Transient pockets on XIAP-BIR2: toward the characterization of putative binding sites of small-molecule XIAP inhibitors

Protein-protein interactions are abundant in signal transduction pathways and thus of crucial importance in the regulation of apoptosis. However, designing small-molecule inhibitors for these potential drug targets is very challenging as such proteins often lack well-defined binding pockets. An example for such an interaction is the binding of the anti-apoptotic BIR2 domain of XIAP to the pro-apoptotic caspase-3 that results in the survival of damaged cells. Although small-molecule inhibitors of this interaction have been identified, their exact binding sites on XIAP are not known as its crystal structures reveal no suitable pockets. Here, we apply our previously developed protocol for identifying transient binding pockets to XIAP-BIR2. Transient pockets were identified in snapshots taken during four different molecular dynamics simulations that started from the caspase-3:BIR2 complex or from the unbound BIR2 structure and used water or methanol as solvent. Clustering of these pockets revealed that surprisingly many pockets opened in the flexible linker region that is involved in caspase-3 binding. We docked three known inhibitors into these transient pockets and so determined five putative binding sites. In addition, by docking two inactive compounds of the same series, we show that this protocol is also able to distinguish between binders and nonbinders which was not possible when docking to the crystal structures. These findings represent a first step toward the understanding of the binding of small-molecule XIAP-BIR2 inhibitors on a molecular level and further highlight the importance of considering protein flexibility when designing small-molecule protein-protein interaction inhibitors.     

Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a mutated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH(2)) showed the highest affinity (Kd =17.92 nM) and a large dynamic range (deltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (Ki values) for inhibitors in the FP-based competitive binding assay conditions, and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain.     

Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis

The flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 with apoptotic inhibitors (CrmA and P35) and addition of caspase peptide substrates (Z-VAD-FMK and Z-IETD-FMK) to NS3-transfected cells blocked NS3-induced apoptosis. In cotransfection experiments, NS3 bound to caspase-8 and enhanced caspase-8-mediated apoptosis. NS3 and caspase-8 colocalized in the cytoplasm of transfected cells. Deletion analysis demonstrated that at least two regions of NS3 contribute to its apoptotic activities. The protease and helicase domains are each able to bind to caspase-8, while the protease domain alone induces apoptosis. The protease domain and tetrahelix region of the helicase domain are required for NS3 to augment caspase-8-mediated apoptosis. Thus, the LGT virus NS3 protein is a multifunctional protein that binds to caspase-8 and induces apoptosis.     

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.     

p38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.     

Synergetic Effects of Caspase 3 and μ-Calpain in XIAP-Breakdown upon Focal Cerebral Ischemia

Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to neurodegenerative disorders. The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of apoptosis. This protein binds to and inhibits both initiator caspases and effector caspases such as caspase-3. The aim of this study was to investigate the relationships between XIAP-breakdown, caspase activation in the development of delayed infarct upon ischemia. We demonstrated that endogenous XIAP is cleaved at least into two fragments during reperfusion following the ischemic insult. The two fragments produced seem to be related to caspase-3 and μ-calpain activities, which are massively enhanced in tissues challenged by ischemia. Therefore, degradation of XIAP by μ-calpain in our system may decrease the activation threshold of caspase-3 normally held in check by the IAPs and/or lead to auto-activation of other caspases. Special issue in honor of Naren Banik.     

Design and characterization of bivalent Smac-based peptides as antagonists of XIAP and development and validation of a fluorescence polarization assay for XIAP containing both BIR2 and BIR3 domains

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.     

Targeting XIAP for the treatment of malignancy

X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins family of caspase inhibitors that selectively binds and inhibits caspases-3, -7 and -9, but not caspase-8. As such, XIAP blocks a substantial portion of the apoptosis pathway and is an attractive target for novel therapeutic agents for the treatment of malignancy. Antisense oligonucleotides directed against XIAP are effective in vitro and are currently being evaluated in clinical trials. Small molecule XIAP inhibitors that target the baculovirus IAP repeat (BIR) 2 or BIR 3 domain are in preclinical development and are advancing toward the clinic. This review will discuss the progress being made in developing antisense and small-molecule XIAP inhibitors.     

