The aim of this study was to investigate the apoptosis effect of Jurkat cells induced by ursolic acid (UA) and its molecular mechanism so as to provide the theoretical basis for treatment of hematological malignancies by using UA. The cytotoxic effect of different concentration UA on Jurkat cells and inhibitory effect of caspase-9 inhibitor on cytotoxicity of UA were assayed by using WST-8 method; the Jurkat cells treated with 20 or 40 micromol/L UA for 2 or 4 hours were collected and were stained by Annexin/PI, then the apoptosis rate of Jurkat cells was detected by flow cytometry; the Jurkat cells in logarithmic growth phase were collected after treatment with different concentrations of UA for different times, the cell protein was extracted, then the activation of caspase-9, -3 and cytochrome C as well as phosphorylation level of Akt were determined by Western blot. The results indicated that the cytotoxic effect of UA on Jurkat cells was significant. UA induced apoptosis of Jurkat cells. Caspase-9, caspase-3 and cytochrome C were activated, and the phosphorylation of Akt was inhibited in the Jurkat cell apoptosis process induced by UA. It is concluded that the UA shows significant cytotoxic effect on Jurkat cells, UA can induce apoptosis of Jurkat cells through the mitochondria pathway. The mechanism may be associated with the inhibition of Akt phosphorylation.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cytochromes c ; metabolism ; Humans ; Jurkat Cells ; Triterpenes ; pharmacology

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

Apoptotic protease activating factor-1 (Apaf1) is an essential factor in intrinsic mitochondrial pathway of apoptosis activation. Apaf1 leads to the formation of apoptosome, which then proteolytically activates caspase-9. The activated caspase-9 opens the downstream signal of caspases to execute programmed cell death. Apaf-1 is important for tumor suppression and drug resistance because it plays a central role in DNA damage-induced apoptosis. Inactivation of the Apaf-1 gene is implicated in disease progression and chemoresistance of some malignancies. Further research on the Apaf-1 will contribute to develop a new type of approach to anti-cancer drugs, which might have good prospect in clinical practice. In this paper, the structure and function of Apaf-1, the mechanism involved in Apaf-1 signaling pathway, and application of Apaf-1 in tumor therapy were reviewed.
Apoptosis ; Apoptotic Protease-Activating Factor 1 ; metabolism ; Caspase 9 ; metabolism ; Humans ; Neoplasms ; therapy ; Signal Transduction

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 investigate the apoptosis and proliferation effect of matrine on human medulloblastoma cell line D341 in vitro and the effect of the expression of the related caspase 3 and caspase 9 proteins.

METHODSThe D341 cells were cultivated successfully in vitro. Then the cells were divided into 5 groups according to the concentration of matrine (0.5 mg/mI group, 1.0 mg/ml group, 1.5 mg/ml group, 2.0 mg/ml group and the control group was 0 mg/ml). All the experiments were repeated three times. The cell morphologic and structure change was observed with the optical microscope and the transmission electron microscope. The proliferation of D341 cell was analyzed using Cell Counting Kit-8 assay. Apoptosis was detected by Annexin V-FITC/PI double staining. The expression of Caspase3 and Caspase9 was detected by Western blot.

RESULTSWith the effect of matrine, the proliferation inhibition rate gradually increased with drug concentrations increasing, and there was a significant difference (P < 0.01). The inhibitory effect of matrine on cell proliferation was different with the different treatment time, there was a significant difference between the 24 h to 72 h groups (P < 0.01). The apoptotic rate increased with matrine concentrations increasing. There were significant differences between the group of 0.5 mg/mI or 1.0 mg/mI to the group of 1.5 mg/mI or 2.0 mg/mI (P < 0.05). The apoptotic rate increased with the prolonged treatment time. There were significant differences between the group of 24 h or 48 h to the group of 72 h ( P < 0.05). With the increase of matrine concentration, the expression of Caspase 3 and Caspase 9 increased (P < 0.01).

CONCLUSIONMatrine induces the apoptosis, and inhibits the proliferation of human medulloblastoma D341 cells in vitro by up-regulation of the expression level of Caspase3, Caspase9.

Alkaloids ; pharmacology ; Apoptosis ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cerebellar Neoplasms ; metabolism ; pathology ; Humans ; Medulloblastoma ; metabolism ; pathology ; Quinolizines ; pharmacology ; Up-Regulation

OBJECTIVETo observe the expressions of caspase-8 and caspase-9 mRNA, and explore the changes of apoptosis of bone marrow hematopoietic cells in patients with chronic mountain sickness (CMS).

METHODSOf 18 CMS patients and 16 controls were enrolled in this study. The apoptotic index (AI) of bone marrow mononuclear cells (BMMNC) was measured by TUNEL technique, the levels of caspase-8 and caspase-9 mRNA in BMMNC of CMS patients and controls were determined by RT-PCR. Results (1)The AI of BMMNC in patients with CMS (8.51 ± 3.35)% was lower than that in controls (16.00 ± 4.28)% (P < 0.01); (2) The values of caspase-8 and caspase-9 mRNA were (0.28 ± 0.07) and (0.23 ± 0.08) respectively, in CMS patients, which were significantly lower than those of (0.45 ± 0.09) and (0.41 ± 0.09) respectively, in the controls (both P < 0.01); (3) Hemoglobin (Hb) value was negatively correlated with levels of caspase-8 and caspase-9 mRNA (r values were -0.52 and -0.61 respectively, both P < 0.05) in CMS patients. There was a negative correlation between AI and Hb (r value was -0.89, P < 0.01) in CMS patients. However, the significant relationship was not found between AI and level of caspase-8 or caspase-9 mRNA (P > 0.05).

