Objective:To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer ceils.Methods:Human non-small-cell lung carcinoma cell lines H460...Objective:To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer ceils.Methods:Human non-small-cell lung carcinoma cell lines H460,AS49 and H157 were cultured in vitro.The cytotoxic activities were determined using MTT assay.The apoptotic cells death was examined by flow cytometry using PI staining and DMA agarose gel electrophoresis.The activities of caspase-3, -8 and -9 were measured via ELISA.Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential.Bcl-2/Bcl-XL/ XIAP/Bid/ DR5 and DR4 proteins were analyzed using western blot.Results:The concentrations required for a 50%decrease in cell growth(IC<sub>50</sub>) ranged from 1.8 to 3.2 Jt M.Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway,including the depolarization of the mitochondrial membrane,release of cytochrome c from mitochondria,activation of procaspase-9 and -3,and increase of DNA fragments.Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zOEVD-FMK suppressed casticin-induced apoptosis.In addition,casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage.In H157 cell line,casticin increased expression of DRS at protein levels but not affect the expression of DR4.The prelreatmenl with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells.No correlation was found between cell sensitivity to casticin and that to p53 status,suggesting that casticin induce a p53- independent apoptosis.Conclusions:Our results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DRS in human lung cancer cells.展开更多
AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of c...AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide(MTT) assay.The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay(ELISA) detection kit,flow cytometry(FCM) after propidium iodide(PI) staining and DNA agarose gel electrophoresis.The caspase activities were measured using ELISA.Reactive oxygen species(ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCFH-DA) probe labeling.Intracellular glutathione(GSH) content was measured using a glutathione assay kit.The expression of death receptor(DR)4 and DR5 proteins was analyzed by Western blotting and FCM.RESULTS:Casticin significantly inhibited the growth of human HCC(PLC/PRF/5 and Hep G2) cells in a dosedependent manner(P < 0.05).Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner.The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil(26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h.Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3,-8 and-9 in a concentration-dependent manner(P < 0.05).Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder.Casticin reduced the GSH content(P < 0.05),but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells.The thiol antioxidants,acetylcysteine(NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis.In contrast,the nonthiol antioxidants,butylated hydroxyanisole and mannitol failed to do so.In the HCC cells treated with casticin for 24 h,DR5 protein level was increased.The expression of DR5 protein induced by casticin was inhibited by NAC.Pretreatment with DR5/Fc chimera protein,a blocking antibody,effectively attenuated the induction of apoptosis by casticin.CONCLUSION:Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.展开更多
Objective： The aim of the study was to investigate the effect of Casticin on proliferation inhibition of human cervical cancer HeLa cells in vitro and to unravel the associated mechanisms. Methods： Human cervical He...Objective： The aim of the study was to investigate the effect of Casticin on proliferation inhibition of human cervical cancer HeLa cells in vitro and to unravel the associated mechanisms. Methods： Human cervical HeLa cells were cultured in vitro. The inhibitory effect of Casticin on the viability of human cervical cancer HeLa ceils was evaluated by the MTT assay. The colony formation ability was detected by plate colony formation assay. Distribution of cell cycle was analyzed by flow cytometry. The protein expression levels were analyzed by Western blot. Results： Casticin significantly inhibited the growth of human cervical cancer HeLa cells in a dose- and time-dependent manner, and the IC50 was 2.82 μg/mL. The colony-forming rate was reduced drastically compared with control group （P 〈 0.05）. The cells were markedly arrested at G2/M phase after the treatment of Casticin for 48 h. Western blot showed that the expression of p21 protein was up-regulated and protein level of Cyctin B1 was depressed by Casticin in a concentration dependent manner. Conclusion： Casticin could inhibit the cell growth and lead to cell arrest in human cervical cancer HeLa cells, and the down-regulation of Cyclin B1 protein expression and activation of p21 protein might contribute to Casticin induced cell arrest in human cervical cancer HeLa cells.展开更多
