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PubMed2006

Polymethoxylated flavones induce Ca(2+)-mediated apoptosis in breast cancer cells.

Flavonoids, polyphenolic phytochemicals which include flavones and isoflavones, are present in the common human diet. It has been suggested that these compounds may exert anticancer activity; however, the mechanisms involved remain unknown. We have recently shown (Sergeev, 2004, Biochem Biophys Res Commun 321: 462-467) that isoflavones can activate the novel apoptotic pathway mediated by cellular Ca(2+). Here, we report that polymethoxyflavones (PMFs) derived from sweet orange (Citrus sinensis L.) inhibit growth of human breast cancer cells via Ca(2+)-dependent apoptotic mechanism. The treatment of MCF-7 breast cancer cells with 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (3'-OH-TtMF) induced a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from both depletion of the endoplasmic reticulum Ca(2+) stores and Ca(2+) influx from the extracellular space. This increase in [Ca(2+)](i) was associated with the activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 peptide substrates and antibodies to active (cleaved) forms of the enzymes. Corresponding non-hydroxylated PMFs, 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF) and 5,6,7,3',4'-pentamethoxyflavone (PtMF), were dramatically less active in inducing Ca(2+)-mediated apoptosis. Our results strongly suggest that the cellular Ca(2+) modulating activity of flavonoids underlies their apoptotic mechanism and that hydroxylation of PMFs is critical for their ability to induce an increase in [Ca(2+)](i) and, thus, activate Ca(2+)-dependent apoptotic proteases.

Apoptosis↗
PubMed2006

ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation.

Expanded polyglutamine 72 repeat (polyQ72) aggregates induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and vesicular formation (autophagy). We examined this relationship and the molecular mechanism of autophagy formation. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent microtubule-associated protein 1 (MAP1) light chain 3 (LC3) conversion from LC3-I to -II, which plays a key role in autophagy. The eucaryotic translation initiation factor 2 alpha (eIF2alpha) A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, autophagy formation is a cellular defense mechanism against polyQ72-induced ER-stress-mediated cell death by degrading polyQ72 aggregates, with PERK/eIF2alpha phosphorylation being involved in polyQ72-induced LC3 conversion.

Adenine↗
PubMed2006

Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors.

Molecular mechanisms underlying apoptosis in retinitis pigmentosa, as in other neurodegenerative diseases, are still elusive, and this fact hampers the development of a cure for this blinding disease. We show that two apoptotic pathways, one from the mitochondrion and one from the endoplasmic reticulum, are coactivated during the degenerative process in an animal model of retinitis pigmentosa, the rd1 mouse. We found that both AIF and caspase-12 translocate to the nucleus of dying photoreceptors in vivo and in an in vitro cellular model. Translocation of both apoptotic factors depends on changes in intracellular calcium homeostasis and on calpain activity. Knockdown experiments defined that AIF plays the major role in this apoptotic event, whereas caspase-12 has a reinforcing effect. This study provides a link between two executor caspase-independent apoptotic pathways involving mitochondrion and endoplasmic reticulum in a degenerating neuron.

Animals↗
PubMed2000

Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma.

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1beta and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes. We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon-gamma strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon-gamma up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1beta. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon-gamma and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.

Base Sequence↗
PubMed2006

Induction of endoplasmic reticulum stress in retinal pericytes by glucose deprivation.

Diabetic retinopathy is one of the major microvascular complications associated with diabetes mellitus, and the selective degeneration of retinal capillary pericytes is considered to be a hallmark of early retinopathy. Because glucose fluctuations commonly occur in diabetes, we hypothesized that these fluctuations will increase the endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR) in retinal pericytes. To study whether ER stress and the UPR can be induced in retinal pericytes, rat retinal capillary pericytes were cultured in different concentrations of glucose. Hypoglycemia but not hyperglycemia was found to activate UPR-specific enzymes in pericytes. Strong UPR activation leading to apoptosis was also observed when pericytes were cultured in glucose concentrations that were reduced from high to low or no glucose. These results indicate that induction of UPR is related not only to absolute concentrations but also to a shifting from higher to lower concentrations of glucose.

