DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals

Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway.     

Microglial secreted cathepsin B induces neuronal apoptosis

Activated microglia release a number of substances that can influence neuronal signalling and survival. Here we report that microglia stimulated with the peptide chromogranin A (CGA), secreted the cysteine protease, cathepsin B. Conditioned medium from CGA exposed microglia was neurotoxic to the HT22 hippocampal cell line and to primary cultures of cerebellar granule neurones. In both neuronal cell types, the neurotoxicity could be significantly attenuated with z-FA-fmk or by depletion of microglial conditioned medium with cathepsin B antibody. Conditioned medium from activated microglia or cathepsin B alone induced neuronal apoptosis and caspase 3 activation. Our data indicate that CGA-activated microglia can trigger neuronal apoptosis and that this may be mediated through the secretion of cathepsin B. Since cathepsins may also play a role in the amyloidogenic processing of amyloid precursor protein, these results may have significance for tissue damage and neuronal loss in the neuropathology of Alzheimer's disease.     

Dopaminochrome induces caspase-independent apoptosis in the mesencephalic cell line, MN9D

Parkinson's disease is characterized by a deficiency in motor cortex modulation due to degeneration of pigmented dopaminergic neurons of the substantia nigra projecting to the striatum. These neurons are particularly susceptible to oxidative stress, perhaps because of their dopaminergic nature. Like all catecholamines, dopamine is easily oxidized, first to a quinone intermediate and then to dopaminochrome (DAC), a 5-dihydroxyindole tautomer, that is cytotoxic in an oxidative stress-dependent manner. Here we show, using the murine mesencephalic cell line MN9D, that DAC causes cell death by apoptosis, illustrated by membrane blebbing, Annexin V, and propidium iodide labeling within 3 h. In addition, DAC causes oxidative damage to DNA within 3 h, and positive terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescence by 24 h. DAC, however, does not induce caspase 3 activation and its cytotoxic actions are not prevented by the pan-caspase inhibitor, Z-VAD-fmk. DAC-induced cytotoxicity is limited by the PARP1 inhibitor, 5-aminoisoquinolinone, supporting a role for apoptosis-inducing factor (AIF) in the apoptotic process. Indeed, AIF is detected in the nuclear fraction of MN9D cells 3 h after DAC exposure. Taken together these results demonstrate that DAC induces cytotoxicity in MN9D cells in a caspase-independent apoptotic manner, likely triggered by oxidative damage to DNA, and involving the translocation of AIF from the mitochondria to the nucleus.     

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.     

High-dose rapamycin induces apoptosis in human cancer cells by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1

mTOR, the mammalian target of rapamycin, has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anti-cancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nano-molar concentrations, however at higher micro-molar doses, rapamycin induces apoptosis in several human cancer cell lines. While much is known about the effect of low dose rapamycin treatment, the mechanistic basis for the apoptotic effects of high-dose rapamycin treatment is not understood. We report here that the apoptotic effects of high-dose rapamycin treatment correlate with suppressing phosphorylation of the mTOR complex 1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). Consistent with this observation, ablation of eIF4E also resulted in apoptorsis in MDA-MB 231 breast cancer cells. We also provide evidence that the differential dose effects of rapamycin are correlated with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. In contrast with MDA-MB-231 cells, MCF-7 breast cancer cells survived rapamycin-induced suppression of 4E-BP1 phosphorylation. We show that survival correlated with a hyper-phosphorylation of Akt at S473 at high rapamycin doses, the suppression of which conferred rapamycin sensitivity. This study reveals that the apoptotic effect of rapamycin requires doses that completely dissociate Raptor from mTORC1 and suppress that phosphorylation of 4E-BP1 and inhibit eIF4E.     

Sangivamycin induces apoptosis by suppressing Erk signaling in primary effusion lymphoma cells

Sangivamycin, a structural analog of adenosine and antibiotic exhibiting antitumor and antivirus activities, inhibits protein kinase C and the synthesis of both DNA and RNA. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients and HIV-infected homosexual males. PEL cells are derived from post-germinal center B cells, and are infected with KSHV. Herein, we asked if sangivamycin might be useful to treat PEL. We found that sangivamycin killed PEL cells, and we explored the underlying mechanism. Sangivamycin treatment drastically decreased the viability of PEL cell lines compared to KSHV-uninfected B lymphoma cell lines. Sangivamycin induced the apoptosis of PEL cells by activating caspase-7 and -9. Further, sangivamycin suppressed the phosphorylation of Erk1/2 and Akt, thus inhibiting activation of the proteins. Inhibitors of Akt and MEK suppressed the proliferation of PEL cells compared to KSHV-uninfected cells. It is known that activation of Erk and Akt signaling inhibits apoptosis and promotes proliferation in PEL cells. Our data therefore suggest that sangivamycin induces apoptosis by inhibiting Erk and Akt signaling in such cells. We next investigated whether sangivamycin, in combination with an HSP90 inhibitor geldanamycin (GA) or valproate (valproic acid), potentiated the cytotoxic effects of the latter drugs on PEL cells. Compared to treatment with GA or valproate alone, the addition of sangivamycin enhanced cytotoxic activity. Our data thus indicate that sangivamycin may find clinical utility as a novel anti-cancer agent targeting PEL.     

Elevated testosterone induces apoptosis in neuronal cells

Testosterone plays a crucial role in neuronal function, but elevated concentrations can have deleterious effects. Here we show that supraphysiological levels of testosterone (micromolar range) initiate the apoptotic cascade. We used three criteria, annexin V labeling, caspase activity, and DNA fragmentation, to determine that apoptotic pathways were activated by testosterone. Micromolar, but not nanomolar, testosterone concentrations increased the response in all three assays of apoptosis. In addition, testosterone induced different concentration-dependent Ca2+ signaling patterns: at low concentrations of testosterone (100 nm), Ca2+ oscillations were produced, whereas high concentrations (1-10 microm) induced a sustained Ca2+ increase. Elevated testosterone concentrations increase cell death, and this effect was abolished in the presence of either inhibitors of caspases or the inositol 1,4,5-trisphosphate receptor (InsP3R)-mediated Ca2+ release. Knockdown of InsP3R type 1 with specific small interfering RNA also abolished the testosterone-induced cell death and the prolonged Ca2+ signals. In contrast, knockdown of InsP3R type 3 modified neither the apoptotic response nor the Ca2+ signals. These results support our hypothesis that elevated testosterone alters InsP3R type 1-mediated intracellular Ca2+ signaling and that the prolonged Ca2+ signals lead to apoptotic cell death. These effects of testosterone on neurons will have long term effects on brain function.     

Cholestanol induces apoptosis of cerebellar neuronal cells

Cerebrotendinous xanthomatosis (CTX) is a hereditary lipid storage disease characterized by hyper-cholestanolemia, cerebellar ataxia, xanthoma, and cataract. We hypothesized that cholestanol in serum of CTX patients might induce neuronal cell death in the cerebellum and eventually lead to cerebellar ataxia. To gain support for this hypothesis we developed hyper-cholestanolemia rats by feeding cholestanol. Neuronal cells, especially Purkinje cells in the cerebellum were stained by Sudan black B only in the cholestanol-fed rats, indicating the deposit of cholestanol in cerebellum. To examine effects of cholestanol in vitro, cerebellar neuronal cells were cultured with cholestanol. The cholestanol concentration increased and the viability decreased in cells cultured with cholestanol. Apoptosis was evident in cells cultured with cholestanol more frequently than in control cells, determined using the terminal deoxynucleotidyl transferase (TdT) dUTP nick end-labeling (TUNEL) method. As activities of interleukin-1beta-converting enzyme (ICE) and CPP32 protease were increased in cells cultured with cholestanol, all these data taken together suggest that cholestanol induced apoptosis of cerebellar neuronal cells. Our observations may explain the mechanism of cerebellar ataxia of CTX patients.     

Suppression of uracil-DNA glycosylase induces neuronal apoptosis

A chronic imbalance in DNA precursors, caused by one-carbon metabolism impairment, can result in a deficiency of DNA repair and increased DNA damage. Although indirect evidence suggests that DNA damage plays a role in neuronal apoptosis and in the pathogenesis of neurodegenerative disorders, the underlying mechanisms are poorly understood. In particular, very little is known about the role of base excision repair of misincorporated uracil in neuronal survival. To test the hypothesis that repair of DNA damage associated with uracil misincorporation is critical for neuronal survival, we employed an antisense (AS) oligonucleotide directed against uracil-DNA glycosylase encoded by the UNG gene to deplete UNG in cultured rat hippocampal neurons. AS, but not a scrambled control oligonucleotide, induced apoptosis, which was associated with DNA damage analyzed by comet assay and up-regulation of p53. UNG mRNA and protein levels were decreased within 30 min and were undetectable within 6-9 h of exposure to the UNG AS oligonucleotide. Whereas UNG expression is significantly higher in proliferating as compared with nonproliferating cells, such as neurons, the levels of UNG mRNA were increased in brains of cystathionine beta-synthase knockout mice, a model for hyperhomocysteinemia, suggesting that one-carbon metabolism impairment and uracil misincorporation can induce the up-regulation of UNG expression.     