Objective： The aim of this study was to explore the mechanisms by which the flavonoid casticin enhances tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）-induced apoptosis in colon cancer cells. Meth...Objective： The aim of this study was to explore the mechanisms by which the flavonoid casticin enhances tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）-induced apoptosis in colon cancer cells. Methods： Human colon cancer HT-29 cells were treated with TRAIL or casticin. Cytotoxicity was examined by MTT assay, and apoptosis determined by morphological observation and flow cytometric analysis. Death receptor 5 （DRS）, DR4, and endoplasmic reticulum （ER） stress response markers, including glucose regulating protein 78 （GRP78）, activating transcription factor 4 （ATF4） and CHOP （CCAAT/enhancer binding protein homologous protein）, were examined with western blot. Small interfering RNA （siRNA） transfection was employed to knock down CHOP. Results： HT-29 cells were resistance to TRAIL-induced apoptosis, but casticin, at subtoxic concentrations, potentiated HT-29 cells to TRAIL-induced apoptosis. Casticin up-regulated the expression of DR5 time-and dose-dependent manners, but had no effect on the expression of DR4. Also, casticin increased the levels of ER stress response markers （GRP78, ATF4 and CHOP） in a similar way to DR5. Knockdown of CHOP by specific siRNA, or salubrinal, an ER stress inhibitor, abolished the up-regulation of DR5 and enhancement of TRAIL-induced apoptosis by casticin. Conclusion： Casticin enhances TRAIL-induced apoptosis of colon cancer cells by ER stress-mediated up-regulation of DR5.展开更多
Objective:To explore whether casticin(CAS)suppresses stemness in cancer stem-like cells(CSLCs)obtained from human cervical cancer(CCSLCs)and the underlying mechanism.Methods:Spheres from He La and Ca Ski cells were us...Objective:To explore whether casticin(CAS)suppresses stemness in cancer stem-like cells(CSLCs)obtained from human cervical cancer(CCSLCs)and the underlying mechanism.Methods:Spheres from He La and Ca Ski cells were used as CCSLCs.DNA methyltransferase 1(DNMT1)activity and m RNA levels,self-renewal capability(Nanog and Sox2),and cancer stem cell markers(CD133 and CD44)were detected by a colorimetric DNMT activity/inhibition assay kit,quantitative real-time reverse transcription-polymerase chain reaction,sphere and colony formation assays,and immunoblot,respectively.Knockdown and overexpression of DNMT1 by transfection with sh RNA and c DNA,respectively,were performed to explore the mechanism for action of CAS(0,10,30,and 100 nmol/L).Results:DNMT1 activity was increased in CCSLCs compared with He La and Ca Ski cells(P<0.05).In addition,He La-derived CCSLCs transfected with DNMT1 sh RNA showed reduced sphere and colony formation abilities,and lower CD133,CD44,Nanog and Sox2 protein expressions(P<0.05).Conversely,overexpression of DNMT1 in He La cells exhibited the oppositive effects.Furthermore,CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in He La-derived CCSLCs(P<0.05).Interestingly,DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness.As expected,DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in He La cells.Conclusion:CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation,suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.展开更多
目的:对新疆特色药材一枝蒿进行质量标志物预测分析。方法:选用CNKI、万方、维普、中国生物医学文献数据库SinoMed、Pubmed、Web of Science等数据库检索一枝蒿、新疆一枝蒿和Artemisia rupestris L.的化学成分、药理作用和质量控制现状...目的:对新疆特色药材一枝蒿进行质量标志物预测分析。方法:选用CNKI、万方、维普、中国生物医学文献数据库SinoMed、Pubmed、Web of Science等数据库检索一枝蒿、新疆一枝蒿和Artemisia rupestris L.的化学成分、药理作用和质量控制现状,并从植物亲缘性、传统功效及药性、成分可测性、入血成分、方剂配伍、网络药理学等方面对一枝蒿质量标志物(Q-marker)进行预测和分析。结果:一枝蒿中黄酮类、倍半萜类、挥发油类化合物均具备作为一枝蒿质量标志物的潜力。初步推测一枝蒿酮酸、一枝蒿酮酸B、一枝蒿酮酸C、一枝蒿酮酸E、一枝蒿酮酸F、山柰酚、紫花牡荆素、艾黄素等成分可作为一枝蒿的质量标志物。结论:为完善一枝蒿的质量评价体系及临床应用提供参考。展开更多
基金supported,in part,by grants from the National Natural Scientific Foundation of China(Nos.30760248, 81072161,81172139,81060183)Programs for Changjiang Scholars and Innovative Research Team in University(No. IRT1119)+4 种基金Programs for Guangxi Innovative Research Team (No.2011GXNSFF018005)Program of Science and Technology of Guangxi(No.1140003A-16)Project of Scientific Research of Hunan Province the Administration Bureau of Traditional Chinese Medicine(No.2010081)the Project of Scientific Research of Hunan Province the Department of Education (No.10C0975Major Project of Scientific Research of Hunan Province the Department of Education(No.09A054)
