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PubMed2002

Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways.

The chemotherapeutic cisplatin causes renal dysfunction and renal proximal tubular cell (RPTC) apoptosis. The goal of these studies was to examine the role of p53, caspase 3, 8, and 9, and mitochondria in the signaling of cisplatin-induced apoptosis. Cisplatin (50 microM) produced time-dependent apoptosis in RPTCs, causing cell shrinkage, a 50-fold increase in caspase 3 activity, a 4-fold increase in phosphatidylserine externalization, and 5- and 15-fold increases in chromatin condensation and DNA hypoploidy, respectively. Mitochondrial membrane potential and ATP levels did not change at any time during cisplatin exposure. Caspase 8 and 9 activities also did not increase during treatment. Cisplatin increased nuclear p53 expression 4 h after treatment, preceding both caspase 3 activation and chromatin condensation. Treatment with the p53 inhibitor alpha-2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone (PFT) before cisplatin exposure inhibited p53 nuclear expression at 4, 8, and 12 h and inhibited phosphatidylserine externalization and caspase 3 activation at 12 h. Neither DEVD-fmk nor ZVAD-fmk inhibited cisplatin-induced p53 nuclear expression. Both DEVD-fmk and ZVAD-fmk completely inhibited caspase 3 activity but, like PFT, partially inhibited cisplatin-induced chromatin condensation, annexin V labeling, and DNA hypoploidy after 24 h. These data demonstrate that at least 50% of cisplatin-induced apoptosis in RPTC is mediated by p53 and that p53 activates caspase 3 independently of either caspase 9 or 8 or mitochondrial dysfunction. Furthermore, 50% of cisplatin-induced RPTC apoptosis is independent of p53 and caspases 3, 8, and 9.

Adenosine Triphosphate↗
PubMedDate not supplied

Rocaglaol induces apoptosis and cell cycle arrest in LNCaP cells.

Rocaglaol is a cytotoxic cyclopenta[b]benzofuran isolated from the bark of Aglaia crassinervia. It exhibited in vitro cytotoxic activity against Lu1, LNCaP and MCF-7 cells with ED50 values of 13.8, 23.0 and 9.2 nM, respectively. DAPI staining indicated that LNCaP cells treated with rocaglaol underwent apoptosis. In order to determine whether rocaglaol-induced apoptosis is mediated by the mitochondrial pathway, apoptosis-related mitochondrial-associated proteins were studied. Rocaglaol treatment induced Bax expression through 12 to 72 h of exposure, while Bcl-xl expression was slightly decreased through 48 h, and decreased more significantly by 72 h. Cleaved caspase-9 expression was detected at 72 h, and cleaved caspase-7 was increased through 48 to 72 h. Consequently, the large fragment (89 kDa) of PARP resulting from caspase cleavage was detected at 12, 24 and 48 h, and especially at 72 h. Cleaved PARP expression was also detected at 72 h. Since rocaglaol caused dose-dependent G2/M phase arrest of LNCaP cells as indicated by flow cytometric analysis, the protein levels of cell cycle-related genes were measured. Rocaglaol treatment (230 nM) did not change cyclin B after 24- to 60-h treatment. The expression of cdc2 was not changed and phospho-cdc2 (Tyr 15) increased after 36-, 48- and 60-h treatment. In addition, protein phosphatase Cdc25C, which functions as a mitotic activator by dephosphorylation of Cdc2, decreased in a time-dependent manner after rocaglaol treatment. Taken together, these results suggest that rocaglaol is a potent anticancer drug that induces apoptosis of LNCaP cells through the mitochondrial pathway and its G2/M-phase cell cycle arrest is associated with the down-regulation of Cdc25C and the dephosphorylation of Cdc2.

