Activation Of Caspase Proteases Research Articles

Bruceine D inhibits the growth of Spodoptera litura by inducing cell apoptosis in the midgut via an oxidative burst.

Spodoptera litura is one of the most harmful lepidoptera pests in China, and is difficult to control due to its strong resistance to the current frequently used insecticide species. The requirement to develop pesticides with novel toxicology mechanisms to control S. litura is urgent. The quassinoid of bruceine D display outstanding systemic properties and strong insecticidal activity against S. litura, which possess notable application potential for integrative management of S. litura, but the mechanism of toxicity remains unclear. In this study, we found that bruceine D exerts potent growth inhibitory activity against S. litura, disrupting the ecdysone and juvenile hormone titers, and causing long-term adverse effects. Association analysis between transcriptomics and metabolomics suggested that bruceine D affected the digestion and absorption capacity of S. litura larvae by inducing a strong oxidative stress response and cell apoptosis in the intestine. Further analysis demonstrated that bruceine D can inhibit the activities of digestive and antioxidant enzymes and induce malondialdehyde (MDA)and reactive oxygen species (ROS) overaccumulation in the midgut. Moreover, the protein level of Bax, cleavage caspase 3, and cytochrome c expressed in cytoplasm (cyto) were up-regulated by bruceine D, while Bcl-2 and cytochrome c expressed in mitochondria (mito) were down-regulated. In addition, there was a noticeable increase in caspase-3 protease activity. Histopathological observations revealed that bruceine D damages the structure of midgut epithelial cells and activates lysosomes, which subsequently disrupts the midgut tissue. Overall, our findings suggested that bruceine D induced excessive ROS accumulation in midgut epithelial cells. The resulting cell apoptosis disrupted midgut tissue, leading ultimately to reduced nutrient digestion and absorption in the midgut and the inhibition of larval growth. © 2024 Society of Chemical Industry.

HSP27 Inhibitory Activity against Caspase-3 Cleavage and Activation by Caspase-9 Is Enhanced by Chaperone O-GlcNAc Modification in Vitro.

One of the O-GlcNAc modifications is the protection of cells against a variety of stressors that result in cell death. Previous experiments have focused on the overall ability of O-GlcNAc to prevent protein aggregation under stress as well as its ability to affect stress-response signaling pathways. Less attention has been paid to the potential role for O-GlcNAc in the direct inhibition of a major cell-death pathway, apoptosis. Apoptosis involves the sequential activation of caspase proteases, including the transfer of cell-stress information from initiator caspase-9 to effector caspase-3. Cells have multiple mechanisms to slow the apoptotic cascade, including heat shock protein HSP27, which can directly inhibit the activation of caspase-3 by caspase-9. We have previously shown that O-GlcNAc modification increases the chaperone activity of HSP27 against amyloid aggregation, raising the question as to whether this modification may play important roles in other facets of HSP27 biology. Here, we use protein chemistry to generate different versions of O-GlcNAc modified HSP27 and demonstrate that the modification enhances this antiapoptotic function of the chaperone, at least in an in vitro context. These results provide additional molecular insight into how O-GlcNAc functions as a mediator of cellular stress with important implications for human diseases like cancer and neurodegeneration.

Transcriptional activity and splicing factors are preserved during physiological apoptosis

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism’s life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

A systematic approach to decode the mechanism of Cornus in the treatment of hepatocellular carcinoma (HCC)

Cornus Officinalis (Cornus), the dried pulp of mature Cornus, is used to treat liver diseases. However, the pharmacological mechanism of Cornus in the treatment of hepatocellular carcinoma (HCC) has not been systematically studied. The chemical compounds and the bioactive chemical compounds of Cornus were screened through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Gene Cards database was used to explore the targets in liver cancer pathogenesis. The disease-drug Venn diagram was constructed using the VENN 2.1 and the STRING database was used to analyze protein-protein Interaction Network (PPI). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed using the R package. Molecular docking was performed using Discovery Studio were assessed using Pymol and Discovery Studio 2016. Cell survival of BEL-7404 cells treated by Hydroxygenkwanin (HGK) were valued through CCK-8 assay. Expressions of caspase-3 and cleaved PARP was detected through Western blot. Pharmacological network diagrams of the Cornus compound-target network and HCC-related target network were successfully constructed. A total of 20 active compounds, 1841 predicted biological targets of Cornus, and 7100 HCC-related targets were identified. 37 target genes between Cornus and HCC were screened trough the network pharmacology. Molecular docking studies suggested that HGK has the highest affinity with caspase-3. HGK could induce apoptosis of HCC cells and significantly activate the caspase-3 protease activity in BEL-7404. This study systematically elaborated the mechanism of Cornus in the treatment of HCC and provided a new perspective to exploit Antineoplastic from Cornus.

