Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells

Abstract

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion - exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.     

Treatment of cultured pancreatic B-cells with streptozotocin induces cell death by apoptosis

Treatment of cultured pancreatic B-cells (HIT-T15 and RINm5F) with the diabetogenic drug Streptozotocin resulted in a significant increase in the number of cells that became detached from the substrate during a subsequent culture period. Examination of the detached cells by fluorescence microscopy after staining with acridine orange or by electron microscopy revealed evidence of chromatin condensation and margination. Isolation and fractionation of DNA from these cells revealed a pattern of oligonucleosomal fragmentation that was not evident in untreated cells. All of these features are characteristic of entry of the cells into apoptosis and the results suggest that the diabetogenic action of Streptozotocin involves induction of apoptosis in pancreatic B-cells.     

Continuous germinal center invasion contributes to the diversity of the immune response

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Cathepsin-regulated apoptosis

Apoptosis can be mediated by different mechanisms. There is growing evidence that different proteolytic enzymes are involved in the regulation of apoptosis. Cathepsins are proteases which, under physiologic conditions, are localized intralysosomally. In response to certain signals they are released from the lysosomes into the cytoplasm where they trigger apoptotic cell death via various pathways, including the activation of caspases or the release of proapoptotic factors from the mitochondria. Here, we review different mechanisms that induce the release of lysosomal enzymes, and the functional relevance of defined cathepsins in defined models of apoptosis.     

Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion--exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.     

BAX contributes to Doppel-induced apoptosis of prion-protein-deficient Purkinje cells

Research efforts to deduce the function of the prion protein (PrPc) in knock-out mouse mutants have revealed that large deletions in the PrPc genome result in the ectopic neuronal expression of the prion-like protein Doppel (Dpl). In our analysis of one such line of mutant mice, Ngsk Prnp0/0 (NP0/0), we demonstrate that the ectopic expression of Dpl in brain neurons induces significant levels of cerebellar Purkinje cell (PC) death as early as six months after birth. To investigate the involvement of the mitochondrial proapoptotic factor BAX in the Dpl-induced apoptosis of PCs, we have analyzed the progression of PC death in aging NP0/0:Bax-/- double knockout mutants. Quantitative analysis of cell numbers showed that significantly more PCs survived in NP0/0:Bax-/- double mutants than in the NP0/0:Bax+/+ mutants. However, PC numbers were not restored to wildtype levels or to the increased number of PCs observed in Bax-/- mutants. The partial rescue of NP0/0 PCs suggests that the ectopic expression of Dpl induces both BAX-dependent and BAX-independent pathways of cell death. The activation of glial cells that is shown to be associated topographically with Dpl-induced PC death in the NP0/0:Bax+/+ mutants is abolished by the loss of Bax expression in the double mutant mice, suggesting that chronic inflammation is an indirect consequence of Dpl-induced PC death.     

溶酶体与细胞凋亡

在特定条件下,包括活性氧、鞘氨醇、细胞凋亡效应因子Bax等在内的多种刺激因子均可诱发溶酶体膜通透,之后溶酶体内含的蛋白酶（如组织蛋白酶等）及其他水解酶从溶酶体释放至胞浆中,通过剪切效应分子、激活包括凋亡酶在内的其他水解酶而启动细胞凋亡程序的执行。简要概括了引发溶酶体膜通透的可能机制及溶酶体参与细胞凋亡的主要途径。     

4-hydroxynonenal contributes to NGF withdrawal-induced neuronal apoptosis

Reactive oxygen species are a necessary triggering event for apoptosis of sympathetic neurons after nerve growth factor (NGF) withdrawal. Reactive oxygen species can lead to the generation of 4-hydroxynonenal (HNE), a highly reactive aldehyde that forms adducts with proteins. This covalent modification can activate or inhibit signal transduction pathways involved in the induction of apoptosis. This process may be clinically relevant because HNE-adduct immunoreactivity increases in several disease states. Here we evaluate the role of HNE-adducts in sympathetic neurons undergoing NGF-deprivation-induced apoptosis, a model of developmental programmed cell death. We show that HNE-adduct immunoreactivity is dramatically increased after NGF-withdrawal in an NADPH oxidase-dependent manner. Moreover, HNE-adducts appear to contribute to NGF-deprivation-induced apoptotic signal transduction because microinjected HNE-adduct antiserum protects sympathetic neurons from NGF withdrawal. In conclusion, this report suggests the direct contribution of endogenously generated HNE in the stimulation of apoptotic signal transduction in neurons.     

