苦参碱调节Bax和Bcl-2蛋白表达诱导肺腺癌A549细胞凋亡的机制

目的:探讨Bax和Bcl-2蛋白在苦参碱诱导A549细胞凋亡中的作用。方法:MTT法检测苦参碱对A549细胞生长的抑制作用,流式细胞仪检测细胞凋亡及Bcl-2、Bax和Caspase-3蛋白的表达情况。结果:不同浓度的苦参碱对肺腺癌细胞生长均有抑制作用,抑制率与浓度呈正相关。苦参碱随时间进行性降低Bcl-2蛋白的表达,相应地稳步提高Bax蛋白的表达,同时促进Bax从胞浆内向线粒体移位,紧接着降解Caspase-3蛋白。结论:苦参碱通过调节细胞内Bax和Bcl-2蛋白的表达经线粒体信号转导途径诱导A549细胞的凋亡。     

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3

Aim of the study

Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated.

Materials and Methods

Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunoflurenscence method. Activity of caspase-3 enzyme was measured by spectrofluorometry.

Results

MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Florescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate.

Conclusions

Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.     

茅莓提取物对大鼠局灶性脑缺血再灌注神经细胞凋亡及其相关蛋白表达的影响

目的：探讨茅莓提取物(RP)对缺血性脑损伤的神经保护机制。方法：采用大鼠大脑中动脉阻塞造成局灶性脑缺血2 h再灌注24 h模型,TUNEL标记和免疫组化的方法观察茅莓提取物5,10 g·kg^-1ig对神经细胞凋亡数目及其相关蛋白Bcl-2,Bax表达的变化。结果：茅莓提取物5,10 g·kg^-1显著抑制神经细胞凋亡,增加Bcl-2阳性细胞的表达,降低Bax阳性细胞的表达,Bcl-2/Bax的表达比例增加。结论：茅莓提取物抗凋亡机制可能与增加Bcl-2阳性细胞的表达,降低Bax阳性细胞的表达,提高Bcl-2/Bax有关。     

Selective induction of apoptosis through activation of caspase-8 in human leukemia cells (Jurkat) by dandelion root extract

Aim of study

Dandelion extracts have been used in traditional Native American Medicine and Traditional Chinese Medicine (TCM) for treatment of leukemia and breast cancer; however, the mechanism of action remains unknown. Today, DRE is mainly marketed for management of gastrointestinal and liver disorders. The current study aims to determine the anti-cancer activity of dandelion root extract (DRE) against human leukemia, and to evaluate the specificity and mechanism of DRE-induced apoptosis.

Materials and methods

The effect of DRE on cell viability was evaluated using the colorimetric-based WST-1 assay. Apoptotic cell death was monitored by nuclear condensation and confirmed by exposure of phosphatidylserine to outer leaflet of plasma membrane. Activation of caspases was detected using a fluorogenic substrate specific to either caspase-8 or -3. Loss of mitochondrial membrane potential was observed by microscopy using JC-1 dye. The apoptotic effect of DRE was also evaluated on a dominant-negative FADD (Fas-associated death domain) cell line and non-cancerous peripheral blood mononuclear cells (PBMCs).

Results

Aqueous DRE effectively induces apoptosis in human leukemia cell lines in a dose and time dependent manner. Very early activation of caspase-8 and the subsequent activation of caspase-3 indicate that DRE may be inducing extrinsic or receptor-mediated apoptosis. Caspase inhibition rendered this extract ineffective, thus DRE-induced apoptosis is caspase-dependent. Moreover, the dominant-negative FADD cells that are unable to form a complete DISC (death-inducing signaling complex) were resistant to DRE treatment, which further confirms our hypothesis that DRE induces receptor-mediated apoptosis. Interestingly, non-cancerous peripheral blood mononuclear cells (PBMCs) exposed to aqueous DRE under the same treatment conditions as leukemia cells were not significantly affected.

Conclusion

Our results suggest that aqueous DRE contains components that act to induce apoptosis selectively in cultured leukemia cells, emphasizing the importance of this traditional medicine and thus presents a potential novel non-toxic alternative to conventional leukemia therapy.     

