Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are known to play an important role in the proliferation and viability of vascular smooth muscle cells. In this study, we determined the effects of increased superoxide dismutase and catalase activity on fetal pulmonary arterial smooth muscle cell (FPASMC) proliferation and viability using EUK-134, a superoxide dismutase/catalase mimetic. Treatment of FPASMC with EUK-134 or with a combination of superoxide dismutase and catalase enzymes decreased superoxide and hydrogen peroxide levels as detected by the fluorescent dyes dihydroethidium and dichlorodihydrofluorescein diacetate, respectively. EUK-134 (5 microM) attenuated serum-induced FPASMC proliferation, whereas 50 microM EUK-134 decreased the number of viable cells, suggesting cell death. Conversely, combined superoxide dismutase and catalase enzyme activity equivalent to 50 microM EUK-134 prevented proliferation but did not reduce the number of viable FPASMC. The loss of mitochondrial membrane potential after 18 h, an increase in caspase-9 and caspase-3 activity after 24 h, and the subsequent appearance of TdT-mediated dUTP nick end labeling-positive nuclei were detected in FPASMC after treatment with 50 microM EUK-134. This indicates an induction of programmed rather than necrotic cell death and suggests that prolonged removal of ROS is required to stimulate apoptosis. Compounds such as EUK-134 may, therefore, prove more effective than enzymic antioxidants over longer periods, especially when the aim is to decrease the number of smooth muscle cells in diseases resulting from excessive muscularization.     

In vitro and in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through ROS generation and mitochondrial death pathway

Pancreatic cancer is one of the most common invasive malignancies and the fourth leading cause of cancer related mortality in U.S., thus developing new strategies to control pancreatic cancer is an important mission. We investigated the mechanism of capsaicin, the major pungent ingredient of red-chili pepper, in inducing apoptosis in pancreatic cancer cells. Treatment of AsPC-1 and BxPC-3 cells with capsaicin resulted in a dose-dependent inhibition of cell-viability and induction of apoptosis which was associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. These effects were significantly blocked when the cells were pretreated with a general antioxidant N-acetyl cysteine (NAC). Exposure of AsPC-1 and BxPC-3 cells to capsaicin was also associated with increased expression of Bax, down-regulation of bcl-2, survivin and significant release of cytochrome c and AIF in the cytosol. On the contrary, above-mentioned effects were not observed in the normal acinar cells in response to capsaicin-treatment. Capsaicin-treatment resulted in the activation of JNK and JNK inhibitor SP600125 afforded protection against capsaicin-induced apoptosis. Furthermore, capsaicin when given orally markedly suppressed the growth of AsPC-1 pancreatic tumor xenografts in athymic nude mice, without side effects. Tumors from capsaicin treated mice demonstrated increased apoptosis, which was related to the activation of JNK and increased cytosolic protein expression of Bax, cytochrome c, AIF and cleaved caspase-3, as compared with controls. Taken together, these results show that capsaicin is an effective inhibitor of in vitro and in vivo growth of pancreatic cancer cells. These findings provide the rationale for further clinical investigation of capsaicin against pancreatic cancer. Ruifen Zhang and Ian Humphreys contributed equally to this work.     

Cinnabarinic acid generated from 3-hydroxyanthranilic acid strongly induces apoptosis in thymocytes through the generation of reactive oxygen species and the induction of caspase

3-Hydroxyanthranilic acid (3HAA) is one of the tryptophan metabolites along the kynurenine pathway and induces apoptosis in T cells. We investigated the mechanism of 3HAA-induced apoptosis in mouse thymocytes. The optimal concentration of 3HAA for apoptosis induction was 300-500 microM. The induction of apoptosis by a suboptimal concentration (100 microM) of 3HAA was enhanced by superoxide dismutase (SOD) as well as MnCl2 and further promoted in the presence of catalase. The 3HAA-mediated generation of intracellular reactive oxygen species (ROS) was enhanced by SOD or MnCl2 and inhibited by catalase. Corresponding to apoptosis induction, the generation of cinnabarinic acid (CA) through the oxidation of 3HAA was enhanced by SOD or MnCl2 in the presence of catalase. The synthesized CA possessed more than 10 times higher apoptosis-inducing activity than 3HAA. The intracellular ROS generation was induced by CA within 15 min and decreased to the control levels within 4 h, whereas the 3HAA-induced ROS generation increased gradually up to 4 h. Corresponding to ROS generation, the mitochondrial membrane potential was downregulated within 15 min and retained by the CA treatment. Apoptosis induction by 3HAA or CA was dependent on caspases, and caspase-3 was much more strongly activated by CA than 3HAA. In conclusion, the CA generated from 3HAA possesses a strong apoptosis-inducing activity in thymocytes through ROS generation, the loss of mitochondrial membrane potential, and caspase activation.     

