation of molecular surfaces. The latter is supported by the results from Laurdan spectroscopy of large unilamellar vesicles providing data on hydration at the glycerol backbones of glycerophospholipides. Higher degree of lipid order is reported for POPC membranes compared to PDPC. While resveratrol mostly enhances the hydration of PDPC membranes, increasing POPC dehydration is reported upon treatment with the polyphenol. Dehydration of the polyunsaturated lipid bilayers is measured only at the highest phytoalexin content studied (resveratrol/lipid 0.5 mol/mol) and is less pronounced than the effect reported for POPC membranes. The polyphenol effect on membrane mechanics is probed by thermal shape fluctuation analysis of quasispherical giant unilamellar vesicles. Markedly different trend of the bending elasticity with increasing resveratrol concentration is reported for the two types of phospholipid bilayers studied. POPC membranes become more rigid in the presence of resveratrol, whereas PDPC-containing bilayers exhibit softening at lower concentrations of the polyphenol followed by a slight growth without bilayer stiffening even at the highest resveratrol content explored. The new data on the structural organization and membrane properties of resveratrol-treated phosphatidylcholine membranes may underpin the development of future liposomal applications of the polyphenol in medicinal chemistry.

Resurrection Plants-A Valuable Source of Natural Bioactive Compounds: From Word-of-Mouth to Scientifically Proven Sustainable Use.

Resurrection plant species are a group of higher plants whose vegetative tissues are able to withstand long periods of almost full desiccation and recover quickly upon rewatering. Apart from being a model system for studying desiccation tolerance, resurrection plant species appear to be a valuable source of metabolites, with various areas of application. A significant number of papers have been published in recent years with respect to the extraction and application of bioactive compounds from higher resurrection plant species in various test systems. Promising results have been obtained with respect to antioxidative and antiaging effects in various test systems, particularly regarding valuable anticancer effects in human cell lines. Here, we review the latest advances in the field and propose potential mechanisms of action of myconoside-a predominant secondary compound in the European members of the Gesneriaceae family. In addition, we shed light on the possibilities for the sustainable use of natural products derived from resurrection plants.

2-Alkyl-Substituted-4-Amino-Thieno[2,3-d]Pyrimidines: Anti-Proliferative Properties to In Vitro Breast Cancer Models.

Thienopyrimidines are structural analogs of quinazolines, and the creation of new 2-alkyl derivatives of ethyl 4-aminothienopyrimidine-6-carboxylates for the study of their anti-proliferative properties is of great pharmacological interest. Some 2-alkyl-4-amino-thieno[2,3-d]pyrimidines 2-5 were synthesized, and their cyto- and phototoxicity against BALB 3T3 cells were established by an in vitro 3T3 NRU test. The obtained results indicate that the tested compounds are not cytotoxic or phototoxic, and that they are appropriate to be studied for their anti-proliferative and anti-tumor properties. The anti-proliferative potential of the compounds was investigated on MCF-7 and MDA-MB-231 cancer cells, as well as a MCF-10A cell line (normal human mammary epithelial cells). The most toxic to MCF-7 was thienopyrimidine 3 with IC50 13.42 µg/mL (IC50 0.045 µM), followed by compound 4 (IC50 28.89 µg/mL or IC50 0.11 µM). The thienopyrimidine 4 revealed higher selectivity to MCF-7 and lower activity (IC50 367 µg/mL i.e., 1.4 µM) than compound 3 with MCF-10A cells. With respect to MDA-MB-231 cells, ester 2 manifested the highest effect with IC50 52.56 µg/mL (IC50 0.16 µM), and 2-ethyl derivative 4 revealed IC50 62.86 µg/mL (IC50 0.24 µM). It was estimated that the effect of the substances on the cell cycle progression was due to cell cycle arrest in the G2 stage for MDA-MB-231, while arrest in G1 was detected for the estrogen (ER)-positive MCF-7 cell line. The tested compound's effects on the change of the zeta potential in the tumorigenic cells utilized in this study were determined. The calculation which we performed of the physicochemical properties and pharmacokinetic parameters influencing the biological activity suggested high intestinal absorption, as well as drug-likeness.

Antitumor Effect of Iscador on Breast Cancer Cell Lines with Different Metastatic Potential.

