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.

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.

Effect of Quercetin and Fingolimod, Alone or in Combination, on the Sphingolipid Metabolism in HepG2 Cells.

Combinations of anti-cancer drugs can overcome resistance to therapy and provide new more effective treatments. In this work we have analyzed the effect of the polyphenol quercetin and the anti-cancer sphingosine analog fingolimod on the sphingolipid metabolism in HepG2 cells, since sphingolipids are recognized as mediators of cell proliferation and apoptosis in cancer cells. Treatment of hepatocellular carcinoma HepG2 cells with quercetin and fingolimod, alone or in combination, induced different degrees of sphingomyelin (SM) reduction and a corresponding activation of neutral sphingomyelinase (nSMase). Western blot analysis showed that only treatments containing quercetin induced up-regulation of nSMase expression. The same treatment caused elevation of ceramide (CER) levels, whereas the observed alterations in sphingosine (SPH) content were not statistically significant. The two tested drugs induced a reduction of the pro-proliferative sphingolipid, sphingosine 1 phosphate (S1P), in the following order: quercetin, fingolimod, quercetin + fingolimod. The activity of the enzyme responsible for CER hydrolysis, alkaline ceramidase (ALCER) was down-regulated only in the incubations involving quercetin and fingolimod did not affect this activity. The enzyme, maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was down-regulated by incubations in the following order: quercetin, fingolimod, quercetin + fingolimod. Western blot analysis showed down-regulation in SK1 expression upon quercetin but not upon fingolimod treatment. Studies on the effect of quercetin and fingolimod on the two proteins associated with apoptotic events, AKT and Bcl-2, showed that only quercetin, alone or in combination, down-regulated the activity of the two proteins. The reported observations provide information which can be useful in the search of novel anti-tumor approaches, aiming at optimization of the therapeutic effect and maximal preservation of healthy tissues.

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.

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.

Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models.

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.

Dimethylsphingosine and miltefosine induce apoptosis in lung adenocarcinoma A549 cells in a synergistic manner.

Lung cancer is one of the most common and lethal types of oncological diseases. Despite the advanced therapeutic approaches, the prognosis for lung cancer still remains poor. Apparently, there is an imperative need for more efficient therapeutic strategies. In this work we report that concurrent treatment of human adenocarcinoma A549 cells with specific concentrations of two antitumor agents, the sphingosine kinase 1 inhibitor N, N dimethylsphingosine (DMS) and the alkylphosphocholine miltefosine, induced synergistic cytotoxic effect, which was confirmed by calculation of the combination index. The simultaneous action of these agents, induced significant decrease of A549 cell number, as well as pronounced morphological alterations. Combined drugs caused substantial apoptotic events, and significant reduction of the pro-survival marker sphingosine- 1-phosphate (S1P), when compared to the individual treatments with each of the anticancer drugs alone. Miltefosine is known to affect the synthesis of choline-containing phospholipids, including sphingomyelin, but we report for the first time that it also reduces S1P. Here we suggest a putative mechanism underlying the effect of miltefosine on sphingosine kinase 1, involving miltefosine-induced inhibition of protein kinase C. In conclusion, our findings provide a possibility for treatment of lung cancer cells with lower concentrations of the two antitumor drugs, DMS and miltefosine, which is favorable, regarding their potential cytotoxicity to normal cells.

Statins and Alkylphospholipids as New Anticancer Agents Targeting Lipid Metabolism.

