Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Involvement of protein kinase Cδ in induction of apoptosis by cationic liposomes in macrophage-like RAW264.7 cells

We have recently demonstrated that reactive oxygen species (ROS) play an important role in RAW264.7 cell apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes). In this study, we investigated whether protein kinase Cδ PKCδ) is involved in apoptosis induced by cationic liposomes. Tyrosine phosphorylation, nuclear localization, and cleavage of PKCδ were observed following the treatment of cells with SA-liposomes, suggesting that SA-liposomes activate PKCδ. Rottlerin, a specific inhibitor of PKCδ, inhibited ROS generation and also suppressed apoptosis. Cell surface proteoglycans may contribute to PKCδ activation by SA-liposomes. These findings suggest that PKCδ is strongly associated with apoptosis induced by SA-liposomes.     

Involvement of actin cytoskeleton in macrophage apoptosis induced by cationic liposomes

We clarified whether actin cytoskeleton is involved in the macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes). Externalization of phosphatidylserine induced by SA-liposomes was suppressed by cytochalasin D, a specific inhibitor of polymerization of F-actin. Furthermore, activation of PKCδ and reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, were inhibited by cytochalasin D. Microscopical observation revealed the co-localization of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled SA-liposomes and fluorescein-labeled phalloidin, which specifically binds to F-actin, and this co-localization was also inhibited by cytochalasin D. Co-localization of SA-liposomes and F-actin was also inhibited by the pre-treatment of cells with chondroitinase ABC. These findings could be the first observation concerning the contribution of the proteoglycan-actin cytoskeleton-ROS generation pathway to apoptosis induced by SA-liposomes in macrophages.     

Cationic liposomes induce macrophage apoptosis through mitochondrial pathway

To clarify the mechanism of apoptosis of the macrophage-like cell line RAW264.7 induced by cationic liposomes, we focused on the mitochondria and investigated the changes in mitochondrial membrane potential and the release of cytochrome c following treatment of cationic liposomes composed of stearylamine (SA-liposomes). SA-liposomes induced mitochondrial membrane depolarization and also the release of cytochrome c from mitochondria. Caspase-3 was also activated by SA-liposome treatment. Pretreatment of cells with N-acetylcysteine, a scavenger of reactive oxygen species (ROS), conferred resistance to the induction of the membrane depolarization, cytochrome c release, and caspase-3 activation by SA-liposomes. These results indicated that SA-liposomes caused the apoptosis in RAW264.7 cells through the mitochondrial pathway, and ROS generation was required for this phenomenon.     

The antioxidant action of ketoconazole and related azoles: comparison with tamoxifen and cholesterol

The azole antifungal drug ketoconazole was found to inhibit Fe(III)-ascorbate dependent lipid peroxidation using either rat liver microsomes or ox-brain phospholipid liposomes as the substrate. It also inhibited microsomal peroxidation induced by the Fe(III)-ADP/NADPH system. The related azoles, miconazole and clotrimazole, were much weaker inhibitors than ketoconazole. Ketoconazole was approximately equipotent with the triphenylethylene anticancer drug tamoxifen in the microsomal system and was almost as effective as 4-hydroxytamoxifen in the liposomal system. Ketoconazole introduced into phospholipid liposomes during their preparation inhibited Fe(III)-ascorbate induced lipid peroxidation to a greater extent than similarly introduced cholesterol, ergosterol or tamoxifen. Miconazole and clotrimazole were again poor inhibitors of lipid peroxidation in this system. These antioxidant effects of ketoconazole may be due to membrane stabilization in the systems used. The implications of our findings for the clinical applications of these drugs are discussed.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

TNF-alpha and IL-1alpha induce apoptosis in subconfluent rat mesangial cells. Evidence for the involvement of hydrogen peroxide and lipid peroxidation as second messengers

