Hck Promotes Neuronal Apoptosis Following Intracerebral Hemorrhage

The hematopoietic cell kinase (Hck) is a member of the Src family protein kinases which regulates many signal transduction pathways including cell growth, proliferation, differentiation, migration, and apoptosis. However, the expression and function of Hck after intracerebral hemorrhage (ICH) are unknown. Western blot, immunohistochemistry, and immunofluorescence showed that Hck was obviously up-regulation in neurons adjacent to the hematoma after ICH. In addition, the temporary raise of Hck expression was paralleled with the expression of p53, Bax, and active caspase-3, suggesting that Hck was involved in neuronal apoptosis. Hck siRNA dramatically decrease hemin-induced expression of p53, Bax, and active caspase-3 as well as the amount of apoptotic SH-SY5Y cells in vitro. Furthermore, Hck interacted with p53. Hence, Hck might promote neuronal apoptosis via p53 signaling pathway after ICH.     

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.     

Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-alpha in IEC-6 cells

Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-alpha (TNF-alpha). TNF-alpha or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-alpha, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with alpha-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-alpha/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-alpha-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.     

Assessment on pollution of Ochratoxin A in grain in China and its apoptosis effect on vitro-cultured human tubular kidney cells

Ochratoxin A (OTA) is nephrotoxic, immunosuppressive, and teratogenic in many species and is a possible human carcinogen. In this study, we investigated the OTA pollution situations of grains in northern China and the signaling pathway that mediated OTA-induced apoptosis in human tubular kidney cells (HKCs). Samples of grains collected from three representative areas were determined by using high-performance liquid chromatography fluorescence method. The effects of OTA on cell apoptosis, caspase-3, Bax, and Bcl-2 expression, and phosphorylation of c-Jun NH(2) terminal kinase (JNK) were detected in cultured HKCs via flow cytometry (FCM), Hoechst 33258 staining, and Western blot. It showed that OTA pollution of edible grains was very common in north China. OTA could affect caspase-3, Bax, and Bcl-2 expression and increased cell apoptosis in cultured HKCs. The JNK signalling pathway might play an important role during these cellular events.     

The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase

Targeted gene disruption studies have established that the c-Jun NH(2)-terminal kinase (JNK) signaling pathway is required for stress-induced release of mitochondrial cytochrome c and apoptosis. Here we demonstrate that activated JNK is sufficient to induce rapid cytochrome c release and apoptosis. However, activated JNK fails to cause death in cells deficient of members of the Bax subfamily of proapoptotic Bcl2-related proteins. Furthermore, exposure to stress fails to activate Bax, cause cytochrome c release, and induce death in JNK-deficient cells. These data demonstrate that proapoptotic members of the Bax protein subfamily are essential for JNK-dependent apoptosis.     

Osmotic and glutamate receptor regulation of c-Jun NH(2)-terminal protein kinase in neuroendocrine cells

Expression of a c-Jun NH(2)-terminal protein kinase (JNK), also known as stress-activated protein kinase (SAPK) in rodents, has been implicated in the ability of cells to respond to a variety of stressors. In nonmammalian cells, JNK participates in the regulation of cell volume in response to hyperosmotic stress. To explore the possibility that JNK may participate in the transduction of osmotic information in mammals, we evaluated the expression of JNK immunoreactivity in neuroendocrine cells of the supraoptic nucleus. Low basal expression of JNK-2 (SAPK-alpha) and JNK-3 (SAPK-beta) was seen in vivo and in vitro. During water deprivation, JNK-2 increased in the supraoptic nucleus but not in the cortex. Osmotic or glutamate receptor stimulation in vitro also resulted in an increase in JNK-2 that was tetrodotoxin (TTX) insensitive and paralleled by increased nuclear phospho-c-Jun immunoreactivity. A TTX-sensitive increase in JNK-3 was seen in smaller neurons. Thus different JNK pathways may mediate individual cellular responses to osmotic stress, with JNK-2 linked to osmotic and glutamate receptor stimulation in magnocellular neuroendocrine cells.     