线粒体在细胞凋亡中的介导作用

线粒体是细胞内产生能量的重要细胞器,被认为是细胞生存与死亡的调节中心。Bcl-2家族蛋白、内质网和溶酶体能引起线粒体膜通透性的改变,造成线粒体功能损伤,诱导细胞凋亡。本文主要综述线粒体在Bcl-2家族蛋白、内质网和溶酶体诱导细胞凋亡中作用的研究进展。     

Cardosin A endocytosis mediated by integrin leads to lysosome leakage and apoptosis of epithelial cells

Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study, we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans. Such a process, which has been called the Trojan Horse mechanism, is believed to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores.     

Autophagy contributes to lysosomal storage disorders

Degradation in the lysosome/vacuole is not the final step of autophagy. In particular, for starvation-induced autophagy it is necessary to release the breakdown products back into the cytosol. However, some researchers ignore this last step and simply refer to the endpoint of autophagy as degradation, or perhaps even cargo delivery. In many cases this is not a serious issue; however, the analysis of autophagy's role in certain diseases makes clear that this can be a significant error.     

Autophagy contributes to lysosomal storage disorders

Degradation in the lysosome/vacuole is not the final step of autophagy. In particular, for starvation-induced autophagy it is necessary to release the breakdown products back into the cytosol. However, some researchers ignore this last step and simply refer to the endpoint of autophagy as degradation, or perhaps even cargo delivery. In many cases this is not a serious issue; however, the analysis of autophagy’s role in certain diseases makes clear that this can be a significant error.     

Immunobiology of chicken germinal center: II. Accumulation of apoptotic cells within the germinal center

The germinal center (GC) develops after antigen stimulation and is thought to occur at the site of various immune responses. We observed apoptotic cells within the GC using in situ end labeling (TUNEL), small amount DNA ladder assay, and RT-PCR analysis of Bcl-2 mRNA expression. Apoptosis was detected within GCs at all phases of the GC reaction by both TUNEL and DNA ladder assays. The number of TUNEL⁺ nuclei within the GC did not increase over the course of the GC reaction. However, the density of DNA in the ladder assay was higher in later-phase GCs. Bcl-2 mRNA expression was detected within GCs during the early phases of the GC reaction. These results indicate that accumulation of apoptotic cells and rescue from apoptosis occur within chicken GCs. In the present paper, the reasons for the accumulation of apoptotic cells will be discussed.This work was supported by Grants-in-Aid for Scientific Research (Nos. 11670322 and 10306017) from the Ministry of Education, Science, Sport and Culture, and the Ministry of Agriculture, Forestry and Fisheries of Japan (Special Scientific Research and Pioneering Research Project in Biotechnology), as well as from the Bio-oriented Technology Research Advancement Institution (BRAIN)     

p53-independent pRB degradation contributes to a drug-induced apoptosis in AGS cells

The retinoblastoma （RB） tumor suppressor protein, pRB, plays an important role in the regulation of mammalian cell cycle. Furthermore, several lines of evidence suggest that pRB also involves in the regulation of apoptosis. In the present study, the degradation of pRB was observed in apoptotic gastric tumor cells treated with a new potent anti-tumor component, tripchlorolide （TC）. The inhibition of pRB degradation by a general cysteine protease inhibitor IDAM resulted in the reduction of the apoptotic cells. Furthermore, the survival of the gastric tumor cells under the TC treatment was enhanced by an over-expression of exogenous pRB. These results suggest that the pRB degradation of the gastric tumor cells under the TC treatment involves in the apoptotic progression. In addition, the same extent of TCinduced pRB-degradation was detected in the gastric tumor cells containing a p53 dominant-negative construct, indicat- ing that this kind of pRB degradation is p53-independent.     

