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.

Sphingolipid Catabolism and Glycerophospholipid Levels Are Altered in Erythrocytes and Plasma from Multiple Sclerosis Patients.

Multiple sclerosis (MS) is an autoimmune, inflammatory, degenerative disease of the central nervous system. Changes in lipid metabolism have been suggested to play important roles in MS pathophysiology and progression. In this work we analyzed the lipid composition and sphingolipid-catabolizing enzymes in erythrocytes and plasma from MS patients and healthy controls. We observed reduction of sphingomyelin (SM) and elevation of its products-ceramide (CER) and shingosine (SPH). These changes were supported by the detected up-regulation of the activity of acid sphingomyelinase (ASM) in MS plasma and alkaline ceramidase (ALCER) in erythrocytes from MS patients. In addition, Western blot analysis showed elevated expression of ASM, but not of ALCER. We also compared the ratios between saturated (SAT), unsaturated (UNSAT) and polyunsaturated fatty acids and suggest, based on the significant differences observed for this ratio, that the UNSAT/SAT values could serve as a marker distinguishing erythrocytes and plasma of MS from controls. In conclusion, the application of lipid analysis in the medical practice would contribute to definition of more precise diagnosis, analysis of disease progression, and evaluation of therapeutic strategies. Based on the molecular changes of blood lipids in neurodegenerative pathologies, including MS, clinical lipidomic analytical approaches could become a promising contemporary tool for personalized medicine.

Characterization of stitch adhesions: Fibronectin-containing cell-cell contacts formed by fibroblasts.

Fibronectin is a multifunctional, extracellular matrix glycoprotein that exists either as an insoluble multimeric fibrillar component of the extracellular matrix or as a soluble monomer. Cells attach to fibronectin through transmembrane integrin receptors and form a variety of cell-matrix contacts. Here we show that primary fibroblasts can use fibronectin to organize a specific cell-cell contact - "stitch adhesions." This contact is formed by short parallel fibronectin fibrils connecting adjacent cells above the level of the focal adhesions that attach the cells to the substrate. Stitch adhesions contain integrin α5ß1 but not αVß3, align with actin filament bundles, and contain talin, tensin, α-actinin, vinculin, paxillin and a phosphorylated form of focal adhesion kinase. This combination of components differs from the described constituents of the known cell adhesions. Stitch adhesions are organized when protein synthesis and secretion are inhibited by cycloheximide and exogenous fibronectin is provided to the cells. The adhesion stitches described here provide an attractive model system for studying fibronectin fibrillogenesis and the mechanisms governing the formation of cellular adhesions.

Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells: are you a man, or a mouse?

The concept of pluripotency as a prerogative of cells of early mammal embryos and cultured embryonic stem cells (ESC) has been invalidated with the advent of induced pluripotent stem cells. Later, it became clear that the ability to generate all cell types of the adult organism is also a questionable aspect of pluripotency, as there are cell types, such as germ cells, which are difficult to produce from pluripotent stem cells. Recently it has been proposed that there are at least two different states of pluripotency; namely, the naïve, or ground state, and the primed state, which may differ radically in terms of timeline of existence, signalling mechanisms, cell properties, capacity for differentiation into different cell types, etc. Germ-like male and female rodent cells have been successfully produced in vitro from ESC and induced pluripotent stem cells. The attempts to derive primate primordial germ cells (PGC) and germ cells in vitro from pluripotent stem cells, however, still have a low success rate, especially with the female germline. The paper reviews the properties of rodent and primate ESC with regard to their capacity for differentiation in vitro to germ-like cells, outlining the possible caveats to derivation of PGC and germ cells from primate and human pluripotent cells.

Testosterone replacement therapy improves erythrocyte membrane lipid composition in hypogonadal men.

AIM: The aim of this study was to investigate the effects of testosterone replacement therapy (TRT) on erythrocyte membrane (EM) lipid composition and physico-chemical properties in hypogonadal men. METHODS: EM isolated from three patients before and after TRT with injectable testosterone undecanoate or testosterone gel were used for analysis of the phospholipid and fatty acid composition, cholesterol/phospholipid ratio, membrane fluidity, ceramide level and enzyme activities responsible for sphingomyelin metabolism. RESULTS: TRT induced increase of phosphatidylethanolamine (PE) in the EMs and sphingomyelin. Reduction of the relative content of the saturated palmitic and stearic fatty acids and a slight increase of different unsaturated fatty acids was observed in phosphatidylcholine (PC). TRT also induced decrease of the cholesterol/total phospholipids ratio and fluidization of the EM. DISCUSSION: The TRT induced increase of PE content and the reduction of saturation in the PC acyl chains induced alterations in the structure of EM could result in higher flexibility of the erythrocytes. The increase of the SM-metabolizing enzyme neutral sphingomyelinase, which regulates the content of ceramide in membranes has a possible impact on the SM signaling pathway. CONCLUSION: We presume that the observed effect of TRT on the composition and fluidity of the EM contributes for improvement of blood rheology and may diminish the thrombosis risk. Larger studies are needed to confirm the findings of this pilot study.

Cell interactions with three-dimensional matrices.