Comparison of the effects of the superoxide dismutase mimetics EUK-134 and tempol on paraquat-induced nephrotoxicity

Paraquat-induced nephrotoxicity involves severe renal cell damage caused by reactive oxygen species (ROS), specifically via increasing concentrations of superoxide anions in the kidney. Recently, superoxide dismutase (SOD) mimetics (SODm) have been developed that display safe SOD activities but which also possess additional antioxidant enzyme (e.g., catalase) or ROS-scavenging activities. The aim of this study was to compare the effects of two such SODm, specifically, EUK-134, a SODm with catalase activity, and tempol, a SODm with ROS-scavenging properties, on paraquat-induced nephrotoxicity of renal NRK-52E cells. Incubation with paraquat (1 mM) for 24 h reduced cell viability and increased necrosis significantly. Paraquat also generated significant quantities of superoxide anions and hydroxyl radicals. Both EUK-134 (10-300 microM) and tempol (0.3-1.0 mM) were able to improve cell viability and reduced paraquat-induced cell death significantly via dismutation or scavenging of superoxide anions and reduced hydroxyl radical generation. The data presented here suggest that SODm such as EUK-134 and tempol, which possess additional catalase and/or ROS-scavenging activities, can significantly reduce renal cell damage caused by paraquat. These effects were evident at concentrations which avoid the pro-oxidant activities associated with higher concentrations of SOD. Such SODm could therefore prove to be beneficial as therapies for paraquat nephrotoxicity.     

Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells

Exposure of animals to hyperoxia results in respiratory failure and death within 72 h. Histologic evaluation of the lungs of these animals demonstrates epithelial apoptosis and necrosis. Although the generation of reactive oxygen species (ROS) is widely thought to be responsible for the cell death observed following exposure to hyperoxia, it is not clear whether they act upstream of activation of the cell death pathway or whether they are generated as a result of mitochondrial membrane permeabilization and caspase activation. We hypothesized that the generation of ROS was required for hyperoxia-induced cell death upstream of Bax activation. In primary rat alveolar epithelial cells, we found that exposure to hyperoxia resulted in the generation of ROS that was completely prevented by the administration of the combined superoxide dismutase/catalase mimetic EUK-134 (Eukarion, Inc., Bedford, MA). Exposure to hyperoxia resulted in the activation of Bax at the mitochondrial membrane, cytochrome c release, and cell death. The administration of EUK-134 prevented Bax activation, cytochrome c release, and cell death. In a mouse lung epithelial cell line (MLE-12), the overexpression of Bcl-XL protected cells against hyperoxia by preventing the activation of Bax at the mitochondrial membrane. We conclude that exposure to hyperoxia results in Bax activation at the mitochondrial membrane and subsequent cytochrome c release. Bax activation at the mitochondrial membrane requires the generation of ROS and can be prevented by the overexpression of Bcl-XL.     

Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not retard aging in the nematode Caenorhabditis elegans

According to the oxidative damage theory a primary cause of aging is the accrual of molecular damage from reactive oxygen species (ROS), particularly superoxide and its derivatives. This predicts that treatments that reduce ROS levels should retard aging. Using the nematode Caenorhabditis elegans, we tested the effects on stress resistance and life span of treatment with EUK-8 and EUK-134, synthetic mimetics of the antioxidant enzyme superoxide dismutase (SOD), which neutralises superoxide. Treatment with SOD mimetics elevated in vivo SOD activity levels, particularly in mitochondria, where up to 5-fold increases in SOD activity were recorded. Treatment with exogenous SOD mimetics did not affect endogenous protein SOD levels. Where life span was reduced by the superoxide generators paraquat and plumbagin, EUK-8 treatment increased life span in a dose-dependent fashion. Yet in the absence of a superoxide generator, treatment with EUK-8 or EUK-134 did not increase life span, even at doses that were optimal for protection against pro-oxidants. Thus, an elevation of SOD activity levels sufficient to increase life span when it is limited by superoxide generators does not retard aging in the absence of superoxide generators. This suggests that C. elegans life span is not normally limited by levels of superoxide and its derivatives.     

Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells

The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tert-butyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of SOD isoforms caused a 2- and 3.5-fold elevation in CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria, respectively, and 3-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-SOD expression similarly prevented PC-12 apoptosis, this was associated with increases in SOD activity but not GSH, indicating that cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-SOD overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-3 activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-SOD overexpression and is independent of cellular GSH levels per se.     

Ceramide-induced apoptosis: role of catalase and hepatocyte growth factor

The aim of this study was to elucidate cellular mechanisms involved in ceramide-induced apoptosis and its attenuation by hepatocyte growth factor (HGF). Human retinal pigmented epithelial cells (RPE) incubated with C2 ceramide accumulated reactive oxygen species (ROS) in mitochondria and underwent apoptosis in a dose-dependent manner. Ceramide-treated cells showed increased caspase-3 activation and an increase in mitochondrial membrane permeability transition (MPT). Low doses of H2O2 (100 microM) alone induced negligible apoptosis; however, ceramide-induced apoptosis was significantly enhanced by co-incubation with H2O2 (100 microM). Furthermore, ceramide treatment significantly decreased catalase enzymatic activity and protein expression. HGF pretreatment (20 ng/ml) significantly inhibited ceramide-induced apoptosis and reduced the accumulation of ROS, the activation of caspase-3, and the increase in MPT and prevented the reduction in catalase activity and expression. Together, the data suggest that ceramide induces apoptosis in RPE cells by increasing ROS production, MPT, and caspase-3 activation. The ceramide effect is potentiated by H2O2 and associated with a reduction in catalase activity, suggesting that catalase plays a central role in regulating this apoptotic response. The ability of HGF to attenuate these effects demonstrates its effectiveness as an antioxidant growth factor.     

Contribution of reactive oxygen species to the regulation of Glut1 in two hemopoietic cell lines differing in cytokine sensitivity

Glucose transport activity and its possible regulation by reactive oxygen species in two Glut1-expressing megakaryocytic cell lines, MO7e and B1647, differing in cytokine sensitivity were compared. Results show that: (1) In MO7e cells, glucose transport rate increased in response to thrombopoietin, granulocyte-macrophage colony-stimulating factor, or stem cell factor, due to a decreased Km. (2) A higher Vmax value was determined in B1647 cells, owing to the relative higher abundance of Glut1 on the plasmalemma; in these cells no change in glucose transport rate was observed on cytokine treatment. (3) The basal level of intracellular ROS was higher in B1647 than in M07e cells, where ROS production was enhanced upon cytokine exposure. (4) Basal or stimulated ROS production and Glut1 activity were significantly reduced by pretreating both cell lines with EUK-134, a superoxide dismutase and catalase mimetic. (5) In MO7e cells, EUK-134 brought back to control levels the Km values obtained on cytokine treatment, whereas in B1647 cells the antioxidant drastically reduced Vmax by decreasing the Glut1 content of the plasma membrane. Our data suggest that differences in acute regulation of glucose transport activity in the two cell lines may be related to differences in amplitude and spatial organization of ROS production.     

Iron toxicity in organotypic cultures of hippocampal slices: role of reactive oxygen species