文摘Objective:To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer ceils.Methods:Human non-small-cell lung carcinoma cell lines H460,AS49 and H157 were cultured in vitro.The cytotoxic activities were determined using MTT assay.The apoptotic cells death was examined by flow cytometry using PI staining and DMA agarose gel electrophoresis.The activities of caspase-3, -8 and -9 were measured via ELISA.Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential.Bcl-2/Bcl-XL/ XIAP/Bid/ DR5 and DR4 proteins were analyzed using western blot.Results:The concentrations required for a 50%decrease in cell growth(IC<sub>50</sub>) ranged from 1.8 to 3.2 Jt M.Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway,including the depolarization of the mitochondrial membrane,release of cytochrome c from mitochondria,activation of procaspase-9 and -3,and increase of DNA fragments.Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zOEVD-FMK suppressed casticin-induced apoptosis.In addition,casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage.In H157 cell line,casticin increased expression of DRS at protein levels but not affect the expression of DR4.The prelreatmenl with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells.No correlation was found between cell sensitivity to casticin and that to p53 status,suggesting that casticin induce a p53- independent apoptosis.Conclusions:Our results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DRS in human lung cancer cells.
基金Supported by The Scientifi c Research Project of Hunan Provincial Administration Bureau of Traditional Chinese Medicine,No. 2010081Scientific Research Project of Hunan Provincial Health Department,No. B2010-030Major Projects of Scien-tific Research of Hunan Provincial Department of Education,No. 09A054
文摘AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide(MTT) assay.The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay(ELISA) detection kit,flow cytometry(FCM) after propidium iodide(PI) staining and DNA agarose gel electrophoresis.The caspase activities were measured using ELISA.Reactive oxygen species(ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCFH-DA) probe labeling.Intracellular glutathione(GSH) content was measured using a glutathione assay kit.The expression of death receptor(DR)4 and DR5 proteins was analyzed by Western blotting and FCM.RESULTS:Casticin significantly inhibited the growth of human HCC(PLC/PRF/5 and Hep G2) cells in a dosedependent manner(P < 0.05).Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner.The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil(26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h.Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3,-8 and-9 in a concentration-dependent manner(P < 0.05).Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder.Casticin reduced the GSH content(P < 0.05),but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells.The thiol antioxidants,acetylcysteine(NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis.In contrast,the nonthiol antioxidants,butylated hydroxyanisole and mannitol failed to do so.In the HCC cells treated with casticin for 24 h,DR5 protein level was increased.The expression of DR5 protein induced by casticin was inhibited by NAC.Pretreatment with DR5/Fc chimera protein,a blocking antibody,effectively attenuated the induction of apoptosis by casticin.CONCLUSION:Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.
基金Supported by a grant from Hunan Provincial Health Department Research Fund (No.B2007-116)
文摘Objective： The aim of the study was to investigate the effect of Casticin on proliferation inhibition of human cervical cancer HeLa cells in vitro and to unravel the associated mechanisms. Methods： Human cervical HeLa cells were cultured in vitro. The inhibitory effect of Casticin on the viability of human cervical cancer HeLa ceils was evaluated by the MTT assay. The colony formation ability was detected by plate colony formation assay. Distribution of cell cycle was analyzed by flow cytometry. The protein expression levels were analyzed by Western blot. Results： Casticin significantly inhibited the growth of human cervical cancer HeLa cells in a dose- and time-dependent manner, and the IC50 was 2.82 μg/mL. The colony-forming rate was reduced drastically compared with control group （P 〈 0.05）. The cells were markedly arrested at G2/M phase after the treatment of Casticin for 48 h. Western blot showed that the expression of p21 protein was up-regulated and protein level of Cyctin B1 was depressed by Casticin in a concentration dependent manner. Conclusion： Casticin could inhibit the cell growth and lead to cell arrest in human cervical cancer HeLa cells, and the down-regulation of Cyclin B1 protein expression and activation of p21 protein might contribute to Casticin induced cell arrest in human cervical cancer HeLa cells.
