Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.     

Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-l-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.     

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.     

Apoptotic signaling in methylglyoxal-treated human osteoblasts involves oxidative stress, c-Jun N-terminal kinase, caspase-3, and p21-activated kinase 2

Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. MG is cytotoxic through induction of cell death, and elevated MG levels in diabetes patients are believed to contribute to diabetic complications. In this report, we show for the first time that MG treatment triggers apoptosis in human osteoblasts. We further show that MG-induced apoptosis of osteoblasts involves specific apoptotic biochemical changes, including oxidative stress, c-Jun N-terminal kinase (JNK) activation, mitochondrial membrane potential changes, cytochrome C release, increased Bax/Bcl-2 protein ratios, and activation of caspases (caspase-9, caspase-3) and p21-activated protein kinase 2 (PAK2). Treatment of osteoblasts with SP600125, a JNK-specific inhibitor, led to a reduction in MG-induced apoptosis and decreased activation of caspase-3 and PAK2, indicating that JNK activity is upstream of these events. Experiments using anti-sense oligonucleotides against PAK2 further showed that PAK2 activation is required for MG-induced apoptosis in osteoblasts. Interestingly, we also found that MG treatment triggered nuclear translocation of NF-kappaB, although the precise regulatory role of NF-kappaB activation in MG-induced apoptosis remains unclear. Lastly, we examined the effect of MG on osteoblasts in vivo, and found that exposure of rats to dietary water containing 100-200 microM MG caused bone mineral density (BMD) loss. Collectively, these results reveal for the first time that MG treatment triggers apoptosis in osteoblasts via specific apoptotic signaling, and causes BMD loss in vivo.     

ASK1 is activated by arsenic trioxide in leukemic cells through accumulation of reactive oxygen species and may play a negative role in induction of apoptosis

Arsenic trioxide (ATO) is remarkably effective for treating acute promyelocytic leukemia. Here, we find that ATO treatment of NB4 and K562 leukemic cells induces activation of ASK1. ASK1 activation was induced most significantly at low concentrations of ATO, where G2/M arrest but not apoptosis was induced. On the other hand, ATO barely activated ASK1 at high concentrations, where apoptosis as well as activation of JNK and p38 was induced significantly. ATO-induced accumulation of reactive oxygen species (ROS), while the ASK1 activation was suppressed by cotreatment with an antioxidant, N-acetyl-l-cysteine. Murine embryonic fibroblasts (MEFs) from ASK1-deficient mice were more susceptible to ATO-induced apoptosis than control MEFs. Furthermore, ATO at the low concentration induced significant apoptosis in K562 cells when ASK1 was knocked down by siRNA. These results indicate that ASK1 is activated by ATO through ROS accumulation and may negatively regulate apoptosis in leukemic cells without activating p38 and JNK.     

Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells: role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway

Staphylococcus aureus infections can result in septic and toxic shock with depletion of immune cells and massive cytokine production. Recently, we showed that, in S. aureus-infected Jurkat T cells, alpha-toxin is the major mediator of caspase activation and apoptosis. Here, we investigated the mechanisms of cell death induced by alpha-toxin in peripheral blood mononuclear cells (MNC). We show that alpha-toxin is required and sufficient for S. aureus-induced cell death not only in transformed Jurkat T cells but also in MNC. Low alpha-toxin doses (3-30 ng ml-1) dose- and time-dependently induced apoptosis in both cell types, which was completely blocked by the caspase inhibitor zVAD-fmk. In Jurkat T cells and MNC, alpha-toxin induced the breakdown of the mitochondrial membrane potential and the intrinsic activation of caspase-3, -8 and -9. Interestingly, unlike in Jurkat T cells, apoptosis in MNC was additionally mediated by a caspase-9-independent component. MNC, but not Jurkat T cells, produced tumour necrosis factor (TNF)-alpha upon alpha-toxin stimulation. Blocking endogenous TNF-alpha with a TNF-alpha receptor antagonist partially decreased apoptosis in MNC. Our data therefore suggest that, whereas in Jurkat T cells apoptosis is solely mediated by the mitochondrial pathway, in MNC endogenous TNF-alpha and a death receptor-dependent pathway are also involved, which may contribute to depletion of immune cells during S. aureus infection.     

Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death

The molecular basis of myocardial cell death in the ischemia-reperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1(-/-) mice to examine the role of ASK1 in ischemia-reperfusion injury. In the wild-type heart, ischemia-reperfusion resulted in necrotic injury, whereas infarct size was drastically reduced in the ASK1(-/-) heart. The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL-positive cells, DNA fragmentation or the activation of caspase-3. ASK1(-/-) cardiomyocytes were more resistant to H(2)O(2)- or Ca(2+)-induced apoptotic and non-apoptotic cell death compared with wild-type cells. These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.     

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.     

Curcumin prevents methylglyoxal-induced oxidative stress and apoptosis in mouse embryonic stem cells and blastocysts

Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Elevated MG levels in diabetes patients are believed to contribute to diabetic complications. MG is cytotoxic through induction of apoptosis. Curcumin, the yellow pigment of Curcuma longa, is known to have antioxidant and anti-inflammatory properties. In the present study, we examined the effect of curcumin on apoptotic biochemical events caused by incubation of ESC-B5 cells with MG. Curcumin inhibited the MG-induced DNA fragmentation, caspase-3 activation, cleavage of PARP, mitochondrial cytochrome c release, and JNK activation. Importantly, curcumin also inhibited the MG-stimulated increase of reactive oxygen species (ROS) in these cells. In addition, we demonstrated that curcumin prevented the MG-induced apoptosis of mouse blastocysts isolated from pregnant mice. Moreover, curcumin significantly reduced the MG-mediated impairment of blastocyst development from mouse morulas. The results support the hypothesis that curcumin inhibits MG-induced apoptosis in mouse ESC-B5 cells and blastocysts by blocking ROS formation and subsequent apoptotic biochemical events.     

Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.     

The ADP-stimulated NADPH oxidase activates the ASK-1/MKK4/JNK pathway in alveolar macrophages

The role of H2O2 as a second messenger in signal transduction pathways is well established. We show here that the NADPH oxidase-dependent production of O2*(-) and H2O2 or respiratory burst in alveolar macrophages (AM) (NR8383 cells) is required for ADP-stimulated c-Jun phosphorylation and the activation of JNK1/2, MKK4 (but not MKK7) and apoptosis signal-regulating kinase-1 (ASK1). ASK1 binds only to the reduced form of thioredoxin (Trx). ADP induced the dissociation of ASK1/Trx complex and thus resulted in ASK1 activation, as assessed by phosphorylation at Thr845, which was enhanced after treatment with aurothioglucose (ATG), an inhibitor of Trx reductase. While dissociation of the complex implies Trx oxidation, protein electrophoretic mobility shift assay detected oxidation of Trx only after bolus H2O2 but not after ADP stimulation. These results demonstrate that the ADP-stimulated respiratory burst activated the ASK1-MKK4-JNK1/c-Jun signaling pathway in AM and suggest that transient and localized oxidation of Trx by the NADPH oxidase-mediated generation of H2O2 may play a critical role in ASK1 activation and the inflammatory response.     

Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons

We have previously shown that nerve growth factor (NGF) withdrawal-induced death requires the activity of the small GTP-binding protein Cdc42 and that overexpression of an active form of Cdc42 is sufficient to mediate neuronal apoptosis via activation of the c-Jun pathway. Recently, a new mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) which activates both the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and plays pivotal roles in tumor necrosis factor- and Fas-induced apoptosis, has been identified. Therefore, we investigated the role of ASK1 in neuronal apoptosis by using rat pheochromocytoma (PC12) neuronal cells and primary rat sympathetic neurons (SCGs). Overexpression of ASK1-DeltaN, a constitutively active mutant of ASK1, activated JNK and induced apoptosis in differentiated PC12 cells and SCG neurons. Moreover, in differentiated PC12 cells, NGF withdrawal induced a four- to fivefold increase in the activity of endogenous ASK1. Finally, expression of a kinase-inactive ASK1 significantly blocked both NGF withdrawal- and Cdc42-induced death and activation of c-jun. Taken together, these results demonstrate that ASK1 is a crucial element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal apoptosis.     