Apoptosis of mesangial cells (MC) plays a role in glomerulonephritis (GN). In this study we investigated cytokine-induced apoptosis of cultured rat MC by morphological and biochemical features. TNF-alpha and IL-1alpha induced apoptosis in rat MC in a time- and concentration-dependent fashion. RT-PCR experiments revealed that MC express the TNF-receptor 1 (p60) gene constitutively. TNF-alpha as well as IL-1alpha stimulated the production of reactive oxygen species (ROS) and induced lipid peroxidation. Coincubation with catalase inhibited TNF-alpha and IL-1alpha induced apoptosis as well as lipid peroxidation. TNF-alpha, but not IL-1alpha increased the expression of c-jun. These results provide evidence that TNF-alpha and IL-1alpha induce apoptosis in rat MC with hydrogen peroxide and lipid peroxidation as second messengers. Increased c-jun expression may be a downstream intracellular signal of TNF-alpha-, but not IL-1alpha-induced apoptosis.     

Triacontanol inhibits both enzymatic and nonenzymatic lipid peroxidation

The effect of the plant growth regulator, triacontanol (TRIA) on lipid peroxidation was studied in three different systems: (i) isolated chloroplasts of spinach (Spinacea oleracea L.) leaves; (ii) egg lecithin liposomes; and (iii) soybean lipoxygenase (LOX) system. The nonenzymatic lipid peroxidation in isolated chloroplasts and egg lecithin liposomes was measured as the amount of thiobarbituric acid reactive substances (TBARS) formed. Inhibition of Fe2+ and/or light-induced lipid peroxidation by TRIA was observed in both isolated chloroplasts and egg lecithin liposomes. The kinetics of soybean lipoxygenase-1 (LOX-1) was studied using linoleic acid as the substrate. The enzyme was competitively inhibited by TRIA. The Ki for TRIA inhibition of the enzyme was estimated to be 3.2-5.0 microM according to different methods of estimation. TRIA has been known to exhibit anti-inflammatory action in animals and this anti-inflammatory effect of TRIA might be mediated through inhibition of lipid peroxidation. Since LOX inhibitors have been extensively used as therapeutic agents, TRIA, being a natural compound has been suggested to be an effective anti-inflammatory drug.     

Cobaltous ion inhibition of lipid peroxidation in biological membranes.

The effect of cobalt on lipid peroxidation in biological membranes, phospholipid liposomes and fatty acid micelles was investigated. Cobaltous ion, at micromolar concentrations, inhibited iron-ascorbate induced lipid peroxidation in erythrocyte ghosts, microsomes and phosphatidylserine liposomes at pH 7.4. The pH seemed to be important for the anti-peroxidative effect of cobalt, because under slightly acidic conditions cobalt did not inhibit peroxidation. Cobalt was less effective in inhibiting peroxidation stimulated by organic hydroperoxides. Iron-ascorbate induced lipid peroxidation was also inhibited by EDTA. However, certain ratios of EDTA: cobalt in the reaction mixture stimulated peroxidation. Cobalt did not inhibit lipid peroxidation in linoleic acid micelles and phosphatidylethanolamine liposomes. The presence of phosphatidylserine, however, rendered these micelles and liposomes to cobalt inhibition. We conclude that the cobaltous ion is a potent inhibitor of lipid peroxidation in biological membranes and that the binding of cobalt to phosphatidylserine is necessary for the inhibitory effect of this metal ion.     

Glutathione depletion and the production of reactive oxygen species in isolated hepatocyte suspensions

Diethyl maleate (DEM) (5 mM) and ethyl methanesulfonate (EMS) (35 mM) treatments rapidly depleted cellular reduced glutathione (GSH) below detectable levels (1 nmol/10(6) cells), and induced lipid peroxidation and necrotic cell death in freshly isolated rat hepatocytes. In hepatocytes incubated with 2.5 mM DEM and 10 mM EMS, however, the complete depletion of cellular GSH observed was not sufficient to induce lipid peroxidation or cell death. Instead, DEM- and EMS-induced lipid peroxidation and cell death were dependent on increased reactive oxygen species (ROS) production as measured by increases in dichlorofluorescein fluorescence. The addition of antioxidants (vitamin E succinate and deferoxamine) prevented lipid peroxidation and cell death, suggesting that lipid peroxidation is involved in the sequence of events leading to necrotic cell death induced by DEM and EMS. To investigate the subcellular site of ROS generation, the cytochrome P450 inhibitor, SKF525A, was found to reduce EMS-induced lipid peroxidation but did not protect against the loss of cell viability, suggesting a mitochondrial origin for the toxic lipid peroxidation event. In agreement with this conclusion, mitochondrial electron transport inhibitors (rotenone, thenoyltrifluoroacetone and antimycin A) increased EMS-induced lipid peroxidation and cell death, while the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, blocked EMS- and DEM-mediated ROS production and lipid peroxidation. Furthermore, EMS treatment resulted in the significant loss of mitochondrial alpha-tocopherol shortly after its addition, and this loss preceded losses in cellular alpha-tocopherol levels. Treatment of hepatocytes with cyclosporin A, a mitochondrial permeability transition inhibitor, oxypurinol, a xanthine oxidase inhibitor, or BAPTA-AM, a calcium chelator, provided no protection against EMS-induced cell death or lipid peroxidation. Our results indicate that DEM and EMS induce cell death by a similar mechanism, which is dependent on the induction of ROS production and lipid peroxidation, and mitochondria are the major source for this toxic ROS generation. Cellular GSH depletion in itself does not appear to be responsible for the large increases in ROS production and lipid peroxidation observed.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

Membrane lipid peroxidation by ultrasound: Mechanism and implications

Ultrasonic radiation produced a dose-dependent linear increase in lipid peroxidation in the liposomes membrane as reflected in the measurement of conjugated dienes, lipid hydroperoxides and malondialdehydes. Ultrasound induced malondialdehyde production could not be inhibited by any significant degree by superoxide dismutase or histidine or dimethyl furan but was very significantly inhibited by butylated hydroxytoluene, cholesterol, sodium benzoate, dimethyl sulphoxide, sodium formate and EDTA. The scavenger studies indicated the functional role of hydroxyl radicals in the initiation of ultrasound induced lipid peroxidation.     

Acid sphingomyelinase activation requires caspase-8 but not p53 nor reactive oxygen species during Fas-induced apoptosis in human glioma cells

During apoptosis of human glioma cells induced by anti-Fas antibody, ceramide formation with activation of acid, but not neutral sphingomyelinase (SMase), was observed. A potent inhibitor of acid SMase, SR33557, effectively inhibited ceramide formation and apoptosis. Fas-induced apoptosis and ceramide formation proceeded regardless of p53 status. The agents, which modify intracellular levels of reactive oxygen species (ROS) and reduced glutathione (GSH), failed to modulate Fas-induced acid SMase activation and apoptosis. Moreover, expression of functional p53 protein using a temperature-sensitive human p53val(138) induced ceramide generation by activation of neutral SMase but not acid SMase through ROS formation. Peptide inhibitors for caspases-8 (z-IETD-fmk) and -3 (z-DEVD-fmk) suppressed Fas-induced apoptosis. However, activation of acid SMase was inhibited only by z-IETD-fmk. Thus, ceramide generated by acid SMase may take a part in Fas-induced apoptosis of human glioma cells and acid SMase activation may be dependent on caspase-8 activation, but not on p53 nor ROS.     

Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Noxa expression through the formation of S-nitrosohomocysteine

Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice but not in the p53-deficient cells. The NO donor S-nitroso-N-acetylpenicillamine, adenoviral transfer of inducible NO synthase gene, and antioxidants (alpha-tocopherol and superoxide dismutase plus catalase) but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by small interfering RNA-mediated suppression of Noxa expression, indicating that Noxa up-regulation plays an important role in HCy-induced endothelial cell apoptosis. Overexpression of inducible NO synthase increased the formation of S-nitroso-HCy, which was inhibited by the NO synthase inhibitor N-monomethyl-l-arginine. Moreover, S-nitroso-HCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.     

Induction of apoptosis in macrophages by cationic liposomes

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.     

Lipid peroxidation induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system.

Cu,Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H2O2 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system was investigated. When linoleic acids micelles or phosphatidylcholine liposomes were incubated with Cu,Zn-SOD and H2O2, lipid peroxidation was gradually increased in a time-dependent manner. The extent of lipid peroxidation was proportional to Cu,Zn-SOD and H2O2 concentrations. Hydroxyl radical scavengers and copper chelator inhibited lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system. These results suggest that lipid peroxidation is mediated by the Cu,Zn-SOD and H2O2 system via the generation of hydroxyl radicals by a combination of the peroxidative reaction of Cu,Zn-SOD and the Fenton-like reaction of free copper released from oxidatively damaged SOD.     

Electrolyte Leakage, Lipoxygenase, and Lipid Peroxidation Induced in Tomato Leaf Tissue by Specific and Nonspecific Elicitors from Cladosporium fulvum

Glycoprotein nonspecific elicitor (NSE) and a specific elicitor preparation from intercellular fluids (SE) of tomato (Lycopersicon esculentum Mill. cv Bonny Best or Potentate) infected with race 2.4.5 of Cladosporium fulvum Cooke [syn. Fulvia fulva (Cooke) Ciferri] were injected into cv Sonatine (resistant to race 2.4.5) to compare electrolyte leakage, lipoxygenase activity, and lipid peroxidation induced in response to these elicitors. Increased electrolyte leakage was induced by NSE or SE; the leakage due to NSE but not to SE was inhibited by the nonsteroidal antiinflammatory drug (NSAID) piroxicam. Under normal photoperiod conditions, higher levels of lipoxygenase activity were detected 6 hours after injection with either elicitor. This activity peaked by 12 hours with both elicitors and declined to control levels by 24 hours when visible necrosis could be detected. Both NSE and SE-induced lipoxygenase was inhibited by piroxicam in vitro. Lipid peroxidation in elicitor-treated tissue was also assayed at 6, 12, and 24 hours after injection using the TBA test for malonaldehyde. Increased peroxidation was detected in response to NSE or SE at 12 hours with similar values obtained at 24 hours. With plants incubated in the dark, lipoxygenase, and lipid peroxidation were similarly induced in SE-injected tissue whereas necrosis induction by SE was light dependent.     

Involvement of beta 2-integrin in ROS-mediated neutrophil apoptosis induced by Entamoeba histolytica

Cellular adhesion through beta 2-integrin (CD18) is an important step in signal transduction leading to apoptosis of human neutrophils, and NADPH oxidase-derived reactive oxygen species (ROS) are essential for neutrophil apoptosis induced by Entamoeba histolytica. Therefore, we investigated the role of beta 2-integrin-mediated signals in ROS-dependent neutrophil apoptosis induced by E. histolytica. Entamoeba-induced apoptosis was inhibited by pre-incubation of cells with mAb to CD18, but not CD29, suggesting that beta )-integrin plays an important role in this response. Moreover, Entamoeba-induced ROS generation in neutrophils was inhibited by mAbs against CD18 or CD11b, but not by mAbs against CD11a, CD11c, or CD29. A combination of d-galactose plus anti-CD18 mAb had a larger inhibitory effect than d-galactose alone on Entamoeba-induced apoptosis and ROS generation. Furthermore, Entamoeba-induced apoptosis and ROS generation were inhibited by pre-treatment of cells with an inhibitor of phosphatidylinositol-3-kinase (PI-3-kinase). These results indicate that beta 2-integrin and PI-3-kinase are crucial signaling molecules in ROS-dependent apoptosis of neutrophils induced by E. histolytica.     

Comparison of iron-catalyzed DNA and lipid oxidation

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.