Cytotoxic and Anticancer Effects of ICD-85 (Venom Derived Peptides) in Human Breast Adenocarcinoma and Normal Human Dermal Fibroblasts.

ICD-85 (venom derived peptides) has anti-proliferative effect and anti- angiogenesis activity on cancer cells. This study was performed to test the effect of ICD-85, on Human breast adenocarcinoma (MCF-7) and normal Human Dermal Fibroblasts (HDF) cell lines. In this experimental study, Mitochondrial activity, Neutral red uptake, Lactate dehydrogenase (cell necrosis), and cell morphology were assessed under unexposed and ICD-85 exposed conditions. Caspase-9 colorimetric assay kit was used to determine caspase protease activity. Morphological changes in MCF-7 cells on treatment with ICD-85 compared with untreated MCF-7 cells are consistent with characterizing the features of apoptosis such as granulation and cell rounding which finally results in the generation of apoptotic bodies. In contrast, this difference was not observed in normal cells. In MTT assay, ICD-85 induced dose dependent manner cytotoxic effects on MCF-7 cells which were confirmed by neutral red assay. The results showed that inhibitory concentration 50% (IC50) value of ICD-85 for MCF-7 cells at 24 h was 36.45 ± 0.38 μg/mL. However, when HDF cells were exposed to ICD-85, no significant elevation of LDH release were observed at concentrations below 20 μg/mL. The apoptosis-induction of ICD-85 on MCF-7 cell was found to be through activation of caspase-9 which was 13 fold greater than unexposed cell. This study showed that ICD-85 induced apoptosis in MCF-7 cell line through caspase activation and hence it can be considered for further investigation to use ICD-85 as a potential therapy for breast cancer.

Mycoplasma genitalium lipoproteins inhibit tumour necrosis factor α-induced apoptosis in HeLa cells

This aim of this study was to investigate the effects of Mycoplasma genitalium lipoproteins on the tumour necrosis factor α (TNF-α)-induced apoptosis in HeLa cells. We separated the M. genitalium lipid-associated membrane proteins (LAMPs) from M. genitalium and used them to treat HeLa cells. Apoptosis was induced by TNF-α treatment. Cell cycle and apoptosis were detected by flow cytometry. Pro-inflammatory cytokine contents were determined by enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential and caspase-3 protease activity were also measured. There was no significant difference in the viable cell percentage in HeLa cells treated with M. genitalium LAMPs at 0–20 μg/mL. However, when treated with 40 μg/mL LAMPs, cytocidal activity was observed during the first 24 h. M. genitalium LAMPs induced cell cycle arrest at the G1 phase in HeLa cells. When apoptosis was induced by TNF-α in HeLa cells, M. genitalium LAMPs significantly reduced the percentage of apoptotic cells. In addition, M. genitalium LAMPs significantly reduced the loss of mitochondrial membrane potential and caspase-3 protease activity in TNF-α-treated HeLa cells. However, the blocking of NF-κB significantly increased the mitochondrial membrane potential and caspase-3 protease activity in TNF-α-induced HeLa cells treated with M. genitalium LAMPs. The obtained results suggest that M. genitalium LAMPs could inhibit TNF-α-induced apoptosis in HeLa cells, which would contribute to the understanding of the pathogenesis of M. genitalium.

