Cytochrome C plays important roles in electron transferring, oxidative stress and apoptosis. In this study, soluble cytochrome C was accumulated in cytoplasm of E. coli by utilizing the co-expression of human cytochrome c and yeast heme lyase from a single plasmid. After ultrasonic disruption of the bacteria, a lot of contaminated proteins were discarded by addition of 350 g/L ammonium sulfate into the supernatant. Then the recombinant human cytochrome C was purified to 80% homogeneity after two times cation exchange chromatography on SP-Sepharose Fast Flow. Yields of cytochrome C greater than 10 mg per liter culture were attained. This efficient system for producing human cytochrome C is helpful for us to understand the roles of this protein in biological processes and therapy of human diseases relevant to apoptosis and oxidative stress.
Cytochromes c ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

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

Apoptotic regulation is critical to organismal homeostasis and protection against many human disease processes such as cancer. Significant research efforts over the past several decades have illuminated many signaling molecules and effecter proteins responsible for this form of programmed cell death. Recent evidence suggests that transfer RNA (tRNA) regulates apoptotic sensitivity at the level of cytochrome c-mediated apoptosome formation. This finding unexpectedly places tRNA at the nexus of cellular biosynthesis and survival. Here we review the current understanding of both the apoptotic machinery and tRNA biology. We describe the evidence linking tRNA and cytochrome c in depth, and speculate on the implications of this link in cell biology.
Animals ; Apoptosis ; genetics ; physiology ; Caspase 9 ; physiology ; Cytochromes c ; physiology ; Humans ; Models, Biological ; Nucleic Acid Conformation ; RNA, Transfer ; chemistry ; genetics ; physiology

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 effects of mild hypothermia on sequential events of neuronal apoptosis following hypoxic-ischemic brain damage (HIBD) in neonatal rats.

METHODSA model of HIBD was prepared by ligating the left common carotid artery in 7-day-old rats, followed by 8% hypoxia exposure. HIBD rats were randomly assigned into a hypothermia group (rectal temperature = 33 centi-degrees) and a normothermia group (rectal temperature = 36 centi-degrees). TUNEL, Haematoxylin and Eosin, and Nissl staining were used to detect neuronal apoptosis. Western blotting, RT-PCR and enzyme activity measurement were used to evaluate the changes of plasma and mitochondrial cytochrome c (Cyt c), caspase-3 mRNA expression and caspase-3 enzyme activity, respectively.

RESULTSThe number of apoptotic cells in the ipsilateral hemisphere of the hypothermia group was significantly reduced compared with that of the normothermia group at 72 hrs post-HI (6.4 +/- 1.7% vs 25.3 +/- 1.5%) (P < 0.01). Analysis of Western blotting showed that Cyt c levels increased in the cytosolic fraction, but decreased significantly in the mitochondrial fraction in the ipsilateral hemisphere of the hypothermia group at 24, 48 and 72 hrs of HI insult compared with the normothermia group (P < 0.05). Caspase-3 mRNA increased significantly after 24 hrs post-HI in the normothermia group, and this change became more pronounced with time. Mild hypothermia treatment decreased significantly caspase-3 mRNA expression at 24, 48 and 72 hrs post-HI (P < 0.05). Caspase-3 activity gradually increased 2 hrs after HI insult and peaked at 24 hrs in the normothermia group. Mild hypothermia treatment resulted in a significant reduction in caspase-3 activity in the ipsilateral hemisphere, with an optimal effect produced at 24 hrs post-HI (2.42 +/- 0.5 RFU vs 34.7 +/- 3.2 RFU; P < 0.01).

CONCLUSIONSMild hypothermia treatment attenuates neuronal apoptosis following HIBD, possibly through a reduction in Cyt c release from mitochondria and an inhibition of caspase-3 mRNA expression and its enzyme activity.

Animals ; Apoptosis ; Brain ; pathology ; Caspase 3 ; genetics ; metabolism ; Cytochromes c ; secretion ; Female ; Hypothermia, Induced ; Hypoxia-Ischemia, Brain ; pathology ; therapy ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

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

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

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

OBJECTIVETo investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2, Bax, Bad, Bcl-xL, caspase-3, poly ADP-ribose polymerase (PARP), and survivin of HT-29 cells.