Actins↗
PubMed2005

Biphasic changes in the levels of poly(ADP-ribose) polymerase-1 and caspase 3 in the immature brain following hypoxia-ischemia.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair-associated enzyme that has multiple roles in cell death. This study examined the involvement of PARP-1 in ischemic brain injury in the 7-day old rat, 0.5-48 h after unilateral carotid artery ligation and 2 h of 7.8% oxygen. This experimental paradigm produced a mild to moderate injury; 40-67% of animals in the ligated groups had histological evidence of neuronal death. Ipsilateral cortical injury was seen at all survival times, while mild contralateral cortical injury was seen only at the 1h survival time. Hippocampal injury was delayed relative to the cortex and did not show a biphasic pattern. Immunohistochemical staining for PARP showed bilateral increased staining as early as 1 h post-hypoxia. PARP staining at early time periods was most intense in layer V of cortex, but did not demonstrate a pattern of cell clusters or columns. Ipsilateral PARP-1 levels quantified by western blotting showed a biphasic pattern of elevation with peaks at 0.5 and 12 h post-hypoxia. Contralateral PARP-1 levels were also elevated at 0.5 and 24 h. PARP activity as determined by immunoreactivity for poly(ADP-ribose) (PAR) was increased ipsilaterally at 0.5, 2 and 12 h survival times. Cortical caspase 3-activity was increased ipsilaterally at 6, 12, and 24 h and contralaterally at 0.5, 1, 2 and 6 h post-hypoxia. There are three main findings in this study. First, changes in the distribution and amount of cell death correlate well with measured PARP-1 levels after hypoxia-ischemia, and both display biphasic characteristics. Second, there are significant early, transient morphological and biochemical changes in the contralateral cortex after neonatal hypoxia-ischemia due to unilateral permanent occlusion of a carotid artery followed by 2 h of systemic hypoxia. Third, variability in the responses of individual pups to hypoxia-ischemia suggests the presence of unidentified confounding factors.

Animals↗
PubMed2003

Time-course induction of apoptosis by wild-type and attenuated strains of rubella virus.

The time-course of rubella virus (RV)-induced apoptosis was studied in RK13 cells. DEVD-specific caspase activity assay and Western blotting for caspase-3 were used to determine the time-course of caspase activation and demonstrated that RV-induced apoptotic changes occur as early as 12 h post-infection (p.i.). Caspase activity followed a cyclic pattern, as seen with apoptotic-inducing drugs, with maximum activity detected at 72 h p.i. Apoptosis caused by wild-type (RN) and attenuated vaccine (Cendehill) strains of RV was compared by TUNEL staining, counting dead floating cells and DNA fragmentation analysis. Although the amount of apoptosis due to the wild-type strain was marginally greater, this was probably due to its faster growth rate.

Apoptosis↗
PubMed2004

Apoptosis by 6-O-palmitoyl-L-ascorbic acid coincides with JNK-phosphorylation and inhibition of Mg2+-dependent phosphatase activity.

6-O-Palmitoyl ascorbic acid (PAA) has recently been used as a substitute for ascorbic acid because of its greater potency as an antioxidant. In detailed concentration response studies distinct cytotoxic effects of PAA at concentrations exceeding 100 microM were reported. Here we examined and further characterized this cytotoxicity. While ascorbic acid was tolerated well up to millimolar concentrations, PAA revealed an LC50 between 125 and 150 microM in rat GH3 tumor cells. Morphological and biochemical observations suggested the induction of apoptosis at concentrations exceeding 125 microM with a prominent activation of caspase 3 at 250 microM after 4 hr. A subsequent pronounced fragmentation of DNA (DNA-ladder) was detected after 6 hr and was further enhanced after 12 hr. The activation of caspases and the cleavage of its substrate PARP was preceded by a distinct increase in the phosphorylation of stress activated JNK-kinases. This observation suggested that the agent affected signal transduction mechanisms regulating protein phosphorylation at serine/threonine residues in the cell. No effect of PAA on protein phosphatase 2A (PP2A)-like activity was observed while magnesium-dependent protein phosphatase activity, presumably PP2C, was inhibited concentration-dependently up to 75% at the respective concentrations. Thus, the cytotoxic, pro-apoptotic effect of PAA might be related to the inhibition of PP2C and the activation of JNK.

Animals↗
PubMed1998

Caspase activation in MCF7 cells responding to etoposide treatment.