芪黄煎剂对缺血-再灌注大鼠肠黏膜上皮细胞Bcl-2、Bax及Caspase-3、9 mRNA表达的影响

目的 观察中药芪黄煎剂对缺血-再灌注(ischemia-reperfusion, I/R)损伤大鼠肠黏膜上皮细胞凋亡基因Bcl-2、Bax mRNA表达和信号转导分子Caspase-3、9 mRNA表达的影响。     

龙葵碱调控Bcl-2与Bax蛋白表达及caspase-3活性诱导HepG2细胞凋亡的研究

目的 探讨龙葵碱诱导HepG2细胞凋亡的作用机制.方法 透射电镜观察凋亡细胞形态变化,原位缺口末端榆测法(TUNEL法)检测DNA断裂情况,流式细胞术检测细胞凋亡率,间接免疫荧光法激光共聚焦扫描显微术检测Bcl-2与Bax蛋白表达,比色法检测caspase-3活性的变化.结果 在透射电镜下观察,龙葵碱组细胞出现细胞固缩,染色质致密,核凝聚固缩,染色体断裂形成核碎块,凋亡小体形成等细胞凋亡特征形态.TUNEL法发现龙葵碱高、中、低剂量组HepG2细胞均有绿色荧光,阴性对照组无荧光.流式细胞术分析表明0.4、2、10μmol/L龙葵碱作用HepG2细胞24 h凋亡率分别为4.0%、8.5%、20.1%.同时,龙葵碱升高caspase-3活性,下调Bcl-2蛋白表达,上调Bax蛋白表达.结论 龙葵碱通过降低Bcl-2/Bax的值,激活caspase-3酶活性诱导HepG2细胞凋亡.     

Cupressus lusitanica (Cupressaceae) leaf extract induces apoptosis in cancer cells

A crude ethanolic extract of Cupressus lusitanica Mill. leaves demonstrate cytotoxicity in a panel of cancer cell lines. Cell death was due to apoptosis, as assessed by morphologic features (chromatin condensation and apoptotic bodies formation) and specific DNA fragmentation detected by in situ end-labeling of DNA breaks (TUNEL). The apoptotic cell death was induced timely in a dose-dependent manner. Despite the absence of changes in the expression levels of antiapoptotic protein Bcl-2, proapoptotic Bax protein variants ω and δ were increased. These results warrant further research of possible antitumor compounds in this plant.     

As2O3对大鼠生精细胞凋亡及其Bcl-2/Bax表达的变化

目的；观察不同剂量的染砷（As2O3）大鼠生精细胞凋亡及其Bcl-2／Bax表达的变化。方法：40只健康雄性sD大鼠随机分成4组，分别为0．375，0．75，1．5mg·kg^-13个染毒组和正常正常组，灌胃法连续给药16周后处死，对各组大鼠进行睾丸脏器系数、精子头计数，并计算每日精子生成量（DSP）。采用TUNEL法检测大鼠生精细胞凋亡，免疫组化法检测大鼠生精细胞Bcl-2／Bax的表达。结果：①与正常组相比，中剂量组（0．75mg·kg^-1）和高剂量组（1．5mg·kg^-1）睾丸精子头计数和DSP均显著降低（P〈0．01），生精细胞凋亡指数（AI）显著升高（P〈0．01），生精细胞Bcl-2阳性产物表达明显降低（P〈0．01），Bax表达明显升高（P〈0．01）；②DSP与生精细胞AI呈负相关（r=-0．563，P〈0．01）；③生精细胞凋亡与Bcl-2表达呈负相关（r=-0．825，P〈0．01），与Bax表达呈正相关（r=0．710，P〈0．01）。结论：一定剂量的As2O3可通过抑制生精细胞Bcl-2和促进Bax的表达，诱导生精细胞凋亡增加而导致精子生成量的减少，产生雄性生殖毒性。     

Swietenia macrophylla King induces mitochondrial-mediated apoptosis through p53 upregulation in HCT116 colorectal carcinoma cells

Ethnopharmacological relevance

Swietenia macrophylla King is a traditional herb used to treat various diseases including hypertension, diabetes and cancer. Previous study demonstrated its anti-tumor effect but the potential mechanisms have not been clearly defined. The current study was to further investigate the underlying mechanism of ethyl acetate fraction of Swietenia macrophylla (SMEAF)-induced anti-proliferative effect and apoptosis in HCT116 colorectal carcinoma cell.

