Neuronal apoptosis inhibitory protein,NAIP, is an inhibitor of procaspase-9

Ability of the full length NAIP and its BIR3 domain in inhibition of the proteases of the intrinsic apoptosis pathway was investigated. Activity of endogenous executioner caspases was drastically reduced by both recombinant NAIP-BIR3 (NBIR3) and the full length protein. Western blotting experiments showed that the full length NAIP and its BIR3 domain inhibited the cleavage of procaspase-3 by apoptosome activated caspase-9. Moreover, full length NAIP inhibited autocatalytic processing of procaspase-9 in the apoptosome complex indicating that unlike other inhibitor of apoptosis proteins (IAPs) human NAIP is an inhibitor of procaspase-9. Furthermore, inhibition of single-chain caspase-9 (human caspase-9, D315, D330/A point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9) by the BIR3 domain indicated that the this domain is the caspase-9 interacting moiety. Consistently, pull-down experiments of single-chain capsase-9 in apoptosome complex by the NBIR3 but not the X-linked inhibitor of apoptosis protein (XIAP)-BIR3 domain confirmed that the protein can associate with procaspase-9 prior to its autoproteolysis upon apoptosome formation. Interaction studies revealed the association of C338W variant of the NBIR3, but not the wild type protein with both SMAC-peptide and the SMAC protein. These data indicate that mutation of C338 to Trp is sufficient to accommodate the interaction of NAIP-BIR3 with SMAC-peptide and protein. Taken together, these results demonstrate that NAIP is evolved to prevent apoptosis right at the initiation stage of apoptosome formation and this inhibition cannot be antagonized by SMAC-type proteins.     

Genetic alterations in caspase-10 may be causative or protective in autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by lymphadenopathy, elevated numbers of T cells with αβ-T cell receptors but neither CD4 nor CD8 co-receptors, and impaired lymphocyte apoptosis in vitro. Defects in the Fas receptor are the most common cause of ALPS (ALPS Ia), but in rare cases other apoptosis proteins have been implicated, including caspase-10 (ALPS II). We investigated the role of variants of caspase-10 in ALPS. Of 32 unrelated probands with ALPS who did not have Fas defects, two were heterozygous for the caspase-10 missense mutation I406L. Like the previously reported ALPS II-associated mutation L285F, I406L impaired apoptosis when transfected alone and dominantly inhibited apoptosis mediated by wild type caspase-10 in a co-transfection assay. Other variants in caspase-10, V410I and Y446C, were found in 3.4 and 1.6% of chromosomes in Caucasians, and in 0.5 and <0.5% of African Americans, respectively. In contrast to L285F and I406L, these variants had no dominant negative effect in co-transfection assays into the H9 lymphocytic cell line. We found healthy individuals homozygous for V410I, challenging the earlier suggestion that homozygosity for V410I alone causes ALPS. Moreover, an association analysis suggested protection from severe disease by caspase-10 V410I in 63 families with ALPS Ia due to dominant Fas mutations (P<0.05). Thus, different genetic variations in caspase-10 can produce contrasting phenotypic effects.     

Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation

The evidence implicating a mode of cell death that either favors or argues against caspase-dependent apoptosis is available in studies that used experimental models of Parkinson’s disease. We sought to investigate the mechanisms by which release of cytochrome c is not linked to caspase activation during rotenone-induced dopaminergic (DA) neurodegeneration. Unlike caspase activation in 6-hydroxydopamine-treated cells, both MN9D DA neuronal cells and primary cultures of mesencephalic neurons showed no obvious signs of caspase activation upon exposure to rotenone. We found that intracellular levels of ATP significantly decreased at the early phase of neurodegeneration (<~24 h) and therefore external addition of ATP to the lysates obtained at this stage reconstituted caspase-3 activity. At a later phase of cell death (>~24 h), both decreased levels of ATP and procaspase-9 contributed to the lack of caspase-3 activation. Under this condition, calpain and the proteasome system were responsible for the degradation of procaspase-9. Consequently, external addition of ATP and procaspase-9 to the lysates harvested at the later phase was required for activation of caspase-3. Similarly, caspase-3 activity was also reconstituted in the lysates harvested from cells co-treated with inhibitors of these proteases and incubated in the presence of external ATP. Taken together, our findings provided a sequential mechanism underlying how DA neurons may undergo caspase-independent cell death, even in the presence of cytoplasmic cytochrome c following inhibition of mitochondrial complex I.     

