Oxidative stress-driven mechanisms of nordihydroguaiaretic acid-induced apoptosis in FL5.12 cells

Nordihydroguaiaretic acid (NDGA), a general lipoxygenase (LOX) enzyme inhibitor, induces apoptosis independently of its activity as a LOX inhibitor in murine pro-B lymphocytes (FL.12 cells) by a mechanism that is still not fully understood. Glutathione depletion, oxidative processes and mitochondrial depolarization appear to contribute to the apoptosis induced by NDGA. The current data demonstrate that NDGA (20 microM)-induced apoptosis in FL5.12 cells is partially protected by N-acetylcysteine (NAC) (10 mM) and dithiothreitol (DTT) (500 microM) pretreatment, confirming a role for oxidative processes. In addition, the treatment of FL5.12 cells with NDGA led to an increase in phosphorylation and activation of the MAP kinases ERK, JNK and p38. Although pretreatment with ERK inhibitors (PD98059 or U0126) abolished ERK phosphorylation in response to NDGA, neither inhibitor had any effect on NDGA-induced apoptosis. SP600125, a JNK inhibitor, did not have any effect on NDGA-induced phosphorylation of JNK nor apoptosis. Pretreatment with the p38 inhibitor SB202190 attenuated NDGA-induced apoptosis by 30% and also abolished p38 phosphorylation, compared to NDGA treatment alone. NAC, but not DTT, also decreased the phosphorylation of p38 and JNK supporting a role for oxidative processes in activating these kinases. Neither NAC nor DTT blocked the phosphorylation of ERK suggesting that this activation is not related to oxidative stress. The release of cytochrome c and activation of caspase-3 induced by NDGA were inhibited by NAC. SB202190 slightly attenuated caspase-3 activation and had no effect on the release of cytochrome c. These data suggest that several independent mechanisms, including oxidative reactions, activation of p38 kinase and cytochrome c release contribute to NDGA-induced apoptosis.     

Fatty acid release and oxidation are factors in lipoxygenase inhibitor-induced apoptosis

MK886, an inhibitor of 5-lipoxygenase activating protein (FLAP), and the lipoxygenase (LOX) inhibitors baicalein and nordihydroguaiaretic acid (NDGA), induce apoptosis by mechanisms independent of both LOX and FLAP. One possible mechanism for these agents is through an effect on the binding of fatty acids to LOX and fatty acid binding proteins resulting in increased intracellular levels of unbound fatty acids, particularly arachidonic acid (AA), that in turn, activate apoptosis signaling pathways either directly or following oxidation. In FL5.12 murine pro-B lymphocytic cells, exogenous fatty acids induced apoptosis proportional to their degree of unsaturation. MK886, baicalein, and NDGA significantly enhanced the release of [3H]-AA two to threefold within 2 h and induced apoptosis by 8 h. Neither MK886-induced AA release, nor apoptosis were affected by quinacrine, a phospholipase A2 inhibitor. The presence of peroxides 1 h after treatment of FL5.12 cells with these agents was evident by a two to threefold increase in the ferrous oxidation-xylenol orange (FOX) assay as well as dichlorofluorescein fluorescence measured with flow cytometry. Isoprostane formation, an additional index of lipid peroxidation, was increased threefold by 2 h, and fourfold at 4 h after MK886 or baicalein, but not after NDGA. Antioxidants were able to protect against NDGA-induced apoptosis but had no effect on baicalein and resulted in enhanced apoptosis with MK886. These data support the hypothesis that release of fatty acids and generation of oxidized species contribute to apoptosis induced by these LOX inhibitors, but that more complex mechanisms are likely involved.     