Innovations in targeting RNA by fragment-based ligand discovery

A subset of functional regions within large RNAs fold into complex structures able to bind small-molecule ligands with high affinity and specificity. Fragment-based ligand discovery (FBLD) offers notable opportunities for discovery and design of potent small molecules that bind pockets in RNA. Here we share an integrated analysis of recent innovations in FBLD, emphasizing opportunities resulting from fragment elaboration via both linking and growing. Analysis of elaborated fragments emphasizes that high–quality interactions form with complex tertiary structures in RNA. FBLD-inspired small molecules have been shown to modulate RNA functions by competitively inhibiting protein binding and by selectively stabilizing dynamic RNA states. FBLD is creating a foundation to interrogate the relatively unknown structural space for RNA ligands and for discovery of RNA-targeted therapeutics.     

Proteolytic activation of protein kinase C-epsilon by caspase-mediated processing and transduction of antiapoptotic signals

Several novel protein kinase C (PKC) isozymes have been identified as substrates for caspase-3. We have previously shown that novel PKCepsilon is cleaved during apoptosis in MCF-7 cells that lack any functional caspase-3. In the present study, we show that in vitro-translated PKCepsilon is processed by human recombinant caspase-3, -7, and -9. Tumor necrosis factor-alpha (TNF) triggered processing of PKCepsilon to a 43-kDa carboxyl-terminal fragment, and cell-permeable caspase inhibitors prevented TNF-induced processing of PKCepsilon in MCF-7 cells. PKCepsilon was cleaved primarily at the SSPD downward arrow G site to generate two fragments with an approximate molecular mass of 43 kDa. It was also cleaved at the DDVD downward arrow C site to generate two fragments with molecular masses of 52 and 35 kDa. Treatment of MCF-7 cells with TNF resulted in the activation of PKCepsilon, and mutation at the SSPD downward arrow G (D383A) site inhibited proteolytic activation of PKCepsilon. Overexpression of wild-type but not dominant-negative PKCepsilon in MCF-7 cells delayed TNF-induced apoptosis, and mutation at the D383A site prevented antiapoptotic activity of PKCepsilon. These results suggest that cleavage of PKCepsilon by caspase-7 at the SSPD downward arrow G site results in the activation of PKCepsilon. Furthermore, activation of PKCepsilon was associated with its antiapoptotic function.     

Screening for caspase-3 inhibitors: a new class of potent small-molecule inhibitors of caspase-3

From the authors' 650,000 compound collection, they have selected approximately 15,000 potential small-molecule protease inhibitors, which were subjected to high-throughput screening against caspase-3. The screening yielded a series of hits that belong to 11 different scaffolds. Based on the structure of one of the hits, a new class of the small-molecule inhibitors with a double electrophilic warhead, 8-sulfonyl-pyrrolo[3,4-c]quinoline-1,3-diones (SPQ), was synthesized and tested in follow-up mechanistic and anti-apoptosis assays. Mechanistic analysis of a representative compound of this class, CD-001-0011, showed that the compound exhibited a high potency (IC (50)=130 nM), was reversible though noncompetitive, and had a broad selectivity profile to other caspases belonging to groups I to III. The compound was effective in preventing staurosporine induced apoptosis in a few cell lines and retinoic acid-induced apoptosis in zebrafish.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line

We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, down-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.     

