辛伐他汀通过线粒体途径诱导K562细胞凋亡

目的观察辛伐他汀诱导K562细胞凋亡不同时间的膜电位(Δψm),caspase-3、9和细胞色素C的改变,以推测其凋亡通路。方法采用浓度为20μmol.L-1的辛伐他汀处理K562细胞24、48、72 h,采用流式细胞技术检测细胞凋亡率和线粒体膜电位,分光光度法检测caspase-3、9蛋白活性,免疫组织化学法检测细胞色素C蛋白。结果浓度为20μmol.L-1辛伐他汀作用K562细胞24、48、72 h后,凋亡率分别为(6.1±0.35)%、(14.15±0.42)%(、30.70±0.65)%,随着凋亡率增加线粒体膜电位降低分别为(39.6±4.80)%,(24.4±2.45)%,(6.0±1.62)%;caspase-3、9蛋白活性与对照组相比上调,细胞浆内细胞色素C升高。结论辛伐他汀诱导K562细胞凋亡时线粒体膜电位下降,caspase-3、9活性增高和细胞色素C释放,推测辛伐他汀诱导K562细胞的凋亡可能经过线粒体凋亡途径。     

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。     

辛伐他汀诱导K562细胞凋亡及对细胞内活性氧和Ca2+动态变化的影响

目的研究辛伐他汀诱导人红白血病细胞株K562细胞凋亡及细胞内活性氧与Ca2+水平的变化,以探讨凋亡机制.方法20 μmol·L-1辛伐他汀处理K562细胞,24 h 后光镜观察细胞形态;流式细胞术检测细胞凋亡率、活性氧和细胞内游离Ca2+水平.结果20 μmol·L-1辛伐他汀作用K562细胞 48 h 后出现核固缩、核碎裂和凋亡小体等形态学改变;AnnexinV-FITC/PI检测细胞早期凋亡率,处理组凋亡率高于对照组,随药物作用时间延长逐渐增大,具有时间依赖性.荧光染料2',7'-二氯荧光乙酰乙酸(2',7'-dichloro fluorescein diacetate, DCFH-DA)检测K562细胞内活性氧,不同时间处理组与对照组比较活性氧均升高,峰值时间为 24 h,与对照组比较发生显著改变.荧光染料Fluo-3AM检测K562细胞内游离Ca2+浓度,不同时间细胞内游离Ca2+浓度均升高,峰值时间为 12 h,与对照组比较发生显著变化.结论辛伐他汀诱导K562细胞凋亡的可能机制是通过提高细胞内活性氧及游离Ca2+水平,从而导致细胞凋亡.     

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。     

蔓荆子活性成分vitexicarpin诱导K562细胞凋亡的机制

目的探讨vitexicarpin对人癌细胞增殖的抑制活性及其抗癌机制。方法以SRB法进行细胞毒测定，观察细胞的形态学变化并用流式细胞术分析癌细胞的凋亡率，DNA琼脂糖凝胶电泳检测凋亡细胞的DNA梯带，以Western blotting法进行蛋白质检测。结果Vitexicarpin对K562等4种人癌细胞表现了较强的增殖抑制活性。处理后的K562细胞表现出典型的凋亡形态特征、出现了剂量依赖性增加的亚二倍体峰并呈现出典型的DNA梯形条带；PARP和caspase-3被剪切、胞浆中细胞色素c增高、Bcl-2表达减少、Bax表达未见明显变化。结论Vitexicarpin通过激活线粒体调控的凋亡通路诱导K562细胞凋亡。     

一叶秋碱诱导K562细胞凋亡

目的研究一叶秋碱（ＳＥＣ）能否诱导Ｋ５６２细胞凋亡。方法细胞增殖抑制采用ＭＴＴ法;形态学研究采用电子和荧光显微镜;流式细胞仪和琼脂糖凝胶电泳检测ＤＮＡ断裂。结果ＳＥＣ１０～１６０ｍｇ·Ｌ－１呈剂量依赖性抑制Ｋ５６２细胞增殖（ｒ＝０９６１３,Ｐ＜００５）;ＳＥＣ作用４８ｈ后,电镜下可见细胞膜完整,核染色质边聚和凋亡小体;荧光显微镜下核染色质凝聚成点状结构;流式细胞仪出现凋亡峰;ＤＮＡ琼脂糖凝胶电泳可见“梯状”图谱。结论ＳＥＣ可诱导Ｋ５６２细胞调亡。     

