ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.     

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.     

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.     

Activation of stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 kinase in calphostin C-induced apoptosis requires caspase-3-like proteases but is dispensable for cell death

Apoptosis was induced in human glioma cell lines by exposure to 100 nM calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis was associated with synchronous down-regulation of Bcl-2 and Bcl-xL as well as activation of caspase-3 but not caspase-1. The exposure to calphostin C led to activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK and that of p38 kinase by SB203580 induced similar effects on the calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.     

Essential role of STAT1 in caspase-independent cell death of activated macrophages through the p38 mitogen-activated protein kinase/STAT1/reactive oxygen species pathway

Unlike other immune cells, activation of macrophages by stimulating agents, such as lipopolysaccharide (LPS), confers significant resistance to many apoptotic stimuli, but the underlying mechanism of this phenomenon remains largely unknown. Here, we demonstrate that LPS-induced early caspase activation is essential for macrophage survival because blocking caspase activation with a pancaspase inhibitor (zVAD [benzyloxycarbonyl-Val-Ala-Asp]) rapidly induced death of activated macrophages. This type of death process by zVAD/LPS was principally mediated by intracellular generation of superoxide. STAT1 knockout macrophages demonstrated profoundly decreased superoxide production and were resistant to treatment with zVAD/LPS, indicating the crucial involvement of STAT1 in macrophage death by zVAD/LPS. STAT1 level and activity were reciprocally regulated by caspase activation and were associated with cell death. Activation of STAT1 was critically dependent upon serine phosphorylation induced by p38 mitogen-activated protein kinase (MAPK) because a p38 MAPK inhibitor nullified STAT1 serine phosphorylation, reactive oxygen species (ROS) production, and macrophage death by zVAD/LPS. Conversely, p38 MAPK activation was dependent upon superoxide and was also nullified in STAT1 knockout macrophages, probably due to impaired generation of superoxide. Our findings collectively indicate that STAT1 signaling modulates intracellular oxidative stress in activated macrophages through a positive-feedback mechanism involving the p38 MAPK/STAT1/ROS pathway, which is interrupted by caspase activation. Furthermore, our study may provide significant insights in regards to the unanticipated critical role of STAT1 in the caspase-independent death pathway.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Suppression of endoplasmic reticulum stress-induced caspase activation and cell death by the overexpression of Bcl-xL or Bcl-2

Continuous endoplasmic reticulum (ER) stress, such as the accumulation of unfolded proteins, results in cell death and relates to the pathogenesis of some neurodegenerative diseases. Treatment of brefeldin A, an inhibitor of transport between the ER and Golgi complex, induced cell death during 24 h, which accompanied activation of caspase-2, caspase-3 and caspase-9, starting at 12 h and increasing time-dependently up to 28 h. Caspase-2 was expressed and activated in not only mitochondria and cytosol, but also in the microsomal fraction containing ER and Golgi. Of note is that overexpression of Bcl-x(L) or Bcl-2 in PC12 cells markedly suppressed brefeldin A-induced activation of caspases and resulting cell death. Delivery of anti-Bcl-2 antibody into the Bcl-2-overexpressed cells again recovered apoptosis. While the brefeldin A-treatment induced the phosphorylation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, overexpression of Bcl-x(L) or Bcl-2 reduced the prolonged phosphorylation of JNK, but not of p38 MAPK. Pretreatment with a JNK inhibitor, SP600125, suppressed the brefeldin A-induced caspase-2 activation and cell death significantly. Thus, our results suggest that protective effects of Bcl-x(L) and Bcl-2 against brefeldin A-induced cell death appear to be dependent on the regulation of JNK activation.     

Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.     

The role of p38 MAPK and JNK in Arsenic trioxide-induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.     

Activation of JNK by vanadate induces a Fas-associated death domain (FADD)-dependent death of cerebellar granule progenitors in vitro

Apoptosis is a highly regulated process that plays a critical role in neuronal development as well as the homeostasis of the adult nervous system. Vanadate, an environmental toxicant, causes developmental defects in the central nervous system. Here, we demonstrated that vanadate induced apoptosis in cultured cerebellar granule progenitors (CGPs). Treatment of cultured CGPs with vanadate activated ERKs and JNKs but not p38 MAPK and also induced c-Jun phosphorylation. In addition, vanadate induced FasL production, Fas (CD95) aggregation, and its association with the Fas-associated death domain (FADD), as well as the activation of caspase-8. Furthermore, vanadate generated reactive oxygen species (ROS) in CGPs; however, ROS was not involved in vanadate-mediated MAPK activation. Vanadate-induced FasL expression was ROS-dependent but JNK-independent. In contrast, vanadate-elicited Fas aggregation and Fas-FADD association, as well as caspase-8 activation, were dependent on JNK activation but were minimally regulated by ROS generation. The hydrogen peroxide scavenger, catalase, blocked vanadate-induced FasL expression and partially mitigated vanadate-induced cell death. On the other hand, dominant negative FADD and caspase-8 inhibitor completely eliminated vanadate-induced apoptosis. Thus, JNK signaling plays a major role in vanadate-mediated activation of the Fas-FADD-caspase-8 pathway that accounts for vanadate-induced apoptosis of CGPs.     

Reactive oxygen species-mediated mitochondrial pathway is involved in Baohuoside I-induced apoptosis in human non-small cell lung cancer

Baohuoside I (also known as Icariside II) is a flavonoid isolated from Epimedium koreanum Nakai. Although Baohuoside I exhibits anti-inflammatory and anti-cancer activities, its molecular targets/pathways in human lung cancer cells are poorly understood. Therefore, in the present study, we investigated the usefulness of Baohuoside I as a potential apoptosis-inducing cytotoxic agent using human adenocarcinoma alveolar basal epithelial A549 cells as in vitro model. The apoptosis induced by Baohuoside I in A549 cells was confirmed by annexin V/propidium iodide double staining, cell cycle analysis and dUTP nick end labeling. Further research revealed that Baohuoside I accelerated apoptosis through the mitochondrial apoptotic pathway, involving the increment of BAX/Bcl-2 ratio, dissipation of mitochondrial membrane potential, transposition of cytochrome c, caspase 3 and caspase 9 activation, degradation of poly (ADP-ribose) polymerase and the over-production of reactive oxygen species (ROS). A pan-caspase inhibitor, Z-VAD-FMK, only partially prevented apoptosis induced by Baohuoside I, while NAC, a scavenger of ROS, diminished its effect more potently. In addition, the apoptotic effect of Baohuoside I was dependent on the activation of ROS downstream effectors, JNK and p38(MAPK), which could be almost abrogated by using inhibitors SB203580 (an inhibitor of p38(MAPK)) and SP600125 (an inhibitor of JNK). These findings suggested that Baohuoside I might exert its cytotoxic effect via the ROS/MAPK pathway.     

Taiwan cobra phospholipase A2‐elicited JNK activation is responsible for autocrine fas‐mediated cell death and modulating Bcl‐2 and Bax protein expression in human leukemia K562 cells

Phospholipase A₂ (PLA₂) from Naja naja atra venom induced apoptotic death of human leukemia K562 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, Bcl‐2 degradation, mitochondrial translocation of Bax, and cytochrome c release were observed in PLA₂‐treated cells. Moreover, PLA₂ treatment increased Fas and FasL protein expression. Upon exposure to PLA₂, activation of p38 MAPK (mitogen‐activated protein kinase) and JNK (c‐Jun NH₂‐terminal kinase) was found in K562 cells. SB202190 (p38 MAPK inhibitor) pretreatment enhanced cytotoxic effect of PLA₂ and led to prolonged JNK activation, but failed to affect PLA₂‐induced upregulation of Fas and FasL protein expression. Sustained JNK activation aggravated caspase8/mitochondria‐dependent death pathway, downregulated Bcl‐2 expression and increased mitochondrial translocation of Bax. SP600125 (JNK inhibitor) abolished the cytotoxic effect of PLA₂ and PLA₂‐induced autocrine Fas death pathway. Transfection ASK1 siRNA and overexpression of dominant negative p38α MAPK proved that ASK1 pathway was responsible for PLA₂‐induced p38 MAPK and JNK activation and p38α MAPK activation suppressed dynamically persistent JNK activation. Downregulation of FADD abolished PLA₂‐induced procaspase‐8 degradation and rescued viability of PLA₂‐treated cells. Taken together, our results indicate that JNK‐mediated autocrine Fas/FasL apoptotic mechanism and modulation of Bcl‐2 family proteins are involved in PLA₂‐induced death of K562 cells. J. Cell. Biochem. 109: 245–254, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Suppression of ERK signaling evokes autocrine Fas‐mediated death in arachidonic acid‐treated human chronic myeloid leukemia K562 cells

Arachidonic acid (AA)‐induced apoptotic death of K562 cells (human chronic myeloid leukemic cells) was characteristic of reactive oxygen species (ROS) generation and mitochondrial depolarization. N‐Acetylcysteine pretreatment rescued viability of AA‐treated cells and abolished mitochondrial depolarization. In contrast to no significant changes in phospho‐JNK and phospho‐ERK levels, AA evoked notable activation of p38 MAPK. Unlike that of JNK and p38 MAPK, ERK suppression further reduced the viability of AA‐treated cells. Increases in Fas/FasL protein expression, caspase‐8 activation, the production of tBid and the loss of mitochondrial membrane potential were noted with K562 cells that were treated with a combination of U0126 and AA. Down‐regulation of FADD attenuated U0126‐evoked degradation of procaspase‐8 and Bid. Abolition of p38 MAPK activation abrogated U0126‐elicited Fas/FasL up‐regulation in AA‐treated cells. U0126 pretreatment suppressed c‐Fos phosphorylation but increased p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun protein expression by siRNA suggested that c‐Fos counteracted the effect of c‐Jun on Fas/FasL up‐regulation. Taken together, our data indicate that AA induces the ROS/mitochondria‐dependent death pathway and blocks the ERK pathway which enhances the cytotoxicity of AA through additionally evoking an autocrine Fas‐mediated apoptotic mechanism in K562 cells. J. Cell. Physiol. 222: 625–634, 2010. © 2009 Wiley‐Liss, Inc.     

ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines

ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth.     

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.     

Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery

Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery.     

Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid.

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 - 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 microM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 - 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 - 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 microM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 microM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 - 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.     

Apoptosis induced by protein phosphatase 2A (PP2A) inhibition in T leukemia cells is negatively regulated by PP2A-associated p38 mitogen-activated protein kinase

Serine/threonine phosphatase regulation of phosphorylation-mediated intracellular signaling controls a number of important processes in mammalian cells. In this study, we show that constitutively active protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase, is essential for T leukemia cell survival. Jurkat and CCRF-CEM T leukemia cells treated with the PP2A-selective inhibitor okadaic acid (OA) showed a dose- and time-dependent induction of apoptosis, as indicated by loss of mitochondrial transmembrane potential (delta psi(m)), cleavage-induced activation of caspase-3, -8, and -9, and DNA fragmentation. In addition, caspase-8 or caspase-9 inhibition with z-IETD-fmk or z-LEHD-fmk, respectively, largely prevented OA-induced apoptosis. Although OA treatment did not affect constitutive Bcl-2 expression, overexpression of Bcl-2 prevented both OA-induced DNA fragmentation and dissipation of delta psi(m). Furthermore, inhibition of caspase-3, -8, or -9 partially protected against OA-induced loss of delta psi(m). In addition, caspase-9 and caspase-3 inhibition largely prevented procaspase-3 and procaspase-8 cleavage, respectively, while caspase-8 inhibition partially interfered with procaspase-9 cleavage in OA-treated T leukemia cells. Thus, PP2A inhibition triggered the intrinsic pathway of apoptosis, which was enhanced by a mitochondrial feedback amplification loop. PP2A has also been implicated in the regulation of p38 mitogen-activated protein kinase (MAPK). Co-immunoprecipitation analysis revealed a physical association between the catalytic subunit of PP2A and p38 MAPK in T leukemia cells. Moreover, OA treatment caused p38 MAPK to be phosphorylated in a dose- and time-dependent fashion, indicating that PP2A prevented p38 MAPK activation. Although p38 MAPK activation usually promotes apoptosis, pharmacologic inhibition of p38 MAPK exacerbated OA-induced DNA fragmentation and loss of delta psi(m) in T leukemia cells, suggesting that, in this instance, the p38 MAPK signaling pathway promoted cell survival. Collectively, these findings indicate that PP2A and p38 MAPK have coordinate effects on signaling pathways that regulate the survival of T leukemia cells.     

cFLIP-L inhibits p38 MAPK activation: an additional anti-apoptotic mechanism in bile acid-mediated apoptosis

In cholestasis, toxic bile acids accumulate within the liver inducing hepatocyte apoptosis, which exacerbates liver injury. Although bile acids activate both death receptors and mitogen-activated kinase (MAPK) pathways, the mechanistic link between death receptor signaling and MAPK activation in bile acid apoptosis remains unclear. The aim of this study was to ascertain if MAPKs contribute to bile acid cytotoxicity. Although deoxycholate induced apoptosis and activated all three classic mediators of the MAPK pathways including JNK 1/2, p38, and p42/44, only p38 MAPK inhibition attenuated apoptosis. Suppressing FADD expression with siRNA or employing a caspase inhibitor, zVAD-fmk, did not block p38 MAPK activation suggesting its activation was not death receptor-dependent. Unexpectedly, expression of cFLIP-L in a stably transfected cell line blocked apoptosis and p38 MAPK phosphorylation. Based on these data we postulated a direct effect of cFLIP on p38 MAPK activation. The nonphosphorylated but not the phosphorylated/active form of p38 MAPK co-immunoprecipitated with cFLIP-L. In reverse immunoprecipitation experiments, cFLIP-L long but not cFLIP-S co-immunoprecipitate with p38 MAPK. In conclusion, these data suggest that cFLIP-L exerts its anti-apoptotic activity, in part, by inhibiting p38 MAPK activation, an additional anti-apoptotic effect for this protein.