In order to investigate the apoptosis triggered by porcine circovirus type 2 (PCV2) in lymphocytes and the underlying mechanism, the levels of apoptosis and the expression levels of miRNA were examined by flow cytometry, Western blotting and real-time PCR (qPCR). The mimics or inhibitors of miR-125a-5p, an apoptosis-related miRNA, were transfected into PK-15 cells, and the apoptosis rate was examined upon overexpression or inhibition of mir-125a-5p. The target gene of mir-125a-5p was predicted by bioinformatics method, and the regulation of mir-125a-5p on the target gene was analyzed by luciferase reporter assay. The expressions of Bcl-2, Bax, cytochrome C and caspase-3 were detected by Western blotting. The results showed that exosomes secreted by PK-15 cells infected with PCV2 significantly increased the lymphocyte apoptosis rate, which was dose-dependent in certain concentration range. The expression of miR-125a-5p was dramatically increased. The apoptosis rate was increased significantly in the cells transfected with miR-125a-5p. It was predicted that there were binding sites of miR-125a-5p at Bcl-2 3'UTR by TargetScan. The luciferase activity of wild-type pmir-Bcl-2 3'UTR was inhibited significantly by miR-125a-5p mimics, but that of mutant pmir-Bcl-2 3'UTR was not changed. By Western blotting, Bcl-2 was reduced significantly, while Bax, cytochrome C and caspase-3 increased significantly, and the ratio of Bcl-2/Bax was significantly decreased. These results showed that PCV2 up-regulated the expression of miR-125a-5p through exosomes, then inhibited the expression of Bcl-2 at both mRNA and protein level, activated mitochondrial apoptosis pathway and induced apoptosis in lymphocytes.
3' Untranslated Regions ; Animals ; Apoptosis/genetics* ; Caspase 3/genetics* ; Cell Proliferation/genetics* ; Circovirus/genetics* ; Cytochromes c/genetics* ; Luciferases/genetics* ; Lymphocytes/metabolism* ; MicroRNAs/metabolism* ; Swine ; bcl-2-Associated X Protein/genetics*

OBJECTIVETo investigate the mechanism underlying the inhibitory effect of STOML-2 overexpression on apoptosis of human cervical squamous carcinoma Siha cells.

METHODSSiha cells were transfected with an adenoviral vector carrying STOML-2, and 72 h later STOML-2 expression and the proliferation of the cells were detected by Western blotting and MTT assay. The transfected cells were treated with IC50 Cisplatin for 24 h, and the morphological changes of cells were observed using fluorescence, and the cell apoptosis was analyzed using flow cytomerty; the expression levels of proteins related with mitochondrial apoptosis pathway, including caspase-3, cleaved caspase-3, Bcl-2, Bax and cytochrome C (Cyt C), were detected by Western blotting.

RESULTSWestern blotting showed a significantly increased STOML-2 expression in the transfected cells. Overexpression of STOML-2 obviously promoted the proliferation of Siha cells. The STOML-2-overexpressing cells exhibited an obvious resistance to IC50 Cisplatin-induced apoptosis as shown by both fluorescence microscopy and flow cytometry and presented with decreased expressions of cleaved caspase-3, Bax, and cytosol Cyt C and increased expressions of caspase-3, Bcl-2, and mitochondrial Cyt C.

CONCLUSIONSOverexpression of STOML-2 can enhance the proliferation of Siha cells by inhibiting cell apoptosis possibly through the mitochondrial apoptosis pathway.

Apoptosis ; Apoptosis Regulatory Proteins ; Blood Proteins ; genetics ; Carcinoma, Squamous Cell ; metabolism ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Cytochromes c ; metabolism ; Female ; Flow Cytometry ; Humans ; Membrane Proteins ; genetics ; Mitochondria ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the expression of the C-terminal truncated human apoptosis-inducing factor (AIF) and its biological effect on MCF-7 cells.

METHODSPcDNA3.0-FDT-AIFδ1-480 was transfected into human breast carcinoma MCF-7 cells with lipofectamine. The expression of the truncated AIF gene was detected by Western blotting, and its effects on the biological behaviors of MCF-7 cells and on the expression of cytochrome c (cytC) were evaluated using flow cytometry, MTT assay, colony-forming assay, and mitochondrial membrane potential measurement.

