Objective Cucurbitacins are the highly oxygenated tetracyclic triterpenes,which are predominantly found in the Cucurbitaceae family but are also present in several other families of the plant kingdom.A number of compo...Objective Cucurbitacins are the highly oxygenated tetracyclic triterpenes,which are predominantly found in the Cucurbitaceae family but are also present in several other families of the plant kingdom.A number of compounds of this group have been investigated for their cytotoxic,hepatoprotective,anti-inflammatory,cardiovascular and anti-diabetic activities.In China,the cucurbitacin preparation,which contains mostly cucurbitacin B and cucurbitacin E,has been clinically used for the treatment of the primary liver carcinoma.It has been previously reported that cucurbitacin E could produce cytotoxicity against a variety of cancer cells,and various mechanisms were implicated in its cytotoxic effect.The present study is to investigate the effect of cucurbitacin E on hepatoma cells in vitro and in vivo and to study their potential mechanisms of action.Methods The MTT assay was used to assess the viability of human HepG2 and BEL7402 hepatoma cells in vitro after treatment with different concentrations of cucurbitacin E.The cell cycle distribution was determined by flowcytometric analysis after propidium iodide(PI)staining.The cell cycle-related proteins were detected using western blotting analysis.Implanted mouse hepatoma H22 model was built to evaluate the growth inhibitory effect of cucurbitacin E in vivo in mice.Results Our studies found that cucurbitacin E(10-300 nM)produced anti-proliferative effect on human HepG2 and BEL7402 hepatoma cells in vitro without cytotoxicity.According to flowcytometric analysis,cucurbitacin E arrested the cell cycle at G2/M phase in both HepG2 and BEL7402 hepatoma cells after 24 h treatment.Cucurbitacin E induced the decrease in the level of CDK1 protein and the increase in the level of p21 protein,but had no effect on the levels of cyclin A,cyclin B1 and Cdc25C protein.In in vivo anti-tumor experiment,cucurbitacin E had significant inhibitory effects on the growth of mouse H22 hepatoma cells.Conclusions Cucurbitacin E inhibited the proliferation of hepatoma cells in vitro and in vivo,at least in part,through induction of cell cycle arrest at G2/M phase,which was mediated by concomitant upregulation of p21 and downregulation of CDK1.We consider that cucurbitacin E may be useful in the treatment of liver cancer.展开更多
Objective Flavans are a set of naturally occurring flavonoids possessing a 2-phenylchroman nucleus,which are widely distributed in the plant kingdom.A number of flavan compounds exhibit antitumor activities.In our pre...Objective Flavans are a set of naturally occurring flavonoids possessing a 2-phenylchroman nucleus,which are widely distributed in the plant kingdom.A number of flavan compounds exhibit antitumor activities.In our previous report,a straightforward synthetic procedure for 2(±)-7,8,3',4',5'-pentamethoxyflavan(PMF)was developed.To be more important,PMF showed growth inhibitory effect on various human tumor cell lines,especially against HL60 cells.In the present study,we aim to investigate the molecular mechanisms of action of PMF in HL60 cells.This is the first report of the molecular mechanisms on anti-tumor effect of flavan compounds.Methods Trypan blue exclusion experiment was used for cell growth inhibition assay.Cell apoptosis,cell cycle distribution and the mitochondrial membrane potential(MMP)were assessed by flowcytometric analysis after AO/EB,PI and Rh123 flurescence staining,respectively.Cell cycle-and apoptosis-related proteins were detected using western blotting analysis.Results PMF(1-30 μM)inhibited the growth of HL60 cells in a time-and concentration-dependent manner.Antiproliferative effect of PMF on HL60 cells was associated with G2/M cell cycle arrest,which was mediated by regulating the expression of p21,Cdc25C and cyclin A proteins and inhibiting the phosphorylation of Cdc2 at Thr161.The prolonged PMF treatment also induced apoptosis of HL60 cells,which was characterized by DNA fragmentation,cleavage of poly(ADP-ribose)polymerase,caspase-3,caspase-8 and caspase-9,changes of Bcl-2 and Bax expression and a decrease in the mitochondrial membrane potential(MMP).Furthermore,caspase-3 inhibitor,not caspase-8 inhibitor and caspase-9 inhibitor,completely blocked PMF-caused apoptosis.Conclusions PMF inhibited the growth of HL60 cells via induction of G2/M arrest and apoptosis.Blockade of cell cycle was associated with the downregulation of Cdc2 complex activity.Both death receptor and mitochondrial apoptotic pathways explained PMF-caused apoptosis.展开更多