Free iron has been assumed to potentiate oxygen toxicity by generating reactive oxygen species (ROS) via the iron-catalyzed Haber-Weiss reaction, leading to oxidative stress. ROS-mediated iron cytotoxicity may trigger apoptotic cell death. In the present study, we used iron treatment of organotypic cultures of hippocampal slices to study potential mechanisms involved in iron-induced neuronal damage. Exposure of mature hippocampal slices to ferrous sulfate resulted in concentration- and time-dependent cell death. After iron treatment, markers of ROS formation and lipid peroxidation, i.e. intensity of dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS), were significantly increased. Levels of cytochrome c were increased while levels of pro-caspase-9 and pro-caspase-3 were decreased in cytosolic fractions of iron-treated hippocampal slice cultures. Treatment of cultured slices with a synthetic catalytic ROS scavenger, EUK-134, provided between 50 and 70% protection against various parameters of cell damage and markers of oxidative stress. In addition, inhibition of caspase-3 activity by Ac-DEVDcho partially protected cells from iron toxicity. The combination of EUK-134 and Ac-DEVDcho resulted in an almost complete blockade of iron-induced damage. These results indicate that iron elicits cellular damage predominantly by oxidative stress, and that ROS-mediated iron toxicity may involve cytochrome c- and caspase-3-dependent apoptotic pathways.     

A comparative proteomic analysis for capsaicin-induced apoptosis between human hepatocarcinoma (HepG2) and human neuroblastoma (SK-N-SH) cells

The endogenous ROS levels were increased during HepG2 apoptosis, whereas they were decreased during SK-N-SH apoptosis in response to capsaicin treatments. We used 2-DE-based proteomics to analyze the altered protein levels in both cells, with special attention on oxidative stress proteins before and after capsaicin treatments. The 2-DE analysis demonstrated that 23 proteins were increased and 26 proteins were decreased significantly (fold change>1.4) in capsaicin-treated apoptotic HepG2 and SK-N-SH cells, respectively. The distinct effect of capsaicin-induced apoptosis on the expression pattern of HepG2 proteins includes the downregulation of some antioxidant enzymes including aldose reductase (AR), catalase, enolase 1, peroxiredoxin 1, but upregulation of peroxiredoxin 6, cytochrome c oxidase, and SOD2. In contrast, most antioxidant enzymes were increased in SK-N-SH cells in response to capsaicin, where catalase might play a pivotal role in maintenance of low ROS levels in the course of apoptosis. The global gene expression for oxidative stress and antioxidant defense genes revealed that 84 gene expressions were not significantly different in HepG2 cells between control and capsaicin-treated cells. In contrast, a number of oxidative genes were downregulated in SK-N-SH cells, supporting the evidence of low ROS environment in apoptotic SK-N-SH cells after capsaicin treatment. It was concluded that the different relationship between endogenous ROS levels and apoptosis of two cancer cells presumably resulted from complicated expression patterns of many oxidative stress and antioxidant genes, rather than the individual role of some classical antioxidant enzymes such as SOD and catalase.     

Induction of apoptosis in a human leukemic cell line via reactive oxygen species modulation by antioxidants

In the human acute myeloid leukemia cell line M07e, the growth factor interleukin-3 (IL-3) induces ROS formation, positively affecting Glut1-mediated glucose uptake and cell survival. The effect of IL-3 and exogenous hydrogen peroxide on cell viability seems to be mediated through inhibition of the cell death commitment, as shown by apoptotic markers such as caspase activities, apoptotic nuclei, and changes in the amount of proteins belonging to the Bcl-2 family. The pivotal role of ROS is confirmed using various antioxidants, such as EUK-134, ebselen, TEMPO, and hydroxylamine probe. In fact, these antioxidants, acting through different mechanisms, decrease glucose transport activity and cell proliferation activated by IL-3 or by low concentrations of hydrogen peroxide. Moreover, antioxidants foster programmed cell death commitment, as shown by the cited apoptotic parameters. EUK-134, a combined superoxide dismutase/catalase mimetic, opposes the effects of IL-3 and H₂O₂, decreasing phosphorylation levels of signaling enzymes such as Akt, Src tyrosine kinase, and ERK. Results show that ROS production induced by IL-3 can protect leukemic cells from apoptosis, the effect being counteracted by antioxidants. This mechanism may play an important role in supporting acute myeloid leukemia treatment, thus representing a novel therapeutic strategy.     

Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression

Pancreatic β cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in β cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic β cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.     

