Palmitoyl protein thioesterase 1 protects against apoptosis mediated by Ras-Akt-caspase pathway in neuroblastoma cells

Palmitoyl protein thioesterase (PPT) 1 is an enzyme involved in deacylation of palmitoylated proteins. A deficiency in PPT1 results in a genetic disease, infantile neuronal ceroid lipofuscinosis, associated with massive death of cortical neurons. The role of PPT1 in neuronal survival and apoptosis was studied in human neuroblastoma (LA-N-5) cells overexpressing PPT1. Overexpression of PPT1 was shown both by the 200-350% increase in depalmitoylating activity over basal level (as determined by an in vitro PPT assay) and by western blot analysis of transiently expressed epitope-tagged PPT1. Overexpressed PPT1 showed the same acidic pH optimum (pH 4.0) as the endogenous enzyme, when assayed with a P0-derived octapeptide substrate, and reduced the growth rate by 30%. LA-N-5 cells underwent apoptosis, as evidenced by increased caspase 3-like activity and increased DNA fragmentation, when challenged with either C2-ceramide or a phosphatidylinositol 3-kinase inhibitor (LY294002). Overexpression of PPT1 inhibited this C2-ceramide- or LY294002-mediated activation of caspase-3 by 50%. There was also a concomitant decrease in DNA fragmentation and cell death. Consistent with increased resistance to apoptosis, we found increased phosphorylation of the antiapoptotic protein Akt (protein kinase B) in PPT1-overexpressing cells. p21Ras is known to be dynamically palmitoylated and depalmitoylated and is involved in both growth and cell death. The C2-ceramide-induced membrane association of p21Ras was reduced by 30-50% in PPT1-overexpressing cells compared with control. PPT overexpression also led to reduced membrane association of another palmitoylated protein, GAP-43, a neuron-specific protein. Our studies suggest that protein palmitoylation could be a physiological regulator of apoptosis.     

Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.     

Nitroalkenes induce rat aortic smooth muscle cell apoptosis via activation of caspase-dependent pathways

Nitroalkene derivatives of nitro-linoleic acid (LNO₂) and nitro-oleic acid (OA-NO₂) are nitrated unsaturated fatty acids that can be detected in healthy human plasma, red blood cells and urine. It has been shown that nitroalkenes have potent anti-inflammatory properties in multiple disease models. In the present study, we are the first to investigate the apoptotic effects of nitroalkenes in rat aortic smooth muscle cells (RASMCs). We observed that nitroalkenes induce RASMCs apoptosis in a dose-dependent manner. In addition, nitroalkenes stimulate extrinsic caspase-8 and intrinsic caspase-9 activity to trigger the caspase-3 apoptotic signaling cascade, resulting in RASMCs death. Furthermore, the pro-apoptotic protein, Bad was upregulated and antiapoptotic protein, Bcl-xl was downregulated during nitroalkene-induced apoptosis. These results demonstrate that nitroalkenes can induce RASMCs apoptosis via stimulation of caspase activity and the regulation of apoptotic protein expression levels.     

c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.     

Nitric oxide protects thymocytes from gamma-irradiation-induced apoptosis in correlation with inhibition of p53 upregulation and mitochondrial damage

Apoptosis plays a crucial role in clonal deletion in the thymus, and NO has been shown to prevent apoptosis in some cell types. Therefore, we examined the effect of NO on gamma-irradiation-induced thymocyte apoptosis. Treatment of 5 Gy gamma-irradiated thymocytes with 1 mM SNAP reduced cell death from 78 to 49% after 8 h incubation (spontaneous cell death in medium control cells was 26%). Coincubation with ZVAD blocked both the spontaneous cell death and the cell death induced by SNAP or gamma-irradiation. The gamma-irradiation-induced increase in caspase 3 and 6 activities was inhibited in the presence of SNAP. The increase in cytosolic cytochrome c as well as the decrease in mitochondrial membrane potential after gamma-irradiation was inhibited in the presence of SNAP. SNAP treatment also decreased the p53 upregulation in gamma-irradiated cells. In summary, we found that NO exerts a protective effect on mouse thymocyte apoptosis induced by gamma-irradiation. The mechanism of this protective effect may involve inhibition of p53 upregulation and reduction in mitochondrial damage, with subsequent inhibition of downstream caspase activation.     

