Mechanism of apoptosis induced by a new topoisomerase inhibitor through the generation of hydrogen peroxide

TAS-103, a new anticancer drug, induces DNA cleavage by inhibiting the activities of topoisomerases I and II. We investigated the mechanism of TAS-103-induced apoptosis in human cell lines. Pulsed field gel electrophoresis revealed that in the leukemia cell line HL-60 and the H(2)O(2)-resistant subclone, HP100, TAS-103 induced DNA cleavage to form 1-2-Mb fragments at 1 h to a similar extent, indicating that the DNA cleavage was induced independently of H(2)O(2). TAS-103-induced DNA ladder formation in HP100 cells was delayed compared with that seen at 4 h in HL-60 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded increases in mitochondrial membrane potential (DeltaPsim) and caspase-3 activation. Inhibitors of poly(ADP-ribose) polymerase (PARP) prevented both TAS-103-induced H(2)O(2) generation and DNA ladder formation. The levels of NAD(+), a PARP substrate, were significantly decreased in HL-60 cells after a 3-h incubation with TAS-103. The decreases in NAD(+) levels preceded both increases in DeltaPsim and DNA ladder formation. Inhibitors of NAD(P)H oxidase prevented TAS-103-induced apoptosis, suggesting that NAD(P)H oxidase is the primary enzyme mediating H(2)O(2) formation. Expression of the antiapoptotic protein, Bcl-2, in BJAB cells drastically inhibited TAS-103-induced apoptosis, confirming that H(2)O(2) generation occurs upstream of mitochondrial permeability transition. Therefore, these findings indicate that DNA cleavage by TAS-103 induces PARP hyperactivation and subsequent NAD(+) depletion, followed by the activation of NAD(P)H oxidase. This enzyme mediates O(2)(-)-derived H(2)O(2) generation, followed by the increase in DeltaPsim and subsequent caspase-3 activation, leading to apoptosis.     

Mechanism of oxyhemoglobin oxidation induced by hydrogen peroxide]

The process of oxyhemoglobin oxidation initiated by hydrogen peroxide in low (10(-7) M) concentrations was investigated. It was found, that H2O2 in this concentration is able to induce the process of chain oxidation of oxyhemoglobin to methemoglobin. The following observations indicate that the process is essentially the chain reaction: 1) The amount of the methemoglobin in haem groups, produced in the reaction, exceed by 20 times the quantity of hydrogen, added initially, to induce the oxidation. 2) Catalase stopped this process at any stage of the reaction. This fact implies that the chain process involves generation of new molecules of H2O2 in the course of oxidation of oxyhemoglobin. The chain reaction proceeded only in the presence of oxygen. But if oxygen was introduced into hemoglobin solution, preincubated with H2O2 in vacuum, than again the oxidation of hemoglobin developed. Apparently, H2O2 in low concentrations appears, mainly, as an inductor of the oxyhemoglobin autooxidation.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Caspase-dependent and -independent events in apoptosis induced by hydrogen peroxide

To define the role of caspase-3 in H2O2-induced apoptosis, we introduced caspase-3 cDNA into MCF-7 breast carcinoma cells that otherwise lack caspase-3 expression. H2O2 treatment induced DNA fragmentation and nuclear condensation in the caspase-3-expressing cells, but not in the caspase-3-deficient cells. This indicated that caspase-3 is essential for nuclear events. However, H2O2 induced an externalization of membrane phosphatidylserine (PS) and cell death regardless of caspase-3 expression. These events were not suppressed by Ac-DEVD-CHO and Z-VAD-fmk, which inhibit DEVD-specific caspases and a broad spectrum of caspases, respectively. In Jurkat T cells, these inhibitors abolished H2O2-induced PS relocalization, but not cell death. Therefore, caspases appear to be dispensable for lethality by H2O2, but required for PS redistribution in a cell-type-specific manner.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Mechanism for generation of hydrogen peroxide and change of mitochondrial membrane potential during rotenone-induced apoptosis

Rotenone, an inhibitor of NADH dehydrogenase complex, is a naturally occurring insecticide, which is capable of inducing apoptosis. Rotenone-induced apoptosis is considered to contribute to its anticancer effect and the etiology of Parkinson's disease (PD). We demonstrated that rotenone induced internucleosomal DNA fragmentation, DNA ladder formation, in human cultured cells, HL-60 (promyelocytic leukemia) and BJAB cells (B-cell lymphoma). Flow cytometry showed that rotenone induced H2O2 generation, followed by significant changes in the mitochondrial membrane potential (DeltaPsim). Caspase-3 activity increased in HL-60 cells in a time-dependent manner. These apoptotic events were delayed in HP100 cells, an H2O2-resistant clone of HL-60, confirming the involvement of H2O2 in apoptosis. Expression of anti-apoptotic protein, Bcl-2, in BJAB cells drastically inhibited DeltaPsim change and DNA ladder formation but not H2O2 generation, confirming the participation of mitochondrial dysfunction in apoptosis. NAD(P)H oxidase inhibitors prevented H2O2 generation and DNA ladder formation. These results suggest that rotenone induces O2(-)-derived H2O2 generation through inhibition of NADH dehydrogenase complex and/or activation of NAD(P)H oxidase, and H2O2 generation causes the disruption of mitochondrial membrane in rotenone-induced apoptosis.     

Ginsenoside-Rd potentiates apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through the mitochondrial pathway

Our previous studies showed that ginsenoside-Rd, a purified component from Panax notoginseng, inhibited cell proliferation and reversed basilar artery remodeling. The aim of this study was to investigate whether ginsenoside- Rd influences H₂O₂-induced apoptosis in basilar artery smooth muscle cells (BASMCs). The results showed that ginsenoside-Rd significantly potentiated H₂O₂-induced cell death and cell apoptosis. This resulted in a concentration-dependent reduction of the cell viability. Ginsenoside-Rd further increased cytochrome C release and caspase-9/caspase-3 activations, and reduced the stability of mitochondrial membrane potential (MMP) and the ratio of Bcl-2/Bax. Cyclosporine A, an inhibitor of mitochondrial-permeability transition, inhibited alteration of mitochondrial permeability induced by H₂O₂ and reversed the effect of ginsenoside-Rd on MMP. Our data strongly suggest that ginsenoside-Rd potentiated H₂O₂-induced apoptosis of BASMCs through the mitochondria-dependent pathway.     

Maltol inhibits apoptosis of human neuroblastoma cells induced by hydrogen peroxide

To analyze the effect of Maltol on the apoptosis of Human Neuroblastoma Cells (SH-SY5Y) treated by free radical which was generated from Hydrogen Peroxide (H2O2), flow cytometry analysis on Phosphatidylserine (PS) inverting percentage was applied to determine the apoptosis. MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was employed to analyze the cell viability. DNA electrophoresis was used to detect DNA fragmentation. Moreover intracellular calcium of concentration ([Ca2+]i) was measured by fluorescence emission. Flow cytometry analysis on the function of mitochondria and Western blot analysis of NF-kappaB. The results showed that the pretreatment with maltol for 2 hours could prevent the H2O2-induced apoptosis. Maltol could reduce the inverting percentage of PS, DNA fragmentation and [Ca2+]i, and enhance the cellular function of mitochondria. NF-kappaB activated by H2O2 is reduced. The experiments suggest that maltol could effectively inhibit the apoptosis induced by H2O2. As a novel anti-oxidant, maltol is a new promising drug in protecting the neurological cells from the damage by free radical.     

Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H₂O₂) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H₂O₂ alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 μmol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 μmol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.     

Induction of apoptosis by takrisodokyeum through generation of hydrogen peroxide and activation of caspase-3 in HL-60 cells

Takrisodokyeum (TRSDY), a Chinese herbal medicine, has been known to exert anti-tumoral activity in Korea. However, its molecular mechanism of action is not understood. In this study, we found that TRSDY induced apoptosis in HL-60 cells as evidenced by both a characteristic ladder pattern of discontinuous DNA fragments and an increase of annexin V+/PI- stained cell population. Our data demonstrated that TRSDY-induced apoptotic cell death was accompanied by activation of caspase-3 and cleavages of its substrates, poly(ADP-ribose) polymerase (PARP) and RhoGDP dissociation inhibitor (RhoGDI-2; also called D4-GDI) in a time- and concentration-dependent manner. Caspase-3 inhibitor, but not caspase-1 inhibitor, prevented TRSDY-induced apoptosis. Furthermore, treatment with TRSDY increased the production of intracellular hydrogen peroxide and pretreatment of cells with anti-oxidants conferred complete protection against hydrogen peroxide generation and subsequent caspase-3 activation. Taken together, these results suggest that TRSDY induces hydrogen peroxide generation, which, in turn, causes activation of caspase-3, degradation of PARP and D4-GDI, and eventually leads to apoptotic cell death.     

Mechanism of superoxide and hydrogen peroxide generation by human electron-transfer flavoprotein and pathological variants

Reactive oxygen species production by mitochondrial enzymes plays a fundamental role both in cellular signaling and in the progression of dysfunctional states. However, sources of reactive oxygen species and the mechanisms by which enzymes produce these reactive species still remain elusive. We characterized the generation of reactive oxygen species by purified human electron-transfer flavoprotein (ETF), a mitochondrial enzyme that has a central role in the metabolism of lipids, amino acids, and choline. The results showed that ETF produces significant amounts of both superoxide and hydrogen peroxide in the presence of its partner enzyme medium-chain acyl-CoA dehydrogenase (MCAD). ETF-mediated production of reactive oxygen species is partially inhibited at high MCAD/ETF ratios, whereas it is enhanced at high ionic strength. Determination of the reduction potentials of ETF showed that thermodynamic properties of the FAD cofactor are changed upon formation of a complex between ETF and MCAD, supporting the notion that protein:protein interactions modulate the reactivity of the protein with dioxygen. Two pathogenic ETF variants were also studied to determine which factors modulate the reactivity toward molecular oxygen and promote reactive oxygen species production. The results obtained show that destabilized conformations and defective protein:protein interactions increase the ability of ETF to generate reactive oxygen species. A possible role for these processes in mitochondrial dysfunction in metabolic disorders of fatty acid β-oxidation is discussed.     