文摘Objective： The aim of this study was to explore the mechanisms by which the flavonoid casticin enhances tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）-induced apoptosis in colon cancer cells. Methods： Human colon cancer HT-29 cells were treated with TRAIL or casticin. Cytotoxicity was examined by MTT assay, and apoptosis determined by morphological observation and flow cytometric analysis. Death receptor 5 （DRS）, DR4, and endoplasmic reticulum （ER） stress response markers, including glucose regulating protein 78 （GRP78）, activating transcription factor 4 （ATF4） and CHOP （CCAAT/enhancer binding protein homologous protein）, were examined with western blot. Small interfering RNA （siRNA） transfection was employed to knock down CHOP. Results： HT-29 cells were resistance to TRAIL-induced apoptosis, but casticin, at subtoxic concentrations, potentiated HT-29 cells to TRAIL-induced apoptosis. Casticin up-regulated the expression of DR5 time-and dose-dependent manners, but had no effect on the expression of DR4. Also, casticin increased the levels of ER stress response markers （GRP78, ATF4 and CHOP） in a similar way to DR5. Knockdown of CHOP by specific siRNA, or salubrinal, an ER stress inhibitor, abolished the up-regulation of DR5 and enhancement of TRAIL-induced apoptosis by casticin. Conclusion： Casticin enhances TRAIL-induced apoptosis of colon cancer cells by ER stress-mediated up-regulation of DR5.
基金Supported by the National Natural Science Foundation of China(No.82074075)Scientific Research Fund of Hunan Provincial Education Department(No.20C1340)+1 种基金the Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province,Hunan Normal University(No.2017TP1020)the Huxiang High-Level Talent Innovation Team(No.2018RS3072)。
文摘Objective:To explore whether casticin(CAS)suppresses stemness in cancer stem-like cells(CSLCs)obtained from human cervical cancer(CCSLCs)and the underlying mechanism.Methods:Spheres from He La and Ca Ski cells were used as CCSLCs.DNA methyltransferase 1(DNMT1)activity and m RNA levels,self-renewal capability(Nanog and Sox2),and cancer stem cell markers(CD133 and CD44)were detected by a colorimetric DNMT activity/inhibition assay kit,quantitative real-time reverse transcription-polymerase chain reaction,sphere and colony formation assays,and immunoblot,respectively.Knockdown and overexpression of DNMT1 by transfection with sh RNA and c DNA,respectively,were performed to explore the mechanism for action of CAS(0,10,30,and 100 nmol/L).Results:DNMT1 activity was increased in CCSLCs compared with He La and Ca Ski cells(P<0.05).In addition,He La-derived CCSLCs transfected with DNMT1 sh RNA showed reduced sphere and colony formation abilities,and lower CD133,CD44,Nanog and Sox2 protein expressions(P<0.05).Conversely,overexpression of DNMT1 in He La cells exhibited the oppositive effects.Furthermore,CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in He La-derived CCSLCs(P<0.05).Interestingly,DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness.As expected,DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in He La cells.Conclusion:CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation,suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.
文摘目的:对新疆特色药材一枝蒿进行质量标志物预测分析。方法:选用CNKI、万方、维普、中国生物医学文献数据库SinoMed、Pubmed、Web of Science等数据库检索一枝蒿、新疆一枝蒿和Artemisia rupestris L.的化学成分、药理作用和质量控制现状,并从植物亲缘性、传统功效及药性、成分可测性、入血成分、方剂配伍、网络药理学等方面对一枝蒿质量标志物(Q-marker)进行预测和分析。结果:一枝蒿中黄酮类、倍半萜类、挥发油类化合物均具备作为一枝蒿质量标志物的潜力。初步推测一枝蒿酮酸、一枝蒿酮酸B、一枝蒿酮酸C、一枝蒿酮酸E、一枝蒿酮酸F、山柰酚、紫花牡荆素、艾黄素等成分可作为一枝蒿的质量标志物。结论:为完善一枝蒿的质量评价体系及临床应用提供参考。