Studies of the biochemical mechanisms evoked by conventional treatments for neoplastic diseases point to apoptosis as a key process for elimination of unwanted cells. Although the pathways through which chemotherapeutics promote cell death remain largely unknown, caspase proteases play a central role in the induction of apoptosis in response to a variety of stimuli including tumor necrosis factor, fas ligand, and growth factor deprivation. In this article, we demonstrate the induction of caspase protease activity in MCF7 human breast carcinoma cells exposed to the topoisomerase inhibitor, etoposide. Caspase protease activity was assessed by incubating cell lysates with the known caspase substrates, acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin or acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin. We observed maximal cleavage of acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin within 6 hr following etoposide addition, a time that precedes cell death. In contrast, acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin was resistant to cleavage activity. This substrate cleavage specificity implies that a caspase-3-like protease is activated in response to DNA damage. Consistent with the lysate protease activity, an intracellular marker of caspase activation, poly-ADP ribose polymerase (PARP), was cleaved in a concentration- and time-dependent manner after etoposide-treatment. PARP cleavage followed caspase activation and reached maximum cleavage between 12 and 16 hr. Incubation of the cells with the peptidic caspase inhibitor z-valine-alanine-asparagine-CH2F prevented caspase activation, inhibited PARP cleavage, and inhibited cell death. Thus, etoposide killing of MCF7 cells requires a caspase-3-like protease.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Caspase-3 activation is required for Leydig cell apoptosis induced by ethane dimethanesulfonate.

Previous studies have shown that ethane dimethanesulfonate (EDS) causes the apoptotic death of Leydig cells. The molecular mechanism by which EDS elicits its effect remains uncertain. The present study tested the hypothesis that caspase-3 is involved in the EDS-induced death of rat Leydig cells. Leydig cells were isolated from adult Sprague Dawley at 3, 6, 12, or 24 h after the rats received an EDS injection. Low mol wt DNA fragments that are characteristic of apoptosis were evident by 12 h post-EDS, and the ladder pattern was more pronounced at 24 h. During this same time period, the number of terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling (TUNEL)-positive cells increased. Western blot analysis revealed that procaspase-3 was present only at low levels in control Leydig cells, and increased through 6 h post-EDS. By 12 h, procaspase-3 was reduced, whereas the cleaved, active caspase-3 forms appeared at 12 h and increased through 24 h post-EDS. Caspase-3 activity was blocked by caspase-3 inhibitor. In vitro, EDS treatment induced Leydig cell apoptosis. In the presence of cell-permeable caspase-3 inhibitor, however, apoptosis was significantly suppressed, providing further evidence for the involvement of caspase-3 in EDS-induced Leydig cell apoptotic death. Immunohistochemical analysis revealed weak staining for caspase-3 in the cytoplasm of control Leydig cells. From 12-24 h post-EDS, the time interval during which the active forms of caspase-3 appeared, caspase-3 immunoreactivity increased and became localized to the nuclei. Apoptosis and caspase-3 were colocalized in Leydig cells by a histological method that combined TUNEL and caspase-3 immunohistochemistry. In these studies, TUNEL-positive cells all exhibited intense nuclear caspase-3 immunoreactivity, whereas TUNEL-negative cells exhibited weak caspase-3 immunoreactivity in the cytoplasm. Taken together, these results indicate that Leydig cell apoptosis induced by EDS is mediated by caspase-3 activation, and suggest that the translocation of the active caspase-3 forms to the nucleus may be involved.

Animals↗
PubMed2002

Huperzine A attenuates amyloid beta-peptide fragment 25-35-induced apoptosis in rat cortical neurons via inhibiting reactive oxygen species formation and caspase-3 activation.

Huperzine A, a novel Lycopodium alkaloid originally discovered in the Chinese herb Qian Ceng Ta (Huperzia serrata), is a reversible, potent, and selective acetylcholinesterase (AChE) inhibitor and has been extensively used for the treatment of Alzheimer's disease (AD) in China. The present studies were designed to investigate effects of huperzine A on amyloid beta-peptide fragment 25-35 (Abeta25-35)-induced neuronal apoptosis and potential mechanisms in primary cultured rat cortical neurons. After exposure of the cells to Abeta25-35 (20 microM), apoptotic cell death was observed as evidenced by a significant decrease in cell viability, alteration of neuronal morphology, and DNA fragmentation. Pretreatment of the cells with huperzine A (0.01-10 microM) prior to Abeta25-35 exposure significantly elevated the cell survival and reduced Abeta25-35-induced nuclei fragmentation. Reactive oxygen species (ROS)-based fluorescence, caspase-3-like fluorogenic cleavage, and Western blot analysis demonstrated that huperzine A reduced Abeta25-35-induced ROS formation in a dose-dependent manner, and 1 microM of huperzine A attenuated Abeta25-35-induced caspase-3 activity at 6, 12, 24, and 48 hr posttreatment. Our results provide the first direct evidence that huperzine A protects neurons against Abeta25-35-induced apoptosis via the inhibition of ROS formation and caspase-3 activity.

