通关藤提取物体外对Jurkat、Raji、RPMI8226细胞的抑制作用研究

目的探讨通关藤提取物对Jurkat、Raji、RPMI8226细胞的抑制作用及可能机制。方法以不同浓度通关藤提取物处理Jurkat、Raji、RPMI8226细胞,MTT法检测其对3种细胞的抑制作用,Annexin V/PI双染法观察细胞形态并检测细胞的凋亡率,JC-1染色法检测细胞线粒体跨膜电位(ΔΨm)水平。结果通关藤提取物对Jurkat细胞的抑制作用最强,对RPMI8226细胞最不敏感。25,50μL/mL的通关藤提取物能降低Jurkat、Raji细胞内ΔΨm,并促进其凋亡。100μL/mL的通关藤提取物也可降低RPMI8226细胞ΔΨm而诱导其凋亡。结论通关藤提取物体外对部分淋巴细胞白血病细胞株和骨髓瘤细胞株有不同程度的诱导凋亡作用,可能是通过降低ΔΨm途径触发这些细胞凋亡。     

通关藤对U937细胞凋亡相关基因表达谱的影响

目的利用细胞凋亡基因芯片比较通关藤作用前后U937细胞基因表达谱的改变,探讨通关藤诱导U937细胞凋亡的可能机制。方法利用流式细胞仪检测细胞凋亡率。提取经和未经50μL/mL通关藤处理的U937细胞的mRNA,逆转录为cDNA,然后用含89个目的基因的细胞凋亡基因芯片(APH-012)进行杂交,GEArray软件分析,通过比较得到通关藤处理前后表达有差异的基因。结果通关藤作用前后表达有差异的基因共36条(占芯片基因总数的44.4%),其中28条(28/89,31.5%)基因表达上调,8条(8/89,8.9%)基因表达下调。这些改变的基因主要包括肿瘤坏死因子配体和受体家族、bcl-2家族、caspase家族、P53家族成员。结论通关藤主要通过上调促凋亡基因及下调凋亡抑制基因来诱导U937细胞凋亡,其中死亡受体途径活化可能在凋亡过程中发挥重要作用。     

大黄素诱导白血病U937细胞凋亡及机制初探

目的研究中药大黄素(Emodin)对人髓系白血病细胞株U937细胞增殖及凋亡的影响,探讨bcl-2/bax比值变化和procaspase-3(CPP32)的激活在其中的作用。方法MTT法绘制细胞生长曲线;克隆形成试验观察大黄素对U937细胞增殖的影响;线粒体膜电位检测、DNA倍体分析及DNA凝胶电泳分析细胞凋亡;PCR及检测大黄素作用前后bcl-2/bax基因及蛋白表达的变化;流式细胞术测定大黄素作用前后caspase-3活性变化及检测CPP32的蛋白表达的变化。结果大黄素能抑制U937细胞增殖,作用72h的半数抑制浓度(IC50)约为30μmol·L-1;线粒体膜电位、亚G1峰(凋亡峰)及DNA片段化的检出证实大黄素能诱导U937细胞凋亡,并呈量效关系。大黄素作用后G0/G1期细胞比例较对照组增高,S期比例下降,且与药物浓度呈正相关。大黄素作用后U937细胞bcl-2/bax基因及蛋白的表达水平比值下降,被激活的caspase-3阳性细胞增多,CPP32蛋白表达水平下降,并呈时效关系。结论大黄素能通过诱导凋亡来抑制U937细胞增殖,bcl-2/bax比值的降低及CPP32的活化可能参与了大黄素抑制U937细胞增殖和诱导凋亡的过程。     

