Glial cell type-specific responses to menadione-induced oxidative stress

Glial cell types in the central nervous system are continuously exposed to reactive oxygen species (ROS) due to their high oxygen metabolism and demonstrate differential susceptibility to certain pathological conditions believed to involve oxidative stress. The purpose of the current studies was to test the hypothesis that mtDNA damage could contribute to the differential susceptibility of glial cell types to apoptosis induced by oxidative stress. Primary cultures of rat astrocytes, oligodendrocytes, and microglia were utilized, and menadione was used to produce the oxidative stress. Apoptosis was detected and quantitated in menadione-treated oligodendrocytes and microglia (but not astrocytes) using either positive annexin-V staining or positive staining for 3'-OH groups in DNA. The apoptotic pathway that was activated involved the release of cytochrome c from the intermitochondrial space and activation of caspase 9. Caspase 8 was not activated after exposure to menadione in any of the cells. Using equimolar concentrations of menadione, more initial damage was observed in mtDNA from oligodendrocytes and microglia. Additionally, using concentrations of menadione that resulted in comparable initial mtDNA damage, more efficient repair was observed in astrocytes compared to either oligodendrocytes or microglia. The differential susceptibility of glial cell types to oxidative damage and apoptosis did not appear related to cellular antioxidant capacity, because under the current culture conditions astrocytes had lower total glutathione content and superoxide dismutase activity than oligodendrocytes and microglia. These results show that the differential susceptibility of glial cell types to menadione-induced oxidative stress and apoptosis appears to correlate with increased oxidative mtDNA damage and support the hypothesis that mtDNA damage could participate in the initiation of apoptosis through the enhanced release of cytochrome c and the activation of caspase 9.     

Apoptosis and adaptive responses to oxidative stress in human endothelial cells exposed to cyclosporin A correlate with BCL-2 expression levels.

Treatment of transplanted patients with cyclosporin A (CSA) may cause adverse effects such as nephrotoxicity and hypertension. As CSA is known to induce oxidative stress in several tissues, it may cause vascular problems by triggering oxidative stress in endothelial cells (EC). However, oxidative stress has been reported for acute exposure to CSA concentrations exceeding its clinical range, whereas immunosuppression requires life-long treatment with therapeutic concentrations. We therefore compared the effects of 21 h pharmacological (200 microM) vs. 8 days clinical (0.5-2.5 microM) doses of CSA on cultured human EC. Pharmacological doses of CSA cause a decrease in cell density via apoptosis and a down-regulation of the antiapoptotic protein Bcl-2. However, these effects are independent of CSA-induced oxidative stress. In contrast, therapeutic concentrations of CSA cause Bcl-2 up-regulation and modification of EC morphology, both effects blocked by antioxidants. Therefore, a low level of oxidants may act in EC as second messengers that up-regulate Bcl-2, thus promoting survival of impaired EC. Our data suggest that the oxidative stress induced by clinical concentrations of CSA may be involved in the adverse effects of the drug on the vascular system of transplanted patients via an adaptive response involving Bcl-2 up-regulation rather than an apoptotic process     

In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line

Abstract: Amyloid β-peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that Aβ25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25-35 were specific because Aβ1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1-40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25-35. Damage and death of ECs induced by Aβ25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.     

Severity of oxidative stress generates different mechanisms of endothelial cell death

