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

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 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 expression of Caspase-3 and Hsp70 in rabbits after severe traumatic brain injury (TBI) and to explore the feasibility of its application in estimation of injury time in forensic medicine.

METHODSA rabbit model of heavy TBI was developed by high velocity impact on the parietal bone with an iron stick. Totally 8 healthy adult New Zealand white rabbits were randomly divided into control group (n equal to 2) and injury group (n equal to 6). Four hours after injury, tissue specimens from the parietal lobe, temporal lobe, occipital lobe, cerebellum and brainstem were harvested to detect the expression of Hsp70 and Caspase-3 by immunohistochemistry. Besides, the gray values of cells positive for Hsp70 and Caspase-3 were analyzed with an image analyzer.

RESULTSImmunohistochemistry staining demonstrated a low level of Caspase-3 and Hsp70 expression in normal control group. While in injury group, both the Caspase-3 and Hsp70 expression was significantly elevated (P less than 0.05). Positive cells gathered around the lesion focus. Occipital lobe and cerebellum had fewer positive cells while temporal and brainstem had the fewest.

CONCLUSIONThe expression of Caspase-3 and Hsp70 at an early stage following severe TBI is characteristic and can be applied to estimate the time of injury.

Animals ; Brain Injuries ; metabolism ; Caspase 3 ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Immunohistochemistry ; Rabbits ; Random Allocation

OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.
Mice ; Animals ; Alocasia/metabolism* ; MAP Kinase Signaling System ; Caspase 3/metabolism* ; Apoptosis ; RNA, Messenger/metabolism*

OBJECTIVETo investigate the apoptosis of J774A.1 cells induced by Leptospira interrogans and the effect of caspase-3, -6 activation on the apoptosis.

METHODSMouse monocyte-macrophage like cell line J774A.1 was infected by L.interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae Lai strain 56601. The apoptosis or necrosis of infected cells was examined by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The activity of caspase-3, -6, and their cleaved substrates PARP and Lamin A/C were measured by fluorometry and Western Blotting, respectively.

RESULTL. interrogans strain Lai was able to induce apoptosis of J774A.1 cells and the maximal apoptotic rate was(48.81+/-5.95)% when microbe: cell ratio was 100: 1. The maximal activities of caspase-3 and -6 in the infected J774A.1 cells were (1453.41+/-36.07) and (618.65+/-39.82) FU, respectively, which were 16.38- and 9.98-fold of those uninfected cells. PARP and Lamin A/C in the infected cells were detected. Caspase-3 and -6 inhibitors remarkably blocked the L. interrogans-induced apoptosis in J774A.1 cells.

CONCLUSIONL. interrogans is able to induce the apoptosis of J774A.1 cells and intracellular caspase-3 and -6 are closely associated with the apoptosis.

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Caspase 6 ; metabolism ; Cell Line ; Leptospira interrogans ; pathogenicity ; Macrophages ; enzymology ; microbiology ; pathology ; Mice

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

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 effects of CD45 expression on induction of apoptosis in multiple myeloma cells.

METHODSMelphalan was used to induce myeloma cell line U266 apoptosis. Serum-free culture was used to induce CD45RB gene or empty plasmid transfected U266 apoptosis. The glucose-free culture was used to induce high CD45 (CD45(hi)) or low CD45 (CD45(low)) expression AMO1 apoptosis. Intraperitoneal inoculation was used to compare the survival of CD45(-) or CD45(+) U266 cells in mice. The number of apoptotic cells and mitochondrial membrane potential (MMP) was detected by flow cytometry. Western blotting was used to detect the cytochrome C release from mitochondrial and caspase-9 activation.

RESULTSMelphalan treatment induced 45% of CD45(+) and 30% of CD45(-) U266 cells apoptosis. Compared with the CD45(low) AMO1 cells, CD45(hi) cells were more susceptible to apoptosis. In serum-free culture for five days, 60% of CD45RB transferred U266 cells underwent apoptosis, while in the empty plasmid transfected ones, apoptotic cell number was not significantly increased. The survival time of CD45(-) U266 cells in the SCID-hIL-6 mice was 5 times that of CD45(+) cells. After melphalan treatment, 60% of the CD45(+) U266 cells lost MMP, while only 30% of CD45(-) U266 cells, and 10% of control cells did so. After UV irradiation, CD45(+) U266 cells mitochondria released more cytochrome C, leading to more caspase-9 activation.

CONCLUSIONCD45 expression is involved in mitochondria-mediated apoptotic process and increases apoptotic sensitivity of myeloma cells under a variety of stimulation.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Mice, SCID ; Mitochondria ; Multiple Myeloma ; metabolism

OBJECTIVETo study the effects of electromagnetic pulse (EMP), S-band high power microwave (S-HPM), and X-band high power microwave (X-HPM) on the Ca(2+) concentration and caspase-3 expression in Raji cells and the relationship between Ca(2+) concentration and caspase-3 expression, and to investigate the regulatory mechanism of electromagnetic radiation damage.

METHODSRaji cells were cultured conventionally. Some cells were irradiated by EMP, S-HPM, and X-HPM in the logarithmic growth phase for 6 hours and then collected; others received sham irradiation as a control. The Ca(2+) concentration in the cells was measured by laser scanning confocal microscope; the caspase-3 expression in the cells was evaluated by Western blot.

RESULTSCompared with the control group (Ca(2+) fluorescence intensity = 43.08 ± 2.08; caspase-3 expression level = 0.444 ± 0.13), the EMP,S-HPM, and X-HPM groups had significantly increased Ca(2+) concentrations, with Ca(2+) fluorescence intensities of 69.56 ± 1.71, 50.06 ± 1.89, and 70.68 ± 1.59, respectively (P < 0.01), and had upregulated caspase-3 expression, with expression levels of 0.964 ± 0.12, 0.586 ± 0.16, and 0.970 ± 0.07, respectively (P < 0.01). Each of the EMP and X-HPM groups had significantly higher Ca(2+) fluorescence intensity and caspase-3 expression level than the S-HPM group (P < 0.01), but there were no significant differences between the EMP and X-HPM groups. The linear regression analysis showed that the caspase-3 expression was upregulated as the Ca(2+) concentration increased, with a positive correlation between them (P < 0.01).

CONCLUSIONEMP, S-HPM, and X-HPM cause damage probably by increasing the Ca(2+) concentration in cells and in turn inducing caspase-3 overexpression.

Calcium ; metabolism ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Electromagnetic Radiation ; Humans