RESULTSPcDNA3.0-FDT-AIFδ1-480 enhanced AIF expression in MCF-7 cells, obviously inhibited the cell proliferation, and significantly reduced the mitochondrial membrane potentials (P<0.05). Transfection of the cells with PcDNA3.0-FDT-AIFδ1-480 promoted the expression of cytC and resulted in significantly increased apoptosis of MCF-7 cells (P<0.05).

CONCLUSIONThe expression of C-terminal truncated human AIF gene can induce apoptosis of human MCF-7 cells by promoting cytC release from mitochondria.

Apoptosis ; Apoptosis Inducing Factor ; genetics ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; Cell Proliferation ; Cytochromes c ; genetics ; metabolism ; Female ; Humans ; MCF-7 Cells ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.
Antineoplastic Agents ; chemistry ; pharmacology ; Apoptosis ; drug effects ; genetics ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Caspase 8 ; genetics ; metabolism ; Caspases ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cell Size ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cytochromes c ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Fucus ; chemistry ; G1 Phase Cell Cycle Checkpoints ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; MCF-7 Cells ; Microscopy, Fluorescence ; Molecular Structure ; Polysaccharides ; chemistry ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; bcl-2-Associated X Protein ; genetics ; metabolism

OBJECTIVE: To investigate the changes of mitochondrion by transferring antisense oligodeoxynucleotide Stat3 into laryngeal carcinoma Hep-2 cell, for elucidating the mechanism of laryngeal carcinoma Hep-2 cell apoptosis, for developing more effective treatment for laryngeal cancer. METHOD: The designed Stat3 ASODN was transferred into laryngeal carcinoma Hep-2 cell by lipofection. Mitochondrion membrane potential, VDAC and Cyt-c were detected for determining the changes of mitochondrion. RESULT: MMP was fell, Cyt-c and VDAC were increased with the heighten concentration of ASODN. CONCLUSION Mitochondria approach play an important role in the apoptosis mechanism of human Hep-2 cell by Stat3. This research elucidated the regulating mechanism of Hep-2 cell proliferation by Stat3, provided a new research focus for clinical therapy.
Apoptosis ; Cell Proliferation ; Cytochromes c ; metabolism ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism ; Oligodeoxyribonucleotides, Antisense ; genetics ; STAT3 Transcription Factor ; genetics ; Transfection ; Voltage-Dependent Anion Channels ; metabolism

OBJECTIVEIn order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells, the effects of PPARγ and POX-induced ROS were explored.

METHODS[3- (4, 5)-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V and PI staining using FACS, plasmid transfection, ROS formation detected by DCFH staining, RNA interference, RT-PCR & RT-QPCR, and Western blotting analyses were employed to investigate the apoptotic effect of troglitazone and the potential role of PPARγ pathway and POX-induced ROS formation in HT29 cells.

RESULTSTroglitazone was found to inhibit the growth of HT29 cells by induction of apoptosis. During this process, mitochondria related pathways including ROS formation, POX expression and cytochrome c release increased, which were inhibited by pretreatment with GW9662, a specific antagonist of PPARγ. These results illustrated that POX upregulation and ROS formation in apoptosis induced by troglitazone was modulated in PPARγ-dependent pattern. Furthermore, the inhibition of ROS and apoptosis after POX siRNA used in troglitazone-treated HT29 cells indicated that POX be essential in the ROS formation and PPARγ-dependent apoptosis induced by troglitazone.

