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).

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

Structural organization of plasma membrane lipids isolated from cells cultured as a monolayer and in tissue-like conditions.

Complementary biophysical approaches were used to study the structural organization of plasma membrane lipids obtained from fibroblasts cultured as two-dimensional (2D) monolayer and in tissue-like three-dimensional (3D) conditions. Fluorescence microscopy experiments demonstrated different domain patterns for 2D and 3D plasma membrane lipid extracts. ESR demonstrated that 3D lipid extract is characterized with lower order parameter than 2D in the deep hydrophobic core of the lipid bilayer.

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.

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.

Surface properties and behavior of lipid extracts from plasma membranes of cells cultured as monolayer and in tissue-like conditions.

The differences in the surface active properties of native lipids extracted from plasma membranes of cells cultured as a monolayer and in three-dimensional (3D) matrix were investigated. This experimental model was chosen because most of the current knowledge on cellular physiological processes is based on studies performed with conventional monolayer two-dimensional (2D) cell cultures, where cells are forced to adjust to unnaturally rigid surfaces that differ significantly from the natural matrix surrounding cells in living organisms. Differences between monolayer and 3D cells were observed in the lipid composition of plasma membranes and especially in the level of the two major microdomain-forming lipids--sphingomyelin (SM) and cholesterol, which were significantly elevated in 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.

beta1 integrin cytoplasmic domain residues selectively modulate fibronectin matrix assembly and cell spreading through talin and Akt-1.

The integrin beta(1) cytoplasmic domain (tail) serves as a scaffold for numerous intracellular proteins. The mechanisms by which the tail coordinates these proteins to facilitate extracellular matrix assembly and cell spreading are not clear. This study demonstrates that the beta(1) cytoplasmic domain can regulate cell spreading on fibronectin and fibronectin matrix assembly through Akt- and talin-dependent mechanisms, respectively.

Determination of Akt/PKB signaling.

This unit provides a basic protocol for assaying the level of activity, as well as the activation capacity and dynamics of inhibition of Akt/PKB in cultured cells. All these data can be obtained in a single nonradioactive experiment by standard techniques including immunoblotting and immunodetection with phosphospecific antibodies. This unit also provides a support protocol for assaying the membrane translocation of Akt.

Non-radioactive quantification of fibronectin matrix assembly.

This unit provides a basic protocol for non-radioactive determination of the rate of incorporation of biotinylated fibronectin into the insoluble matrix organized by cultured cells. The protocol provides quantification of changes in matrix assembly due to different experimental conditions with concomitant determinations of the activation state of various signaling molecules that may be involved in the process of matrix assembly. This unit also provides a support protocol for biotinylation of purified fibronectin.

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.

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 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.

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.

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.

Cholesterol distribution in plasma membranes of beta1 integrin-expressing and beta1 integrin-deficient fibroblasts.

The effect of integrin receptors on the level and transmembrane localization of cholesterol molecules was investigated in beta1 integrin-expressing (beta1) and beta1 integrin-deficient (beta1 null) cells. We found that the content of specific raft components-cholesterol, sphingomyelin, and caveolin-was increased in integrin-expressing cells. Integrin presence affected as well the transmembrane distribution of cholesterol-a higher percent was found in the plasma membrane outer monolayer of beta1 compared to beta1 null cells.