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.

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.

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.

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.

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.

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. To identify these mechanisms, we characterized GD25 cells expressing the beta(1) integrin cytoplasmic domain mutants W775A and R760A. Although cell spreading appears normal in R760A mutant-integrin cells compared with wild type, it is inhibited in W775A mutant cells. In contrast, both mutant cell lines show defective fibronectin matrix assembly. Inhibition of cell spreading, but not matrix assembly, in the W775A mutant cells is due to a specific defect in Akt-1 activation. In addition, we find that both W775A and R760A mutant integrins have reduced surface expression of the 9EG7 epitope that correlates with reduced recruitment of talin to beta(1) integrin cytoplasmic complexes. Down-regulation of talin with small interfering RNA or expression of green fluorescent protein-talin head domain inhibits matrix assembly in beta(1) wild-type cells, mimicking the defect seen with the W775A and R760A mutant cells. These results demonstrate distinct mechanisms by which integrins regulate cell spreading and matrix assembly through the beta(1) integrin cytoplasmic tail.

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.

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.

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.

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.

Specific beta1 integrin site selectively regulates Akt/protein kinase B signaling via local activation of protein phosphatase 2A.

Integrin transmembrane receptors generate multiple signals, but how they mediate specific signaling is not clear. Here we test the hypothesis that particular sequences along the beta(1) integrin cytoplasmic domain may exist that are intimately related to specific integrin-mediated signaling pathways. Using systematic alanine mutagenesis of amino acids conserved between different beta integrin cytoplasmic domains, we identified the tryptophan residue at position 775 of human beta(1) integrin as specific and necessary for integrin-mediated protein kinase B/Akt survival signaling. Stable expression of a beta(1) integrin mutated at this amino acid in GD25 beta(1)-null cells resulted in reduction of Akt phosphorylation at both Ser(473) and Thr(308) activation sites. As a consequence, the cells were substantially more sensitive to serum starvation-induced apoptosis when compared with cells expressing wild type beta(1) integrin. This inactivation of Akt resulted from increased dephosphorylation by a localized active population of protein phosphatase 2A. Both Akt and protein phosphatase 2A were present in beta(1) integrin-organized cytoplasmic complexes, but the activity of this phosphatase was 2.5 times higher in the complexes organized by the mutant integrin. The mutation of Trp(775) specifically affected Akt signaling, without effects on other integrin-activated pathways including phosphoinositide 3-kinase, MAPK, JNK, and p38 nor did it influence activation of the integrin-responsive kinases focal adhesion kinase and Src. The identification of Trp(775) as a specific site for integrin-mediated Akt signaling supports the concept of specificity of signaling along the integrin cytoplasmic domain.