Mechanism of ricin-induced apoptosis in human cervical cancer cells

The mechanism of ricin-induced apoptosis in human cervical cancer cell line HeLa was studied. The present study demonstrated that ricin induces apoptosis of human cervical cancer cells (HeLa) in a time dependent manner with an IC(50) for cell viability of 1 microg/ml. Ricin treatment resulted in a time dependent increase in LDH leakage, DNA fragmentation, percent apoptotic cells, generation of reactive oxygen species and depletion of intracellular glutathione levels. DNA agarose gel electrophoresis showed typical oligonucleosomal length DNA fragmentation. Additionally, DNA diffusion assay was performed to confirm DNA damage and apoptosis. Ricin activated caspase-3 as evidenced by both proteolytic cleavage of procaspase-3 into 20 and 18 kDa subunits, and increased protease activity. Caspase activity was maximum at 4h and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the 85 kDa cleavage product. Ricin-induced caspase-3 activation also resulted in cleavage of DNA fragmentation factor-45 (DFF45/ICAD) and DFF40 or caspase-activated DNase in HeLa cells. Activation of caspase-3, cleavage of PARP and DNA fragmentation was blocked by pre-treatment with caspase-3 specific inhibitor Ac-DEVD-CHO (100 microM) and broad-spectrum caspase inhibitor Z-VAD-FMK (40 microM). Ricin-induced DNA fragmentation was inhibited by pre-treatment with PARP inhibitors 3-aminobenzamide (100 microM) and DPQ (10 microM). Our results indicate that ricin-induced cell death was mediated by generation of reactive oxygen species and subsequent activation of caspase-3 cascade followed by down stream events leading to apoptotic mode of cell death.     

Proteolytic loss of bcl-x(L) in FL5.12 Cells undergoing apoptosis induced by MK886

Apoptosis induced in the IL3-dependent murine pro-B lymphocytic (FL5.12) cell line by the 5-lipoxygenase activating protein inhibitor MK886 is accompanied by the rapid loss of the anti-apoptotic bcl-x(L) and bcl-2, but not the proapoptotic bax proteins (Datta et al., J. Biol. Chem. 273, 28163-28169, 1998). Since several reports indicate important roles for noncaspase proteases in apoptosis, the participation of lysosomes, as well as serine, cysteine, or aspartic acid proteases, in the effects of MK886 were investigated. Consistent with the involvement of various proteases, lysosomal degranulation was evident, as observed by a decrease in acridine orange fluorescence at 2 h and an increase in cytosolic beta-hexosaminidase activity at 4 h after treating FL5.12 cells with 10 microM MK886. The disappearance of bcl-x(L) from FL5.12 cells upon MK886 treatment was prevented in a dose-dependent manner by pretreatment with leupeptin, pepstatin, phenylmethylsulfonyl fluoride, or the broad-spectrum caspase inhibitor Boc-D-FMK. Each of the noncaspase protease inhibitors partially inhibited MK886-induced apoptosis as measured by phosphatidylserine externalization and DNA fragmentation. The noncaspase inhibitors also blocked about half of the increase in caspase-3-like activity. Boc-D-FMK completely inhibited this enzyme and prevented apoptosis. None of the inhibitors were able to directly inhibit activated caspase-3 in cell lysates, suggesting their effects were upstream of caspase activation. These observations suggest the involvement of various proteases, possibly originating from lysosomes, upstream of active caspase-3, in the loss of bcl-x(L) protein and in the signaling pathway of MK886-induced apoptosis in FL5.12 cells. This pathway may be unique to MK886 since these same protease inhibitors had only minimal effects on etoposide-induced apoptosis and the accompanying moderate loss of bcl-x(L) in FL5.12 cells.     

Mechanism of tert-butylhydroperoxide induced cytotoxicity in U-937 macrophages by alteration of mitochondrial function and generation of ROS.

tert-Butylhydroperoxide has been reported to inhibit growth and induce apoptosis in number of cell types, but little is known about the molecular mechanism mediating these effects. In the present study, we determined the molecular pathways that lead to apoptosis after treatment of cells with t-BOOH. The cells were exposed to different concentrations of t-BOOH (100-750 microM) for 1-4 h and various parameters such as cytotoxicity, ROS (reactive oxygen species) generation, MMP (mitochondrial membrane potential), intracellular Ca++ levels and expression of various proteins involved in apoptosis were determined. Exposure of U-937 cells to t-BOOH induced cytotoxicity in a time dependent manner with about 50% toxicity at 400 microM t-BOOH in 4h. t-BOOH treatment resulted in a time dependent increase in reactive oxygen species levels, Ca++ influx and annexin V positive cells. There was a significant fall in MMP following exposure to t-BOOH with time. t-BOOH treatment of U-937 cells leads to apoptosis, which is accompanied by activation of caspase-3. The caspase-3 inhibitor (Ac-DEVD-CHO) inhibits the cytotoxicity induced by t-BOOH, indicating a direct link between caspase-3 activation and cell death. This activation of apoptosis is accompanied by release of cytochrome c, down regulation of anti-apoptotic protein Bcl-2 levels with concurrent increase in pro-apoptotic proteins Bax and Bad levels. These observations indicate that t-BOOH induces cell death in U-937 macrophages by apoptosis, which is mediated through mitochondrial pathway.     