METHODSHT-29 cells were treated with curcumin (0 approximately 80 micromol/L) for 24 h. The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax, Bcl-2, Bcl-xL, Bad, caspase-3, PARP, and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSCurcumin significantly induced the growth inhibition and apoptosis of HT-29 cells. A decrease in expressions of Bcl-2, Bcl-xL and survivin was observed after exposure to 10 approximately 80 micromol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cytochrome c, the activation of caspase-3, and the cleavage of PARP in a dose-dependent manner.

CONCLUSIONThese data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitochondria-mediated pathway.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Cell Nucleus ; drug effects ; Cell Survival ; drug effects ; Curcumin ; pharmacology ; Cytochromes c ; secretion ; HT29 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Membrane Potential, Mitochondrial ; drug effects ; Microtubule-Associated Proteins ; genetics ; Mitochondria ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; RNA, Messenger ; analysis ; bcl-2-Associated X Protein ; genetics ; bcl-X Protein ; genetics

OBJECTIVEIt has been reported that neuronal apoptosis plays a critical role in pathology of hypoxic-ischemic encephalopathy (HIE). Cytochrome C (CytC) is an important apoptotic protease activating factor. Inosine might have a neuroprotective effect against cerebral ischemia reperfusion injury by inhibiting the neuronal apoptosis and the expression of CytC mRNA in adult rats. This study examined the effects of inosine on neuronal apoptosis and CytC mRNA expression following hypoxic-ischemic brain damage (HIBD) in order to investigate the neuroprotectivity of inosine against cerebral ischemia injury in neonatal rats and the possible mechanism.

METHODSA total of 140 healthy 7-day-old Sprague-Dawley rat pups were randomly assigned into Control (n=40), HIBD (n=50) and Inosine treatment groups (n=50). HIBD rat models were established by ligating the left common carotid artery, followed by 8% O2 hypoxia exposure for 2 hrs in the HIBD and Inosine treatment groups. The Control group was not subjected to hypoxia-ischemia (HI). The Inosine treatment and the HIBD groups were randomly divided into 5 sub-groups sacrificed at 6 and 12 hrs, and 1, 3 and 7 days post- HI (n=10 each). The Control group rats were sacrificed at the corresponding time points (n=8 each). Inosine was administered to the Inosine treatment group by intraperitoneal injection immediately after HIBD at the dosage of 100 mg/kg twice daily for 7 days. TUNEL staining and in situ hybridization method was used to detect neuronal apoptosis and CytC mRNA expression respectively.

RESULTSFew apoptotic cells and CytC mRNA positive cells were found in brain tissues of the Control group. In the HIBD group, the number of apoptotic cells and the CytC mRNA expression in the cortical and hippocampal gyrum CA1 areas increased 6 hrs after HI, peaking at 1 day after HI and then decreased gradually. Until the 7th day, the number of apoptotic cells and the CytC mRNA expression in the cortical and hippocampal gyrum CA1 areas in the HIBD group remained significantly higher than in the Control group. Inosine treatment decreased the apoptotic cells and the CytC mRNA expression in both areas from 6 hrs to 7 days after HI compared with the HIBD group. The linear correlation analysis demonstrated that the number of apoptotic cells was positively correlated to the CytC mRNA expression in neonatal rats with HIBD (r=0.88, P < 0.01) .

CONCLUSIONSInosine can reduce the number of apoptotic cells and down-regulate the expression of CytC mRNA following HIBD in neonatal rats. The decreased number of apoptotic cells was positively correlated to the decreased CytC mRNA expression after inosine treatment, suggesting that inosine offered neuroprotectivity against HIBD possibly through inhibiting the CytC mRNA expression and resulting in a decrease of cell apoptosis.

Animals ; Apoptosis ; drug effects ; Cytochromes c ; genetics ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; In Situ Nick-End Labeling ; Inosine ; pharmacology ; therapeutic use ; Neurons ; drug effects ; Neuroprotective Agents ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley