Surfactant phospholipid DPPC downregulates monocyte respiratory burst via modulation of PKC

Pulmonary surfactant phospholipids have been shown previously to regulate inflammatory functions of human monocytes. This study was undertaken to delineate the mechanisms by which pulmonary surfactant modulates the respiratory burst in a human monocytic cell line, MonoMac-6 (MM6). Preincubation of MM6 cells with the surfactant preparations Survanta, Curosurf, or Exosurf Neonatal inhibited the oxidative response to either lipopolysaccharide (LPS) and zymosan or phorbol 12-myristate 13-acetate (PMA) by up to 50% (P < 0.01). Preincubation of MM6 cells and human peripheral blood monocytes with dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid component of surfactant, inhibited the oxidative response to zymosan. DPPC did not directly affect the activity of the NADPH oxidase in a MM6 reconstituted cell system, suggesting that DPPC does not affect the assembly of the individual components of this enzyme into a functional unit. The effects of DPPC were evaluated on both LPS/zymosan and PMA activation of protein kinase C (PKC), a ubiquitous intracellular kinase, in MM6 cells. We found that DPPC significantly inhibited the activity of PKC in stimulated cells by 70% (P < 0.01). Western blotting experiments demonstrated that DPPC was able to attenuate the activation of the PKCdelta isoform but not PKCalpha. These results suggest that DPPC, the major component of pulmonary surfactant, plays a role in modulating leukocyte inflammatory responses in the lung via downregulation of PKC, a mechanism that may involve the PKCdelta isoform.     

Curosurf modulates cAMP accumulation in human monocytes through a membrane-controlled mechanism

The cellular mechanisms by which pulmonary surfactant exerts its effects, including anti-inflammatory or proinflammatory effects, have remained elusive. To address the issue of whether plasma membrane modifications represent a target for these mechanisms, we designed an experimental protocol involving the determination of changes in cAMP levels under membrane-dependent or -independent stimulatory pathways. The effects of a modified natural porcine surfactant, Curosurf, and the major surfactant protein A were evaluated on resting and stimulated cAMP levels of human monocytes. We found that agents that elevate intracellular cAMP exhibit different susceptibilities toward a preexposure to Curosurf. The rise in cAMP induced by membrane-active agents such as cholera toxin or the diterpene forskolin was significantly inhibited by monocyte preexposure to Curosurf. In contrast, the rise in cAMP induced by the membrane-permeant phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine or by the Bordetella pertussis toxin adenylate cyclase-hemolysin was unaffected by Curosurf. Surfactant protein A did not affect either cAMP levels or the inhibitory capacity of Curosurf. We suggest that a plasma membrane-associated event affecting the mechanism underlying the effects of cholera toxin or forskolin is involved in the inhibition of cAMP accumulation caused by Curosurf.     

Impact of chronic smoking on traumatic brain microvascular injury: An in vitro study

Traumatic brain injury (TBI) is a major reason of cerebrovascular and neurological damage. Premorbid conditions such as tobacco smoking (TS) can worsen post-TBI injuries by promoting vascular endothelial impairments. Indeed, TS-induced oxidative stress (OS) and inflammation can hamper the blood-brain barrier (BBB) endothelium. This study evaluated the subsequence of chronic TS exposure on BBB endothelial cells in an established in vitro model of traumatic cell injury. Experiments were conducted on confluent TS-exposed mouse brain microvascular endothelial cells (mBMEC-P5) following scratch injury. The expression of BBB integrity–associated tight junction (TJ) proteins was assessed by immunofluorescence imaging (IF), Western blotting (WB) and quantitative RT-PCR. We evaluated reactive oxygen species (ROS) generation, the nuclear factor 2–related (Nrf2) with its downstream effectors and several inflammatory markers. Thrombomodulin expression was used to assess the endothelial haemostatic response to injury and TS exposure. Our results show that TS significantly decreased Nrf2, thrombomodulin and TJ expression in the BBB endothelium injury models while increased OS and inflammation compared to parallel TS-free cultures. These data suggest that chronic TS exposure exacerbates traumatic endothelial injury and abrogates the protective antioxidative cell responses. The downstream effect was a more significant decline of BBB endothelial viability, which could aggravate subsequent neurological impairments.     

Tensile stress induces alpha-adaptin C production in mouse calvariae in an organ culture: possible involvement of endocytosis in mechanical stress-stimulated osteoblast differentiation

We previously demonstrated that tensile stress (TS)-induced osteoblast differentiation eventually led to osteogenesis in an organ culture of mouse calvarial sutures. In the present study, we employed RNA-fingerprinting using an arbitrarily primed polymerase chain reaction (RAP-PCR) to identify alpha-adaptin C, a component of the endocytosis machinery AP2, as a TS-inducible gene. Protein production, as well as the gene expression of alpha-adaptin C, was induced by TS as early as 3 h following the initiation of loading. In situ hybridization and immunohistochemical analysis revealed that the induction of alpha-adaptin C mostly occurred in fibroblastic cells in the sutures, suggesting that it precedes TS-induced osteoblast differentiation. Consistent with this result, TS significantly increased the number of coated pits (CPs) and coated vesicles (CVs) in the undifferentiated fibroblastic cells but not in the osteoblastic cells around calvarial bones. Further, TS-induced osteoblast differentiation was suppressed when endocytosis was inhibited by potassium depletion. These results, taken together, suggest that TS accelerates osteoblast differentiation and osteogenesis, possibly through the induction of the alpha-adaptin C expression and consequent activation of receptor-mediated endocytosis.     

Overexpression of HSP25 reduces the level of TNFα-induced oxidative DNA damage biomarker, 8-hydroxy-2′-deoxyguanosine,in L929 cells

Previously we and others have demonstrated that oxidative stress involving generation of reactive oxygen species (ROS) is responsible for the cytotoxic action of TNFα. Protective effect of small heat shock proteins (HSP) against diverse oxidative stress conditions has been suggested. Although overexpression of small HSP was shown to provide an enhanced survival of TNFα-sensitive cells when challenged with TNFα, neither the nature of TNFα-induced cytotoxicity nor the protective mechanism of small HSP has been completely understood. In this study, we have attempted to determine whether TNFα induces oxidative DNA damage in TNFα-sensitive L929 cells. We chose to measure the level of 8-hydroxy-2′-deoxyguanosine (8 ohdG), which has been increasingly recognized as one of the most sensitive markers of oxidative DNA damage. Our results clearly demonstrated that the level of 8 ohdG increased in L929 cells in a TNFα dose-dependent manner. Subsequently, we asked whether small HSP has a protective effect on TNFα-induced oxidative DNA damage. To accomplish this goal, we have stably transfected into L929 cells, which are devoid of endogenous small HSP, with the mouse small hsp cDNA (hsp25). We found that TNFα-induced 8 ohdG was decreased in cells overexpressing exogenous small HSP25. We also found that the cell-killing activity of TNFα was decreased in these cells as measured by clonogenic survival. Taken together, results from the current study show that a cytotoxic mechanism of TNFα involves oxidative damage of DNA, and that overexpression of the small HSP25 reduces this oxidative damage. We suggest that the reduction of oxidative DNA damage is an important protective mechanisms of small HSP against TNFα. J. Cell. Physiol. 174:27–34, 1998. © 1998 Wiley-Liss, Inc.     

