We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margi...We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margination and DNA ladder were observed. After incubating isolated mitochondria in some hypotonic solutions, and centrifuging these mixtures at high speed, we got mitochondrial supernatants. It was found that in the absence of cytosolic factor, the supernatant alone was able to induce apoptotic changes in nuclei. The effective components were partly of protein. DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC- YVADCHO. Meanwhile, caspase inhibitors fully blocked chromatin condensation. Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted. It was found that this endonuclease is different from endonuclease G, cytochrome c-induced nuclease, or Ca2+-activated endonuclease.展开更多
Voltage-dependent anion channel （VDAC）I is the main channel of the mitochondrial outer membrane （MOM） and it has been proposed to be part of the permeability transition pore （PTP）, a putative multiprotein comple...Voltage-dependent anion channel （VDAC）I is the main channel of the mitochondrial outer membrane （MOM） and it has been proposed to be part of the permeability transition pore （PTP）, a putative multiprotein complex candidate agent of the mitochondrial permeability transition （MPT）. Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane （MIM）. Conversely, silencing VDAC1 ex- pression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and BcI-XL, indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome e release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDACl-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.展开更多
AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by ...AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a doseand timedependent manner. It did notaffect the cell cycle, but induced apoptosis. PE significantly decreased ΔΨm at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a doseand time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.展开更多
Objective： To 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. Methods： HT-29 ce...Objective： To 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. Methods： HT-29 cells were treated with curcumin （0-80 μmol/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）. Results： Curcumin 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 10-80 μmol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cyto- chrome c, the activation ofcaspase-3, and the cleavage of PARP in a dose-dependent manner. Conclusion： These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitoehondria-mediated pathway.展开更多
基金the National Natural Science Fundation of China(Grant No. 39800075 and 19890380) and the special Funds for Major StateBasic
文摘We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margination and DNA ladder were observed. After incubating isolated mitochondria in some hypotonic solutions, and centrifuging these mixtures at high speed, we got mitochondrial supernatants. It was found that in the absence of cytosolic factor, the supernatant alone was able to induce apoptotic changes in nuclei. The effective components were partly of protein. DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC- YVADCHO. Meanwhile, caspase inhibitors fully blocked chromatin condensation. Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted. It was found that this endonuclease is different from endonuclease G, cytochrome c-induced nuclease, or Ca2+-activated endonuclease.
文摘Voltage-dependent anion channel （VDAC）I is the main channel of the mitochondrial outer membrane （MOM） and it has been proposed to be part of the permeability transition pore （PTP）, a putative multiprotein complex candidate agent of the mitochondrial permeability transition （MPT）. Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane （MIM）. Conversely, silencing VDAC1 ex- pression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and BcI-XL, indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome e release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDACl-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.
基金Supported by The National Natural Science Foundation of China (No. 30872481)the Scientific and Technological Planning Foundation of Shaanxi Province (No. 2006K09-G7-1)
文摘AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a doseand timedependent manner. It did notaffect the cell cycle, but induced apoptosis. PE significantly decreased ΔΨm at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a doseand time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.
基金Project (No.2006C12098) supported by the Science and TechnologyDepartment of Zhejiang Province, China
文摘Objective： To 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. Methods： HT-29 cells were treated with curcumin （0-80 μmol/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）. Results： Curcumin 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 10-80 μmol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cyto- chrome c, the activation ofcaspase-3, and the cleavage of PARP in a dose-dependent manner. Conclusion： These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitoehondria-mediated pathway.
