OBJECTIVETo study the apoptosis and mechanism of Fipronil on PC12 Cells.

METHODSThe effect of fipronil on the apoptosis and necrosis of PC12 cells of 3.13 x 10(-6), 1.25 x 10(-5) and 5.00 x 10(-5) mol/L three dose groups after 24 h treatment was detected by morphology and the apoptosis rate was detected by flow cytometer (FCM). The expression of Bcl-2 protein in PC12 cells of 3.13 x 10(-6), 1.25 x 10(-5) and 5.00 x 10(-5) mol/L three dose groups after 24 h treatment was measured by immunofluorescence.

RESULTSThe number of apoptotic cells of 3.13 x 10(-6) mol/L group was more than the control group examined by fluorescence microscope, and the number of dead cells of 5.00 x 10(-5) mol/L group was more than the control group. The apoptotic rates of PC12 cells was higher in the 3.13 x 10(-6) mol/L group than the control group measured by FCM, and the dead rates of PC12 cells was higher in the 5.00 x 10(-5) mol/L group than the control group (P < 0.05). Immunofluorescence cytochemistry experiment demonstrated that the level of Bcl-2 expression was significantly lower in the 3.13 x 10(-6) mol/L group than the control group.

CONCLUSIONAt low dosage, fipronil increases the apoptotic rates of PC12 cells possibly by decreasing the expression of Bcl-2 protein while at high dosage, fipronil only increases the amount of necrotic cells.

Animals ; Apoptosis ; drug effects ; PC12 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pyrazoles ; toxicity ; Rats

OBJECTIVETo explore the mechanism of shikonin for inducing the apoptosis of human promyelocytic leukemia cell HL-60.

METHODSThe effects of shikonin on the HL-60 cell proliferation were detected using MTT. The apoptosis rate was analyzed by Annexin-V/PI double staining. The expression level of the bcl-2 gene was detected using semi-quantitative reverse transcriptase PCR (RT-PCR), thus analyzing the correlation between the bcl-2 expression level and the apoptosis of HL-60.

RESULTSShikonin could inhibit the proliferation of HL-60 cells with the concentration range of 1-8 microg/mL in a time- and concentration-dependent manner. Two microg/mL shikonin could induce the apoptosis of HL-60 cells in a time-dependent manner. The expression level of bcl-2 was obviously down-regulated at 2 microg/mL shikonin.

CONCLUSIONSShikonin could induce the apoptosis of HL-60 cells. Its mechanism was correlated with down-regulation of the expression level of bcl-2.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; HL-60 Cells ; Humans ; Naphthoquinones ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

OBJECTIVE: To investigate the effectiveness of cisplatin on the expressions of Bcl-2 and Bax in cochlea and spiral ganglion cells (SGC) of guinea pigs. METHOD: Twenty guinea pigs were randomly divided into cisplatin (n = 10) and control groups (n = 10). Cisplatin group were administrated with a dose of intraperitoneal injection of 16 mg/kg, while the control group were received intraperitoneal injection of normal saline as placebo. Before and 7 days following injections, the ototoxic effect was measured with distortion product otoacoustic emission (DPOAE). Bcl-2, Bax in cochlea were detected by Western Blot. Immunohistochemical staining was used to detect the protein levels of Bcl-2 and Bax in spiral ganglion cells. RESULT: In control and cisplatin group, Bcl-2 protein levels were 0.727 8 ± 0.016 9 and 0.467 6 ± 0.020 1, Bax protein levels were 0.384 8 ± 0. 0217 and 0.735 6 ± 0.022 3 in cochlea respectively, both P < 0.01. In Control and cisplatin group, the grey values of Bcl-2 in SGC were 99.00 ± 2.42 and 149.80 ± 2.37 respectively, the grey values of Bax were 154.50 ± 2.80 and 104.50 ± 3.09 respectively, both P < 0.05. CONCLUSION Decreased expression of Bcl-2 and increased expression of Bax may be involved in cisplatin-induced apoptosis in cochlea and SGC of guinea pigs.
Animals ; Apoptosis ; Cisplatin ; pharmacology ; Cochlea ; metabolism ; Guinea Pigs ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Spiral Ganglion ; drug effects ; metabolism ; bcl-2-Associated X Protein ; metabolism

This study was aimed to clarify whether valproic acid (VPA) induces apoptosis of leukemia HL-60 cell line and its possible mechanism. The effect of different concentrations and treatment time of VPA on HL-60 cell proliferation was assayed by cytotoxicity test (CCK-8 method) and fluorescence microscopy, and flow cytometry was used to detect cell apoptosis. The expressions of telomerase subunit h-tert mRNA and apoptosis-related protein as well as caspase-3 activity were detected by real time-quantitative PCR, Western blot and ELISA respectively. The results indicated that VPA inhibited proliferation of HL-60 cells and induced cell apoptosis in a dose dependent manner (r = -0.87). The expressions of anti-apoptotic protein BCL-2 and h-tert mRNA were significantly decreased while the pro-apoptotic protein BAX and caspase-3 activity increased after treatment with VPA. The apoptosis rate of HL-60 cell was negatively correlated with expression of h-tert mRNA. It is concluded that VPA can inhibit leukemia HL-60 cell proliferation and induce apoptosis. The VPA displays anti-leukemia activity possibly through reducing h-tert mRNA and BCL-2 protein expression, increasing BAX expression and activity of caspase-3.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; HL-60 Cells ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Telomerase ; metabolism ; Valproic Acid ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the effects of paraquat on microRNA expressions in PCl2 cells, and to explore the regulatory mechanism of bcl-2.

