脂蟾毒配基通过线粒体途径诱导人肝癌Bel-7402细胞凋亡的研究

目的：探讨中药蟾酥主要成份之一脂蟾毒配基（Resihufogenin，RG）诱导人肝癌Bel-7402细胞凋亡的作用机制。方法：体外培养Bel-7402细胞．采用酸性磷酸酶法检测RG对细胞增殖抑制作用：吖啶橙荧光染色观察细胞形态变化；流式细胞仪检测细胞凋亡率及线粒体膜电位（△ψm）变化；Westem Blot检测与细胞调亡相关蛋白表达的变化。结果：RG显著抑制细胞增殖，其作用24、48、72h的IC50分别为3．8、1．8、1．2μmol／L；经10μmol／LRG处理24h后，癌细胞出现凋亡的形态变化，其凋亡率明显高于对照组细胞；RC引起△ψm下降，释放入胞质中的细胞色素c（cytochrome c，CytC）增多，促进caspase-3蛋白活化，抑制Bel-2蛋白表达。诱导细胞凋亡。结论：RG诱导人肝癌Bel-7402细胞凋亡．其诱导凋亡作用可能通过线粒体通路实现。     

BaX在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用

背景与目的:探讨Bax蛋白在维生素E琥珀酸酯(vitamin E succinate,VES)诱导人胃癌SGC-7901细胞凋亡中的作用.材料与方法:用吖啶橙/溴化乙啶(AO/EB)染色观察细胞凋亡的形态学改变;Westem Blot法检测不同剂量VES对人胃癌SGC-7901细胞中Bax蛋白表达量的影响和VES对Bax蛋白与细胞色素c(cytochrome c,Cyt c)蛋白在细胞内分布的影响;用Mito Tracker RedCMXRos荧光染色观察线粒体膜电位(△Ψm)的变化.结果:VES可引起SGC-7901细胞发生凋亡,提高Bax蛋白表达水平,并使Bax蛋白从胞浆转移到线粒体;VES作用后发生线粒体膜电位下降,Cyt c从线粒体释放到胞浆.结论:VES可能通过Bax蛋白来启动线粒体凋亡途径,诱导SGC-7901细胞发生凋亡.     

线粒体通透性改变与细胞凋亡

细胞凋亡受线粒体的调控,线粒体通透性改变(MPT)在凋亡中起着关键作用.Bcl-2家族蛋白等许多因子作用于线粒体,使线粒体膜上的通透性改变孔(PTP)开放,线粒体跨膜电位(ΔΨm)降低甚至崩解,线粒体释放出细胞色素c、凋亡诱导因子(AIF)等凋亡相关因子,激活caspase级联反应,导致细胞凋亡.     

Puma基因转染对胃癌SGC-7901细胞的促凋亡作用及机制

[目的]探讨puma基因转染对人胃癌SGC-7901细胞株增殖和凋亡的影响及其作用机制。[方法]应用脂质体介导重组真核表达载体pEGFP-C1-PUMA瞬时转染至SGC-7901细胞。分别用荧光显微镜和RT—PCR法检测外源基因的表达，MTF比色法测定细胞增殖的抑制，Hoechst33342染色法检测细胞凋亡，流式细胞仪检测细胞周期和线粒体膜电位的变化，Westernblot检测细胞色素C（CytC）和凋亡诱导因子（AIF）的转位。[结果]外源性puma基因在pEGFP—C1-PUMA转染的SGC-7901细胞中实现了表达。PUMA表达使SGC-7901细胞的增殖能力降低并诱导凋亡，转染24、48、72h的生长抑制率分别为19．3％、34．7％、42．2％，凋亡率分别为19．6％、35．4％、46．6％。PUMA表达的SGC-7901细胞DNA合成受到抑制，周期阻滞在GgG1期；线粒体膜电位明显下降，CytC、AIF从线粒体进入胞浆。[结论]puma基因转染可有效抑制胃癌SGC-7901细胞的增殖，促进其凋亡。促凋亡机制主要是线粒体途径，与线粒体膜电位降低和CytC、AIF从线粒体释放有关。     