Cell cycle effects and caspase-dependent and independent death of HL-60 and Jurkat cells treated with the inhibitor of NF-kappaB parthenolide

The sesquiterpene parthenolide (PRT) is an active component of Mexican-Indian medicinal plants and also of the common herb of European origin feverfew. PRT is considered to be a specific inhibitor of NF-kappaB. Human leukemic HL-60, Jurkat, and Jurkat IkappaBalphaM cells, the latter expressing a dominant-negative IkappaBalpha and thus having non-functional NF-kappaB, were treated with PRT and activation of caspases, plasma membrane integrity, DNA fragmentation, chromatin condensation (probed by DNA susceptibility to denaturation), and changes in cell morphology were determined. As a positive control for apoptosis cells were treated with topotecan (TPT) and H2O2. At 2-8 microM concentration PRT induced transient cell arrest in G2 and M followed by apoptosis. A narrow range of PRT concentration (2-10 microM) spanned its cytostatic effect, induction of apoptosis and induction of necrosis. In fact, necrotic cells were often seen concurrently with apoptotic cells at the same PRT concentration. Atypical apoptosis was characterized by loss of plasma membrane integrity very shortly after caspases activation. In contrast, a prolonged phase of caspase activation with preserved integrity of plasma membrane was seen during apoptosis induced by TPT or H2O2. Necrosis induced by PRT was also atypical, characterized by rapid rupture of plasma membrane and no increase in DNA susceptibility to denaturation. Using Jurkat cells with inactive NF-kappaB we demonstrate that cell cycle arrest and the mode of cell death induced by PRT were not caused by inhibition of NF-kappaB. The data suggest that regardless of caspase activation PRT targets plasma membrane causing its destruction. A caution, therefore, should be exercised in interpreting data of the experiments in which PRT is used with the intention to specifically prevent activation of NF-kappaB.     

Diagnosis of cell death induced by methylglyoxal, a metabolite derived from glycolysis, in Saccharomyces cerevisiae

Methylglyoxal (MG) is a ubiquitous metabolite derived from glycolysis; however, this aldehyde kills all types of cell. We analyzed the properties of MG-induced cell death of the budding yeast Saccharomyces cerevisiae. The MCA1 gene encodes a caspase homologue that is involved in H2O2-induced apoptosis in yeast, although the disruption of MCA1 did not repress sensitivity to MG. In addition, the intracellular oxidation level did not increase under conditions in which MG kills the cell. Furthermore, the disruption of genes encoding antioxidant enzymes did not affect the susceptibility to MG. Here, we demonstrate that yeast cells killed by MG do not exhibit the characteristics of apoptosis in a TUNEL assay or an annexin V staining, but show those of necrosis upon propidium iodide staining. We demonstrate that MG at high concentrations provokes necrotic cell death without the generation of reactive oxygen species in S. cerevisiae.     

Fructose-induced peroxynitrite production is mediated by methylglyoxal in vascular smooth muscle cells

Methylglyoxal (MG), a highly reactive molecule, has been implicated in the development of insulin resistance. We investigated whether fructose, a precursor of MG, induced ONOO(-) generation and whether this process was mediated via endogenously increased MG formation. Fructose significantly increased MG generation in vascular smooth muscle cells (VSMCs) in a concentration and time dependent manner. The intracellular production of MG was increased by 153+/-23% or 259+/-28% after cells were treated 6 h with fructose (15 mM or 30 mM), compared with production from untreated cells (p<0.01, n=4 for each group). A significant increase in the production of ONOO(-), NO, and O(2)(*-), was found in the cells treated with fructose (15 mM) or MG (10 microM). Fructose- or MG-induced ONOO(-) generation was significantly inhibited by MG scavengers, including reduced glutathione or N-acetyl-l-cysteine, and by O(2)(*-) or NO inhibitors, such as diphenylene iodonium, superoxide dismutase or N-nitro-l-arginine methyl ester. Moreover, an enhanced iNOS expression was also observed in the cells treated directly with MG which was significantly inhibited when co-application with N-acetyl-l-cysteine. Our results demonstrated that fructose is capable of inducing a significant increase in ONOO(-) production, which is mediated by an enhanced formation of endogenous MG in VSMCs.     