Materials and methods

Cell viability was evaluated in HCT116 cells by trypan blue exclusion assay. Apoptotic cell death was detected by Hoechst 33342/propidium iodide (PI) staining and intracellular reactive oxygen species (ROS) was analyzed by flow cytometry. The apoptotic gene and protein expression were determined by Real-time quantitative PCR (q-PCR) and immunofluorescence staining using flow cytometry, respectively.

Results

SMEAF significantly inhibited HCT116 cell viability and induced apoptosis in a dose-dependent manner. SMEAF-induced apoptosis was triggered by the activation of p53 and intracellular reactive oxygen species (ROS) production. Moreover, the significant increase in p53 was accompanied by a decrease murine double minute 2 (MDM2) expression. SMEAF significantly increased the expression of the Bax protein resulting in a markedly elevated Bax/Bcl-2 ratio which may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3/7 and caspase-9 activation.

Conclusion

These results suggested that SMEAF exerts its antitumor activity in HCT116 cells by activating proapoptotic signaling pathway through intracellular ROS formation triggering the mitochondrial-mediated pathway via p53 activation.     

穴位注射对脑缺血再灌注大鼠海马回Bcl-2、Bax蛋白表达以及血液流变学的影响

目的:探讨穴位注射对脑缺血的改善作用及其机制.方法:48只Wistar大鼠随机分为假手术组、模型组、穴位注射组.采用线栓法制备大鼠大脑中动脉缺血再灌注模型.穴位注射组分别在造模后6、12、24 h在"百会""大椎"穴进行复方当归注射液穴位注射.运用免疫组化法观察再灌注24 h后各组大鼠脑组织缺血区海马回Bcl-2、Ba...     

白藜芦醇对K562/ADM细胞促凋亡作用研究

目的:探讨白藜芦醇诱导K562/ADM细胞凋亡的效应与细胞活性氧水平的关系。方法:应用噻唑蓝(MTT)比色法、光镜、电镜和流式细胞术(FCM)检测细胞凋亡;FCM测定细胞内活性氧(ROS)水平。结果:白藜芦醇显著抑制K562/ADM细胞增殖,形态学呈现典型的凋亡改变;FCM分析显示S期细胞数明显增多,出现S/G2期阻滞;ROS水平升高。结论:白藜芦醇诱导K562/ADM胞凋亡与细胞内活性氧水平升高有关。     

JR6, a new compound isolated from Justicia procumbens, induces apoptosis in human bladder cancer EJ cells through caspase-dependent pathway

Ethnopharmacological relevance

Numerous efforts have been conducted in searching for effective agents against cancer, in particular from herbal medicines. Justicia procumbens is a traditional herbal remedy which was produced in the south-western and southern provinces of China and Taiwan province used to treat fever, pain, and cancer. Here, we identified a new compound 6′-hydroxy justicidin A (JR6) from Justicia procumbens, which showed obvious anti-cancer effects.

Materials and methods

The cytotoxicity activity was assayed using MTT and SRB. Intracellular ROS visualization and quantification were acquired by using a laser scanning confocal microscopy. Apoptosis was measured using a propidium iodide (PI) apoptosis detection kit by flow cytometry. Activation of caspases (caspase-3, caspase-8, and caspase-9) was evaluated respectively using GloMax luminescence detector and Caspase-Glo 3,8,9 assay kits. Loss of mitochondrial membrane potential was observed by microscopy using JC-1 dye. Quantitative real-time PCR analysis was employed to detect the expression of protein associated with cell death.

Results

JR6 remarkably inhibited growth in human bladder cancer EJ cells by decreasing cell proliferation, reduced the SOD activity, increased the content of reactive oxygen species (ROS), and induced apoptosis. Activation of caspase-8, caspase-9, and the subsequent activation of caspase-3 indicated that JR6 may be inducing intrinsic and extrinsic apoptosis pathways. Caspase-3, caspase-8, and caspase-9 inhibition rendered this extract ineffective, thus JR6-induced apoptosis is caspase-dependent. JR6 also disrupted the mitochondrial membrane potential (Δψm) and unregulated the Bax and p53 expressions in EJ cells.

Conclusion

These observations suggest that JR6 induce apoptosis through caspase-dependent pathway in human bladder cancer EJ cells, emphasizing the importance of this traditional medicine and thus presents a potential novel alternative to bladder cancer therapy.     

