Iressa和Gleevec增敏Lexatumumab诱导肝癌细胞凋亡的研究

摘 要：［目的］ 研究酪氨酸激酶抑制剂Gleevec或Iressa增敏DR5激动剂Lexatumumab诱导肝癌细胞凋亡的效果及机制。［方法］ 体外培养肝癌LH86细胞株。分为对照组、TKI处理组、Lexatumumab 处理组、TKI+Lexatumumab处理组。用亚致死剂量的TKI或者Lexatumumab或者TKI+Lexatumumab同时处理细胞,通过荧光显微镜观察对照组和处理组细胞内细胞核凋亡形态变化情况。然后计数凋亡细胞,统计凋亡率。免疫印迹（Western blot）方法检测细胞凋亡标志性蛋白Caspase 3 切割条带在各组中的表达,促凋亡蛋白Bim、Bax以及抗凋亡蛋白在各处理组中的表达,以评估TK抑制作用对死亡受体信号通路诱导细胞凋亡的影响。［结果］在荧光显微镜下,可以观察到Gleevec、Iressa或Lexatumumab单独处理细胞不能够诱导LH86肝癌细胞核出现固缩凝聚现象。抑制剂Gleevec或Iressa 与Lexatumumab联合应用能够显著逆转诱导的肝癌细胞凋亡(Gleevec和Lexatumumab诱导凋亡率：39.23%,P<0.001;Iressa和Lexatumumab诱导凋亡率：37.5%,P<0.0001)。TKI和Lexatumumab诱导的细胞凋亡为Caspase依赖性。TKI和Lexatumumab诱导的细胞凋亡特异性诱导Bcl-2家族蛋白Bim上调。对Bax和Bcl-xl没有影响。［结论］ 肝癌细胞对Gleevec/Iressa或Lexatumumab单一制剂具有凋亡抗性。酪氨酸激酶抑制剂均能够增敏Lexatumumab诱导的细胞凋亡。这种联合诱导的细胞凋亡作用具有Caspase依赖性。TKI增敏的死亡受体介导的凋亡可能与促凋亡蛋白Bim上调有关。     

YM155增敏Lexatumumab诱导肝癌细胞株LH86凋亡的实验研究

目的 探讨小分子化合物YM155增敏死亡受体单克隆抗体Lexatumumab激活诱导肝癌细胞株LH86凋亡的效果及分子机制。方法 体外培养肝癌LH86细胞株,分为对照组（DMSO）、YM155处理组（1 μmol／L）、Lexatumumab 处理组（1 μg／ml）和YM155（1 μmol／L）联合Lexatumumab（1 μg／ml）组。荧光显微镜观察上述各组细胞核凋亡形态变化,计数凋亡细胞,计算凋亡率。Western blotting检测各组中细胞凋亡标志性蛋白caspase-3和Bax的表达。结果 荧光显微镜下,1 μmol／L YM155或1 μg／ml Lexatumumab单独处理均未能诱导LH86细胞出现胞核固缩凝聚或染色质断裂,而1 μmol／L YM155预处理细胞30 min能够逆转Lexatumumab诱导的肝癌细胞核固缩凝聚和染色质断裂。YM155和Lexatumumab联合处理 12 h,细胞凋亡率达60％,高于其余3组（P＜0.05）。Western blotting 检测显示,仅YM155联合Lexatumumab组出现明显的caspase-3蛋白切割条带;YM155和Lexatumumab单独处理均未能诱导Bax构象变化;经1 μmol/L YM155预处理后,Lexatumumab能够有效诱导Bax构象变化激活。结论 YM155能够增敏单克隆抗体Lexatumumab诱导肝癌细胞株LH86细胞凋亡,YM155和Lexatumumab联合处理诱导凋亡可能与Bax构象变化激活相关。     