Nordihydroguaiaretic acid elevates osteoblastic intracellular Ca2+

Nordihydroguaiaretic acid (NDGA) is widely used as a pharmacological tool to inhibit lipoxygenases; however, recent evidence suggests that it increases renal intracellular [Ca2+]i via novel mechanisms. Here the effect of NDGA on Ca2+ signaling in MG63 osteoblastic cells was explored using fura-2 as a Ca2+ indicator. NDGA (2-50 microM) increased [Ca2+]i in a concentration-dependent manner. The signal comprised an initial rise and an elevated phase over a time period of 4 min. Removing extracellular Ca2+ reduced 2-50 microM NDGA-induced signals by 62+/-2%. After incubation with 50 microM NDGA in Ca2+-free medium for several minutes, addition of 3 mM CaCl2 induced an increase in [Ca2+]i. NDGA (50 microM)-induced [Ca2+]i increases were not changed by pretreatment with 10 microM of verapamil, diltiazem, nifedipine, nimodipine and nicardipine. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (1 microM) inhibited 50 microM NDGA-induced [Ca2+]i increases by 69+/-3%. Inhibition of phospholipase C with 2 microM U73122 had little effect on 50 microM NDGA-induced Ca2+ release. Several other lipoxygenase inhibitors had no effect on basal [Ca2+]i. At a concentration that did not increase basal [Ca2+]i, NDGA (1 microM) did not alter 10 microM ATP- or 1 microM thapsigargin-induced [Ca2+]i increases. Alteration of protein kinase C activity with 1 nM phorbol 12-myristate 13-acetate or 2 microM GF 109203X did not affect 50 microM NDGA-induced [Ca2+]i increases. Together, the results show that NDGA increased [Ca2+]i in osteoblasts in a lipoxygenase-independent manner, by releasing stored Ca2+ in a fashion independent of phospholipase C activity, and by causing Ca2+ influx.     

Cocaine induces apoptosis in human coronary artery endothelial cells

This study was designed to determine the direct cytotoxic effect of cocaine on human coronary artery endothelial cells (HCAECs). Cocaine treatment of cultured HCAECs induced a time- and dose-dependent increase in apoptotic cell death in HCAECs. Cocaine-induced surface exposure of phosphatidylserine in HCAECs was seen as early as at 6 h. With prolonged treatment < or =72 h, cocaine (10-500 microM) produced a dose-dependent increase in apoptosis in the cells. Corresponding DNA fragmentation induced by cocaine was demonstrated in situ by terminal deoxynucleotidyl transferase (Tdt) UTP nick end-labeling TUNEL assay and by electrophoresis of labeled DNA fragments, showing the characteristic apoptotic ladders. Both caspase-9 (Z-LEHD-FMK) and caspase-3 (Ac-DEVD-CHO) inhibitors blocked cocaine-induced apoptosis. In addition, cyclosporin A inhibited cocaine-induced apoptosis in a concentration-dependent manner with a median inhibitory concentration (IC50) of 0.3 microM. The maximum of 62% inhibition was obtained with 3 microM cyclosporin A. Cocaine-induced apoptosis also was blocked by naloxone and nifedipine in a dose-dependent manner. These findings suggest that cocaine induces apoptosis in cultured HCAECs, which may be mediated by opioid receptors. The release of cytochrome c from the mitochondria and its subsequent activation of caspase-9 and caspase-3 may play a key role in cocaine-induced apoptosis.     