Caspase-3 regulates catalytic activity and scaffolding functions of the protein tyrosine phosphatase PEST, a novel modulator of the apoptotic response

The protein tyrosine phosphatase PEST (PTP-PEST) is involved in the regulation of the actin cytoskeleton. Despite the emerging functions attributed to both PTPs and the actin cytoskeleton in apoptosis, the involvement of PTP-PEST in apoptotic cell death remains to be established. Using several cell-based assays, we showed that PTP-PEST participates in the regulation of apoptosis. As apoptosis progressed, a pool of PTP-PEST localized to the edge of retracting lamellipodia. Expression of PTP-PEST also sensitized cells to receptor-mediated apoptosis. Concertedly, specific degradation of PTP-PEST was observed during apoptosis. Pharmacological inhibitors, immunodepletion experiments, and in vitro cleavage assays identified caspase-3 as the primary regulator of PTP-PEST processing during apoptosis. Caspase-3 specifically cleaved PTP-PEST at the (549)DSPD motif and generated fragments, some of which displayed increased catalytic activity. Moreover, caspase-3 regulated PTP-PEST interactions with paxillin, leupaxin, Shc, and PSTPIP. PTP-PEST acted as a scaffolding molecule connecting PSTPIP to additional partners: paxillin, Shc, Csk, and activation of caspase-3 correlated with the modulation of the PTP-PEST adaptor function. In addition, cleavage of PTP-PEST facilitated cellular detachment during apoptosis. Together, our data demonstrate that PTP-PEST actively contributes to the cellular apoptotic response and reveal the importance of caspases as regulators of PTPs in apoptosis.     

Limited proteolysis of filamin is catalyzed by caspase-3 in U937 and Jurkat cells

Members of the caspase family have been implicated as key mediators of apoptosis in mammalian cells. However, few of their substrates are known to have physiological significance in the apoptotic process. We focused our screening for caspase substrates on cytoskeletal proteins. We found that an actin binding protein, filamin, was cleaved from 280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135, 120, and 110 kDa major C-terminal fragments when apoptosis was induced by etoposide in both the human monoblastic leukemia cell line U937, and the human T lymphoblastic cell line Jurkat. The cleavage of filamin was blocked by a cell permeable inhibitor of caspase-3-like protease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ac-YVAD-cho. These results suggest that filamin is cleaved by a caspase-3-like protease. To examine whether caspase-3 cleaves filamin in vitro, we prepared a recombinant active form of caspase-3 directly using a Pichia pastoris overexpression system. When we applied recombinant active caspase-3 to the cell lysate of U937 and Jurkat cells, filamin was cleaved into the same fragments seen in apoptosis-induced cells in vivo. Platelet filamin was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-terminal fragments, and the cleavage pattern was the same as observed in apoptotic human cells in vivo. These results suggest that filamin is an in vivo substrate of caspase-3.     

Requirement of both the second and third BIR domains for the relief of X-linked inhibitor of apoptosis protein (XIAP)-mediated caspase inhibition by Smac

The inhibitor of apoptosis proteins (IAP) are endogenous caspase inhibitors in the metazoan and characterized by the presence of baculoviral IAP repeats (BIR). X-linked IAP (XIAP) contains three BIR domains and directly inhibits effector caspases such as caspase-7 via a linker_BIR2 fragment and initiator caspases such as caspase-9 via the BIR3 domain. A mitochondrial protein Smac/DIABLO, which is released during apoptosis, antagonizes XIAP-mediated caspase inhibition by interacting directly with XIAP. Here, using glutathione S-transferase pulldown and caspase activity assay, we show that Smac is ineffective in relieving either caspase-7 or caspase-9 inhibition by XIAP domain fragments. In addition, Smac forms a ternary complex with caspase-7 and linker_BIR2, suggesting that Smac/linker_BIR2 interaction does not sterically exclude linker_BIR2/caspase-7 interaction. However, Smac is effective in removing caspase-7 and caspase-9 inhibition by XIAP fragments containing both the BIR2 and BIR3 domains. Surface plasmon resonance measurements show that Smac interacts with the BIR2 or BIR3 domain in micromolar dissociation constants. On the other hand, Smac interacts with an XIAP construct containing both BIR2 and BIR3 domains in a subnanomolar dissociation constant by the simultaneous interaction of the Smac dimer with the BIR2 and BIR3 domains of a single XIAP molecule. This 2:1 Smac/XIAP interaction not only possesses enhanced affinity but also sterically excludes XIAP/caspase-7 interaction, demonstrating the requirement of both BIR2 and BIR3 domains for Smac to relieve XIAP-mediated caspase inhibition.     

