BACKGROUND/AIMS: The aim of this study was to investigate the immunohistochemical expression of bcl-2, bcl-xL, bax, and p53 proteins according to the pathological parameters such as grade of dysplasia, histological type, depth of invasion, lymph node metastasis, and TNM stage in the gastric adenoma and gastric adenocarcinoma. METHODS: Immunohistochemical staining using monoclonal bcl-2, bcl-xL, bax, p53 antibodies were performed on paraffin embedded specimens from forty-one gastric adenomas and 100 gastric adenocarcinomas. RESULTS: The expression rate of bcl-2 was higher in adenomas (34.2%), especially in high grade dysplasia (52.4%), than adenocarcinomas (2.0%). The expression rate of bcl-xL was higher in adenocarcinomas (55.0%) than adenomas (22%). The expression rate of the bax was higher in adenocarcinomas (58.0%) than adenomas (14.6%). In the adenocarcinoma, the bax expression was significantly related with the depth of invasion, lymph node metastasis, and TNM stage. The expression rate of p53 was higher in adenocarcinomas (64.0%) than adenomas (14.6%). CONCLUSIONS: Bcl-2 protein would be related with the development of gastric adenoma, especially with high grade dysplasia. Bcl-xL and p53 proteins would be involved in the development of relatively early stage of gastric adenocarcinoma but not in tumor progression. Bax protein would be involved in the development of gastric adenocarcinoma and related with depth of invasion, lymph node metastasis, and TNM stage.
Adenocarcinoma/*metabolism/pathology ; Adenoma/*metabolism/pathology ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2/*metabolism ; Stomach Neoplasms/*metabolism/pathology ; Tumor Suppressor Protein p53/*metabolism ; bcl-2-Associated X Protein/*metabolism ; bcl-X Protein/*metabolism

In order to investigate the role of melatonin in inhibiting the proliferation of murine gastric cancer and the underlying molecular mechanism, we performed an in vivo study by inoculating murine foregastric carcinoma (MFC) cells in mice, and then tumor-bearing mice were treated with different concentrations of melatonin (i.p.). The changes of Bcl-2, Bax, p21 and p53 expressions in tumor tissue were detected by using real-time fluorescence quantitative RT-PCR and Western blot. We found that: (1) melatonin resulted in reductions of tumor's volume and weight in the gastric cancer-bearing mice and thus showed anti-cancer effect; (2) melatonin reduced Bcl-2 expression, but increased the expression of Bax, p53 and p21 in tumor tissue. Our results suggest that melatonin could inhibit the growth of tumors in gastric cancer-bearing mice through accelerating the apoptosis of tumor cells.
Animals ; Apoptosis ; Melatonin ; pharmacology ; Mice ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; drug therapy ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism

The permeability of mitochondrial outer membrane (MOM) is regulated by the proteins of the Bcl-2 family via their interactions at the membrane. While pro-apoptotic Bax protein promotes MOM permeabilization (MOMP) releasing cytochrome c after activation by BH3-only protein, anti-apoptotic Bcl-2 protein protects MOM. However both Bax and Bcl-2 can form pores in model membranes. Unlike Bax pore that has been extensively studied and reported to be directly linked to MOMP, Bcl-2 pore is much less known; thus we investigated the pore-forming property of recombinant Bcl-2 lacking the C-terminal transmembrane sequence (Bcl-2deltaTM) in liposomal membranes of MOM lipids. We found that: (1) Bcl-2 formed pores at acidic pH that induced the association of Bcl-2 with liposome; (2) Bcl-2 pore size was dependent on Bcl-2 concentration, suggesting that oligomerization is involved in Bcl-2 pore formation; (3) Unlike Bax pore that could release large molecules up to 2 mega-Da, Bcl-2 pore was smaller and could only release the molecules of a few kilo-Da. Therefore, Bcl-2 and Bax may form different size pores in MOM, and while the large pore formed by Bax may release cytochrome c during apoptosis, the small pore formed by Bcl-2 may maintain the normal MOM permeability.
BH3 Interacting Domain Death Agonist Protein ; metabolism ; Cell Membrane Permeability ; Cytochrome c Group ; metabolism ; Humans ; Hydrogen-Ion Concentration ; Liposomes ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism

OBJECTIVETo investigate the effects of adenoviral vector-mediated over-expression of Wnt3 on the apoptosis of hepatic progenitor cells in vitro.

METHODSHepatic progenitor cells transfected with Ad-GFP-Wnt3 vector or the control vector Ad-GFP were examined for cell apoptosis under fluorescence microscopy with Hoechst 33342 staining, and the proportion of apoptotic cells were determined by flow cytometric analysis with Annexin-PE/7-ADD staining. The mRNA and protein expressions of Bax, Bcl-2 and Bcl-xl in the cells were detected by real-time PCR and Western blotting, respectively.

RESULTSReal-time PCR and Western blotting showed a high expression of Wnt3 in Ad-GFP-Wnt3-transfected hepatic progenitor cells, which exhibited significantly decreased cell apoptosis as compared with the control group. The expressions of Bcl-2 and Bcl-xl mRNA and proteins increased significantly while Bax expression decreased obviously in Ad-GFP-Wnt3-transfected cells (P<0.05).

