DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Microcystin-RR induced apoptosis in tobacco BY-2 suspension cells is mediated by reactive oxygen species and mitochondrial permeability transition pore status

When tobacco BY-2 cells were treated with 60 mu g/mL MC-RR for 5 d, time-dependent effects of MC-RR on the cells were observed. Morphological changes such as abnormal elongation, evident chromatin condensation and margination, fragmentation of nucleus and formation of apoptotic-like bodies suggest that 60 mu g/mL MC-RR induced rapid apoptosis in tobacco BY-2 cells. Moreover, there was a significant and rapid increase of ROS level before the loss of mitochondrial membrane potential (Delta Psi(m)) and the onset of cell apoptosis. Ascorbic acid (AsA), a major primary antioxidant, prevented the increase of ROS generation, blocked the decrease in Delta Psi(m) and subsequent cell apoptosis, indicating a critical role of ROS in serving as an important signaling molecule by causing a reduction of Delta Psi(m) and MC-RR-induced tobacco BY-2 cell apoptosis. In addition, a specific mitochondrial permeability transition pores (PTP) inhibitor, cyclosporin A (CsA), significantly blocked the MC-RR-induced ROS formation, loss of Delta Psi(m), as well as cell apoptosis when the cells were MC-RR stressed for 3 d, suggesting that PTP is involved in 60 mu g/mL MC-RR-induced tobacco cell apoptosis signalling process. Thus, we concluded that the mechanism of MC-RR-induced apoptosis signalling pathways in tobacco BY-2 cells involves not only the excess generation of ROS and oxidative stress, but also the opening of PTP inducing loss of mitochondrial membrane potential. (C) 2007 Published by Elsevier Ltd.

Microcystin-RR-induced apoptosis in tobacco BY-2 cells

Our previous studies showed that microcystin-RR could induce oxidative damage in plant cells as they do with animal cells. However, whether microcystin can induce plant cell apoptosis is still unknown. In this study, the morphological changes of tobacco BY-2 suspension cells exposed to microcystin-RR were observed under light microscopy and transmission electron microscopy, and apoptosis was clearly distinguished by intense perinuclear chromatin margination, condensation of nuclear chromatin after 6d exposure of 50 mg/L (about 50 mu M) microcystin-RR. We also found that microcystin-RR can induce tobacco cell apoptosis in a dose- and time-dependent manner with flow cytometry analysis. Our study provides the first evidence that microcystins can induce plant cell apoptosis. (c) 2006 Elsevier Ltd. All rights reserved.

potential mechanisms involved in resistant phenotype of MCF-7breast carcinoma cells to ionizing radiation induced apoptosis

In the present study, we investigated the mechanisms of apoptosis resistance and the roles of the phosphorylation of BRCA1, p21, the Bax/Bcl-2 protein ratio and cell cycle arrest in IR-induced apoptosis in MCF-7 cells. X-irradiation, in particular at low dose (1 Gy), but not carbon ion irradiation, had a significant antiproliferative effect on the growth of MCF-7 cells. 1 Gy X-irradiation resulted in G1 and G2 phase arrest, but 4 Gy induced a significant G1 block. In contrast, carbon ion irradiation resulted in a significant accumulation in the G2 phase. Concomitant with the phosphorylation of H2AX induced by DNA damage,carbon ion irradiation resulted in an approximately 1.9–2.8-fold increase in the phosphorylation of BRCA1 on serine residue 1524, significantly greater than that detected for X-irradiation. Carbon ion irradiation caused a dramatic increase in p21 expression and drastic decrease in Bax expression compared with X-irradiation. The data implicated that phosphorylation of BRCA1 on serine residue 1524 might,at least partially, induce p21 expression but repress Bax expression. Together, our results suggested that the phosphorylation of BRCA1 at Ser-1524 might contribute to the G2 phase arrest and might be an upstream signal involved in preventing apoptosis signal via upregulation of p21 and downregulation of the Bax/Bcl-2 ratio.

Emodin-induced apoptosis in human breast cancer BCap-37 cells through the mitochondrial signaling pathway

Emodin, a natural anthraquinone compound isolated from the rhizome of rhubarb, is reported to suppress the growth of tumor in many clinical situations. In this study, we focused on the effect of emodin in human breast cancer BCap-37 cells and further understand the underlying molecular mechanism in treating breast cancer. Using MTT assay and flow cytometry, we demonstrated the critical role of emodin in the suppression of the proliferation of BCap-37 cells based on a concentration- and time-dependent manner. The increase of apoptotic rate was also observed after incubation of BCap-37 cells on emodin at 20 mu M and 50 mu M for 48 h. The cells exhibited typical apoptotic features including cellular morphological change, chromatin condensation and membrane blebbing. The results of the study further showed that Bcl-2 level decreased, while Bax and cytosolic cytochrome c levels in sample cells increased after the emodin treatment by using Western blot. The decline in the Bcl-2/Bax ratio and the increase of cytosolic cytochrome c concentration were consistent with the increase of the apoptotic ratio. The results strongly suggest that the disruption of the mitochondrial signaling pathway was involved in emodin-induced apoptosis in BCap-37 cells.

Mitochondrion-mediated apoptosis induced by Rana grylio virus infection in fish cells

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Delta psi m collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-kappa B activation and intracellular Ca2+ increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells.