Activating Transcription Factor 4↗
PubMed2004

Dexamethasone induces apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt-phosphatidylinositol 3'-kinase signaling pathway.

Although glucocorticoids are known to induce apoptosis in chondrocytes, the mechanisms for this effect and the potential antiapoptotic role of IGF-I are unknown. To address this, we studied the effects of dexamethasone (Dexa) on apoptosis in the HCS-2/8 chondrocytic cell line. Dexa (25 microm) increased apoptosis (cell death ELISA) by 39% and 45% after 48 and 72 h, respectively (P < 0.01 and P < 0.05, respectively). IGF-I (100 ng/ml) decreased Dexa-induced apoptosis to levels similar to control cells. Apoptosis was associated with cleavage of poly-ADP-ribose polymerase (PARP) and alpha-fodrin and activation of caspases-8, -9, and -3 (Western), an effect that was counteracted when chondrocytes were cocultured with Dexa + IGF-I. Inhibitors for caspases-8, -9, and -3 (50 microm each) equally suppressed Dexa-induced apoptosis (P < 0.01). Time-response experiments showed that caspase-8 was activated earlier (at 12 h) than caspase-9 (at 36 h). We studied the phosphatidylinositol 3'-kinase (PI3K) pathway to further investigate the mechanisms of Dexa-induced apoptosis. Dexa decreased Akt phosphorylation by 93% (P < 0.001) without affecting total Akt and increased the p85alpha subunit 4-fold. The Akt inhibitor SH-6 (10 microm) increased apoptosis by 54% (P < 0.001). When combining Dexa with SH-6, apoptosis was not further increased, showing that Dexa-induced apoptosis is mediated through inhibition of the PI3K pathway. Addition of IGF-I to SH-6- or Dexa + SH-6-treated cells decreased apoptosis by 21.2% (P < 0.001) and 20.6% (P < 0.001), respectively. We conclude that Dexa-induced apoptosis is caspase dependent with an early activation of caspase-8. IGF-I can rescue chondrocytes from Dexa-induced apoptosis partially through the activation of other pathways than the PI3K signaling pathway. Based on our in vitro data, we speculate that in vivo treatment with glucocorticoids may diminish longitudinal growth by increasing apoptosis of proliferative growth plate chondrocytes.

Apoptosis↗
PubMed2006

Antiproliferative and proapoptotic effects of histone deacetylase inhibitors on gastrointestinal neuroendocrine tumor cells.

Treatment options of advanced neuroendocrine tumors (NETs) are unsatisfactory. Hence, innovative therapeutic approaches are urgently needed. Inhibition of histone deacetylases (HDACs) is a promising new approach in cancer therapy. While several HDAC inhibitors have already entered clinical trials, the effect of HDAC inhibition on NET has not been investigated. Therefore, we evaluated the antineoplastic effects of three different HDAC inhibitors, trichostatin A (TSA), sodium butyrate (NaB), and MS-275, on growth and apoptosis of the gastrointestinal NET cell lines CM and BON. We could demonstrate that HDAC inhibition dose-dependently inhibited proliferation of both cell lines with IC50 values varying from the millimolar (NaB) to the micromolar (MS-275) and the nanomolar range (TSA). Moreover, HDAC inhibition potently induced apoptosis, which was accompanied by DNA-fragmentation, an up to 12-fold caspase-3 activation and downregulated Bcl-2 expression. Furthermore, HDAC inhibition resulted in cell cycle arrest at the G1-S-transition, which was associated with the suppression of cyclin D1 expression and induction of p21 and p27 expression. For BON cells, we observed an additional block in the G2/M phase, which was aligned with a downregulation of cyclin B1. In addition, combined treatment with MS-275 and somatostatin or the synthetic somatostatin analog octreotide was evaluated. Neither somatostatin nor its stable analog octreotide augmented the antiproliferative effect of MS-275 in NET cells. To conclude, our data show that HDAC inhibition is a promising new approach in the treatment of NET disease, which should be evaluated in clinical studies.