The role of GSH efflux in staurosporine-induced apoptosis in colonic epithelial cells

Staurosporine (STP) was shown to induce cell apoptosis through formation of reactive oxygen species, but a role for cellular redox has not been defined. In this study, we report that STP (2 microM) caused apoptosis (24+/-3% at 24 h) of human colon adenocarcinoma epithelial cell line HT29 that was preceded by significant glutathione (GSH) and glutathione disulfide (GSSG) efflux (6 h), but independent of changes in cellular glutathione/glutathione disulfide (GSH/GSSG) redox status. The blockade of GSH efflux by gamma-glutamyl glutamate (gamma-GG) or ophthalmic acid was associated with apoptosis attenuation; however, gamma-GG administration after peak GSH efflux (8 h) did not confer cytoprotection. Moreover, lowering cellular GSH through inhibition of its synthesis prevented extracellular GSH accumulation and cell apoptosis, thus validating a link between cellular GSH export and the trigger of cell apoptosis. Inhibition of gamma-glutamyl transferase (GGT1, EC 2.3.2.2)-catalyzed extracellular GSH degradation with acivicin significantly blocked GSH efflux, suggesting that GSH breakdown is a driving force for GSH export. Interestingly, acivicin treatment enhanced extracellular GSSG accumulation, consistent with GSH oxidation. STP-induced HT29 cell apoptosis was associated with caspase-3 activation independent of caspase-8 or caspase-9 activity; accordingly, inhibitors of the latter caspases were without effect on STP-induced apoptosis. STP similarly induced GSH efflux and apoptosis in a non-malignant human NCM460 colonic cell line in association with caspase-3 activation. Collectively, our results demonstrate that STP induction of apoptosis in malignant and non-malignant colonic cells is temporally linked to the export of cellular GSH and the activation of caspase-3 without caspase-8 or -9 involvement.     

Arsenic trioxide-induced apoptosis in H9c2 cardiomyocytes: implications in cardiotoxicity

Arsenic trioxide (As(2)O(3)) achieved dramatic remissions in patients with acute promyelocytic leukaemia. Clinical reports have shown that treatment was associated with cardiotoxicity. We investigated the toxic mechanisms of As(2)O(3) in H9c2 cardiomyocytes. Clinically relevant concentrations of As(2)O(3) (2-10 microM) reduced the viability of H9c2 cells in a concentration-dependent manner. The decreased cell viability was because As(2)O(3) induced cell apoptosis (cell shrinkage, nuclear alterations and caspase-3 activation), or even necrosis at higher concentrations. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-CHO, suppressed apoptosis induced by As(2)O(3). In addition, reactive oxygen species formation and cellular Ca(2+) overload were observed in H9c2 cells exposed to As(2)O(3), which was partly inhibited by vitamin E and verapamil. These results suggest that As(2)O(3)-induced cardiotoxicity is mediated, at least in part, by activation of caspase-3 pathway, which may be triggered by reactive oxygen species formation and intracellular Ca(2+) overload.     

Effect of nordihydroguaiaretic acid on intracellular Ca(2+) concentrations in hepatocytes.

The effect of nordihydroguaiaretic acid (NDGA) on Ca(2+) signaling in human hepatoma cells (HA22/VGH) has been investigated. NDGA (5-50 microM) increased [Ca(2+)](i) concentration-dependently. The [Ca(2+)](i) increase comprised an initial rise and an elevated phase over a time period of 4 min. Removal of extracellular Ca(2+) reduced 10-50 microM NDGA induced [Ca(2+)](i) signals by 45+/-5%. Consistently, the 50 microM NDGA-induced [Ca(2+)](i) increase in Ca(2+)-containing medium was reduced by 41+/-2% by 10 microM of La(3+), nifedipine or verapamil. In Ca(2+)-free medium, pretreatment with 20 microM NDGA for 6 min abolished the [Ca(2+)](i) increase induced by the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM). Conversely, 20 microM NDGA failed to increase [Ca(2+)](i) after 1 microM thapsigargin had depleted the endoplasmic reticulum Ca(2+) store. Inhibition of phospholipase C with 2 microM U73122 had little effect on 20 microM NDGA-induced Ca(2+) release. Several other lipoxygenase inhibitors had no effect on basal [Ca(2+)](i). Together, the data suggest that NDGA increased [Ca(2+)](i) in hepatocytes in a lipoxygenase-independent manner, by releasing Ca(2+) from the endoplasmic reticulum and causing Ca(2+) influx.     

