Non-redundant function of the MEK5-ERK5 pathway in thymocyte apoptosis

The mitogen-activated protein kinases (MAPKs) ERK1/2, p38, and JNK are thought to determine survival-versus-death fate in developing thymocytes. However, this view was challenged by studies using 'MEK1-ERK1/2-specific' pharmacological inhibitors, which block both positive and negative selection. Recently, these inhibitors were also shown to affect MEK5, an upstream activator of ERK5, another class of MAPK with homology to ERK1/2. To define the contribution of the MEK5-ERK5 pathway in T-cell development, we retrovirally expressed dominant-negative or constitutively activated form of MEK5 to inhibit or activate the MEK5-ERK5 pathway. We demonstrate that MEK5 regulates apoptosis of developing thymocytes but has no function in positive selection. ERK5 activity correlates with the levels of Nur77 family members but not that of Bim, two effector pathways of thymocyte apoptosis. These results illustrate the critical involvement of the MEK5-ERK5 pathway in thymocyte development distinct from that of ERK1/2 and highlight the importance of the MAPK network in mediating differential effects pertaining to T-cell differentiation and apoptosis.     

Acacetin inhibits neuronal cell death induced by 6-hydroxydopamine in cellular Parkinson’s disease model

Acacetin (5,7-dihydroxy-4′-methoxyflavone), a flavonoid compound isolated from Flos Chrysanthemi Indici, chrysanthemum, safflower, and Calamintha and Linaria species has been shown to have anti-cancer activity, indicating its potential clinical value in cancer treatment. In this study, we sought to study the potentials of acacetin in preventing human dopaminergic neuronal death via inhibition of 6-hydroxydopamine (6-OHDA)-induced neuronal cell death in the SH-SY5Y cells. Our results suggest that acacetin was effective in preventing 6-OHDA-induced neuronal cell death through regulation of mitochondrial-mediated cascade apoptotic cell death. Pretreatment with acacetin significantly inhibited neurotoxicity and neuronal cell death through reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) dysfunction. Acacetin also markedly acted on key molecules in apoptotic cell death pathways and reduced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinases (PI3K)/Akt, and glycogen synthase kinase-3beta (GSK-3β). These results suggested that acacetin could inhibit 6-OHDA-induced neuronal cell death originating from ROS-mediated cascade apoptosis pathway. Thus, the results of our study suggest that acacetin is a potent therapeutic agent for PD progression.     

The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.     

Role of ROS in the protective effect of silibinin on sodium nitroprusside-induced apoptosis in rat pheochromocytoma PC12 cells

Abstract

Silibinin mostly has been used as hepatoprotectants, but it has other interesting activities, e.g. anti-cancer, cardial protective and brain-protective activities. A previous study demonstrated that silibinin protected amyloid β (Aβ)-induced mouse cognitive disorder by behavioural pharmacological observation. This study assessed the effect of silibinin on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. Subsequent morphologic observation, flow cytometric analysis and Western blot analysis indicated that treatment with SNP significantly induced apoptosis in PC12 cells. However, silibinin eliminated the apoptotic effect by reactive oxygen species (ROS) generation, especially hydroxyl free radical. Silibinin-induced autophagy through ROS generation when exerting a protective effect and silibinin-induced autophagy also enhanced the ROS generation since 3-methyladenine (3-MA), a specific autophagy inhibitor, decreased the ROS generation and rapamycin, an autophagy inducer, enhanced the ROS generation. Therefore, there exists a positive feedback loop between autophagy and ROS generation. Autophagy prevented SNP-induced apoptosis, since the addition of 3-MA significantly eliminated the protective effect of silibinin. This protective effect was attributed to the generation of ROS and its two downstream Ras/PI3K/NF-κB and Ras/Raf/MEK/ERK pathways. Both prevented PC12 cells from apoptosis. The PI3K/NF-κB pathway induced autophagy to protect PC12 cells, but the Raf/MEK/ERK pathway directly protected PC12 cells bypassing the autophagic effect.     