Antineoplastic Agents, Hormonal↗
PubMed2005

Endoplasmic reticulum mediated necrosis-like apoptosis of HeLa cells induced by Ca2+ oscillation.

Apoptosis and necrosis are distinguished by modality primarily. Here we show an apoptosis occurred instantly, induced by 300 muM W-7 ((N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride), inhibitor of calmodulin), which demonstrated necrotic modality. As early as 30 min after W-7 addition, apoptotic (sub-diploid) peak could be detected by fluorescence-activated cell sorter (FACS), "DNA ladders" began to emerge also at this time point, activity of caspase-3 elevated obviously within this period. Absence of mitochondrial membrane potential (MMP) reduction and cytochrome c, AIF (apoptosis inducing factor) release, verified that this rapid apoptosis did not proceed through mitochondria pathway. Activation of caspase-12 and changes of other endoplasmic reticulum (ER) located proteins ascertained that ER pathway mediated this necrosis-like apoptosis. Our findings suggest that it is not credible to judge apoptosis by modality. Elucidation of ER pathway is helpful to comprehend the pathology of diseases associated with ER stress, and may offer a new approach to the therapy of cancer and neurodegenerative diseases.

Apoptosis↗
PubMed2005

[Preparation and identification of hammerhead ribozyme in vitro against caspase-12 mRNA fragments].

OBJECTIVE: To design and synthesize ribozymes targeting 138 and 218 sites of the mRNA nucleotide of mouse caspase-12, a key intermedium of ER stress mediated apoptosis, and to identify their activities through in vitro transcription and cleavage. METHODS: The mouse caspase-12 gene fragment was obtained by RT-PCR and cloned into the PGEM-T vector under the control of T7 RNA polymerase promoter. The transcription product of the target was labeled with a-32P UTP, while ribozymes were not labeled. Ribozyme and target RNA were incubated for 90 min at 37 degree C in a reaction buffer to perform the cleavage reaction. RESULTS: It was found that under a condition of 37 degree C, pH 7.5 and with Mg2+ in a concentration of 10 mmol/L, Rz138 and Rz218 both cleaved targets at predicted sites, and the cleavage efficiency of Rz138 was 100%. CONCLUSION: Rz138 and Rz218 prepared in vitro possess the perfect specific catalytic cleavage activity. Rz138 has excellent cleavage efficiency. It may be a promising tool to prevent ER stress induced apoptosis through catalytic cleavage of caspase-12 mRNA in vivo. It also can be used to verify whether caspase-12 is necessary in ER stress induced apoptosis.

Animals↗
PubMed2005

[Caspase-12 expression and activation in the pathogenesis of acute hepatic failure induced by lipopolysaccharide and D-galactosamine].

OBJECTIVE: To study the role of caspase-12 expression on hepatocyte apoptosis in an experimental model of acute hepatic failure (AHF). METHODS: A mouse experimental model of AHF was developed by intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-Gal). Hepatocyte apoptosis was examined by DNA agarose gel and liver pathology. Caspase-12 mRNA expression in liver was detected by reverse transcriptase PCR (RT-PCR) method. The expression of caspase-12, GRP78 proteins in livers was determined by Western blot. RESULTS: Caspase-12 mRNA expression in the livers increased significantly from 5 to 7 hours after administration of LPS and D-Gal. Typical manifestation of hepatocyte apoptosis appeared at 5 hours after the drug administration. After 5 hours the level of serum ALT and AST were remarkably increased, and they reached the peak at 7 hours. The expression of procaspase-12 protein decreased obviously at 7 hours. Seven hours after the drug administration, hepatocyte apoptosis and necrosis both started. The marker of endoplasmic reticulum (ER) stress, Bip/GRP78 was activated during the development of hepatocyte apoptosis. The level of Bip/GRP78 protein was gradually increased at 5 hours after the drug induction. CONCLUSION: Hepatocyte apoptosis plays an important role in the pathogenesis of AHF. Caspase-12 induced ER stress involves in hepatocyte apoptosis. It suggests that inhibition of caspase-12 activation might be a potential strategy in the treatment of AHF in the future.