ABT-737对顺铂诱导乳腺癌T47D细胞凋亡的增敏作用

目的 探讨Bcl-2抑制剂ABT-737联合顺铂对乳腺癌T47D细胞株凋亡的影响.方法 常规培养T47D细胞,采用四甲基偶氮唑蓝(MTT)法检测ABT-737联合顺铂对T47D细胞增殖的影响,采用Western blot法检测凋亡相关蛋白的表达,采用荧光染色法观察T47D细胞凋亡的形态学变化,采用流式细胞仪检测T47D细胞的凋亡率.结果 MTT法检测结果显示,ABT-737可明显降低顺铂在T47D细胞中的IC50[由顺铂单药的(26.00±1.41) μmol/L降为联合用药的(13.00±1.11)μmol/L].ABT-737增加顺铂对T47D细胞的抑制作用,并呈剂量依赖性,而ABT-737单药对T47D细胞的抑制作用不明显.Western blot检测多聚ADP核糖聚合酶(PARP)的剪切结果显示,ABT-737明显降低了顺铂诱导T47D细胞凋亡的浓度,加快了凋亡诱导时间,ABT-737对顺铂诱导T47D细胞凋亡的增敏作用呈剂量依赖性,单药对T47D细胞的作用不明显.与单药组相比,ABT-737与顺铂联合时T47D细胞中PARP和caspase3的剪切明显增加,而Bcl-2、Bcl-XL和Bax的表达无明显变化.荧光染色和流式细胞术检测结果均显示,ABT-737联合顺铂时,T47D细胞的凋亡率明显增加.结论 ABT-737可显著提高顺铂对乳腺癌T47D细胞的凋亡诱导作用.     

自噬在索拉菲尼诱导肝癌细胞凋亡中的作用

目的：研究索拉菲尼（Sorafenib）诱导的自噬在肝癌细胞凋亡中的作用。方法：体外培养肝癌细胞Huh7和 LH86细胞株。分为对照组、Sorafenib 处理组、Bafilomycin 处理组、Sorafenib + Bafilomycin 处理组。将构建的质粒表达载体 pEGFP - N1- LC3分别转染 Huh7和 LH86细胞株,用索拉菲尼或者索拉菲尼加 Bafilo-mycin 同时处理细胞,通过荧光显微镜观察对照组和处理组细胞内细胞自噬小体形成情况。免疫印迹（West-ern blot）方法检测细胞自噬作用标志蛋白 LC3以及细胞凋亡蛋白 Caspase 3切割条带在各组中的表达, Heochst 33342染料对各组细胞核进行染色,以评估细胞自噬作用对索拉菲尼诱导细胞凋亡的影响。结果：在荧光显微镜下,可以观察到 Sorafenib 能够显著诱导两种肝癌细胞株发生自噬作用。抑制剂 Balfilomycin 能够显著抑制 Sorafenib 诱导的自噬作用。Balfilomycin 抑制自噬能够显著增强 Sorafenib 诱导的细胞凋亡。结论：Sorafenib 能够诱导肝癌细胞产生自噬作用。这种自噬作用在 Sorafenib 诱导的肝癌细胞凋亡中起到负调控作用。     

ABT-199体外对急性T淋巴细胞白血病细胞株Molt4细胞增殖凋亡影响及其机制研究

目的 大多数肿瘤细胞对化疗诱导细胞凋亡的耐受与Bcl-2家族蛋白有关,ABT-199是针对抗凋亡蛋白Bcl-2的选择性拮抗剂.本研究旨在探讨ABT-199体外对急性T淋巴细胞白血病Molt4细胞株增殖凋亡的影响及其机制.方法 体外培养Molt4细胞,CCK8法检测不同浓度ABT-199对Moh4的增殖抑制作用,DAPI细胞核染色后荧光显微镜观察细胞凋亡特征,Annexin Ⅴ/PI双染法检测不同浓度ABT-199对Molt4细胞的诱导凋亡作用,JC-1染色法检测不同浓度ABT-199作用于Moh4细胞后线粒体膜电位的变化,蛋白质印迹法检测经ABT-199处理后线粒体信号通路相关蛋白(Bcl-2、PARP和cleaved Caspase-3蛋白)表达水平的变化.结果 ABT-199对Molt4细胞具有抑制增殖的作用,48 h的IC50值为(4.63±0.15) μmol/L.荧光显微镜观察显示,ABT-199处理Moh4细胞后细胞核染色质染色浓聚,细胞核碎裂,出现凋亡小体,且随着药物浓度的增加上述形态变化更加明显;Annexin Ⅴ/PI检测细胞凋亡结果显示,0、1、2、4、8 μmol/L的ABT-199诱导细胞凋亡的百分比分别为(3.09±1.16)％、(11.59±5.58)％、(25.37±7.42)％、(46.38±7.05)％和(80.60±11.18)％,给药组与对照组的凋亡比例差异有统计学意义,x2=18.286,v=4,P=0.001.JC-1检测结果显示,ABT-199能促使Molt4细胞的线粒体膜电位下降,呈浓度依赖性,给药组与对照组的线粒体膜电位下降比例差异有统计学意义,x2 =17.386,v=4,P=0.002.蛋白质印迹法检测结果显示,给药组线粒体通路中Bcl-2表达水平显著下降(x2=9.024,v=3,P=0.029),并出现Caspase-3下游底物多聚ADP核糖聚合酶(poly ADP ribose poly-merase,PARP)的裂解,同时出现了Caspase-3裂解片段的累积.结论 ABT-199在体外能抑制急性T淋巴细胞白血病Molt4细胞的增殖及诱导其凋亡,其机制与激活线粒体信号通路相关.     