维生素E琥珀酸酯诱导K562/ADM细胞凋亡过程中线粒体结构和功能的改变

目的：观察维生素E琥珀酸酯（vitamin E succinate，VES）诱导K562／ADM耐药细胞凋亡过程中线粒体形态及功能的改变。方法：采用annexin V／PI双标记法和透射电镜检测K562／ADM细胞凋亡；电镜观察凋亡细胞线粒体形态结构变化；流式细胞仪（FCM）检测线粒体跨膜电位（△ψm）和caspase-3活性变化；激光共聚焦显微镜分析细胞色素C（cytochrome c，Cyt c）的释放。结果：20μmol／L和40μmol／L VES处理K562／ADM细胞12～24h，annexin V／PI染色检测发现早期凋亡细胞明显增加，并呈现典型的凋亡形态改变。线粒体内外膜融合、缩小、碎片化，嵴减少且紊乱；线粒体△ψm降低，细胞质内Cytc释放增多；caspase-3活性显著增强。结论：VES可能通过改变线粒体的形态结构和稳定性，导致线粒体膜电位降低，Cytc释放，caspase-3激活而诱发K562／ADM耐药细胞凋亡。     

冬凌草甲素诱导食管癌细胞凋亡过程中线粒体结构与膜电位的改变

目的:研究冬凌草甲素(Oridonin,ORI)诱导食管癌细胞凋亡的过程中线粒体超微结构和功能的变化。方法:采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法和透射电镜法检测细胞凋亡和超微结构改变;罗丹明123(Rhodamine 123,Rho123)荧光探针标记流式细胞仪检测和分析线粒体跨膜电位(MTP,△Ψm)的改变。结果:32μg/ml ORI作用2h后电镜下SHEEC细胞线粒体增多,4h后线粒体肿胀空泡化、内部结构消失,8h后细胞核染色质成块状边集,细胞凋亡。ORI作用24h后,代表线粒体膜电位的Rho123荧光强度降低。结论:在ORI诱导下,SHEEC细胞线粒体有明显的形态和功能改变伴随线粒体△Ψm降低,线粒体改变可能是ORI诱导食管癌细胞凋亡过程的重要环节。     

新型多胺缀合物NNAMB诱导K562细胞凋亡及其机制

目的 探讨新型多胺缀合物NNAMB诱导K562细胞凋亡及其分子机制.方法 采用二苯基溴化四氮唑蓝(MTT)法、台盼监拒染法检测细胞活力;Hoechst33258染色观察细胞形态变化;流式细胞仪检测细胞周期变化、凋亡率、线粒体膜电位的变化;Western blot检测caspase-3、caspase-8、caspase-9和cytochrome c的表达.结果 NNAMB抑制K562细胞的生长,并呈现剂量及时间依赖性.随着NNAMB浓度的增加和作用时间的延跃,Sub-G1期细胞明显增加,线粒体膜电位下降.经NNAMB处理后的K562细胞中,可见到明显的凋亡细胞.蛋白印迹检测表明,NNAMB可诱导caspase-3、easpase-9的活化及线粒体cytochrome c释放到胞浆中,但caspase-8的表达无明显变化.结论 NNAMB能显著抑制K562细胞增殖,并可通过内源性线粒体途径诱导细胞凋亡.     

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.     

小白菊内酯对胃癌BGC823细胞凋亡表型的影响

目的:探讨小白菊内酯对胃癌BGC823凋亡表型影响.方法:MTT法检测BGC823增殖能力的变化,同时在倒置显微镜下观察细胞形态;采用流式细胞仪检测细胞线粒体膜电位的变化; ELISA法检测caspase-3活性;western blot法检测细胞Bcl-2表达.结果:Par以浓度和时间依赖方式抑制胃癌细胞增殖,镜下可见细胞凋亡形态学改变 .经Par 处理后,随着Par浓度增加,细胞线粒体膜电位逐渐下降,经100μmol/L Par处理6h后,细胞caspase-3活性增加, 24h后下降, 至48h仍保持在高水平,western blot法检测到细胞Bcl-2表达下调.结论: Par通过下调Bcl-2和上调caspase-3活性来诱导细胞凋亡.     

