OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compoun...OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.展开更多
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
Cucurbitacin E(CuE) is previously reported to exhibit antitumor effect by several means.In this study, CuE acted as a tyrosine kinase inhibitor interfering with the epidermal growth factor receptor/mitogen-activated p...Cucurbitacin E(CuE) is previously reported to exhibit antitumor effect by several means.In this study, CuE acted as a tyrosine kinase inhibitor interfering with the epidermal growth factor receptor/mitogen-activated protein kinase(EGFR/MAPK) signaling pathway and subsequently induced apoptosis and cell cycle arrest in non-small-cell lung cancer(NSCLC) cell line A549.The apoptosis regulators, cleaved Caspases-3 and Caspases-9, were observed to be increased with the treatment of CuE.The activated transcription factor STAT3 and the apoptosis inhibitor protein survivin were also observed to be reduced.The cell cycle regulators, CyclinA2, cylinB1, CyclinD1 and CyclinE, were also investigated and the results suggested that the cell cycle was arrested at G1/G0 phase.Treatment of CuE also altered the existence status of most of the participants in the EGFR/MAPK signaling.Phosphorylation of EGFR enhanced significantly, leading to the alteration of members downstream, either total amount or phosphorylation level, notably,MEK1/2 and ERK1/2.Moreover, the results of molecular simulation brought an insight on the interaction mechanism between CuE and EGFR.In summary, CuE exhibited anti-proliferative effect against A549 cells by targeting the EGFR/MAPK signaling pathway.展开更多
This study explored the effects of cucurbitacin E （CUE）, a bioactive compound from Cucurbitaceae, on the metabolism/ pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression ...This study explored the effects of cucurbitacin E （CUE）, a bioactive compound from Cucurbitaceae, on the metabolism/ pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression in rats. Liquid chro- matography-（tandem） mass spectrometry （LC-MS/MS） assay was used to detect tolbutamide as well as 4-hydroxytolbutamide, and then successfully applied to the pharmacokinetic study of tolbutamide in rats. The effect of CuE on CYP2C11 expression was determined by western blot. CuE （1.25-100μmol· L-1） competitively inhibited tolbutamide 4-hydroxylation （CYP2C11） activity only in concentration-dependent manner with a Ki value of 55.5 μmol L-1 in vitro. In whole animal studies, no signifi- cant difference in metabolism/pharmacokinetic of tolbutamide was found for the single pretreatment groups. In contrast, mul- tiple pretreatments of CuE （200 μg kg-1 d-1, 3 d, i.p.） significantly decreased tolbutamide clearance （CL） by 25% and pro- longed plasma half-time （T1/2） by 37%. Moreover, CuE treatment （50-200 pg kg-l d-1, i.p.） for 3 d did not affect CYP2C11 expression. These findings demonstrated that CuE competitively inhibited the metabolism of CYP2C11 substrates but had no effect on rat CYP2C11 expression. This study may provide a useful reference for the reasonable and safe use of herbal or nat- ural products containing CuE to avoid unnecessary drug-drug interactions.展开更多
基金The project supported by National Natural Science Foundation of China(81260497,81460564)Science and Technology Department of Jilin Province Youth Scientific Research Fund Project(201201075)
文摘OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.
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.
基金supported by the National Natural Science Foundation of China(Nos.31871717,31972160,and31601534)the Science and Technology Development Project Foundation of Jilin Province(Nos.20200201204JC and 20160441003SC)the National Key Research and Development Program of China(Nos.2018YFD0300201 and 2016YFD0300103)。
文摘Cucurbitacin E(CuE) is previously reported to exhibit antitumor effect by several means.In this study, CuE acted as a tyrosine kinase inhibitor interfering with the epidermal growth factor receptor/mitogen-activated protein kinase(EGFR/MAPK) signaling pathway and subsequently induced apoptosis and cell cycle arrest in non-small-cell lung cancer(NSCLC) cell line A549.The apoptosis regulators, cleaved Caspases-3 and Caspases-9, were observed to be increased with the treatment of CuE.The activated transcription factor STAT3 and the apoptosis inhibitor protein survivin were also observed to be reduced.The cell cycle regulators, CyclinA2, cylinB1, CyclinD1 and CyclinE, were also investigated and the results suggested that the cell cycle was arrested at G1/G0 phase.Treatment of CuE also altered the existence status of most of the participants in the EGFR/MAPK signaling.Phosphorylation of EGFR enhanced significantly, leading to the alteration of members downstream, either total amount or phosphorylation level, notably,MEK1/2 and ERK1/2.Moreover, the results of molecular simulation brought an insight on the interaction mechanism between CuE and EGFR.In summary, CuE exhibited anti-proliferative effect against A549 cells by targeting the EGFR/MAPK signaling pathway.
基金supported by the National Natural Science Foundation of China (81301908)the Science and Technology Commission of Shanghai Municipality (13ZR1412600, 14DZ2270100)
文摘This study explored the effects of cucurbitacin E （CUE）, a bioactive compound from Cucurbitaceae, on the metabolism/ pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression in rats. Liquid chro- matography-（tandem） mass spectrometry （LC-MS/MS） assay was used to detect tolbutamide as well as 4-hydroxytolbutamide, and then successfully applied to the pharmacokinetic study of tolbutamide in rats. The effect of CuE on CYP2C11 expression was determined by western blot. CuE （1.25-100μmol· L-1） competitively inhibited tolbutamide 4-hydroxylation （CYP2C11） activity only in concentration-dependent manner with a Ki value of 55.5 μmol L-1 in vitro. In whole animal studies, no signifi- cant difference in metabolism/pharmacokinetic of tolbutamide was found for the single pretreatment groups. In contrast, mul- tiple pretreatments of CuE （200 μg kg-1 d-1, 3 d, i.p.） significantly decreased tolbutamide clearance （CL） by 25% and pro- longed plasma half-time （T1/2） by 37%. Moreover, CuE treatment （50-200 pg kg-l d-1, i.p.） for 3 d did not affect CYP2C11 expression. These findings demonstrated that CuE competitively inhibited the metabolism of CYP2C11 substrates but had no effect on rat CYP2C11 expression. This study may provide a useful reference for the reasonable and safe use of herbal or nat- ural products containing CuE to avoid unnecessary drug-drug interactions.