Alkaloids↗
PubMed2003

Pro-apoptotic signaling in neuronal cells following iron and amyloid beta peptide neurotoxicity.

In a previous report, we characterized several oxidative stress parameters during the course of amyloid beta (Abeta) peptide/Fe2+-induced apoptotic death in neuronal cells. In extending these findings, we now report a marked decrease in protein kinase C (PKC) isoforms, reduced Akt serine/threonine kinase activity, Bcl 2-associated death promoter (BAD) phosphorylation and enhanced p38 mitogen-activated protein kinase (MAPK) and caspase-9 and -3 activation, 12 h after addition of both 5 micro m Abeta and 5 micro m Fe2+. These activities reminiscent for a pro-apoptotic cellular course were blocked in the presence of the iron chelator deferroxamine. Abeta alone, increased PKC isoform levels between three- and four-fold after 12 h, enhanced Akt activity approximately eight-fold and Ser136 BAD phosphorylation two-fold, suggesting that by itself is not toxic. Fe2+ alone transiently enhanced p38 MAPK and caspase-9 and -3 enzymes indicative for cell damage, but was not sufficient to cause cell death as previously indicated. GF, a PKC inhibitor or wortmannin, a blocker of the Akt pathway enhanced Abeta/Fe2+-induced toxicity, while SB, a p38 MAPK inhibitor, prevented cell damage and apoptosis. These findings further support the hypothesis that metal ion chelation and inhibitors of pro-apoptotic kinase cascades may be beneficial for Alzheimer's disease therapy.

Amyloid beta-Peptides↗
PubMed2004

[Experimental study on the induction of endothelial apoptosis by burn serum and subeschar tissue fluid].

OBJECTIVE: To explore the molecular mechanism of apoptosis of the human umbilical vein endothelial cells (HUVECs) induced by burn serum and subeschar tissue fluid. METHODS: In vitro cultured HUVECs were randomly divided into A (treated by normal serum, n = 6), B (treated by burn serum, n = 6) and C (treated by subeschar tissue fluid, n = 6) groups. The change in cellular nuclear morphology was determined by Hochst 33258 fluorescent staining, and the apoptotic rate was calculated at 24th and 36th post treatment hours (PTHs). The DNA ladder strips were observed with agarose electrophoresis at 12th and 24th PTH. The activity of caspase-3, 8, 9 was assayed by caspase colorimetric assay kit at 12 PTH. RESULTS: It was found that nuclei appeared to be condensed and highly fluorescent granular in form in B and C groups. The nucleic apoptotic rate in B and C groups was (38.9 +/- 7.3)% and (49.5 +/- 6.5)%, respectively, which was evidently higher than that in A group (P < 0.01). The DNA ladder strips could be found by agarose electrophoresis in B and C groups. The activity of caspase-3, 8, 9 in B and C groups was significantly higher than that in A group after 12 PTHs (P < 0.01). CONCLUSION: Burn serum and subeschar tissue fluid might induce apoptosis of HUVEC by means of the activation of death receptor and mitochondrial signal pathways simultaneously.

Adolescent↗
PubMedDate not supplied

Cataractogenesis in neonatal Sprague-Dawley rats by N-methyl-N-nitrosourea.