葛根提取物诱导K562细胞凋亡

目的 探讨葛根提取物(PL)对慢性粒细胞自血病(CML)细胞株K562的凋亡诱导作用及其可能的分子机制.方法 以不同浓度(0、2.5、5、10和20 mg/ml)PL处理K562细胞,24 h后光镜下观察形态学改变,MTT法测定细胞增殖抑制,Hoechest 33258荧光染色及FITC-Annexin V/PI双染法检测凋亡率变化,半定量RT-PCR及Western blot方法检测Bcr/abl、Bcl-2、p53、Fas/FasL蛋白表达.结果 PL对K562细胞有明显的增殖抑制作用,并观察到典型的细胞凋亡形态变化,细胞凋亡率与浓度呈正相关;不同浓度PL干预后Bcr/abl基因在mRNA和蛋白水平呈浓度依赖性下调,而 Bcl-2基因则无明显变化;p53表达呈浓度依赖性上调;Fas/FasL表达无明显变化.结论 不同浓度PL能有效诱导K562细胞凋亡;其机制可能通过在mRNA和蛋白水平下调Bcr/abl基因,上调p53基因表达而实现.     

辛伐他汀对K562细胞端粒酶活性及人端粒酶逆转录酶表达的影响

目的探讨辛伐他汀治疗慢性粒细胞白血病可能的作用机制。方法辛伐他汀10和20μmol.L-1与K562细胞作用48或72 h后,用流式细胞术AnnexinⅤ-FITC/PI双染法检测细胞凋亡百分率;提取细胞端粒酶,用PCR-ELISA检测端粒酶活性;实时荧光定量PCR检测人端粒酶逆转录酶(hTERT),c-myc和bcl2-mRNA表达。结果辛伐他汀10和20μmo.lL-1与K562细胞作用48 h后,细胞凋亡率分别为(6.24±0.18)%和(9.41±0.22)%,与对照组(1.88±0.14)%比较明显增加;作用72 h后细胞凋亡率分别为(12.41±0.32)%和(19.08±0.26)%,与对照组(4.20±0.19)%比较明显增加。辛伐他汀10和20μmo.lL-1与K562细胞作用48或72 h后,端粒酶活性,hTERT,c-myc和bcl-2 mRNA表达明显低于对照组。结论辛伐他汀可使K562细胞端粒酶活性降低,其机制可能与下调hTERT mRNA表达有关。     

志苓胶囊通过激活caspase-3抑制K562细胞增殖和诱导细胞凋亡

目的研究志苓胶囊(ZLJN,抗癌复方Ⅱ号)对人慢性髓系白血病K562细胞株增殖及凋亡的影响。方法将志苓胶囊按其不同中西药成分比例配制成中药、西药和复方组,与K562细胞共培养后,采用MTT法、集落形成实验分别检测细胞存活率和集落形成率;Annexin V-FITC/PI标记法、DNA倍体分析及DNA片段化分析检测细胞凋亡;流式细胞仪检测caspase-3活性;Western blot法检测caspase-3酶原(pro-caspase-3)表达。结果不同药物组与K562细胞共培养后,细胞生长受抑制,集落形成率降低。Annexin V-FITC/PI法检测到早期凋亡细胞;DNA倍体分析可见亚二倍体峰(凋亡峰);琼脂糖电泳见典型的DNA梯状带。流式细胞检测caspase-3活性增强,Western blot检测pro-caspase-3表达减弱。结论志苓胶囊可有效抑制K562细胞增殖,诱导其凋亡,其作用机制可能与caspase-3活性增强有关。     