The role of reactive oxygen species (ROS) in the pathogenesis of vascular diseases is well established, but few data exist on the mechanisms by which ROS induce endothelial cell (EC) death. We examined the conditions and the mechanisms by which oxidative stress induces EC death, using cultured confluent bovine aortic ECs exposed for 30 min to different concentrations of hydroxyl radicals (HO*) generated by hydrogen peroxide (H(2)O(2)) in the presence of 100 microM ferrous sulfate (FeSO(4)). Cell viability assays, Hoechst DNA staining, TUNEL (TDT-mediated dUTP-biotin nick end-labeling) analysis, agarose gel electrophoresis and annexin V assay were used to determine the effect of HO* on the viability of ECs, and to distinguish between apoptosis and necrosis. The results showed that at concentrations of up to 0.1 mM H(2)O(2)/FeSO(4), the large majority of cells are viable, except for approximately 12.5% death, which occurs by apoptosis. At a concentration of 0.2 mM H(2)O(2), the cell viability is reduced to 66%, while EC apoptosis remained at comparable values (14%). At high oxidative stress (0.5 mM H(2)O(2)), the cell viability was drastically reduced (approximately 39%), and the prevalent form of death was necrosis; apoptosis accounted for only approximately 17%. Together, these data indicate that: (1) HO* induce EC death either by apoptosis or necrosis and (2) the mechanisms of EC death differ as a function of the concentration of HO. Thus, the same insult can cause apoptosis and/or necrosis, as a function of the intensity rather than the nature of the insult.     

Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells

Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells.     

Smokeless tobacco, oxidative stress, apoptosis, and antioxidants in human oral keratinocytes

We have investigated the effects of a smokeless tobacco extract (STE) on lipid peroxidation, cytochrome c reduction, DNA fragmentation and apoptotic cell death in normal human oral keratinocyte cells, and assessed the protective abilities of selected antioxidants. The cells, isolated and cultured from human oral tissues, were treated with STE (0-300 microl;g/ml) for 24 h. Superoxide anion production was determined by cytochrome c reductase. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, whereas apoptotic cell death was assessed by flow cytometry. STE-induced fragmentation of genomic DNA was also determined by gel electrophoresis. The comparative protective abilities of vitamin C (75 microM), vitamin E (75 microM), a combination of vitamins C & E (75 microM each), and a novel grape seed proanthocyanidin (IH636) extract (GSPE) (100 microg/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatment of the cells with 300 microg STE/ml 1.5-7.6-fold increases in lipid peroxidation, cytochrome c reduction and DNA fragmentation were observed. The addition of the antioxidants to cells treated with STE provided 10-54% decreases in these parameters. Approximately 9, 29, and 35% increases in apoptotic cell death were observed following treatment with 100, 200, and 300 microg STE/ml, respectively, and 51-85% decreases in apoptotic cell death were observed with the antioxidants. The results demonstrate that STE produces oxidative tissue damage and apoptosis, which can be attenuated by antioxidants including vitamin C, vitamin E, a combination of vitamins C plus E and GSPE. GSPE exhibited better protection against STE than vitamins C and E, singly and in combination.     

The role of Bax in glutamate-induced nerve cell death

The role of the Bax gene product was examined in three forms of cortical nerve cell death in primary cultures. These include spontaneous cell death, oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake resulting in toxic oxidative stress, and ionotropic glutamate receptor-mediated excitotoxicity following a brief exposure to 10 microM glutamate. Primary cortical and hippocampal neuron cultures were established from embryos of Bax -/+ x Bax -/+ matings and the embryos genotyped and assayed for cell death in the three experimental paradigms. Cell death induced by oxidative glutamate toxicity and glutamate-mediated excitotoxicity was not altered in the Bax -/- homozygous knockout animals. In contrast, there was an approximately 50% inhibition of spontaneous cell death. These results suggest that a classical Bax-dependent apoptotic pathway contributes to the spontaneous cell death that takes place when nerve cells are initially exposed to cell culture conditions. A Bax-dependent programmed cell death pathway is not, however, utilized in oxidative glutamate toxicity and NMDA receptor-mediated excitotoxicity following a brief exposure to low concentrations of glutamate.     

Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction

One of the earliest changes observed in retinal microvessels in diabetic retinopathy is the selective loss of intramural pericytes. We tested the hypothesis that AGE might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the signaling pathway leading to cell death. Chronic exposure of pericytes to methylglyoxal-modified bovine serum albumin (AGE-BSA) (3 microM) leads to a 3-fold increase of apoptosis (8.9 +/- 1.1%), associated with an increase in cellular ceramide (185 +/- 12%) and diacylglycerol (194 +/- 9%) levels. Ceramide formation was almost inhibited (95%) by an acidic sphingomyelinase inhibitor, desipramine (0.3 microM). Dual inhibition of ceramide (95%) and diacylglycerol (80%) production was observed with a phosphatidylcholine-phospholipase C inhibitor, D609 (9.4 microM). Taken together, these results suggest activation of phosphatidylcholine-phospholipase C coupled to acidic sphingomyelinase. However, both inhibitors only partially protected pericytes against apoptosis, suggesting another apoptotic pathway independent of diacylglycerol/ceramide production. Treatments with various antioxidants completely inhibited pericyte apoptosis, suggesting oxidative stress induction during this apoptotic process. Inhibition of diacylglycerol/ceramide production by N-acetyl-L-cysteine suggests that oxidative stress acts upstream of the two metabolic pathways. AGE treated with metal chelators were also able to induce pericyte apoptosis, suggesting a specific effect of AGE on intracellular oxidative stress independent of redox-active metal ions bound to AGE. In conclusion, these results identify new biochemical targets involved in pericyte loss, which can provide new therapeutic perspectives in diabetic retinopathy.     

Protective effects of riluzole on dopamine neurons: involvement of oxidative stress and cellular energy metabolism

Riluzole is neuroprotective in patients with amyotrophic lateral sclerosis and may also protect dopamine (DA) neurons in Parkinson's disease. We examined the neuroprotective potential of riluzole on DA neurons using primary rat mesencephalic cultures and human dopaminergic neuroblastoma SH-SY5Y cells. Riluzole (up to 10 microM:) alone affected neither the survival of DA neurons in primary cultures nor the growth of SH-SY5Y cells after up to 72 h. Riluzole (1-10 microM:) dose-dependently reduced DA cell loss caused by exposure to MPP(+) in both types of cultures. These protective effects were accompanied by a dose-dependent decrease of intracellular ATP depletion caused by MPP(+) (30-300 microM:) in SH-SY5Y cells without affecting intracellular net NADH content, suggesting a reduction of cellular ATP consumption rather than normalization of mitochondrial ATP production. Riluzole (1-10 microM:) also attenuated oxidative injury in both cell types induced by exposure to L-DOPA and 6-hydroxydopamine, respectively. Consistent with its antioxidative effects, riluzole reduced lipid peroxidation induced by Fe(3+) and L-DOPA in primary mesencephalic cultures. Riluzole (10 microM) did not alter high-affinity uptake of either DA or MPP(+). However, in the same cell systems, riluzole induced neuronal and glial cell death with concentrations higher than those needed for maximal protective effects (> or =100 microM:). These data demonstrate that riluzole has protective effects on DA neurons in vitro against neuronal injuries induced by (a) impairment of cellular energy metabolism and/or (b) oxidative stress. These results provide further impetus to explore the neuroprotective potential of riluzole in Parkinson's disease.     

Arachidonic acid cytotoxicity in leukocytes: implications of oxidative stress and eicosanoid synthesis

