辛伐他汀诱导K562细胞凋亡过程中对线粒体膜电位的影响

目的:观察辛伐他汀诱导人红白血病K562细胞凋亡时线粒体膜电位(Δψm)的变化及其与时间的关系。方法:常规培养细胞24h,辛伐他汀(20μmol.L-1)处理K562细胞12、24、48、72h,观测细胞形态学改变;MTT法检测细胞增殖抑制情况;流式细胞术检测细胞凋亡率和线粒体膜电位改变,并与溶剂对照组比较。结果:与对照组比较,辛伐他汀作用K562细胞48h后细胞出现核固缩、核碎裂和凋亡小体等形态学改变;作用12、24、48、72h后细胞凋亡率分别增加(2.55±0.35)%、(6.1±0.35)%、(14.15±0.42)%、(30.70±0.65)%,K562细胞凋亡率随着药物作用时间延长而增加;细胞膜电位降低百分率分别为(0.7±0.24)%、(39.6±4.80)%、(24.4±2.45)%、(6.0±1.62)%,24h时膜电位降低百分率最大。结论:线粒体跨膜电位降低是凋亡发生的早期事件,辛伐他汀诱导K562细胞凋亡的可能机制是通过使线粒体跨膜电位崩溃,从而导致细胞凋亡。     

辛伐他汀通过内质网应激途径诱导K562细胞凋亡

探讨内质网应激在辛伐他汀诱导K562细胞凋亡中的作用。采用荧光显微镜观察凋亡细胞的形态变化，AnnexinV-FITC/PI双染法检测细胞凋亡率，激光扫描共聚焦显微镜检测细胞内Ｃa²⁺浓度，RT-PCR检测葡萄糖调节蛋白78(glucose regulated protein 78，GRP78)、钙蛋白酶(calpain)基因mRNA表达水平，Western blotting检测GRP78、 calpain、 caspase-3， -6， -7， -9， -12蛋白水平。结果显示，10、 20、 30 μmol·L^-1辛伐他汀(simvastatin，Sim)作用K562细胞72 h后，细胞出现典型的凋亡形态，凋亡率分别为12.41%、 19.08%和23.41%；细胞内Ca²⁺浓度增加，荧光强度分别为43、54和64；GRP78、calpain基因mRNA表达上调；calpain、 caspase-3， -6， -7， -9， -12蛋白剪切活化、GRP78蛋白表达增强。以上结果表明，内质网作为细胞凋亡的重要途径参与了辛伐他汀诱导K562细胞的凋亡。辛伐他汀将可能被用于临床治疗白血病。     

α-维尼非林经线粒体凋亡途径诱导K562细胞凋亡

目的旨在研究α-维尼非林(α-viniferin)对人慢性髓系白血病细胞K562的作用与相关机制。方法 MTT法评价α-viniferin对K562细胞的细胞毒活性。采用细胞形态学和生物化学方法检测细胞凋亡。通过化学荧光法对细胞内线粒体膜电位、caspase-9、caspase-3活性分析。通过半定量RT-PCR表达分析来确定Bcl-2家族相关基因在α-viniferin诱导K562细胞凋亡中的作用。结果α-viniferin能抑制K562细胞增殖,呈剂量和时间依赖性,IC50为13.61 mg·L-1。α-viniferin引起K562细胞出现死亡并伴随有染色质聚集、核破碎、凋亡小体等典型的凋亡形态学特征;此外还伴随有线粒体膜电位显著降低,caspase-9、caspase-3活性升高等现象;α-viniferin(2~32 mg·L-1)引起K562细胞caspase-3 mRNA表达持续升高,Bax、Bad、Bim、Bid促凋亡基因mRNA表达增加,而Bcl-2、Bcl-xL抗凋亡基因mRNA表达持续下降。结论α-viniferin通过线粒体途径诱导K562细胞凋亡。     