木犀草素对K562细胞caspase-3活化的影响

目的:探讨木犀草素对K562细胞增殖和凋亡的影响.方法:采用MTT法检测K562细胞的增殖,流式细胞术和Hoechst33258/PI荧光染色分析K562细胞的凋亡,比色法测定caspase-3的相对活性,半定量RT-PCR检测caspase-3 mRNA水平的改变,Western-blot分析caspase-3酶原的变化.结果:木犀草素处理细胞24 h后的IC50值为(104.6±13.5)μmol·L-1.浓度为10,20,40μmol·L-1木犀草素处理细胞24 h后均可诱导K562细胞发生凋亡,各处理组的细胞凋亡率均明显高于对照组(P＜0.01).随着浓度及时间的增加,各个木犀草素处理组K562细胞的caspase-3活性升高(P＜0.01),其作用具浓度及时间依赖性.而随着木犀草素浓度的增加,K562细胞中caspase-3的mRNA水平逐渐增加,caspase-3酶原的蛋白水平逐渐减少.结论:木犀草素可通过诱导K562细胞凋亡而抑制其增殖,其诱导K562细胞凋亡可能与激活caspase-3有关.     

Autophagic and apoptotic mechanisms of curcumin-induced death in K562 cells

Curcumin (1), a natural polyphenolic compound, has shown strong antioxidant and anticancer activities. Several molecular mechanisms have been attributed to its inhibitory effects on a wide range of tumor cells. In this study, the response of the chronic myeloid leukemia cell line K562 cells to 1 is investigated. Curcumin inhibited the viability of K562 cells in a dose- and time-dependent manner. Furthermore, curcumin-induced cell death was associated with the formation of the apoptosome complex, the collapse of the mitochondrial membrane potential, and caspase-3 activation. Curcumin treatment also induced Bid cleavage and downregulated the expression of Bcl-2 protein. Surprisingly, even with these molecular features of apoptosis, we showed that 1 stimulated autophagy, which was evidenced by microtubule-associated protein light chain 3 (LC3) immunoreactivty. Curcumin also increased the protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitor bafilomycin A1 and the pan-caspase inhibitor Z-VAD-fmk suppressed curcumin-induced K562 cell death. Overall, these results suggest that curcumin induces autophagic and apoptotic death of K562 cells. These findings suggest that both apoptotic and autophagic mechanisms contribute to the curcumin-induced K562 cell death.     

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.     

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。     

3,5-dibenzyloxy-4'-hydroxystilbene induces early caspase-9 activation during apoptosis in human K562 chronic myelogenous leukemia cells

A series of 22 stilbene derivatives based on resveratrol were synthesized incorporating acetoxy-, benzyloxy-, carboxy-, chloro-, hydroxy- and methoxy functional groups. We examined the cytotoxicity of these 22 stilbenes in human K562 chronic myelogenous leukemia cells. Only four compounds were cytotoxic namely 4'-hydroxy-3-methoxystilbene (15), 3'-acetoxy-4-chlorostilbene (19), 4'-hydroxy-3,5-dimethoxystilbene or pterostilbene (3) and 3,5-dibenzyloxy-4'-hydroxystilbene (28) with IC(50)s of 78 μM, 38 μM, 67 μM and 19.5 μM respectively. Further apoptosis assessment on the most potent compound, 28, confirmed that the cells underwent apoptosis based on phosphatidylserine externalization and loss of mitochondrial membrane potential. Importantly, we observed a concentration-dependent activation of caspase-9 as early as 2 hr with resultant caspase-3 cleavage in 28-induced apoptosis. Additionally, a structure-activity relationship (SAR) study proposed a possible mechanism of action for compound 28. Taken together, our data suggests that the pro-apoptotic effects of 28 involve the intrinsic mitochondrial pathway characterized by an early activation of caspase-9.     