Differential effect of surfactant and its saturated phosphatidylcholines on human blood macrophages

Blood monocyte-derived macrophages invading the alveolus encounter pulmonary surfactant, a phospholipoprotein complex that changes composition during lung development. We tested the hypothesis that characteristic phosphatidylcholine (PC) components differentially influence macrophage phenotype and function, as determined by phagocytosis of green fluorescent protein-labeled Escherichia coli and alphaCD3-induced T cell proliferation. Human macrophages were exposed to surfactant (Curosurf(R)), to two of its characteristic phosphadidylcholine (PC) components (dipalmitoyl-PC and palmitoylmyristoyl-PC), and to a ubiquituous PC (palmitoyloleoyl-PC) as control. Interaction of Curosurf and PC species with macrophages was assessed using Lissaminetrade mark-dihexadecanoyl-phosphoethanolamine-labeled liposomes. Curosurf and both saturated surfactant PC species downregulated CD14 expression and upregulated CD206. HLA-DR and CD80 were upregulated by Curosurf and palmitoylmyristoyl-PC, whereas dipalmitoyl-PC showed no effect. The latter upregulated TLR2 and TLR4 expression, whereas Curosurf and palmitoylmyristoyl-PC had no effect. PC species tested were incorporated in comparable amounts by macrophages. Curosurf and PC species inhibited phagocytosis of E. coli. Scavenger receptor CD36, CD68, SR-A, and LOX-1 mRNA expression was upregulated by Curosurf, whereas PC species only upregulated SR-A. Curosurf and palmitoylmyristoyl-PC inhibited alphaCD3-induced T cell proliferation by 50%, whereas dipalmitoyl-PC and palmitoyloleoyl-PC showed no effect. These data identify individual surfactant PC species as modifiers of macrophage differentiation and suggest differential effects on innate and adaptive immune functions.     

Effect of surfactant on pulmonary expression of type IIA PLA(2) in an animal model of acute lung injury

We previously showed that the seminatural surfactant Curosurf inhibits the in vitro synthesis of secretory type IIA phospholipase A(2) (sPLA(2)-IIA) in alveolar macrophages (AM). These cells are the main source of sPLA(2)-IIA in a guinea pig model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Here, we investigate the effect of Curosurf on the pulmonary synthesis of sPLA(2)-IIA in this ALI model. Our results showed that intratracheal administration of LPS (330 microg/kg) induced an increase in pulmonary expression of sPLA(2)-IIA, which was inhibited when animals received Curosurf (16 mg/guinea pig) 30 min or 8 h after LPS instillation. When AM were isolated from LPS-treated animals and cultured in conditioned medium, they expressed higher levels of sPLA(2)-IIA than AM from saline-treated animals. This ex vivo sPLA(2)-IIA expression was significantly reduced when guinea pigs received Curosurf 30 min after LPS instillation. Finally, we examined the effect of Curosurf on pulmonary inflammation measured 8 or 24 h after LPS administration. Curosurf instillation 30 min or 8 h after LPS reversed the increase in tumor necrosis factor-alpha expression, polymorphonuclear cell extravasation, and protein concentration in bronchoalveolar lavage fluids. Curosurf also decreased the bronchial reactivity induced by LPS. We conclude that Curosurf inhibits the pulmonary expression of sPLA(2)-IIA and exhibits palliative anti-inflammatory effects in an animal model of ALI.     

Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by alpha-tocopheryl hemisuccinate

We investigated the mechanism of cell toxicity of alpha-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O(2)(-) generated via the oxidase system activated with TS.     

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O2*-) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O2*- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O2*- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O2*- than those from controls. Nifedipine treatment considerably decreased O2*- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O2*- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O2*- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.     

Different efficiency of heat shock proteins (HSP) to activate human monocytes and dendritic cells: superiority of HSP60