ABT263对PHA739358增敏作用的分子机制

目的 探讨ABT263对PHA739358增敏作用的分子机制.方法 培养人乳腺癌细胞株MDA-MB-231,采用PHA739358及ABT263进行处理.免疫荧光法观察多核细胞形成,PI单染流式细胞术观察多倍体形成,FITC-Annexin V和PI双染流式细胞术及荧光染料法显示不同组别细胞凋亡情况;Western Blot检测单药和联合用药不同组别对p-Histone H3,抗凋亡蛋白Bcl-xl、Bcl-2、Mcl-1及促凋亡蛋白Bax、PARP的影响.结果 免疫荧光和PI单染流式细胞术结果显示,1μM PHA739358作用于MDA-MB-231细胞,48 h后出现多核现象及多倍体细胞形成.Western Blot、流式细胞术、荧光染料结果显示,ABT263可促进多倍体细胞快速凋亡,ABT263与PHA739358联合组的细胞凋亡率为(43.70±0.78)%,高于对照组、ABT263组、PHA739358组、MLN8054组,以及ABT263与MLN8054联合组.Western Blot结果显示,ABT263与PHA739358联合主要抑制Aurora B和Bcl-xl/2,对Mcl-1的影响很小,对Aurora A无影响;ABT263与PHA739358联合作用于MDA-MB-231细胞,48 h后的细胞凋亡数目明显增加,凋亡率从(0.26±0.02)%增加到(42.30±0.23)%,且荧光染料法显示此组凋亡细胞明显增多.结论 ABT263对PHA739358的增敏作用可为乳腺癌的治疗提供新思路.     

环王巴明诱导DU145细胞凋亡的机制研究

目的:研究环王巴明诱导DU145细胞凋亡作用及其对Gli-1和Bcl-2 mRNA表达的影响,探讨环王巴明诱导DU145细胞凋亡的机制.方法:不同浓度环王巴明处理DU145细胞后,吖啶橙染色,荧光显微镜下观察细胞形态变化.流式细胞术观察细胞凋亡率变化,RT-PCR检测5、10μmol/L环王巴明作用72h后的实验组和对照组Gli-1、Bcl-2 mRNA表达水平差异.结果:当环王巴明浓度大于5μmol/L作用72h后,荧光显微镜下可见细胞核固缩或碎裂,核仁变形等典型的凋亡形态学改变.环王巴明5μmol/L组Gli-1、Bcl-2mRNA表达减弱,但同对照组差异无统计学意义(P>0.05),与空白及DMSO对照组相比10μmol/L环王巴明可以显著下调Gli-1、Bcl-2 mRNA表达(P<0.01).结论:环王巴明可诱导人前列腺癌DU145细胞株凋亡,其作用机制可能与下调Gli-1和Bcl-2mRNA表达,活化细胞凋亡的线粒体途径有关.     

Apogossypolone对乳腺癌MCF-7细胞株放射增敏的体外实验研究

[目的]探讨Apogossypolone(ApoG2)对乳腺癌MCF-7细胞株的放射增敏作用及其作用机制。[方法]①MTT法检测ApoG2单药对MCF-7细胞的抑制率,确立细胞半数抑制浓度(IC50)。②根据MTT结果选取低浓度(相似文献   