辛伐他汀诱导人红白血病K562细胞凋亡的实验研究

目的:探讨辛伐他汀诱导K562细胞凋亡及其机制。方法:取浓度为0(阴性对照组)、10、20、30μmol·L-1的辛伐他汀作用K562细胞72h后,双染色法检测细胞凋亡率;DNALadder实验检测细胞凋亡条带;用线粒体分离试剂分离出胞浆蛋白、微粒体蛋白、线粒体蛋白;逆转录-聚合酶链反应法检测GRP78、caspase-9、caspase-3、GADD153mRNA表达;Western blot法检测GRP78、caspase-12、caspase-9、caspase-3、GADD153、细胞色素C蛋白水平。结果:与阴性对照组比较,10、20、30μmol·L-1辛伐他汀作用K562细胞72h后出现典型的凋亡条带,凋亡率分别为12.41%、19.08%、23.41%(P<0.01),GRP78、caspase-9、caspase-3和GADD153 mRNA表达上调(P<0.05),caspase-12、caspase-9、caspase-3蛋白水平下降,caspase-12定位于内质网,细胞色素C从线粒体释放,GRP78、GADD153蛋白表达上调。结论:辛伐他汀可通过内质网和线粒体途径诱导K562细胞凋亡。     

辛伐他汀诱导K562细胞凋亡时caspase-3，8，9蛋白活性与mRNA的动态变化

目的:研究辛伐他汀诱导K562细胞凋亡过程中caspase-3,8,9活性与mRNA的动态变化,探讨其凋亡通路。方法:20μmol.L-1辛伐他汀处理K562细胞,48 h观察细胞形态学;24,48和72 h流式细胞技术检测细胞凋亡率;比色法测定caspase-3,8和9活性;半定量RT-PCR检测caspase-3,8,9的mRNA变化。结果:20μmol.L-1辛伐他汀作用K562细胞48 h后细胞出现核固缩、核碎裂和凋亡小体等形态学改变;24,48和72 h辛伐他汀处理组细胞凋亡率较对照组的增加值分别为(6.10±0.35)%,(14.15±0.42)%和(30.70±0.65)%,差异均具有显著性(P<0.01);不同时间辛伐他汀处理组的caspase-3,8,9活性与mRNA表达量均比对照组明显升高(P<0.01)。结论:caspase-3,8,9蛋白活性与mRNA上调是辛伐他汀诱导K562细胞凋亡的重要原因和机制。     

线粒体与细胞凋亡的研究进展

细胞凋亡 (apoptosis)是一种普遍的生理现象 ,是指在一定的生理或病理条件下 ,按程序进行的细胞死亡过程 ,亦称程序性细胞死亡 (programmedcelldeath ,PCD) ,对于维持多细胞机体的完整性和自身稳态具有重要作用。线粒体是所有真核细胞内腺苷三磷酸(ATP)产生中心 ,对维持细胞能量代谢和正常功能活动起重要作用。新近研究发现 ,线粒体内包含一些与细胞凋亡有密切关系的物质 ,如细胞色素C(CytC、凋亡诱导因子 (AIF)、Ca2 + 和活性氧自由基 (ROS)等。在凋亡信号的刺激下 ,线粒体膜通透性增加 ,由此产生一系列关键性变化 ,包括CytC的释放…     

镉诱发HEK293细胞凋亡的线粒体途径

目的研究镉引起的HEK293细胞凋亡与线粒体的关系。方法分别以2′,7′-二氯荧光素二酯和若丹明123为荧光探针,经流式细胞仪检测胞浆内活性氧含量和线粒体膜电位的变化;Western印迹法检测线粒体细胞色素c的释放。结果在镉诱导HEK293细胞的凋亡过程中,胞浆内的活性氧水平升高,但能被抗氧化剂N-乙酰-L-半胱氨酸(NAC)抑制;同时,镉使线粒体膜电位下降和线粒体细胞色素c释放进入胞浆,二者都被NAC加剧。结论镉诱导HEK293细胞的凋亡可能通过线粒体途径。     

