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
Cyclin-dependent kinase 1 (CDK1) plays an essential role in cell cycle regulation.However,as mouse Cdk1embryos die early,the role of CDK1 in regulating the cell cycle and embryo development remains unclear.Here,we sho...Cyclin-dependent kinase 1 (CDK1) plays an essential role in cell cycle regulation.However,as mouse Cdk1embryos die early,the role of CDK1 in regulating the cell cycle and embryo development remains unclear.Here,we showed that zebrafish cdk1^(-/-)embryos exhibit severe microphthalmia accompanied by multiple defects in S phase entry,M phase progression,and cell differentiation but not in interkinetic nuclear migration.We identified Top2a as a potential downstream target and cyclin A2 and cyclin B1 as partners of Cdk1 in cell cycle regulation via an in silico analysis.While depletion of either cyclin A2 or Top2a led to the decreased S phase entry in zebrafish retinal cells,the depletion of cyclin B1 led to M phase arrest.Moreover,phosphorylation of Top2a at serine 1213 (S1213) was nearly abolished in both cdk1 and ccna2mutants,but not in ccnb1 mutants.Furthermore,overexpression of TOP2A^(S1213D),the phosphomimetic form of human TOP2A,rescued S phase entry and alleviated the microphthalmia defects in both cdk1^(-/-)and ccna2^(-/-)embryos.Taken together,our data suggest that Cdk1 interacts with cyclin A2 to regulate S phase entry partially through Top2a phosphorylation and interacts with cyclin B1 to regulate M phase progression.展开更多
OBJECTIVE:To investigate the mechanism underlying the anticancer effect of Artemisia species through the inhibition of cell growth and induction of apoptosis in breast carcinoma cells.METHODS:To evaluate the anticance...OBJECTIVE:To investigate the mechanism underlying the anticancer effect of Artemisia species through the inhibition of cell growth and induction of apoptosis in breast carcinoma cells.METHODS:To evaluate the anticancer activity of methanol extracts of eight Artemisia species(Artemisia stolonifera,Artemisia selengensis,Artemisia japonica,Artemisia Montana,Artemisia capillaris,Artemisia sylvatica,Artemisia keiskeana,and Artemisia scoparia),we first investigated the proliferation of estrogen receptor(ER)-positive MCF-7breast carcinoma cells exposed to 5 or 200 g/mL for72 h.Apoptosis induction was assessed by an Annexin V binding assay in cells exposed to extracts at a high concentration(200 g/mL).To verify the mechanism of apoptosis,ER expression and its related signaling was investigated using an immunoblot assay under the same conditions.RESULTS:MCF-7 cells showed the strongest antiproliferative response to the tested extracts.Howev-er,a biphasic effect was observed:the extracts inhibited proliferation at high concentrations whereas they stimulated it at low ones.ER expression was similarly modulated by the extracts.However,all of the extracts induced apoptosis at a high concentration(200 g/mL).Compared to the control level,exposure to the extracts resulted in a remarkable increase in the shift of cell populations.CONCLUSION:The present study suggests that the tested Artemisia species exerted their anticancer effects through the induction of apoptosis via an ER-related pathway.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
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supported by grants from the National Key Research and Development Program of China (2017YFA0104600)the National Natural Science Foundation of China (31970767, 31771597 and 31571515)+1 种基金the National Basic Research Program of China (973 Program 2012CB966601 and 2013CB945300)the Ministry of Science and Technology of the People’s Republic of China (2011DFB30010)。
文摘Cyclin-dependent kinase 1 (CDK1) plays an essential role in cell cycle regulation.However,as mouse Cdk1embryos die early,the role of CDK1 in regulating the cell cycle and embryo development remains unclear.Here,we showed that zebrafish cdk1^(-/-)embryos exhibit severe microphthalmia accompanied by multiple defects in S phase entry,M phase progression,and cell differentiation but not in interkinetic nuclear migration.We identified Top2a as a potential downstream target and cyclin A2 and cyclin B1 as partners of Cdk1 in cell cycle regulation via an in silico analysis.While depletion of either cyclin A2 or Top2a led to the decreased S phase entry in zebrafish retinal cells,the depletion of cyclin B1 led to M phase arrest.Moreover,phosphorylation of Top2a at serine 1213 (S1213) was nearly abolished in both cdk1 and ccna2mutants,but not in ccnb1 mutants.Furthermore,overexpression of TOP2A^(S1213D),the phosphomimetic form of human TOP2A,rescued S phase entry and alleviated the microphthalmia defects in both cdk1^(-/-)and ccna2^(-/-)embryos.Taken together,our data suggest that Cdk1 interacts with cyclin A2 to regulate S phase entry partially through Top2a phosphorylation and interacts with cyclin B1 to regulate M phase progression.
基金Supported by Priority Research Centers Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education,Science and Technology(NRF-2009-0094017 and NRF-2011-0017017)
文摘OBJECTIVE:To investigate the mechanism underlying the anticancer effect of Artemisia species through the inhibition of cell growth and induction of apoptosis in breast carcinoma cells.METHODS:To evaluate the anticancer activity of methanol extracts of eight Artemisia species(Artemisia stolonifera,Artemisia selengensis,Artemisia japonica,Artemisia Montana,Artemisia capillaris,Artemisia sylvatica,Artemisia keiskeana,and Artemisia scoparia),we first investigated the proliferation of estrogen receptor(ER)-positive MCF-7breast carcinoma cells exposed to 5 or 200 g/mL for72 h.Apoptosis induction was assessed by an Annexin V binding assay in cells exposed to extracts at a high concentration(200 g/mL).To verify the mechanism of apoptosis,ER expression and its related signaling was investigated using an immunoblot assay under the same conditions.RESULTS:MCF-7 cells showed the strongest antiproliferative response to the tested extracts.Howev-er,a biphasic effect was observed:the extracts inhibited proliferation at high concentrations whereas they stimulated it at low ones.ER expression was similarly modulated by the extracts.However,all of the extracts induced apoptosis at a high concentration(200 g/mL).Compared to the control level,exposure to the extracts resulted in a remarkable increase in the shift of cell populations.CONCLUSION:The present study suggests that the tested Artemisia species exerted their anticancer effects through the induction of apoptosis via an ER-related pathway.
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.