抑制上游转录因子2的表达对胃癌BGC-823细胞增殖和凋亡的影响

目的 探讨上游转录因子2（USF2）对人胃癌BGC-823细胞增殖和凋亡的影响。方法 使用Lipofectamine?3000转染试剂将USF2 siRNA转染至BGC-823细胞（siRNA-USF2组）中,同时设空白对照组和阴性对照组（siRNA-NC组）。实时荧光定量PCR法检测转染后的BGC-823细胞中USF2mRNA的表达。Western blot实验检测转染后BGC-823细胞中USF2蛋白的表达。CCK-8和平板克隆实验检测每组BGC-823细胞的增殖能力和克隆形成能力。流式细胞术检测各组胃癌细胞的凋亡情况。Western blot实验检测各组BGC-823细胞增殖相关蛋白增殖细胞核抗原（PCNA）及凋亡相关蛋白Bax和Bcl-2的表达水平。结果 与空白对照组和siRNA-NC组相比,siRNA-USF2组细胞USF2 mRNA和蛋白表达显著降低（均P<0.05）。转染后72 h,siRNA-USF2组的吸光度值低于空白对照组（P<0.05）。与空白对照组和siRNA-NC组比较,siRNA-USF2组BGC-823细胞中的克隆数明显较少（P<0.05）。空白对照组、siRNA-NC组和siRNA-USF2组胃癌BGC-823细胞的凋亡率差异具有统计学意义（P<0.05）。与空白对照组和siRNA-NC组比较,siRNA-USF2组BGC-823细胞中PCNA和Bcl-2蛋白表达减少,Bax蛋白表达增加（P<0.05）。结论 抑制USF2表达能够抑制人胃癌细胞的增殖,诱导其凋亡。USF2抑制剂可能在胃癌治疗中具有重要价值。     

TR3/Nur77 in colon cancer cell apoptosis

The orphan nuclear receptor TR3/Nur77 has emerged as a viable candidate in the coordinate regulation of cell proliferation and apoptosis, essential for maintaining normal architecture in rapidly renewing tissues such as the colonic mucosa. TR3 induces apoptosis in a number of cell lineages exposed to proapoptotic stimuli by directly targeting the mitochondria, inducing cytochrome c release. Here we report a distinctly different mechanism of TR3-mediated apoptosis in colon cancer cells. Nucleus-to-cytoplasm translocation of a green fluorescent protein-TR3 construct, but not its direct mitochondrial targeting, was associated with apoptosis induced by the short-chain fatty acid, butyrate. Similar results were observed for the nonsteroidal anti-inflammatory drug, sulindac, and the chemotherapeutic drug, 5-fluorouracil. A mutant TR3 construct lacking DNA-binding ability exerted a potent proapoptotic effect in colon cancer cells that was associated with cytochrome c release, an action dependent upon cytoplasmic localization of the construct, but, again, not its direct mitochondrial targeting. We identified a potential role for BAX recruitment to the mitochondria, secondary to cytoplasmic translocation of TR3, in inducing cytochrome c release and in mediating apoptosis. Therefore, TR3 translocation from the nucleus may initiate the apoptotic cascade in colon cancer cells by stimulating other cytosolic proapoptotic molecules to associate with mitochondria.     

Bax membrane insertion during Fas(CD95)-induced apoptosis precedes cytochrome c release and is inhibited by Bcl-2

Ligation of the Fas cell surface receptor leads to activation of caspases and subsequent apoptosis. Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that the pro-apoptotic protein, Bax, translocates from the cytosol specifically to the mitochondria following Fas ligation in MCF10A1 breast epithelial cells. Bax translocation was dependent on caspase activation, and preceded the release of cytochrome c and loss of mitochondrial respiratory activity. Bax translocation occurred in concert with activation of downstream caspases as determined by cleavage of a synthetic substrate, proteolysis of poly(ADP-ribose) polymerase, and processing of procaspase-3 and -7. Overexpression of the anti-apoptotic protein, Bcl-2, prevented Bax insertion, cytochrome c release, complete processing of caspase-3 and -7, and full activation of DEVD-specific cleavage activity. These data establish a role for Bax mitochondrial insertion during Fas-mediated apoptosis, and support a model in which Bax insertion amplifies the Fas apoptotic cascade through cytochrome c release and complete processing of caspases-3 and -7. In addition, our findings indicate that prevention of Bax insertion into the mitochondria represents a novel mechanism by which Bcl-2 inhibits Fas-induced apoptosis.     