氨肽酶抑制剂—Bestatin诱导人白血病细胞凋亡的研究

目的：研究国产氨肽酶N抑制剂Bestatin对人白血病细胞的作用及其机理,方法：应用MTT比色法观察bestatin对细胞的生长抑制作用,流式细胞仪检测细胞表面CD13的表达,光学显微镜观察细胞形态结构的改变。DNA凝胶电泳,DNA片段原位末端标记及流式细胞仪（FCM）检测,以分析细胞凋亡,Rhodamin(Rh)123染色后,FCM检测细胞线粒体跨膜电位,结果：（1）Bestatin明显抑制HL60细胞的生长,半数抑制（IC50）约为13．03ug/ml,而K562细胞的敏感性较差,IC50大于400ug/ml,其抑制作用均呈剂量－时间效应关系。：（2）典型的细胞形态改变,DNA片段化,DNA末端原位标记的检出及ＦＣＭ结果,均证实bstatin能诱导白血病细胞的凋亡,（３） 10ug/ml bestatin作用２４h即可明显诱导ＨＬ６０细胞凋亡,Ｋ５６２细胞的凋亡敏感性显著低于HL60细胞,两者凋亡率均呈剂量和时间依赖性。（4）经bestatin作用后,细胞出现G1期阻滞;（5）经bestatin处理的HL60细胞出现线粒体跨膜电位（Δφm）下降,结论：Bestatin能抑制K562,HL60细胞生长,诱导凋亡是其机制之一,该效应可能与线粒体Δφm下降有关。 wtetrg ,xlw     

manumycin诱导舌鳞癌Tca8113细胞凋亡的作用及机制

目的研究manumycin抑制Tca8113细胞的生长和诱导细胞凋亡的作用及机制。方法细胞毒作用以MTT法测定;凋亡细胞形态以Hoechst33258染色后荧光显微镜观察;凋亡率的检测以AnnexinV染色,流式细胞仪检测;细胞内活性氧(ROS)和线粒体跨膜电位(ΔΨm)分别用DCFH-DA和DiOC6荧光探针标记,流式细胞仪检测;蛋白质定量检测以Westernblot法。结果manumycin浓度依赖性抑制Tca8113细胞的生长,IC50为(11.33±0.63)μmol.L-1;manumycin可浓度和时间依赖性诱导Tca8113细胞的凋亡,过氧化物清除剂N-乙酰半胱氨酸能清除manumycin介导的细胞活性氧的增加,抑制线粒体跨膜电位降低及细胞凋亡。结论manumycin体外显著抑制舌鳞癌Tca8113的生长及诱导细胞凋亡,其机制可能通过激活线粒体依赖性凋亡通路有关。     

苞叶香茶菜庚素诱导人白血病细胞系HL-60凋亡的分子机制研究

目的：观察苞叶香茶菜庚素（melissoidesin G,MOG）对多种肿瘤细胞系的细胞毒活性,并研究MOG诱导白血病细胞系HL．60凋亡的相关分子机制。方法：用MTT法评价化合物对多种肿瘤细胞的增殖抑制活性;通过流式细胞术PI单染法观察化合物对肿瘤细胞内DNA含量的影响,以分析周期变化;Annexin V染色法分析凋亡;荧光底物发光法检测caspase活性;膜电位依赖性结合的荧光探针JC-1标记法检测线粒体膜电位（△ⅢIn）;Western blotting检测蛋白表达。结果：（1）MOG对多种肿瘤细胞系均有一定的增殖抑制作用;（2）MOG诱导HL-60细胞发生剂量依赖性凋亡;（3）在MOG作用下,HL-60细胞出现时间依赖性的△ψm降低;（4）HL-60细胞在MOG作用6～12h后caspase-3和caspase-7均被明显激活,且在抑制剂Z-VAD-FMK的作用下,MOG诱导的caspase激活和细胞凋亡被完全抑制;（5）抗氧化剂NAC可以抑制MOG诱导的△ψm降低、细胞凋亡及caspase-3激活。结论：MOG能够明显抑制肿瘤细胞增殖,并通过诱导细胞凋亡发挥其抗肿瘤作用。推测MOG可能通过引起细胞内氧化还原状态失平衡,进一步破坏线粒体功能,并激活caspase通路引起肿瘤细胞凋亡。     