Arachidonic acid (AA)-induced cytotoxicity was evaluated in leukocytes: the human leukemia cell lines HL-60, Jurkat and Raji and in rat lymphocytes. Such cytotoxicity was dose- and time-dependent. At concentrations below 5 microM, AA was not toxic; at 10-400 microM, AA induced apoptosis and at concentrations beyond 400 microM, necrosis. The minimum exposure time to trigger cell death was of around 1 h, but the effect was increased by longer exposure times until 6-24 h. Apoptosis was morphologically characterized by a decrease in cell and nuclear volume, chromatin condensation and DNA fragmentation and the presence of lipid bodies, without changes in organelle integrity. Biochemically, AA-induced apoptosis was associated with internucleosomal fragmentation and caspase activation, evaluated by PARP cleavage and the use of a caspase inhibitor. Necrosis was characterized by increased cell volume, presence of loose chromatin, appearance of vacuoles, loss of membrane integrity and of the definition of organelles. The apoptotic effect of AA was studied as to oxidative-reductive imbalance and the participation of eicosanoids. Apoptotic AA treatment was accompanied by an increase in the quantity of thiobarbituric acid reactive substances (TBARS), low-level chemiluminescence and in the glutathione disulfide/reduced glutathione ratio, indicating oxidative stress. The addition of tocopherol, ascorbate, prostaglandin E2 and lipoxygenase inhibitors delayed cell death, whereas the inhibition of cyclooxygenase promoted AA-induced cell death. Cell treatment with AA was accompanied by increased cellular production of LTB4. AA, therefore, is cytotoxic at physiological and supraphysiological concentrations, causing apoptosis and necrosis. Cell treatment with apoptotic concentrations of AA involves oxidative stress and changes in eicosanoid biosynthesis.     

Kinetics of HL-60 cell entry to apoptosis during treatment with TNF-alpha or camptothecin assayed by the stathmo-apoptosis method

BACKGROUND: Duration of apoptosis, from onset to final disintegration of the cell, is often short and variable. The apoptotic index (AI), as a snapshot of a transient event of variable length, does not truly represent incidence of apoptosis in the studied cell population. We recently proposed to estimate the cumulative apoptotic index (CAI) by inducing stathmo-apoptosis. A fluorescent inhibitor of caspases (FLICA) FAM-VAD-FMK is used to arrest the process of apoptosis and thereby prevent cell disintegration. Simultaneously, the arrested/apoptotic cells become FLICA-labeled. In the present study, this approach was applied to measure kinetics of HL-60 cell entrance into apoptosis induced via cell surface death receptor or a mitochondria-initiated pathway. Materials and Methods Cultures of HL-60 cells were treated with either TNF-alpha or camptothecin (CPT) in the absence or constant presence of 10-50 microM FLICA. The CAI was measured at different time points for up to 48 h by flow cytometry. Bivariate analysis of DNA content and cell labeling with FLICA was used to correlate apoptosis with the cell-cycle position. RESULTS: Selective loss of apoptotic cells seen in HL-60 cell cultures exposed to either TNF-alpha or CPT alone was prevented in cultures containing FLICA. Addition of FLICA alone had no effect on cell viability. The percentage of FLICA-labeled cells was plotted as a function of time after addition of TNF-alpha or CPT. The rate of cell entry to apoptosis was subsequently estimated from the slopes of the stathmo-apoptotic plot. The slopes revealed that the TNF-alpha or CPT-treated cells asynchronously underwent apoptosis with a stochastic-like kinetics and at two different rates. About 50% of cells in the TNF-alpha-treated cultures underwent apoptosis during the initial 6 h at a rate of approximately 8% of cells per hour; the remaining cells were undergoing apoptosis at a rate of approximately 2.5% of cells per hour for up to 24 h. Also, about 50% of the CPT-treated cells, predominantly those in S phase of the cell cycle, underwent apoptosis within the initial 8 h of CPT exposure, at a rate of approximately 7% of cells per hour. Remaining cells were undergoing apoptosis at a rate of approximately 1% of cells per hour during up to 48 h exposure to CPT. Spontaneous apoptosis in the untreated cultures occurred at a rate of 0.2% of cells per hour. CONCLUSIONS: This approach provides a means for measuring the kinetics of cell entrance to apoptosis (caspase activation) in large populations of cells in relation to the cell-cycle position.     