Rapid cold-hardening protects <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> from cold-induced apoptosis

The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to ‒7°C for 2 h, whereas significantly more flies in the RCH group survived exposure to ‒7°C for 2 h after a 2-h exposure to 5°C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5°C, 30 min), and frozen (‒20°C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of ∼150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.     

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.     

Hyperbaric oxygen induces apoptosis via a mitochondrial mechanism

During therapeutic hyperbaric oxygenation lymphocytes are exposed to high partial pressures of oxygen. This study aimed to analyze the mechanism of apoptosis induction by hyperbaric oxygen. For intervals of 0.5–4 h Jurkat-T-cells were exposed to ambient air or oxygen atmospheres at 1–3 absolute atmospheres. Apoptosis was analyzed by phosphatidylserine externalization, caspase-3 activation and DNA-fragmentation using flow cytometry. Apoptosis was already induced after 30 min of hyperbaric oxygenation (HBO, P < 0.05). The death receptor Fas was downregulated. Inhibition of caspase-9 but not caspase-8 blocked apoptosis induction by HBO. Hyperbaric oxygen caused a loss of mitochondrial membrane potential and caspase-9 induction. The mitochondrial pro-survival protein Bcl-2 was upregulated, and antagonizing Bcl-2 function potentiated apoptosis induction by HBO. In conclusion, a single exposure to hyperbaric oxygenation induces lymphocyte apoptosis by a mitochondrial and not a Fas-related mechanism. Regulation of Fas and Bcl-2 may be regarded as protective measures of the cell in response to hyperbaric oxygen.     

Overexpression of 4EBP1, p70S6K,Akt1 or Akt2 differentially promotes Coxsackievirus B3-induced apoptosis in HeLa cells

Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24 h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6 h p. i. but lower at 24 h p. i., and the expression of Bax protein were higher at 6 and 24 h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis.     

Combination of all-<Emphasis Type="Italic">trans</Emphasis> retinoic acid and taxol regressed glioblastoma T98G xenografts in nude mice

Glioblastoma is the most prevalent and highly malignant brain tumor that continues to defy current treatment strategies. This investigation used all-trans retinoic acid (ATRA) and taxol (TXL) as a combination therapy for controlling the growth of human glioblastoma T98G xenografted in athymic nude mice. Histopathological examination revealed that ATRA induced differentiation and combination of ATRA and TXL caused more apoptosis than either treatment alone. Combination therapy decreased expression of telomerase, nuclear factor kappa B (NFκВ), and inhibitor-of-apoptosis proteins (IAPs) indicating suppression of survival factors while upregulated Smac/Diablo. Combination therapy also changed expression of Bax and Bcl-2 proteins leading to increased Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9. Increased activities of calpain and caspase-3 degraded 270 kD α-spectrin at the specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. Further, increased activity of caspase-3 cleaved inhibitor-of-caspase-activated DNase (ICAD). In situ double immunofluorescent labelings showed overexpression of calpain, caspase-12, caspase-3, and AIF during apoptosis, suggesting involvement of both caspase-dependent and caspase-independent pathways for apoptosis. Our investigation revealed that treatment of glioblastoma T98G xenografts with the combination of ATRA and TXL induced differentiation and multiple molecular mechanisms for apoptosis.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Vaccinia Virus Protein F1L Is a Caspase-9 Inhibitor

Apoptosis plays important roles in host defense, including the elimination of virus-infected cells. The executioners of apoptosis are caspase family proteases. We report that vaccinia virus-encoded F1L protein, previously recognized as anti-apoptotic viral Bcl-2 family protein, is a caspase-9 inhibitor. F1L binds to and specifically inhibits caspase-9, the apical protease in the mitochondrial cell death pathway while failing to inhibit other caspases. In cells, F1L inhibits apoptosis and proteolytic processing of caspases induced by overexpression of caspase-9 but not caspase-8. An N-terminal region of F1L preceding the Bcl-2-like fold accounts for caspase-9 inhibition and significantly contributes to the anti-apoptotic activity of F1L. Viral F1L thus provides the first example of caspase inhibition by a Bcl-2 family member; it functions both as a suppressor of proapoptotic Bcl-2 family proteins and as an inhibitor of caspase-9, thereby neutralizing two sequential steps in the mitochondrial cell death pathway.     

Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells

Objective To investigate the pro-apoptotic effect of tissue factor pathway inhibitor (TFPI) gene transfer mediated by adenovirus on vascular smooth muscle cells (VSMCs). Methods Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. Apoptosis of VSMCs was determined by electron microscopy and flow cytometry. The expression of cytochrome c, procaspase-3, cleaved caspase-3, cleaved caspase-9 and inhibitor of apoptosis protein-1(IAP-1) were examined by western blot and RT-PCR. Results TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th day after gene transfer, the apoptotic rates in the TFPI group were 11.95%, 71.96% and 37.83%, respectively, whereas those in the LacZ group were 1.34%, 1.83% and 6.37%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. Cytochrome c, cleaved caspase-3 and cleaved caspase-9, which are all involved in mitochondrial pathway, were detected in the cytoplasm on the 3rd, 5th and 7th day after TFPI gene transfer. Procaspase-3 expression was significantly decreased over time in the TFPI group (each P < 0.05), which were not seen in the Ad-LacZ and DMEM groups. The expression of IAP-1 mRNA in the TFPI group was also decreased compared with the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd and 7th day after gene transfer. Conclusion The results demonstrated that overexpression of TFPI gene might induce VSMC apoptosis in vitro through the mitochondrial pathway; meanwhile, IAP-1 expression is decreased. These findings indicated that TFPI might inhibit restenosis by inducing apoptosis in VSMCs.     

Wnt inhibitors enhance chondrogenesis of human mesenchymal stem cells in a long-term pellet culture

The long-term effects (~3 weeks) of two Wnt inhibitors (dickkopf [DKK]-1 and secreted frizzled-related protein [sFRP]-1), on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was determined. Wnt inhibitors significantly increased the amount of glycosaminoglycan (GAG) in treated pellets (P < 0.05). The gene expression of COL2A1 increased and COL1A1 decreased while the gene expression of SOX-9 and COL10A1 did not change significantly after three weeks of in vitro culture. The protein expression of type II collagen significantly increased (P < 0.05) and that of type I collagen significantly decreased (P < 0.05) while SOX-9 and type X collagen protein expression was unaffected. These findings suggest that Wnt inhibitors promote the chondrogenic differentiation of hMSCs when treated for three weeks.     

Arsenic induced apoptosis in malignant melanoma cells is enhanced by menadione through ROS generation, p38 signaling and p53 activation

Introduction  Resistance to apoptosis is a prominent feature of melanoma. Pharmacological concentration of arsenic in combination with a widely known oxidant, menadione was explored in this study to synergistically sensitize malignant melanoma cells to apoptosis. The molecular mechanism of apoptosis and the signaling-pathways involved were thoroughly investigated. Materials methods and results  Menadione synergized NaAsO₂ to significantly increase ROS generation and facilitate the major apoptotic signaling events: alteration of mitochondrial membrane potential, cytochrome c release and anti-apoptotic protein Bcl-2 down-regulation and subsequent activation of caspase-9 and caspase-3 followed by poly-ADP-ribose polymerase-1 cleavage. Antioxidant N-acetyl-l-cysteine antagonized these events. Investigation of the signaling-pathway revealed significant suppression of AP-1 activity but not NF-κB upon NaAsO₂ and menadione application. An increase in p38 phosphorylation and p53 protein expression did also dictate the apoptotic response. Suppression of p38 activation with SB203580 and inhibition of p53 expression by siRNA attenuated apoptosis. Transfection of p53, in p53 null HCT cells augmented the apoptotic events. Moreover, the treatment also led to tumor size reduction in BALB/c mice developed by intra-dermal B16 mouse melanoma cell injection; however, it had no detectable pro-proliferative or pro-apoptotic effect on non-tumor keratinocytes, normal fibroblasts or PBMC. Conclusion  This study thus provides an insight into innovative mechanisms of melanoma sensitization, a proper cure against which is still elusive. Taken together, our data also provides the first evidence of arsenic activity accentuation by menadione through modulation of specific signaling-pathways.     