Ceramide increases oxidative damage due to inhibition of catalase by caspase-3-dependent proteolysis in HL-60 cell apoptosis

We investigated through which mechanisms ceramide increased oxidative damage to induce leukemia HL-60 cell apoptosis. When 5 microm N-acetylsphingosine (C(2)-ceramide) or 20 microm H(2)O(2) alone induced little increase of reactive oxygen species (ROS) generation as judged by the 2'-7'-dichlorofluorescin diacetate method, 20 microm H(2)O(2) enhanced oxidative damage as judged by ROS accumulation, and thiobarbituric acid-reactive substance production after pretreatment with 5 microm C(2)-ceramide at least for 12 h. The treatment with a catalase inhibitor, 3-amino-1h-1,2,4-triazole, increased oxidative damage and apoptosis induced by H(2)O(2), and in contrast, purified catalase inhibited the enhancement of oxidative damage by H(2)O(2) in ceramide-pretreated cells, suggesting that the oxidative effect of ceramide is involved in catalase regulation. Indeed, C(2)-ceramide inhibited the activity of immunoprecipitated catalase and decreased the levels of catalase protein in a time-dependent manner. Moreover, acetyl-Asp-Met-Gln-Asp-aldehyde, which dominantly inhibited caspase-3 and blocked the increase of oxidative damage and apoptosis due to C(2)-ceramide-induced catalase depletion at protein and activity levels. In vitro, active and purified caspase-3, but not caspase-6, -8, and -9, inhibited catalase activity and induced the proteolysis of catalase protein whereas these in vitro effects of caspase-3 were blocked by acetyl-Asp-Met-Gln-Asp-aldehyde. Taken together, it is suggested that H(2)O(2) enhances apoptosis in ceramide-pretreated cells, because ceramide increases oxidative damage by inhibition of ROS scavenging ability through caspase-3-dependent proteolysis of catalase.     

Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis

Our objective is to clarify the role of reactive oxygen species (ROS) in the atrophying tail of anuran tadpoles (tail apoptosis). Changes in catalase, superoxide dismutase (SOD) and caspase activity, genomic DNA, and nitric oxide (NO) generation were investigated biochemically using Rana japonica tadpole tails undergoing regression during thyroid hormone enhancement. DNA fragmentation and ladder formation with concomitant shortening of tadpole tail were induced by DL-thyroxine (T4) in culture medium. Catalase activity was also decreased by T4 treatment. T4 was also found to increase NO synthase (NOS) activity in cultured tadpole tail with concomitant increase in the concentration of NO2- plus NO3- (NOx) in the culture medium. Additional treatment with N-monomethyl-L-arginine (NMMA), a potent inhibitor of NOS, suppressed the enhancing effects of T4 on tail shortening and catalase activity reduction. It was also found that treatment with isosorbide dinitrate (ISDN), a NO generating drug, alone also had an enhancing effect on tail shortening and catalase activity reduction similar to that seen with T4. Both NO and an NO donor (ISDN) strongly suppressed catalase activity. Kinetic analysis revealed that catalase activity decreased and caspase-3-like activity increased during normal tadpole tail atrophy (apoptosis). These results suggested that T4 enhances NO generation, thereby strongly inhibiting catalase activity, resulting in an increase in hydrogen peroxide, and that the oxidative stress elicited by excess hydrogen peroxide might activate cysteine-dependent aspartate-directed protease-3 (caspase-3-like protease), which is thought to cause DNA fragmentation, leading to apoptosis.     

Direct current electrical fields induce apoptosis in oral mucosa cancer cells by NADPH oxidase-derived reactive oxygen species

The presence of more than one dental alloy in the oral cavity often causes pathological galvanic currents and voltage resulting in superficial erosions of the oral mucosa and eventually in the emergence of oral cancer. In the present study the mechanisms of apoptosis of oral mucosa cancer cells in response to electromagnetic fields was investigated. Direct current (DC) electrical fields with field strengths between 2 and 16 V/m, applied for 24 h to UM-SCC-14-C oral mucosa cancer cells, dose-dependently resulted in decreased cell proliferation as evaluated by Ki-67 immunohistochemistry and upregulation of the cyclin-dependent kinase (CDK) inhibitors p21(cip1/waf1) and p27(kip1), which are associated with cell cycle arrest. Electrical field treatment (4 V/m, 24 h) increased apoptosis as evaluated by immunohistochemical analysis of cleaved caspase-3 and poly-(ADP-ribose)-polymerase-1 (PARP-1). Furthermore, robust reactive oxygen species (ROS) generation, increased expression of NADPH oxidase subunits as well as Hsp70 was observed. Electrical field treatment (4 V/m, 24 h) resulted in increased expression of Cu/Zn superoxide dismutase and decreased intracellular concentration of reduced glutathione (GSH), whereas the expression of catalase remained unchanged. Pre-treatment with the free radical scavenger N-acetyl cysteine (NAC) and the superoxide dismutase mimetic EUK-8 abolished caspase-3 and PARP-1 induction, suggesting that apoptosis in oral mucosa cancer cells is initated by ROS generation in response to DC electrical field treatment.     

Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells.

Recent studies have demonstrated that induction of apoptosis is related to the cell growth inhibition potential of Salvia Miltiorrhiza (SM), a traditional herbal medicine. In the present study, we further explore the mechanistic pathway involved in SM-induced apoptosis in human hepatoma HepG2 cells. A rapid decline of intracellular glutathione (GSH) and protein thiol content was found in SM-treated cells. Moreover. SM exposure resulted in mitochondrial dysfunction as demonstrated by: (i) the onset of mitochondrial permeability transition (MPT); (ii) the disruption of mitochondrial membrane potential (MMP); and (iii) the release of cytochrome c from mitochondria into the cytosol. Subsequently, elevated level of intracellular reactive oxygen species (ROS) was observed prior to the onset of DNA fragmentation. However, no caspase-3 cleavage was observed throughout the whole period of SM treatment, while a caspase-3-independent poly(ADP-ribose) polymerase (PARP) cleavage was noted at the late stage in SM-induced apoptosis. Pretreatment of cells with N-acetylcysteine (NAC), the GSH synthesis precursor, conferred complete protection against MMP loss, ROS generation and apoptosis induced by SM. MPT inhibitors, cyclosporin A plus trifluoperazine, partially restored intracellular GSH content, and reduced SM-induced ROS formation and subsequently inhibited cell death. Moreover, antioxidants NAC, deferoxamine and catalase had little effect on GSH depletion and mitochondrial dysfunction, yet still were able to completely protect cells from SM-induced apoptosis. Taken together, our results suggest that SM deplete intracellular thiols, which, in turn, causes MPT and subsequent increase in ROS generation, and eventually apoptotic cell death.     

Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.     

Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: dependency on reactive oxygen species, Akt, Bid, cytochrome and caspase pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cells, but the mechanism of this effect has not been fully elucidated. We report that diclofenac, a NSAID, induces growth inhibition and apoptosis of HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS), Akt, caspase-8, and Bid. ROS generation occurs in an early stage of diclofenac-induced apoptosis preceding cytochrome c release, caspase activation, and DNA fragmentation. N-Acetyl-L-cysteine, an antioxidant, suppresses ROS generation, Akt inactivation, caspase-8 activation, and DNA fragmentation. Cyclic AMP, an inducer of Akt phosphorylation, suppresses Akt inactivation, Bid cleavage, and DNA fragmentation. LY294002, a PI3 kinase inhibitor, enhances Akt inactivation and DNA fragmentation. Ac-IETD-CHO, a caspase-8 inhibitor, suppresses Bid cleavage and DNA fragmentation. z-VAD-fmk, a universal caspase inhibitor, but not cyclosporin A (CsA), an inhibitor of mitochondrial membrane permeability transition, suppresses DNA fragmentation. These results suggest the sequential mechanism of diclofenac-induced apoptosis of HL-60 cells: ROS generation suppresses Akt activity, thereby activating caspase-8, which stimulates Bid cleavage and induces cytochrome c release and the activation of caspase-9 and-3 in a CsA-insensitive mechanism. Furthermore, we found that 2-methoxyestradiol (2-ME), a superoxide dismutase inhibitor, significantly enhances diclofenac-induced apoptosis; that is, diclofenac combined with 2-ME may have therapeutic potential in the treatment of human leukemia.     

The Roles of ROS and Caspases in TRAIL-Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells

Death signaling provided by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS) are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either N-acetyl-L-cysteine (NAC), a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI)⁺ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1), and siRNA-mediated knockdown of RIP3 decreased the annexin V^-/PI⁺ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process.