Signaling and other cellular functions differ in three-dimensional compared with two-dimensional systems. Cell adhesion structures can evolve in vitro towards in-vivo-like adhesions with distinct biological activities. In this review, we examine recent advances in studies of interactions of fibroblasts with collagen gels and fibronectin-containing matrices that mimic in vivo three-dimensional microenvironments. These three-dimensional systems are illuminating mechanisms of cell-matrix interactions in living organisms.

A Rac switch regulates random versus directionally persistent cell migration.

Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3'-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

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.

Photobiomodulation with 590 nm Wavelength Delays the Telomere Shortening and Replicative Senescence of Human Dermal Fibroblasts In Vitro.

Background: Cellular senescence is one of the major factors contributing to the aging process. Photobiomodulation (PBM) is known to trigger an array of cellular responses, but there are no data on how it affects the process of cellular senescence. In this study, we analyze the effect of PBM on the cellular senescence and telomere dynamics. Methods: Human dermal fibroblasts were irradiated by a panel of light-emitting diodes with 590 nm and dose 30 J/cm2 accumulated over 1200 sec repeated in 4-day cycle within 40 days. After the last cycle of PBM treatment, the difference in number of senescent cells between PBM treated groups end nontreated control groups was measured by senescent sensitive ß-galactosidase assay, and the difference in average telomere length between the experimental end control groups was analyzed using relative human telomere length quantitative Polymerase Chain Reaction (qPCR) assay. Results: After 10 cycles of irradiation, the percentage of senescent cells in PBM-treated cultures was 19.7% ± 4.5%, p < 0.05 smaller than the percentage of senescent cells in the control group, and their relative telomere length was 1.19 ± 0.09-fold, p < 0.05 greater than nontreated controls. Conclusions: Our study demonstrates for the first time that PBM with appropriate parameters can delay the attrition of the telomeres and the entry of cells into senescence, suggesting a potential involvement of telomerase reactivation. A hypothetical mechanism for this light-induced antiaging effect is discussed.

Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol.

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and "matrix" cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.

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.

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.

Alterations of the composition and metabolism of pulmonary surfactant phospholipids induced by experimental peritonitis in rats.

Pulmonary complications often accompany the development of acute peritonitis. In this study, we analyzed the alterations of alveolar surfactant phospholipids in rats with experimentally induced peritonitis. The results showed a reduction of almost all phospholipid fractions in pulmonary surfactant of experimental animals. The most abundant alveolar phospholipids-phosphatidylcholine and phosphatidylglycerol were reduced significantly in surfactant of rats with experimental peritonitis. In addition, analysis of the fatty acid composition of these two phospholipids revealed marked differences between experimental and control animals. The activity of phospholipase A2, which is localized in the hydrophyllic phase of alveolar surfactant, was higher in rats with experimental peritonitis compared to sham-operated ones. Also, a weak acyl-CoA:lysophospholipid acyltransferase activity was detected in alveolar surfactant of rats with experimental peritonitis, whereas in control animals this activity was not detectable. The lipid-transfer activity was quite similar in pulmonary surfactant of control and experimental rats. The total number of cells and the percentage of neutrophils were strongly increased in broncho-alveolar lavage fluid from rats with peritonitis. Thus, our results showed that the development of peritonitis was accompanied by pulmonary pathophysiological processes that involved alterations of the phospholipid and fatty acid composition of alveolar surfactant. We suggest that the increased populations of inflammatory cells, which basically participate in internalization and secretion of surfactant components, contributed to the observed alterations of alveolar phospholipids. These studies would be useful for clarification of the pathogenic mechanisms underlying the occurrence of pulmonary disorders that accompany acute inflammatory conditions, such as peritonitis and sepsis.

Live-cell biosensor for assessment of adhesion qualities of biomaterials.

The present study describes a live-cell biosensor, suitable for general evaluation of adhesion qualities of different substrates. It is based on NIH/3T3 fibroblast cell line stably expressing fusion fluorescently tagged proteins mCherry-vinculin and GFP-tensin as quantifiable markers for assessment not only of focal but also of fibrillar contacts. Four measurable parameters - spreading, polarization and development of focal and fibrillar adhesions were used to standardize the adhesion of biosensor cells after plating on five substrates of natural origin - fibronectin, vitronectin, laminin-111, laminin-521 and collagen type I. The obtained set of values (adhesion quality map) were utilized to describe the default biosensor behavior and as a standard for evaluation of surface biocompatibility of materials with unknown adhesive properties. To demonstrate the applicability of the biosensor we studied two PDMS-based artificial materials. The results demonstrated the superior adhesive properties of the poly(acrylic acid)-containing polymer (PDMS-PAA) over that of poly(vinyl pyrrolidone) copolymer (PDMS-PVP), and pointed out the formation of focal adhesions as a parameter for possible further improvements.

Inhibition of rho GTPases by RNA interference.