Studies were performed for the first time on the effect of Iscador Qu and Iscador M on phototoxicity, cytotoxicity, antiproliferative activity, changes in ξ-potential of cells, membrane lipid order, actin cytoskeleton organization and migration on three breast cancer lines with different metastatic potential: MCF10A (control), MCF-7 (low metastatic) and MDA-MB231 (high metastatic) cells. The tested Iscador Qu and M did not show any phototoxicity. The antiproliferative effect of Iscador species appeared to be dose-dependent and was related to the metastatic potential of the tested cell lines. A higher selectivity index was obtained for Iscador Qu and M towards the low metastatic MCF-7 cell line compared to the high metastatic MDA-MB-231. Iscador Qu demonstrated higher selectivity for both cancer cell lines compared to Iscador M. The malignant cell lines exhibited a decrease in fibril number and thickness regardless of the type of Iscador used. The strongest effect on migration potential was observed for the low metastatic cancer cell line MCF-7 after Iscador treatment. Both Iscador species induced a slight increase in the percentage of cells in early apoptosis for the low and high metastatic cell lines, MCF-7 and MDA-MB-231, unlike control cells. Changes in the zeta potential and membrane lipid order were observed for the low metastatic MCF-7 cell line in contrast to the high metastatic MDA-MB-231 cells. The presented results reveal a higher potential of Iscador as an antitumor agent for the low metastatic cancer cell line MCF-7 compared to the high metastatic one. Iscador Qu appears to be more potent compared to Iscador M, but at this point, the exact mechanism of action is still unclear and needs further investigations.

Valorphins alter physicochemical characteristics of phosphatidylcholine membranes: Datasets on lipid packing, bending rigidity, specific electrical capacitance, dipole potential, vesicle size.

Endogenous hemorphins are being intensively investigated as therapeutic agents in neuropharmacology, and also as biomarkers in mood regulation, inflammation and oncology. The datasets collected herein report physicochemical parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes in the presence of VV-hemorphin-5 (Val-Val-Tyr-Pro-Trp-Thr-Gln) and analogues, modified at position 1 and 7 by the natural amino acid isoleucine or the non-proteinogenic 2-aminoisobutyric, 2,3-diaminopropanoic or 2,4-diaminobutanoic amino acids. These peptides have been previously screened for nociceptive activity and were chosen accordingly. The present article contains fluorescence spectroscopy data of Laurdan- and di-8-ANEPPS- labelled large unilamellar vesicles (LUV) providing the degree of hydration and dipole potential of lipid bilayers in the presence of VV-hemorphin-5 analogues. Lipid packing is accessible from Laurdan intensity profiles and generalized polarization datasets reported herein. The data presented on fluorescence intensity ratios of di-8-ANEPPS dye provide dipole potential values of phosphatidylcholine-valorphin membranes. Vesicle size and electrophoretic mobility datasets included refer to the effect of valorphins on the size distribution and ζ -potential of POPC LUVs. Investigation of physicochemical properties of peptides such as diffusion coefficients and heterogeneous rate constant relates to elucidation of transport mechanisms in living cells. Voltammetric data of valorphins are presented together with square-wave voltammograms of investigated peptides for calculation of their heterogeneous electron transfer rate constants. Datasets from the thermal shape fluctuation analysis of quasispherical 'giant' unilamellar vesicles (GUV) are provided to quantify the influence of hemorphin incorporation on the membrane bending elasticity. Isothermal titration calorimetric data on the thermodynamics of peptide-lipid interactions and the binding affinity of valorphin analogues to phosphatidylcholine membranes are reported. Data of frequency-dependent deformation of GUVs in alternating electric field are included together with the values of the specific electrical capacitance of POPC-valorphin membranes. The datasets reported in this article can underlie the formulation and implementation of peptide-based strategies in pharmacology and biomedicine.

Resveratrol Affects Sphingolipid Metabolism in A549 Lung Adenocarcinoma Cells.

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity-apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)-in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies.

Design, Cytotoxicity and Antiproliferative Activity of 4-Amino-5-methyl-thieno[2,3-d]pyrimidine-6-carboxylates against MFC-7 and MDA-MB-231 Breast Cancer Cell Lines.

Novel 4-amino-thieno[2,3-d]pyrimidine-6-carboxylates substituted at the second position were prepared by cyclocondensation of 2-amino-3-cyano-thiophene and aryl nitriles in an acidic medium. The design of the target compounds was based on structural optimization. The derivatives thus obtained were tested in vitro against human and mouse cell lines. The examination of the compound effects on BLAB 3T3 and MFC-10A cells showed that they are safe, making them suitable for subsequent experiments to establish their antitumor activity. The photoirritancy factor of the compounds was calculated. Using the MTT test, the antiproliferative activity to MCF-10A, MCF-7 and MDA-MB-231 cell lines was estimated. The best antiproliferative effect in respect to the MCF-7 cell line revealed compound 2 with IC50 4.3 ± 0.11 µg/mL (0.013 µM). The highest selective index with respect to MCF-7 cells was shown by compound 3 (SI = 19.3), and to MDA-MB-231 cells by compound 2 (SI = 3.7). Based on energy analysis, the most stable conformers were selected and optimized by means of density functional theory (DFT). Ligand efficiency, ligand lipophilicity efficiency and the physicochemical parameters of the target 4-amino-thienopyrimidines were determined. The data obtained indicated that the lead compound among the tested substances is compound 2.