一氧化氮诱导PC12细胞凋亡及人参皂苷Rg1的保护作用

目的　探讨一氧化氮诱导PC12细胞凋亡及人参皂苷Rg1保护作用的可能机制。 方法　DNA凝胶电泳观察DNA的断裂情况 ,流式细胞仪检测线粒体跨膜电位 ,West ernblotting检测胞浆细胞色素C和活化型半胱氨酸蛋白水解酶caspase 3P2 0 水平。结果　一氧化氮供体SNAP(5 0 0μmol·L-1)可诱导PC12细胞凋亡 ,细胞线粒体跨膜电位明显下降、胞浆细胞色素C水平增加及caspase 3得到激活 ;预先经过 5 0、10和 2 0 μmol·L-1等浓度人参皂苷Rg1处理后 ,SNAP诱导的PC12细胞凋亡明显减少 ,同时明显减弱SNAP对细胞线粒体跨膜电位、胞浆细胞色素C水平及cas pase 3激活的影响。 结论　人参皂苷Rg1可抑制一氧化氮诱导PC12细胞凋亡 ,其作用机制可能与其稳定细胞线粒体跨膜电位、减少线粒体细胞色素C向胞浆释放及抑制cas pase 3的激活有关     

白花蛇舌草提取物诱导U937细胞凋亡的实验研究

目的探讨中草药白花蛇舌草(HDW)醇提取物在体外对人白血病细胞株的增殖抑制和凋亡作用。方法以白血病U937细胞为研究对象,以不同浓度和不同时间HDW醇提取物为干预因素,用MTT法检测HDW醇提取物对U937细胞的抑制作用;用细胞形态学、流式细胞术、DNA电泳观察细胞凋亡情况。结果U937细胞经HDW醇提取物作用后,呈时间和剂量依懒性。光镜观察可见细胞膜完整、核碎裂、凋亡小体等形态学特征,DNA电泳呈现典型的梯形条带,流式细胞仪分析表明0.5,1,2,4 mg/mL的HDW醇提取物使U937细胞发生凋亡率分别为4.18%,56.96%,82.43%,56.41%。结论HDW醇提取物抗肿瘤效应强,同时抑制U937细胞的增殖及诱导细胞凋亡。     

白花蛇舌草提取物诱导U937细胞凋亡的实验研究

目的探讨中草药白花蛇舌草(HDW)醇提取物在体外对人白血病细胞株的增殖抑制和凋亡作用。方法以白血病U937细胞为研究对象,以不同浓度和不同时间HDW醇提取物为干预因素,用MTT法检测HDW醇提取物对U937细胞的抑制作用;用细胞形态学、流式细胞术、DNA电泳观察细胞凋亡情况。结果U937细胞经HDW醇提取物作用后,呈时间和剂量依懒性。光镜观察可见细胞膜完整、核碎裂、凋亡小体等形态学特征,DNA电泳呈现典型的梯形条带,流式细胞仪分析表明0.5,1,2,4 mg/mL的HDW醇提取物使U937细胞发生凋亡率分别为4.18%,56.96%,82.43%,56.41%。结论HDW醇提取物抗肿瘤效应强,同时抑制U937细胞的增殖及诱导细胞凋亡。     

熊果酸对HL60细胞作用机制的初步研究

目的探讨熊果酸在体外对急性早幼粒白血病HL60细胞增殖抑制和诱导凋亡作用。方法采用体外细胞培养技术,荧光显微镜观察细胞结构变化、MTT法观察细胞生长抑制作用、流式细胞仪检测细胞凋亡及周期变化。结果 MTT法证实熊果酸对HL60细胞具有增殖抑制和杀伤效应,并呈浓度和时间依赖性。显微镜下可见HL60细胞出现典型凋亡细胞形态学改变。流式细胞仪检测细胞周期阻滞于G0/G1期。结论熊果酸在体外可抑制急性早幼粒白血病HL60细胞增殖,诱导细胞凋亡。     