CONCLUSIONSAdenoviral vector-mediated over-expression of Wnt3 can suppress apoptosis of hepatic progenitor cells possibly through the Bcl-2 pathway.

Apoptosis ; Cells, Cultured ; Genetic Vectors ; Hepatocytes ; cytology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stem Cells ; cytology ; Transfection ; Wnt3 Protein ; metabolism ; bcl-X Protein ; metabolism

The efficiency and safe range of Lipofectamine2000 (LF2000)/bcl-xl applied in human keratocytes, the optimal ratio of LF2000/bcl-xl and the bcl-xl gene expression in human keratocytes were investiaged. By using trypan-blue staining, the effects of LF2000 and bcl-xl on the survival rate of the cultured human keratocytes were measured respectively. By using semi-quantitative RT-PCR, the efficiency and the expression of LF2000-mediated bcl-xl transfection into keratocytes were examined. The results showed that the survival rate of human keratocytes had no signficant change in the presence of LF2000 (20 microg/ml) or bcl-xl (10 microg/ml) for 24 h. LF2000 could effectively mediate the transfection of exogenous gene bcl-xl into human keratocytes. The best transfection efficiency could be obtained when the ratio of bcl-xl/LF2000 was 1:8. One day after transfection, the positive cells for bcl-xl could be detectable, and the positive rate reached the peak-on the posttransfection day 3 (48.3%), then gradually decreased. Fifteen days after transfection, there were few positive cells. It was suggested that LF2000 could effectively transfer the exogenous gene bcl-xl into human keratocytes without obvious toxicity during a concentration range. LF2000/bcl-xl may be likely to play an important role in gene therapy of human keratocytes.
Cations/administration & dosage ; Cornea/cytology ; Gene Therapy ; Keratinocytes/cytology ; Keratinocytes/*metabolism ; Liposomes ; Transfection ; bcl-X Protein/biosynthesis ; bcl-X Protein/*genetics

OBJECTIVETo investigate the changes of bcl-2 gene family and the molecular mechanism of neuronal apoptosis following traumatic brain injury (TBI) in rats.

METHODSMale Sprague-Dawley (SD) rats were subjected to lateral fluid percussion brain injury (FPBI) of moderate severity. The bcl-x(L) and bax mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). In addition to morphological evidence of apoptosis, terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling (TUNEL) histochemistry was used to identify the DNA fragmentation in situ at both light and electron microscope levels, whereas characteristic internucleosomal DNA fragmentation of apoptosis was demonstrated by DNA gel electrophoresis.

RESULTSThe apoptotic response to trauma was regionally distinct and may be involved in both acute and delayed cell death. The bcl-x(L) mRNA expression of the impact site was significantly lower (67.42%+/-7.54%) than that of the ipsilateral hemisphere at 6 hours after injury (P<0.01). The decrease of bcl-x(L) mRNA expression preceded apoptosis at 24 hours after injury. The bax mRNA expression rose slowly, doubled at 3 days after injury and returned to the sham level slowly.

CONCLUSIONSDecreased expression of bcl-x(L) mRNA and increased expression of bax mRNA coincides with apoptosis following brain injury. The bcl-2 gene family is involved in neuronal apoptosis after TBI, and the changes of mRNA expression of the family members lead the neuronal cells to apoptosis.

Animals ; Apoptosis ; physiology ; Brain Injuries ; metabolism ; DNA Fragmentation ; In Situ Nick-End Labeling ; Male ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; bcl-X Protein

OBJECTIVETo study the mechanisms of pseudolaric acid B-induced apoptosis on A375-S2 cells.

METHODMTT, fluorescence microscope observation, DNA agarose gel electrophoresis and Western blot analysis wereused.

RESULTPseudolaric acid Binduces A375-S2 cell apoptosis in a time and dose-dependent manner. Apoptotic bodies and DNA ladder were observed in 5 micromol x L(-1) pseudolaric acid B-treated A375-S2 cells for 36 h. The expression of Bcl-2, Bcl-xL and ICAD was reduced time dependently, whereas the expression of Bax was increased.

CONCLUSIONThe major cause of pseudolaric acid B induced cytotoxicity on A375-S2 cells was apoptosis. Mitochondria proteins and ICAD might be involved in the apoptotic pathways of pseudolaric acid B-treated A375-S2 cells.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Cell Line, Tumor ; Diterpenes ; isolation & purification ; pharmacology ; Dose-Response Relationship, Drug ; Humans ; Melanoma ; metabolism ; pathology ; Pinaceae ; chemistry ; Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; bcl-X Protein