Although previous studies showed that the principal oncoprotein encoded by Epstein-Barr virus, latent membrane protein 1 (LMP1), could induce the nasopharyngeal carcinoma cells in G2/M phase increased, littleis known ...Although previous studies showed that the principal oncoprotein encoded by Epstein-Barr virus, latent membrane protein 1 (LMP1), could induce the nasopharyngeal carcinoma cells in G2/M phase increased, littleis known about the target molecules and mechanisms. The present study demonstrated that LMP1 couldinduce the accumulation of p53 protein and upregulate its transactivity in a dose dependent manner, whichresulted in the decrease of the kinase activity of cdc2/cyclin B complex and inducing arrest at G2/M phasethrough the activation of NF-kB and AP-1 signaling pathways, and the effect of NF-kB was more obviousthan that of AP-1. This study provided some significant evidence for further elucidating the molecular mechanisms that LMP1 had effects on the surveillance mechanism of cell cycle and promoting the survivalof transformed cells and tumorigenesis.展开更多
As a cell cycle regulator, the Myb-related CDC5 protein was reported to be essential for the G2 phase of the cell cycle in yeast and animals, but little is known about its function in plants. Here we report the functi...As a cell cycle regulator, the Myb-related CDC5 protein was reported to be essential for the G2 phase of the cell cycle in yeast and animals, but little is known about its function in plants. Here we report the functional characterization of the CDC5 gene in Arabidopsis thaliana. Arabidopsis CDC5 （AtCDC5） is mainly expressed in tissues with high cell division activity, and is expressed throughout the entire process of embryo formation. The AtCDC5 loss-of-function mutant is embryonic lethal. In order to investigate the function of AtCDC5 in vivo, we generated AtCDC5-RNAi plants in which the expression of AtCDC5 was reduced by RNA interference. We found that the G2 to M （G2/M） phase transition was affected in the AtCDC5-RNAi plants, and that endoreduplication was increased. Additionally, the maintenance of shoot apical meristem （SAM） function was disturbed in the AtCDC5-RNAi plants, in which both the WUSCHEL （WUS）- CLAVATA （CLV） and the SHOOT MERISTEMLESS （STM） pathways were impaired. In situ hybridization analysis showed that the expression of STMwas greatly reduced in the shoot apical cells of the AtCDC5-RNAi plants. Moreover, cyclinB1 or Histone4 was found to be expressed in some of these cells when the transcript of STM was undetectable. These results suggest that AtCDC5 is essential for the G2/M phase transition and may regulate the function of SAM by controlling the expression ofSTMand WUS.展开更多
文摘Objective Cucurbitacins are the highly oxygenated tetracyclic triterpenes,which are predominantly found in the Cucurbitaceae family but are also present in several other families of the plant kingdom.A number of compounds of this group have been investigated for their cytotoxic,hepatoprotective,anti-inflammatory,cardiovascular and anti-diabetic activities.In China,the cucurbitacin preparation,which contains mostly cucurbitacin B and cucurbitacin E,has been clinically used for the treatment of the primary liver carcinoma.It has been previously reported that cucurbitacin E could produce cytotoxicity against a variety of cancer cells,and various mechanisms were implicated in its cytotoxic effect.The present study is to investigate the effect of cucurbitacin E on hepatoma cells in vitro and in vivo and to study their potential mechanisms of action.Methods The MTT assay was used to assess the viability of human HepG2 and BEL7402 hepatoma cells in vitro after treatment with different concentrations of cucurbitacin E.The cell cycle distribution was determined by flowcytometric analysis after propidium iodide(PI)staining.The cell cycle-related proteins were detected using western blotting analysis.Implanted mouse hepatoma H22 model was built to evaluate the growth inhibitory effect of cucurbitacin E in vivo in mice.Results Our studies found that cucurbitacin E(10-300 nM)produced anti-proliferative effect on human HepG2 and BEL7402 hepatoma cells in vitro without cytotoxicity.According to flowcytometric analysis,cucurbitacin E arrested the cell cycle at G2/M phase in both HepG2 and BEL7402 hepatoma cells after 24 h treatment.Cucurbitacin E induced the decrease in the level of CDK1 protein and the increase in the level of p21 protein,but had no effect on the levels of cyclin A,cyclin B1 and Cdc25C protein.In in vivo anti-tumor experiment,cucurbitacin E had significant inhibitory effects on the growth of mouse H22 hepatoma cells.Conclusions Cucurbitacin E inhibited the proliferation of hepatoma cells in vitro and in vivo,at least in part,through induction of cell cycle arrest at G2/M phase,which was mediated by concomitant upregulation of p21 and downregulation of CDK1.We consider that cucurbitacin E may be useful in the treatment of liver cancer.