Ochratoxin A induces apoptosis in neuronal cells

The mycotoxin ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is a frequently present contaminant of food and feedstuffs. OTA exhibits a wide range of toxic activities including nephro- and hepatotoxicity. However, little is known regarding potential neurotoxic effects of OTA. In the present study primary neurons as well as SH-SY5Y neuronal cells were incubated with increasing concentrations of OTA (0.1–2.5 μmol/L). OTA treatment resulted in a dose-dependent increase in cytotoxicity in both neuronal cell types. Caspase-9 and caspase-3 were activated in response to OTA treatment. Furthermore, caspase inhibitors were effective in partly counteracting OTA induced neurocytotoxicity. OTA induced apoptosis was accompanied by a loss of mitochondria membrane potential. Overall, present data indicated that OTA is neurotoxic at relatively low concentrations. OTA induced neurotoxicity seems to be, at least party, mediated by apoptosis. OTA may contribute to the pathogenesis of neurodegenerative diseases (e.g. Alzheimer’s and Parkinson’s disease) in which apoptotic processes are centrally involved.     

Inhibition of neuronal apoptosis by the cyclin-dependent kinase inhibitor GW8510: identification of 3' substituted indolones as a scaffold for the development of neuroprotective drugs

Increasing evidence suggests that neuronal apoptosis is triggered by the inappropriate activation of cyclin-dependent kinases leading to an abortive re-entry of neurons into the cell cycle. Pharmacological inhibitors of cell-cycle progression may therefore have value in the treatment of neurodegenerative diseases in humans. GW8510 is a 3' substituted indolone that was developed recently as an inhibitor of cyclin-dependent kinase 2 (CDK2). We found that GW8510 inhibits the death of cerebellar granule neurons caused by switching them from high potassium (HK) medium to low potassium (LK) medium. Although GW8510 inhibits CDK2 and other CDKs when tested in in vitro biochemical assays, when used on cultured neurons it only inhibits CDK5, a cytoplasmic CDK that is not associated with cell-cycle progression. Treatment of cultured HEK293T cells with GW8510 does not inhibit cell-cycle progression, consistent with its inability to inhibit mitotic CDKs in intact cells. Neuroprotection by GW8510 is independent of Akt and MEK-ERK signaling. Furthermore, GW8510 does not block the LK-induced activation of Gsk3beta and, while inhibiting c-jun phosphorylation, does not inhibit the increase in c-jun expression observed in apoptotic neurons. We also examined the effectiveness of other 3' substituted indolone compounds to protect against neuronal apoptosis. We found that like GW8510, the VEGF Receptor 2 Kinase Inhibitors [3-(1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one], {(Z)-3-[2,4-Dimethyl-3-(ethoxycarbonyl)pyrrol-5-yl)methylidenyl]indol-2-one} and [(Z)-5-Bromo-3-(4,5,6,6-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one], the Src family kinase inhibitor SU6656 and a commercially available inactive structural analog of an RNA-dependent protein kinase inhibitor 5-Chloro-3-(3,5-dichloro-4-hydroxybenzylidene)-1,3-dihydro-indol-2-one, are all neuroprotective when tested on LK-treated neurons. Along with our recent identification of the c-Raf inhibitor GW5074 (also a 3' substituted indolone) as a neuroprotective compound, our findings identify the 3' substituted indolone as a core structure for the designing of neuroprotective drugs that may be used to treat neurodegenerative diseases in humans.     