METHODSWe used PC12 cells as a popular in vitro cell model system for characterizing the dopaminergic neuron. After 24 h treatment with different concentrations of PQ (0, 62.5 ümol/L), expression difference of microRNA was detected by microarray and examined by real-time quantitative PCR (RT-PCR). Cell apoptosis was analyzed with flow cytometry (FCM) and the relative levels of miR-34a, miR-Let-7e were measured by RT-RCR following the PCl2 cells treatment with 0, 62.5, 125, 250, 500, 1000 ümol/L PQ. Meanwhile, the protein expression of bcl-2 was evaluated by western blot according to forecasting targets analysis databases.

RESULTSCell viability decreased and cell apoptosis increased with increasing PQ concentrations (from 125 to 1000 ümol/L) in a dose-dependent manner (P < 0.05 or P < 0.01). MiRNA microarray showed that after 62.5 ümol/L PQ treatment, 11 miRNAs were significantly up-regulated while 8 miRNAs were down-regulated compared with control (P < 0.01). We chose miR-34a, miR-Let-7e which appeared most remarkable changes in microarray to examine by RT-PCR. It revealed that the level of miR-34a gradually ascended while miR-Let-7e declined after PQ treatment, which are accordant to the microarray results. The protein expression of bcl-2 treated with PQ significantly decreased compared with control and presented a negative correlation with the expression of miR-34a (P < 0.05 or P < 0.01).

CONCLUSIONThe alteration of miRNAs expression may be involved in the neurotoxicity of PQ. Especially, mir-34a negatively regulated the level of bcl-2, and thus plays a key role in PQ-induced cell apoptosis.

Animals ; Apoptosis ; drug effects ; MicroRNAs ; genetics ; PC12 Cells ; Paraquat ; toxicity ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats

In order to investigate the mechanisms of phenylhexyl isothiocyanate (PHI) inhibiting the proliferation of multiple myeloma cell RPMI8226 in vitro, the RPMI8226 cells were co-cultured with PHI of various concentrations. The inhibition of proliferation was measured by MTT test and the cell apoptosis was assayed by DAPI staining. The changes of Notch1, Jagged2, BCL-2 and p-Akt proteins in the PHI-treated cells were detected by Western blot. The results showed that PHI inhibited RPMI8226 cell proliferation in certain concentration range and induced their apoptosis. The inhibiting effect caused by PHI showed a concentration-and time-dependent manner. The PHI decreased expressions of Notch1 and Jagged2 proteins in a concentration-and time-dependent manners, the levels of BCL-2 and p-Akt declined at the same time. It is concluded that PHI can inhibit proliferation of RPMI8226 cells, and induce their apoptosis. The cell apoptosis is associated with the inhibition of Notch signaling and downstream targets BCL-2 and p-Akt proteins of RPMI8226 cells, PHI may be a new Notch signaling inhibitor and a promising therapeutic drug for multiple myeloma.
Cell Line, Tumor ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Isothiocyanates ; pharmacology ; Jagged-2 Protein ; Membrane Proteins ; metabolism ; Multiple Myeloma ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Receptor, Notch1 ; metabolism ; Signal Transduction ; drug effects

This study was aimed to explore the apoptotic effect of Resveratrol (RES) on KG-1 cells and the expression of bcl-2/bax in vitro, and to clarify the possible mechanism of apoptotic effect of RES on leukemia cells. After treating with different concentrations of RES, the suppressive effect of RES on proliferation of KG-1 cells was analyzed by MTT method. Transmission electron microscope technique were used to detect the apoptosis status of KG-1 cells. The cell cycle and apoptosis percentage of KG-1 cells treated with RES were detected by flow cytometry. The expressions of bcl-2, bax mRNA and protein were assessed by semiquantitative RT-PCR and flow cytometry. The results showed that RES could obviously inhibit proliferation of KG-1 cells (p < 0.05). Compared with the control group, the cell number in S phase of KG-1 cells treated with RES increased (p < 0.05), the apoptosis rate enhanced significantly (p < 0.01) and the expression level was down-regulated, while expression level of bax was obviously up-regulated (p < 0.01). It is concluded that RES significantly induces apoptosis of KG-1 cells in vitro, which is probably related to the down-regulation of bcl-2 expression and up-regulation of bax expression.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stilbenes ; pharmacology ; bcl-2-Associated X Protein ; metabolism

