PURPOSE: To investigate the pathways of radiation induced apoptosis and the effect of cysteamine (beta-mercaptoethylamine), as a radioprotector, on it. MATERIALS AND METHODS: HL-60 cells were assigned to control, irradiated, and cysteamine (1 mM, 10mM) pretreated groups, Irradiation was given in a single fraction of 10 Gy (6 MV x-ray) and cysteamine was administered 1 hour before irradiation. The activities of caspase-8 were measured in control and irradiated group to evaluate its relation to the radiation induced apoptosis. To evaluate the role of cysteamine in radiation induced apoptosis, the number of viable cells, the expression and activity of caspase-3, and the expression of poly (ADP-ribose) polymerase (PARP) were measured and compared after irradiation the HL-60 cells with cysteamine pretreatment or not. RESULTS: The intracellular caspase-8 activity, known to be related to the death receptor induced apoptosis, was not affected by irradiation (p>0.05). The number of viable cells began to decrease from 6 hours after irradiation (p>0.05), but the number of viable cells in 1 mM cysteamine pretreated group was not decreased afger irradiation and was similar to those in the control group. In caspase-3 analyses, known as apoptosis executioner, its expression was not different but its activity was increased by irradiation (p>0.05). However, this increase of activity was suppressed by the pretreatment of 1mM crysteamine. The cleavage of PARP, thought to be resulted from caspase-3 activation, occurred after irradiation, which was attenuated by the pretreatment of 1mM cysteamine. CONCLUSION: these results show that radiation induced apoptotic process is somewhat different from death receptor induced one and the pretreatment of 1 mM cysteamine has a tendency to decrease the radiation-induced apoptosis in HL-60 cells.
Apoptosis* ; Caspase 3 ; Caspase 8 ; Cysteamine* ; HL-60 Cells ; Humans

Cordyceps species including Cordyceps bassiana are a notable anti-cancer dietary supplement. Previously, we identified several compounds with anti-cancer activity from the butanol fraction (Cb-BF) of Cordyceps bassiana. To expand the structural value of Cb-BF-derived anti-cancer drugs, we employed various chemical moieties to produce a novel Cb-BF-derived chemical derivative, KTH-13-amine-monophenyl [4-isopropyl-2-(1-phenylethyl) aniline (KTH-13-AMP)], which we tested for anti-cancer activity. KTH-13-AMP suppressed the proliferation of MDA-MB-231, HeLa, and C6 glioma cells. KTH-13-AMP also dose-dependently induced morphological changes in C6 glioma cells and time-dependently increased the level of early apoptotic cells stained with annexin V-FITC. Furthermore, the levels of the active full-length forms of caspase-3 and caspase-9 were increased. In contrast, the levels of total forms of caspases-3, caspase-8, caspase-9, and Bcl-2 were decreased in KTH-13-AMP treated-cells. We also confirmed that the phosphorylation of STAT3, Src, and PI3K/p85, which is linked to cell survival, was diminished by treatment with KTH-13-AMP. Therefore, these results strongly suggest that this compound can be used to guide the development of an anti-cancer drug or serve as a lead compound in forming another strong anti-proliferative agent.
Apoptosis ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Survival ; Cordyceps* ; Dietary Supplements ; Glioma ; Phosphorylation

OBJECTIVETo detect whether the murine T-cell lymphoma cell line EL4 could be infected by murine cytomegalovirus (MCMV), and to observe the morphological changes and apoptosis of El4 cells before and after infection.

METHODSEL4 cells were infected with MCMV smith strain with 1, 10 and 100 multiplicity of infection (MOI) respectively. The morphology of the cells was observed by light microscopy and Wright's-Giemsa staining. The survival rate was calculated by trypan blue staining. RT-PCR-based assay was used to detect the copy number of MCMV-DNA in the infected ELA cells. Flow cytometry was used to detect apoptosis. RT-qPCR was used to detect the mRNA expression levels of P53, P21, cFlip and Caspase 8. The protein expression levels of Caspase8, P53, BAX, BCL-2 and Cleaved Caspase3 proteins were detected by Western blot.

RESULTSAfter Wright-Giemsa staining, it was found that the infected EL4 cells displayed larger volume, irregular nuclei and the folded twist under light microscopy. Compared with the normal control group, the survival rate of EL4 cells decreased, and the apoptosis rate statistically significantly (P<0.05) increased with the increasing MOI and the infected time in each group. While, the level of apoptosis protein P53, BAX/BCL-2, Cleaved-caspase3 and Caspase8 were up-regulated. And the survival rate, apoptosis rate and the apoptosis protein level of infected EL4 cells with MOI=10 were the most obvious at the 5day. Compared with MCMV infection group (MOI=60), the content of MCMV DNA in EL4 cells was decreased in MCMV+GGV group ［MOI=60, GCV 25 (g/ml)］, and the cell apoptosis rate and apoptosis protein expression of P53, Caspase8, BAX/BCL-2 were decreased (P<0.05).