One essential immunoregulatory function of heat shock protein (HSP) is activation of the innate immune system. We investigated the activation of human monocytes and monocyte-derived dendritic cells (DC) by recombinant human HSP60, human inducible HSP72, and preparations of human gp96 and HSP70 under stringent conditions, in the absence of serum and with highly purified monocytes. HSP60 induced human DC maturation and activated human DC to secrete proinflammatory cytokines. HSP72 induced DC maturation to a lesser extent, but activated human monocytes and immature DC as efficiently as HSP60 to release proinflammatory cytokines. The independence of the effects of HSP60 and HSP72 from endotoxin or another copurifying bacterial component was shown by the resistance of these effects to polymyxin B, their sensitivity to heat treatment, the inactivity of endotoxin controls at concentrations up to 100-fold above the endotoxin contents of the HSP, and the inactivity of a recombinant control protein. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73, induced only marginal cytokine release from monocytes. The gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations were not susceptible to gp96 signaling under the stringent conditions applied in this study. The biological activities of gp96 and HSP70 preparations were confirmed by their peptide binding activity. These findings show that HSP can differ considerably in the capacity to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system.     

Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress

Heat shock proteins (HSP) or stress proteins serve as biomarkers to identify the contribution of stress situations underlying the pathogenesis of degenerative diseases of the CNS. We have analyzed by immunoblot technique the constitutive and inducible occurrence of stress proteins in cultured rat brain oligodendrocytes subjected to heat shock or oxidative stress exerted by hydrogen peroxide, or a combination of both. The data demonstrate that oligodendrocytes constitutively express HSP32, HSP60 and the cognate form of the HSP70 family of proteins, HSC70. After heat shock, HSP25, alpha B-crystallin and HSP70 were up-regulated, while after oxidative stress the specific induction of HSP32 and alpha B-crystallin was observed. HSP32 represents heme oxygenase 1 (HO-1), a small stress protein with enzymatic activity involved in the oxidative degradation of heme which participates in iron metabolism. The presence of the iron chelators phenanthroline or deferoxamine (DFO), which previously has been shown to protect oligodendrocytes from oxidative stress-induced onset of apoptosis, caused a marked stimulation of HSP32 without affecting HSP70. This indicates that DFO possibly exerts its protective role by directly influencing the antioxidant capacity of HO-1. In summary, HSP in oligodendrocytes are differentially stimulated by heat stress and oxidative stress. Heme oxygenase-1 has been linked to inflammatory processes and oxidative stress, its specific up-regulation after oxidative stress in oligodendrocytes suggests that it is an ideal candidate to investigate the involvement of oxidative stress in demyelinating diseases.     

Metals accelerate production of the aberrant splicing isoform of the presenilin-2

Oxidative stress is a major risk factor for Alzheimer's disease (AD) and other neurodegenerative disorders. Metals are known to be one of the factors that contribute to oxidative stress. Recently, we reported that the aberrant splicing isoform (PS2V) generated by skipping exon5 of the presenilin-2 (PS2) gene is a diagnostic feature of sporadic AD (SAD). PS2V is inducible by exposure of human neuroblastoma to hypoxia. We examined whether this aberrant splicing was caused by metal-induced oxidative stress, such as exposure to aluminum. As a result, we demonstrated that exposure to aluminum accelerated PS2V production induced by hypoxia. This acceleration of the production of PS2V to hypoxia was caused by chronic aluminum exposure, but was not related to the intracellular content of aluminum. HMGA1a is a mediator of PS2V production, and it was induced by aluminum as well as by hypoxia. Induction of HMGA1a was increased by chronic exposure to aluminum, and a nuclear extract containing HMGA1a bound to a specific sequence on exon5 of PS2 pre-mRNA, as reported previously. Finally, the acceleration of PS2V production induced by aluminum under hypoxic conditions reflected, but has not yet been directly shown to cause, vulnerability to endoplasmic reticulum stress. These results suggest that exposure to some metals can accelerate and enhance PS2V generation, and that hypoxia plus chronic exposure to metals may promote the development of AD.     