大蒜提取物Z-ajoene对U937细胞增殖抑制作用机制的研究

目的　研究大蒜含硫化合物Z ajoene抗肿瘤作用的分子机制。方法　应用MTT法测定Z ajoene对不同白血病细胞株的生长抑制作用。采用流式细胞技术 (FACS)分析Z ajoene对U937细胞周期的影响以及对该细胞的诱导凋亡作用。用Western blot的方法检测Z ajoene对凋亡相关蛋白以及微管蛋白表达的影响。结果　Z ajoene对两种白血病细胞系HL6 0和U937有着相近的生长抑制作用 ,IC50 值均约为10 μmol·L-1。细胞周期分析发现 ,在给药初期和药物浓度较低时G2 /M期细胞数量明显升高 ;2 0 μmol·L-1Z ajoene作用 2 4h时 ,G2 /M期细胞比对照组升高约15 2 5 %。Z ajoene可以诱导U937细胞凋亡 ,并呈浓度和时间依赖性。实验结果表明 ,细胞凋亡过程中的关键蛋白酶caspase 3的激活及其底物PARP蛋白的裂解随着给药浓度和时间的增加而增加。此外 ,Z ajoene也产生以出现 2 3ku裂解带为特征的Bcl 2裂解。另外 ,实验浓度的Z ajoene对β 微管蛋白的表达有抑制作用 ,而对α 微管蛋白表达无明显改变。结论　Z ajoene能明显抑制U937细胞株的生长并可阻断U937细胞周期于G2 /M期 ,且这种阻断早于细胞凋亡。Z ajoene抗肿瘤机制与诱导细胞凋亡和干扰微管蛋白聚合相关。     

Cytotoxic and apoptogenic effect of Swietenia mahagoni (L.) Jacq. leaf extract in human leukemic cell lines U937, K562 and HL-60

The apoptogenic activity of Swietenia mahagoni leaf extract (SMLE) was investigated against three human leukemic cell lines – U937, K562 and HL-60. SMLE inhibited cell growth and metabolic activity of the leukemic cells and showed characteristic features of apoptosis. Flow-cytometric analysis showed that SMLE arrested U937 and K562 cell populations in the G2-M phase and the HL-60 cell population in the G1 phase of cell cycle. SMLE induced apoptosis was found to be mediated through mitochondrial intrinsic pathway involving the release of cytochrome c into the cytosol and activation of caspase-9 and caspase-3. Two flavonoids, catechin and quercetin-3-O-glucoside, isolated from SMLE, were found to inhibit the growth and metabolic activity of U937, K562 and HL-60 cells at much lower concentrations thus indicating that these two flavonoids might be the active ingredients responsible for the anti-leukemic activity of SMLE.     

番荔枝内酯诱导白血病细胞凋亡的机理

目的　研究番荔枝内酯(squamocin)诱导白血病细胞凋亡的机理。方法　DNA凝胶电泳法、荧光染色等检测细胞凋亡。试剂盒检测caspase-3的活性。Westernblot法检测PARP和caspase-3的剪切片断和磷酸化的SAPK/JNK量的变化。结果　Squamocin处理HL-60细胞后,导致染色质浓缩、片断化,DNA梯形条带出现,完整PARP的116ku条带逐渐减少,而85ku的片断逐渐增加,caspase-3酶的活性也增加。Caspase-3的特异性抑制剂DEVD-CHO预处理HL-60细胞,可阻止squamocin诱导的DNA片断化、PARP的剪切和细胞死亡。Squamocin激活HL-60细胞中SAPK/JNK。结论　Squamocin诱导HL-60细胞凋亡依赖caspase-3途径的激活,squamocin激活caspase-3可能与SAPK/JNK的激活相关     

Compound MMH01 possesses toxicity against human leukemia and pancreatic cancer cells

MMH01 is a compound isolated from Antrodia cinnamomea. MMH01 markedly inhibited growth of human leukemia U937 and pancreatic cancer BxPC3 cells. It resulted in distinct patterns of cell cycle distribution in U937 (G2/M, sub-G1 and polyploidy) and BxPC3 cells (G0/G1 and sub-G1). The modes of cell death in U937 cells include apoptosis and mitotic catastrophe, whereas apoptosis-associated events or necrosis in BxPC3 cells. Neither mitochondrial membrane permeabilization nor caspase dependence was noted. Proteins involving mitotic catastrophe-associated cell death such as cyclin B1 and checkpoint kinase 2 were activated in U937 cells. Only slight to moderate viability inhibition was noted to human monocytes, the normal counterpart of these myeloid leukemic cells. In conclusion, MMH01 possesses cytotoxicity against human leukemia and pancreatic cancer cells.     