CONCLUSIONThe findings from this study showed that troglitazone-induced apoptosis was mediated by POX-induced ROS formation, at least partly, via PPARγ activation.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Chromans ; pharmacology ; Cytochromes c ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; HT29 Cells ; Humans ; PPAR gamma ; metabolism ; Proline Oxidase ; metabolism ; Reactive Oxygen Species ; metabolism ; Thiazolidinediones ; pharmacology

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsim), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.
Apoptosis/*drug effects ; Caspase 3/metabolism ; Chalcones/metabolism/*pharmacology ; Chemoprevention ; Cytochromes c/biosynthesis/secretion ; Down-Regulation ; Gene Expression ; HL-60 Cells ; Humans ; Immunoblotting ; Leukemia, Myeloid/*enzymology/pathology ; Membrane Potential, Mitochondrial/drug effects ; Mitochondria/enzymology ; Ornithine Decarboxylase/antagonists & inhibitors/genetics/*metabolism ; Reactive Oxygen Species/analysis/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

The complementary oligonucleotides, each with two consensus estrogen response element (ERE)-sequences and 5'-Hind III and 3'-Sph I sticky ends were artificially synthesized. A solution with both the complementary DNA sequences was heated to 95'C and cooled down to room temperature to form double strand DNA (dsDNA). The set was cloned into the corresponding sites of CYC1 promoter of the pERE-CYC-yEGFP to yield pERE-CYCalpha-yEGFP vector. The two different reporter vectors, pERE-CYC-yEGFP and pERE-CYCalpha-yEGFP, the 2ERE, were placed in the CYC1 promoter. The former promoter downstream ERE contains alpha and beta-TATA boxes and the latter has only alpha-TATA box. The two different reporter vectors were transformed into the yeast cells that express human estrogen receptor alpha (ERalpha). Incubation of the recombinant yeasts with the six estrogenic compounds for 4 hours showed that the recombinant cell containing pERE-CYCalpha-yEGFP would give very poor dose-response curves, in contrast to the recombinant cell containing pERE-CYC-yEGFP which produced well-shaped dose-response curves. So it is necessary for this bioassay that alpha and beta-TATA boxes in the minimal CYC1 promoter when the promoter is used as a rapid and high throughput system for screening estrogenic chemical products.
Base Sequence ; Cytochromes c ; biosynthesis ; genetics ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogens ; genetics ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics ; TATA-Box Binding Protein ; genetics

Activated protein C (APC) is known to be beneficial on ischemia reperfusion injury in myocardium. However, the protection mechanism of APC is not fully understood. The purpose of this study was to investigate the effects and possible mechanisms of APC on myocardial ischemic damage. Artificially ventilated anaesthetized Sprague-Dawley rats were subjected to a 30 min of left anterior descending coronary artery occlusion followed by 2 hr of reperfusion. Rats were randomly divided into four groups; Sham, I/R, APC preconditioning and postconditioning group. Myocardial infarct size, apoptosis index, the phosphorylation of ERK1/2, Bcl-2, Bax and cytochrome c genes and proteins were assessed. In APC-administrated rat hearts, regardless of the timing of administration, infarct size was consistently reduced compared to ischemia/reperfusion (I/R) rats. APC improved the expression of ERK1/2 and anti-apoptotic protein Bcl-2 which were significantly reduced in the I/R rats. APC reduced the expression of pro-apoptotic genes, Bax and cytochrome c. These findings suggest that APC produces cardioprotective effect by preserving the expression of proteins and genes involved in anti-apoptotic pathways, regardless of the timing of administration.
Animals ; Apoptosis ; Cytochromes c/genetics/metabolism ; Hemodynamics/physiology ; Male ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Myocardial Reperfusion Injury/metabolism/pathology/*prevention & control ; Myocardium/*metabolism/pathology ; Phosphorylation ; Protein C/*therapeutic use ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; bcl-2-Associated X Protein/metabolism

An abrupt increase of intracellular Ca2+ is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca2+ triggers apoptotic cell death through mitochondrial swelling and activation of Ca2+-dependent enzymes. Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca2+-overload. Overexpression of TG2 inhibited the A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca2+-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca2+-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.
*Apoptosis ; Apoptosis Regulatory Proteins/metabolism ; Calcimycin/pharmacology ; Calcium/*metabolism ; Caspases/metabolism ; Cell Death ; Cell Survival ; Cytochromes c/metabolism ; Down-Regulation ; GTP-Binding Proteins/*metabolism ; HEK293 Cells ; Hela Cells ; Humans ; Ionophores/pharmacology ; Mitochondria/metabolism ; Transglutaminases/*metabolism ; bcl-2-Associated X Protein/genetics/*metabolism