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.     

Induction of microsomal epoxide hydrolase by sulfur amino acid deprivation via the pathway of C-Jun N-terminal kinase and its extracellular exposure during cell death

Microsomal epoxide hydrolase (mEH), an epoxide detoxifying enzyme and putative cell surface autoantigen, is inducible by xenobiotics and by certain pathophysiological conditions (e.g., tumorigenesis and protein-calorie malnutrition). The present study was designed to determine mEH expression in H4IIE cells during cell death initiated by sulfur amino acid deprivation (SAAD) and to identify the signaling pathway for the enzyme induction. SAAD induced cell death at 48-72 h with translocation of Bax to mitochondria and increased mitochondrial permeability with cytochrome c release, both of which were prevented by SB203580 or by dominant-negative JNK1 [JNK1(-)] stable transfection. Caspase-3 activity was only marginally increased by SAAD. Neither genomic DNA fragmentation nor poly(ADP-ribose) polymerase cleavage was observed during SAAD-induced cell death. Thus, SAAD induced cell death independent of caspase activation. This was supported by the observation that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a general caspase inhibitor, did not prevent cell death. The levels of mEH mRNA and protein were notably increased in cells under SAAD for 48-72 h. The induction of mEH occurred in parallel with cell death. Whereas SAAD-induced cell death resulted from both JNK1 and p38 kinase activation, mEH induction was decreased only by JNK1(-) transfection. Immunocytochemistry revealed that mEH protein was intensely stained in dying cells, cellular fragments and cell debris. Furthermore, the number of cells positive for surface mEH substantially increased by SAAD, as evidenced by flow cytometry analysis. These results demonstrated that SAAD induced nonapoptotic cell death with Bax translocation to mitochondria and mitochondrial cytochrome c release, but not through caspase-3 activation, and that mEH was induced by SAAD via the pathway of JNK1, but not ERK1/2 or p38 kinase, in parallel with cell death.     

Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death. This work was supported by grant from Association pour la Recherche sur le Cancer (CNRS6543/ARC). S. Cagnol is supported by a fellowship from the Ligue Nationale contre le Cancer.     

Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton

Resolution of neutrophil mediated inflammation is achieved, in part, through induction of neutrophil apoptosis. This constitutively expressed programme can be delayed by inflammatory mediators and induced by ligation of the Fas receptor. However, functional activation of the neutrophil results in resistance to Fas signalled death. We evaluated the effects of Fas antibody engagement on caspase activation and mitochondrial permeability, and the impact of co-stimulation by lipopolysaccharide (LPS) or granulocyte macrophage-colony stimulating factor (GM-CSF) on these events. Fas engagement by an agonistic anti-Fas antibody resulted in enhanced caspase 3 and 8 activity and increased mitochondrial permeability. Studies with pharmacological inhibitors of caspase activity showed that activation of caspase 8 occurred before, and activation of caspase 3 occurred after mitochondrial disruption. The mitochondrial stabilising agent bongkrekic acid also inhibited caspase activation and apoptosis. LPS, GM-CSF and increased glutathione stabilised the mitochondria and inhibited caspase 3. Caspase 8 activity was also inhibited by co-stimulation through a mechanism independent of mitochondrial stabilisation. Glutathione directly inhibited caspase 3 and 8 activity. We conclude inhibition of Fas antibody induced apoptosis by inflammatory proteins is associated with augmented mitochondrial stability and reduced caspase 3 activity that may be glutathione mediated.     

Constitutive ERK MAPK activity regulates macrophage ATP production and mitochondrial integrity

A unique feature of human alveolar macrophages is their prolonged survival in the face of a stressful environment. We have shown previously that the ERK MAPK is constitutively active in these cells and is important in prolonging cell survival. This study examines the role of the ERK pathway in maintaining mitochondrial energy production. The data demonstrate that ATP levels in alveolar macrophages depend on intact mitochondria and optimal functioning of the electron transport chain. Significant levels of MEK and ERK localize to the mitochondria and inhibition of ERK activity induces an early and profound depletion in cellular ATP coincident with a loss of mitochondrial transmembrane potential. The effect of ERK suppression on ATP levels was specific, since it did not occur with PI3K/Akt, p38, or JNK suppression. ERK inhibition led to cytosolic release of mitochondrial proteins and caspase activation. Both ERK inhibition and mitochondrial blockers induced loss of plasma membrane permeability and cell death. The cell death induced by ERK inhibition had hallmarks of both apoptotic (caspase activation) and necrotic (ATP loss) cell death. By blocking ERK inhibition-induced reactive oxygen species, caspase activation was prevented, although necrotic pathways continued to induce cell death. This suggests that mitochondrial dysfunction caused by ERK inhibition generates both apoptotic and necrotic cell death-inducing pathways. As a composite, these data demonstrate a novel mitochondrial role for ERK in maintaining mitochondrial membrane potential and ATP production in human alveolar macrophages.     

Loss of caspase-2-dependent apoptosis induces autophagy after mitochondrial oxidative stress in primary cultures of young adult cortical neurons

Mitochondrial dysfunctions have been associated with neuronal apoptosis and are characteristic of neurodegenerative conditions. Caspases play a central role in apoptosis; however, their involvement in mitochondrial dysfunction-induced neuronal apoptosis remains elusive. In the present report using rotenone, a complex I inhibitor that causes mitochondrial dysfunction, we determined the initiator caspase and its role in cell death in primary cultures of cortical neurons from young adult mice (1-2 months old). By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor that irreversibly binds to and traps the active caspase, we identified caspase-2 as an initiator caspase activated in rotenone-treated primary neurons. Loss of caspase-2 inhibited rotenone-induced apoptosis; however, these neurons underwent a delayed cell death by necrosis. We further found that caspase-2 acts upstream of mitochondria to mediate rotenone-induced apoptosis in neurons. The loss of caspase-2 significantly inhibited rotenone-induced activation of Bid and Bax and the release of cytochrome c and apoptosis inducing factor from mitochondria. Rotenone-induced downstream activation of caspase-3 and caspase-9 were also inhibited in the neurons lacking caspase-2. Autophagy was enhanced in caspase-2 knock-out neurons after rotenone treatment, and this response was important in prolonging neuronal survival. In summary, the present study identifies a novel function of caspase-2 in mitochondrial oxidative stress-induced apoptosis in neurons cultured from young adult mice.     

Lack of X-linked inhibitor of apoptosis protein leads to increased apoptosis and tissue loss following neonatal brain injury

Neurological deficits caused by H-I (hypoxia-ischaemia) to the perinatal brain are often severely debilitating and lead to motor impairment, intellectual disability and seizures. Perinatal brain injury is distinct from adult brain injury in that the developing brain is undergoing the normal process of neuronal elimination by apoptotic cell death and thus the apoptotic machinery is more easily engaged and activated in response to injury. Thus cell death in response to neonatal H-I brain injury is partially due to mitochondrial dysfunction and activation of the apoptosome and caspase 3. An important regulator of the apoptotic response following mitochondrial dysfunction is XIAP (X-linked inhibitor of apoptosis protein). XIAP inhibits apoptosis at the level of caspase 9 and caspase 3 activation, and lack of XIAP in vitro has been shown to lead to increased apoptotic cell death. In the present study we show that mice lacking the gene encoding the XIAP protein have an exacerbated response to neonatal H-I injury as measured by tissue loss at 7 days following the injury. In addition, when the XIAP-deficient mice were studied at 24 h post-H-I we found that the increase in injury correlates with an increased apoptotic response in the XIAP-deficient mice and also with brain imaging changes in T2-weighted magnetic resonance imaging and apparent diffusion coefficient that correspond to the location of apoptotic cell death. These results identify a critical role of XIAP in regulating neuronal apoptosis in vivo and demonstrate the enhanced vulnerability of neurons to injury in the absence of XIAP in the developing brain.     

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.     

Antimycin A-induced killing of HL-60 cells: apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9.

BACKGROUND: Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested. METHODS: Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy. RESULTS: AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell. CONCLUSIONS: The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.     

Nuclear translocation of X-linked inhibitor of apoptosis (XIAP) determines cell fate after hypoxia ischemia in neonatal brain

The inhibitors of apoptosis (IAPs) are emerging as key proteins in the control of cell death. In this study, we evaluated the expression and subcellular distribution of the antiapoptotic protein X-linked IAP (XIAP), and its interactions with the XIAP-associated factor 1 (XAF1) in neonatal rat brain following hypoxia-ischemia (HI). HI triggered the mitochondrial release of cytochrome c , Smac/DIABLO, and caspase 3 activation. Confocal microscopy detected XIAP-specific immunofluorescence in the cytoplasm under normal condition, which exhibited a diffuse distribution at 6 h post-HI and by 12 h the majority of XIAP was redistributed into the nucleus. XIAP nuclear translocation was confirmed by subcellular fractionations and by expressing FLAG-tagged XIAP in primary cortical neurons. Over-expression of XIAP significantly reduced, whereas XIAP gene silencing further enhanced cell death, demonstrating a specific requirement of cytoplasmic XIAP for cell survival. An elevated level of cytosolic XIAP was also evident under the conditions of neuroprotection by fibroblast growth factor-1. XAF1 expression was increased temporally and there was increased nuclear co-localization with XIAP in hypoxic-ischemic cells. XIAP co-immunoprecipitated > 9-fold XAF1 protein concurrent with decreased association with caspases 9 and 3. This is evidenced by the enhanced caspase 3 activity and neuronal death. Our findings implicate XIAP nuclear translocation in neuronal death and point to a novel mechanism in the regulation of hypoxic-ischemic brain injury.     

Muscarinic receptor activation protects cells from apoptotic effects of DNA damage,oxidative stress,and mitochondrial inhibition

The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50-200 microm H(2)O(2) caused the activation of caspase-3 beginning after 2-3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H(2)O(2)-induced caspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H(2)O(2) or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate neurotransmission may exacerbate neuronal loss.     

Bacterial GroEL-like heat shock protein 60 protects epithelial cells from stress-induced death through activation of ERK and inhibition of caspase 3

Bacterial heat shock proteins (hsps) can have various effects on human cells. We investigated whether bacterial hsp60s can protect epithelial cells from cell death by affecting the mitogen-activated protein kinase (MAPK) signal pathways. Cell protection was studied by adding bacterial hsp60s to skin keratinocyte cultures (HaCaT cell line) before UV radiation. The results show that hsp60 significantly protected against UV radiation-induced cell death. Effects of UV radiation and exogenous hsp60 on phosphorylation of MAPKs and on activation of caspase 3 were examined by Western blot analysis. UV radiation strongly induced phosphorylation of p38 MAPK and formation of active caspase 3. A p38 inhibitor, SB 203580, totally blocked UV radiation-mediated activation of caspase 3. Preincubation with hsp60 strongly induced phosphorylation of ERK1/2 and inhibited UV radiation-mediated activation of caspase 3. PD 98059, a specific inhibitor of the ERK1/2 pathway, blocked this inhibitory effect of exogenous hsp60. Studies on the association between activity of MAPKs or caspase 3 and cell death showed that the ERK1/2 pathway inhibitor reversed protective effect of hsp60 while specific inhibition of p38 and caspase 3 reduced cell death. These results indicate that in HaCaT cells UV radiation mediates cell death through activation of p38 followed by caspase 3 activation. Exogenous hsp60 partially protects against UV radiation-mediated epithelial cell death through activation of ERK1/2, which inhibits caspase 3 activation.     

Neuronal cell death caused by inhibition of intracellular cholesterol trafficking is caspase dependent and associated with activation of the mitochondrial apoptosis pathway

An elevated level of cholesterol in mitochondrial membranes of Niemann-Pick disease type C1 (NPC1) mouse brains and neural cells has been found to cause mitochondrial dysfunction. In this study, we demonstrate that inhibition of intracellular cholesterol trafficking in primary neurons by class 2 amphiphiles, which mimics the major biochemical and cellular feature of NPC1, led to not only impaired mitochondrial function but also activation of the mitochondrial apoptosis pathway. In activation of this pathway both cytochrome c and Smac/Diablo were released but apoptosis-inducing factor (AIF) was not involved. Treatment of the neurons with taurine, a caspase 9-specific inhibitor, could prevent the amphiphile-induced apoptotic cell death, suggesting that formation of apoptosome, followed by caspase 9 and caspase 3 activation, might play a critical role in the neuronal death pathway. Taken together, the mitochondria-dependent death cascade induced by blocking intracellular cholesterol trafficking was caspase dependent. The findings provide clues for both understanding the molecular basis of neurodegeneration in NPC1 disease and developing therapeutic strategies for treatment of this disorder.     

Involvement of ERK MAP kinase in endoplasmic reticulum stress in SH-SY5Y human neuroblastoma cells

Endoplasmic reticulum (ER) stress has increasingly come into focus as a factor contributing to neuronal injury. Although caspase-dependent mechanisms have been implicated in ER stress, the signaling pathways involved remain unclear. In this study, we examined the role of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase pathway that is highly conserved in many systems for balancing cell survival and death. Prolonged treatment of the human neuroblastoma cell line SH-SY5Y with thapsigargin, an inducer of ER stress, increased cell death over 24-48 h, as measured by LDH release. Caspases were involved; increased levels of active caspase-3 and cleaved caspase substrate PARP were detected, and treatment with Z-VAD-FMK reduced thapsigargin-induced cytotoxicity. In contrast, inhibition of calpain was not protective, although calpain was activated following thapsigargin treatment. An early and transient phosphorylation of ERK1/2 occurred after thapsigargin-induced ER stress, and targeting this pathway with the MEK inhibitors U0126 or PD98059 significantly reduced cell death. Similar cytoprotection was obtained against brefeldin A, another ER stress agent. However, protection against ER stress via ERK inhibition was not accompanied by amelioration of caspase-3 activation, PARP cleavage, or DNA laddering. These data indicate that ERK may contribute to non-caspase-dependent pathways of injury after ER stress.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

Molecular signaling cascade in DNA bisintercalator, echinomycin-induced apoptosis of HT-29 cells: evidence of the apoptotic process via activation of the cytochrome c-ERK-caspase-3 pathway

The DNA-interactive drug, echinomycin, is a potent antitumor agent, which is able to induce apoptosis in a multitude of cancer cell lines. Previously, we showed that echinomycin strongly inhibited the growth of a variety of cancer cell lines, and the activation of mitogen-activated protein kinases (MAPK) in human colon cancer cells (HT-29). However, little information currently exists regarding the details of intracellular signaling pathways such as the MAPK, mitochondrial, and caspase pathways. In order to clarify this issue, we verified the plausible molecular signaling cascade by performing an immunobiochemical apoptosis experiment involving the mitochondrial and caspase pathways. The apoptotic process of HT-29 cells was accompanied by the activation of procaspase-9, -3 and cytochrome c release. Both caspase and MAPK inhibitors were used in the determination of the specific roles of MAPK and caspase in echinomycin-induced apoptosis. ERK (PD98059) or caspase-3-specific (Z-DEVD-FMK) inhibitors were discovered to significantly attenuate echinomycin-induced apoptosis. PD98059 treatment or overexpression of kinase-inactive ERK did not alter the echinomycin-induced cytochrome c release into the cytosol, but did diminish the activation of procaspase-3. Also, Z-DEVD-FMK was found to have no effect on either cytochrome c release or ERK activation. Taken together, these results indicate that cytochrome c release, and the activation of ERK and caspase-3 in the final apoptosis pathway are all relevant factors in echinomycin-induced apoptosis. To our knowledge, this study is the first to delineate the echinomycin's direct detrimental effects on colon cancer cells.     

3-Hydroxykynurenine, an Endogenous Oxidative Stress Generator, Causes Neuronal Cell Death with Apoptotic Features and Region Selectivity

Abstract: 3-Hydroxykynurenine (3-HK) is a potential endogenous neurotoxin whose increased levels have been described in several neurodegenerative disorders. Here, we characterized in vitro neurotoxicity of 3-HK. Of the tested kynurenine pathway metabolites, only 3-HK, and to a lesser extent 3-hydroxyanthranilic acid, were toxic to primary cultured striatal neurons. 3-HK toxicity was inhibited by various antioxidants, indicating that the generation of reactive oxygen species is essential to the toxicity. 3-HK-induced neuronal cell death showed several features of apoptosis, as determined by the blockade by macromolecule synthesis inhibitors, and by the observation of cell body shrinkage with nuclear chromatin condensation and fragmentation. In addition, 3-HK toxicity was dependent on its cellular uptake via transporters for large neutral amino acids, because uptake inhibition blocked the toxicity. Cortical and striatal neurons were much more vulnerable to 3-HK toxicity than cerebellar neurons, which may be attributable to the differences in transporter activities of these neurons. These results indicate that 3-HK, depending on transporter-mediated cellular uptake and on intracellular generation of oxidative stress, induces neuronal cell death with brain region selectivity and with apoptotic features, which may be relevant to pathology of neurodegenerative disorders.     

Decreases in phosphoinositide-3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling activate components of spinal motoneuron death

Motoneuron dependence on target-derived trophic factors during development is well established, with loss of trophic support leading to the death of these cells. A complete understanding of the intracellular signal transduction machinery associated with extracellular survival signals requires the examination of individual pathways in various cellular and environmental contexts. In cells deprived of trophic support, and hence compromised for survival, phosphoinositide-3-kinase (PI3K) is decreased when compared with healthy cells supplied with trophic support. Extracellular signal-regulated kinase 1/2 (ERK1/2) signaling is dramatically decreased in deprived cells. We have examined the role of these two pathways to understand how changes in their activity regulate motoneuron survival and death. Pharmacological inhibition of PI3K attenuated motoneuron survival and was important in the regulation of Bcl-2 serine phosphorylation, limited release of cytochrome c into the cytoplasm and caspase activation. Bax translocation from cytoplasm to mitochondria was not altered when PI3K was inhibited. High levels of ERK1/2 inhibition robustly attenuated motoneuron survival in cells supplied with trophic support, whereas moderate inhibition of ERK1/2 activation had little effect. ERK1/2 inhibition in these cells decreased Bcl-2 phosphorylation and resulted in release of cytochrome c from the mitochondria. Bax translocation and caspase activation were not affected by ERK1/2 inhibition. These data reveal that changes in PI3K and ERK1/2 signaling lead to individual and overlapping effects on the cell-death machinery. Characterizing the role of these pathways is critical for a fundamental understanding of the development and degeneration of specific neuronal populations.     

SM-20 is a novel mitochondrial protein that causes caspase-dependent cell death in nerve growth factor-dependent neurons

Sympathetic neurons undergo protein synthesis-dependent apoptosis when deprived of nerve growth factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived sympathetic neurons, and ectopic SM-20 is sufficient to promote neuronal death in the presence of NGF. We now report that SM-20 is a mitochondrial protein that promotes cell death through a caspase-dependent mechanism. SM-20 immunofluorescence was present in the cytoplasm in a punctate pattern that colocalized with cytochrome oxidase I and with mitochondria-selective dyes. Analysis of SM-20/dihydrofolate reductase fusion proteins revealed that the first 25 amino acids of SM-20 contain a functional mitochondrial targeting sequence. An amino-terminal truncated form of SM-20 was not restricted to mitochondria but instead localized throughout the cytosol and nucleus. Nevertheless, the truncated SM-20 retained the ability to induce neuronal death, similar to the wild type protein. SM-20-induced death was accompanied by caspase-3 activation and was blocked by a general caspase inhibitor. Additionally, overexpression of SM-20, under conditions where cell death is blocked by a general caspase inhibitor, did not result in widespread release of cytochrome c from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal-mediated cell death.