CONCLUSIONMurine T-cell lymphoma cell line EL4 can be infected by MCMV and displayes an obvious apoptosis phenomenon. MCMV may up-regulate the expression levels of apoptosis protein P53, BAX-BCL-2, Cleaved-caspase3 and Caspase8 in EL4 cells. The drug ganciclovir reduces the copy mumber of MCMV DNA in infected EL4 cells and inhibited the killing effect of MCMV on EL4 cells.

PURPOSE: We investigated the effect of ceramide on keratocyte apoptosis and pathway of ceramide-induced keratocyte apoptosis. METHODS: Cultured Newzealand White Rabbit keratocytes were exposed to various concentrations of ceramide type II, VI and phytoceramide type II, VI. LDH level was measured for the evaluation of time and concentration related apoptosis. Keratocytes were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK, diffuse caspase inhibitor), specific caspase-8 inhibitor (IETD-CHO) and specific caspase-9 inhibitor (Z-LEHD-FMK), then were exposed to 20 micro M of 4 types of ceramide. Cytochome C immune stainining was done after exposure of keratocyte to 4 types of ceramide. RESULTS: The lower effective dose of 4 types of ceramide was 20 micro M. Apoptosis of keratocytes was dependent on ceramide exposure time. Ceramide induced keratocyte apoptosis was inhibited by CPP32-like protease inhibitor, specific caspase-8 inhibitor and specific caspase-9 inhibitor. Apoptotic keratocytes induced by ceramide were immune-stained with cytochrome C antibody. CONCLUSIONS: Ceramide induced apoptosis in cultured corneal keratocytes. This apoptosis developed according caspase cascade, especially via mitochondria.
Apoptosis* ; Caspase 8 ; Caspase 9 ; Corneal Keratocytes ; Cytochromes c ; Mitochondria ; Protease Inhibitors

PURPOSE: To evaluate the effect of variable ceramides on the apoptosis of corneal endothelial cell and then, if ceramide induce the apoptosis in endothelial cells, via which pathway apoptosis occur. METHODS: Corneal endothelial cells were isolated from fresh rabbit cornea and cultured. Cultured corneal endothelial cells were exposed to 10, 20, 40 and 80 micro M of ceramide type II, VI and phytoceramide type II, VI. And then, apoptosis was evaluated with Hoechst staining and flow cytometric analysis with Annexin V for evaluation of apoptotic response. Corneal endothelial cells were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK(R)), specific caspase-8 inhibitor(IETD-CHO(R)) and specific caspase-9 inhibitor (Z-LEHD-FMK(R)), then treated with 20 M of 4 types of ceramide. 12 hours later, LDH assay was done. Cytochrome c immunostaining was done after exposure to 4 types of ceramide. RESULTS: Shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation were observed on Hoechst staining. In flow cytometric analysis, early apoptotic responses were identified. Apoptotic response increased significantly at the concentration of 10M and more 12 hours later. CPP32-like protease inhibitor, caspase-8, 9 inhibitor reduced the LDH activity. Apoptotic endothelial cells induced by ceramide were stained with cytochrome c antibody. CONCLUSIONS: Ceramide induced apoptosis in cultured corneal endothelial cells. This apoptosis developed via caspase and mitochondrial pathway.
Annexin A5 ; Apoptosis* ; Caspase 8 ; Caspase 9 ; Ceramides ; Cornea ; Cytochromes c ; Cytoplasm ; Endothelial Cells* ; Protease Inhibitors

Cells undergo apoptosis mainly via two pathways-the mitochondrial pathway and the cytosolic pathway. It has been well documented that activation of the mitochondrial pathway promotes mitochondrial fragmentation and inhibition of mitochondrial fragmentation partly represses cell death. However, the mitochondrial events following activation of the cytosolic pathway are less understood. In this study, we treated Fas-activating antibody and found mitochondrial fragmentation without cell death in hippocampal primary neurons and HT-22 cell lines. Fas antibody treatment, in fact, promoted rapid activation of caspase-8, while executioner caspase-3 activation was not observed. Furthermore, blockage of caspase-8 efficiently prevented Fas antibody-induced mitochondrial fragmentation. These results suggest that the cytosolic pathway induced by death receptor activation promotes caspase-8-dependent mitochondrial fission.
Apoptosis ; Caspase 3 ; Caspase 8* ; Cell Death ; Cell Line ; Cytosol ; Mitochondrial Dynamics* ; Neurons*