The Kv2.1 channels mediate neuronal apoptosis induced by excitotoxicity

Chronic loss of intracellular K⁺ can induce neuronal apoptosis in pathological conditions. However, the mechanism by which the K⁺ channels are regulated in this process remains largely unknown. Here, we report that the increased membrane expression of Kv2.1 proteins in cortical neurons deprived of serum, a condition known to induce K⁺ loss, promotes neuronal apoptosis. The increase in I _K current density and apoptosis in the neurons deprived of serum were inhibited by a dominant negative form of Kv2.1 and MK801, an antagonist to NMDA receptors. The membrane level of Kv2.1 and its interaction with SNAP25 were increased, whereas the Kv2.1 phosphorylation was inhibited in the neurons deprived of serum. Botulinum neurotoxin, an agent known to prevent formation of soluble N -ethylmaleimide-sensitive factor attachment protein receptor complex, suppressed the increase in I _K current density. Together, these results suggest that NMDA receptor-dependent Kv2.1 membrane translocation is regulated by a soluble N -ethylmaleimide-sensitive factor attachment protein receptor-dependent vesicular trafficking mechanism and is responsible for neuronal cell death induced by chronic loss of K⁺.     

Fructose-1,6-bisphosphatase aggravates oxidative stress-induced apoptosis in asthma by suppressing the Nrf2 pathway

Asthma is a chronic airway disease that causes excessive inflammation, oxidative stress, mucus production and bronchial epithelial cell apoptosis. Fructose-1,6-bisphosphatase (Fbp1) is one of the rate-limiting enzymes in gluconeogenesis and plays a critical role in several cancers. However, its role in inflammatory diseases, such as asthma, is unclear. Here, we examined the expression, function and mechanism of action of Fbp1 in asthma. Gene Expression Omnibus (GEO) data sets revealed that Fbp1 was overexpressed in a murine model of asthma and in interleukin (IL)-4- or IL-13-stimulated bronchial epithelial cells. We confirmed the findings in an animal model as well as Beas-2B and 16HBE cells. In vitro investigations revealed that silencing of Fbp1 reduced apoptosis and the proportion of cells in the G2/M phase, whereas overexpression led to increases. Fbp1 knock-down inhibited oxidative stress by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, whereas Fbp1 overexpression aggravated oxidative stress by suppressingthe Nrf2 pathway. Moreover, the Nrf2 pathway inhibitor ML385 reversed the changes caused by Fbp1 inhibition in Beas-2B and 16HBE cells. Collectively, our data indicate that Fbp1 aggravates oxidative stress-induced apoptosis by suppressing Nrf2 signalling, substantiating its potential as a novel therapeutic target in asthma.     

Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.     

Carbon monoxide induces murine thymocyte apoptosis by a free radical-mediated mechanism

Carbon monoxide (CO) induces acute or chronic toxicity, according to the level and duration of the exposure. Since chronic CO exposure was shown to have immunosuppressive effects (as it decreases the frequency of rat splenic immunocompetent cells and immunoglobulin production), we investigated the effect of CO on thymocytes, since these are the most sensitive cells to oxidative damage from the lymphoid lineage. We exposed thymocytes to CO, then determined their apoptotic index after 6 h of incubation at 37°C using the fluorochrome Hoechst 33342 and electron microscopy and found an increase of apoptosis in CO-exposed thymocytes. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an antioxidant vitamin E analog, decreased CO-induced thymocyte apoptosis unlike methylene blue, L-nitroarginine methyl ester or pyrrolidine dithiocarbamate. We also observed that lipid peroxidation was increased in the CO-exposed thymocytes and that it was inhibited by Trolox. Our results suggest that CO induces thymocyte apoptosis by a free radical-mediated mechanism which can be inhibited by Trolox but which does not involve the activation of the guanylyl cyclase–cGMP pathway.     

Treatment of cultured pancreatic B-cells with streptozotocin induces cell death by apoptosis

Treatment of cultured pancreatic B-cells (HIT-T15 and RINm5F) with the diabetogenic drug Streptozotocin resulted in a significant increase in the number of cells that became detached from the substrate during a subsequent culture period. Examination of the detached cells by fluorescence microscopy after staining with acridine orange or by electron microscopy revealed evidence of chromatin condensation and margination. Isolation and fractionation of DNA from these cells revealed a pattern of oligonucleosomal fragmentation that was not evident in untreated cells. All of these features are characteristic of entry of the cells into apoptosis and the results suggest that the diabetogenic action of Streptozotocin involves induction of apoptosis in pancreatic B-cells.     

Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 microM MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30-50%. Interestingly, administration of 1-10 microM MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 microM MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome c, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 microM MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.