The aim of this study was to investigate apoptotic effect of 2-methoxyestradiol (2-ME2) on K562 cells and its mechanism. K562 cells were treated with different concentrations of 2-ME2. MTT assay was used to examine the effect inducing growth inhibition. DNA fragmentation assay and Annexin V-FITC/PI staining were used to detect the effect of apoptosis. The change of mitochondrial transmembrane potential was analyzed by flow cytometry. The expressions of related gene mRNA and/or proteins were detected by RT-PCR and Western blot respectively. The results indicated that the 2-ME2 inhibited proliferation of K562 cells in a time- and dose-dependent manners and the concentration of 50% growth inhibition (IC(50)) was 2 micromol/L at 48 hours. 2-ME2 induced DNA ladder and significantly increased apoptosis in K562 cells when exposed to 2 micromol/L of 2-ME2 for 24, 48 and 72 hours, the result of Annexin-V/PI staining showed that rates of the apoptotic cells were 13.78%, 22.32% and 29.43% respectively, which was remarkably higher than that of control (1.78%) (p < 0.05). The FCM analysis showed that the mitochondrial transmembrane potential in K562 cells lowered after exposed to 1, 2 and 4 micromol/L of 2-ME2 for 24 hours. 2-ME2 down-regulated the expression of bcr/abl and bcl-2, up-regulated the expression of bax mRNA, and down-regulated protein expressions of bcl-2, procaspase-3, procaspase-9, PARP (116 kD) and p-Akt, and up-regulated expression of cytoplasmic Cyto-C and PARP 85 kD apoptosis-related cleavage fragment protein, but had no effect on total Akt protein in K562 cells after treated with 2 micromol/L of 2-ME2 for 24, 48 and 72 hours. It is concluded that the 2-ME2 can induce the apoptosis and inhibit the proliferation of K562 cells by increasing the ratio of bax/bcl-2, reducing the mitochondrial transmembrane potential, releasing cytochrome C to cytoplasm, initiating the mitochondrial apoptosis pathway and leading in turn to caspase-3 activation. These findings suggest that interfere PI3K/Akt signal pathway via down-regulating the expression of bcr/abl mRNA is implicated in the effect of 2-ME2 on K562 cells.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cytochromes c ; metabolism ; Down-Regulation ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; genetics

OBJECTIVETo investigate the effects of Matrine on apoptosis of fibroblasts and the expression of apoptotic modulation related protein in the hypertrophic scar.

METHODSHypertrophic scar was produced on the ear of 24 New Zealand white rabbits, which were employed as the model, and were randomly and equally divided into control (CC) and Matrine (M) groups (12 in each group). Matrine (50 g/L) was injected into the ear scar in M group and with normal saline in C group once every four days. At 2, 4, 6, 8 and 12 weeks after the injection, the apoptotic fibroblast count in the scar was determined by TUNEL method, and the expressions of apoptosis related modulation proteins p53, bcl-2, bax were detected by immunohistochemistry method.

RESULTSThe apoptotic fibroblast count was much larger in M group than that in C group at all test time points (P < 0.05). Furthermore, the bax expression was increased and that of p53 and bcl-2 was decreased significantly in M group. In adding, the scar became flat in M group.

CONCLUSIONMatrine might obviously enhance the fibroblast apoptosis in rabbit ear hypertrophic scar, and up-regulate the expression of apoptosis related modulation protein bax and down-regulate the expression of p53 and Bcl-2.

Alkaloids ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Female ; Fibroblasts ; drug effects ; Immunohistochemistry ; Male ; Proto-Oncogene Proteins ; analysis ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Quinolizines ; Rabbits ; Tumor Suppressor Protein p53 ; analysis ; bcl-2-Associated X Protein

OBJECTIVETo study DNA damage, Bcl-2 and Bax expression, and ultrastructure change in spermatogenic cell of mice by cadmium exposure.

METHODSTwenty-four male mice were divided into 4 groups: 3 groups treated with cadmium chloride of 1, 5, 10 micromol x kg(-1) x d(-1) i.p. respectively for 5 days, and one normal saline control group. The DNA damage of spermatogenic cell by single-cell gel electrophoresis technology was detected. The expression positive rate of Bcl-2, Bax protein in spermatogenic cell by the immunohistochemical method was assayed, and the ultrastructural change of spermatogenic cell by the transmission electron microscope was observed.

RESULTSDNA damage rates of of spermatogenic cell in 1, 5, 10 micromol/kg cadmium chloride groups were higher than that of normal group (P < 0.001). Bcl-2 protein expression positive rates were lower than that of normal group (P < 0.001). Bax protein positive expression rate in 5 micromol/kg group was higher than those in normal group, and 1, 10 micromol/kg groups. The ultrastructure of karyotis, karyotheca, mitochondria, endoplasmic reticulum in three treated groups had different degree of damage and the degree of ultrastructural change was increasing with rising concentration of cadmium.

CONCLUSIONCadmium exposure will cause the DNA break, Bcl-2 and Bax protein abnormal expression and ultrastructural change in spermatogenic cell.

Animals ; Apoptosis ; Cadmium Chloride ; toxicity ; DNA Damage ; Male ; Mice ; Mice, Inbred ICR ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Spermatozoa ; drug effects ; metabolism ; ultrastructure ; bcl-2-Associated X Protein ; metabolism