Reactive oxygen species are downstream mediators of p53-dependent apoptosis.

Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis. Conversely, experiments under hypoxic conditions have suggested that apoptosis could occur in the absence of ROS. We sought to determine whether a central modulator of apoptosis, p53, regulates the levels of intracellular ROS and whether a rise in ROS levels is required for the induction of p53-dependent apoptosis. We transiently overexpressed wild-type p53, using adenoviral gene transfer, and identified cell types that were sensitive or resistant to p53-mediated apoptosis. Cells sensitive to p53-mediated apoptosis produced ROS concomitantly with p53 overexpression, whereas cells resistant to p53 failed to produce ROS. In sensitive cells, both ROS production and apoptosis were inhibited by antioxidant treatment. These results suggest that p53 acts to regulate the intracellular redox state and induces apoptosis by a pathway that is dependent on ROS production.     

Liposomal encapsulation diminishes daunorubicin-induced generation of reactive oxygen species, depletion of ATP and necrotic cell death in human leukaemic cells

In this study, we tested the mechanisms of daunorubicin (DNR)- and the liposomal encapsulated daunorubicin (DaunoXome or DNX)-induced killing in three human leukaemic cell lines, K562, K/Bax and CEM. DNX showed less cytotoxicity in leukaemic cells than conventional DNR. The intracellular accumulation of DNX was 10 times less than conventional DNR during exposure to drugs for up to 5 h. Cell cycle analysis indicated that DNR induced concentration-dependent G2/M arrest, apoptosis and necrosis. However, DNX induced G2/M arrest and apoptosis but not necrotic cell death, even at a higher concentration. DNR- or DNX-induced activation of caspase-9 and -3 was detected at concentrations that induced apoptosis and necrosis. The sensitivity of leukaemic cells to DNR- and DNX-induced apoptosis correlated with the activation of caspases and the reduction of mitochondrial membrane potential (DeltaPsim), but not the depletion of ATP and the generation of reactive oxidative species (ROS). DNX did not provoke ROS generation and ATP depletion in leukaemic cells. We conclude that the liposomal encapsulation of DNR restricts the intracellular accumulation speed and therefore diminishes ROS generation, ATP depletion and necrotic cell death. This may have implications for the cause of cardiotoxicity seen with DNR, its main dose-limiting step.     

Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization

OBJECTIVE: Increased chondrocyte nitric oxide (NO) and peroxynitrite production appears to modulate decreased matrix synthesis and increased mineralization in osteoarthritis (OA). Because NO inhibits mitochondrial respiration, this study was undertaken to directly assess the potential role of chondrocyte mitochondrial oxidative phosphorylation (OXPHOS) in matrix synthesis and mineralization. METHODS: We studied cultured human articular chondrocytes and immortalized costal chondrocytes (TC28 cells). We also assessed the effects of antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) on chondrocyte mitochondrial respiration, ATP synthesis, and inorganic pyrophosphate (PPi) generation, and the mineralizing potential of released matrix vesicles (MV). RESULTS: Articular chondrocytes and TC28 cells respired at comparable rates. Peroxynitrite and NO donors markedly suppressed respiration and ATP generation in chondrocytes. Because NO exerts multiple effects on chondrocytes, we investigated the primary functions of mitochondrial respiration and OXPHOS. To do so, we identified minimally cytotoxic doses of antimycin and oligomycin, which both induced intracellular ATP depletion (by 50-80%), attenuated collagen and proteoglycan synthesis, and blocked transforming growth factor beta from increasing intracellular ATP and elaboration of PPi, a critical inhibitor of hydroxyapatite deposition. Antimycin and oligomycin also abrogated the ability of the ATP-hydrolyzing enzyme plasma cell membrane glycoprotein 1 (PC-1) to increase chondrocyte PPi generation. Finally, MV from cells treated with antimycin or oligomycin contained less PPi and precipitated >50% more 45Ca. CONCLUSION: Chondrocyte mitochondrial reserve, as NO-sensitive mitochondrial respiration-mediated ATP production, appears to support matrix synthesis and PPi elaboration and to regulate MV composition and mineralizing activity. NO-induced depression of chondrocyte respiration could modulate matrix loss and secondary cartilage mineralization in OA.     