白藜芦醇对Hepa 1-6肝癌细胞凋亡和ROS的影响

目的:观察白藜芦醇对小鼠肝癌Hepa 1-6细胞凋亡及活性氧(Reactive oxygen species,ROS)作用。方法:MTT比色法检测白藜芦醇对Hepa 1-6细胞增殖的影响;Hoechst 33258染色检测细胞凋亡形态;流式细胞术检测细胞凋亡;Western Blot法检测活化型Caspase-3;2',7'-二氯氢化荧光素乙二脂(DCF-DA)染色法结合荧光酶标仪检测细胞内ROS的产生。结果:与对照组比较,20、40、80μmol/L白藜芦醇作用24、48、72 h均可显著抑制肝癌Hepa1-6细胞增殖,呈一定的时间与剂量依赖性。不同浓度白藜芦醇作用48 h可促使Hepa 1-6细胞凋亡,呈现细胞凋亡形态改变,同时伴有活化型Caspase-3及ROS的产生。结论:白藜芦醇可以抑制Hepa 1-6细胞增殖,诱导肝癌细胞凋亡,可能与Caspase-3活化及细胞内ROS水平升高相关。     

双氢青蒿素通过调节凋亡相关蛋白的表达及活性氧的产生而抑制胰腺癌JF-305细胞的增殖

探讨双氢青蒿素诱导人胰腺癌JF-305细胞凋亡作用及活性氧在双氢青蒿素诱导JF-305细胞凋亡中的作用。采用MTT法考察不同浓度双氢青蒿素对人胰腺癌JF-305细胞增殖的影响,流式细胞术检测细胞周期,Hochest 333258荧光染色法观察细胞凋亡形态,Annexin V荧光染色法检测JF-305细胞凋亡的变化,DCFH-DA检测凋亡过程中活性氧(ROS)的变化。Western blot检测细胞内Bax,Bcl-2,Cleaved caspase-3,Cleaved caspase-9和Cyto C蛋白表达的变化。与对照相比,双氢青蒿素作用JF-305细胞48 h,细胞增殖受到明显抑制(P0.05);细胞被阻滞于G2/M期;细胞出现核浓缩聚集、碎裂的凋亡形态,细胞凋亡比例升高(P0.05);DCFH-DA检测双氢青蒿素给药组细胞ROS明显升高(P0.05);Western blot结果显示,双氢青蒿素作用后细胞内Bcl-2蛋白表达下调,Bax蛋白表达上调,Bax/Bcl-2蛋白表达比例升高,Cleaved caspase-3,Cleaved caspase-9和Cyto C蛋白表达升高。双氢青蒿素能诱导JF-305细胞凋亡,其凋亡过程可能与ROS的生成增加相关。     

The ethyl acetate fraction of Polytrichum commune L.ex Hedw induced cell apoptosis via reactive oxygen species in L1210 cells

Ethnopharmacological relevance

Polytrichum commune L.ex Hedw is a traditional Chinese herb for treatment of fever, hemostatic, uterine prolapse and especially for leukemia. Previous studies indicated its anti-leukemia effect but the potential mechanisms have not been fully explained.

Aims of the study

The present study was further to investigate the underlying mechanism of ethyl acetate extract of Polytrichum commune L.ex Hedw (EEF)-induced toxicity and apoptosis in L1210 cells.

Materials and methods

Viability, DNA damage and apoptotic protein expressions of L1210 cells were analyzed by ViaCount, comet assay and western blot, respectively. At different times after EEF treatment, Bax redistribution in L1210 cells was examined using confocal microscopy; loss of mitochondrial membrane potential (MMP) was monitored by fluorescence microscope using rhodamine 123 staining; Intracellular reactive oxygen species (ROS) generation and DNA fragmentation were measured by flow cytometry using fluorescent dye, DCFH-DA and PI, respectively.

Results

EEF significantly inhibited L1210 cell survival, promoted Bax translocation onto mitochondria, stimulated caspase-9 activation and subsequent DNA damage in L1210 cells. Abundant ROS was detected in L1210 cells after EEF treatment, and the ROS scavenger NAC significantly relieved EEF-induced cell viability decline, MMP loss, and DNA fragmentation.

Conclusion

EEF could induce mitochondria-dependent cell apoptosis in L1210 cells, and ROS may play an important role in this action.     

小檗碱对HeLa细胞凋亡及其凋亡相关蛋白表达的影响

目的 研究小檗碱对人宫颈癌HeLa细胞株增殖及凋亡的影响及其发生机制.方法 采用MTT法观察小檗碱对HeLa细胞增殖的影响,DNA ladder、流式细胞仪等方法进行细胞凋亡检测,用Western blotting方法检测凋亡过程中相关蛋白Bel-2和Bax表达的变化.结果 小檗碱在体外试验中能明显抑制HeLa细胞的增殖,在一定的时间、剂量范围内呈时间-剂量依赖关系.20、40 mg/L小檗碱作用48 h后,细胞DNA片段分析可看到凋亡时降解形成的梯带,流式细胞仪检测到了细胞凋亡峰,各组的凋亡率分别是(16.7±2.8)%、(29.6±4.4)%,与对照组(1.9±0.6)%和5 mg/L小檗碱组(2.3±0.8)%相比差异显著(P＜0.01).在凋亡过程中,Bax蛋白表达明显增高,而Bcl-2蛋白表达下凋.结论 小檗碱体外能抑制HeLa细胞增殖并诱导其发生凋亡,其机制可能与Bax蛋白表达明显增高,而Bcl-2蛋白表达下调有关.     

烟酸对缺血/再灌注损伤大鼠心肌细胞凋亡及Bax、Bcl-2和Fas表达的影响

目的研究大鼠心肌缺血/再灌注损伤时心肌凋亡以及烟酸的干预作用。方法采用结扎左冠状动脉前降支方法制备大鼠缺血/再灌注模型。健康Sprage-Dawley大鼠60只随机分成4组:正常对照组(A组,n=15),心肌缺血/再灌注组(B组,n=15),心肌缺血/再灌注+生理盐水组(C组,n=15),心肌缺血/再灌注+烟酸组(D组,n=15),心电图记录肢体Ⅱ导联ST段的变化。光镜和电镜检测大鼠心肌缺血再灌注损伤形态学凋亡,流式细胞仪检测心肌细胞凋亡率。Western Blot方法和RT-PCR方法检测凋亡相关基因蛋白Bax,Bcl-2,Fas的表达。结果B组光镜和电镜可以发现心肌的凋亡,B组心肌凋亡率较A组有显著性差异(P0.05),Bax增高,Bcl-2下降,Bcl-2/Bax下降及Fas升高(P均0.01),C组各项指标与B组比较均无显著性差异。D组的各项指标较B组明显改善,心肌细胞凋亡减少,Bax下降,Bcl-2升高,Bcl-2/Bax升高及Fas下降。结论心肌再灌注损伤引起心肌细胞凋亡增多,Bax增高,Bcl-2下降及Bcl-2/Bax下降,Fas升高;烟酸能够减轻以上变化,说明烟酸对心肌缺血/再灌注损伤有保护作用。     

Physalin F from Physalis minima L. triggers apoptosis-based cytotoxic mechanism in T-47D cells through the activation caspase-3- and c-myc-dependent pathways

Ethnopharmacological relevance

Physalin F (a secosteroid derivative), is well recognized as a potent anticancer compound from Physalis minima L., a plant that is traditionally used to treat cancer. However, the exact molecular anticancer mechanism remains to be elucidated.

Aim of the study

We have recently reported the apoptosis-based cytotoxic effect of the chloroform extract of this plant. Here, we investigated the cytotoxicity and possible cell death mechanism elicited by the active constituent, physalin F on human breast T-47D carcinoma.

Materials and methods

Cytotoxic-guided fractionation of the chloroform extract of Physalis minima has led to the isolation of physalin F. The cytotoxicity activity was assayed using MTS assay. The effect of the compound to induce apoptosis was determined by biochemical and morphological observations through DeadEnd Colorimetric and annexin V assays, respectively, and RT-PCR analysis of mRNA expression of the apoptotic-associated genes.

Results

Cytotoxicity screening of physalin F displayed a remarkable dose-dependant inhibitory effect on T-47D cells with lower EC₅₀ value (3.60 μg/ml) than the crude extract. mRNA expression analysis revealed the co-regulation of c-myc- and caspase-3-apoptotic genes in the treated cells with the peak expression at 9 and 12 h of treatment, respectively. This apoptotic mechanism is reconfirmed by DNA fragmentation and phosphatidylserine externalization.

Conclusion

These findings indicate that physalin F may potentially act as a chemopreventive and/or chemotherapeutic agent by triggering apoptosis mechanism via the activation of caspase-3 and c-myc pathways in T-47D cells.