文摘Objective Flavans are a set of naturally occurring flavonoids possessing a 2-phenylchroman nucleus,which are widely distributed in the plant kingdom.A number of flavan compounds exhibit antitumor activities.In our previous report,a straightforward synthetic procedure for 2(±)-7,8,3',4',5'-pentamethoxyflavan(PMF)was developed.To be more important,PMF showed growth inhibitory effect on various human tumor cell lines,especially against HL60 cells.In the present study,we aim to investigate the molecular mechanisms of action of PMF in HL60 cells.This is the first report of the molecular mechanisms on anti-tumor effect of flavan compounds.Methods Trypan blue exclusion experiment was used for cell growth inhibition assay.Cell apoptosis,cell cycle distribution and the mitochondrial membrane potential(MMP)were assessed by flowcytometric analysis after AO/EB,PI and Rh123 flurescence staining,respectively.Cell cycle-and apoptosis-related proteins were detected using western blotting analysis.Results PMF(1-30 μM)inhibited the growth of HL60 cells in a time-and concentration-dependent manner.Antiproliferative effect of PMF on HL60 cells was associated with G2/M cell cycle arrest,which was mediated by regulating the expression of p21,Cdc25C and cyclin A proteins and inhibiting the phosphorylation of Cdc2 at Thr161.The prolonged PMF treatment also induced apoptosis of HL60 cells,which was characterized by DNA fragmentation,cleavage of poly(ADP-ribose)polymerase,caspase-3,caspase-8 and caspase-9,changes of Bcl-2 and Bax expression and a decrease in the mitochondrial membrane potential(MMP).Furthermore,caspase-3 inhibitor,not caspase-8 inhibitor and caspase-9 inhibitor,completely blocked PMF-caused apoptosis.Conclusions PMF inhibited the growth of HL60 cells via induction of G2/M arrest and apoptosis.Blockade of cell cycle was associated with the downregulation of Cdc2 complex activity.Both death receptor and mitochondrial apoptotic pathways explained PMF-caused apoptosis.
文摘Although previous studies showed that the principal oncoprotein encoded by Epstein-Barr virus, latent membrane protein 1 (LMP1), could induce the nasopharyngeal carcinoma cells in G2/M phase increased, littleis known about the target molecules and mechanisms. The present study demonstrated that LMP1 couldinduce the accumulation of p53 protein and upregulate its transactivity in a dose dependent manner, whichresulted in the decrease of the kinase activity of cdc2/cyclin B complex and inducing arrest at G2/M phasethrough the activation of NF-kB and AP-1 signaling pathways, and the effect of NF-kB was more obviousthan that of AP-1. This study provided some significant evidence for further elucidating the molecular mechanisms that LMP1 had effects on the surveillance mechanism of cell cycle and promoting the survivalof transformed cells and tumorigenesis.
基金Acknowledgments The authors thank Dr Liying Du （Peking University, China） for technical help on the flow cytometric analysis. The authors also thank Dr Zhongchi Liu （University of Maryland, USA）, Dr Chun-Ming Liu （Institute of Botany CAS, China）, Dr Terry Matthew （University of Southampton, UK）, Professor Daochun Kong （Peking University, China） and Dr Naomi Nakayama （Yale University, USA） for critical comments and valuable discussion. This work was supported by the National Natural Science Foundation of China （GN 30625002 to L-J Qu）.
文摘As a cell cycle regulator, the Myb-related CDC5 protein was reported to be essential for the G2 phase of the cell cycle in yeast and animals, but little is known about its function in plants. Here we report the functional characterization of the CDC5 gene in Arabidopsis thaliana. Arabidopsis CDC5 （AtCDC5） is mainly expressed in tissues with high cell division activity, and is expressed throughout the entire process of embryo formation. The AtCDC5 loss-of-function mutant is embryonic lethal. In order to investigate the function of AtCDC5 in vivo, we generated AtCDC5-RNAi plants in which the expression of AtCDC5 was reduced by RNA interference. We found that the G2 to M （G2/M） phase transition was affected in the AtCDC5-RNAi plants, and that endoreduplication was increased. Additionally, the maintenance of shoot apical meristem （SAM） function was disturbed in the AtCDC5-RNAi plants, in which both the WUSCHEL （WUS）- CLAVATA （CLV） and the SHOOT MERISTEMLESS （STM） pathways were impaired. In situ hybridization analysis showed that the expression of STMwas greatly reduced in the shoot apical cells of the AtCDC5-RNAi plants. Moreover, cyclinB1 or Histone4 was found to be expressed in some of these cells when the transcript of STM was undetectable. These results suggest that AtCDC5 is essential for the G2/M phase transition and may regulate the function of SAM by controlling the expression ofSTMand WUS.
