Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway

Surfactin has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in surfactin-induced apoptosis remain poorly understood. The present study was undertaken to elucidate the underlying network of signaling events in surfactin-induced apoptosis of human breast cancer MCF-7 cells. In this study, surfactin caused reactive oxygen species (ROS) generation and the surfactin-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) and catalase, suggesting involvement of ROS generation in surfactin-induced cell death. Surfactin induced a sustained activation of the phosphorylation of ERK1/2 and JNK, but not p38. Moreover, surfactin-induced cell death was reversed by PD98059 (an inhibitor of ERK1/2) and SP600125 (an inhibitor of JNK), but not by SB203580 (an inhibitor of p38). However, the phosphorylation of JNK rather than ERK1/2 activation by surfactin was blocked by NAC/catalase. These results suggest that the action of surfactin on MCF-7 cells was via ERK1/2 and JNK, but not via p38, and the ERK1/2 and JNK activation induce apoptosis through two independent signaling mechanisms. Surfactin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade reaction. The NAC and SP600125 blocked these events induced by surfactin. Moreover, the general caspase inhibitor z-VAD-FMK inhibited the caspase-6 activity and exerted the protective effect against the surfactin-induced cell death. Taken together, these findings suggest that the surfactin induces apoptosis through a ROS/JNK-mediated mitochondrial/caspase pathway.     

Apomorphine attenuates 6-hydroxydopamine-induced apoptotic cell death in SH-SY5Y cells

Abstract

Enhanced oxidative stress is implicated in the pathogenesis of Parkinson's disease. The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS), leading to neuronal cell death. On the other hand, apomorphine, a dopamine D1/D2 receptor agonist and known as a potent antioxidant, has been reported to have a neuroprotective effect. In the present study, we investigated the effect of apomorphine on 6-OHDA-induced apoptotic cell death using the human dopaminergic neuroblastoma cell line, SH-SY5Y. The co-treatment of cells with apomorphine significantly attenuated 6-OHDA-induced ROS generation, the phosphorylation of c-Jun N-terminal kinase (JNK), DNA fragmentation and subsequent apoptotic cell death. In addition, pretreatment with apomorphine for 24 h and the following concomitant treatment enhanced the protective effects against 6-OHDA-induced toxicity except for the attenuation of JNK phosphorylation. We also demonstrated that pretreatment alone with apomorphine for 24 h prior to the exposure confers resistance against 6-OHDA-induced cell toxicity. These findings suggested that apomorphine acts principally as a radical scavenger to suppress the level of ROS and ROS-stimulated apoptotic signaling pathway, whereas the other mechanisms might be involved in the protective effects.     

Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1α-JNK and mitochondrial apoptotic pathway

An increased intracellular methylglyoxal (MGO) under hyperglycemia led to pancreatic beta cell death. However, its mechanism in which way with MGO induced beta cell death remains unknown. We investigated both high glucose and MGO treatment significantly inclined intracellular MGO concentration and inhibited cell viability in vitro. MGO treatment also triggered intracellular advanced glycation end products (AGEs) formation, declined mitochondrial membrane potential (MMP), increased oxidative stress and the expression of ER stress mediators Grp78/Bip and p-PERK; activated mitochondrial apoptotic pathway, which could mimic by Glo1 knockdown. Aminoguanidine (AG), a MGO scavenger, however, prevented AGEs formation and MGO-induced cell death by inhibiting oxidative stress and ER stress. Furthermore, both antioxidant N-acetylcysteine (NAC) and ER stress inhibitor 4-phenylbutyrate (4-PBA) could attenuate MGO-induced cell death through ameliorating ER stress. MGO treatment down-regulated Ire1α, a key ER stress mediator, increased JNK phosphorylation and activated mitochondrial apoptosis; down-regulated Bcl-2 expression which could be attenuated by the JNK inhibitor SP600125 and further inhibited cytochrome c leakage from mitochondria and blocked the conversion of pro caspase 3 into cleaved caspase 3, all these might contribute to the inhibition of INS-1 cell apoptosis. Ire1α down-regulation by Ire1α siRNAs mimicked MGO-induced cytotoxicity by activating the JNK phosphorylation and mitochondrial apoptotic pathway. In summary, we demonstrated that increased intracellular MGO induced cytotoxicity in INS-1 cells primarily by activating oxidative stress and further triggering mitochondrial apoptotic pathway, and ER stress-mediated Ire1α-JNK pathway. These findings may have implication on new mechanism of glucotoxicity-mediated pancreatic beta-cell dysfunction.     

Amplification loop cascade for increasing caspase activity induced by docetaxel

The hierarchy of events accompanying induction of apoptosis by the microtubule inhibitor docetaxel was investigated in HL-60 human leukemia cells. Treatment of HL-60 cells with docetaxel resulted in the production of reactive oxygen species (ROS), activation of caspase-3 (-like) protease, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation, bcl-2 phosphorylation and apoptosis. Docetaxel elicited ROS production from NADPH oxidase as demonstrated by specific oxidase inhibitor diphenylene iodonium (DPI). ROS mediated the caspase-3 activation and apoptosis in HL-60 cells. The caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) effectively inhibited JNK/SAPK activation, bcl-2 phosphorylation and partially attenuated the ROS production induced by docetaxel. Docetaxel-induced bcl-2 phosphorylation was completely blocked by expression of dominant negative JNK or the JNK/SAPK inhibitor SP600125. Overexpression of bcl-2 partially prevented docetaxel-mediated ROS production and subsequent caspase-3 activation, thereby inhibiting apoptotic cell death. It is thus conferred that such sequent events as ROS production, caspase activation, JNK/SAPK activation, bcl-2 phosphorylation and the further generation of ROS should be parts of an amplification loop to increase caspase activity, thereby facilitating the apoptotic cell death process.     

Direct interaction of the molecular scaffolds POSH and JIP is required for apoptotic activation of JNKs

A sequential pathway (the JNK pathway) that includes activation of Rac1/Cdc42, mixed lineage kinases, MAP kinase kinases 4 and 7, and JNKs plays a required role in many paradigms of apoptotic cell death. However, the means by which this pathway is assembled and directed toward apoptotic death has been unclear. Here, we report that propagation of the apoptotic JNK pathway requires the cooperative interaction of two molecular scaffolds, POSH and JIPs. POSH (plenty of SH3s) is a multidomain GTP-Rac1-interacting protein that binds and promotes activation of mixed lineage kinases. JIPs are reported to bind MAP kinase kinases 4/7 and JNKs. We find that POSH and JIPs directly associate with one another to form a multiprotein complex, PJAC (POSH-JIP apoptotic complex), that includes all of the known kinase components of the pathway. Our observations indicate that this complex is required for JNK activation and cell death in response to apoptotic stimuli.     

Piceatannol attenuates hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells by blocking down-regulation of Bcl-XL and activation of JNK

There is mounting evidence implicating the accumulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. Recently, considerable attention has been focused on identifying naturally occurring antioxidants that are able to reduce excess ROS and RNS, thereby protecting against oxidative stress and neuron death. The present study investigated the possible protective effects of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene), which is present in grapes and other foods, on hydrogen-peroxide- and peroxynitrite-induced oxidative cell death. PC12 rat pheochromocytoma (PC12) cells treated with hydrogen peroxide or SIN-1 (a peroxynitrite-generating compound) exhibited apoptotic death, as determined by nucleus condensation and cleavage of poly(ADP-ribose)polymerase (PARP). Piceatannol treatment attenuated hydrogen-peroxide- and peroxynitrite-induced cytotoxicity, apoptotic features, PARP cleavage and intracellular ROS and RNS accumulation. Treatment of PC12 cells with hydrogen peroxide or SIN-1 led to down-regulation of Bcl-X(L) and activation of caspase-3 and -8, which were also inhibited by piceatannol treatment. Hydrogen peroxide or SIN-1 treatment induced phosphorylation of the c-Jun-N-terminal kinase (JNK), which was inhibited by piceatannol treatment. Moreover, SP600125 (a JNK inhibitor) significantly inhibited hydrogen-peroxide- and peroxynitrite-induced PC12 cell death, revealing inactivation of the JNK pathway as a possible molecular mechanism for the protective effects of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells. Collectively, these findings suggest that the protective effect of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells is associated with blocking the activation of JNK and the down-regulation of Bcl-XL.     

7-Ketocholesterol and cholestane-triol increase expression of SMO and LXRα signaling pathways in a human breast cancer cell line

Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of 7-ketocholesterol (7-KC), cholestane-3β-5α-6β-triol (triol), and a mixture of 5α-cholestane-3β,6β-diol and 5α-cholestane-3β,6α-diol (diol) to promote cell death in a human breast cancer cell line (MDA-MB-231). We determined cell viability, after 24-h incubation with oxysterols. These oxysterols promoted apoptosis. At least part of the observed effects promoted by 7-KC and triol arose from an increase in the expression of the sonic hedgehog pathway mediator, smoothened. However, this increased expression was apparently independent of sonic hedgehog expression, which did not change. Moreover, these oxysterols led to increased expression of LXRα, which is involved in cellular cholesterol efflux, and the ATP-binding cassette transporters, ABCA1 and ABCG1. Diols did not affect these pathways. These results suggested that the sonic hedgehog and LXRα pathways might be involved in the apoptotic process promoted by 7-KC and triol.     

5-Hydroxy-7-Methoxyflavone Triggers Mitochondrial-Associated Cell Death via Reactive Oxygen Species Signaling in Human Colon Carcinoma Cells

Plant-derived compounds are an important source of clinically useful anti-cancer agents. Chrysin, a biologically active flavone found in many plants, has limited usage for cancer chemotherapeutics due to its poor oral bioavailability. 5-Hydroxy-7-methoxyflavone (HMF), an active natural chrysin derivative found in various plant sources, is known to modulate several biological activities. However, the mechanism underlying HMF-induced apoptotic cell death in human colorectal carcinoma cells in vitro is still unknown. Herein, HMF was shown to be capable of inducing cytotoxicity in HCT-116 cells and induced cell death in a dose-dependent manner. Treatment of HCT-116 cells with HMF caused DNA damage and triggered mitochondrial membrane perturbation accompanied by Cyt c release, down-regulation of Bcl-2, activation of BID and Bax, and caspase-3-mediated apoptosis. These results show that ROS generation by HMF was the crucial mediator behind ER stress induction, resulting in intracellular Ca²⁺ release, JNK phosphorylation, and activation of the mitochondrial apoptosis pathway. Furthermore, time course study also reveals that HMF treatment leads to increase in mitochondrial and cytosolic ROS generation and decrease in antioxidant enzymes expression. Temporal upregulation of IRE1-α expression and JNK phosphorylation was noticed after HMF treatment. These results were further confirmed by pre-treatment with the ROS scavenger N-acetyl-l-cysteine (NAC), which completely reversed the effects of HMF treatment by preventing lipid peroxidation, followed by abolishment of JNK phosphorylation and attenuation of apoptogenic marker proteins. These results emphasize that ROS generation by HMF treatment regulates the mitochondrial-mediated apoptotic signaling pathway in HCT-116 cells, demonstrating HMF as a promising pro-oxidant therapeutic candidate for targeting colorectal cancer.     

ROS-Induced JNK and p38 Signaling Is Required for Unpaired Cytokine Activation during Drosophila Regeneration

Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. We genetically induced cell death in wing imaginal discs, monitored the production of ROS and analyzed the signals required for repair. We found that cell death generates a burst of ROS that propagate to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. Our findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration.     

Apomorphine attenuates 6-hydroxydopamine-induced apoptotic cell death in SH-SY5Y cells

Enhanced oxidative stress is implicated in the pathogenesis of Parkinson's disease. The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS), leading to neuronal cell death. On the other hand, apomorphine, a dopamine D1/D2 receptor agonist and known as a potent antioxidant, has been reported to have a neuroprotective effect. In the present study, we investigated the effect of apomorphine on 6-OHDA-induced apoptotic cell death using the human dopaminergic neuroblastoma cell line, SH-SY5Y. The co-treatment of cells with apomorphine significantly attenuated 6-OHDA-induced ROS generation, the phosphorylation of c-Jun N-terminal kinase (JNK), DNA fragmentation and subsequent apoptotic cell death. In addition, pretreatment with apomorphine for 24 h and the following concomitant treatment enhanced the protective effects against 6-OHDA-induced toxicity except for the attenuation of JNK phosphorylation. We also demonstrated that pretreatment alone with apomorphine for 24 h prior to the exposure confers resistance against 6-OHDA-induced cell toxicity. These findings suggested that apomorphine acts principally as a radical scavenger to suppress the level of ROS and ROS-stimulated apoptotic signaling pathway, whereas the other mechanisms might be involved in the protective effects.     

The Nox/Duox family of ROS-generating NADPH oxidases

Reactive oxygen species (ROS) generated by the NADPH oxidases are conventionally thought to be cytotoxic and mutagenic and at high levels induce an oxidative stress response. The phagocyte NADPH oxidase catalyzes the NADPH-dependent reduction of molecular oxygen to generate superoxide O2-., which can dismute to generate ROS species. Together, these ROS participate in host defence by killing or damaging invading microbes. Flavocytochrome b558 is the catalytic core of the phagocyte NADPH oxidase and consists of a large glycoprotein gp91phox or Nox-2 and a small protein p22phox. The other components of the NADPH oxidase are cytosolic proteins, namely p67phox, p47phox, p40phox and Rac. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections. Evidence is rapidly accumulating that low level of ROS were produced by NADPH oxidase homologs in non-phagocytic cells. To date, six human homologs (Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 and Duox-2) have been recently identified in a variety of non-phagocytic cells. The identification of Nox-1 was quickly followed by the cloning of Nox-3, Nox-4, and Nox-5. In parallel, two very large members of the Nox family were discovered, namely Duox-1 and Duox-2, initially also referred to as thyroid oxidases. The physiological functions of Nox-dependent ROS generation are in progress and still require detailed characterization. Activation mechanisms and tissue distribution of the different members of the Nox family are very different, suggesting distinct physiological functions. Nox family enzymes are likely to be involved in a variety of physiological events including cell proliferation, host defence, differentiation, apoptosis, senescence and activation of growth-related signaling pathways. An increase and a decrease in the function of Nox enzymes can contribute to a wide range of pathological processes.     

Regulation of cytochrome P450 2e1 expression by ethanol: role of oxidative stress-mediated pkc/jnk/sp1 pathway

CYP2E1 metabolizes ethanol leading to production of reactive oxygen species (ROS) and acetaldehyde, which are known to cause not only liver damage but also toxicity to other organs. However, the signaling pathways involved in CYP2E1 regulation by ethanol are not clear, especially in extra-hepatic cells. This study was designed to examine the role of CYP2E1 in ethanol-mediated oxidative stress and cytotoxicity, as well as signaling pathways by which ethanol regulates CYP2E1 in extra-hepatic cells. In this study, we used astrocytic and monocytic cell lines, because they are important cells in central nervous system . Our results showed that 100 mM ethanol significantly induced oxidative stress, apoptosis, and cell death at 24 h in the SVGA astrocytic cell line, which was rescued by a CYP2E1 selective inhibitor, diallyl sulfide (DAS), CYP2E1 siRNA, and antioxidants (vitamins C and E). Further, we showed that DAS and vitamin C abrogated ethanol-mediated (50 mℳ) induction of CYP2E1 at 6 h, as well as production of ROS at 2 h, suggesting the role of oxidative stress in ethanol-mediated induction of CYP2E1. We then investigated the role of the protein kinase C/c-Jun N-terminal kinase/specificity protein1 (PKC/JNK/SP1) pathway in oxidative stress-mediated CYP2E1 induction. Our results showed that staurosporine, a non-specific inhibitor of PKC, as well as specific PKCζ inhibitor and PKCζ siRNA, abolished ethanol-induced CYP2E1 expression. In addition, inhibitors of JNK (SP600125) and SP1 (mithramycin A) completely abrogated induction of CYP2E1 by ethanol in SVGA astrocytes. Subsequently, we showed that CYP2E1 is also responsible for ethanol-mediated oxidative stress and apoptotic cell death in U937 monocytic cell lines. Finally, our results showed that PKC/JNK/SP1 pathway is also involved in regulation of CYP2E1 in U937 cells. This study has clinical implications with respect to alcohol-associated neuroinflammatory toxicity among alcohol users.     

An antiapoptotic protein, c-FLIPL, directly binds to MKK7 and inhibits the JNK pathway

Inhibition of NF-kappaB activation increases susceptibility to tumor necrosis factor (TNF)alpha-induced cell death, concurrent with caspases and prolonged c-Jun N-terminal kinase (JNK) activation, and reactive oxygen species (ROS) accumulation. However, the detailed mechanisms are unclear. Here we show that cellular FLICE-inhibitory protein (c-FLIP) is rapidly lost in NF-kappaB activation-deficient, but not wild-type fibroblasts upon TNFalpha stimulation, indicating that NF-kappaB normally maintains the cellular levels of c-FLIP. The ectopic expression of the long form of c-FLIP (c-FLIPL) inhibits TNFalpha-induced prolonged JNK activation and ROS accumulation in NF-kappaB activation-deficient fibroblasts. Conversely, TNFalpha induces prolonged JNK activation and ROS accumulation in c-Flip-/- fibroblasts. Moreover, c-FLIPL directly interacts with a JNK activator, MAP kinase kinase (MKK)7, in a TNFalpha-dependent manner and inhibits the interactions of MKK7 with MAP/ERK kinase kinase 1, apoptosis-signal-regulating kinase 1, and TGFbeta-activated kinase 1. This stimuli-dependent interaction of c-FLIPL with MKK7 might selectively suppress the prolonged phase of JNK activation. Taken that ROS promote JNK activation and activation of the JNK pathway may promote ROS accumulation, c-FLIPL might block this positive feedback loop, thereby suppressing ROS accumulation.     

A critical role for IkappaB kinase beta in metallothionein-1 expression and protection against arsenic toxicity

Arsenic is a widespread environmental toxic agent that has been shown to cause diverse tissue and cell damage and at the same time to be an effective anti-cancer therapeutic agent. The objective of this study is to explore the signaling mechanisms involved in arsenic toxicity. We show that the IkappaB kinase beta (IKKbeta) plays a crucial role in protecting cells from arsenic toxicity. Ikkbeta(-)(/)(-) mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKKbeta-reconstituted Ikkbeta(-)(/)(-) cells, IKKbeta-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH(2)-terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikkbeta(-)(/)(-) cells prevented JNK activation; moreover, NAC pretreatment, MT1 expression, MKK4 ablation, and JNK inhibition all protected cells from death induced by arsenic. Our data show that two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-kappaB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract ROS accumulation, and second, when this pathway fails, excessive ROS leads to activation of the MKK4-JNK pathway, resulting in apoptosis.     

cAMP-guanine exchange factor protection from bile acid-induced hepatocyte apoptosis involves glycogen synthase kinase regulation of c-Jun NH2-terminal kinase

Cholestatic liver disorders are accompanied by the hepatic accumulation of cytotoxic bile acids that induce cell death. Increases in cAMP protect hepatocytes from bile acid-induced apoptosis by a cAMP-guanine exchange factor (cAMP-GEF)/phosphoinositide-3-kinase (PI3K)/Akt pathway. The aim of these studies was to identify the downstream substrate in this pathway and to determine at what level in the apoptotic cascade cytoprotection occurs. Since inhibitory phosphorylation of glycogen synthase kinase-3 (GSK) occurs downstream of PI3K/Akt and this phosphorylation has been implicated in cell survival, we conducted studies to determine whether GSK was downstream in cAMP-GEF/PI3K/Akt-mediated cytoprotection. Our results show that treatment of hepatocytes with the cAMP-GEF-specific analog, 4-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cAMP, results in PI3K-dependent phosphorylation of GSK. Direct chemical inhibition of GSK in rat hepatocytes or human HUH7-NTCP cells with several structurally and functionally distinct inhibitors including bromoindirubin-3'-oxime (BIO), maleimides (SB216763, SB415286), thiadiazolidine derivatives, and LiCl attenuates apoptosis induced by glycochenodeoxycholate (GCDC). In addition, genetic silencing of the GSK β isoform with small interfering RNA attenuates GCDC apoptosis in HUH7-NTCP cells. Adenoviral inhibition of the Rap1 blocks both cAMP-GEF-mediated cytoprotection against GCDC-induced apoptosis and Akt/GSK3β phosphorylation. GCDC-induced phosphorylation of the proapoptotic kinase, c-Jun NH(2)-terminal kinase (JNK) is inhibited by GSK inhibition or cAMP-GEF activation. GCDC-induced apoptosis is accompanied by phosphorylation of the endoplasmic reticulum stress markers pIEF2α and IRE-1, and pretreatment with the cAMP-GEF analog or GSK inhibitors prevents this phosphorylation. Collectively, our results support the presence of a cAMP/cAMP-GEF/Rap1/PI3K/Akt/GSKβ survival pathway in hepatocytes that inhibits bile acid-induced JNK phosphorylation.     

Role of NADPH oxidase in the apoptotic death of cultured cerebellar granule neurons

Cerebellar granule neurons (CGN) cultured in a medium containing 25 mM KCl and treated with staurosporine (ST) or transferred to a medium with 5 mM KCl (K5) die apoptotically. CGN death is mediated by an increase in reactive oxygen species (ROS) production. When CGN are treated with antioxidants all apoptotic parameters and cell death are markedly diminished, showing a central role for ROS in this process. Recently, it has been suggested that a possible ROS source involved in cell death is a NADPH oxidase. In that regard, we found expression in CGN of the components of NADPH proteins, p40phox, p47phox and p67phox, and p22phox, as well as three homologues of the catalytic subunit of this complex, NOX1, 2, and 4. The inhibition of NADPH oxidase with diphenylene iodonium or 4-(2-aminoethyl)benzenesulfonyl fluoride significantly reduced ROS production, NADPH oxidase activity, all the apoptotic events, and cell death induced by both K5 and ST. We conclude that ROS could be an early signal of apoptotic neuronal death and that NADPH oxidase, including NOX1, 2, and/or 4, could have a central role in apoptotic death induced by different conditions in these neurons.     

NRAGE,a p75 neurotrophin receptor-interacting protein,induces caspase activation and cell death through a JNK-dependent mitochondrial pathway

The p75 neurotrophin receptor (p75NTR) mediates signaling events leading to activation of the JNK pathway and cell death in a variety of cell types. We recently identified NRAGE, a protein that directly interacts with the p75NTR cytosolic region and facilitates p75NTR-mediated cell death. For the present study, we developed an inducible recombinant NRAGE adenovirus to dissect the mechanism of NRAGE-mediated apoptosis. Induced NRAGE expression resulted in robust activation of the JNK pathway that was not inhibited by the pharmacological mixed lineage kinase (MLK) inhibitor CEP1347. NRAGE induced cytosolic accumulation of cytochrome c, activation of Caspases-3, -9 and -7, and caspase-dependent cell death. Blocking JNK and c-Jun action by overexpression of the JNK-binding domain of JIP1 or dominant-negative c-Jun ablated NRAGE-mediated caspase activation and NRAGE-induced cell death. These findings identify NRAGE as a p75NTR interactor capable of inducing caspase activation and cell death through a JNK-dependent mitochondrial apoptotic pathway.     

Endoplasmic reticulum (ER) stress triggers Hax1-dependent mitochondrial apoptotic events in cardiac cells

Cardiomyocyte apoptosis is a major process in pathogenesis of a number of heart diseases, including ischemic heart diseases and cardiac failure. Ensuring survival of cardiac cells by blocking apoptotic events is an important strategy to improve cardiac function. Although the role of ER disruption in inducing apoptosis has been demonstrated, we do not yet fully understand how it influences the mitochondrial apoptotic machinery in cardiac cell models. Recent investigations have provided evidences that the prosurvival protein HCLS1-associated protein X-1 (Hax1) protein is intimately associated with the pathogenesis of heart disease, mitochondrial biology, and protection from apoptotic cell death. To study the role of Hax1 upon ER stress induction, Hax1 was overexpressed in cardiac cells subjected to ER stress, and cell death parameters as well as mitochondrial alterations were examined. Our results demonstrated that the Hax1 is significantly downregulated in cardiac cells upon ER stress induction. Moreover, overexpression of Hax1 protected from apoptotic events triggered by Tunicamycin-induced ER stress. Upon treatment with Tunicamycin, Hax1 protected from mitochondrial fission, downregulation of mitofusins 1 and 2 (MFN1 and MFN2), loss of mitochondrial membrane potential (?Ψm), production of reactive oxygen species (ROS) and apoptotic cell death. Taken together, our results suggest that Hax1 inhibits ER stress-induced apoptosis at both the pre- and post-mitochondrial levels. These findings may offer an opportunity to develop new agents that inhibit cell death in the diseased heart.     

Mixed lineage kinase 3 connects reactive oxygen species to c-Jun NH2-terminal kinase-induced mitochondrial apoptosis in genipin-treated PC3 human prostate cancer cells

It has been reported that genipin, the aglycone of geniposide, induces apoptotic cell death in human hepatoma cells via a NADPH oxidase-reactive oxygen species (ROS)-c-Jun NH(2)-terminal kinase (JNK)-dependent activation of mitochondrial pathway. This continuing work aimed to define that mixed lineage kinase 3 (MLK3) is a key mediator, which connect between ROS and JNK in genipin-induced cell death signaling. In PC3 human prostate cancer cells, genipin stimulated MLK3 activity in concentration- and time-dependent manner. The PC3 cells stably transfected with dominant-negative form of MLK3 was less susceptible to population of the sub-G1 apoptotic cells, activation of caspase, collapse of mitochondrial membrane potential, and release of cytochrome c triggered by genipin, suggesting a crucial role of MLK3 in genipin signaling to apoptotic cell death. Diphenyleneiodonium (DPI), a specific inhibitor of NADPH oxidase, markedly inhibited ROS generation and MLK3 phosphorylation in the genipin-treated cells. Pretreatment with SP0600125, a specific inhibitor of JNK but neither U0126, a specific inhibitor of MEK1/2 nor PD169316, a specific inhibitor of p38 suppressed genipin-induced apoptotic cell death. Notably, both the phosphorylation of JNK and induction of c-Jun induced by genipin were markedly inhibited in PC3-EGFP-MLK3 (K144R) cells expressing a dominant-negative MLK3 mutant. Taken together, our observations suggest genipin signaling to apoptosis of PC3 cells is mediated via activation of ROS-dependent MLK3, which leads to downstream activation of JNK.     

alpha-Synuclein protects against oxidative stress via inactivation of the c-Jun N-terminal kinase stress-signaling pathway in neuronal cells.

The expression of alpha-synuclein, a synaptic molecule implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body disease is increased upon injury to the nervous system, indicating that it might play a role in regeneration and plasticity; however, the mechanisms are unclear. Because c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, plays an important role in stress response, the main objective of the present study was to better understand the involvement of this pathway in the signaling responses associated with resistance to injury in cells expressing alpha-synuclein. For this purpose, the JNK-signaling pathway was investigated in alpha-synuclein-transfected neuronal cell line glucose transporter (GT) 1-7 under oxidative stress conditions. Although hydrogen peroxide challenge resulted in JNK activation and cell death in cells transfected with vector control or beta-synuclein, alpha-synuclein-transfected cells were resistant to hydrogen peroxide, and JNK was not activated. The inactivation of JNK in the alpha-synuclein-transfected cells was associated with increased expression and activity of JNK-interacting protein (JIP)-1b/islet-brain (IB)1, the scaffold protein for the JNK pathway. Similarly, cells transfected with JIP-1b/IB1 were resistant to hydrogen peroxide associated with inactivation of the JNK pathway. In these cells, expression of endogenous alpha-synuclein was significantly increased at the protein level. Furthermore, alpha-synuclein was co-localized with JIP-1b/IB1 in the growth cones. Taken together, these results suggest that increased alpha-synuclein expression might protect cells from oxidative stress by inactivation of JNK via increased expression of JIP-1b/IB1. Furthermore, interactions between alpha-synuclein and JIP-1b/IB1 may play a mutual role in the neuronal response to injury and neurodegeneration.