Cataract was induced by a single intraperitoneal injection of 100 mg/kg N-methyl-N-nitrosourea (MNU) to 0-, 5-, 10-, 15-, or 20-day-old male and female Sprague-Dawley rats. In day 0, 5, 10, and 15 MNU-treated rats, mature cataracts were constantly seen 7, 14, 14, and 30 days after dosing, respectively. In the day 20 MNU-treated rats, only subcapsular cataract was seen 30 days after dosing. Therefore, the rats exposed to MNU at an earlier age caused cataract more rapidly and severely. In the day 0 MNU-treated rats, 7-methyldeoxyguanosine DNA adduct was detected in the lens epithelial nuclei 12 hours after MNU dosing, followed by apoptosis, which was confirmed by morphology, by TUNEL signals, and by DNA ladder and peaked 3 days after MNU dosing. In the apoptosis cascade, upregulation of Bax, downregulation of Bcl-2, and increased CPP32 protease (caspase-3) activity were seen 12 hours after MNU dosing. Therefore, the pathogenesis of MNU-induced cataract was associated with DNA adduct formation in the lens epithelial cell nuclei leading to apoptosis by upregulation of Bax protein, downmodulation of Bcl-2 protein, and activation of caspase-3.

Animals↗
PubMed2003

[Tumor necrosis factor-alpha, caspase-3 expression and hepatocyte apoptosis in fulminanting hepatic failure].

OBJECTIVE: To study the role of tumor necrosis factor-alpha (TNFalpha) and caspase-3 expression on hepatocyte apoptosis in experimental model of fulminant hepatic failure (FHF). METHODS: Mouse experimental model of FHF was induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Serum TNFalpha level and TNFalpha mRNA expression in liver were tested by ELISA and reverse transcriptase PCR (RT-PCR) method, respectively. The expression of caspase-3 in liver tissue was determined by in situ hybridization. Hepatocyte apoptosis was examined by DNA agarose gel electrophoresis and TUNEL method. TNFalpha, caspase-3 and hepatocyte apoptosis were observed in different stages after drug administration. In addition, we observed the changes of above variables after pretreatment with anti-TNFalpha IgG1. RESULTS: TNFalpha mRNA expression in liver increased significantly (0.91 +/- 0.75, that of normal control was 0.32 +/- 0.10) 2 to 4 hours after administration of LPS and D-GalN. The level of serum TNFalpha increased [(320.50 +/- 86.57) ng/L; that of normal control was (16.66 +/- 7.01) ng/L] and caspase-3 expressed to a slight extent, too. Typical manifestation of hepatocyte apoptosis appeared 8h after drug administration. 8 hours after drug administration the level of serum ALT and total bilirubin (TBil) remarkably increased [(560.66 +/- 60.20) U/L and (163.66 +/- 34.51) micro mol/L, respectively, that of normal control was (23.56 +/- 8.03) U/L and (14.90 +/- 4.80) micro mol/L, respectively] and the expression of caspase-3 reached the peak. 12 hours after drug administration, hepatocyte apoptosis and necrosis coexisted, and the level of serum ALT and TBil reached a peak [(668.30 +/- 78.69) U/L and (203.17 +/- 19.92) micro mol/L, respectively] whereas the expression of caspase-3 already decreased. Hepatocyte apoptosis and liver injury and the expression of caspase-3 could be blocked by antagonizing TNFalpha. CONCLUSIONS: TNFalpha played an important role in hepatocyte apoptosis and liver injury in fulminant hepatic failure. The hepatocyte apoptosis induced by TNFalpha is correlated with the activation of caspase-3. Hepatocyte apoptosis occurs earlier than necrosis.

Animals↗
PubMed2003

Temporal ordering of caspase activation and substrate cleavage during antigen receptor-triggered apoptosis in Ramos-Burkitt lymphoma B cells.