Inhibition of the MUC1-C oncoprotein induces multiple myeloma cell death by down-regulating TIGAR expression and depleting NADPH

The MUC1-C oncoprotein is aberrantly expressed in most multiple myeloma cells. However, the functional significance of MUC1-C expression in multiple myeloma is not known. The present studies demonstrate that treatment of multiple myeloma cells with a MUC1-C inhibitor is associated with increases in reactive oxygen species (ROS), oxidation of mitochondrial cardiolipin, and loss of the mitochondrial transmembrane potential. The MUC1-C inhibitor-induced increases in ROS were also associated with down-regulation of the p53-inducible regulator of glycolysis and apoptosis (TIGAR). In concert with the decrease in TIGAR expression, which regulates the pentose phosphate pathway, treatment with the MUC1-C inhibitor reduced production of NADPH, and in turn glutathione (GSH) levels. TIGAR protects against oxidative stress-induced apoptosis. The suppression of TIGAR and NADPH levels thus contributed to ROS-mediated late apoptosis/necrosis of multiple myeloma cells. These findings indicate that multiple myeloma cells are dependent on MUC1-C and TIGAR for maintenance of redox balance and that targeting MUC1-C activates a cascade involving TIGAR suppression that contributes to multiple myeloma cell death.     

Mitochondrial membrane sensitivity to depolarization in acute myeloblastic leukemia is associated with spontaneous in vitro apoptosis, wild-type TP53, and vicinal thiol/disulfide status.

Nonresponse to remission-induction chemotherapy, which remains a major problem in acute myeloblastic leukemia (AML), has been linked to cellular resistance to apoptosis. Because the apoptosis induced by chemotherapeutic drugs is mediated by loss of mitochondrial transmembrane potential (MTP), it was postulated that sensitivity to mitochondrial membrane depolarization might be heterogeneous in AML. Using the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (mClCCP), the mitochondrial membrane sensitivity to depolarization (mClCCP concentrations that inhibit 50% of the transmembrane potential [IC(50)]) in AML blasts was measured and demonstrated marked interclonal heterogeneity, with the existence of comparatively sensitive (median mClCCP IC(50), 4 microM) and resistant (median mClCCP IC(50), 10 microM) clones. Furthermore, the mClCCP IC(50) was inversely associated with spontaneous in vitro apoptosis (P =.001). It was high in cases with mutant TP53 and correlated with the total cellular level of the multidrug resistance-associated protein (P =.019) but not of bcl-2, bax, or bcl-x. It was also found that the dithiol oxidant diamide, in contrast to the monovalent thiol oxidant diethyl maleate, increased the sensitivity of mitochondrial membranes to mClCCP. To confirm that TP53 directly affects MTP in leukemic cells and to establish the role of vicinal thiol oxidation in the TP53-dependent pathway, CEM 4G5 leukemia cells with forced, temperature-dependent expression of TP53 were studied. Monobromobimane, which inhibits mitochondrial membrane depolarization by preventing dithiol cross-linking, inhibited depolarization and apoptosis in 4G5 cells. It was concluded that in leukemia, TP53 and vicinal thiol/disulfide status are determinants of mitochondrial membrane sensitivity to depolarization, which is in turn associated with spontaneous apoptosis.     

BCL-2 expression and mitochondrial activity in leukemic cells with different sensitivity to glucocorticoid-induced apoptosis

The present study investigates the relationship between mitochondrial activity and the expression of the BCL-2 gene in a panel of six human and murine leukemia/lymphoma cell lines. The cell lines all contained normal glucocorticoid receptors but differed widely in sensitivity to dexamethasone, ranging from very sensitive S49 lymphoma to completely resistant HL-60 acute leukemia cells. In this panel, 10- to 15-fold differences in basal adenosine triphosphate (ATP) content and adenosine diphosphate (ADP)/ATP ratio were correlated with up to fivefold differences in bcl-2 protein (in human cells) and approximately 25-fold difference in bcl-2 mRNA content (all cell lines). Moreover, ATP content and BCL-2 gene expression were inversely correlated with glucocorticoid sensitivity and cell cycle length. In resistant cell lines, sensitivity to dexamethasone was restored by the mitochondrial inhibitors rotenone and meta-iodobenzylguanidine. This sensitization was not accompanied by detectable reductions in bcl-2 mRNA or protein content, suggesting that the inhibitors were capable of overriding BCL- 2-mediated inhibition of apoptosis. Increased mitochondrial activity and (overexpressed) BCL-2 appeared closely related properties of glucocorticoid-resistant cells, sharing common cellular targets in hormone-induced apoptosis.     

Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts

The mitochondrial theory of aging predicts that functional alterations in mitochondria contribute to the aging process. Whereas this hypothesis implicates increased production of reactive oxygen species (ROS) as a driving force of the aging process, little is known about molecular mechanisms by which mitochondrial impairment might contribute to aging. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between mitochondrial impairment and the appearance of a senescence-like phenotype in young cells. We found that treatment by antimycin A delays proliferation and induces premature senescence in a subset of the cells, associated with increased reactive oxygen species (ROS) production. Quenching of ROS by antioxidants did however not restore proliferation capacity nor prevent premature senescence. Premature senescence is also induced upon chronic exposure to oligomycin, irrespective of ROS production, and oligomycin treatment induced the up-regulation of the cdk inhibitors p16, p21 and p27, which are also up-regulated in replicative senescence. Thus, besides the well-established influence of ROS on proliferation and senescence, a reduction in the level of oxidative phosphorylation is causally related to reduced cell proliferation and the induction of premature senescence.     

Induction of mitochondrial changes in myeloma cells by imexon

Imexon is a cyanoaziridine derivative that has antitumor activity in multiple myeloma. Previous studies have shown that imexon induces oxidative stress and apoptosis in the RPMI 8226 myeloma cell line. This study reports that imexon has cytotoxic activity in other malignant cell lines including NCI-H929 myeloma cells and NB-4 acute promyelocytic leukemia cells, whereas normal lymphocytes and U266 myeloma cells are substantially less sensitive. Flow cytometric experiments have shown that imexon treatment is associated with the formation of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (Deltapsi(m)) in imexon-sensitive myeloma cell lines and NB-4 cells. In contrast, reduction of Deltapsi(m) and increased levels of ROS were not observed in imexon-resistant U266 cells. Treatment of imexon-sensitive RPMI 8226 cells with the antioxidant N-acetyl-L-cysteine (NAC) protects cells against these effects of imexon. Mitochondrial swelling was observed by electron microscopy in RPMI 8226 myeloma cells treated with 180 microM imexon as early as 4 hours. Damage to mitochondrial DNA was detected by a semiquantitative polymerase chain reaction assay in imexon-treated RPMI 8226 cells; however, nuclear DNA was not affected. Finally, partial protection of RPMI 8226 cells against the imexon effects was achieved by treatment with theonyltrifluoroacetone, an inhibitor of superoxide production at mitochondrial complex II. These changes are consistent with mitochondrial oxidation and apoptotic signaling as mediators of the growth inhibitory effects of imexon. Interestingly, oxidative damage and decrease of Deltapsi(m) induced by imexon highly correlates with sensitivity to imexon in several myeloma cell lines and an acute promyelocytic leukemia cell line. (Blood. 2001;97:3544-3551)     

Genipin ameliorates age-related insulin resistance through inhibiting hepatic oxidative stress and mitochondrial dysfunction

Insulin resistance (IR) increases with age and plays a key role in the pathogenesis of type 2 diabetes mellitus. Oxidative stress and mitochondrial dysfunction are supposed to be major factors leading to age-related IR. Genipin, an extract from Gardenia jasminoides Ellis fruit, has been reported to stimulate insulin secretion in pancreatic islet cells by regulating mitochondrial function. In this study, we first investigated the effects of genipin on insulin sensitivity and the potential mitochondrial mechanisms in the liver of aging rats. The rats were randomly assigned to receive intraperitoneal injections of either 25 mg/kg genipin or vehicle once daily for 12 days. The aging rats showed hyperinsulinemia and hyperlipidemia, and insulin resistance as examined by the decreased glucose decay constant rate during insulin tolerance test (kITT). The hepatic tissues showed steatosis and reduced glycogen content. Hepatic malondialdehyde level and mitochondrial reactive oxygen species (ROS) were higher, and levels of mitochondrial membrane potential (MMP) and ATP were lower as compared with the normal control rats. Administration of genipin ameliorated systemic and hepatic insulin resistance, alleviated hyperinsulinemia, hyperglyceridemia and hepatic steatosis, relieved hepatic oxidative stress and mitochondrial dysfunction in aging rats. Furthermore, genipin not only improved insulin sensitivity by promoting insulin-stimulated glucose consumption and glycogen synthesis, inhibited cellular ROS overproduction and alleviated the reduction of levels of MMP and ATP, but also reversed oxidative stress-associated JNK hyperactivation and reduced Akt phosphorylation in palmitate-treated L02 hepatocytes. In conclusion, genipin ameliorates age-related insulin resistance through inhibiting hepatic oxidative stress and mitochondrial dysfunction.     