线粒体介导的细胞凋亡的研究进展

细胞凋亡是由相关基因调控的程序化细胞死亡过程,线粒体在细胞凋亡中起重要的调控作用。该文对Cyt-c、活性氧、Bcl-2及AIF介导的细胞凋亡机制进行综述。     

锰超氧化物岐化酶模拟化合物诱导K562细胞凋亡的机制研究

目的观察锰超氧化物岐化酶模拟化合物(mimics of manganese superoxide dismutase,MnSODm)对人白血病K562细胞凋亡诱导作用,并探讨其分子机制。方法以人白血病K562细胞为靶细胞,四氮唑蓝比色法(MTT法)测定细胞增殖活性;Annexin V/PI双标记和细胞形态学法检测细胞凋亡;RT-PCR检测bcl-2和bax基因mRNA的表达水平;流式细胞术(FCM)测定Bcl-2和Bax蛋白表达水平、线粒体跨膜电位(Δψm)、细胞色素C(Cyt C)释放和Caspase-3活性变化。结果0.5~10mg·mL-1MnSODm明显抑制K562细胞增殖(P<0.01),Annexin V/PI染色显示凋亡细胞明显增多,光学显微镜和透射电镜观察呈现典型的凋亡形态改变;bcl-2基因mRNA和蛋白表达下调,bax基因mRNA和蛋白表达上调,线粒体Δψm降低,Cyt C释放增多,Caspase-3活性增强。结论MnSODm调控Bax/Bcl-2表达,通过线粒体途径诱导K562细胞凋亡。     

锰超氧化物歧化酶模拟化合物通过Fas途径诱导白血病K562细胞凋亡

目的：探讨锰超氧化物歧化酶模拟化合物（mi mics of manganese superoxide dismutase,MnSODm）对人白血病K562细胞的凋亡诱导效应及作用机制。方法：应用MTT比色法、An-nexin V/PI双标记和细胞形态学法观察细胞凋亡;流式细胞术（FCM）测定Fas蛋白表达水平;RT-PCR检测Caspase-3mRNA的表达水平,比色法测定Caspase-3活性变化。结果：MnSODm作用后K562细胞的增殖受到抑制,Annexin V/PI染色显示凋亡细胞明显增多,透射电镜观察呈现典型的凋亡形态改变。同时,Fas蛋白表达水平显著增高,Caspase-3mRNA表达水平明显升高,活性显著增强。结论：MnSODm可能通过Fas途径诱导白血病K562细胞凋亡。     

Cardiotoxin III induces apoptosis in K562 cells through a mitochondrial-mediated pathway

1. Cardiotoxin (CTX) III is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III on human leukaemia K562 cells. 2. Cardiotoxin III was found to inhibit the growth of K562 cells in a time- and dose-dependent manner, with an IC(50) value of 1.7 mug/mL, and displayed several features of apoptosis, including apoptotic body formation, an increase in the sub-G(1) population, DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. 3. Investigation of the mechanism of CTX III-induced apoptosis revealed that treatment of K562 cells with CTX III resulted in the loss of mitochondrial membrane potential, cytochrome c release from mitochondria into the cytosol and activation of caspase-9 and caspase-3 and the subsequent cleavage of the caspase-3 substrate PARP; however, CTX III did not generate reactive oxygen species (ROS). 4. Taken together, the results indicate that CTX III induces apoptosis in K562 cells through an ROS-independent mitochondrial dysfunction pathway.     

Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells

Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC‐1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose‐dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl‐2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl‐2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma.     