特异性环氧合酶-2抑制剂SC236诱导胃癌细胞株凋亡的实验研究

目的：探讨环氧合酶-2（COX-2）特异性抑制剂SC236诱导胃癌细胞株SGC7901凋亡的机制。方法：SC236对人胃癌来源的SGC7901细胞进行凋亡诱导。以吖啶橙染色后荧光显微镜下观察凋亡细胞形态并计算凋亡指数。Western blot法检测凋亡调节蛋白Bcl-2、Bak、Bax与CED-9的表达情况。流式细胞仪检测细胞凋亡率。结果：SGC7901细胞经SC236作用后,细胞中凋亡促进因子Bak的表达水平明显增高、凋亡抑制因子Bcl-2的表达水平显著下降、而凋亡促进因子Bax和凋亡抑制因子CED-9表达水平无明显变化。细胞凋亡指数或凋亡率则随SC236浓度的增加和作用的时间延长而升高。结论：COX-2特异抑制剂SC236可通过上调凋亡促进因子Bak表达和下调凋亡抑制因子Bcl-2表达而诱导胃癌细胞株SGC7901的凋亡。     

特异性环氧合酶-2抑制剂SC236诱导胃癌细胞株凋亡的实验研究

目的:探讨环氧合酶-2(COX-2)特异性抑制剂SC236诱导胃癌细胞株SGC7901凋亡的机制. 方法: SC236对人胃癌来源的SGC7901细胞进行凋亡诱导.以吖啶橙染色后荧光显微镜下观察凋亡细胞形态并计算凋亡指数.Western blot法检测凋亡调节蛋白Bcl-2、Bak、Bax与CED-9的表达情况.流式细胞仪检测细胞凋亡率.结果: SGC7901细胞经SC236作用后,细胞中凋亡促进因子Bak的表达水平明显增高、凋亡抑制因子Bcl-2的表达水平显著下降、而凋亡促进因子Bax和凋亡抑制因子CED-9表达水平无明显变化.细胞凋亡指数或凋亡率则随SC236浓度的增加和作用的时间延长而升高.结论: COX-2特异抑制剂SC236可通过上调凋亡促进因子Bak表达和下调凋亡抑制因子Bcl-2表达而诱导胃癌细胞株SGC7901的凋亡.     

Mus81基因沉默对MDA-MB-231乳腺癌细胞增殖、凋亡及裸鼠成瘤能力的影响北大核心

目的:分析甲磺酸盐及紫外线敏感性81号基因(Mus81)在乳腺癌组织中的表达水平,观察Mus81基因敲减对三阴性乳腺癌细胞MDA-MB-231增殖、凋亡和裸鼠移植瘤形成能力的影响。方法:从TCGA数据库下载乳腺癌样本基因表达数据,应用perl及R软件整理数据筛选出每个样本Mus81基因表达量。通过慢病毒介导的小干扰RNA(short interfering RNA,siRNA)技术构建Mus81基因敲减的MDA-MB-231乳腺癌细胞系(即Mus81敲减组)和阴性对照组MDA-MB-231乳腺癌细胞系,以实时定量PCR法检测Mus81基因的敲减效率,再以MTT检测实验、克隆形成实验、细胞流式检测及实时定量PCR法检测两组MDA-MB-231细胞的生长、增殖、细胞周期分布、凋亡水平及Mus81下游基因表达水平。最后,向BALB/c裸鼠右侧腋下注射Mus81基因敲减组和阴性对照组MDA-MB-231乳腺癌细胞,观察Mus81基因沉默对MDA-MB-231细胞在裸鼠中成瘤能力的影响。结果:Mus81基因在乳腺癌组织中的平均表达量明显高于癌旁组织(P<0.05)。Mus81基因敲减组MDA-MB-231细胞中Mus81基因的表达水平明显低于阴性对照组(P<0.05),Mus81基因的敲减效率达70.7%。较之阴性对照组,Mus81基因敲减组MDA-MB-231细胞的生长速度明显减缓(P<0.05);形成的细胞克隆数也明显下降(P<0.05);细胞凋亡水平、G2/M期细胞比例则明显升高(P<0.05)。STC2等Mus81下游基因的表达水平在两组MDA-MB-231细胞间也有明显差异(P<0.05)。裸鼠成瘤实验显示,Mus81基因敲减组形成的裸鼠瘤体体积和重量均明显低于阴性对照组(P<0.05)。结论:Mus81基因在乳腺癌组织中的表达明显升高,且可能通过调控STC2等下游基因的表达促进三阴性乳腺癌细胞的生长增殖及裸鼠体内成瘤能力并抑制其凋亡,提示其可能是一个潜在的乳腺癌治疗靶点。     

ERK1/2 activation is required for resveratrol-induced apoptosis in MDA-MB-231 cells

Resveratrol (RSVL), a phytoalexin found in abundance in grapes and other grape-related products, has been shown to be antiproliferative and protective against various types of cancers, including breast cancer. However, the precise underlying mechanisms are not well understood. In this study, we show that treatment with RSVL induces growth inhibition and apoptosis in a highly invasive and metastatic breast cancer cell line MDA-MB-231. Cleavage of caspase-3 and PARP and fragmentation of DNA were observed following exposure to RSVL. Co-treatment with pan-caspase inhibitor completely prevents cell death induced by RSVL. We found that RSVL-induced apoptosis correlates with sustained activation of ERK1/2 and suppression of Bcl-2 expression. Inhibition of ERK1/2 activation by its specific inhibitor or small interfering RNA reverses the effect of RSVL on Bcl-2 suppression and inhibits apoptosis, while overexpression of MEK1, which is directly upstream of both ERK1 and ERK2, enhances apoptosis induced by RSVL. Moreover, ERK1/2 was found to act upstream of caspase-3 to induce apoptosis, while it was not directly involved in caspase-3 cleavage. The other closely related MAPK members, p38 and JNK are not involved in apoptosis induced by RSVL in MDA-MB-231 cells. These results suggest that activation of ERK1/2 is required for RSVL-induced apoptosis in MDA-MB-231 cells.     

miR-101、EZH2和MYC调控乳腺癌MDA-MB-231细胞增殖能力的机制

目的:研究miR-101、EZH2和MYC对乳腺癌MDA-MB-231细胞增殖的影响,并探究三者之间的调控机制。方法:以实时荧光定量PCR法检测三阴型乳腺癌组织及相应正常乳腺组织中miR-101、EZH2与MYC的表达,以及乳腺癌细胞系和正常乳腺细胞中miR-101的表达水平。以LipofectamineTM 2000将miR-101 mimics、EZH2 siRNA和MYC siRNA以及相应的阴性对照转染至MDA-MB-231细胞,Western blot法检测EZH2、MYC蛋白的表达,MTT法检测MDA-MB-231细胞的增殖能力。结果:相对于正常乳腺组织,三阴型乳腺癌组织中miR-101 mRNA的表达降低,而EZH2、MYC mRNA表达则显著升高;三阴型乳腺癌细胞中miR-101 mRNA的表达低于正常乳腺细胞MCF-10A。miR-101 mimics转染使MDA-MB-231细胞中miR-101 mRNA含量显著升高。miR-101过表达抑制EZH2、MYC的表达;EZH2敲减也抑制了MYC蛋白的表达。EZH2或MYC敲减可使miR-101 mRNA的表达显著升高。MTT实验结果示,miR-101过表达或敲减EZH2、MYC均可抑制MDA-MB-231细胞的增殖能力。结论:miR-101通过EZH2与MYC形成反馈环路调控MDA-MB-231细胞的增殖能力。     

盐霉素通过靶向调控ALDH影响三阴性乳腺癌细胞MDA-MB- 231增殖和凋亡

目的 探讨盐霉素（salinomycin,Sal）对三阴性乳腺癌细胞MDA-MB-231增殖和凋亡的影响及其作用机制。方法 采用流式细胞术筛选ALDH高表达乳腺癌细胞系;采用MTT法检测不同浓度Sal（1~32 μmol/L）对细胞增殖的影响;采用流式细胞仪检测Sal对细胞凋亡以及肿瘤干细胞表面标志物ALDH表达的影响;采用qRT-PCR、Western blot检测凋亡相关分子Caspase-3、Bax、Bcl-2、PARP mRNA和蛋白表达;TCGA（the Cancer Genome Atlas）数据库下载乳腺癌患者数据集（n=1 218）,采用Pearson法分析ALDH与凋亡相关分子表达的相关性。结果 Sal在24 h、48 h、72 h均可以剂量依赖的方式抑制MDA-MB-231细胞增殖（均P<0.001）;Sal作用MDA-MB-231细胞48 h后,与对照组比较,2 μmol/L组、4 μmol/L组、8 μmol/L组的细胞凋亡率均升高（均P<0.05）;Bcl-2、PARP、Caspase-3 mRNA表达下降（均P<0.05）,Bax mRNA表达升高（均P<0.05）;Bcl-2、Caspase-3 蛋白表达下降（均P<0.05）,PARP、Bax 蛋白表达升高（均P<0.05）;MDA-MB-231乳腺癌干细胞表面标志物ALDH表达下降（均P<0.05）。TCGA数据库分析显示,ALDH与抗凋亡分子Bcl-2、Caspase-3的表达呈正相关（r=0.209,P=0.007;r=0.235,P=0.002）,与Bax、PARP的表达呈负相关（r=-0.326,P＜0.001;r=-0.453,P＜0.001）。结论 Sal可抑制人乳腺癌MDA-MB-231细胞生长,可能通过下调肿瘤干细胞表面标志物ALDH表达实现。     