Reactive oxygen species-mediated caspase-3 pathway involved in cell apoptosis of Karenia mikimotoi induced by linoleic acid

Linoleic acid (LA) secreted by macroalgae has been shown to inhibit harmful algal blooming Karenia mikimotoi. In this study, we investigated the effects of LA on cell proliferation and apoptosis of K. mikimotoi. The results indicated that LA (>500 μg/L) could significantly inhibit the algal proliferation, with the cell cycle blocked at the G2 phase and a clear decrease of photosynthetic pigments. Intracellular reactive oxygen species (ROS) level, malondialdehyde (MDA) content and the activities of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) were obviously elevated, indicating that LA induced oxidative stress of K. mikimotoi. Several indicators for cell apoptosis, including DNA degradation, increased caspase-3 protease activity and phosphatidylserine (PTS) externalization, were observed in K. mikimotoi treated by LA. Addition of the ROS scavenger N‑acetyl‑l‑cysteine (NAC) decreased caspase-3 activity, and reduced cell apoptosis. Caspase-3 inhibitor also decreased cell apoptosis rate. Summarily, the results suggest that LA induced ROS overproduction, which further stimulated caspase-3, and then induced cell apoptosis of K. mikimotoi.

The Role of Apoptotic Signaling in Axon Guidance.

Navigating growth cones are exposed to multiple signals simultaneously and have to integrate competing cues into a coherent navigational response. Integration of guidance cues is traditionally thought to occur at the level of cytoskeletal dynamics. Drosophila studies indicate that cells exhibit a low level of continuous caspase protease activation, and that axon guidance cues can activate or suppress caspase activity. We base a model for axon guidance on these observations. By analogy with other systems in which caspase signaling has non-apoptotic functions, we propose that caspase signaling can either reinforce repulsion or negate attraction in response to external guidance cues by cleaving cytoskeletal proteins. Over the course of an entire trajectory, incorrectly navigating axons may pass the threshold for apoptosis and be eliminated, whereas axons making correct decisions will survive. These observations would also explain why neurotrophic factors can act as axon guidance cues and why axon guidance systems such as Slit/Robo signaling may act as tumor suppressors in cancer.

Relationship between blastocoel cell-free DNA and day-5 blastocyst morphology.

Cell-free DNA (cfDNA) which is present in the blastocoel cavity of embryos is believed to result from physiological apoptosis during development. This study assessed cfDNA content and caspase-3 protease activity in day-5 IVF blastocysts to determine if there was a correlation with embryo morphology. Day-5 IVF blastocysts were scored according to the Gardner and Schoolcraft system (modified to generate a numerical value) and cfDNA was collected following laser-induced blastocoel collapsing prior to cryopreservation in 25μL of media. cfDNA was quantified via fluorospectrometry and apoptotic activity was assessed via a caspase-3 protease assay using a fluorescent peptide substrate. Data were compared by linear regression. A total of 32 embryos were evaluated. There was a significant (p < 0.01) and positive correlation (cfDNA = 104.753 + (11.281 × score); R2 = 0.200) between embryo score and cfDNA content. A significant (p < 0.05) and positive correlation (cfDNA = 115.9 + (0.05 × caspase-3); R2= 0.128) was observed between caspase-3 activity and cfDNA levels. There was no significant relationship between caspase-3 activity and embryo morphology score. This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA present is from intracellular contents of embryonic cells that underwent apoptosis. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship with cfDNA content, embryo morphology, and chromosomal ploidy status plus implantation potential.

Routes to cell death in animal and plant kingdoms: from classic apoptosis to alternative ways to die—a review

Programmed cell death is fundamental for multicellular organisms either in animal or plant kingdom. Classic apoptosis, which represents the best studied form of cell death, is dependent on caspase protease activity in animals. These proteases are not present in plants, where caspase-like activities, including metacaspases, are involved in the execution of plant cell death. Beyond apoptosis, various non-apoptotic forms of cell death also exist, including autophagy, necroptosis, pyroptosis, and ferroptosis. These types of cell death can be activated independently of apoptosis and sometimes occur when apoptosis is inhibited. Non-apoptotic forms of cell death are best characterized in animals, whereas, in plants, the literature is less extensive, but the molecular executioners of plant cell death are becoming clearer. The aim of this review is to describe different types of cell death and the mechanisms involved in animals and plants, highlighting similarities and differences in the two kingdoms. Moreover, implications of cell death pathways in cancer are discussed.

N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton.

Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, α-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of α-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.

Non-apoptotic cell death in animal development.

