This study was aimed to investigate the mechanism of phosphatidylserine exposure of human erythrocytes induced by high concentrated glucose. After exposure to high concentrated glucose, the phosphatidylserine (PS) exposure and forward scatter value were analyzed by flow cytometry; the activities of caspase-3 and caspase-8 were detected; The inhibitory effect of leupeptin on cell PS exposure induced by high concentrated glucose was observed by flow cytometry and fluorescent microscopy. The results showed that the high concentrated glucose could induce PS exposure of erythrocytes and this inducing efficiency was dependent on the glucose concentrations. With increase of the glucose concentrations, the percentages of cells with exposed PS also increased. When the glucose concentration was 0.8 mol/L, the PS exposure was over 80%. However, caspase-3 and caspase-8 were not activated during PS exposure of cells induced by high concentrated glucose, but leupeptin could significantly inhibit PS exposure and volume shrinkage induced by high concentrated glucose. With increase of the leupeptin concentrations, the percentage of cells with exposed PS decreased and the cell volume increased. It is concluded that the high concentrated glucose can result in serious PS exposure, which does not depend on caspase. It can be hypothesized that the PS exposure of erythrocytes induced by high concentrated glucose may be controlled by an unknown pathway sensitive to leupeptin.
Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Erythrocytes ; drug effects ; metabolism ; Flow Cytometry ; Glucose ; administration & dosage ; pharmacology ; Humans ; Phosphatidylserines ; biosynthesis

OBJECTIVETo investigate the effects of Mycobacterium tuberculosis on apoptosis of mouse dendritic cells (DC 2. 4) and the activation of caspase-3, caspase-8.

METHODSMycobacterium tuberculosis H37Rv strain was co-cultured with DC 2. 4 cells. The morphological changes of DC 2. 4 cells were observed with fluorescence microscope after DAPI staining and transmission electron microscope. The apoptosis of DC 2. 4 cells were examined by DNA agarose gel electrophoresis. The activities of caspase-3 and caspase-8 were detected by colorimetric assay.

RESULTSBacterial invasion was observed while DC 2. 4 cells and H37Rv were co-cultured for 2 h; and the rates of invasion were (16.1 ± 4.3)%, (35.8 ± 5.1)%, (50.2 ± 5.7)%, (58.3 ± 6.2)% and(65.9 ± 6.9)% at 4, 6, 8,10, 12 h, respectively. The phenomenon of nuclear condensation and marginalization were shown by DAPI staining and transmission electron microscope in DC 2. 4 cells at 6 h of co-cultivation with H37Rv. The characteristic bands of apoptosis by DNA electrophoresis were detected. The activities of caspase-3 and caspase-8 were increased in a time-dependent manner. The rates of DC 2. 4 cell apoptosis were (6.4 ± 2.5)%, (11.8 ± 5.3)% and (31.1 ± 8.7)% at 6 h,12 h and 24 h after co-cultivation with H37Rv, respectively. The maximal activities of intracellular caspase-3 and caspase-8 at 10 h and 6 h were (2.01 ± 0.09) U/μg and (2.40 ± 0.07)U/μg, respectively, which was significantly different compared with the control groups(P<0.05). The activation of caspase-8 was earlier than that of caspase-3.

CONCLUSIONMycobacterium tuberculosis can induce the apoptosis of DC 2. 4 cells, which is associated with the activation of intracellular caspase-3 and caspase-8.

Animals ; Apoptosis ; physiology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cells, Cultured ; Dendritic Cells ; metabolism ; pathology ; Mice ; Mycobacterium tuberculosis

OBJECTIVETo investigate the effects of snakegourd root polysaccharide on apoptosis of human breast cancer cells (MCF-7 cells).

METHODSColorimetric MTT assay was used to measure the inhibition of snakegourd root polysaccharide on MCF-7 cells. The morphological changes of MCF-7 cells were observed by fluorescence microscope after DAPI staining and transmission electron microscope. The apoptosis of MCF-7 cells was examined by DNA agarose gel electrophoresis analysis of DNA fragmentation amd flow cytometry. The activity of Caspase-3 and Caspase-8 was detected by colorimetric assay.

RESULTSPolysaccharide of snakegourd root significantly inhibited MCF-7 cells in a dose-and time-dependent manner. The nuclear condensation and marginalization were observed by DAPI staining and transmission electron microscope. The characteristic ladder of apoptosis in DNA electrophoresis was detected in MCF-7 cells treated with 10.0 μmol/L polysaccharide of snakegourd root at d 2. The activities of Caspase-3 and Caspase-8 were increased in a time-dependent manner. The rates of apoptosis in MCF-7 cells were (5.2 ±1.3)%, (13.1 ±4.7)%, (27.6 ±6.8)% and (43.8 ±9.8)% treated with 1.0,5.0,10.0 and 20.0 μmol/L snakegourd root polysaccharide at d 2,respectively. The maximal activities of intracellular Caspase-3 and Caspase-8 were (2.32 ±0.12)U/μg and (1.92 ±0.11)U/μg at d 2 and d 1, respectively when MCF-7 cells were treated with 10.0 μmol/L.

CONCLUSIONThe polysaccharide of snakegourd root can induce the apoptosis of MCF-7 cells,which is associated with the activation of intracellular Caspase-3 and Caspase-8.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Humans ; MCF-7 Cells ; Plant Roots ; chemistry ; Polysaccharides ; pharmacology ; Trichosanthes ; chemistry

OBJECTIVETo observe the change of caspase-8, caspase-9 activity and apoptosis rates in the process of trichloroethylene-induced damage in keratinocytes, and explore the tentative mechanism of apoptosis.

METHODSHuman keratinocytes were exposed to 0.125, 0.250, 0.500, 1.000 and 2.000 mmol/L trichloroethylene for 4, 8, 12 and 24 h. The inhibitive groups were pretreated with 100 micromol/L Z-LEHD-FMK (a specific inhibitor of caspase-9) for 1 h, and were stimulated with 2.000 mmol/l TCE for 12 h. MTT assay was used to detect the viability of different cells; The activity of caspase were calculated according to spectrophotometry; Change of the apoptotic rates was assessed by flow cytometer (FCM) after double-stained with Annexin V-FITC and propidium iodide (PI).

RESULTS(1) The minimum effective concentration for cell viability reduction was 0.125 mmol/L at 12 h and the shortest time required to produce a change was 4 h at a concentration of 2.000 mmol/L (compared with control group, P < 0.01). Cell viability in all the groups markedly decreased from 12 h to 24 h (P < 0.05). (2) The activity of caspase-8 in the various dosage groups at different times had no statistical difference compared with the control group, P > 0.01. (3) At 8 h, 1.000 and 2.000 mmol/L TCE groups could significantly enhance caspase-9 activity (P < 0.05). The caspase-9 activity in all the groups showed differences and was significantly higher than those of control cells when time was over 12 h (P < 0.05). (4) After exposing to different dosages of TCE for 12 h, the rate of apoptosis rose to (80.43 +/- 4.21)% with the increase of dosage, compared with the control group, (9.40 +/- 2.98)%, which showed a dose-effect relationship. (5) The cells pre-treated with caspase-9 inhibitor resulted in a decrease in the caspase-9 activity and apoptosis rates (compared with 2.000 mmol/L TCE exposed group, P < 0.01). However, there was no statistical significance in comparison with the control group (P > 0.05).

CONCLUSIONCaspase-9 may be an important mediator of apoptosis in keratinocytes induced by trichloroethylene.

Apoptosis ; drug effects ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cells, Cultured ; Humans ; Keratinocytes ; drug effects ; enzymology ; pathology ; Trichloroethylene ; toxicity

OBJECTIVETo evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells.

METHODSF344 rats at postnatal day 3 or 4 were used for the study in cochlear organotypic cultures. The cochlear basilar membrane was micro-dissected out and cultured overnight, and then treated with 1 mmol/L streptomycin for 24 hours. Before the termination, the activity of caspase-8, 9 or 6 were detected with FAM-peptide-FMK labeled caspase-8, 9 or 6, respectively. The stereocilia and cuticular plate of hair cells were stained with TRITC conjugated phalloidin, and the nuclei were stained with Topro-3 DNA probe. The specimens were observed and photographed under confocal fluorescent microscope.

RESULTSStreptomycin with 1 mmol/L causes about 80% cochlear hair cells missing in the basal turn and 10% hair cell loss in the apex. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in most cochlear hair cells, and the caspase-8, caspase-9 and caspase-6 were greatly activated.

CONCLUSIONSApoptosis is involved in the cochlear hair cells death induced by Streptomycin in vitro. The caspase activities in upstream and downstream are maybe the major apoptotic pathway.

Animals ; Apoptosis ; drug effects ; Caspase 6 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cells, Cultured ; Hair Cells, Auditory ; cytology ; drug effects ; Rats ; Rats, Inbred F344 ; Streptomycin ; adverse effects

This study was aimed to investigate the apoptosis-inducing effect of gambogic acid (GA) on K562 cell line and its mechanism. The K562 cells were treated with GA at different concentrations and times, the inhibition rates were detected by MTT assay. Apoptosis induced by GA was assayed by Annexin-V/PI doubling staining. The influence of GA on cell cycle was studied by propidium iodide method. The mitochondrial membrane potential was measured by JC assay. The levels of caspase 3, caspase 8 and caspase 9 activated by fluorescein in living K562 cells were measured by caspGLOW(TM) fluorescein staining kit. The results showed that after incubation with GA, K562 cell proliferation was dramatically inhibited in concentration- and time-dependent manners. K562/A02 cells need higher GA concentration (> 2 microg/ml) to show antiproliferative effect, compared with that of K562 cells (> 0.5 microg/ml). Apoptosis could be induced by GA but the influence on cell cycle was not significant. GA could decrease the mitochondrial membrane potential and increase the activated caspase 3, caspase 8, caspase 9 positive cell levels by 2.19%, -1.95%, 34.01% in 24 hr and 60.4%, 71.3%, 77.7% in 48 hr respectively. It is concluded that the GA can significantly inhibit the proliferation of K562 cells without influence on cell cycles. The GA triggers K562 cell apoptosis through both intrinsic and extrinsic pathways.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Humans ; K562 Cells ; Membrane Potential, Mitochondrial ; Xanthones ; pharmacology

OBJECTIVETo study the role of apoptosis and the expression of apoptosis-related genes Fas ligand (FasL), Fas, caspase-3 and caspase-8 in an animal model of non-alcoholic steatohepatitis (NASH).

METHODSAn experimental progressive NASH model was established by feeding male C57BL6/J mice with a high fat, methionine-choline deficient (MCD-) diet for two days, five days, ten days, three weeks and eight weeks. Control mice were fed methionine-choline supplemented (MCD+) diet. Hepatic steatosis, inflammation and fibrosis were graded by examining their H and E stained liver sections. Hepatocyte apoptosis was detected by TUNEL assay. Expressions of mRNA and protein of FasL, Fas and caspase-8 were performed by quantitative real time RT-PCR and Western blot. Caspase-3 activity assay was conducted using ApoAlert caspase-3 assay kit.

RESULTSIn MCD- mice, minimal hepatic steatosis was observed at day 5, and by day 10, mild steatosis with inflammatory infiltration was found. Severe steatohepatitis was noted at week 3, and fibrosis at week 8. TUNEL assay showed that apoptotic index in MCD- group was higher than that in MCD+ group at week 3 (15.59%+/-4.87% vs 5.17%+/-3.19%, P less than 0.05) and at week 8 (11.29%+/-3.22% vs 5.41%+/-1.54%, P less than 0.05). Compared to MCD+ group, the expression of FasL was dramatically increased on day 10 and in week 3 in MCD- mice both at the mRNA and protein levels (P less than 0.05 and P less than 0.01). Expression of Fas mRNA was up-regulated in weeks 3 and 8 (P less than 0.01), and expression of Fas in protein level was higher at week 8 (P less than 0.01) in MCD- group. Expression of caspase-8 significantly increased at the mRNA level at week 3 and week 8 (P less than 0.01 and P less than 0.05 respectively) and at the protein level at week 8 (P less than 0.05) in MCD- group. In all of the time points except for day 5, caspase-3 activities were significantly more enhanced in MCD- group than that in MCD+ group (P less than 0.05).

CONCLUSIONSIn our experimental NASH model, hepatic apoptosis was frequently detected. Increased apoptosis was probably attributable to up-regulation of apoptosis-related genes, such as FasL/Fas system, and activation of the caspase pathway. These changes may provoke hepatic apoptosis and the development of inflammation and fibrosis.

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Fas Ligand Protein ; metabolism ; Fatty Liver ; genetics ; metabolism ; pathology ; Hepatocytes ; metabolism ; Liver ; pathology ; Male ; Mice ; Mice, Inbred C57BL

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cells, Cultured ; HSP70 Heat-Shock Proteins ; metabolism ; Heat Stress Disorders ; metabolism ; Myocytes, Cardiac ; metabolism ; Rats ; Rats, Wistar

As a classic fluorescent detect technique, fluorescence resonance energy transfer (FRET) has been widely used in biological researches. Researchers have developed a series of fluorescence detect probes which were based on FRET. Caspase family plays an important role in apoptosis pathway, especially Caspase-8 which located, at the initial of death receptor mediated apoptosis pathway, whose its activation can trigger subsequent precaspases' activation and lead to apoptosis. So it is of great significance to detect the activation of Caspase-8 in apoptosis assay. In this study, a fluorescent probe based on FRET has been designed which can detect the activity change of Caspase-8 in cells. To identify the effectiveness and specificity of the probe, we measure the Caspase-8 activity under the Caspase-8 specifically activated apoptosis inducer RGD-TRAIL with the flow cytometry FRET detection platform. The results show that the probe can respond to the activity change of Caspase-8 in apoptotic cells, and the change can be quantified rapidly by flow cytometry. The study provides a more efficient and convenient detection method of Caspase-8 activity in living cells.
Apoptosis ; Caspase 8 ; metabolism ; Flow Cytometry ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes ; Humans

The aim of this study was to investigate the effects of the traditional Chinese medicine psoralen (PSO) plus long wave ultraviolet A (PUVA) on apoptosis in HL-60 leukemia cells and its mechanism. The effect of PUVA on HL-60 cell growth was assayed by MTT method and the changes of ultrastructure of cells were observed by electron microscopy. The apoptosis ratios, changes of mitochondrial membrane potential, expression of Fas and FasL protein and Fas and FasL mRNA were detected by FCM and fluorescent quantitation RT-PCR respectively. The expression of Caspase 8 and Caspase 3 protein were detected by immunocytochemistry (ICC). The results showed that the growth of HL-60 cells were inhibited by PUVA in time-and concentration-dependent manner through inducing cell apoptosis. When the irradiation time of long wave ultraviolet A lasted 15 minutes and the concentration of PSO was 80 microg/ml, the inhibition of HL-60 cell proliferation and apoptosis ratios reached the peak. There were obvious apoptotic ultrastructure changes and decrease of mitochondrial membrane potential in HL-60 cells after treatment with PUVA. The expression of Fas mRAN increased and expression of FasL mRNA decreased after treating with PUVA for 4 hours, and the same results of Fas, FasL expression on protein level were obtained also after treating with PUVA for 24 hours. The expression of caspase 8 and caspase 3 protein enhanced and reached the peak after treating with PUVA for 8 hours. It is concluded that the PUVA can inhibit the growth of HL-60 cells and induce apoptosis of these cells. The possible mechanism is supposed to be up-regulating Fas, down-regulating FasL levels and then activating the levels of caspase 8 and caspase 3. The decreasing of mitochondrial membrane potential may be involved in this process probably.
Apoptosis ; drug effects ; radiation effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Fas Ligand Protein ; metabolism ; Ficusin ; pharmacology ; HL-60 Cells ; Humans ; Ultraviolet Rays ; fas Receptor ; metabolism