Fas-mediated apoptosis eliminates B cells that acquire self-reactivity during the germinal center response to NP

C57Bl/6 mice with the lpr mutation of Fas (CD95) were tested for deviation from the genetically restricted antibody response to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). lambda1+ germinal centers (GC) with the canonical v186.2 V(H) gene element develop in lpr/lpr mice with the same time course as in wild-type (+/+) mice. In contrast to +/+ mice, however, lambda1+ GC persist in the spleens of lpr/lpr mice 25 days after immunization. Virtually all of the lambda1+ GC are reactive with NP 10 days after immunization. Sixteen days after immunization, however, many of the lambda1+ GC are not reactive with NP, and few of the lambda1+ GC are reactive with NP 25 days after immunization. The V(H) gene elements of three lambda1+NP- GC 25 days after immunization are derived by somatic mutation of v186.2, but have lost reactivity with NP. The mutated VDJs from these GC react with cells in spleen sections from +/+ and lpr/lpr mice, indicating that they represented secondary antibody responses induced by self antigens that are available as presented antigen. These data indicate that Fas-mediated apoptosis serves to eliminate a (limited) population of B cells that acquire reactivity to "self antigens" by somatic mutation of VDJs in the GC.     

Variations of thioredoxin system contributes to increased susceptibility to apoptosis in cardiomyocytes of type 2 diabetic rats

Cardiac complications are the leading cause of death in diabetes. However, the mechanism of diabetes in inducing myocardial injury and apoptosis, and whether the thioredoxin （Trx） system is involved remain unclear. In this study, male Sprague-Dawley rats were randomly divided into two groups： the control and the diabetes groups, and then were randomly divided into five different timepoints （the 1st, 2nd, 4th, 12th, and 24th week）. The results showed that diabetes-induced cardiac injury was enhanced in the type 2 diabetes rats, as evidenced by aggravated cardiac dysfunction, biochemical indicators, and increased myocardial apoptosis （TUNEL and caspase-3 activity）. The activity of myocardial Trx and Trx reductase （TR） in diabetic rats was significantly decreased from the second week and continually aggravated with the disease progression. In diabetic rats, the mRNA expression of Trxl, Trx2, TR1, and TR2 was decreased first and then increased after the fourth week. Meanwhile, the protein expression of these Trx system members was significantly increased at the 12th week. Trx nitration was cleared, the Trx/ASK1 interaction was significantly decreased, and the activity of p38 was significantly enhanced in cardiac tissues at the 12th week. These results demonstrated that diabetes may cause myocardial injury and apoptosis, and the extent of which was accompanied with the development of the disease. The mechanism is associated with the development of diabetes and the decreased activity of Trx and TR. The reasons for decreased Trx activity may include： decrease of Trx and TR protein expression; nitration modification of Trx; and up-regulation of TXNIP expression.     

Differential regulation of survivin by p53 contributes to cell cycle dependent apoptosis

Recent studies indicate that cell-cycle checkpoints are tightly correlated with the regulation of apoptosis, in which p53 plays an important role. Our present works show that the expression of E6/E7 oncogenes of human papillomavirus in HeLa cells is inhibited in the presence of anti-tumor reagent tripchlorolide (TC), which results in the up-regulation of p53 in HeLa cells. Interestingly, under the same TC-treatment, the cells at the early S-phase are more susceptible to apoptosis than those at the middle S-phase although p53 protein is stabilized to the same level in both situations. Significant difference is exhibited between the two specified expression profiles. Further analysis demonstrates that anti-apoptotic gene survivin is up-regulated by p53 in the TC-treated middle-S cells, whereas it is down-regulated by p53 in the TC-treated early-S cells. Taken together, the present study indicates that the differential p53-regulated expression of survivin at different stages of the cell cycle results in different cellular outputs under the same apoptosis-inducer.     

Apoptotic cell fragments locally activate tingible body macrophages in the germinal center

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