Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli.To determine a specific substrate of Cyclin A-Cdk2 for apoptosis,in this study,we carried out an...Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli.To determine a specific substrate of Cyclin A-Cdk2 for apoptosis,in this study,we carried out an in vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source,and recombinant protein GST-Bad as a substrate.Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro.To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo,we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn,a dominant negative version of Cdk2,in Hela cells and determined the phosphorylation status of protein Bad.The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock transfectent.Moreover,etoposide also caused the phosphorylation of endogenetic Bad.In conclusion,here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.展开更多
Background:Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health.Substantial evidence has revealed that preventing vascular smooth muscle ...Background:Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health.Substantial evidence has revealed that preventing vascular smooth muscle cell proliferation using a drug-eluting stent is an effective approach to improve restenosis.Cucurbitacins have been demonstrated to exert an anti-proliferation effect in various tumors and a hypoten-sive effect.This study aims to investigate the role of cucurbitacins extracted from Cucumis melo L.(CuECs)and cucurbitacin B(CuB)on restenosis.Methods:C57BL/6 mice were subjected to left carotid artery ligation and subcu-taneously injected with CuECs or CuB for 4 weeks.Hematoxylin-Eosin,immuno-fluorescence and immunohistochemistry staining were used to evaluate the effect of CuECs and CuB on neointimal hyperplasia.Western blot,real-time PCR,flow cytometry analysis,EdU staining and cellular immunofluorescence assay were em-ployed to measure the effects of CuECs and CuB on cell proliferation and the cell cycle in vitro.The potential interactions of CuECs with cyclin A2 were performed by molecular docking.Results:The results demonstrated that both CuECs and CuB exhibited significant inhibitory effects on neointimal hyperplasia and proliferation of vascular smooth muscle cells.Furthermore,CuECs and CuB mediated cell cycle arrest at the S phase.Autodocking analysis demonstrated that CuB,CuD,CuE and CuI had high binding en-ergy for cyclin A2.Our study also showed that CuECs and CuB dramatically inhibited FBS-induced cyclin A2 expression.Moreover,the expression of cyclin A2 in CuEC-and CuB-treated neointima was downregulated.Conclusions:CuECs,especially CuB,exert an anti-proliferation effect in VSMCs and may be potential drugs to prevent restenosis.展开更多
目的研究Cyc lin D1和Bc l-2在口腔鳞癌组织和正常口腔粘膜中的表达及意义。方法用免疫组织化学S-P法对62例口腔鳞癌手术切除标本和10例正常口腔粘膜标本中Cyc lin D1和Bc l-2基因蛋白的表达情况进行检测。结果口腔鳞癌组织中Cyc lin D...目的研究Cyc lin D1和Bc l-2在口腔鳞癌组织和正常口腔粘膜中的表达及意义。方法用免疫组织化学S-P法对62例口腔鳞癌手术切除标本和10例正常口腔粘膜标本中Cyc lin D1和Bc l-2基因蛋白的表达情况进行检测。结果口腔鳞癌组织中Cyc lin D1表达率67.7%(42/62),正常口腔粘膜组织中未见表达,两组间差异有显著性(P<0.05);Bc l-2在正常口腔粘膜组织中也未见表达,在癌组织中Bc l-2蛋白阳性表达率58.1%(36/62),两组间差异也有显著性(P<0.05)。结论Cyc lin D1和Bc l-2两种蛋白在癌组织中存在过表达,口腔鳞癌组织中Cyc lin D1与Bc l-2的表达呈正相关。过表达的Cyc lin D1与Bc l-2可作为评价口腔鳞癌组织分级的参考指标之一。展开更多
基金Supported by the National Natural Science Foundation of China(Nos.30370294and30640064).
文摘Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli.To determine a specific substrate of Cyclin A-Cdk2 for apoptosis,in this study,we carried out an in vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source,and recombinant protein GST-Bad as a substrate.Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro.To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo,we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn,a dominant negative version of Cdk2,in Hela cells and determined the phosphorylation status of protein Bad.The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock transfectent.Moreover,etoposide also caused the phosphorylation of endogenetic Bad.In conclusion,here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.
基金Scientific Research Fund Project of Liaoning Provincial Department of Education,Grant/Award Number:LJKMZ20221267,LJKZ0840 and LJKZ0847National Natural Science Foundation of China Grants,Grant/Award Number:81900267。
文摘Background:Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health.Substantial evidence has revealed that preventing vascular smooth muscle cell proliferation using a drug-eluting stent is an effective approach to improve restenosis.Cucurbitacins have been demonstrated to exert an anti-proliferation effect in various tumors and a hypoten-sive effect.This study aims to investigate the role of cucurbitacins extracted from Cucumis melo L.(CuECs)and cucurbitacin B(CuB)on restenosis.Methods:C57BL/6 mice were subjected to left carotid artery ligation and subcu-taneously injected with CuECs or CuB for 4 weeks.Hematoxylin-Eosin,immuno-fluorescence and immunohistochemistry staining were used to evaluate the effect of CuECs and CuB on neointimal hyperplasia.Western blot,real-time PCR,flow cytometry analysis,EdU staining and cellular immunofluorescence assay were em-ployed to measure the effects of CuECs and CuB on cell proliferation and the cell cycle in vitro.The potential interactions of CuECs with cyclin A2 were performed by molecular docking.Results:The results demonstrated that both CuECs and CuB exhibited significant inhibitory effects on neointimal hyperplasia and proliferation of vascular smooth muscle cells.Furthermore,CuECs and CuB mediated cell cycle arrest at the S phase.Autodocking analysis demonstrated that CuB,CuD,CuE and CuI had high binding en-ergy for cyclin A2.Our study also showed that CuECs and CuB dramatically inhibited FBS-induced cyclin A2 expression.Moreover,the expression of cyclin A2 in CuEC-and CuB-treated neointima was downregulated.Conclusions:CuECs,especially CuB,exert an anti-proliferation effect in VSMCs and may be potential drugs to prevent restenosis.
文摘目的研究Cyc lin D1和Bc l-2在口腔鳞癌组织和正常口腔粘膜中的表达及意义。方法用免疫组织化学S-P法对62例口腔鳞癌手术切除标本和10例正常口腔粘膜标本中Cyc lin D1和Bc l-2基因蛋白的表达情况进行检测。结果口腔鳞癌组织中Cyc lin D1表达率67.7%(42/62),正常口腔粘膜组织中未见表达,两组间差异有显著性(P<0.05);Bc l-2在正常口腔粘膜组织中也未见表达,在癌组织中Bc l-2蛋白阳性表达率58.1%(36/62),两组间差异也有显著性(P<0.05)。结论Cyc lin D1和Bc l-2两种蛋白在癌组织中存在过表达,口腔鳞癌组织中Cyc lin D1与Bc l-2的表达呈正相关。过表达的Cyc lin D1与Bc l-2可作为评价口腔鳞癌组织分级的参考指标之一。