Chronic Melatonin Administration Reduced Oxidative Damage and Cellular Senescence in the Hippocampus of a Mouse Model of Down Syndrome

Previous studies have demonstrated that melatonin administration improves spatial learning and memory and hippocampal long-term potentiation in the adult Ts65Dn (TS) mouse, a model of Down syndrome (DS). This functional benefit of melatonin was accompanied by protection from cholinergic neurodegeneration and the attenuation of several hippocampal neuromorphological alterations in TS mice. Because oxidative stress contributes to the progression of cognitive deficits and neurodegeneration in DS, this study evaluates the antioxidant effects of melatonin in the brains of TS mice. Melatonin was administered to TS and control mice from 6 to 12 months of age and its effects on the oxidative state and levels of cellular senescence were evaluated. Melatonin treatment induced antioxidant and antiaging effects in the hippocampus of adult TS mice. Although melatonin administration did not regulate the activities of the main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase) in the cortex or hippocampus, melatonin decreased protein and lipid oxidative damage by reducing the thiobarbituric acid reactive substances (TBARS) and protein carbonyls (PC) levels in the TS hippocampus due to its ability to act as a free radical scavenger. Consistent with this reduction in oxidative stress, melatonin also decreased hippocampal senescence in TS animals by normalizing the density of senescence-associated β-galactosidase positive cells in the hippocampus. These results showed that this treatment attenuated the oxidative damage and cellular senescence in the brain of TS mice and support the use of melatonin as a potential therapeutic agent for age-related cognitive deficits and neurodegeneration in adults with DS.     

Effects of antioxidants on surfactant peroxidation by stimulated human polymorphonuclear leukocytes

Production of oxygen radicals by stimulated phagocytes followed by surfactant lipid peroxidation (LPO) and loss of surfactant function have all been implicated in the pathogenesis of acute lung injury. We studied the interactions between natural lung surfactant (Curosurf) and neutrophils in vitro , and compared various antioxidants; (superoxide dismutase (SOD), vitamin E, vitamin C, ebselen and melatonin), or combinations of them in duplicate and triplicate regarding their ability to decrease superoxide production and the peroxidation level of surfactant caused by activated phagocytes. The superoxide production of neutrophils activated by Candida albicans was measured with the nitroblue tetrazolium (NBT) test. The subsequent LPO was estimated as the content of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE). We found that lung surfactant decreased the superoxide production by activated neutrophils (29.7%) and that Curosurf was peroxidized with elevated MDA/4-HNE values. With supplements of antioxidants (except vitamin C), superoxide radical production and the surfactant LPO level fell in a dose-dependent manner. The protective effect of the antioxidants differed in each test. SOD had a slight effect in both tests. The findings with vitamin E, melatonin and ebselen were similar. The best combination was that of a natural and a synthetic antioxidant (melatonin-ebselen) with a 60% decrease in comparison to the corresponding control. These findings suggest that antioxidants, particularly in combination, prevent LPO of lung surfactant.     

Effects of Antioxidants on Surfactant Peroxidation by Stimulated Human Polymorphonuclear Leukocytes

Production of oxygen radicals by stimulated phagocytes followed by surfactant lipid peroxidation (LPO) and loss of surfactant function have all been implicated in the pathogenesis of acute lung injury. We studied the interactions between natural lung surfactant (Curosurf) and neutrophils in vitro, and compared various antioxidants; (superoxide dismutase (SOD), vitamin E, vitamin C, ebselen and melatonin), or combinations of them in duplicate and triplicate regarding their ability to decrease superoxide production and the peroxidation level of surfactant caused by activated phagocytes. The superoxide production of neutrophils activated by Candida albicans was measured with the nitroblue tetrazolium (NBT) test. The subsequent LPO was estimated as the content of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE). We found that lung surfactant decreased the superoxide production by activated neutrophils (29.7%) and that Curosurf was peroxidized with elevated MDA/4-HNE values. With supplements of antioxidants (except vitamin C), superoxide radical production and the surfactant LPO level fell in a dose-dependent manner. The protective effect of the antioxidants differed in each test. SOD had a slight effect in both tests. The findings with vitamin E, melatonin and ebselen were similar. The best combination was that of a natural and a synthetic antioxidant (melatonin-ebselen) with a 60% decrease in comparison to the corresponding control. These findings suggest that antioxidants, particularly in combination, prevent LPO of lung surfactant.     