The partial efficacy and high toxicity of the current anticancer chemotherapeutics as well as the development of multiple drug resistance are the major problems in cancer therapy. Therefore, there is an emergency need for the development of novel well-tolerated anticancer agents with different mode of action that could be successfully used in combination with other drugs as an adjuvant therapy. The inhibition of intracellular signaling pathways associated with cancer growth and invasiveness is a main therapeutic approach in cancer treatment. It is well known that lipid metabolism is involved in the regulation of key cellular processes such as proliferation, differentiation and apoptosis. Statins and alkylphospholipids are both relatively new synthetic agents with considerable anticancer properties that disturb lipid metabolism and subsequently modulate proliferation and cell survival signaling pathways, leading to apoptosis. Numerous in vitro and in vivo studies have shown promising results for the use of statins and alkylphospholipids as potential therapeutic agents in the treatment of various human malignancies. However, more investigations and clinical trials are needed to assess their optimal safe dose and maximal efficacy and better understand the molecular mechanisms underlying the antitumor effects of these drugs.

Antitumor Lipids--Structure, Functions, and Medical Applications.

Cell proliferation and metastasis are considered hallmarks of tumor progression. Therefore, efforts have been made to develop novel anticancer drugs that inhibit both the proliferation and the motility of tumor cells. Synthetic antitumor lipids (ATLs), which are chemically divided into two main classes, comprise (i) alkylphospholipids (APLs) and (ii) alkylphosphocholines (APCs). They represent a new entity of drugs with distinct antiproliferative properties in tumor cells. These compounds do not interfere with the DNA or mitotic spindle apparatus of the cell, instead, they incorporate into cell membranes, where they accumulate and interfere with lipid metabolism and lipid-dependent signaling pathways. Recently, it has been shown that the most commonly studied APLs inhibit proliferation by inducing apoptosis in malignant cells while leaving normal cells unaffected and are potent sensitizers of conventional chemo- and radiotherapy, as well as of electrical field therapy. APLs resist catabolic degradation to a large extent, therefore accumulate in the cell and interfere with lipid-dependent survival signaling pathways, notably PI3K-Akt and Raf-Erk1/2, and de novo phospholipid biosynthesis. They are internalized in the cell membrane via raft domains and cause downstream reactions as inhibition of cell growth and migration, cell cycle arrest, actin stress fibers collapse, and apoptosis. This review summarizes the in vitro, in vivo, and clinical trials of most common ATLs and their mode of action at molecular and biochemical levels.

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.

Fluorescent labeling techniques for investigation of fibronectin fibrillogenesis (labeling fibronectin fibrillogenesis).

Fibronectin fibrillogenesis is a cell-mediated, step-wise process that converts soluble fibronectin into insoluble fibronectin matrix. The deposition of fibronectin fibrils occurs at specific sites on the cell surface and depends on the unfolding of the fibronectin dimer. Fibronectin matrix provides positional information for cell migration during early embryogenesis and plays an important role in cell growth, differentiation, survival, and oncogenic transformation. Here we present simple techniques, based on the use of fluorescently labeled fibronectin and species-specific antifibronectin antibodies that allow determination of the fibronectin fibril growth in conventional in vitro cell cultures and in three-dimensional matrix environment.

Three-dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway.

Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion-mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three-dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady-state levels of ERK1/2 activity in primary human fibroblasts, induced by cell-derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5-fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo-like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.

Halothane affects focal adhesion proteins in the A 549 cells.

Halothane is a volatile anaesthetic, which is known to induce alterations in cellular plasma membranes, modulating the physical state of the membrane lipids and/or interacting directly with membrane-bound proteins, such as integrin receptors. Integrin-mediated cell adhesion is a general property of eukaryotic cells, which is closely related to cell viability. Our previous investigations showed that halothane is toxic for A 549 lung carcinoma cells when applied at physiologically relevant concentrations and causes inhibition of adhesion to collagen IV. The present study is focused on the mechanisms underlying halothane toxicity. Our results imply that physiologically relevant concentrations of halothane disrupt focal adhesion contacts in A 549 cells, which is accompanied with suppression of focal adhesion kinase activity and paxillin phosphorylation, and not with proteolytic changes or inhibition of vinculin and paxillin expression.We suggest that at least one of the toxic effects of halothane is due to a decreased phosphorylation of the focal contact proteins.