雷公藤红素通过活性氧诱导结肠癌SW620细胞凋亡

目的:探讨雷公藤红素(celestrol)诱导KRAS驱动的结肠癌SW620细胞产生凋亡的作用和机制。方法:四甲基偶氮唑蓝(MTT)法和台盼蓝拒染法检测细胞增殖;免疫印迹法检测蛋白表达;流式细胞仪和荧光显微镜检测细胞凋亡、细胞周期、线粒体膜电位;荧光显微镜检测细胞内活性氧水平(reactive oxygen species,ROS)。结果:雷公藤红素明显抑制SW620细胞的增殖活性;雷公藤红素下调SW620胞内的p-Akt、NF-κB、Survivin表达,激活caspase-7、caspase-3 和PARP;雷公藤红素增加SW620细胞内的ROS、降低线粒体膜电位、阻滞细胞周期于G₂/M期和诱导凋亡。抗氧化剂N-乙酰半胱氨酸(NAC)抑制雷公藤红素引起的上述作用。结论:通过诱导细胞内ROS的累积导致细胞内线粒体膜电位的下降进而触发细胞发生凋亡是雷公藤红素诱导SW620细胞凋亡的作用机制之一。     

Rimonabant-induced apoptosis in leukemia cell lines: Activation of caspase-dependent and -independent pathways

Rimonabant (SR141716), a cannabinoid CB1 receptor antagonist known for anti-obesity activity, has more recently been shown to inhibit tumor cell growth. Here we demonstrated the antitumor potential of SR141716 in leukemia-derived cell lines and its low toxicity in normal cells (PBMC). SR141716 (1-20 μM range of doses) reduced Jurkat and U937 cell number by activating death signals as well as affecting cell cycle progression. The most prominent response in U937 to SR141716 was a G₀/G₁ block, while in Jurkat cells there was activation of cell death processes. SR141716-treated cells exhibited the morphological and biochemical features of apoptosis and to some extent necrosis. Apoptotic mode of cell death was confirmed in both cell lines by analysis of cell morphology, phosphatidylserine exposure and DNA fragmentation. Moreover, the drug was found to induce an early and robust mitochondrial membrane depolarization. In Jurkat cells the apoptotic process was typically caspase-dependent, while in U937 caspase-independent pathways were also activated. The contribution of PARP activation to SR141716-induced apoptosis in U937 was suggested by protein PARylation, AIF release and apoptosis reversal by PARP inhibitors. Moreover, SR141716 negatively modulated, especially in U937, the PI3K/AKT pathways. In conclusion, our data indicate that SR141716 elicits alternative response and/or cell death pathways depending on the cell type affected.     

Molecular mechanisms of ZD1839 （Iressa）-induced apoptosis in human leukemic U937 cells

Aim： To investigate the molecular mechanisms of ZD 1839-induced apoptosis in human leukemic U937 cells. Methods： The inhibition of human leukemic U937 cell growth was assessed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphnyl-2H-tetrazolim bromide （MTT） assays, lactate dehydrogenase （LDH） release, and cell cycle distribution. The expression of anti- and pro-apoptotic proteins was detected by Western blot analysis. Results： This study demonstrated that ZD1839 induced apoptosis in leukemic U937 cells by the downregulation of Bcl-2, caspase activation and subsequent apoptotic features. Cotreatment with ZD 1839 and the caspase- 3 inhibitor z-DEVD-fmk blocked apoptosis, indicating that caspase-3 activation is at least partially responsible for ZD 1839-induced apoptosis. The ectopic expression of Bcl-2 attenuated caspase-3 activation, PARP cleavage, and subsequent indicators of apoptosis, including sub-G1 DNA content and LDH release. These results indicate that the downregulation of Bcl-2 plays a major role in the initiation of ZD1839-induced apoptosis, and that the activation of a caspase cascade is involved in the execution of apoptosis. Furthermore, ZD1839 treatment triggered the activation of p38 mitogen-activated protein kinase （MAPK） and the downregulation of c-Jun-N-terminal kinase （JNK）, extracellular signal-regulated kinase （ERK） and phosphatidyl inositol 3-kinase （PI3K）/Akt. The inhibition of the ERK and PI3K/Akt pathways also significantly increased cellular death. Conclusion： ZD 1839 activated caspase-3 and the inhibited Bcl-2 in human leukemic U937 cells through the downregulation of the ERK and PI3K/Akt pathways.     