Animals↗
PubMed2006

[Hypoxic preconditioning induces endoplasmic reticulum stress-related cardioprotection mediated by p38 mitogen-activated protein kinase].

Calreticulin (CRT), an important Ca(2+)-binding molecular chaperone in the endoplasmic reticulum (ER), and caspase-12, a pivotal molecule mediating ER-initiated apoptosis, are involved in the ER stress (ERS). Using primary cultured neonatal cardiomyocytes, CRT and caspase-12 expression and activation during hypoxic preconditioning (HPC) and hypoxia/reoxygenation (H/R) were studied to explore the role of ERS in cardioprotection by HPC. And by using SB203580 and SP600125 [the specific inhibitors of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK)] separately, the role of p38 MAPK in HPC-induced ERS was also detected. Neonatal cardiomyocytes were prepared from Sprague-Dawley rats aged 24 h, and cultured in DMEM medium containing 10% fetal bovine serum, and then randomly divided into six groups as follows: H/R, HPC+H/R, SB203580+HPC+H/R, SP600125+HPC+H/R, HPC and control groups. H/R was produced by 2-hour hypoxia/14-hour reoxygenation, and HPC by 20-minute hypoxia/24-hour reoxygenation. Morphological studies, estimation of lactate dehydrogenase (LDH) leakage and flow cytometry were employed to assess cell apoptosis and necrosis. CRT and caspase-12 expression and activation, levels of phospho-p38 MAPK and phospho-JNK were detected by Western blot. All experiments were repeated at least four separate times. The results obtained are as follows: (1) HPC relieved the cell injury caused by H/R. Compared with that in H/R group, cellso survival rate in HPC+H/R group increased by 6.4%, and the apoptosis rate and LDH leakage in the cell culture medium decreased by 6.6% and 70.0%, respectively. (2) H/R induced caspase-12 activation (33.2-fold increase in comparison with control) and CRT expression (8.1-fold increase in comparison with control). HPC itself resulted in mild CRT up-regulation (2.6-fold increase in comparison with control), but the extent of up-regulation was lower than that induced by H/R. HPC before H/R was found to relieve the over-expression of CRT induced by H/R (72.4% decrease), and to inhibit the activation of caspase-12 (59.6% decrease). (3) The protection of HPC and HPC-induced up-expression of CRT and inhibition of caspase-12 activation were almost eliminated when the inhibitor of p38 MAPK, not of JNK, was present before HPC. These results suggest that HPC protects the neonatal cardiomyocytes from severe ERS-induced apoptosis during sustained H/R through pre-invoking proper ERS response. Mild up-expression of CRT and inhibition of caspase-12 activation induced by HPC, which are important protection factors, are mediated by p38 MAPK, not by JNK.

Animals↗
PubMed2005

Cyclic AMP alleviates endoplasmic stress and programmed cell death induced by lipopolysaccharides in human endothelial cells.

The possible protection provided by enhancement of the cAMP signal in the process of lipopolysaccharide (LPS)-induced endothelial cell death has been addressed, with special emphasis on the endoplasmic initiation of caspase-12-mediated apoptosis. Human umbilical vein endothelial cells were challenged with LPS to reduce viability after 12 h to less than 20% that of the control. Cell death was preceded by ultrastructural disintegration at the endoplasmic reticulum, PERK-phosphorylation, degradation of caspase-12-like protein and cleavage of caspase 9, resulting in apoptosis through the activation of caspase 3. Treatment with a cell-permeable cAMP analogue led to a dose-dependent reduction of cell death over time, mitigated endoplasmic reticulum disturbances, reduced phosphorylation of PERK, and the degradation of caspases 12, 9 and 3. The selective inhibition of caspase 9 completely supplanted the anti-apoptotic effects obtained by cAMP, while being without any influence on caspase 12 degradation. The data suggest that cAMP positively modulates early endoplasmic alterations and caspase activation in LPS-induced apoptosis.

Apoptosis↗
PubMed2002

Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway.

ARHI, an imprinted putative tumor suppressor gene, encodes a M(r) 26,000 GTP-binding protein that is 60% homologous to ras and rap but has a dramatically different function. ARHI expression is down-regulated in a majority of breast and ovarian cancers. Using a dual adenovirus system, we have reexpressed ARHI in ovarian cancer and breast cancer cells that have lost ARHI expression. Reexpression of ARHI inhibited growth, decreased invasiveness, and induced apoptosis. At 5 days after infection with ARHI adenovirus, 30-45% of MDA-MB-231 breast cancer cells and 5-11% of SKOv3 ovarian cancer cells were apoptotic as judged by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay and by Annexin V staining with flow cytometric analysis. Although poly(ADP-ribose) polymerase could be detected immunohistochemically in the nuclei of apoptotic cells, no activation of the effector caspases (caspase 3, 6, 7, or 12) or the initiator caspases (caspase 8 or 9) could be detected in cell lysates using Western blotting. When gene expression was analyzed on a custom cDNA array that contained 2304 known genes, infection with ARHI adenovirus up-regulated 15 genes relative to control cells infected with LacZ adenovirus. The greatest degree of mRNA up-regulation was observed in a Homo sapiens calpain-like protease. On Western blot analysis, calpain protein was increased 2-3-fold at 3-5 days after infection with ARHI adenovirus. No increase in calpain protein was observed after LacZ adenovirus infection. Calpain cleavage could be detected after ARHI reexpression, and inhibitors of calpain, but not inhibitors of caspase, partially prevented ARHI-induced apoptosis. Consequently, reexpression of ARHI in breast and ovarian cancer cells appears to induce apoptosis through a caspase-independent, calpain-dependent mechanism.

Adenoviridae↗
PubMed2004

The immunosuppressant drug FK506 prevents Fas-induced apoptosis in human hepatocytes.

FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12 h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3 h with immunosuppressant drugs. A significant reduction (ca. 30-40%) of caspase-3 activation by 5 microM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia-reperfusion injury following liver transplantation.

Adult↗
PubMed2001

Role of p53-dependent activation of caspases in chronic obstructive uropathy: evidence from p53 null mutant mice.

Chronic obstructive uropathy (COU) created by unilateral ureteric ligation is associated with increased renal cell apoptosis and p53 expression. Genetically engineered mice were used to examine the role of p53 in renal cell apoptosis in COU and the involved molecular pathways. Obstructed kidneys in p53+/+, p53+/-, and p53-/- mice were examined at days 4, 7, 15, 20, and 30 for apoptosis, and mRNA were examined for p53, members of the bcl-2 family, the death receptor family, and the common effectors of apoptosis. Obstructed kidneys in p53+/- and p53-/- mice exhibited equal attenuation of tubular and interstitial cell apoptosis (70 and 50%, respectively), compared with p53+/+ mice. However, p53 gene deficiency did not confer complete protection from apoptosis. Obstructed kidneys from p53-/- mice did not express p53 mRNA, whereas those from p53+/- and p53+/+ mice displayed mild and marked increase in their expression, respectively. Obstructed kidneys in p53+/+, p53+/-, and p53-/- mice displayed upregulation of mRNA for members of the bcl-2 family and most of the death receptor family, except for a lower level of tumor necrosis factor receptor-1, TRAIL, and FAP in p53+/+ mice. Obstructed kidneys in p53-/- and p53+/- mice showed virtual absence of caspase 11 and marked attenuation of caspases 1 and 12, contrasted with their strong expression in p53+/+ kidneys. These data suggest that apoptosis in obstructed kidneys involves p53-dependent as well as p53-independent pathways. The p53-dependent pathway may involve activation of caspases 1, 11, and 12, whereas the p53-independent pathway may involve activation of members of the bcl-2 and death receptor families.