C1q-like inhibits p53-mediated apoptosis and controls normal hernatopoiesis during zebrafish embryogenesis

Except for the complement C1q, the immunological functions of other C1q family members have remained unclear. Here we describe zebrafish C1q-like, whose transcription and translation display a uniform distribution in early embryos, and are restricted to mid-hind brain and eye in later embryos. In vitro studies showed that C1q-like could inhibit the apoptosis induced by ActD and CHX in EPC cells, through repressing caspase 3/9 activities. Moreover, its physiological roles were studied by morpholino-mediated knockdown in zebrafish embryogenesis. In comparison with control embryos, the C1q-like knockdown embryos display obvious defects in the head and cramofacial development mediated through p53-induced apoptosis, which was confirmed by the in vitro transcribed C1q-like mRNA or p53 MO co-injection. TUNEL assays revealed extensive cell death, and caspase 3/9 activity measurement also revealed about two folds increase in C1q-like morphant embryos, which was inhibited by p53 MO co-injection. Real-time quantitative PCR showed the up-regulation expression of several apoptosis regulators such as p53, mdm2, p21, Box and caspase 3, and down-regulation expression of hbae1 in the C1q-like morphant embryos. Knockdown of C1q-like in zebrafish embryos decreased hemoglobin production and impaired the organization of mesencephalic vein and other brain blood vessels. Interestingly, exposure of zebrafish embryos to UV resulted in an increase in mRNA expression of C1q-like, whereas over-expression of C1q-like was not enough resist to the damage. Furthermore, C1q-like transcription was up-regulated in response to pathogen Aeromonas hydrophila, and embryo survival significantly decreased in the C1q-like morphants after exposure to the bacteria. The data suggested that C1q-like might play an antiapoptotic and protective role in inhibiting p53-dependent and caspase 3/9-mediated apoptosis during embryogenesis, especially in the brain development, and C1q-like should be a novel regulator of cell survival during zebrafish embryogenesis. (c) 2008 Elsevier Inc. All rights reserved.

Surface-Relevant Regulable DNA Toroids Induced by Dopamine

Dopamine (2-(3,4-dihydroxyphenyl)ethylamine) is known as a natural chemical neurotransmitter and is also a cytotoxic and genotoxic molecule for cell apoptosis. In this work, the interaction of DNA with dopamine was investigated. Though the electrostatic interaction of DNA and dopamine was weak in aqueous solution, dopamine condensed circular pBR322 DNA into toroids on the mica surface cooperatively with ethanol. The formed DNA toroids came from the shrinking of DNA that was driven by ethanol-enhanced DNA-dopamine electrostatic interaction. The size of the DNA toroids could be modulated by varying the concentration of dopamine. This study offers useful information about the DNA condensation induced by monovalent cations and the sample preparation for AFM measurement and application.

Enhanced apoptotic action of trichosanthin in HIV-1 infected cells

Trichosanthin (TCS) is a type 1 ribosome-inactivating protein (RIP) effective against HIV-1 replication. The mechanism is not clear. Present results suggested that the antiviral action tray be partly mediated through enhanced apoptosis on infected cells. TCS induced apoptosis in normal H9 cells and this action was more potent in those infected with HIV-1. In flow cytometry study, TCS induced larger population of apoptotic H9 cells chronically infected with HIV-1 in a dose-dependent manner. At TCS concentration of 25 mu g/ml. 8.4% of normal H9 cells were found to be apoptotic whereas the same concentration induced 24.5% in HIV-1 chronically infected cells. Such difference was not found in the control experiments without TCS treatment. Two other studies supported this action. Cytotoxic study showed that cell viability was always lower in HIV-1 infected cells after TCS treatment, and DNA fragmentation studs confirmed more laddering in infected cells. The mechanism of TCS induced apoptosis in normal or infected H9 cells is not clear. Results in this study demonstrated that TCS is snore effective in inducing apoptosis in HIV-1 infected cells. This may explain in part the antiviral action of TCS. (c) 2005 Elsevier Inc. All rights reserved.

Characterization and expression analysis of Paralichthys olivaceus voltage-dependent anion channel (VDAC) gene in response to virus infection

Voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is acknowledged to play an important role in stress-induced mammalian apoptosis. In this study, Paralichthys olivaceus VDAC (PoVDAC) gene was identified as a virally induced gene from Scophthalmus Maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). The full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one alpha-helix, 13 transmembrane beta-strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells. This is the first report on the inductive expression of VDAC by viral infection, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis. (c) 2007 Elsevier Ltd. All rights reserved.

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.)

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 mu g L-1) for 2, 4, 8, 16 and 24 h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC50 of microcystins in carp hepatocytes was 169.2 mu g L-1. The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 mu g L-1) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 mu g L-1) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 mu g L-1), they died in the form of necrosis. (C) 2006 Elsevier Inc. All rights reserved.

Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L-1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L-1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L-1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L-1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD. (C) 2009 Elsevier B.V. All rights reserved.

TRAIL in the mandarin fish Siniperca chuatsi: Gene and its apoptotic effect in HeLa cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the CYS231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPaLp, NF-kappa B and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis. (c) 2007 Elsevier Ltd. All rights reserved.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.