CONCLUSIONSThe results showed a decrease apoptosis of BMMNCs and reduced levels of caspase-8 and caspase-9 mRNA in CMS patients, the latter might be involved in the change of BMMNCs apoptosis.

Adult ; Altitude Sickness ; metabolism ; pathology ; Apoptosis ; Bone Marrow Cells ; metabolism ; Case-Control Studies ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Humans ; Male ; Middle Aged

OBJECTIVETo investigate the roles of cytochrome c (Cyt-c), caspase-9, and caspase-3 in pentavalent vanadium-induced neuronal apoptosis and to provide a basis for mechanism research.

METHODSNeurons from rats aged 1-3 days were cultured and treated with vanadium pentoxide (V2O5) at 5, 10, or 20 mmol/L. Neuronal apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL). The protein levels of Cyt-c, caspase-9, and caspase-3 were determined by Western blot.

RESULTSApoptosis bodies were detected in the nuclei of neurons by TUNEL. The number of neurons with apoptosis bodies increased with increasing dose of V2O5 The apoptosis index (AI) was significantly higher in the 10 and 20 mm/L exposure groups than in the control group (P < 0.05 or P < 0.01). Western blot showed that the protein expression levels of Cyt-c and caspase-3 significantly increased in the 5 mmol/L exposure group as compared with the control group (P < 0.05). In the 10 and 20 mmol/L exposure groups, the protein expression of Cyt-c, caspase-9, and caspase-3 all increased as compared with the control group (P < 0.01). Neuronal AI was positively correlated with Cyt-c, caspase-9, and caspase-3 (r = 0.954, P < 0.01; r = 0.938, P < 0.01; r = 0.943, P < 0.01).

CONCLUSIONPentavalent vanadium may induce neuronal apoptosis. The protein expression of Cyt-c, caspase-9, and caspase-3 may play an important role in neuronal apoptosis induced by pentavalent vanadium.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cytochromes c ; metabolism ; Neurons ; drug effects ; metabolism ; Rats ; Vanadium ; toxicity ; Vanadium Compounds ; toxicity

The purpose of this study was to investigate the effect of bone marrow mesenchymal stem cells (BMMSC) from patients with chronic myeloid leukemia (CML) in blastic phase (Bp) on K562 cells and the primary CML-Bp cells, and to explore its potential mechanisms. K562 cells and primary CML-Bp cells were co-cultured with BMMSC of different groups; the cell proliferation was detected by MTT method, the cell apoptosis rate and mitochondrial membrane potential were measured by flow cytometry, the expression levels of Caspase-8, Caspase-9, and activated Caspase-3 in cells were measured by Western blot. The results showed that the CML-Bp BMMSC could enhance the survival rate of K562 cells treated with adviamycin (ADM) and display protective effect on K562 cells and primary CML-Bp mononuctear cells, inhibited ADM-induced leukimia cell apoptosis (P < 0.05); as compared with CML-chronic phase (CML-Cp) BMMSC and normal BMMSC, the CML-Bp BMMSC showed the highest protective effect on leukemic cells, the mitochondrial membrane potential of co-cultured cells slightly droped (P < 0.05). In the CML-Bp BMMSC cultured with K562 cells, the expression level of caspase-3 was more down-regulated than that in K562 alone plus ADM group, while the expression of caspase-9 significantly increased (P < 0.05). It is concluded that the CML-Bp BMMSC down-regulates ADM-induced leukemia cell appoptosis, its mechanism may relate with the inhibition of mitochondrial membrane potential drop, the stabilization of unactive expression of caspase-9 and down-regulation of caspase-3 expression.
Apoptosis ; Bone Marrow ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Proliferation ; Cells, Cultured ; Down-Regulation ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Mesenchymal Stromal Cells ; cytology ; metabolism

OBJECTIVE: To evaluate the inhibited effect of matrine on human nasopharyngeal carcinoma CNE2 cells and the expression of apoptosis-related gene Caspase-3 mRNA, Caspase-3, Caspase-8, Caspase-9 protein. And to explore the inhibiting effect of matrine on the apoptosis of human nasopharyngeal carcinoma CNE2 cells. METHOD: In vitro experiments, MTT assay was used to detect the effect of matrine on CNE2 cell proliferation with different concentration. The expression levels of Caspase-3 were detected by real-time PCR. Western Blot was used to gauge the levels of Caspase-3, Caspase-8 and Caspase-9 protein. RESULT: MTT results showed significant inhibitory action of matrine on CNE2 cells proliferation in does-dependent manner, which could up-regulate the expression of Caspase-3 mRNA and Caspase-3, Caspase-8, Caspase-9 protein in a dose-dependent manner. CONCLUSION Matrine could inhibit CNE2 cells proliferation in a does-dependent,that was closely related to the up-regulation of Caspase-3 mRNA and Caspase-3, Caspase-8 and Caspase-9 protein expression, and to the cascade reaction of Caspase.
Alkaloids ; pharmacology ; Apoptosis ; drug effects ; Carcinoma ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Caspases ; metabolism ; Cell Line, Tumor ; Humans ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; enzymology ; pathology ; Quinolizines ; pharmacology