PURPOSE: The purpose of this study was to determine if ceramide, which is known as secondary messenger of programmed cell death (apoptosis), can cause apoptosis in lens epithelial cell (LEC) and if so, to identify the pathway by which apoptosis occurs. METHODS: After LECs were exposed to various concentrations of ceramide and phytoceramide, we evaluated the resulting apoptosis response using the Hoechst-EthD stain and Annexin stain. To search for the apoptosis pathway, LECs were preincubated in various concentrations of CPP32-like protease inhibitor, specific caspase-8 inhibitor, and specific caspase-9 inhibitor, then treated with ceramide and phytoceramide. We performed LDH assay 12 hours later. Cytochrome c immunostaining was done after exposure to the ceramide and phytoceramide. RESULTS: All kinds of ceramide induced time and concentration dependent apoptosis in LEC. Caspase 8 inhibitor and caspase 9 inhibitor reduced the apoptosis in ceramide VI, phytoceramide II, and phytoceramide VI. In all ceramides, cytochrome c staining was positive. CONCLUSIONS: Ceramide and phytoceramide can cause apoptosis in LEC. Ceramide and phytoceramide may be used to prevent the posterior capsular opacity after cataract surgery.
Apoptosis* ; Caspase 8 ; Caspase 9 ; Cataract ; Cell Death ; Ceramides ; Cytochromes c ; Epithelial Cells* ; Protease Inhibitors

BACKGROUND: Although deferasirox (DFX) is reported to have anti-tumor effects, its anti-leukemic activity remains unclear. We evaluated the effect of DFX treatment on two murine lymphoid leukemia cell lines, and clarified the mechanisms underlying its potential anti-leukemic activity. METHODS: L1210 and A20 murine lymphoid leukemia cell lines were treated with DFX. Cell viability and apoptosis were evaluated by the 3-(4,5-dimethylthaizol-2-yl)-5-(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and fluorescence-activated cell sorting (FACS) analysis, respectively. Immunoblotting was performed to detect the expression of key apoptotic proteins. RESULTS: In dose- and time-dependent manner, DFX decreased viability and increased apoptosis of murine leukemic cells. Fas expression was significantly higher in A20 cells than in L1210 cells at all DFX concentrations tested. Although both cell lines exhibited high caspase 3 and caspase 9 expression, a critical component of the intrinsic mitochondrial apoptotic pathway, expression was greater in L1210 cells. In contrast, caspase 8, a key factor in the extrinsic apoptotic pathway, showed greater expression in A20 cells. Cytochrome c expression was significantly higher in L1210 cells. In both cell lines, co-treatment with ferric chloride and DFX diminished the expression of these intracellular proteins, as compared to DFX treatment alone. CONCLUSION: Treatment with DFX increased caspase-dependent apoptosis in two murine lymphoid leukemia cell lines, with differing apoptotic mechanisms in each cell line.
Apoptosis* ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Line* ; Cell Survival ; Cytochromes c ; Flow Cytometry ; Immunoblotting ; Leukemia* ; Leukemia, Lymphoid

Chronic hypoxia has been associated with change in neurovascular behavior, mediated, in part, by erythropoietin (EPO). EPO, a hematopoietic growth factor, could act as a neurotrophic factor. In the present study, we investigated the characteristics of EPO and erythropoietin receptor (EPOR) expressions by cortical neuron in vivo and in vitro and tested the hypothesis that EPO serves protective functions under chronic hypoxia. E18, P5 and P7 mice for 3 days and primary cultured neurons for 6 days were incubated in hypoxic conditions consisted of a mixture of 10% O2, 5% CO2, 85% N2. To study expressions of EPO, EPOR, caspases, pAKT, pERK, and PARP, immunohistochemical stainning and western blotting were carried out. In addition to expressing EPO and EPOR under normoxic conditions, neurons increased their expression of EPO and EPOR under hypoxia. The effects of recombinant EPO appeared to be mediated via the phosphatidylinositol (PI) 3- kinase-AKT pathway, correlated directly with activation of caspase 3. Also recombinant EPO decreased expression of caspase 8, but not caspase 9. Finally, recombinant EPO decreased apoptosis of cultured neurons as evaluated by expression of PARP. These data support a role for EPO in maintenance of cortical neuron under chronic hypoxia.
Animals ; Anoxia* ; Apoptosis ; Blotting, Western ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Caspases ; Erythropoietin ; Mice ; Neurons* ; Phosphatidylinositols ; Receptors, Erythropoietin

Columbianadin (CBN), a natural coumarin from Angelica decursiva (Umbelliferae), is known to have various biological activities including anti-inflammatory and anti-cancer effects. In this study, the anti-proliferative mechanism of actions mediated by CBN was investigated in HCT-116 human colon cancer cells. CBN effectively suppressed the growth of colon cancer cells. Low concentration (up to 25 μM) of CBN induced apoptosis, and high concentration (50 μM) of CBN induced necroptosis. The induction of apoptosis by CBN was correlated with the modulation of caspase-9, caspase-3, Bax, Bcl-2, Bim and Bid, and the induction of necroptosis was related with RIP-3, and caspase-8. In addition, CBN induced the accumulation of ROS and imbalance in the intracellular antioxidant enzymes such as SOD-1, SOD-2, catalase and GPx-1. These findings demonstrate that CBN has the potential to be a candidate in the development of anti-cancer agent derived from natural products.
Angelica ; Apoptosis* ; Biological Products ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Catalase ; Cell Proliferation* ; Colon* ; Colonic Neoplasms* ; Humans ; Oxidative Stress