Apoptotic effects of 25-hydroxycholesterol in immature rat Sertoli cells: prevention by 17beta-estradiol

The aim of the present study was to determine whether or not apoptosis occurs in Sertoli cells in presence of 25-hydroxycholesterol, an oxysterol derived from cholesterol-containing foods or endogenous oxidation. Here, we provide evidence that 25-hydroxycholesterol can induce cultured Sertoli cells of immature rat to undergo apoptosis. The cell death was identified by analysis of fragmented DNA detected using enzyme-immunoassay. After 48 h of treatment with 50 microM of 25-hydroxycholesterol, apoptosis increased by 70% in Sertoli cells. Moreover, 50 microM of 25-hydroxycholesterol inhibited the incorporation of [14C] acetate into cholesterol by 70%. Addition of mevanolate to prevent isoprenoid deficiency do not inhibit the apoptosis generated by 25-hydroxycholesterol. In contrast, this increase of DNA fragmentation was reversed by addition of caspase-3 inhibitors as Ac-DEVD-CHO or Ac-ESMD-CHO. Bcl-2 mRNA level in the Sertoli cells decreased by 60% after 24 h exposure to 25-hydroxycholesterol. In parallel, Bax mRNA level increased by 40% in the Sertoli cells incubated in presence of 50 microM of 25-hydroxycholesterol. Physiological concentrations of 17beta-estradiol (10 or 100 nM) elicited a significant protection on apoptosis generated by 25-hydroxycholesterol in Sertoli cells. Our results show that the 25-hydroxycholesterol would control the cholesterol synthesis without toxic effect in immature rat Sertoli cells, these cells being able to protect themselves by estradiol production.     

Effect of fraxetin and myricetin on rotenone-induced cytotoxicity in SH-SY5Y cells: comparison with N-acetylcysteine

The purpose of this study was to investigate the potential neuroprotective effects of myricetin (flavonoid) and fraxetin (coumarin) on rotenone-induced apoptosis in SH-SY5Y cells, and the possible signal pathway involved in a neuronal cell model of Parkinson's disease. These two compounds were compared to N-acetylcysteine. The viability of cells was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cytotoxicity was assayed by lactate dehydrogenase (LDH) released into the culture medium. Parameters related to apoptosis, such as caspase-3 activity, the cleavage of poly(ADP-ribose) polymerase and the levels of reactive oxygen species were also determined. Rotenone caused a time- and dose-dependent decrease in cell viability and the degree of LDH release was proportionally to the effects on cell viability. Cells were pretreated with fraxetin, myricetin and N-acetylcysteine at different concentrations for 30 min before exposure to rotenone. Cytotoxicity of rotenone (5 microM) for 16 h was significantly diminished as well as the release of LDH into the medium, by the effect of fraxetin, myricetin and N-acetylcysteine, with fraxetin (100 microM) and N-acetylcysteine (100 microM) being more effective than myricetin (50 microM). Rotenone-induced apoptosis in SH-SY5Y cells was detected by an increase in caspase-3 activity and in the cleavage of poly(ADP-ribose) polymerase. After exposing these cells to rotenone, a significant increase in reactive oxygen species preceded apoptotic events. Fraxetin (100 microM) and N-acetylcysteine (100 microM) not only reduced rotenone-induced reactive oxygen species formation, but also attenuated caspase-3 activity and poly(ADP-ribose) polymerase cleavage at 16 h against rotenone-induced apoptosis. The effect of fraxetin in both experiments was similar to that of N-acetylcysteine. These results demonstrated the protective action of fraxetin and suggest that it can reduce apoptosis, possibly by decreasing free radical generation in SH-SY5Y cells. Myricetin at 100 microM was without any preventive effect.     

Kainic acid-induced neuronal cell death in cerebellar granule cells is not prevented by caspase inhibitors

1. We examined the role of non-NMDA receptors in kainic acid (KA)-induced apoptosis in cultures of rat cerebellar granule cells (CGCs). KA (1 - 500 microM) induced cell death in a concentration-dependent manner, which was prevented by NBQX and GYKI 52466, non-NMDA receptor antagonists. Moreover, AMPA blocked KA-induced excitotoxicity, through desensitization of AMPA receptors. 2. Similarly, KA raised the intracellular calcium concentration of CGCs, which was inhibited by NBQX and GYKI 52466. Again, AMPA (100 microM) abolished the KA (100 microM)-induced increase in intracellular calcium concentration. 3. KA-induced cell death in CGCs had apoptotic features, which were determined morphologically, by DNA fragmentation, and by expression of the prostate apoptosis response-4 protein (Par-4). 5. KA (500 microM) slightly (18%) increased caspase-3 activity, which was strongly enhanced by colchicine (1 microM), an apoptotic stimulus. However, neither Z-VAD.fmk, a pan-caspase inhibitor, nor the more specific caspase-3 inhibitor, Ac-DEVD-CHO, prevented KA-induced cell death or apoptosis. In contrast, both drugs inhibited colchicine-induced apoptosis. 5. The calpain inhibitor ALLN had no effect on KA or colchicine-induced neurotoxicity. 6. Our findings indicate that colchicine-induced apoptosis in CGCs is mediated by caspase-3 activation, unlike KA-induced apoptosis.     