Programmed cell death (PCD) is an important process in the development of multicellular organisms. Apoptosis, a form of PCD characterized morphologically by chromatin condensation, membrane blebbing, and cytoplasm compaction, and molecularly by the activation of caspase proteases, has been extensively investigated. Studies in Caenorhabditis elegans, Drosophila, mice, and the developing chick have revealed, however, that developmental PCD also occurs through other mechanisms, morphologically and molecularly distinct from apoptosis. Some non-apoptotic PCD pathways, including those regulating germ cell death in Drosophila, still appear to employ caspases. However, another prominent cell death program, linker cell-type death (LCD), is morphologically conserved, and independent of the key genes that drive apoptosis, functioning, at least in part, through the ubiquitin proteasome system. These non-apoptotic processes may serve as backup programs when caspases are inactivated or unavailable, or, more likely, as freestanding cell culling programs. Non-apoptotic PCD has been documented extensively in the developing nervous system, and during the formation of germline and somatic gonadal structures, suggesting that preservation of these mechanisms is likely under strong selective pressure. Here, we discuss our current understanding of non-apoptotic PCD in animal development, and explore possible roles for LCD and other non-apoptotic developmental pathways in vertebrates. We raise the possibility that during vertebrate development, apoptosis may not be the major PCD mechanism.

Neuroprotective effects of 2,3,5,4′-tetrahydoxystilbene-2-O-β-D-glucoside from Polygonum multiflorum against glutamate-induced oxidative toxicity in HT22 cells

Ethnopharmacological relevanceSince ancient times, Polygonum multiflorum Thunb. has been used to treat premature grey hair, dizziness, and blurred vision in East Asia. A major bioactive constituent of this medicinal herb, 2,3,5,4'-tetrahydoxystilbene-2-O-β-D-glucoside (THSG), has antioxidant activity and exerts beneficial effects on cognition and memory. Aim of the studyThe purpose of the current study was to determine if THSG affects hippocampal neuronal cell death and mitochondrial function following exposure to oxidative stress. Materials and methodsHT22 hippocampal cells with or without THSG pretreatment were exposed to glutamate, and the effects on cell viability and expression of molecules related to apoptotic cell death were examined using biochemical techniques, flow cytometry, western immunoblotting, and real-time polymerase chain reaction. ResultsPretreatment with THSG significantly attenuated glutamate-induced loss of cell viability and release of lactate dehydrogenase as well as apoptotic cell death. THSG inhibited generation of reactive oxygen species (ROS), expression of heme oxygenase-1, and activation of caspase-3 and calpain-1 proteases, all of which were increased by glutamate. THSG inhibited glutamate-induced disruption of mitochondrial membrane potential (MMP) and voltage-dependent anion channel-1. It also regulated the ratio of Bax to Bcl-2. ConclusionsThese results indicate that THSG has a marked neuroprotective effect against glutamate-induced hippocampal damage by decreasing ROS production and stabilizing MMP. These findings suggest the potential of THSG as a new therapeutic agent for the treatment of cognitive disorders.

Inflammasome Involvement in Alzheimer's Disease.

Inflammasomes are responsible for the maturation of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-18, and IL-33 and activation of inflammatory cell death, pyroptosis. They assemble in response to cellular infection and stress or to tissue damage, promote inflammatory reactions, and are important in regulating innate immunity particularly by acting as platforms for activation of caspase proteases. They appear to be involved in several pathological processes activated by microbes including Alzheimer's disease (AD). Best characterized in microbial pathogenesis is the nucleotide-binding domain and leucine-rich repeat (NLR)-protein 3 (NLRP3) inflammasome. AD is a neurodegenerative condition in which the neuropathological hallmarks are the deposition of amyloid-β (Aβ) and hyperphosphorylated tau protein coated neurofibrillary tangles. For decades, the role of the innate immune system in the etiology of AD was considered less important, but the recently discovered inflammatory genes by genome-wide association studies driving inflammation in this disease has changed this view. Innate immune inflammatory activity in the AD brain can result from the pathological hallmark protein Aβ as well as from specific bacterial infections that tend to possess weak immunostimulatory responses for peripheral blood myeloid cell recruitment to the brain. The weak immunostimulatory activity is a consequence of their immune evasion strategies and survival. In this review we discuss the possibility that inflammasomes, particularly via the NLR family of proteins NLRP3 are involved in the pathogenesis of AD. In addition, we discuss the plausible contribution of specific bacteria playing a role in influencing the activity of the NLRP3 inflammasome to AD progression.