右美托咪定对感染性休克大鼠脾脏及胸腺caspase-3、caspase-8、caspase-9表达及淋巴细胞凋亡的影响

目的研究右美托咪定对内毒素诱导感染性休克大鼠细胞免疫的作用。方法选取12周龄雄性Walse大鼠18只,采用数字表法随机分为空白组(S组)、内毒素组(E组)和右美托咪定治疗组(D组),每组6只。S组静脉推注1.0 mL/kg 0.9%生理盐水后,持续泵入同浓度的生理盐水,E组、D组均采用静脉注射内毒素(LPS)建立感染性休克大鼠模型,D组在注射内毒素后持续微泵右美托咪定。持续监测心率(HR)、呼吸、有创动脉血压(ABP)及平均动脉压(MAP),用药后0.5、1、2、3、4、6 h检测血清中IL-4(白介素-4)、IL-10、IFN-γ(γ干扰素)和IL-2的浓度,6 h后处死大鼠,检测大鼠脾脏及胸腺caspase-3、caspase-8、caspase-9活性,测定淋巴细胞凋亡情况及Th1/Th2。结果①与S组相比,E组、D组大鼠血清的抗炎因子IL-4、IL-10和促炎因子IFN-γ、IL-2均显著提高,其差异均有统计学意义(P<0.05)。随着治疗时间延长,E组的IL-4、IL-10水平降低,IFN-γ、IL-2水平升高;D组IL-4、IL-10水平升高,IFN-γ、IL-2水平降低;两组比较差异有统计学意义(P<0.05)。②与E组相比,D组大鼠脾脏及胸腺caspase-3、caspase-8、caspase-9表达及淋巴细胞凋亡的阳性细胞数均显著减少,差异有统计学意义(P<0.05)。③D组大鼠脾脏及胸腺Th1/Th2比率均显著高于E组,差异有统计学意义(P<0.05)。结论右美托咪定对感染性休克大鼠具有减轻炎症反应和改善免疫功能的作用,有利于改善预后。     

Contributions of autophagic and apoptotic mechanisms to CrTX-induced death of K562 cells.

Previous studies reported that the neurotoxin, Crotoxin, isolated from the venom of South American rattlesnake had potent anti-tumor activity. Here, we investigated the involvement of autophagy and apoptosis in the Crotoxin-induced death of chronic myeloid leukemia cell line K562 cells. The neurotoxin dose dependently inhibited the viability of K562 cells. Crotoxin stimulated the autophagic activity as evidenced by the appearance of punctuate monodansylcadaverine (MDC) fluorescence staining in the cytoplasm and increased the formation of autophagosomes. Crotoxin caused the collapse of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-3. Caspase inhibitors attenuated Crotoxin-induced K562 cell death, while blockage of autophagy maturation with 3-methyladenine (3-MA) and NH4Cl potentiated the neurotoxin's cytotoxicity. These results suggest that an apoptotic mechanism contributes to the Crotoxin-induced death of K562 cells, while the activation of autophagy delays neurotoxin-induced apoptosis.     

c-FLIP knockdown induces ligand-independent DR5-, FADD-, caspase-8-, and caspase-9-dependent apoptosis in breast cancer cells

Cellular-FLICE inhibitory protein (c-FLIP) is an inhibitor of apoptosis downstream of the death receptors Fas, DR4, and DR5, and is expressed as long (c-FLIP_L) and short (c-FLIP_S) splice forms. We found that the knockdown of c-FLIP using small interfering RNA (siRNA) triggered ligand-independent caspase-8- and -9-dependent spontaneous apoptosis and decreased the proliferation of MCF-7 breast cancer cells. Further analysis revealed that an apoptotic inhibitory complex (AIC) comprised of DR5, FADD, caspase-8, and c-FLIP_L exists in MCF-7 cells, and the absence of c-FLIP_L from this complex induces DR5- and FADD-mediated caspase-8 activation in the death inducing signaling complex (DISC). c-FLIP_S was not detected in the AIC, and using splice form-specific siRNAs we showed that c-FLIP_L but not c-FLIP_S is required to prevent spontaneous death signaling in MCF-7 cells. These results clearly show that c-FLIP_L prevents ligand-independent death signaling and provides direct support for studying c-FLIP as a relevant therapeutic target for breast cancers.