Organophosphorus compound-induced apoptosis in SH-SY5Y human neuroblastoma cells

Organophosphorus (OP) compounds have been shown to be cytotoxic to SH-SY5Y human neuroblastoma cell cultures. The mechanisms involved in OP compound-induced cell death (apoptosis versus necrosis) were assessed morphologically by looking at nuclear fragmentation and budding using the fluorescent stain Hoechst 33342 (10 microgram/ml). Hoechst staining revealed significant paraoxon (1 mM), parathion (1 mM), phenyl saligenin phosphate (PSP, 10 and 100 microM), tri-ortho-tolyl phosphate (TOTP, 100 microM and 1 mM), and triphenyl phosphite (TPPi, 1 mM) induced time-dependent increases in traditional apoptosis (p < 0.05). In many cells, PSP and TOTP (1 mM) also induced nuclear condensation with little fragmentation or budding. Pretreatment with cyclosporin A (500 nM, 30 h) decreased apoptosis following 1 mM parathion and TOTP exposures. Apoptotic nuclear changes were verified by DNA gel electrophoresis. Activation of caspase-3, a cysteine aspartate protease, was also monitored. OP compounds induced significant time-dependent increases in caspase-3 activation following paraoxon (1 mM), parathion (100 microM, 1 mM), PSP (10 microM, 100 microM, 1 mM), TOTP (100 microM, 1 mM), and TPPi (1 mM) exposure (p < 0.05). Pretreatment with cyclosporin A (500 nM, 30 h) significantly decreased caspase-3 activation during extended incubations with paraoxon, parathion, and TPPi (p < 0.05). In addition, pretreatment with the caspase-3 inhibitor Ac-DEVD-CHO and the caspase-8 inhibitor Ac-IETD-CHO (25 microM, 8 h) significantly decreased caspase-3 activation following exposure to 1 mM PSP and parathion (p < 0.05). Pretreatment with the serine protease inhibitor phenylmethyl sulfonyl fluoride (PMSF; 1 mM, 8 h) also significantly decreased caspase activation following 1 mM PSP and TOTP exposures (p < 0.05). Alteration of OP compound-induced nuclear fragmentation or caspase-3 activation by pretreatment with cyclosporin A, Ac-IETD-CHO, or PMSF suggested that OP compound-induced cytotoxicity may be modulated through multiple sites, including mitochondrial permeability pores, receptor-mediated caspase pathways, or serine proteases.     

Schizandrin protects primary cultures of rat cortical cells from glutamate-induced excitotoxicity

The neuroprotective effect of schizandrin on the glutamate (Glu)-induced neuronal excitotoxicity and its potential mechanisms were investigated using primary cultures of rat cortical cells. After exposure of primary cultures of rat cortical cells to 10 microM Glu for 24 h, cortical cell cultures exhibited remarkable apoptotic death. Pretreatment of the cortical cell cultures with schizandrin (10, 100 microM) for 2 h significantly protected cortical neurons against Glu-induced excitotoxicity. The neuroprotective activity of schizandrin was the most potent at the concentration of 100 microM. Schizandrin reduced apoptotic characteristics by DAPI staining in Glu-injured cortical cell cultures. In addition, schizandrin diminished the intracellular Ca2+ influx, inhibited the subsequent overproduction of nitric oxide (NO), reactive oxygen species (ROS), and cytochrome c, and preserved the mitochondrial membrane potential. Furthermore, schizandrin also increased the cellular level of glutathione (GSH) and inhibited the membrane lipid peroxidation malondialdehyde (MDA). As indicated by Western blotting, schizandrin attenuated the protein level changes of procaspase-9, caspase-9, and caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Taken together, these results suggest that schizandrin protected primary cultures of rat cortical cells against Glu-induced apoptosis through a mitochondria-mediated pathway and oxidative stress.     