PEDF induces apoptosis in human endothelial cells by activating p38 MAP kinase dependent cleavage of multiple caspases

We examined how pigment epithelium derived factor (PEDF), an effective endogenous antiangiogenic protein, decreases survival of primary cultures of human umbilical vein endothelial cells (HUVECs) in a low serum environment supplemented with the endothelial cell growth factor (VEGF). We provide evidence that induction of apoptosis by PEDF is associated with activation of p38 followed by cleavage of caspases 3, 8, and 9 by treatment with PEDF, and PEDF's actions are caspase dependent. A key mediator in the executioner effects of PEDF is p38 since the inhibition of p38 activity blocked apoptosis and prevented cleavage of caspases 3, 8, and 9. Although PEDF-induced phosphorylation of JNK1, the inhibition of JNK1 had no effect on apoptosis, even though it prevented phosphorylation of JNK1 by PEDF. Based on these findings, we propose that the antiangiogenic action of PEDF is dependent on activation of p38 MAPkinase which regulates cleavage of multiple caspases cascades.     

X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway

The X protein from a chronic strain of hepatitis B virus (HBx) was determined to inhibit Fas-mediated apoptosis and promote cell survival. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. Experiments demonstrated that cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This effect was also observed in mouse erythroleukemia cells that lacked p53, indicating that protection against Fas-mediated apoptosis was independent of p53. Components of the signal transduction pathways involved in this protection were studied. The SAPK/JNK pathway has previously been suggested to be a survival pathway for some cells undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK activity was highly up-regulated in cells expressing the HBx protein. Normal mouse fibroblasts expressing HBx were protected from death, whereas identical fibroblasts lacking the SEK1 component from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx was present or not. Assays showed that caspase 3 and 8 activities and the release of cytochrome c from mitochondria were inhibited, in the presence of HBx, following stimulation with anti-Fas antibodies. Coprecipitation and confocal immunofluorescence microscopy experiments demonstrated that HBx localizes with a cytoplasmic complex containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and protection from Fas-mediated apoptosis.     

Arsenic Trioxide Selectively Induces Early and Extensive Apoptosis via the APO2/Caspase-8 Pathway Engaging the Mitochondrial Pathway in Myeloma Cells with Mutant p53

Arsenic trioxide (ATO) is effective in the treatment of acute promyelocytic leukemia (APL) and induces apoptosis in APL cells and in a great variety of other cancer cells. We have previously shown that ATO induces apoptosis in myeloma cells in two different modes depending on p53 status in the cells. In cells expressing mutated p53, ATO induced, G2/M arrest and activation caspase 8 and 3 and rapid and extensive apoptosis. Myeloma cells expressing w.t. p53, ATO induced G1 arrest and delayed apoptosis with activation of caspase 9 and 3. APO2/TRAIL receptor expression was induced in both cell types and APO2/TRAIL synergized with ATO in the induction of apoptosis. Here we tested the effect of ATO on mitochondrial membrane potential (MMP) in myeloma cells expressing mutated or w.t. p53. In myeloma cells expressing mutated p53, depolarization of MMP occurred early, concomitant with induction of APO2/TRAIL, activation of BID and release of AIF, preceding apoptosis. However, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with cytochrome c release and apoptosis. These results explain the greater sensitivity to ATO of cells with mutated p53 and suggest perhaps a general mechanism for ATO-induced apoptosis.     

Arsenic trioxide selectively induces early and extensive apoptosis via the APO2/caspase-8 pathway engaging the mitochondrial pathway in myeloma cells with mutant p53

Arsenic trioxide (ATO) is effective in the treatment of acute promyelocytic leukemia (APL) and induces apoptosis in APL cells and in a great variety of other cancer cells. We have previously shown that ATO induces apoptosis in myeloma cells in two different modes depending on p53 status in the cells. In cells expressing mutated p53, ATO induced, G2/M arrest and activation caspase 8 and 3 and rapid and extensive apoptosis. Myeloma cells expressing w.t. p53, ATO induced G1 arrest and delayed apoptosis with activation of caspase 9 and 3. APO2/TRAIL receptor expression was induced in both cell types and APO2/TRAIL synergized with ATO in the induction of apoptosis. Here we tested the effect of ATO on mitochondrial membrane potential (MMP) in myeloma cells expressing mutated or w.t. p53. In myeloma cells expressing mutated p53, depolarization of MMP occurred early, concomitant with induction of APO2/TRAIL, activation of BID and release of AIF, preceding apoptosis. However, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with cytochrome c release and apoptosis. These results explain the greater sensitivity to ATO of cells with mutated p53 and suggest perhaps a general mechanism for ATO-induced apoptosis.     