HMGB1 Promotes Mitochondrial Dysfunction–Triggered Striatal Neurodegeneration via Autophagy and Apoptosis Activation

Impairments in mitochondrial energy metabolism are thought to be involved in many neurodegenerative diseases. The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces striatal pathology mimicking neurodegeneration in vivo. Previous studies showed that 3-NP also triggered autophagy activation and apoptosis. In this study, we focused on the high-mobility group box 1 (HMGB1) protein, which is important in oxidative stress signaling as well as in autophagy and apoptosis, to explore whether the mechanisms of autophagy and apoptosis in neurodegenerative diseases are associated with metabolic impairment. To elucidate the role of HMGB1 in striatal degeneration, we investigated the impact of HMGB1 on autophagy activation and cell death induced by 3-NP. We intoxicated rat striata with 3-NP by stereotaxic injection and analyzed changes in expression HMGB1, proapoptotic proteins caspase-3 and phospho-c-Jun amino-terminal kinases (p-JNK). 3-NP–induced elevations in p-JNK, cleaved caspase-3, and autophagic marker LC3-II as well as reduction in SQSTM1 (p62), were significantly reduced by the HMGB1 inhibitor glycyrrhizin. Glycyrrhizin also significantly inhibited 3-NP–induced striatal damage. Neuronal death was replicated by exposing primary striatal neurons in culture to 3-NP. It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1. These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.     

An autophagic mechanism is involved in apoptotic death of rat striatal neurons induced by the non-N-methyl-D-aspartate receptor agonist kainic acid

Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome- associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, upregulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and upregulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.     

Cadmium Induces PC12 Cells Apoptosis via an Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase-Mediated Mitochondrial Apoptotic Pathway

To investigate the role of mitogen-activated protein kinase (MAPK) and downstream events in cadmium (Cd)-induced neuronal apoptosis executed via the mitochondrial apoptotic pathway, this study used the PC-12 cell line as a neuronal model. The result showed that Cd significantly decreased cell viability and the Bcl-2?/?Bax ratio and increased the percentage of apoptotic cells, release of cytochrome c, caspase-3, and poly(ADP-ribose) polymerase cleavage, and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G. In addition, exposure to Cd-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2?/?Bax ratio and cytochrome c release and suppressed caspase-3 and poly(ADP-ribose) polymerase cleavage and AIF and endonuclease G nuclear translocation. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathway played an important role in Cd-induced PC12 cells apoptosis.     

NH2-terminal BH4 domain of Bcl-2 is functional for heterodimerization with Bax and inhibition of apoptosis.

The Bcl-2 family proteins comprise pro-apoptotic as well as anti-apoptotic members. Heterodimerization between members of the Bcl-2 family proteins is a key event in the regulation of apoptosis. We report here that Bcl-2 protein was selectively cleaved by active caspase-3-like proteases in CTLL-2 cell apoptosis in response to interleukin-2 deprivation. Structural and functional analyses of the cleaved fragment revealed that the NH2-terminal region of Bcl-2 (1-34 amid acids) was required for its anti-apoptotic activity and heterodimerization with pro-apoptotic Bax protein. Site-directed mutagenesis of the NH2-terminal region showed that substitutions of hydrophobic residues of BH4 domain resulted in the loss of ability to form a heterodimer with Bax. Particularly instructive was that the V15E mutant of Bcl-2, which completely lost the ability to form a heterodimer with Bax, failed to inhibit Bax- and staurosporine-induced apoptosis. Our results suggest that the BH4 domain of Bcl-2 is critical for its heterodimerization with Bax and for exhibiting anti-apoptotic activity. Therefore, agents interferring with the critical residues of the BH4 domain may provide a new strategy in cancer therapy by impairing Bcl-2 function.     

JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins

Targeted gene disruption studies have established that the c-Jun NH2-terminal kinase (JNK) is required for the stress-induced release of mitochondrial cytochrome c and apoptosis, and that the Bax subfamily of Bcl-2-related proteins is essential for JNK-dependent apoptosis. However, the mechanism by which JNK regulates Bax has remained unsolved. Here we demonstrate that activated JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax. Phosphorylation of 14-3-3 led to dissociation of Bax from this protein. Expression of phosphorylation-defective mutants of 14-3-3 blocked JNK-induced Bax translocation to mitochondria, cytochrome c release and apoptosis. Collectively, these results have revealed a key mechanism of Bax regulation in stress-induced apoptosis.     

FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line

Protein phosphorylation in a human glioblastoma cell line, T98G, was examined after exposure to oxidative stress in vitro. Hydrogen peroxide (1 mM) markedly induced tyrosine phosphorylation of focal adhesion kinase (FAK) and serine phosphorylation of Akt at 1 h after stimulation. Concommitantly, the association of FAK with phosphatidylinositide 3'-OH-kinase (PI 3-kinase) was also observed by the hydrogen peroxide stimulation. When T98G cells were incubated with wortmannin, a PI 3-kinase inhibitor, both PI 3-kinase activity and phosphorylation of Akt were inhibited, whereas apoptosis by oxidative stress was accelerated. Concomitant with apoptosis, elevated level of CPP32 protease activity (caspase-3) was observed, with decreases in Bcl-2 protein and increases in Bax protein. These results suggested that in the signal transduction pathway from FAK to PI 3-kinase, Akt promotes survival. Thus, it became apparent that FAK is the upstream signal protein of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis in T98G cells.     