Selective down-modulation or silencing of individual members of the Rho-GTPase family is now practical using RNA interference. Transfection of mammalian cells with an individual siRNA duplex or siRNA pools can suppress expression of a specific isoform to understand its function. By adjusting the dose of siRNA, intermediate levels of suppression can be attained to test the biological role of different levels of a GTPase such as Rac. Nevertheless, there are significant potential pitfalls, including "off-target" effects of the siRNA on other genes. Besides demonstrating successful, noncytotoxic suppression of protein and activity levels of a specific GTPase, controls are essential to establish specificity. In this chapter, we provide methods for selective knockdown of expression by siRNA and confirmation of the effectiveness of Rho GTPase silencing, as well as descriptions and some examples of controls for specificity that include evaluations of dose-response, negative and positive controls, GTPase specificity, confirmation by using more than one siRNA for the same gene, rescue by a mutated siRNA-resistant cDNA encoding the target gene, and complementary supporting evidence. Selective silencing of specific Rho family GTPases should provide increasing insight into the regulatory and functional roles of each isoform in a wide variety of biological processes.

The plasma membrane lipid composition affects fusion between cells and model membranes.

Investigations were carried out on the effect of plasma membrane lipid modifications on the fusogenic capacity of control and ras-transformed fibroblasts. The plasma membrane lipid composition was modified by treatment of cells with exogenous phospholipases C and D, sphingomyelinase and cyclodextrin. The used enzymes hydrolyzed definite membrane lipids thus inducing specific modifications of the lipid composition while cyclodextrin treatment reduced significantly the level of cholesterol. The cells with modified membranes were used for assessment of their fusogenic capacity with model membranes with a constant lipid composition. Treatment with phospholipases C and D stimulated the fusogenic potential of both cell lines whereas the specific reduction of either sphingomyelin or cholesterol induced the opposite effect. The results showed that all modifications of the plasma membrane lipid composition affected the fusogenic capacity irrespective of the initial differences in the membrane lipid composition of the two cell lines. These results support the notion that the lipid composition plays a significant role in the processes of membrane-membrane fusion. This role could be either direct or through modulation of the activity of specific proteins which regulate membrane fusion.

Epirubicin loading in poly(butyl cyanoacrylate) nanoparticles manifests via altered intracellular localization and cellular response in cervical carcinoma (HeLa) cells.

OBJECTIVE: Drug loading into nanocarriers is used to facilitate drug delivery to target cells and organs. We have previously reported a change in cellular localization of epirubicin after loading to poly(butyl cyanoacrylate) (PBCA) nanoparticles. We aimed to further investigate the altered cellular localization and cellular responses to the described drug formulation. MATERIALS AND METHODS: HeLa cells were treated with epirubicin-loaded PBCA nanoparticles prepared by the pre-polymerization method. A systematic study was performed to evaluate the formulation cytotoxicity. Cellular localization and uptake of the formulation as well as cellular response to the treatment were evaluated. RESULTS: Our studies revealed decreased cytotoxicity of the nanoparticle-formulated epirubicin compared to the free drug as well as a noticeable change in the drug's intracellular localization. Epirubicin-loaded nanoparticles were internalized via endocytosis, accumulated inside endosomal vesicles and induced a two-fold stronger pro-apoptotic signal when compared to the free drug. The level of the tumor suppressor protein p53 in HeLa cells increased significantly upon treatment with free epirubicin, but remained relatively unchanged when cells were treated with equivalent dose of nanoparticle-loaded drug, suggesting a possible shift from p53-dependent DNA/RNA intercalation-based induction of cytotoxicity by free epirubicin to a caspase 3-induced cell death by the epirubicin-loaded PBCA formulation.

Effect of halothane on lung carcinoma cells A 549.

The halogenated hydrocarbons, such as halothane, are widely used as anesthetics in clinical practice; however their application is often accompanied with metabolic, cardiovascular and respiratory complications. One of the possible factors for this negative outcome might be the severe toxicity of these agents. In this paper, we investigate in vitro effects of halothane on human lung carcinoma A 549 cells, namely on their cytotoxicity, adhesive properties and metabolic activity. The cytotoxicity response of lung carcinoma A 549 cells to halothane was determined by lactate dehydrogenase (LDH) assay (for cytotoxicity), by detachment assay after adhesion to type IV collagen (for cell adhesive properties) and by surface tension measurements of culture medium (for cell metabolic activity). Regarding the cytotoxicity, the determined maximal non-toxic concentration of halothane on A 549 cells, given here as volume percentages (vol.%) was 0.7 vol.% expressed as aqueous concentration in the culture medium. Direct measurement of the actual halothane concentration in the culture medium showed that 0.7 vol.% corresponds to 1.05 mM and 5.25 aqueous-phase minimum alveolar concentration (MAC). Concentrations equal or higher than 1.4 vol.% (2.1 mM; 10.5 MAC) of halothane provoked complete detachment (cell death), or reduction of initial adhesion to collagen IV in half of the cell population. Surfactant production of A 549 cells, registered up to 48 h after halothane treatment, was inhibited by halothane concentrations as low as 0.6 vol.% (0.9 mM; 4.5 MAC). Our results demonstrate that sub toxic halothane concentrations of 0.6 vol.% inhibits surfactant production; concentrations in the range 0.8-1.4 vol.% induce membrane damages and concentrations equal and higher than 1.4 vol.%--cell death of approximately 50% of the cells.

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.