Light-induced intracellular hydrogen peroxide generation through genetically encoded photosensitizer KillerRed-SOD1

Abstract

Hydrogen peroxide (H₂O₂) plays an important role in various biological processes in numerous organisms. Depending on the concentration and the distribution within the cell, it can act as stressor or redox signalling molecule. To analyse the effects of H₂O₂ and its diffusion within the cell we developed the new genetically encoded photosensitizer KillerRed-SOD1 which enables a light-induced spatially and temporally controlled generation of H₂O₂ in living cells. The KillerRed-SOD1 is a fusion protein of the photosensitizer KillerRed (KR) and the cytosolic superoxide dismutase isoform 1 (SOD1) connected by a helix-forming peptide linker. Light irradiation at a wavelength of 560?nm induced superoxide radical formation at the KR domain which was transformed to H₂O₂ at the SOD1 domain. H₂O₂ was specifically detected under live cell conditions using the fluorescent sensor protein HyPer. Genetically encoded photosensitizers have the advantage that appropriate tag sequences can determine the localisation of the protein within the cell. Herein, it was exemplarily shown that the peroxisomal targeting sequence 1 directed the photosensitizer KR-SOD1 to the peroxisomes and enabled H₂O₂ formation specifically in these organelles. In summary, with the photosensitizer KR-SOD1 a new valuable tool was established which allows a controlled intracellular H₂O₂ generation for the analysis of H₂O₂ effects on a subcellular level.     

ClC-3 chloride channel prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through mitochondria dependent pathway

ClC-3 Cl⁻ channel plays an important role in cell volume regulation and cell cycle. In vascular smooth muscle cells, we have found that ClC-3 was involved in ET-1 induced cell proliferation. The present study was designed to further investigate the role of ClC-3 Cl⁻ channel in H₂O₂-induced apoptosis and its underlying mechanisms in rat basilar arterial smooth muscle cell (BASMCs). By using ClC-3 cDNA and small interference RNA (siRNA) transfection strategy, it was found that overexpression of ClC-3 significantly decreased the apoptotic rate of H₂O₂-treated BASMCs and increased the cell viability, whereas silencing of ClC-3 with siRNA produced opposite effects and increased the apoptotic rate. ClC-3 overexpression decreased cytochrome C release and caspase-3 activation, and increased both the stability of mitochondrial membrane potential and the ratio of Bcl-2/Bax, whereas silencing of ClC-3 produced opposite effect. Furthermore, we demonstrated that overexpression of ClC-3 attenuated, whereas silencing of ClC-3 facilitated, the degradation of LaminA, one of the structural matrix proteins, in BASMCs. Our data suggest that ClC-3 Cl⁻ channel can modulate H₂O₂-induced apoptosis in BASMCs via the intrinsic, mitochondrial pathway.     

Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H(2)O(2) by N-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H(2)O(2) generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53-/-) cells. These results demonstrate that vanadate induces p53 activation mainly through H(2)O(2) generation, and this activation is required for vanadate-induced apoptosis.     

Oxidative modification of ferritin induced by hydrogen peroxide

Excess free iron generates oxidative stress that may contribute to the pathogenesis of various causes of neurodegenerative diseases. In this study, we assessed the modification of ferritin induced by H(2)O(2). When ferritin was incubated with H(2)O(2), the degradation of ferritin L-chain increased with the H(2)O(2) concentration whereas ferritin H-chain was remained. Free radical scavengers, azide, thiourea, and N-acetyl-(L)-cysteine suppressed the H(2)O(2)-mediated ferritin modification. The iron specific chelator, deferoxamine, effectively prevented H(2)O(2)-mediated ferritin degradation in modified ferritin. The release of iron ions from ferritin was increased in H(2)O(2) concentration-dependent manner. The present results suggest that free radicals may play a role in the modification and iron releasing of ferritin by H(2)O(2). It is assumed that oxidative damage of ferritin by H(2)O(2) may induce the increase of iron content in cells and subsequently lead to the deleterious condition.     

Fragmentation of human ceruloplasmin induced by hydrogen peroxide

We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H₂O₂) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca²⁺/Mg²⁺-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca²⁺/Mg²⁺-supplemented medium. In Ca²⁺/Mg²⁺-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 µm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H₂O₂ (84.40±0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 µm) induced apoptosis and pronounced generation of intracellular H₂O₂ (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H₂O₂ level (81.20±1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H₂O₂ in rat eggs.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H(2)O(2)) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca(2+)/Mg(2+)-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca(2+)/Mg(2+)-supplemented medium. In Ca(2+)/Mg(2+)-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 microm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H(2)O(2) (84.40+/-0.50 ng/egg) when compared to control eggs (80.46+/-1.34 ng/egg). The higher concentration of calcium ionophore (1.6 microm) induced apoptosis and pronounced generation of intracellular H(2)O(2) (92.43+/-0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H(2)O(2) level (81.20+/-1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H(2)O(2) in rat eggs.     

Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide

Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.