Cadmium-induced apoptosis in murine macrophages is antagonized by antioxidants and caspase inhibitors

Cadmium is a toxic heavy metal that accumulates in the environment and is commonly found in cigarette smoke and industrial effluents. This study was designed to determine the role of reactive oxygen species (ROS) generation, and its antagonism by antioxidants, in cadmium-mediated cell signaling and apoptosis in murine macrophage cultures. Cadmium-generated ROS production was observed in J774A.1 cells at 6 h, reverting to control levels at 16 and 24 h. The ROS production was concentration related between 20 and 500 microM cadmium. Activation of caspase-3 was observed at 8 h and DNA fragmentation at 16 h in the presence of 20 microM cadmium, suggesting that caspase-3 activation is a prior step to DNA fragmentation in cadmium-induced apoptosis. Inhibitors of caspase-3, -8, -9, and a general caspase inhibitor suppressed cadmium-induced caspase-3 activation and apoptosis indicating the importance of caspase-3 in cadmium-induced toxicity in these cells. Protection against the oxidative stress with N-acetylcysteine (NAC) and silymarin (an antioxidant flavonoid) blocked cadmium-induced apoptosis. Pretreatment of cells with NAC and silymarin prevented cadmium-induced cell injury, including growth arrest, mitochondrial impairment, and necrosis, and reduced the cadmium-elevated intracellular calcium ([Ca2+]i), suggesting that the oxidative stress is a source of increased [Ca2+]i. NAC inhibited cadmium-induced activation of mitogen-activated protein kinases, the c-Jun NH2-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK). However, silymarin provided only a partial protection for JNK activation, and only at the low concentration did it inhibit cadmium-induced ERK activation. Inhibition of caspase-3 protected oxidative stress produced by cadmium, suggesting that the activation of caspase-3 also contributes to generation of reactive oxygen species (ROS). Results emphasized the role of ROS, Ca2+ and mitogen-activated protein kinases in cadmium-induced cytotoxicity in murine macrophages.     

Reactive oxygen species and mitochondria mediate the induction of apoptosis in human hepatoma HepG2 cells by the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid

We have previously shown that one of the most potent rodent hepatocarcinogens, perfluorooctanoic acid (PFOA), induces apoptosis in human HepG2 cells in a dose- and time-dependent manner. In this study we have investigated the involvement of reactive oxygen species (ROS), mitochondria, and caspase-9 in PFOA-induced apoptosis. Treatment with 200 and 400 microM PFOA was found to cause a dramatic increase in the cellular content of superoxide anions and hydrogen peroxide after 3 h. Measurement of the mitochondrial transmembrane potential (Delta Psi(m)) after PFOA treatment showed a dissipation of Delta Psi(m) at 3 h. Caspase-9 activation was seen at 5 h after treatment with 200 microM PFOA. In order to evaluate the importance of these events in PFOA-induced apoptosis, cells were cotreated with PFOA and N-acetylcysteine (NAC), a precursor of glutathione, or Cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MPT pore). NAC reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced ROS. In addition, CsA also reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced opening of the MPT pore. In summary, we have delineated a ROS and mitochondria-mediated pathway for induction of apoptosis by PFOA.     

Lovastatin-induced cardiac toxicity involves both oncotic and apoptotic cell death with the apoptotic component blunted by both caspase-2 and caspase-3 inhibitors

The objective of this study was to evaluate the cardiac toxicity of the HMG-CoA reductase inhibitors by testing the hypothesis that lovastatin induces apoptotic and/or oncotic cell death in the myocyte element of the heart and further that cell death is mediated through interruption of the mevalonate pathway and that apoptosis is induced through activation of caspase-2 and caspase-3. Cardiomyocytes were cultured from embryonic chick heart. Lovastatin-induced apoptosis in these cells was demonstrated by three independent techniques, namely (1) FACS analysis of low DNA content by propidium iodide (PI); (2) microscopic assessment for cellular changes of apoptosis; and (3) FACS analysis of cells stained with PI and fluorescein diacetate. Lovastatin produced a concentration-dependent increase in apoptotic cell death and 100 microM lovastatin showed over a 4-fold increase in apoptosis compared to control. Lovastatin also induced oncotic cell death, as there was a 2.5-fold increase in the amount of oncotic cell death compared to control. Lovastatin-induced apoptosis operated, in part, through the mevalonate pathway. The caspase-2 inhibitor z-VDVAD-fmk and the caspase-3 inhibitor Ac-DEVD-CHO reduced the extent of lovastatin-induced cardiac apoptosis. In contrast, lovastatin-induced oncosis was not only insensitive to these caspase-2 or -3 inhibitors but occurred through a mevalonate-independent mechanism of action. In summary, lovastatin-induced cardiotoxicity is complex and represents the sum of two distinct modes of cell death operating in part through the mevalonate pathway with the apoptotic component subject to modification by inhibitors of the initiator caspase, caspase-2, as well as the effector caspase, caspase-3.     

Oxidative and non-oxidative mechanisms of neuronal cell death and apoptosis by L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine

1. The present study was designed to evaluate the nature of intervening agents in L-DOPA- and dopamine-induced neurotoxicity in Neuro-2A cells. 2. In the absence of cells and in conditions of light protection, at 37 degrees C, L-DOPA or dopamine (1 mM) in culture medium degraded spontaneously in a time-dependent manner, this being prevented by ascorbic acid (200 microM) and other antioxidants, namely glutathione (1 mM), N-acetyl-L-cysteine (1 mM), sodium metabisulphite (200 microM), but not N-ter-butyl-alpha-phenylnitrone (1 mM) and deferoxamine (100 microM). 3. The viability of Neuro-2A cells declined following treatment with L-DOPA or dopamine in a concentration- and time-dependent manner. The decrease in cell viability by L-DOPA (10+/-4% of control) or dopamine (15+/-4% of control) was markedly attenuated by antioxidants (ascorbic acid, glutathione, N-acetyl-L-cysteine and sodium metabisulphite). Autoxidation of L-DOPA or dopamine was accompanied by the formation of H(2)O(2) in a time-dependent manner, this being completely prevented by ascorbic acid at 24 h or markedly reduced at 48 h. 4. Protective effects of 100 U ml(-1) catalase (40+/-1% of control) against L-DOPA-induced cell death were lower than those conferred by 200 microM ascorbic acid (70+/-3% of control). Catalase-induced protection (59+/-5% of control) against dopamine-induced cell death was similar to that conferred by 200 microM ascorbic acid (57+/-4% of control). L-DOPA-induced neuronal cell death was also accompanied by increases in caspase-3 activity, this being insensitive to ascorbic acid. Dopamine-induced increase in caspase-3 activity occurred only when autoxidation of the amine was prevented by ascorbic acid. 5. It is suggested that in addition to generation of H(2)O(2) and quinone formation, L-DOPA- and dopamine-induced cell death may result from induction of apoptosis, as evidenced by increases in caspase-3 activity. Dopamine per se induces apoptosis by a mechanism independent of oxidative stress, as evidenced by the fact that increases in caspase-3 activity occurred only when autoxidation of the amine was prevented.     

Zinc inhibition of caspase-3 activation does not protect HeLa cells from apoptotic cell death

Zinc is proposed to be antiapoptotic for it has been shown to inhibit late events of apoptotic pathways such as Ca(2+)/Mg(2+)-dependent endonuclease cleavage of chromatin DNA, poly-ADP ribose polymerase cleavage, and caspase-3 activity. Because caspase-3 is a critical executioner caspase in apoptosis, this study was undertaken to examine specifically a correlation between zinc inhibition of caspase-3 activation and apoptosis in HeLa cells. Cultured HeLa cells were exposed to 100 microM ZnCl(2) for 1 h prior to 12 h treatment with 1.0 microM doxorubicin (DOX), an important anticancer agent that causes apoptosis in a wide variety of tumor cells. Western blot analysis of HeLa cells treated with DOX for 12 h revealed that DOX caused proteolytic activation of caspase-3 and zinc inhibited this activation. Interestingly, zinc did not inhibit DOX-induced apoptosis as measured by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Furthermore, a microculture tetrazolium assay confirmed that cell death occurred in the presence of zinc. These results demonstrate that zinc specifically inhibits DOX-induced activation of caspase-3 in HeLa cells, but does not suppress DOX-induced apoptosis or otherwise cell death, thus suggesting DOX-induced caspase-3 activation may not play a major role in overall cell death and/or non-caspase-3 pathways are involved in DOX-induced apoptosis in HeLa cells.