Electroporation, electrochemotherapy and electro-assisted drug delivery in cancer. A state-of-the-art review.

This review focuses on electrochemotherapy that consists in the delivery of anti-cancer drugs using high-voltage electrical pulses. Technical issues, choice of drugs, and protocol of drug delivery are still under investigation and no consensus has been achieved yet. The different aspects of electrochemotherapy are discussed in the present paper. It includes interrogations about the choice of the preferred anti-cancer drug and dose to be delivered on the solid tumors. Another promising area is related to the electro-assisted release of nanoparticles (quantum dots) in xenografted solid tumors. Molecular mechanisms of enhanced drug delivery are discussed in terms of high cholesterol level and large fraction of lipid rafts in cancer cells. Electrochemotherapy is a paradigmatic example of cooperation between physicists, biophysicists, chemists, technicians, manufacturers, biologists, clinicians, and patients to improve a very promising treatment delivery in line with the conception of personalized medicine.

Myconoside interacts with the plasma membranes and the actin cytoskeleton and provokes cytotoxicity in human lung adenocarcinoma A549 cells.

Studies have been carried out on the effects of the phenyl glycoside myconoside, extracted from the relict, Balkan endemic resurrection plant Haberlea rhodopensis on the plasma membrane structural organization and the actin cytoskeleton. Because the plasma membrane is the first target of exogenous bioactive compounds, we focused our attention on the influence of myconoside on the membrane lipid order and actin cytoskeleton in human lung adenocarcinoma A549 cells, using fluorescent spectroscopy and microscopy techniques. We found that low myconoside concentration (5 µg/ml) did not change cell viability but was able to increase plasma membrane lipid order of the treated cells. Higher myconoside concentration (20 µg/ml) inhibited cell viability by decreasing plasma membrane lipid order and impairing actin cytoskeleton. We hypothesize that the observed changes in the plasma membrane structural organization and the actin cytoskeleton are functionally connected to cell viability. Biomimetic membranes were used to demonstrate that myconoside is able to reorganize the membrane lipids by changing the fraction of sphingomyelin-cholesterol enriched domains. Thus, we propose a putative mechanism of action of myconoside on A549 cells plasma membrane lipids as well as on actin filaments in order to explain its cytotoxic effect at high myconoside concentration.

Resveratrol alters the lipid composition, metabolism and peroxide level in senescent rat hepatocytes.

Investigations were performed on the influence of resveratrol on the lipid composition, metabolism, fatty acid and peroxide level in plasma membranes of hepatocytes, isolated from aged rats. Hepatocytes were chosen due to the central role of the liver in lipid metabolism and homeostasis. The obtained results showed that the level of sphingomyelin (SM) and phosphatidylserine (PS) was augmented in plasma membranes of resveratrol-treated senescent hepatocytes. The saturated/unsaturated fatty acids ratio of the two most abundant membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was decreased as a result of resveratrol treatment. The neutral sphingomyelinase was found to be responsible for the increase of SM and the decrease of ceramide in plasma membranes of resveratrol-treated senescent hepatocytes. Using labeled acetate as a precursor of lipid synthesis we demonstrated, that resveratrol treatment resulted in inhibition mainly of phospholipid synthesis, followed by fatty acids synthesis. Resveratrol induced reduction of specific membrane-associated markers of apoptosis such as localization of PS in the external plasma membrane monolayer and ceramide level. Finally, the content of lipid peroxides was investigated, because the unsaturated fatty acids, which were augmented as a result of resveratrol treatment, are an excellent target of oxidative attack. The results showed that the lipid peroxide level was significantly lower, ROS were slightly reduced and GSH was almost unchanged in resveratrol-treated hepatocytes. We suggest, that one possible biochemical mechanism, underlying the reported resveratrol-induced changes, is the partial inactivation of neutral sphingomyelinase, leading to increase of SM, the latter acting as a native membrane antioxidant. In conclusion, our studies indicate that resveratrol treatment induces beneficial alterations in the phospholipid and fatty acid composition, as well as in the ceramide and peroxide content in plasma membranes of senescent hepatocytes. Thus, the presented results imply that resveratrol could improve the functional activity of the membrane lipids in the aged liver by influencing specific membrane parameters, associated with the aging process.

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix.

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an in vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress.