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

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

OBJECTIVETo study the effect of ginsenoside on apoptosis of human leukemia-60 (HL-60) cells and its mechanism.

METHODSMTT cytotoxicity assay was used to determine the growth inhibition activity of ginsenoside (100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μmol/L) on HL-60 cells. The apoptosis of HL-60 cells after treatment with ginsenoside (0,5,10 and 20 μmol/L) was determined by Annexin V-FITC/PI staining and flow cytometry. The cleavage of total proteins by caspase-8, caspase-9 and caspase-3 was evaluated by Western blot. The cleavage of caspase-3 protein was detected by Western blot after treatment with 10 μmol/L ginsenoside and caspase-8 and 9 inhibitors.

RESULTSGinsenoside had potent cytotoxicity on HL-60 cells, with an IC50 value of 7.3±1.2 μmol/L. After treatment with ginsenoside (0, 5, 10 and 20 μmol/L) for 48 hours, the apoptotic rate displayed a dose dependency, as shown by flow cytometry, with significant differences between the groups (F=12.67, P<0.01). Western blot showed that there were caspase-9 and caspase-3 cleavage bands, but without caspase-8 cleavage band. The specific inhibitor of caspase-9 Z-LEHD-FMK could block the caspase-3 cleavage induced by 10 μmol/L ginsenoside, but the specific inhibitor of caspase-8 Z-IETD-FMK did not have this effect.

CONCLUSIONSGinsenoside can induce apoptosis of HL-60 cells, which may be related to a mitochondria-dependent pathway.

Apoptosis ; drug effects ; Caspase 9 ; physiology ; Caspase Inhibitors ; pharmacology ; Ginsenosides ; pharmacology ; HL-60 Cells ; Humans

PURPOSE: NS398, a selective COX-2 inhibitor, is known to inhibit the growth of COX-2 expressing hepatocellular carcinoma cells. The present study investigated whether the cytotoxic effect of NS398 was COX-2 dependent and whether caspases were involved in NS398-induced apoptosis in hepatocellular carcinoma cells. MATERIALS AND METHODS: The expressions of COX-2 in SNU 423 and SNU 449 hepatocellular carcinoma cell lines were examined using RT-PCR and Western blot. The cytotoxic effect of NS398 was measured using MTT in the presence or absence of caspase inhibitors. The distribution of the cell cycle and extent of apoptosis were analyzed using flow cytometry and a Cell Death Elisa kit, respectively. RESULTS: The expression of COX-2 was observed in SNU423 cells, but not in SNU 449 cells. NS398 treatment resulted in both dose-and time-dependent growth inhibitions, with increases in apoptotic cells in both cell lines. Treatment with the pan-caspase inhibitor, z-VAD- fmk, or the caspase-3 inhibitor, Ac-DMQD-CHO, showed no attenuation of the cytotoxic effect of NS398 in either cell line. CONCLUSIONS: This study demonstrated that the cytotoxic effect of NS398 was independent of COX-2 expression. Caspases were also shown not to be involved in NS398-induced apoptosis in either SNU 423 or SNU 449 Korean HCC cell lines. Our data suggests the feasibility of preventing hepatocellular carcinoma with the use of COX-2 inhibitors needs to be carefully evaluated.
Apoptosis* ; Blotting, Western ; Carcinoma, Hepatocellular* ; Caspase 3 ; Caspase Inhibitors ; Caspases ; Cell Cycle ; Cell Death ; Cell Line* ; Cyclooxygenase 2 ; Cyclooxygenase 2 Inhibitors ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry

BACKGROUND/AIMS: Peroxisome proliferator-activated receptor (PPAR)-gamma ligand is known to inhibit the growth of several kinds of cancer cells, yet its effect on cholangiocarcinoma is indecisive. We investigated the effect of an endogenous ligand of PPAR-gamma, 15-deoxy-delta (12,14)-prostaglandin J2 (15-deoxy-PGJ2) on cholangiocarcinoma cells that were established from intrahepatic cholangiocarcinoma tissue of Korean patients. METHODS: Four cholangiocarcinoma cell lines, Cho-CK, Choi-CK, JCK and SCK, were studied. The mRNA expression of PPAR-gamma, bcl-2, and bax were examined by RT-PCR. Cell viability was determined by MTT assay. The cell cycle was analyzed by flow cytometry, and apoptosis by cell death detection ELISA kit. Caspase activity was measured by colorimetric assay. The effect of caspase inhibitors on 15-deoxy-PGJ2-induced apoptosis was determined by measuring cell viability using the MTT assay. RESULTS: PPAR-gamma mRNA was expressed in all cholangiocarcinoma cells. 15-deoxy-PGJ2 inhibited proliferation of all cells in a dose- and time-dependent manner. All cells treated with 15-deoxy-PGJ2 showed increased dose-dependent apoptosis. Caspase 3 was activated in all cells and caspase 9 was activated in all but JCK cells after 15-deoxy-PGJ2 treatment. Caspase 8 activity showed no significant change. The pan-caspase inhibitor, Z-VAD-FMK, and the caspase-3 inhibitor, Z-DEVD-FMK, blocked 15-deoxy-PGJ2-induced apoptosis in all cells dose-dependently. The expression of bcl-2 was decreased in Cho-CK, Choi-CK and SCK cells, and bax expression was not changed significantly after 15-deoxy-PGJ2 treatment. CONCLUSIONS: PPAR-gamma mRNA was expressed in all Korean cholangiocarcinoma cells. Our data suggest that 15-deoxy-PGJ2 exerts an antineoplastic effect against cholangiocarcinoma cells by inducing apoptosis through caspase activation.
Amino Acid Chloromethyl Ketones ; Apoptosis ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Caspase Inhibitors ; Cell Cycle ; Cell Death ; Cell Line ; Cell Survival ; Cholangiocarcinoma ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Liver Neoplasms ; Oligopeptides ; Peroxisomes ; PPAR gamma ; Prostaglandin D2 ; RNA, Messenger

PURPOSE: The purpose of this study was to evaluate the effect of mitomycin C on rabbit keratocytes for their potential to modulate corneal stromal wound healing. We also investigated the pathway on which the modulation occurs. METHODS: Keratocytes were isolated from New Zealand White Rabbits and cultured. We used Hoechst stain and flowcytometric analysis with Annexin V to identify the kind of response that mitomycin C induced from the keratocytes. After cultured keratocytes were exposed to 0.005%, 0.01%, 0.02%, 0.04%, and 0.06% mitomycin C, we evaluated the response with LDH assay. Next, after exposing the keratocytes to 0.01% mitomycin C, we evaluated the responses with LDH assay at 6, 12, and 24 hours. Keratocytes were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK(R)), specific caspase-8 inhibitor (Z-IETD-FMK(R)), and specific caspase-9 inhibitor (Z-LEHD-FMK(R)), then treated with 0.01% mitomycin C. Twelve hours later, an LDH assay was performed. Cytochrome C immunostain was done after exposure to 0.01% mitomycin C. RESULTS: We observed shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation on Hoechst staining. In flowcytometric analysis, the cells showed apoptotic change. LDH activities increased significantly at a concentration of 0.005% and greater and were time-dependent until 24 hours. CPP32-like protease inhibitor decreased the LDH activity, but there was no statistical significance. Specific caspase-8 and -9 inhibitors significantly reduced the LDH activities that were induced by mitomycin C. The keratocytes which had been pretreated with mitomycin C were stained with cytochrome C antibody. CONCLUSIONS: Mitomycin C induces apoptosis, rather than necrosis, in cultured corneal keratocytes. This apoptosis occurs via the caspase pathway, and is especially related to the mitochondrial pathway, and caspases 8, and 9.
Annexin A5 ; Apoptosis* ; Caspase 8 ; Caspase 9 ; Caspases ; Corneal Keratocytes ; Cytochromes c ; Cytoplasm ; Mitochondria ; Mitomycin* ; Necrosis ; Protease Inhibitors ; Rabbits ; Wound Healing

Emphysema is characterized by air space enlarge?ment and alveolar destruction. The mechanism responsible for the development of emphysema was thought to be protease/antiprotease imbalance and oxidative stress. A very recent study shows that alveolar cell apoptosis causes lung destruction and emphysematous changes. Thus, this study was per?formed to support the evidence for the role of apoptosis in the development of emphysema by characterizing cigarette smoke extract (CSE)-induced apoptosis in A549 (type II pneumocyte) lung epithelial cells. CSE induced apoptosis at low concentration (10% or less) and both apoptosis and necrosis at high concentration (20%). Apoptosis was demonstrated by DNA fragmentation using FACScan for subG1 fraction. Discrimination between apoptosis and necrosis was done by morphologic analysis using fluorescent microscopy with Hoecst 33342/propium iodide double staing and electron microscopy. Cy?tochrome c release was confirmed by using immuno?fluorescence with monoclonal anti-cytochrome c antibody. However, CSE-induced cell death did not show the activation of caspase 3 and was not blocked by caspase inhibitors. This suggests that CSE-induced apoptosis might be caspase-independent apoptosis. CSE-induced cell death was near com?pletely blocked by N-acetylcystein and bcl-2 over?expression protected CSE-induced cell death. This results suggests that CSE might induce apoptosis through intracellular oxidative stress. CSE also activated p53 and functional knock-out of p53 using stable overexpression of HPV-E6 protein inhibited CSE-induced cell death. The characterization of CSE-induced cell death in lung epithelial cells could support the role of lung cell apoptosis in the patho?genesis of emphysema.
Apoptosis ; Caspase 3 ; Caspase Inhibitors ; Cell Death* ; Discrimination (Psychology) ; DNA Fragmentation ; Emphysema ; Epithelial Cells* ; Lung* ; Microscopy ; Microscopy, Electron ; Necrosis ; Oxidative Stress ; Smoke* ; Tobacco Products*

OBJECTIVEThe loss of caspase-8 expression correlates with unfavorable survival outcomes in neuroblastoma (NB). Caspase-8 gene inactivation is caused by methylation. This study aimed to explore the effect of the demethylation agent 5-azacytidine on caspase-8 expression and whether 5-azacytidine can increase the sensitivity of chemotherapy drug doxorubicin to NB cells.

METHODSCaspase-8 mRNA expression in NB cell lines (SH-SY5Y cells) was examined by RT-PCR before and after 5-azacytidine treatment. Survival rates of SH-SY5Y cells were detected using MTT analysis and compared among the doxorubicin alone treatment, 5-azacytidine along with doxorubicin treatment, and caspase-8 inhibitor+5-azacytidine+doxorubicin treatment groups.

RESULTSCaspase-8 mRNA was not expressed in untreated SH-SY5Y cell lines. Caspase-8 mRNA expression in SH-SY5Y cells was detectable 3 days after 5-azacytidine treatment, and increased significantly 5 days after 5-azacytidine treatment (P < 0.05). Survival rates of SH-SY5Y cells treated with 5-azacytidine along with different concentrations of doxorubicin (0.05, 0.1,0.25, 0.5 microg/mL) were (77.61 +/- 7.30)%, (57.35 +/- 6.64)%, (46.25 +/- 4.46)% and (35.59 +/- 5.12)%, respectively, which were significantly lower than those treated with doxorubicin alone (94.89 +/- 4.15%, 80.60 +/- 8.50%, 64.48 +/- 4.92% and 52.32 +/- 6.71%) (P < 0.01). Caspase-8 inhibitor pretreatment resulted in an increased survival rate of SH-SY5Y cells (92.95 +/- 3.48%, 78.39 +/- 4.28 %, 62.31 +/- 6.50% and 49.92 +/- 5.77%) compared with the 5-azacytidine+doxorubicin treatment group.

CONCLUSIONS5-azacytidine may enhance anti-tumor efficacy of doxorubicin to NB cell lines, possibly through an up-regulation of caspase-8 mRNA expression.

Antineoplastic Agents ; pharmacology ; Azacitidine ; pharmacology ; Caspase 8 ; genetics ; Caspase Inhibitors ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Synergism ; Humans ; RNA, Messenger ; analysis

BACKGROUNDA nonsteroidal anti-inflammatory drug, indomethacin, has been shown to have anti-leukemic activity and induce leukemic cell apoptosis. This study was to elucidate the mechanism of indomethacin-induced K562 cell apoptosis.

METHODSK562 cells were grown in RPMI 1640 medium and treated with different doses of indomethacin (0 micromol/L, 100 micromol/L, 200 micromol/L, 400 micromol/L, 800 micromol/L) for 72 hours. The cells were harvested, and cell viability or apoptosis was analyzed using MTT assay and AO/EB stain, combining laser scanning confocal microscopy (LSCM) technique separately. For the localization and distribution of intracellular caspase-3 or caspase-8 protein, immunofluorescence assay was carried out. To reveal the activation of caspase-3 or caspase-8 in indomethacin-treated cells, Western blot detection was used. The change in intracellular free calcium was determined by Fluo-3/Am probe labeling combined with LSCM.

RESULTSIndomethacin could lead to K562 cell apoptosis and inhibit cell viability in a concentration-dependent manner. An increased expression of intracellular caspase-3 or caspase-8 was observed at higher doses of indomethacin (400 - 800 micromol/L). Western blot results showed upregulation and activation in both caspase-3 and caspase-8 protein. Under indomethacin intervention, the levels of intracellular free calcium showed a significant increase. Blocking the activity of cyclooxygenase did not abolish the effects of indomethacin on K562 cell apoptosis.

CONCLUSIONSActivation and upregulation of caspase-3 or caspase-8 protein were responsible for Indomethacin-induced K562 cell apoptosis. Variation of intracellular free calcium might switch on the apoptotic pathway and the proapoptotic effect of indomethacin might be cyclooxygenase-independent.

Apoptosis ; drug effects ; Calcium ; metabolism ; Caspase 3 ; Caspase 8 ; Caspases ; genetics ; metabolism ; Cyclooxygenase Inhibitors ; pharmacology ; Enzyme Activation ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Indomethacin ; pharmacology ; K562 Cells