榄香烯诱导人白血病K562细胞凋亡

目的研究榄香烯抗肿瘤的作用机制。方法用流式细胞仪和琼脂糖电泳检测DNA断裂,透射电镜观察超微结构变化。结果260μmol·L-1榄香烯孵育K562细胞6、12、24、48h后流式细胞仪检测到逐渐增强的凋亡峰,电泳发现明显的阶梯状条带,电镜观察到染色体聚集、凋亡小体。结论榄香烯能诱导K562细胞凋亡。     

A novel homospermidine conjugate inhibits growth and induces apoptosis in human hepatoma cells

Aim： To elucidate the mechanism responsible for the antiproliferative effects of a novel homospermidine conjugate, anthracenylmethyl homospermidine （ANTMHspd）, in the human hepatoma BEL-7402 cell line. Methods： The viability of the cells was assessed by MTT assay and the trypan blue dye exclusion method. Morphological changes were observed by fluorescence microscopy with Hoechst 33258 staining. Cell cycle distribution, apoptosis, and mitochondrial membrane potential were measured by flow cytometry. Protein expression was detected by Western blot analysis. Results： ANTMHspd strongly decreased BEL-7402 cell proliferation in a dose- and time-dependent manner. Hoechst 33258 staining and the flow cytometry assay showed that ANTMHspd induced cell apoptosis and cell cycle perturbation. Furthermore, ANTMHspd could induce mitochondrial membrane potential loss and cytochrome c release and enhance cleaved caspase-3, cleaved caspase-9, and Bax protein expression without caspase-8 activation. ANTMHspd could also decrease the expression of Bcl-2 and cytochrome c in mitochondria. In addition, the specific inhibitors of caspase-9 and caspase-3 almost abolished the ANTMHspd-induced caspase-9 and caspase-3 activation, respectively. Conclusion： ANTMHspd could induce BEL-7402 cell apoptosis via the mitochondrial/caspase-dependent pathway and the Bcl-2 family was involved in the control of apoptosis.     

新型蒽醌类衍生物HG251诱导人白血病K562/DOX细胞凋亡的机制

目的探讨新型蒽醌类衍生物HG251诱导K562/DOX细胞凋亡的机制。方法以MTT法检测细胞活力,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P-gp及凋亡相关蛋白如半胱天冬蛋白酶-3、-8、-9、p53、Bcl-xL、cytochrome C的表达。结果HG251剂量依赖性的抑制K562/DOX细胞的生长、降低线粒体膜电位并诱导其凋亡;上调p53并下调Bcl-xL;诱导半胱天冬蛋白酶-3、-8、-9的活化及cytochrome C的释放,但对P-gp的表达无影响。结论HG251通过干扰p53及Bcl-xL的表达而克服K562/DOX细胞的多药耐药,并通过细胞膜死亡受体途径及线粒体途径诱导其细胞凋亡。     

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

冬凌草甲素通过PI3K/Akt通路诱导MDA-MB-231细胞的凋亡

目的：研究冬凌草甲素对人乳腺癌MDA-MB-231细胞增殖产生的影响,初步探讨其作用机理。方法：体外培养人乳腺癌MDA-MB-231细胞,采用6、12、24μmol/L冬凌草甲素对其进行处理,采用倒置显微镜进行细胞形态学观察,MTT比色法检测细胞存活率,流式细胞术检测细胞凋亡率,Western blotting检测凋亡相关蛋白procaspase-3、PARP及Akt、p-Akt、p-GSK3β表达的变化。结果：冬凌草甲素作用MDA-MB-231细胞24h后,可观察到细胞凋亡的形态学改变,以24μmol/L组最为明显。实验组与对照组相比,细胞存活率显著降低、凋亡率显著升高（P〈0.01）,具有时间和剂量依赖性,凋亡相关蛋白procaspase-3下调,caspase-3底物PARP被逐步剪切,并伴随p-Akt、p-GSK3β蛋白水平下调（P〈0.05）。结论：冬凌草甲素可有效抑制人乳腺癌MDA-MB-231细胞的增殖,诱导其凋亡,机制与PI3K/Akt通路的抑制有关。