The purpose of study was to explore the possible functions of Bcl-xL in the glucosamine sulfate-induced apoptosis of chronic myeloid leukemia K562 cells. Light microscopy and Wright-Giemsa staining were used to investigate the morphologic evidences for apoptosis of K562 cells induced by glucosamine sulfate (GS); immunofluorescence was used to observe the translocation of cathepsin D and cytochrome C during the apoptosis; Western blot was performed to detect the expression of Bcl-xL, Bid, Bax in K562 cells treated by GS. The results showed that many vacuoles were observed in the cytoplasma of the K562 cells treated by GS; fluorescent signals of cathepsin D and cytochrome were fransformed from granules to disperse form by using immunofluorescence; the expression of Bcl-xL was found down-regulated in K562 cells treated by GS, but not in the cells pre-treated with pepstatin A; the significant changes were not detected in expression of Bax and Bid protein before or after apoptosis. It is concluded that Bcl-xL protein may mediate relationship between cathepsin D and mitochondia pathway, Cathepsin D may play an important role in the GS inducing apoptosis of K562 cells through downregulation of Bcl-xL expression.
Apoptosis ; drug effects ; physiology ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Blotting, Western ; Cathepsin D ; metabolism ; Cytochromes c ; metabolism ; Fluorescent Antibody Technique ; Glucosamine ; pharmacology ; Humans ; K562 Cells ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism ; physiology

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.
Oxygen ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/genetics* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Autophagy ; Ubiquitin-Protein Ligases ; Microtubule-Associated Proteins

OBJECTIVEDuring the critical period of brain development, insufficiency of thyroid hormone results in severe mental retardation and learning deficit. This study was designed to investigate the effects of hypothyroidism on apoptosis and the expression of Bcl-2 and Bax gene in the developing rat hippocampus neurons and to explore the mechanism of brain development regulated by thyroid hormone.

METHODHypothyroidism was induced by administration of propylthiouracil (PTU, 50 mg/d) solution to the dams from gestational day 15 by gavage. Pups from both hypothyroid and control groups were harvested at postnatal day 1 (P1), P5, P10 and P15, respectively. Blood samples were collected at the time of death for the determination of thyroid hormone. Serum free tri-iodothyronine (FT(3)) and free thyroxine (FT(4)) were measured by using chemoluminescence. Hippocampus collected from the control and hypothyroid pups were examined under light and transmissional electron microscopy. Measurement of DNA fragmentation was carried out by agarose gel electrophoresis. The expression of Bcl-2 and Bax protein in the developing rat hippocampus neurons was performed by Western blotting.

RESULTSSignificantly lower circulating FT(4) and FT(3) levels confirmed the hypothyroid status of the experimental pups. The shrunken and contracted degenerations increased in hippocampus neurons of hypothyroid pups under light microscopy. Enhanced apoptotic cells were found in hippocampus neurons of hypothyroid pups under transmission electron microscopy, especially at P10 and P15. Extensive DNA fragmentation was seen throughout development in hippocampus of hypothyroid pups, but not in the euthyroid controls except for basal level at P10. The expression of Bcl-2 in the hippocampus neurons of hypothyroid pups was significantly lower than that of euthyroid controls at all stages of development (P1: 1.95 +/- 0.27 vs. 2.59 +/- 0.19, P < 0.05, P5: 1.86 +/- 0.24 vs. 2.47 +/- 0.17, P < 0.05, P10: 1.29 +/- 0.22 vs. 1.86 +/- 0.28, P < 0.05 and P15: 1.21 +/- 0.27 vs. 2.18 +/- 0.17, P < 0.01, respectively). The relative amount of expression varied significantly with age in the control pups. The level of Bcl-2 was high in hippocampus neurons of euthyroid at P1, P5, and decreased significantly at P10, and showed a trend of recovery at P15. Similar age-related variation in the expression of Bcl-2 gene was observed in the hypothyroid group at P1, P5 and P10, but the level was maintained low at P15. The expression of Bax in the hippocampus neurons of hypothyroid pups was significantly higher than that of control pups at all stages of development (P1: 1.69 +/- 0.14 vs. 1.24 +/- 0.23, P < 0.05, P5: 1.78 +/- 0.16 vs. 1.29 +/- 0.17, P < 0.05, P10: 1.92 +/- 0.18 vs. 1.45 +/- 0.14, P < 0.05 and P15: 1.86 +/- 0.14 vs. 1.51 +/- 0.12, P < 0.05, respectively). The ratio of Bcl-2/Bax in hippocampus neurons of hypothyroid pups was lower than that of age-matched controls (P1: 1.16 +/- 0.17 vs. 2.12 +/- 0.35, P < 0.05, P5: 1.05 +/- 0.16 vs. 1.94 +/- 0.36, P < 0.05, P10: 0.68 +/- 0.17 vs. 1.29 +/- 0.16, P < 0.05 and P15: 0.67 +/- 0.19 vs. 1.45 +/- 0.22, P < 0.01, respectively).

CONCLUSIONThyroid hormone significantly prevents apoptosis of hippocampus neurons. Congenital hypothyroidism increases not only the extent but also the duration of apoptosis by down-regulation of the anti-apoptotic gene Bcl-2 and maintaining a high level of the pro-apoptotic gene Bax.

Animals ; Animals, Newborn ; Apoptosis ; physiology ; Down-Regulation ; Hippocampus ; metabolism ; Hypothyroidism ; physiopathology ; Neurons ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; bcl-2-Associated X Protein ; metabolism