Heat shock proteins induction reduces stress kinases activation, potentially improving insulin signalling in monocytes from obese subjects

Induction of heat shock proteins (Hsp) 72 and 27 can improve insulin signalling in obesity and type 2 diabetes via inhibition of key stress kinases. In metabolic disease, altered insulin signalling, as illustrated by increased serine phosphorylation of insulin receptor substrate (IRS)-1 (Ser312), is not confined to muscle or liver and can also affect other tissues and cell types, potentially impairing their primary biological function. This study specifically investigated insulin-stimulated glucose metabolism in monocytes and examined the impact of HSP induction on insulin signalling. Control (CG, BMI < 25 kg/m(2)) or obese (OG, BMI > 30 kg/m(2)) participants were included in the study. Glucose transporter (GLUT)4 expression on monocytes, phosphorylated JNK, IKK-β and IRS-1, as well as Hsp27 and Hsp72, were measured in monocytes under fasting conditions. GLUT4 expression was also measured during an oral glucose tolerance test (OGTT). HSP induction as well as JNK, IKK-β activation and IRS-1 serine phosphorylation was investigated following heat stress. Obese patients showed lower GLUT4 levels on monocytes during the OGTT. pJNK, pIKK-β and pIRS-1 levels were increased in OG with pJNK and pIKK-β levels positively correlated with serine pIRS-1 and negatively with GLUT4 supporting their role in insulin resistance. Heat exposure induced Hsp72 and Hps27, but only in CG for the latter, and decreased pJNK, pIKK-β and pIRS-1. Our results show that induction of Hsp72 and 27 via heat stress is associated with inactivation of stress kinases and reduced serine pIRS-1 in monocytes from obese participants. This indicates that metabolic diseases can also affect monocyte metabolism via cellular stress that can be modulated via HSP induction.     

Enhancement of Nitric Oxide and Superoxide Generations by α-Tocopheryl Succinate and Its Apoptotic and Anticancer Effects

Tocopheryl succinate (TS), a succinyl ester of alpha-tocopherol (alpha-T), has been reported to have various biological activities. In this communication, we review the current findings about TS including our recent studies of its effects on nitric oxide (NO) and superoxide (O2-) generations implicated in cancer and atherosclerosis. First, we investigated the effect of TS on NO production in vascular smooth muscle cells (VSMC) under atherosclerosis-like conditions using lipopolysaccharide (LPS) and interferon-gamma (IFN). TS enhanced LPS/IFN-dependent NO production, but alpha-T itself did not. The enhancement by TS of NO production was inhibited by alpha-T but not by antioxidants such as ascorbic acid and 2[3]-t-butyl-4-hydroxyanisole (BHA). TS enhanced the amount of protein kinase Calpha (PKCalpha) in VSMC, and PKC inhibitors inhibited TS-enhanced NO production, suggesting that the enhancing effect of TS on NO production is caused by up-regulation of PKC. Second, we found that TS induced apoptosis in VSMC associated with increase in O2- generation via NADPH-dependent oxidase. We further observed that a mouse breast cancer cell line C127I was more susceptible for TS-induced apoptosis than a mouse breast normal cell line NmuMG, and that superoxide dismutase, alpha-T, and BHA inhibited TS-caused morphological cell damage in C127I. From these results, O2- itself and/or other reactive oxygen species are assumed to associate with TS-induced cell toxicity, and antioxidative defense systems are supposed to be lowered in cancer cells. Finally, we found that intravenous injection of TS vesicles completely inhibited the growth of melanoma cells B16-F1 inoculated on the back of hairless mice and enhanced their survival time.     

Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by α-tocopheryl hemisuccinate

We investigated the mechanism of cell toxicity of α-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O₂⁻ generated via the oxidase system activated with TS.