Inhibition of extracellular signal regulated kinase (ERK) leads to apoptosis inducing factor (AIF) mediated apoptosis in epithelial breast cancer cells: the lack of effect of ERK in p53 mediated copper induced apoptosis

Recent studies have shown that MEK/ERK-mediated signals play a major role in regulation of activity of p53 tumor suppressor protein. In this study, we investigated whether or not there is functional interaction between p53 and MEK/ERK pathways in epithelial breast cancer cells exposed to copper or zinc. We demonstrated that expression of wild-type p53 induced by copper or zinc significantly reduced phosphorylation of extracellular signal regulated kinase (ERK) in epithelial breast cancer MCF7 cells. Mutation or suppression of p53 in MDA-MB231 and MCF7-E6 cells, respectively, resulted in a strong ERK phosphorylation in the presence of metals. Weak ERK phosphorylation in MCF7 cells induced by copper or zinc was linked to mitochondrial disruption and apoptosis. Furthermore, inhibition of ERK through addition of PD98059 stimulated p53 activation in MCF7 cells and also led to upregulation of p53 downstream targets, p21 and Bax, which is a proapototic member of Bcl-2 family triggering mitochondrial pore opening. Moreover, blockage of the MEK/ERK pathway caused a breakdown of the mitochondrial membrane potential accompanied by an elevation in the ROS production. Disruption of p53 expression attenuated the depolarization of the mitochondrial membrane and ROS generation. Furthermore, PD98059 initiated apoptosis inducing factor (AIF) translocation from mitochondria to the nucleus in MCF7 cells; which are depleted in caspase 3. Interestingly, repression of MEK/ERK pathway did not intensify the cell stress caused by metal toxicity. Therefore, these findings demonstrate that MEK/ERK pathway plays an important role in downregulation of p53 and cell survival. Inhibition of ERK can lead to apoptosis via nuclear relocation of AIF. However, metal-induced activation of p53 and mitochondrial depolarization appears to be independent of ERK. Our data suggest that copper induces apoptosis through depolarization of mitochondrial membrane with release of AIF, and this process is MEK/ERK independent.     

Phosphorylation of MEK1 by cdk5/p35 down-regulates the mitogen-activated protein kinase pathway.

Cyclin-dependent protein kinase 5 (cdk5), a member of the cdk family, is active mainly in postmitotic cells and plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission. In this study we investigated the relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway. We report that cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 resulted in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) -/- mice, which lack appreciable cdk5 activity, we observed an increase in the phosphorylation of NF-M subunit of neurofilament proteins that correlated with an up-regulation of MEK1 and ERK1/2 activity. The activity of a constitutively active MEK1 with threonine 286 mutated to alanine (within a TPXK cdk5 phosphorylation motif in the proline-rich domain) was not affected by cdk5 phosphorylation, suggesting that Thr286 might be the cdk5/p35 phosphorylation-dependent regulatory site. These findings support the hypothesis that cdk5 and the MAP kinase pathway cross-talk in the regulation of neuronal functions. Moreover, these data and the recent studies of Harada et al. (Harada, T., Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459) have prompted us to propose a model for feedback down-regulation of the MAP kinase signal cascade by cdk5 inactivation of MEK1.     

Neuroprotective Effects of 3α-Acetoxyeudesma-1,4(15),11(13)-trien-12,6α-olide Against Dopamine-Induced Apoptosis in the Human Neuroblastoma SH-SY5Y Cell Line

Dopamine (DA), as a neurotoxin, can elicit severe Parkinson’s disease-like syndrome by elevating intracellular reactive oxygen species (ROS) levels and apoptotic activity. We examined the inhibitory effects of 3α-acetoxyeudesma-1,4(15),11(13)-trien-12,6α-olide (AETO), purified from the leaves of Laurus nobilis L., on DA-induced apoptosis and α-synuclein (α-syn) formation in dopaminergic SH-SY5Y cells. AETO decreased the active form of caspase-3 and the levels of p53, which were accompanied by increased levels of Bcl-2 in a dose-dependent manner. Flow cytometric and Western blot analysis showed that AETO significantly inhibited DA-induced apoptosis along with suppression of intracellular tyrosinase activity, ROS generation, quinoprotein, and α-syn formation (P < 0.01). These results indicate that AETO inhibited DA-induced apoptosis, which is closely related to the suppression of intracellular tyrosinase activity and the formation of α-syn, ROS, and quinoprotein in SH-SY5Y cells.     

p38 mitogen-activated protein kinase is a key regulator of 5-phenylselenyl- and 5-methylselenyl-methyl-2'-deoxyuridine-induced apoptosis in human HL-60 cells

Two novel, modified thymidine nucleosides, 5-phenylselenyl-methyl-2'-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2'-deoxyuridine (MeSe-T), trigger reactive oxygen species (ROS) generation and DNA damage and thereby induce caspase-mediated apoptosis in human HL-60 cells; however, the mechanism leading to caspase activation and apoptotic cell death remains unclear. Therefore, we investigated the signaling molecules involved in nucleoside derivative-induced caspase activation and apoptosis in HL-60 cells. PhSe-T/MeSe-T treatment activated two mitogen-activated protein kinases (MAPKs), extracellular-receptor kinase (ERK) and p38, and induced the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2. In addition, the selective p38 inhibitor SB203580 suppressed PhSe-T/MeSe-T-induced apoptosis and activation of caspase-3, -9, -8, and -2, whereas the jun amino-terminal kinase (JNK) inhibitor SP600125 and the ERK inhibitor PD98059 had no effect. SB203580 and an ROS scavenger, tiron, inhibited PhSe-T/MeSe-T-induced histone H2AX phosphorylation, which is a DNA damage marker. Moreover, tiron inhibited PhSe-T/MeSe-T-induced phosphorylation of p38 and enhanced p38 MAP kinase activity, indicating a role for ROS in PhSe-T/MeSe-T-induced p38 activation. Taken together, our results suggest that PhSe-T/MeSe-T-induced apoptosis is mediated by the p38 pathway and that p38 serves as a link between ROS generation and DNA damage/caspase activation in HL-60 cells.     

Cellular events involved in butyric acid-induced T cell apoptosis

We have previously demonstrated that butyric acid induces cytotoxicity and apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Therefore, to determine the apoptotic signaling pathway induced by butyric acid, we investigated the contribution of reactive oxygen species (ROS), mitochondria, ceramide, and mitogen-activated protein kinases in butyric acid-induced human Jurkat cell apoptosis. After exposure of cells to butyric acid, a pronounced accumulation of ROS was seen. Pretreatment of cells with the antioxidant N-acetyl-cysteine or 3-aminobenzamide attenuated butyric acid-induced apoptosis through a reduction of ROS generation. Cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases protein release from mitochondria into the cytosol were detected shortly after butyric acid treatment. Exposure of cells to butyric acid resulted in an increase in cellular ceramide in a time-dependent fashion. In addition, butyric acid-induced apoptosis was inhibited by DL-threo-dihidrosphingosine, a potent inhibitor of sphingosine kinase. Using anti-extracellular signal-regulated kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and anti-p38 phosphospecific Abs, we showed a decrease in ERK, but not in JNK and p38 phosphorylation after treatment of cells with butyric acid. Pretreatment of cells with the JNK inhibitor SP600125 attenuated the effect of butyric acid on apoptosis, whereas no effect was seen with the p38 inhibitor SB202190 or the ERK inhibitor PD98059. Taken together, our results indicate that butyric acid-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Effect of microRNA-27a-5p on apoptosis and inflammatory response of pancreatic acinar cells in acute pancreatitis by targeting PTEN

To investigate the apoptosis and inflammatory response of microRNA-27a-5p (miR-27a-5p) in pancreatic acinar cells of acute pancreatitis (AP) and its related mechanisms. Rat pancreatic acinar cell line AR42J was treated with caerulein (10nmol/L) to construct an acute pancreatitis cell model. Quantitative real-time polymerase chain reaction was performed to measure the expression of miR-27a-5p; The miR-27a-5p mimic was transfected into cell, and the apoptosis rate of the cells was detected by flow cytometry; The levels of TNF-α, IL-1, and IL-6 in the culture supernatant were determined by enzyme-linked immunosorbent assay; TargetScans database predicted and dual luciferase reporter gene assay verified the relationship between miR-27a-5p and the phosphatase and tensin homolog deleted on chromosome 10 (PTEN); The recovery experiment explored the apoptosis and the effects of inflammatory responses. The expression of miR-27a-5p decreased gradually (P < 0.05) and the expression of PTEN increased gradually (P < 0.05) with the prolongation of acting time. Upregulation of miR-27a-5p significantly promoted cell apoptosis (P < 0.05) and inhibited inflammatory response (P < 0.05); The TargetScans database predicted that the 3'UTR of PTEN contains a base complementary to the miR-27a-5p seed region. Cotransfection of wild-type vector (PTEN-WT) with miR-27a-5p mimic or miR-27a-5p inhibitor significantly affected the relative activity of luciferase (P < 0.05), and no significant impact was observed in mutant PTEN-MUT. Compared with miR-27a-5p + pcDNA group, transfection of miR-27a-5p mimic and pcDNA-PTEN significantly increased the expression of PTEN (P < 0.05), decreased the apoptotic rate (P < 0.05), and increased the inflammatory response (P < 0.05). miR-27a-5p induced apoptosis and inhibited the inflammatory response of pancreatic acinar cells in AP by targeting PTEN.     

ERK5/MAPK is activated by TGFbeta in hepatocytes and required for the GSK-3beta-mediated Snail protein stabilization

Extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase, specifically activated by MEK5, and involved in the regulation of many cellular functions including proliferation, survival, differentiation and apoptosis. MEK5/ERK5 module is an important element of different signal transduction pathways. The aim of this study was to investigate whether ERK5 participates to the signalling of the multifunctional cytokine TGFbeta, known to play an important role in the regulation of hepatic growth. Here, we reported that ERK5 is phosphorylated and activated by TGFbeta in hepatocytes, with a rapid and sustained kinetic, through a Src-dependent pathway. Moreover, we demonstrated that ERK5 participates to the TGFbeta-induced Snail protein regulation being required for its stabilization. We also found that the functional inactivation of ERK5 impedes the TGFbeta-mediated glycogen synthase kinase-3beta inactivation suggesting this as mechanism responsible for ERK5-mediated Snail stabilization. Thus, results presented in this study uncovered for the first time a role for ERK5 in the TGFbeta-induced cellular responses.     

NADPH oxidase-derived reactive oxygen species-mediated activation of ERK1/2 is required for apoptosis of human neutrophils induced by Entamoeba histolytica

The extracellular tissue penetrating protozoan parasite Entamoeba histolytica has been known to induce host cell apoptosis. However, the intracellular signaling mechanism used by the parasite to trigger apoptosis is poorly understood. In this study, we investigated the roles of reactive oxygen species (ROS), and of MAPKs in the Entamoeba-induced apoptosis of human neutrophils. The neutrophils incubated with live trophozoites of E. histolytica revealed a marked increase of receptor shedding of CD16 as well as phosphatidylserine (PS) externalization on the cell surface. The Entamoeba-induced apoptosis was effectively blocked by pretreatment of cells with diphenyleneiodonium chloride (DPI), a flavoprotein inhibitor of NADPH oxidase. A large amount of intracellular ROS was detected after exposure to viable trophozoites, and the treatment with DPI strongly inhibited the Entamoeba-induced ROS generation. However, a mitochondrial inhibitor rotenone did not attenuate the Entamoeba-induced ROS generation and apoptosis. Although E. histolytica strongly induced activation of ERK1/2 and p38 MAPK in neutrophils, the activation of ERK1/2 was closely associated with ROS-mediated apoptosis. Pretreatment of neutrophils with MEK1 inhibitor PD98059, but not p38 MAPK inhibitor SB202190, prevented Entamoeba-induced apoptosis. Moreover, DPI almost completely inhibited Entamoeba-induced phosphorylation of ERK1/2, but not phosphorylation of p38 MAPK. These results strongly suggest that NADPH oxidase-derived ROS-mediated activation of ERK1/2 is required for the Entamoeba-induced neutrophil apoptosis.     

PB1 domain-dependent signaling complex is required for extracellular signal-regulated kinase 5 activation

MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38.     

The structure of a full turn of an A-DNA duplex d(CGCGGGTACCCGCG)₂

Fms-like tyrosine kinase-3 (FLT3) is a growth factor receptor normally expressed on hematopoietic progenitor cells. Approximately one third of all patients with AML carry an activating mutation in FLT3 that drives proliferation and survival of the leukemic cells. The most common activating mutation is the so-called internal tandem duplication (ITD), which involves an in-frame duplication of a segment of varying length in the region of the FLT3 gene that encodes the juxtamembrane domain. The pathways downstream of FLT3-ITD are partially known but further knowledge regarding the downstream signal transduction molecules is important in order to develop alternative strategies for pharmacological intervention.In this paper we have studied the role of MEK/ERK5 in FLT3-ITD mediated transformation. We have found that both wild-type FLT3 and FLT3-ITD activate MEK5 leading to the activation of ERK5. By use of the selective inhibitor of MEK5, BIX02188, we have shown that activation of AKT downstream of FLT3 is partially dependent on ERK5. Furthermore, inhibition of MEK5/ERK5 induces apoptosis of both FLT3-ITD transfected Ba/F3 cells as well as the FLT3-ITD carrying leukemic cell lines MV4-11 and MOLM-13. These results suggest that MEK5/ERK5 is important for FLT3-ITD induced hematopoietic transformation and may thus represent an alternative therapeutic target in the treatment of FLT3-ITD positive leukemia.     

Advanced glycation endproduct induces ROS accumulation, apoptosis, MAP kinase activation and nuclear O-GlcNAcylation in human cardiac myocytes

Accumulation of advanced glycation endproduct (AGE) has been implicated in the pathogenesis of diabetic complications. However, the precise role and mechanism behind AGE-associated diabetic heart injury are not fully clear. This study was designed to evaluate the effect of AGE on accumulation of reactive oxygen species (ROS), apoptosis, mitogen-activated protein kinase (MAPK) activation and nuclear O-GlcNAcylation in fetal human cardiac myocytes. Myocytes were maintained for 24-72 h in a defined culture medium containing high glucose, the AGE carbon precursor methylglyoxal (MG), and MG-AGE derived from MG and bovine serum albumin (BSA). Generation of ROS was detected by 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by caspase-3 activity and quantitative DNA fragmentation. Both high glucose (25.5 mM) and MG (200 microM) significantly enhanced ROS and AGE formation with greater effects elicited by MG. Both high glucose and MG-AGE significantly facilitated apoptosis with a more predominant effect from MG-AGE. In addition, phosphorylation of MAPK cascade [extracellular signal-regulated kinase-1/2 (ERK1/2) and p38] and nuclear O-GlcNAcylation were enhanced in MG-AGE-treated myocytes, similar to those elicited by high glucose. MG-AGE-induced phosphorylation of ERK1/2 and p38 was nullified by neutralizing AGE with specific anti-AGE antibody but not nonspecific antiserum. Collectively, these results indicated that AGE or its precursor MG may trigger ROS generation, apoptosis, MAPK activation and nuclear O-GlcNAcylation in human cardiac myocytes, in a manner reminiscent of high extracellular glucose.     

Fluazinam targets mitochondrial complex I to induce reactive oxygen species-dependent cytotoxicity in SH-SY5Y cells

Although the underlying cause of Parkinson's disease (PD) is not well characterized, epidemiological studies suggest that exposure to agricultural chemicals is a risk factor for PD. Fluazinam (FZN) is a new active ingredient for the control of grey mould, belonging to the novel broad spectrum phenylpyridinamine fungicides. We used human neuroblastoma SH-SY5Y cells to investigate mechanisms of dopaminergic cell death in response to FZN. FZN treatment produced dose-dependent cytotoxicity, and decreased the tyrosine hydroxylase (TH) expression in SH-SY5Y cells. We provided evidence for the occurrence of oxidative stress and oxidative damage during FZN exposure on dopaminergic cells through the measurement of reactive oxygen species (ROS) in cells with DCFH-DA. The cytotoxic effects of FZN appear to involve an increase in ROS generation since pretreatment with N-acetyl cysteine (NAC), an anti-oxidant, reduced cell death. After FZN treatment, dopamine (DA) levels decreased in both cell and culture media, and oxidative effects of FZN were blocked by NAC pretreatment. We show that cell death in response to FZN was due to apoptosis since FZN exposure results in an increased in cytochrome c release into the cytosol and activated caspase-3 through p38 and JNK signaling. Furthermore, the blocking of p38 or JNK signaling inhibits FZN-induced cell death. Phosphorylation of mitogen-activated protein kinases precedes cytochrome c release and caspase-3 activation. This cellular response is characteristic of mitochondrial dysfunction. Therefore, we also investigated the effect of FZN on mitochondrial complex I activity in FZN-treated cell. Interestingly, we show that FZN inhibited the complex I activity. Thus in this study, we report a new mode of action by which the fungicide FZN could triggers apoptosis.     

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.