Methylglyoxal mediates vascular inflammation via JNK and p38 in human endothelial cells

Methylglyoxal (MGO) is a reactive metabolite of glucose. Since the plasma concentration of MGO is increased in diabetic patients, MGO is implicated in diabetes-associated vascular endothelial cells (ECs) injury, which might be responsible for atherosclerosis. In the present study, we examined effects of treatment of human umbilical vein ECs with MGO on EC morphology and inflammatory responses. MGO (24 h) induced cytotoxic morphological changes in a concentration-dependent manner (0-420 microM). MGO induced mRNA and protein expression of cyclooxygenase (COX)-2 in a concentration (0-420 microM)- and time (6-24 h)-dependent manner. COX-2 induction was associated with increased PGE(2) release. Acute treatment with MGO (20 min) induced concentration-dependent (0-420 microM) activation of JNK and p38 MAP kinase but not ERK or NF-kappaB. Both the JNK inhibitor SP600125 and the p38 inhibitor SB203580 prevented the MGO induction of COX-2. However, inhibiting JNK and p38 or COX-2 was ineffective to the morphological damage by MGO (420 microM, 24 h). EUK134, a synthetic combined superoxide dismutase/catalase mimetic, had no effect on MGO-induced COX-2. Present results indicated that MGO mediates JNK- and p38-dependent EC inflammatory responses, which might be independent of oxidative stress. On the other hand, MGO-induced morphological cell damage seems unlikely to be associated with COX-2-PGE(2).     

N-methyl bases of ethanolamine prevent apoptotic cell death induced by oxidative stress in cells of oligodendroglia origin

A major reason for brain tissue vulnerability to oxidative damage is the high content of polyunsaturated fatty acids (PUFAs). Oligodendroglia-like OLN 93 cells lack PUFAs and are relatively insensitive to oxidative stress. When grown in serum-free defined medium in the presence of 0.1 mM docosahexaenoic acid (DHA; 22:6 n-3) for 3 days, OLN 93 cells release in the medium 2.6-fold more thiobarbituric acid-reactive substances (TBARS) after a 30-min exposure to 0.1 mM H2O2 and 50 microM Fe2+. Release of TBARS was substantially decreased by approximately 20 and 30% on coincubation with either 1 mM N-monomethylethanolamine or N,N'-dimethylethanolamine (dEa), respectively. The protective effect of dEa was concentration- and time-dependent and was still visible after dEa removal, suggesting a long-lasting mechanism of protection. After 24 h following H2O2-induced stress, cell death monitored by cell sorting showed 16% of the cells in the sub-G1 area, indicative of apoptotic cell death. DHA-supplemented cultures showed 35% cell death, whereas cosupplements with dEa reduced cell death to 12%, indicating cell rescue. Although the exact mechanism for this protection is not known, the nature of the polar head group and the degree of unsaturation may determine the ultimate resistance of nerve cells to oxidative stress.     

Diazoxide triggers cardioprotection against apoptosis induced by oxidative stress

Although mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels have been reported to reduce the extent of apoptosis, the critical timing of mitoK(ATP) channel opening required to protect myocytes against apoptosis remains unclear. In the present study, we examined whether the mitoK(ATP) channel serves as a trigger of cardioprotection against apoptosis induced by oxidative stress. Apoptosis of cultured neonatal rat cardiomyocytes was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33342. Mitochondrial membrane potential (DeltaPsi) was measured by flow cytometry of cells stained with rhodamine-123 (Rh-123). Exposure to H(2)O(2) (500 microM) induced apoptosis, and the percentage of apoptotic cells increased progressively and peaked at 2 h. This H(2)O(2)-induced apoptosis was associated with the loss of DeltaPsi, and the time course of decrease in Rh-123 fluorescence paralleled that of apoptosis. Pretreatment of cardiomyocytes with diazoxide (100 microM), a putative mitoK(ATP) channel opener, for 30 min before exposure to H(2)O(2) elicited transient and mild depolarization of DeltaPsi and consequently suppressed both apoptosis and DeltaPsi loss after 2-h exposure to H(2)O(2). These protective effects of diazoxide were abrogated by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microM) but not by the sarcolemmal K(ATP) channel blocker HMR-1098 (30 microM). Our results suggest for the first time that diazoxide-induced opening of mitoK(ATP) channels triggers cardioprotection against apoptosis induced by oxidative stress in rat cardiomyocytes.     

Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine

Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.     

Effects of an anti-oxidative ACAT inhibitor on apoptosis/necrosis and cholesterol accumulation under oxidative stress in THP-1 cell-derived foam cells

THP-1 cell-derived foam cells were exposed to oxidative stress through combined treatment with acetylated LDL (acLDL) and copper ions (Cu2+). The foam cells showed caspase-dependent apoptotic changes on exposure to oxidative stress for 6 h, and necrotic changes with the leakage of LDH after 24 h. KY-455, an anti-oxidative ACAT inhibitor, and ascorbic acid (VC) but not YM-750, an ACAT inhibitor, prevented apoptotic and necrotic changes. These preventive effects of KY-455 and VC were accompanied by the inhibition of lipid peroxidation in culture medium containing acLDL and Cu2+, suggesting the involvement of oxidized acLDL in apoptosis and necrosis. Foam cells accumulated esterified cholesterol (EC) for 24 h in the presence of acLDL without Cu2+, which was suppressed by KY-455 and YM-750. Foam cells showed necrotic changes and died in the presence of acLDL and Cu2+. KY-455 but not YM-750 prevented cell death and reduced the amount of EC accumulated. The foam cells treated with VC further accumulated EC without necrotic changes for 24 h even in the presence of acLDL and Cu2+. YM-750 as well as KY-455 inhibited lipid accumulation when co-incubated with VC in foam cells exposed to oxidative stress. It is concluded that an anti-oxidative ACAT inhibitor or the combination of an antioxidant and an ACAT inhibitor protects foam cells from oxidative stress and effectively reduces cholesterol levels, which would be a promising approach in anti-atherosclerotic therapy.     

Selenium protection against mercury-induced apoptosis and growth inhibition in cultured K-562 cells

Selenium and mercuric chloride (MC) interactions regarding effects on cell growth and cell death have been studied. Human K-562 cells were pretreated or simultaneously treated with either selenite (5 or 50 μM) or selenomethionine (10 or 50 μM) and with MC (35 or 50 μM). The 35-μM MC treatments resulted in a clear inhibition of cell growth with no obvious difference between mercury-treated and mercury-selenium-treated cells. Furthermore, the apoptotic frequency was similar at all observations for all selenium treatments with 35 μM MC. In the simultaneously treated selenite and 50-μM MC combinations, a selenite-dependent protection was shown both by increased cell growth and by lower apoptotic frequency at 48 and 96 h of exposure. Both treatments with selenomethionine showed protection observed as an increased cell growth at 48 and 96 h and as decreased apoptotic frequency at 96 h of exposure.     

Puerarin protects PC12 cells against beta-amyloid-induced cell injury

beta-Amyloid protein (Abeta), a major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. In this study, the protective effect of puerarin, an isoflavone purified from the radix of the Chinese herb Pueraria lobata, on Abeta-induced rat pheochromocytoma (PC12) cultures was investigated. Although exposure of PC12 cells to 50 microM Abeta25-35 caused significant viability loss and apoptotic rate increase, pretreatment of the cells with puerarin for 24h reduced the viability loss and apoptotic rate. Puerarin (1 microM) significantly inhibited Abeta25-35-induced apoptosis of PC12 cells. Preincubation of the cell with puerarin also restored the ROS and mitochondrial membrane potential levels that had been altered as a result of Abeta25-35 treatment. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce caspase-3 activation. These results suggest that puerarin could attenuate Abeta25-35-induced PC12 cell injure and apoptosis and could also promote the survival of PC12 cells. Therefore, puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta25-35-induced cell injury.