Oxidant hypersensitivity of Fanconi anemia type C-deficient cells is dependent on a redox-regulated apoptotic pathway

Fanconi anemia is a genetic disorder characterized by bone marrow failure. Significant evidence supports enhanced apoptosis of hematopoietic stem/progenitor cells as a critical factor in the pathogenesis of bone marrow failure in Fanconi anemia. However, the molecular mechanism(s) responsible for the apoptotic phenotype are incompletely understood. Here, we tested whether alterations in the activation of a redox-dependent pathway may participate in the pro-apoptotic phenotype of primary Fancc -/- cells in response to oxidative stress. Our data indicate that Fancc -/- cells are highly sensitive to oxidant stimuli and undergo enhanced oxidant-mediated apoptosis compared with wild type controls. In addition, antioxidants preferentially enhanced the survival of Fancc -/- cells. Because oxidative stress activates the redox-dependent ASK1 pathway, we assessed whether Fancc -/- cells exhibited increased oxidant-induced ASK1 activation. Our results revealed ASK1 hyperactivation in H2O2-treated Fancc -/- cells. Furthermore, using small interfering RNAs to decrease ASK1 expression and a dominant negative ASK1 mutant to inhibit ASK1 kinase activity, we determined that H2O2-induced apoptosis was ASK1-dependent. Collectively, these data argue that the predisposition of Fancc -/- hematopoietic stem/progenitor cells to apoptosis is mediated in part through altered redox regulation and ASK1 hyperactivation.     

β-Elemene piperazine derivatives induce apoptosis in human leukemia cells through downregulation of c-FLIP and generation of ROS

β-Elemene is an active component of the herb medicine Curcuma Wenyujin with reported antitumor activity. To improve its antitumor ability, five novel piperazine derivatives of β-elemene, 13-(3-methyl-1-piperazinyl)-β-elemene (DX1), 13-(cis-3,5-dimethyl-1-piperazinyl)-β-elemene (DX2), 13-(4-ethyl-1-piperazinyl)-β-elemene (DX3), 13-(4-isopropyl-1-piperazinyl)-β-elemene (DX4) and 13-piperazinyl-β-elemene (DX5), were synthesized. The antiproliferative and apoptotic effects of these derivatives were determined in human leukemia HL-60, NB4, K562 and HP100-1 cells. DX1, DX2 and DX5, which contain a secondary amino moiety, were more active in inhibiting cell growth and in inducing apoptosis than DX3 and DX4. The apoptosis induction ability of DX1 was associated with the generation of hydrogen peroxide (H(2)O(2)), a decrease of mitochondrial membrane potential (MMP), and the activation of caspase-8. Pretreatment with the antioxidants N-acetylcysteine and catalase completely blocked DX1-induced H(2)O(2) production, but only partially its activation of caspase-8 and induction of apoptosis. HL-60 cells were more sensitive than its H(2)O(2)-resistant subclone HP100-1 cells to DX1-induced apoptosis. The activation of caspase-8 by these compounds was correlated with the decrease in the levels of cellular FLICE-inhibitory protein (c-FLIP). The proteasome inhibitor MG-132 augmented the decrease in c-FLIP levels and apoptosis induced by these derivatives. FADD- and caspase-8-deficient Jurkat subclones have a decreased response to DX1-induced apoptosis. Our data indicate that these novel β-elemene piperazine derivatives induce apoptosis through the decrease in c-FLIP levels and the production of H(2)O(2) which leads to activation of both death receptor- and mitochondrial-mediated apoptotic pathways.     

Recruitment of tumor necrosis factor receptor-associated factor family proteins to apoptosis signal-regulating kinase 1 signalosome is essential for oxidative stress-induced cell death

Apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in oxidative stress-induced cell death. Reactive oxygen species disrupt the interaction of ASK1 with its cellular inhibitor thioredoxin and thereby activates ASK1. However, the precise mechanism by which ASK1 freed from thioredoxin undergoes oligomerization-dependent activation has not been fully elucidated. Here we show that endogenous ASK1 constitutively forms a high molecular mass complex including Trx ( approximately 1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H(2)O(2) treatment, the ASK1 signalosome forms a higher molecular mass complex at least in part because of the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Consistent with our previous findings that TRAF2 and TRAF6 activate ASK1, H(2)O(2)-induced ASK1 activation and cell death were strongly reduced in the cells derived from Traf2-/- and Traf6-/- mice. A novel signaling complex including TRAF2, TRAF6, and ASK1 may thus be the key component in oxidative stress-induced cell death.