Mitochondrial hyperpolarization and ATP depletion in patients with systemic lupus erythematosus

OBJECTIVE: Peripheral blood lymphocytes (PBLs) from systemic lupus erythematosus (SLE) patients exhibit increased spontaneous and diminished activation-induced apoptosis. We tested the hypothesis that key biochemical checkpoints, the mitochondrial transmembrane potential (deltapsim) and production of reactive oxygen intermediates (ROIs), mediate the imbalance of apoptosis in SLE. METHODS: We assessed the deltapsim with potentiometric dyes, measured ROI production with oxidation-sensitive fluorochromes, and monitored cell death by annexin V and propidium iodide staining of lymphocytes, using flow cytometry. Intracellular glutathione levels were measured by high-performance liquid chromatography, while ATP and ADP levels were assessed by the luciferin-luciferase assay. RESULTS: Both deltapsim and ROI production were elevated in the 25 SLE patients compared with the 25 healthy subjects and the 10 rheumatoid arthritis patients. Intracellular glutathione contents were diminished, suggesting increased utilization of reducing equivalents in SLE. H2O2, a precursor of ROIs, increased deltapsim and caused apoptosis in normal PBLs. In contrast, H2O2-induced apoptosis and deltapsim elevation were diminished, particularly in T cells, and the rate of necrotic cell death was increased in patients with SLE. The intracellular ATP content and the ATP:ADP ratio were reduced and correlated with the deltapsim elevation in lupus. CD3:CD28 costimulation led to transient elevation of the deltapsim, followed by ATP depletion, and sensitization of normal PBLs to H2O2-induced necrosis. Depletion of ATP by oligomycin, an inhibitor of F0F1-ATPase, had similar effects. CONCLUSION: T cell activation and apoptosis are mediated by deltapsim elevation and increased ROI production. Mitochondrial hyperpolarization and the resultant ATP depletion sensitize T cells for necrosis, which may significantly contribute to inflammation in patients with SLE.     

Ergosterol Peroxide Inhibits Porcine Epidemic Diarrhea Virus Infection in Vero Cells by Suppressing ROS Generation and p53 Activation

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that causes severe watery diarrhea in piglets with high morbidity and mortality, resulting in serious economic losses to the farming industry. Ergosterol peroxide (EP) is a sterol with diverse biological activities including antiviral activity. In this study, we explored whether EP extracted from the fruiting body of the mushroom Cryptoporus volvatus had the potential to inhibit PEDV infection in Vero cells. The results revealed that EP had a remarkable inhibitory effect on PEDV infection. It could significantly inhibit multiple stages of the PEDV life cycle, including internalization, replication and release, and could directly inactivate PDCoV infectivity. However, it did not affect PEDV attachment. Furthermore, EP alleviated PEDV-induced apoptosis and mitigated the decrease in mitochondrial membrane potential caused by PEDV infection. It suppressed ROS generation and p53 activation caused by PEDV infection. The ROS scavenger N-acetyl-l-cysteine (NAC) and the p53 specific inhibitor Pifithrin-α (PFT-α) suppressed PEDV-induced apoptosis and impeded viral replication, suggesting that ROS and p53 play an important role in PEDV-induced apoptosis and viral replication. Collectively, EP can prevent PEDV internalization, replication and release, possesses the ability to directly inactivate PEDV, and can inhibit PEDV-induced apoptosis by interfering with PEDV-induced ROS production and p53 activation. These findings highlight the therapeutic potential of EP against PEDV infection.     

Mitochondria in eosinophils: functional role in apoptosis but not respiration

In most eukaryotic cells, mitochondria use the respiratory chain to produce a proton gradient, which is then harnessed for the synthesis of ATP. Recently, mitochondrial roles in regulation of apoptosis have been discovered in many cell types. Eosinophils (Eos) die by apoptosis, but the presence and function of mitochondria in Eos are unknown. This study found that Eos contain mitochondria in small numbers, as shown by labeling with membrane potential-sensitive dyes and in situ PCR for a mitochondrial gene. Eos generate mitochondrial membrane potential from hydrolysis of ATP rather than from respiration, as shown by mitochondrial respiratory inhibitors and mitochondrial uncouplers. The mitochondria provide insignificant respiration but can induce apoptosis, as shown by using the mitochondrial F(1)F(0)-ATPase inhibitor oligomycin and translocation of cytochrome c. Thus during differentiation of Eos, although respiration is lost, the other central role of mitochondria, the induction of apoptosis, is retained.     

Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL

Previous studies showed that chronic lymphocytic leukemia (CLL) cells exhibit certain mitochondrial abnormalities including mtDNA mutations, increased superoxide generation, and aberrant mitochondrial biogenesis, which are associated with impaired apoptosis and reduced sensitivity to fludarabine. Here we report that CLL cells and multiple myeloma cells are highly sensitive to brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport currently being developed as a novel anticancer agent in a prodrug formulation. Of importance, brefeldin A effectively induced apoptosis in fludarabine-refractory CLL cells. Disruption of protein trafficking by brefeldin A caused the sequestration of the prosurvival factors APRIL and VEGF in the ER, leading to abnormal ER swelling and a decrease in VEGF secretion. Such ER stress and blockage of secretory protein traffic eventually resulted in Golgi collapse, activation of caspases, and cell death. Notably, the cellular sensitivity to this compound appeared to be independent of p53 status. Taken together, these findings suggest that malignant B cells may be highly dependent on ER-Golgi protein transport and that targeting this process may be a promising therapeutic strategy for B-cell malignancies, especially for those that respond poorly to conventional treatments.     

Intracellular ATP is required for mitochondrial apoptotic pathways in isolated hypoxic rat cardiac myocytes

OBJECTIVES: The present study examined the possibility that intracellular ATP levels dictate whether hypoxic cardiac myocytes die by apoptosis or necrosis. BACKGROUND: Although apoptosis and necrosis may appear to be distinct forms of cell death, recent studies suggest that the two may represent different outcomes of a common pathway. In ischemic myocardium, apoptosis appears early, while energy stores are presumably still available, followed only later by necrosis. METHODS: Neonatal rat cardiac myocytes were exposed to continuous hypoxia, during which the intracellular ATP concentration was modulated by varying the glucose content in the medium. The form of cell death was determined at the end of the hypoxic exposure. RESULTS: Under total glucose deprivation, ATP dropped precipitously and cell death occurred exclusively by necrosis as determined by nuclear staining with ethidium homodimer-1 and smearing on DNA agarose gels. However, with increasing glucose concentrations (10, 20, 50, 100 mg/dl) cellular ATP increased correspondingly, and apoptosis progressively replaced necrosis until it became the sole form of cell death, as determined by nuclear morphology, DNA fragmentation on agarose gels, and caspase-3 activation. The data showed a significantly positive correlation between myocyte ATP content and the percentage of apoptotic cells. Hypoxia resulted in lactate production and cellular acidification which stimulates apoptosis. However, acidification-induced apoptosis was also increased in an ATP-dependent fashion. Loss of mitochondrial membrane potential and cytochrome c release from the mitochondria was observed in both the apoptotic and necrotic cells. Furthermore, translocation of Bax from cytosol into mitochondria preceded these events associated with mitochondrial permeability transition. Increased lactate production and a lack of effect by the mitochondrial inhibitor oligomycin indicated that ATP was generated exclusively through glycolysis. CONCLUSIONS: We demonstrate that ATP, generated through glycolysis, is a critical determinant of the form of cell death in hypoxic myocytes, independently of cellular acidification. Our data suggest that necrosis and apoptosis represent different outcomes of the same pathway. In the absence of ATP, necrosis prevails. However, the presence of ATP favors and promotes apoptosis.     

Role of reactive oxygen species and Bax in oxidized low density lipoprotein-induced apoptosis of human monocytes

This study investigated the proapoptotic effects of oxidized low density lipoprotein (oxLDL), which plays a key role in atherogenesis, on normal fresh human monocytes isolated from peripheral blood (PBMs), on human monocyte-derived macrophages, and on U937 monocytic cell line. OxLDL were generated by hypochlorous acid (HOCl) treatment of native LDL. We demonstrated that HOCl–oxLDL (200 μg/ml) induced apoptosis in PBMs and U937 cells via the mitochondrial pathway, whereas it failed to induce apoptosis in human monocyte-derived macrophages. OxLDL-induced U937 cells apoptosis involved ROS generation, mitochondrial Bax translocation with a disruption of mitochondrial membrane potential, cytosolic liberation of cytochrome c and subsequently activation of caspases-9 and -3. The interference of ROS scavengers N-acetylcysteine and catalase with HOCl–oxLDL-induced apoptosis further supports the importance of mitochondrial ROS production in this process. Bcl-2 overexpression prevented Bax translocation whereas it failed to prevent ROS generation indicating that ROS is an upstream signal for inducing mitochondrial apoptotic damages. Because monocyte apoptosis could limit early atheroma formation, it will be interesting to identify the signaling pathway(s) induced by HOCl–oxLDL leading to ROS generation. In contrast, monocyte-derived macrophages, which resist to HOCl–oxLDL-induced oxidative stress, may promote atherosclerosis.     