A novel indoloquinoline derivative, IQDMA, induces S-phase arrest and apoptosis in promyelocytic leukemia HL-60 cells

N′-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine (IQDMA), an indoloquinoline compound, was identified in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells. Cell cycle analysis showed S-phase arrest and induction of apoptosis in HL-60 cells following 24 h exposure to IQDMA. Analysis of the cell cycle regulatory proteins demonstrated that IQDMA did not change the steady-state levels of cyclin B1, cyclin D3, and p21, but decreased the protein levels of Cdk1, Cdk2, and cyclin A. IQDMA also caused a marked increase in apoptosis, which was accompanied by increased levels of Bax, activated caspase-3, -8, and -9, and cleaved PARP. These molecular alterations provide an insight into IQDMA-caused growth inhibition, S-phase arrest, and apoptotic death of HL-60 cells.     

Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.     

Mitochondria-targeted antioxidant SkQR1 reduces TNF-induced endothelial permeability <Emphasis Type="Italic">in vitro</Emphasis>

Prolonged or excessive increase in the circulatory level of proinflammatory tumor necrosis factor (TNF) leads to abnormal activation and subsequent damage to endothelium. TNF at high concentrations causes apoptosis of endothelial cells. Previously, using mitochondria-targeted antioxidants of SkQ family, we have shown that apoptosis of endothelial cells is dependent on the production of reactive oxygen species (ROS) in mitochondria (mito-ROS). Now we have found that TNF at low concentrations does not cause cell death but activates caspase-3 and caspase-dependent increase in endothelial permeability in vitro. This effect is probably due to the cleavage of β-catenin–an adherent junction protein localized in the cytoplasm. We have also shown that extracellular matrix metalloprotease 9 (MMP9) VE-cadherin shedding plays a major role in the TNF-induced endothelial permeability. The mechanisms of the caspase-3 and MMP9 activation are probably not related to each other since caspase inhibition did not affect VE-cadherin cleavage and MMP9 inhibition had no effect on the caspase-3 activation. Mitochondria-targeted antioxidant SkQR1 inhibited TNF-induced increase in endothelial permeability. SkQR1 also inhibited caspase-3 activation, β-catenin cleavage, and MMP9-dependent VE-cadherin shedding. The data suggest that mito-ROS are involved in the increase in endothelial permeability due to the activation of both caspase-dependent cleavage of intracellular proteins and of MMP9-dependent cleavage of the transmembrane cell-to-cell contact proteins.     

Mechanism of Activation of Caspase-9 and Caspase-3 during Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca<Superscript>2+</Superscript>-Influx

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, the executioner of programmed cell death. We have also shown that cerebral hypoxia results in high affinity Ca²⁺–ATPase-dependent increase in nuclear Ca²⁺-influx in the cerebral cortex of newborn piglets. The present study tests the hypothesis that inhibiting nuclear Ca²⁺-influx by pretreatment with clonidine, an inhibitor of high affinity Ca²⁺–ATPase, will prevent the hypoxia-induced increase in caspase-9 and caspase-3 activity in the cerebral cortex of newborn piglets. Thirteen newborn piglets were divided into three groups, normoxic (Nx, n = 4), hypoxic (Hx, n = 4), and hypoxic treated with clonidine (100 mg/kg) (Hx–Cl, n = 5). Anesthetized, ventilated animals were exposed to an FiO₂ of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Caspase-9 and -3 activity were determined spectrofluoro-metrically using specific fluorogenic synthetic substrates. ATP (μmoles/g brain) was 4.6 ± 0.3 in Nx, 1.7±0.4 in Hx (P < 0.05 vs. Nx), and 1.5 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). PCr (μmoles/g brain) was 3.6 ± 0.4 in Nx, 1.1 ± 0.3 in Hx (P < 0.05 vs. Nx), and 1.0 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). Caspase-9 activity (nmoles/mg protein/h) was 0.548 ± 0.0642 in Nx and increased to 0.808 ± 0.080 (P < 0.05 vs. Nx and Hx–Cl) in the Hx and 0.562 ± 0.050 in the Hx–Cl group (p = NS vs. Nx). Caspase-3 activity (nmoles/mg protein/h) was 22.0 ± 1.3 in Nx and 32 ± 6.3 in Hx (P < 0.05 vs. Nx) and 18.8 ± 3.2 in the Hx–Cl group (P < 0.05 vs. Hx). The data demonstrate that clonidine administration prior to hypoxia prevents the hypoxia-induced increase in the activity of caspase-9 and caspase-3. We conclude that the high afinity Ca²⁺–ATPase-dependent increased nuclear Ca²⁺ during hypoxia results in increased caspase-9 and caspase-3 activity.     