A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells

Purpose

According to the World Health Organization (WHO), breast cancer is the most common cancer affecting women worldwide. In the USA ~12.3 % of all women are expected to be diagnosed with various types of breast cancer, exhibiting varying degrees of therapeutic response rates. Therefore, the identification of novel anti-breast cancer drugs is of paramount importance.

Methods

The 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore was incorporated into a number of cytotoxins. Three of the resulting dienones, 2a, 2b and 2c, were tested for their anti-neoplastic potencies in a variety of human breast cancer-derived cell lines, including the triple negative MDA-MB-231 cell line and its metastatic variant, using a live-cell bio-imaging method. Special emphasis was put on dienone 2c, since its anti-cancer activity and its mode of inflicting cell death have so far not been reported.

Results

We found that all three dienones exhibited potent cytotoxicities towards the breast cancer-derived cell lines tested, whereas significantly lower toxicities were observed towards the non-cancerous human breast cell line MCF-10A. The dienones 2b and 2c exhibited the greatest selective cytotoxicity at submicromolar concentration levels. We found that these two dienones induced phosphatidylserine externalization in MDA-MB-231 cells in a concentration-dependent manner, suggesting that their cytotoxic effect might be mediated by apoptosis. This possibility was confirmed by our observation that the dienone 2c can induce mitochondrial depolarization, caspase-3 activation, cell cycle disruption and DNA fragmentation in MDA-MB-231 cells.

Conclusion

Our findings indicate that dienone 2c uses the mitochondrial/intrinsic pathway to inflict apoptosis in triple negative MDA-MB-231 breast cancer-derived cells. This observation warrants further assessment of dienone 2c as a potential anti-breast cancer drug.

     

Genipin, a constituent of Gardenia jasminoides Ellis, induces apoptosis and inhibits invasion in MDA-MB-231 breast cancer cells

Genipin, a constituent of Gardenia jasminoides Ellis, is used in the treatment of hepatic disorders and inflammatory diseases in traditional medicine. Although mounting evidence suggests an anti-tumor activity of genipin in several cancer cell systems, the inhibitory effect of genipin on the growth of breast cancer cells has not been reported yet. The present study aimed to investigate the anti-proliferative activity of genipin in MDA-MB-231 human breast cancer cells. Herein, we showed that genipin efficiently induced apoptosis in MDA-MB-231 cells by the down-regulation of Bcl-2, up-regulation of Bax and proteolytic activation of caspase-3. Activation of JNK and p38 MAPK also increased by genipin. Importantly, genipin significantly inhibited invasive and migratory phenotypes of MDA-MB-231 cells. Taken together, this study demonstrates that genipin induces apoptosis and inhibits invasive/migratory abilities of highly invasive MDA-MB-231 human breast cancer cells, suggesting a potential application of genipin as a chemopreventive agent that may prevent or alleviate metastatic breast cancer.     