Partial cleavage of RasGAP by caspases is required for cell survival in mild stress conditions

Tight control of apoptosis is required for proper development and maintenance of homeostasis in multicellular organisms. Cells can protect themselves from potentially lethal stimuli by expressing antiapoptotic factors, such as inhibitors of apoptosis, FLICE (caspase 8)-inhibitory proteins, and members of the Bcl2 family. Here, we describe a mechanism that allows cells to survive once executioner caspases have been activated. This mechanism relies on the partial cleavage of RasGAP by caspase 3 into an amino-terminal fragment called fragment N. Generation of this fragment leads to the activation of the antiapoptotic Akt kinase, preventing further amplification of caspase activity. Partial cleavage of RasGAP is required for cell survival under stress conditions because cells expressing an uncleavable RasGAP mutant cannot activate Akt, cannot prevent amplification of caspase 3 activity, and eventually undergo apoptosis. Executioner caspases therefore control the extent of their own activation by a feedback regulatory mechanism initiated by the partial cleavage of RasGAP that is crucial for cell survival under adverse conditions.     

BRCA1 185delAG mutation inhibits Akt-dependent, IAP-mediated caspase 3 inactivation in human ovarian surface epithelial cells

BRCA1 mutations have long been associated with altered apoptosis. We have recently reported that caspase 3 activation is increased in human ovarian surface epithelial (OSE) cells expressing a germline N-terminal BRCA1 185delAG mutation. Here, we report increased caspase 3 activity in 185delAG OSE cells associated with decreased expression of cIAP-1 and X-linked inhibitor of apoptosis (XIAP), and decreased ubiquitination of caspase 3. Overexpression of XIAP restored active caspase 3 ubiquitination and lowered levels of caspase 3 activity. Further, the BRCA1 185delAG mutation was associated with reduced levels of phosphorylated Akt1. Transfection with activated Akt1 led to increased cIAP-1 and XIAP levels similar to that seen in BRCA1 185delAG cell lines. Taken together, these data suggest a direct link between the BRCA1 185delAG mutation and alterations in the caspase-mediated apoptotic pathway.     

Modulation of caspase activation and p27(Kip1) degradation in the p53-induced apoptosis in IW32 erythroleukemia cells

To examine the p53-mediated biological activities and signalling pathways, we generated stable transfectants of the p53-null IW32 murine erythroleukemia cells expressing the temperature-sensitive p53 mutant DNA, tsp53(val135). Two clones with different levels of p53 protein expression were selected for further characterization. At permissive temperature, clone 1-5 cells differentiated along the erythroid pathway, and clone 3-2 cells that produced greater levels (3.5-fold) of p53 underwent apoptosis. Apoptosis of 3-2 cells was accompanied by mitochondrial cytochrome c release and caspase activation as well as by cleavage of caspase substrates. Bax protein was induced to a similar extent in these clones by wild-type p53; expression of p21(Cip1/Waf1) and p27(Kip1) proteins was also increased. However, significantly lesser extent of induction for both CDK inhibitors was detected in the apoptotic 3-2 clone. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk) blocked the p53-induced apoptosis in 3-2 cells, with a concomitant elevation of p27(Kip1), suggesting that p27(Kip1) protein underwent caspase-dependent proteolysis in the apoptotic 3-2 cells. Together these results linked a pathway involving cytochrome c release, caspase activation and p27(Kip1) degradation to the p53-induced apoptosis in IW32 erythroleukemia cells.     

Cloning of zebrafish BAD,a BH3-only proapoptotic protein,whose overexpression leads to apoptosis in COS-1 cells and zebrafish embryos

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS(84)AP, corresponding to S(136) in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish alpha-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.     

The mitogen-activated protein kinase cascade promotes myoblast cell survival by stabilizing the cyclin-dependent kinase inhibitor,p21WAF1 protein

During myogenesis, proliferating myoblasts withdraw from the cell cycle and are either eliminated by programmed cell death or differentiate into mature myotubes. Previous studies indicate that mitogen-activated protein kinase (MAPK) activity is significantly induced with the onset of terminal differentiation of C2 myoblasts. We have investigated the part played by the MAPK pathway in the differentiation of C2 myoblasts. Specific activation of MAPK by expression of an active Raf1-estrogen receptor chimera protein reduced significantly the number of myoblasts undergoing programmed cell death in the differentiation medium. Activation of Raf1 prevented the proteolytic activation of the proapoptotic caspase 9-protein during differentiation. The antiapoptotic function of Raf1 correlated with accumulation of the p21WAF1 protein resulting from its increased stability. Antisense expression of p21 was used to determine whether the p21WAF1 protein mediated the antiapoptotic activity of Raf1. Reduction of p21WAF1 protein in muscle cells abolished the antiapoptotic activity of the MAPK pathway. We conclude that MAPK contributes to muscle differentiation by preventing apoptotic cell death of differentiating myoblasts and that this activity is mediated by stabilization of the p21WAF1 protein.     

Antihelminthic benzimidazoles potentiate navitoclax (ABT-263) activity by inducing Noxa-dependent apoptosis in non-small cell lung cancer (NSCLC) cell lines

Background

Evasion of apoptosis is a hallmark of cancer cells. One mechanism to deregulate the apoptotic pathway is by upregulation of the anti-apoptotic Bcl-2 family members. Navitoclax (ABT-263) is a Bcl-2/Bcl-x_L inhibitor that restores the ability of cancer cells to undergo apoptosis.

Methods

In this study we performed a high-throughput screen with 640 FDA-approved drugs to identify potential therapeutic combinations with navitoclax in a non-small cell lung cancer (NSCLC) cell line.

Results

Other than a panel of cancer compounds such as doxorubicin, camptothecin, and docetaxel, four antihelminthic compounds (benzimidazoles) potentiated navitoclax activity. Treatment with benzimidazoles led to induction of the pro-apoptotic protein Noxa at the mRNA and protein level. Noxa binds and antagonizes antiapoptotic protein Mcl-1. siRNA-mediated knock-down of Noxa completely rescued benzimidazole-potentiated navitoclax activity. In addition, inhibiting caspase 3 and 9 partially rescued benzimidazole-potentiated navitoclax activity.

Conclusions

We have identified compounds and mechanisms which potentiate navitoclax activity in lung cancer cell lines. Further validation of the benzimidazole-potentiated navitoclax effect in vivo is required to evaluate the potential for translating this observation into clinical benefit.

Electronic supplementary material

The online version of this article (doi:10.1186/s12935-014-0151-3) contains supplementary material, which is available to authorized users.     

Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

CDK4/6 inhibitor protects chemerin‐induced human granulosa‐lutein cells from apoptosis by inhibiting the p53/p21 waf pathway

Dysregulation of the cell cycle is common in human tumorigenesis. Therefore, CDK4/6 inhibitors targeting the cell cycle have been developed, and their antiapoptotic effects have been highly correlated with potential clinical therapies. The aim of this study was to identify the regulatory effect of the CDK4/6 inhibitor palbociclib on chemerin‐induced apoptosis of immortalized human granulosa‐lutein (hGL) cells and to elucidate its fundamental mechanism of action. Palbociclib enhanced antioxidative enzyme generation and diminished ROS generation in hGL cells. Furthermore, we found that palbociclib suppressed chemerin‐induced apoptotic protein expression, reversing the Bcl‐2/Bax ratio and inhibiting the p53/p21 ^waf pathway. Eventually, palbociclib decreased the level of cleaved caspase‐3 and ‐9, hindering the apoptosis of hGL cells. In general, the antiapoptotic efficacy of palbociclib could be attributed in part to the modulation of the mitochondrial apoptotic pathway in hGL cells.     

Cloning and characterization of a dronc homologue in the silkworm, Bombyx mori

We cloned and characterized a novel Bombyx mori homologue (bm-dronc) of Drosophila melanogaster dronc (dm-dronc), which could encode a polypeptide of 438 amino acid residues. Bm-Dronc shares relatively low amino acid sequence identities of 25% and 26% with Dm-Dronc and Aedes aegypti Dronc (Aa-Dronc), respectively. Bm-Dronc has the sequence QACRG surrounding the catalytic site (C), which is consistent with the QAC(R/Q/G)(G/E) consensus sequence in most caspases but distinct from the sequences PFCRG and SICRG of Dm-Dronc and Aa-Dronc, respectively. Bm-Dronc possesses a long N-terminal prodomain containing a caspase recruitment domain (CARD), a p20 domain and a p10 domain, exhibiting cleavage activities on synthetic substrates Ac-VDVAD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC, which are preferred by human initiator caspases-2, -8 and -9, respectively. Bm-Dronc transiently expressed in insect cells and Escherichia coli cells underwent spontaneous cleavage and caused apoptosis and stimulation of caspase-3-like protease activity in various lepidopteran cell lines, but not in the dipteran cell line D. melanogaster S2. The apoptosis and the stimulation of caspase-3-like protease activity induced by Bm-Dronc overexpression were abrogated upon transfection with either a double-stranded RNA against bm-dronc or a plasmid expressing functional anti-apoptotic protein Hycu-IAP3 encoded by the baculovirus Hyphantria cunea multiple nucleopolyhedrovirus (MNPV). Apoptosis induction in BM-N cells by infection with a p35-defective Autographa californica MNPV or exposure to actinomycin D and UV promoted the cleavage of Bm-Dronc. These results indicate that Bm-Dronc serves as the initiator caspase responsible for the induction of caspase-dependent apoptosis.