Yeast YPK9 deficiency results in shortened replicative lifespan and sensitivity to hydrogen peroxide

YPK9/YOR291W of Saccharomyces cerevisiae encodes a vacuolar membrane protein. Previous research has suggested that Ypk9p is similar to the yeast P5-type ATPase Spf1p and that it plays a role in the sequestration of heavy metals. In addition, bioinformatics analysis has suggested that Ypk9p is a homolog of human ATP13A2, which encodes a protein of the subfamily of P5 ATPases. However, no specific function of Ypk9p has been described to date. In this study, we found, for the first time, that YPK9 is involved in the oxidative stress response and modulation of the replicative lifespan (RLS). We found that YPK9 deficiency confers sensitivity to the oxidative stress inducer hydrogen peroxide accompanied by increased intracellular ROS levels, decreased mitochondrial membrane potential, abnormal mitochondrial function, and increased incidence of early apoptosis in budding yeast. More importantly, YPK9 deficiency can lead to a shortened RLS. In addition, we found that overexpression of the catalase-encoding gene CTA1 can reverse the phenotypic abnormalities of the ypk9Δ yeast strain. Collectively, these findings highlight the involvement of Ypk9p in the oxidative stress response and modulation of RLS.

     

Reactive oxygen species and phosphatidylserine externalization in murine sickle red cells

Due to their role in oxygen transport and the presence of redox active haemoglobin molecules, red blood cells (RBC) generate relatively high levels of reactive oxygen species (ROS). To counteract the potential deleterious effects of ROS, RBCs have a well-integrated network of anti-oxidant mechanisms to combat this oxidative stress. ROS formation is increased in sickle-cell disease (SCD) and our studies in a murine SCD model showed a significant increase in the generation of ROS when compared with normal mice. Our data also indicated that murine sickle RBCs exhibit a significantly increased ATP catabolism, partly due to the increased activity of glucose-6-phosphate dehydrogenase and glutathione reductase to regenerate intracellular glutathione (GSH) levels to neutralize the adverse milieu of oxidative stress. Higher ATP consumption by the murine sickle RBCs, together with the increased ROS formation and impairment of the aminophospholipid translocase or flipase may underlie the exposure of phosphatidylserine on the surface of these cells.     

Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes

Oxidative stress-induced mitochondrial dysfunction has been shown to play a crucial role in the pathogenesis of a wide range of diseases. Protecting mitochondrial function, therefore, is vital for cells to survive during these disease processes. In this study, we demonstrate that melatonin, a chief secretory product of the pineal gland, readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptotic events and death in rat brain astrocytes (RBA-1). The early protection provided by melatonin in mitochondria of intact living cells was investigated by the application of time-lapse conventional, confocal, and multiphoton fluorescent imaging microscopy coupled with noninvasive mitochondria-targeted fluorescent probes. In particular, we observed that melatonin effectively prevented exogenously applied H2O2-induced mitochondrial swelling in rat brain astrocytes at an early time point (within 10 min) and subsequently reduced apoptotic cell death (150 min later). Other early apoptotic events such as plasma membrane exposure of phosphatidyl serine and the positive YOPRO-1 staining of the early apoptotic nucleus were also prevented by melatonin. A mechanistic study at the mitochondrial level related to the early protection provided by melatonin revealed that the indole molecule significantly reduced mitochondrial reactive oxygen species (ROS) formation induced by H2O2 stress. Melatonin also prevented mitochondrial ROS generation caused by other organic hydroperoxides including tert-butyl hydroperoxide and cumene hydroperoxide. This antioxidative effect of melatonin is more potent than that of vitamin E. Via its ability to reduce mitochondrial ROS generation, melatonin prevented H2O2-induced mitochondrial calcium overload, mitochondrial membrane potential depolarization, and the opening of the mitochondrial permeability transition (MPT) pore. As a result, melatonin blocked MPT-dependent cytochrome c release, the downstream activation of caspase 3, the condensation and karyorrhexis of the nucleus and apoptotic fragmentation of nuclear DNA. Thus, the powerful mitochondrial protection provided by melatonin reinforces its therapeutic potential to combat a variety of oxidative stress-induced mitochondrial dysfunctions as well as mitochondria-mediated apoptosis in various diseases.