We demonstrate here that selective activation of endogenous members of the caspase family and cleavage of substrates responsible for the maintenance of nuclear functional and structural integrity are major effectors of antigen receptor (AgR)- and ionomycin-triggered apoptosis in Ramos-Burkitt lymphoma (Ramos-BL) B cells. Ramos-BL B cells express significant proenzyme levels of caspase-2, -3, -7 and -8, low levels of caspase-6 and are caspase-1-negative. However, while anti-IgM and ionomycin trigger for significant activation of caspase-3, -7 and -8 at 12-16 h and at 4 h post-stimulation respectively, both anti-IgM and ionomycin fail to activate caspase-2 indicating that AgR- and ionomycin-triggered Ramos-BL B cell apoptosis is mediated by the selective activation of, at least, caspase-3, -7 and -8. Anti-IgM triggers for cleavage of the resident nuclear proteins poly(ADP-ribose) polymerase (PARP) at 8 h, lamins B1 and B2 from 12 to 16 h; likewise, ionomycin triggers for degradation of PARP at 2 h, lamins B1 and B2 at 4 h. Signal transduction through CD40 rescues Ramos-BL B cells from AgR- and ionomycin-triggered apoptosis at a very early stage of the apoptotic process by inhibiting both the early cleavage of PARP as well as the activation of caspase-3, -7 and -8 and cleavage of lamin B1; CD40-mediated rescue occurs upstream of CD40-induced expression of Bcl-2 and increased expression of Bcl-xL. In such cellular populations subject to regulation through apoptosis, dysregulation of the apoptotic mechanisms can have devastating consequences by contributing to the pathogenesis of malignancy as well as to lymphoproliferative and autoantibody disorders. An understanding of the role played by caspases in the execution of apoptosis may provide insight into the pathogenesis of these disease states and thereby provide targets for novel therapeutic strategy.

Apoptosis↗
PubMed2002

[The relationship between apoptosis of the cardiac myocytes and myocardial dysfunction in severely scalded rats].

OBJECTIVE: To investigate relationship between apoptosis of the cardiac myocytes and myocardial dysfunction in severely scalded rats. METHODS: Wistar rats inflicted by 40% TBSA III degree scalding were employed as the model. The myocardial tissue was obtained from the left ventricle at different postburn time points. Apoptosis was determined by the determination of myocardial tissue Caspase-3 activity and TUNEL staining (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling). The LVSP (left ventricular systolic pressure), LVEDP (left ventricular end-diastolic pressure) and +dp/dtmax (the rate of the rise of left ventricular pressure) and -dp/dtmax (the rate of the fall of left ventricular pressure) were all monitored by four-channel physiological recorder. In addition, myocardial activities of MPO and SOD were detected. RESULTS: The TUNEL staining of rat myocardial cells was positive at 6 postburn hour (PBH) and reached top level at 12 PBH. The change in Caspase-3 activity was earlier than that of apoptotic morphology and reached peak values at 3 and 6 PBHs. The left ventricular function (systolic and diastolic function) was significantly impaired after the scalding and dropped to the lowest levels at 12 PBHs. The increase in myocardial tissue MPO activity was accompanied by the decrease in SOD activity. CONCLUSION: Myocardial cellular apoptosis was one of the reasons of postburn myocardial injury in scalded rats. Caspase protease cascade pathway might be involved in the process of apoptosis, which suggested that the initiation of apoptosis was closely related to the infiltration of neutrophils and to the release of large amounts of oxygen free radicals in myocardial tissue.

Animals↗
PubMed2006

Adeno-associated viral-vector-mediated hypoxia-inducible vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: Exogenous delivery of vascular endothelial growth factor gene (VEGF) may provide a useful approach to the treatment of brain ischemia. We investigated the use of a hypoxia-responsive element to control VEGF expression given for neuroprotection. METHODS: Three groups (n=36) of mice received AAVH9-VEGF, AAVH9-lacZ, or saline injection. Five days after gene transfer, the mice underwent 45 minutes of transient middle cerebral artery occlusion (tMCAO) followed by 1 to 7 days of reperfusion. Infarct volume was determined using cresyl violet staining; neuronal injury was examined using TUNEL, cleaved caspase-3, and fluoro-Jade B staining. RESULTS: Hypoxia-inducible factor-1 (HIF-1) was overexpressed after tMCAO in the ischemic hemisphere in the brain. Expression of lacZ, mediated by AAV-lacZ, was seen before and after tMCAO; however, AAVH9-lacZ-mediated lacZ expression was detected only after tMCAO. Infarct volume was smaller in the AAVH9-VEGF-transduced group compared with AAVH9-lacZ and saline groups (55% reduction, P<0.05) with reduced TUNEL (29+/-5% and 30+/-7% versus 12+/-3%, P<0.05), cleaved caspase-3 (20+/-3% and 21+/-5% versus 13+/-4%, P<0.05) and fluoro-Jade B (23+/-3% and 24+/-5% versus 12+/-5%, P<0.05) -positive neurons, respectively. CONCLUSIONS: Exogenous expression of VEGF through AAVH9-VEGF gene transfer 5 days before the onset of ischemia provides neuroprotection. Hypoxia-responsive element is a viable strategy of restricting VEGF expression to areas of ischemia to minimize adverse effects of therapy on adjacent normal parenchyma.