Tumour necrosis factor-related apoptosis-inducing ligand sequentially activates pro-survival and pro-apoptotic pathways in SK-N-MC neuronal cells

The SK-N-MC neuroblastoma cell line, which expresses surface tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors TRAIL-R2 and TRAIL-R4, was used as a model system to examine the effect of TRAIL on key intracellular pathways involved in the control of neuronal cell survival and apoptosis. TRAIL induced distinct short-term (1-60 min) and long-term (3-24 h) effects on the protein kinase B (PKB)/Akt (Akt), extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-kappaB) and caspase pathways. TRAIL rapidly (from 20 min) induced the phosphorylation of Akt and ERK, but not of c-Jun NH2-terminal kinase (JNK). Moreover, TRAIL increased CREB phosphorylation and phospho-CREB DNA binding activity in a phosphatidylinositol 3-kinase (PI 3K)/Akt-dependent manner. At later time points (from 3 to 6 h onwards) TRAIL induced a progressive degradation of inhibitor of kappaB (IkappaB)beta and IkappaBepsilon, but not IkappaBalpha, coupled to the nuclear translocation of NF-kappaB and an increase in its DNA binding activity. In the same time frame, TRAIL started to activate caspase-8 and caspase-3, and to induce apoptosis. Remarkably, caspase-dependent cleavage of NF-kappaB family members as well as of Akt and CREB proteins, but not of ERK, became prominent at 24 h, a time point coincident with the peak of caspase-dependent apoptosis.     

Apoptotic signaling in methylglyoxal-treated human osteoblasts involves oxidative stress, c-Jun N-terminal kinase, caspase-3, and p21-activated kinase 2

Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. MG is cytotoxic through induction of cell death, and elevated MG levels in diabetes patients are believed to contribute to diabetic complications. In this report, we show for the first time that MG treatment triggers apoptosis in human osteoblasts. We further show that MG-induced apoptosis of osteoblasts involves specific apoptotic biochemical changes, including oxidative stress, c-Jun N-terminal kinase (JNK) activation, mitochondrial membrane potential changes, cytochrome C release, increased Bax/Bcl-2 protein ratios, and activation of caspases (caspase-9, caspase-3) and p21-activated protein kinase 2 (PAK2). Treatment of osteoblasts with SP600125, a JNK-specific inhibitor, led to a reduction in MG-induced apoptosis and decreased activation of caspase-3 and PAK2, indicating that JNK activity is upstream of these events. Experiments using anti-sense oligonucleotides against PAK2 further showed that PAK2 activation is required for MG-induced apoptosis in osteoblasts. Interestingly, we also found that MG treatment triggered nuclear translocation of NF-kappaB, although the precise regulatory role of NF-kappaB activation in MG-induced apoptosis remains unclear. Lastly, we examined the effect of MG on osteoblasts in vivo, and found that exposure of rats to dietary water containing 100-200 microM MG caused bone mineral density (BMD) loss. Collectively, these results reveal for the first time that MG treatment triggers apoptosis in osteoblasts via specific apoptotic signaling, and causes BMD loss in vivo.     

Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 microm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca(2+)-activated neutral protease calpain I was readily detectable after the exposure to N-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.     

The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP^C is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.     

The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP^C is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.     

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.     

Growth factors prevent changes in Bcl-2 and Bax expression and neuronal apoptosis induced by nitric oxide

Recent studies have shown that nitric oxide (NO) donors can trigger apoptosis of neurons, and growth factors such as insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) can protect against NO-induced neuronal cell death. The purpose of this study was to elucidate the possible mechanisms of NO-mediated neuronal apoptosis and the neuroprotective action of these growth factors. Both IGF-1 and bFGF prevented apoptosis induced by NO donors, sodium nitroprusside (SNP) or 3-morpholinosydnonimin (SIN-1) in hippocampal neuronal cultures. Incubation of neurons with SNP induced caspase-3-like activation following downregulation of Bcl-2 and upregulation of Bax protein levels in cultured neurons. Treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by SNP. In addition, treatment of neurons with an inhibitor of caspase-3, Ac-DEVD-CHO, together with SNP did not affect the changes in the protein levels, although it inhibited NO-induced cell death. Pretreatment of cultures with either IGF-1 or bFGF prior to NO exposure inhibited caspase-3-like activation together with the changes in Bcl-2 and Bax protein levels. These results suggest that the changes in Bcl-2 and Bax protein levels followed by caspase-3-like activation are a component in the cascade of NO-induced neuronal apoptosis, and that the neuroprotective actions of IGF-1 and bFGF might be due to inhibition of the changes in the protein levels of the Bcl-2 family.