Real-time imaging of apoptosis induction of human breast cancer cells by the traditional Chinese medicinal herb tubeimu

Traditional Chinese Medicine (TCM) has been used for thousands of years, including treatment for cancer. Use of modern technology and the scientific method to evaluate the efficacy of TCM for cancer should enable its more widespread use. In the present study, the efficacy of the TCM tubeimu, extracted from the tuber of the plant Bolbostemma paniculatum, on the MDA-MB-231 human breast cancer cell line was evaluated. The MDA-MB-231 cell line was engineered to express red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) linked to histone H2B in the nucleus, which allows real-time imaging of nuclear-cytoplasmic dynamics. Apoptosis was readily visualized in these cells by nuclear shape changes and fragmentation. The MDA-MB-231 RFP-GFP cells were cultured either in two-dimensions on plastic or in three-dimensions on Gelfoam?. Cells were treated with a dichloromethane extract of fresh tubeimu. Apoptosis was further monitored by DNA fragmentation determined by gel electrophoresis. Tubeimu induced apoptosis of MDA-MB-231 cells, as observed by fluorescence microscopy, as early as 24 hours of treatment in vitro in two-dimensional culture. By 48 hours' treatment, DNA fragmentation could be observed. The frequency of apoptosis increased through at least 72 hours' treatment, with most of the cells being killed. Tubeimu also induced apoptosis of MDA-MB-231 cells in three-dimensional culture on Gelfoam?, but to a lesser extent than in 2D culture. The results of the present study indicate the potential of tubeimu in breast cancer therapy.     

Functional screening of genes suppressing TRAIL-induced apoptosis: distinct inhibitory activities of Bcl-XL and Bcl-2

TNF-related apoptosis-inducing ligand (TRAIL) is known to selectively induce apoptosis in various tumour cells. However, downstream-signalling of TRAIL-receptor is not well defined. A functional genetic screening was performed to isolate genes interfering with TRAIL-induced apoptosis using cDNA retroviral library. Bcl-X(L) and FLIP were identified after DNA sequencing analysis of cDNA rescued from TRAIL-resistant clones. We found that increased expression of Bcl-X(L), but not Bcl-2, suppressed TRAIL-induced apoptosis in tumour cells. Western blot and immunohistochemical analyses showed that expression of Bcl-X(L), but not Bcl-2, was highly increased in human breast cancer tissues. Exposure of MDA-MB-231 breast tumour cells to TRAIL induced apoptosis accompanied by dissipation of mitochondrial membrane potential and enzymatic activation of caspase-3, -8, and -9. However, SK-BR-3 breast tumour cells exhibiting increased expression level of Bcl-X(L) were resistant to TRAIL, though upon exposure to TRAIL, caspase-8 and Bid were activated. Forced expression of Bcl-X(L), but not Bcl-2, desensitised TRAIL-sensitive MDA-MB-231 cells to TRAIL. Similar inhibitory effects were also observed in other tumour cells such as HeLa and Jurkat cells stably expressing Bcl-X(L), but not Bcl-2. These results are indicative of the crucial and distinct function of Bcl-X(L) and Bcl-2 in the modulation of TRAIL-induced apoptosis.     

曲格列酮增强乳腺癌细胞对表阿霉素敏感性的研究

背景与目的:过氧化物酶增殖体激活受体γ(peroxisomeproliferator-activatedreceptorγ,PPARγ)在乳腺癌组织中呈高表达,其特异性配体噻唑烷二酮(thiazolidinediones,TZD)类药物作用于肿瘤细胞后可抑制细胞增殖、诱导细胞凋亡。本研究探讨TZD类药物曲格列酮的使用与雌激素受体(estrogenreceptor,ER)阴性乳腺癌细胞对表阿霉素化疗敏感性的关系。方法:通过MTT法和流式细胞仪检测曲格列酮、表阿霉素单独和联合使用时,对ER阴性人乳腺癌细胞系MDA-MB-435S和MDA-MB-231增殖、凋亡的影响。Westernblot和划痕实验分别检测不同药物处理下,乳腺癌细胞中Bcl-2蛋白表达水平和细胞迁移能力的变化。结果:在4～24μmol/L浓度范围内,曲格列酮与表阿霉素协同抑制乳腺癌细胞的增殖,IC50是表阿霉素单独使用时的60%。曲格列酮和表阿霉素单独作用于乳腺癌细胞时并无明显的凋亡发生,而两种药物联合作用时,MDA-MB-435S和MDA-MB-231细胞的凋亡率分别为(5.48±0.45)%、(10.08±1.89)%。联合使用曲格列酮下调了Bcl-2蛋白的表达,降低了乳腺癌细胞的迁移能力。结论:曲格列酮不仅促进了表阿霉素对乳腺癌细胞的增殖抑制和凋亡诱导作用,而且明显抑制了乳腺癌细胞的迁移能力,从而增强乳腺癌细胞对表阿霉素的敏感性。