Animals↗
PubMed2006

Activation of the caspase 8 pathway mediates seizure-induced cell death in cultured hippocampal neurons.

In response to harmful stresses, cells induce programmed cell death (PCD) or apoptosis. Seizures can induce neural damage and activate biochemical pathways associated with PCD. Since seizures trigger intracellular calcium overload, it has been presumed that the intrinsic cell death pathway mediated by mitochondrial dysfunction would modulate cell death following seizures. However, previous work suggests that the extrinsic cell death pathway may initiate the damage program. Here we investigate intrinsic versus extrinsic cell death pathway activation using caspase cleavage as a marker for activation of these pathways in a rat in vitro model of seizures. Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40 min. Subjecting rat hippocampal cultures to seizures increased cell death and apoptosis-like DNA fragmentation using TUNEL staining. Seizure-induced cell death was blocked by both MK801 (10 microM) and CNQX (40 microM), which suggests multiple glutamate receptors regulate seizure-induced cell death. Cleavage of the initiator caspases, caspase 8 and 12 were increased 4h following seizure, and cleavage of the quintessential executioner caspase, caspase 3 was increased 4h following seizure. In contrast, caspase 9 cleavage only increased 24h following seizure. Using an affinity labeling approach to trap activated caspases in situ, we show that caspase 8 is the apical caspase activated following seizures. Finally, we show that the caspase 8 inhibitor Ac-IETD-CHO was more effective at blocking seizure-induced cell death than the caspase 9 inhibitor Ac-LEHD-CHO. Taken together, our data suggests the extrinsic cell death pathway-associated caspase 8 is activated following seizures in vitro.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2002

Cisplatin-induced apoptosis in auditory cells: role of death receptor and mitochondrial pathways.

Cisplatin, a commonly used chemotherapeutic agent, has a major limitation due to its ototoxicity. Previous studies have shown that cisplatin induces apoptosis in auditory sensory cells, but the underlying mechanisms remain to be elucidated. In this study, cisplatin was found to induce apoptosis in a cochlear cell line, in a dose- and duration-dependent manner. Specific caspase assays revealed an early (6 h) but transient increase in caspase 8 activity, and a delayed (12 h) increase in caspase 9 activity. The enhanced caspase 8 activity was preceded by upregulation of p53 expression, and coincided with cleavage of Bid to its truncated form. This was followed temporally by activation and mitochondrial translocation of Bax, induction of mitochondrial permeability transition, release of cytochrome c into the cytosol, activation of caspase 9, and entry into the execution phase of apoptosis. Our results indicate the involvement of both the death receptor mechanisms as well as mitochondrial pathways in cisplatin-induced apoptosis of auditory cells in an in vitro model system.

Animals↗
PubMed2004

Increased expression and processing of caspase-12 after traumatic brain injury in rats.

Traumatic brain injury (TBI) disrupts tissue homeostasis resulting in pathological apoptotic activation. Recently, caspase-12 was reported to be induced and activated by the unfolded protein response following excess endoplasmic reticulum (ER) stress. This study examined rat caspase-12 expression using the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase-12 proform (approximately 60 kDa) and processed form (approximately 12 kDa), with peak induction observed within 24 h post-injury in the cortex (418% and 503%, respectively). Hippocampus caspase-12 proform induction peaked at 24 h post-injury (641%), while processed form induction peaked at 6 h (620%). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed elevated caspase-12 mRNA levels after TBI. Injury severity (1.0, 1.2 or 1.6 mm compression) was associated with increased caspase-12 mRNA expression, peaking at 5 days in the cortex (657%, 651% and 1259%, respectively) and 6 h in the hippocampus (435%, 451% and 460%, respectively). Immunohistochemical analysis revealed caspase-12 induction in neurons in both the cortex and hippocampus as well as in astrocytes at the contusion site. This is the first report of increased expression of caspase-12 following TBI. Our results suggest that the caspase-12-mediated ER apoptotic pathway may play a role in rat TBI pathology independent of the receptor- or mitochondria-mediated apoptotic pathways.

Animals↗