Roles of Bcl-2 family members,PI3K and NF-κB pathways in <Emphasis Type="Italic">Escherichia coli</Emphasis>-induced apoptosis in human monocytic U937 cells

We previously showed that infection of human monocytic U937 cells with nonpathogenic Escherichia coli (E. coli) induced rapid apoptosis in a dose- and time-dependent manner. We also found that E. coli increase p38 mitogen-activated protein Kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), and decrease extracellular-Regulated Kinase1/2 (ERK1/2) phosphorylation and increase caspase-3 and -9 activity in U937 cells. The current study determines if Bcl-2, Bax, the phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor kappa B (NF-κB) regulates E. coli–induced U937 cell apoptosis. Studying the underlying mechanisms we found that the E. coli-induced apoptosis in U937 cells was associated with a more prominent reduction in expression of Bcl-2, levels of P-Akt and NF-κB. Because levels of inhibition of apoptosis protein (cIAP), and X-chromosomelinked inhibitor of apoptosis protein (XIAP) are regulated by NF-κB, E. coli decreased the levels of these proteins in U937 cells through inhibition of NF-κB. Moreover, E. coli markedly elevated Bax expression and cytochrome c redistribution. LY294002, PDTC and Embelin, specific inhibitors of PI3K, NF-κB and XIAP, induced U937 cell apoptosis and the apoptosis is dependent on activity of caspase-3 and -9 in E. coli-treated U937 cells. Through using LY294002 and western blotting, we identified NF-κB was the downstream Akt target regulated by E. coli. Taken together, these results clearly indicate reduced activation of NF-κB via impaired PI3K/Akt activation could result in increased apoptosis of U937 cells infected by E. coli. Moreover, E. coli can induce apoptosis with an increased expression of Bax and a reduced expression of Bcl-2, which resulted in increased levels of cytochrome c release and increase caspase-3 and -9 in U937 cells.     

Role of microRNA-195 in cardiomyocyte apoptosis induced by myocardial ischaemia–reperfusion injury

This study aims to investigate microRNA-195 (miR-195) expression in myocardial ischaemia–reperfusion (I/R) injury and the roles of miR-195 in cardiomyocyte apoptosis though targeting Bcl-2. A mouse model of I/R injury was established. MiR-195 expression levels were detected by real-time quantitative PCR (qPCR), and the cardiomyocyte apoptosis was detected by TUNEL assay. After cardiomyocytes isolated from neonatal rats and transfected with miR-195 mimic or inhibitor, the hypoxia/reoxygenation (H/R) injury model was established. Cardiomyocyte apoptosis and mitochondrial membrane potential were evaluated using flow cytometry. Bcl-2 and Bax mRNA expressions were detected by RT-PCR. Bcl-2, Bax and cytochrome c (Cyt-c) protein levels were determined by Western blot. Caspase-3 and caspase-9 activities were assessed by luciferase assay. Compared with the sham group, miR-195 expression levels and rate of cardiomyocyte apoptosis increased significantly in I/R group (both P<0.05). Compared to H/R + negative control (NC) group, rate of cardiomyocyte apoptosis increased in H/R + miR-195 mimic group while decreased in H/R + miR-195 inhibitor group (both P<0.05). MiR-195 knockdown alleviated the loss of mitochondrial membrane potential (P<0.05). MiR-195 overexpression decreased Bcl-2 mRNA and protein expression, increased BaxmRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). While, downregulated MiR-195 increased Bcl-2 mRNA and protein expression, decreased Bax mRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). Our study identified that miR-195 expression was upregulated in myocardial I/R injury, and miR-195 overexpression may promote cardiomyocyte apoptosis by targeting Bcl-2 and inducing mitochondrial apoptotic pathway.