右旋柠烯诱导人胃癌细胞凋亡

目的　探讨右旋柠烯 (D limonene)诱导人胃癌细胞株凋亡作用的机制。方法　采用噻唑蓝 (MTT)比色法、电镜、流式细胞术以及免疫细胞化学法 ,对p5 3和bcl 2在肿瘤细胞内的表达以及细胞凋亡的定性与定量指标进行检测。结果　经D limonene处理的BGC 82 3细胞出现核固缩 ,染色质边集 ,凋亡小体形成。凋亡细胞发生率与药物浓度呈正相关 ,即药物浓度为 0 .2 5 μg/ml(4 8h) ,凋亡细胞的发生率从 (2 .71± 0 .78) %上升至 (31.6 2± 7.81) %。经D limonene处理的BGC 82 3细胞内p5 3蛋白表达较对照组明显增加 ,bcl 2蛋白表达较对照组降低。结论　D limonene对人胃癌细胞的杀伤主要是通过诱导细胞凋亡 ,升提p5 3及降低bcl 2的蛋白表达为其诱发肿瘤细胞凋亡的机制之一。     

Mitochondrial translocation of cofilin-1 promotes apoptosis of gastric cancer BGC-823 cells induced by ursolic acid

The pathogenesis of gastric cancer is characterized by excessive proliferation, abnormal differentiation, and reduced apoptosis. Ursolic acid, extracted from traditional Chinese medicine bearberry, inhibits cell growth and induces apoptosis in gastric cancer. However, the mechanism of the proapoptotic effect of ursolic acid on gastric cancer cells needs further investigation. In our present study, we found in apoptotic gastric cancer BGC-823 cells induced by ursolic acid that a translocation of cofilin-1 protein from the cytoplasm to the mitochondria promoted the release of cytochrome c from the mitochondria to the cytoplasm, thereby activating the caspase cascade and finally inducing gastric cancer cell apoptosis. These results implied that the mitochondrial translocation of cofilin-1 might play a crucial role in the promotion of apoptosis and might be a key target for future treatment of human gastric cancer.     

Nucleophosmin is a novel Bax chaperone that regulates apoptotic cell death

The proapoptotic B-cell lymphoma-2 family protein Bax is a key regulatory point in the intrinsic apoptotic pathway. However, the factors controlling the process of Bax activation and translocation to mitochondria have yet to be fully identified and characterized. We performed affinity chromatography using peptides corresponding to the mitochondrial-targeting region of Bax, which is normally sequestered within the inactive structure. The molecular chaperone nucleophosmin was identified as a novel Bax-binding protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Reciprocal co-immunoprecipitation and proximity assays confirmed the Bax-nucleophosmin protein-protein interaction and verified that nucleophosmin only bound to activated conformationally altered Bax. Confocal microscopy in a cell-based apoptosis model, demonstrated that nucleophosmin translocation from nucleolus to cytosol preceded Bax movement. Specific knockdown of nucleophosmin expression using RNAi attenuated apoptosis as measured by mitochondrial cytochrome c release and activation of the caspase cascade. In a mouse model of ischaemic stroke, subcellular fractionation studies verified that nucleophosmin translocation occurred within 3 h, at a time before Bax translocation but after Bax conformational changes have occurred. Thus, we have elucidated a novel molecular mechanism whereby Bax becomes activated and translocates to the mitochondria to orchestrate mitochondrial dysfunction and apoptotic cell death, which opens new avenues for therapeutic intervention.     

Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis

Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria. However, it is unclear whether Bax translocation is associated with Bax induction by DNA damaging agents. The induction of Bax in response to DNA damaging agents has been considered to be linked with p53. In this study, we used the p53 negative human chronic myeloid leukaemia K562 cell line. Bax up-regulation occurred at the whole cell level after DNA damage induced by etoposide. However, after incubation with etoposide, Bax failed to translocate to mitochondria and as a result, the apoptotic process was blocked. A Bax stable transfectant, the K/Bax cell line, expressed more Bax protein in the cytosol, mitochondria and nuclei. This Bax overexpression induced cytochrome c release, a reduction of cytochrome c oxidase activity and mitochondrial membrane potential (Delta(Psi)m). However, Bax-induced apoptosis was blocked downstream of mitochondria in K562 cells. The increased levels of mitochondrial Bax sensitized cells to etoposide-induced activation of caspases-2, -3 and -9 and apoptosis. However, after transient transfection with the Apaf-1 gene, K/Bax cells were sensitized to etoposide-induced caspase activation and apoptosis to a larger extent compared with Bax or Apaf-1 transfection alone. We therefore conclude that two mechanisms contribute to the resistance of K562 cells to etoposide-induced apoptosis; firstly failure of Bax targeting to mitochondria and, secondly, deficiency of Apaf-1. Uncoupling of Bax translocation from Bax induction can occur in response to etoposide-induced DNA damage.     

表阿霉素提高胃癌BGC823细胞对TRAIL的敏感性

目的：研究表阿霉素对TRAIL诱导胃癌细胞BGC823细胞凋亡的影响,探讨脂筏和死亡受体4（DR4）在TRAIL诱导细胞凋亡中的作用。方法：采用MTT法测定细胞活力,流式细胞仪检测细胞凋亡,免疫荧光显微技术检测脂筏和DR4在细胞膜的分布。结果：（0.1-50）μg/ml表阿霉素处理BGC823细胞24h,抑制细胞增殖50%的药物浓度（IC50）为（4.61±0.62）μg/ml。在BGC823细胞中,100ng/ml的TRAIL导致轻度的增殖抑制和细胞凋亡,TRAIL（100ng/ml）联合表阿霉素（4.61μg/ml）引起明显的增殖抑制和细胞凋亡（P〈0.05）。与对照组相比,100ng/ml的TRAIL作用BGC823细胞24 h,没有引起明显的脂筏聚集或DR4聚集。表阿霉素（4.61μg/ml）明显促进脂筏聚集和DR4聚集,同时观察到DR4和脂筏的共定位。表阿霉素和TRAIL联合作用24 h,同样观察到DR4定位在聚集的脂筏内。结论：表阿霉素通过促进DR4在脂筏聚集增强TRAIL诱导的胃癌BGC823细胞凋亡。     

Dual roles of Nur77 in selective regulation of apoptosis and cell cycle by TPA and ATRA in gastric cancer cells

Nur77 is an orphan receptor. Although Nur77 affects cell proliferation and apoptosis through its capability of binding to a variety of response elements and regulating their transactivation activities, the intrinsic function of Nur77 is not yet fully understood; in particular, its regulation of apoptosis and proliferation has been characterized as cell type-dependent and agent context-dependent. In this study, Nur77 can be seen to regulate apoptosis via its expression and translocation, rather than its transactivation activity in gastric cancer cells. Nur77 was constitutively expressed in BGC-823 cells. The tetradecanoylphorbol-1,3-acetate (TPA) treatment not only resulted in up-regulation of the Nur77 mRNA level, but also led to translocation of Nur77 protein from the nucleus to the mitochondria, and caused the release of cytochrome c. This TPA-induced translocation of Nur77 was in association with the initiation of apoptosis in gastric cancer cells. Although all-trans retinoic acid (ATRA) could not induce apoptosis in BGC-823 cells due to failure of stimulating Nur77 translocation, expression of Nur77 in the nucleus was required for cell growth inhibition by ATRA. Transfection of antisense Nur77 receptor into BGC-823 cells resulted in resistance of cell growth against ATRA inhibition, and the cells were still arrested in the S phase. Furthermore, the action of Nur77 in TPA-induced apoptosis was mediated through a protein kinase C signaling pathway, while mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways were responsible for the regulation of Nur77 mRNA expression. Taken together, the data revealed the dual functioning mechanisms of Nur77 in gastric cancer cells in response to TPA and ATRA.     

Molecular mechanism of black tea polyphenols induced apoptosis in human skin cancer cells: involvement of Bax translocation and mitochondria mediated death cascade

Theaflavins (TF) and thearubigins (TR) are the most exclusive polyphenols of black tea. Even though few previous reports showed the anticancer effects of TF through apoptosis, the potential effect of TR has not been appraised. This study investigated the induction of apoptosis in human skin cancer cells after treatment of TF and TR. We report that both TF and TR could exert inhibition of A431 (human epidermoid carcinoma) and A375 (human malignant melanoma) cell proliferation without adversely affecting normal human epidermal keratinocyte cells. Growth inhibition of A375 cells occurred through apoptosis, as evident from cell cycle arrest at G(0)/G(1) phase, increase in early apoptotic cells, externalization of phosphatidylserine and DNA fragmentation. In our pursuit to dissect the molecular mechanism of TF- and TR-induced apoptosis in A375 cells, we investigated whether cell death is being mediated by mitochondria. In our system, Bax translocation to mitochondria persuaded depolarization of mitochondrial membrane potential, cytochrome c release in cytosol and induced activation of caspase-9, caspase-3 and poly (ADP-ribose) polymerase cleavage. Our intricate investigations on apoptosis also explained that TF and TR augmented Bax:Bcl2 ratio, up-regulated the expression of p53 as well as p21 and inhibited phosphorylation of the cell survival protein Akt. Furthermore, TF and TR elicited intracellular reactive oxygen species generation in A375 cells. These observations raise speculations that TF as well as TR might exert chemopreventive effect through cell cycle arrest and induction of apoptogenic signals via mitochondrial death cascade in human skin cancer cells.