Mitochondria-dependent apoptosis induced by a novel amphipathic photochemotherapeutic agent ZnPcS2P2 in HL60 cells

AIM: To investigate the mechanism underlying the killing effects of a novel amphipathic photosensitizer, disulfonated diphthalimidomethyl phthalocyanine zinc (ZnPcS2P2), mediated photodynamic therapy (ZnPc-PDT) in human myelogenous leukemia HL60 cells. METHODS: After incubation for 5 h with 0.5 mumol/L ZnPcS2P2, the HL60 cells were exposed to a light source of 670 nm wavelength. Thereafter, the cells were detected at different time intervals after PDT. The characteristics of apoptosis were detected by observation of ultrastructure assay, DNA fragmentation assay and terminal deoxynucleotidyl transferase deoxyuridine nick-end labeling method (TUNEL). Mitochondria-dependent apoptosis was determined by the detection of mitochondrial membrane potential (deltaPhim), activities of caspase family protease and of caspase-3, cytosol cytochrome c. Proteins Bcl-2 and Bax were detected by immunoblot analysis. RESULTS: Evident characteristics of apoptosis were observed post-ZnPc-PDT with ultrastructure assay, DNA fragmentation assay and TUNEL staining. TUNEL assay showed that apoptotic rates in the cells collected from 6 h, 12 h and 24 h after PDT were 9.6%, 24.4%, and 33.0%, respectively. HL60 cells underwent mitochondria-dependent apoptosis as a result of cytochrome c release from mitochondria into cytosol accompanied by a reduction of deltaPhim. The activities of caspase family protease and of caspase-3 were elevated. Furthermore, ZnPc-PDT could remarkably down-regulate the Bcl-2 pro-apoptotic protein and up-regulate the anti-apoptotic Bax protein. CONCLUSION: ZnPc-PDT could induce mitochondria-dependent apoptosis in HL60 cells.     

A potent apoptosis-inducing activity of a sesquiterpene lactone, arucanolide, in HL60 cells: a crucial role of apoptosis-inducing factor

Six main sesquiterpene lactones (germacranolides) from Calea urticifolia were evaluated for in vitro cytotoxicity against human tumor cell lines HL60 and SW480 cells. Among them, arucanolide and parthenolide displayed marked cytotoxicity against both cell lines. Arucanolide exhibited a low IC(50) in HL60 cells. The cytotoxic activity of arucanolide was observed at lower concentrations compared to that of parthenolide, which has been reported to be a typical and simple germacranolide. The activity was found to be mainly due to apoptosis that was assessed by morphological findings, DNA ladder formation (24 - 36 h), and flow cytometric analysis in HL60 cells. Western blotting and an apoptosis inhibition assay using caspase inhibitors did not demonstrate the activation of any caspases tested. However, the mitochondrial membrane potential of HL60 cells was lost after 24-h treatment with arucanolide, and concurrently apoptosis-inducing factor (AIF) released from mitochondria was detected by Western blot analysis. The inactivation of nuclear factor-kappaB, which has been commonly shown in parthenolide-induced apoptosis, did not occur in arucanolide-induced apoptosis. Taken together, the findings presented here indicate that arucanolide induced marked apoptosis in HL60 cells mainly by dissipating mitochondrial membrane potential, which would trigger AIF-induced apoptosis.