Discovery and development of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives as Bcl-2/Mcl-1 inhibitors

Bcl-2 family proteins play a vital role for cancer cell in escaping apoptosis, and small-molecule anti-apoptotic Bcl-2 protein inhibitors have been developed as new anticancer therapies. In current study, a series of substituted 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were developed based on the lead compound 1 (K_i = 5.2 µM against Bcl-2 protein). The fluorescence polarization assays suggested that active compounds possessed potent binding affinities to both Bcl-2 and Mcl-1 protein, but had minor or no binding affinities to Bcl-X_L protein. MTT assays showed that these compounds had certain anti-proliferative activities against cancer cells. Furthermore, it was found that active compound 11t could induce cell apoptosis and caspase-3 activation in a dose-dependent manner in Jurkat cells.     

Altered Processing of Amyloid Precursor Protein in Cells Undergoing Apoptosis

Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer''s disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25–35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer''s disease.     

Degradation of focal adhesion proteins paxillin and p130cas by caspases or calpains in apoptotic rat-1 and L929 cells

Immunofluorescence microscopy revealed the rearrangement and gradual dissociation of paxillin from focal adhesion sites during apoptosis. In vitro, cleavage of paxillin by caspase-3 generated a 42-kDa fragment, among other products, while cleavage by calpain generated a different set of fragments. In Rat-1 cells, cleavage of paxillin by caspase-3 was suppressed by zVAD-fmk or zDEVD-cmk, making caspase-3 a likely executioner during etoposide-induced apoptosis. In contrast, the cleavage of paxillin and p130cas in apoptotic L929 cells was blocked by calpain-specific inhibitors, which also reduced the death rate by 23 to 44%. Therefore, The disassembly and degradation of p130cas and paxillin during apoptosis may controlled by both caspases and calpains, depending upon their cellular contexts. Our findings also suggest that focal adhesion proteins paxillin and p130cas take part in integrin-mediated signaling for cell survival, and that their cleavage by caspase and/or calpain may not only disrupt focal adhesion complexes, but may also impede cell survival signaling.     

Caspase-3：治疗神经退行性疾病的新靶点

Caspase-3是caspases家族（一类天冬氨酸特异性酶切半胱氨酸蛋白酶）中的成员,是哺乳动物细胞凋亡的关键蛋白酶.随着研究的深入,发现caspase-3在神经退行性疾病的病理过程中起着很重要的角色.Caspase-3在这些疾病的病理过程中,不仅仅是起着凋亡的效应器作用,还能直接与老年性痴呆症、帕金森氏症、亨廷顿舞蹈病、脊椎小脑失调等疾病的致病蛋白质分子相互作用,参与致病机制.因此,caspase-3是治疗神经退行性疾病的新靶点,寻找caspase-3高效高选择性的抑制剂将为治疗神经退行性疾病提供新的途径.     

Caspase-dependent Apoptosis Induced by Thapsigargin was Prevented by Glycogen Synthase Kinase-3 Inhibitors in Cultured Rat Cortical Neurons

Calcium ion is essential for cellular functions including signal transduction. Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases. Thapsigargin, which inhibits Ca²⁺-ATPase in the endoplasmic reticulum (ER) and blocks the sequestration of calcium by the ER, induced apoptotic cell death (chromatin condensation and nuclear fragmentation) accompanied by GRP78 protein expression and caspase-3 activation in rat fetal cortical neurons (days in vitro 9–10). Blockade of N-methyl-d-aspartate (NMDA) receptors with NMDA antagonists induced apoptosis without GRP78 protein expression. Apoptosis accompanied both caspase-9 and caspase-3 activation. We then examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors. We assayed the effects of selective GSK-3 inhibitors, SB216763, alsterpaullone and 1-azakenpaullone, on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. In addition, GSK-3 inhibitors inhibited caspase-9 and caspase-3 activation accompanied by thapsigargin-induced apoptosis. These results suggest that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3β activation in rat cortical neurons.