Antigen detection and apoptosis in Mongolian gerbil’s kidney experimentally intraperitoneally infected by swine hepatitis E virus

We examined the effect of hepatitis E virus (HEV) on the renal tissue pathogenesis, morphological damages and related molecular mechanisms following swine HEV suspension intraperitoneally inoculation in Mongolian gerbils. The microscopic and ultramicroscopic analyses of kidney tissue structure were carried out at different points after inoculation of HEV. The immunohistochemistry, real-time PCR and Western blot were performed to explore the molecular mechanisms associated with HEV presence in the renal tissues. Real-time PCR revealed that the copies of HEV RNA in the kidney were detected at 7dpi, and peaked at 14dpi at a concentration was 7.18logsg−1, with detection of HEV ORF2 antigen by immunohistochemistry. Hematoxylin and eosin (HE) staining showed pathological lesions including glomerular atrophy, degeneration, edema and necrosis of renal tubular epithelial cells and Mallory and Sirius red staining indicated the presence of collagen fibers and fibrosis in kidney tissues of inoculated gerbils. Ultrastructural studies of basal membrane of renal tubules demonstrated the rough and uneven with mitochondria swelling and vacuolation in the tissues of HEV inoculated animals. Similarly, significantly higher number of (TUNEL)-positive cells were seen in renal tubule tissues compared to control group. Moreover, immuno histochemical results indicated that significant increase expression of the B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), FAS and Caspase-3 in HEV inoculated Mongolian gerbils at each time points. Relative mRNA expression by real-time PCR revealed a significantly higher (P<0.05) mRNA level of BAX, Bcl-2 and caspase-3 transcription in HEV inoculated Mongolian gerbils. Our results demonstrates that activation of mitochondria and Caspase-3 protease might be induced the apoptosis which subsequently cause the necrosis and cell death of renal epithelial cells during acute phase of HEV infection in HEV inoculated Mongolian gerbils.

Killing the Killer: PARC/CUL9 promotes cell survival by destroying cytochrome C.

Balanced amounts of apoptotic cell death are essential for health; its deregulation plays key roles in neurodegeneration, autoimmunity, and cancer. Mitochondria orchestrate apoptosis through a process called mitochondrial outer-membrane permeabilization (MOMP). After MOMP, mitochondrial cytochrome c is released into the cytoplasm, where it binds the adaptor molecule APAF1, triggering caspase protease activation and cell death. In this issue of Science Signaling, Deshmukh and colleagues define a new survival mechanism downstream of mitochondrial permeabilization. Specifically, they identify proteasomal degradation of cytochrome c as a major determinant of cell survival. In an unbiased approach, PARC (also known as CUL9) was found to be the ubiquitin ligase responsible for the ubiquitination and proteasomal degradation of cytochrome c. The consequences of this survival process may be double-edged because both cancer cells and postmitotic cells use PARC/CUL9-mediated cytochrome c degradation to ensure cell survival. Ultimately, differential targeting of this process may promote survival of postmitotic tissue or enhance tumor-specific killing.

Tacrolimus(FK506) Induced Cytotoxicity Via TRAIL-R1 (DR4) or TRAIL-R2 (DR5) in Jurkat Cells.

Purpose: To elucidate the mechanism of Tacrolimus(FK506) induced cytotoxicity via TRAIL(TNF related apoptosis inducing ligand)-R1(DR4) and TRAIL-R2(DR5) in FK506-treated Jurkat T cells. And signal transduction pathway of TNF-related events was studied. Methods: Viability of Jurkat T cells was measure by MTT assay. The catalytic activation of caspase-3 and caspase-9 proteases was determined by digestion of fluorogenic biosubstrates and western blot with anti-caspase-3 and anti-caspase-9 antibodies. The levels of mRNA and proteins for p53, Bax, PUMA, proline oxidase, TRAIL(TNF related apoptosis inducing ligand), TRAIL-R1(DR4), TRAIL-R2(DR5), Fas, FasL, TNF-α, IL-6, and NKκB were measured by RT-PCR and western blot with specific antibodies. Also we further examined the localization of TRAIL family proteins using by fluorescent microscope with specific TRAIL family antibodies. Results: FK506 decreased the viability of Jurkat T cells dose- and time-dependently along with catalytic activation of caspase-3 and caspase-9, p53 phosphorylation, and changes in expression levels of Bax, PUMA, and proline oxidase protein. It caused an increase in expression of TRAIL, TRAIL-R1(DR4), TRAIL-R2(DR5), Fas, and FasL in the levels of mRNA and proteins of Jurkat T cells. Furthermore, FK506 increased extracellular release of TNF-α and IL-6 cytokines in Jurkat T cells. It also induced the transactivation of NKκB through the dephosphrylation of Ser486 residues in Jurkat t cells. Conclusion: These results suggest that FK506 induces apoptotic death of Jurkat cells through activation of caspase family protease, Bcl-2 family protein-related mitochondrial dysfunction, and activation of death-receptor mediated signaling pathways.

Cryptotanshinone Induces Apoptosis of HL-60 Cells via Mitochondrial Pathway

Purpose: To test the effect of Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, on human leukemic cell lines (HL-60).Methods: HL-60 cells were treated with CPT. Cell growth inhibition (%) was quantitated using MTT assay. Apoptosis detection with Annexin V-FITC/propidium iodide staining was followed by flow cytometry. Caspase-3, caspase-8, and caspase-9 colorimetric assay kit was used to determine caspase protease activity. Loss of mitochondrial membrane potential was examined by flow cytometry with JC-1 staining. Bax, PARP, p53, p21 and cytochrome C were determined using Western blot.Results: Morphologic assessment, Annexin V-FITC/propidium iodide staining results and sub-G1 percentage indicate that the cytotoxic effect of CPT was mediated by induction of apoptosis. Furthermore, increased Bax expression, decreased Bcl-2 expression, loss of mitochondria membranepotential (MMP), release of cytochrome C, activation of caspase enzyme, cleavage of PARP and accumulation of p53 and p21 were detected during the apoptotic process. Caspase inhibitor partially abrogated CPT-induced apoptosis.Conclusion: The results show that CPT induced apoptosis of HL-60 cell lines by mitochondria pathway, and suggest that CPT may serve as a potential therapy for leukemia.Keywords: Cryptotanshinone, Salvia miltiorrhiza Bunge, Caspase, Membrane potential, Mitochondrial, Apoptosis, Leukemia, Cytochrome C, Cell cycle

Die another way--non-apoptotic mechanisms of cell death.

Regulated, programmed cell death is crucial for all multicellular organisms. Cell death is essential in many processes, including tissue sculpting during embryogenesis, development of the immune system and destruction of damaged cells. The best-studied form of programmed cell death is apoptosis, a process that requires activation of caspase proteases. Recently it has been appreciated that various non-apoptotic forms of cell death also exist, such as necroptosis and pyroptosis. These non-apoptotic cell death modalities can be either triggered independently of apoptosis or are engaged should apoptosis fail to execute. In this Commentary, we discuss several regulated non-apoptotic forms of cell death including necroptosis, autophagic cell death, pyroptosis and caspase-independent cell death. We outline what we know about their mechanism, potential roles in vivo and define outstanding questions. Finally, we review data arguing that the means by which a cell dies actually matters, focusing our discussion on inflammatory aspects of cell death.

Grass carp reovirus induces apoptosis and oxidative stress in grass carp (Ctenopharyngodon idellus) kidney cell line

Grass carp hemorrhage is an acute contagious disease caused by grass carp reovirus (GCRV). The pathogenesis of GCRV and the relationship between GCRV and the host cells remain unclear. The aim of the present study was to investigate the relations among apoptosis, intracellular oxidative stress and virus replication in GCRV infected-cells. The results showed that GCRV induced activation of caspase proteases as early as 12h, and reached maximum activities at 24h or 48h post-infection in a grass carp kidney cell line (CIK cells). Meanwhile, the levels of tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) also were increased in GCRV-infected CIK cells and showed a statistically significant difference from 24h to 96h post-infection. The infection of GCRV caused the destruction of entire monolayer and the death of host cells. Accompanied by the infection, a severe oxidative stress occurred, which led to extensive loss of antioxidants and formation of lipid peroxidation after 48h post-infection. These data suggested that the apoptosis which was triggered at an early stage (12–24h) in the viral infection cycle, might be independent of virus replication, while the oxidative stress induced by GCRV was mostly related to the virus replication.