Animals↗
PubMed2006

Activation of the endoplasmic reticulum stress pathway is associated with survival of myeloma cells.

The endoplasmic reticulum (ER) is an organelle in which proteins are modified. When unfolded proteins accumulate in the ER under various stresses, ER stress (ERS) pathways, including the induction of chaperones, are activated to protect the cell. However, when ERS is excessive, the cell undergoes apoptosis. This study investigated ERS in multiple myeloma cells (MMCs) because they contain a well-developed ER due to M-protein production. The myeloma cell line 12-PE underwent apoptosis via caspase-3 after treatment with thapsigargin (thap), an ERS inducer, while another cell line, U266, did not. To understand the mechanism regulating this heterogeneity, the induction of chaperones by thap was analysed. Chaperones were up-regulated in U266 cells but down-regulated in 12-PE cells, suggesting that chaperones contribute to cell survival under ERS. Analysis of XBP-1, a transcriptional inducer of chaperones, in freshly isolated MMCs from 22 myeloma cases revealed 10 cases with active XBP-1, who also showed significantly poorer survival (p < 0.05), suggesting that chaperone expression protects MMCs from apoptosis, thereby allowing tumor cell expansion. These results suggest that MMCs are subjected to ERS under certain circumstances and that chaperones are induced to protect the cells against such ERS. Inhibition of chaperones could be a new target for myeloma therapy.

Adult↗
PubMed2003

Inhibiting caspase-8 after injury reduces hypoxic-ischemic brain injury in the newborn rat.

A broad spectrum caspase inhibitor reduces brain injury. Will a caspase-8 inhibitor provide protection? Seven-day-old rat pups had the right carotid artery ligated, then were subjected to 2.5 h of 8% oxygen. Caspase-8 activity in the right cortex was measured enzymatically. Caspase-8 activity was increased at 12 and 24 h after injury and IETD-CHO, (Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Pro-Ile-Glu-Thr-Asp-CHO, CHO is aldehyde) a cell permeable caspase-8 inhibitor, given by i.c.v. injection after the hypoxic period eliminated this increase with significant effect at 15 and 50 microg/pup (1.7 micromol/kg). Thirty pups were randomly assigned to receive 50 microg/pup of IETD-CHO or vehicle i.c.v. immediately after the hypoxic period. The loss of brain weight in the right hemisphere 22 days after injury was 29+/-5% in the vehicle-treated animals and 12+/-5% in the IETD-CHO-treated animals (P<0.05). Inhibiting caspase-8 activity after hypoxic-ischemic brain injury reduces brain injury.

Animals↗
PubMed1998

Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species.

Human neutrophils have a short half-life and are believed to die by apoptosis or programmed cell death both in vivo and in vitro. We found that caspases are activated in a time-dependent manner in neutrophils undergoing spontaneous apoptosis, concomitant with other characteristic features of apoptotic cell death such as morphologic changes, phosphatidylserine (PS) exposure, and DNA fragmentation. The treatment of neutrophils with agonistic anti-Fas monoclonal antibodies (MoAbs) significantly accelerated this process. However, in cells treated with the potent neutrophil activator phorbol 12-myristate 13-acetate (PMA), caspase activity was only evident after pharmacologic inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Similarily, inhibition of the NADPH oxidase in constitutive and Fas/APO-1-triggered apoptosis resulted in increased rather than suppressed levels of caspase activity, suggesting that reactive oxygen species may prevent caspases from functioning optimally in these cells. Moreover, oxidants generated via the NADPH oxidase were essential for PS exposure during PMA-induced cell death, but not for neutrophils undergoing spontaneous apoptosis. We conclude that caspases are an important component of constitutive and Fas/APO-1-triggered neutrophil apoptosis. However, these redox sensitive enzymes are suppressed in activated neutrophils, and an alternate oxidant-dependent pathway is used to mediate PS exposure and neutrophil clearance under these conditions.

Apoptosis↗