光动力疗法对人乳腺癌MCF-7细胞增殖凋亡的影响

目的 研究光动力疗法（PDT）对人乳腺癌MCF-7细胞增殖的影响和诱导凋亡的作用.方法 癌光啉（PSD-007）与细胞共同孵育2h后,以不同能量635 nm激光照射,通过噻唑蓝（MTT）比色法测定PDT后不同时间（0.5、1、3、6、12、24h）细胞的光密度（0D）值及存活率.DAPI染色观察PDT后不同时间细胞凋亡中细胞核形态学的改变.Annexin-V/PI双染法结合流式细胞术分析细胞凋亡率的变化.结果 PDT对MCF-7细胞的增殖有抑制作用,且随着PDT光照能量的提高而增强,PDT作用后细胞存活率随时间延长而逐渐降低.细胞形态学观察结果表明,MCF-7细胞呈典型的凋亡形态特征.Annexin-V/PI双染也证实PDT可以诱导细胞凋亡,且凋亡率随PDT作用后时间的延长而升高.结论 PDT能显著抑制MCF-7细胞增殖,诱导细胞凋亡,且其诱导肿瘤细胞的凋亡是一渐进性的过程,具有光照剂量和时间效应关系.     

青蒿琥酯对乳腺癌MCF-7细胞抗失巢凋亡的影响

目的：探讨青蒿琥酯对乳腺癌细胞抗失巢凋亡的影响。 方法： 采用不同浓度的青蒿琥酯处理乳腺癌MCF-7细胞，采用末端脱氧核苷酸转移酶介导的dUTP切口末端标记法(TUNEL)法检测乳腺癌细胞抗失巢凋亡，采用软琼脂集落形成试验检测细胞的生长增殖。 结果： 青蒿琥酯能有效地抑制乳腺癌MCF-7细胞锚着不依赖性增殖，且呈浓度依赖性。乳腺癌细胞经青蒿琥酯处理后，可促进失巢凋亡，且与时间和浓度相关。 结论： 青蒿琥酯可有效地抑制乳腺癌细胞锚着不依赖性增殖，其机制可能与促进失巢凋亡有关。     

褐藻糖胶对人乳腺癌MCF-7细胞的增殖抑制和凋亡诱导作用

目的：研究褐藻糖胶对体外培养的人乳腺癌MCF-7细胞增殖及凋亡的影响，并探讨其凋亡机制。方法:不同浓度(100 mg/L、300 mg/L、500 mg/L、1 000 mg/L)褐藻糖胶处理MCF-7细胞48h后，应用四甲基偶氮唑蓝（MTT）法检测细胞的增殖；Hoechst 33258染色、琼脂糖凝胶电泳法观察细胞的凋亡的形态学及生化改变；逆转录聚合酶链反应（RT-PCR）和蛋白印迹分别测定细胞〖STBX〗bcl-2〖STBZ〗和bax mRNA及其蛋白的表达。结果:不同实验浓度(100 mg/L、300 mg/L、500 mg/L、1 000 mg/L)的褐藻糖胶均能抑制MCF-7细胞的增殖（P<0.01），抑制率呈浓度依赖性增大；褐藻糖胶诱导MCF-7细胞凋亡数增加，且可见明显的、凋亡特有的DNA梯形条带；在褐藻糖胶存在下，〖STBX〗 bcl-2〖STBZ〗 mRNA和蛋白表达减少，而bax mRNA和蛋白表达增加，Bcl-2/Bax比值下降（P<0.05）。结论:褐藻糖胶可抑制MCF-7细胞增殖，且诱导其凋亡，其凋亡机制是下调Bcl-2和上调Bax蛋白的表达。     

WNT5B过表达对人乳腺癌MCF-7细胞活力及凋亡的影响

目的:检测WNT5B在人正常乳腺上皮细胞和乳腺癌细胞株中的表达,及过表达WNT5B后对人乳腺癌MCF-7细胞活力及凋亡的影响,探讨WNT5B在乳腺癌发生发展中的作用。方法:采用RT-PCR法检测正常乳腺上皮细胞和乳腺癌细胞中WNT5B的mRNA表达水平,将WNT5B的表达载体pc DNA3.1/WNT5B和空白对照载体pc DNA3.1分别瞬时转染人乳腺癌MCF-7细胞,通过real-time PCR和Western blotting法分别在mRNA和蛋白水平验证WNT5B的表达效率;并通过CCK-8的方法检测WNT5B过表达对人乳腺癌MCF-7细胞活力的影响,通过流式细胞术分别检测过表达WNT5B对MCF-7细胞周期分布及凋亡比例的影响。结果:与正常乳腺上皮细胞相比,WNT5B在乳腺癌细胞中低表达;在乳腺癌细胞MCF-7中转染WNT5B表达质粒后,WNT5B表达上调,差异有统计学显著性(P0.05);与对照组相比,过表达WNT5B后,MCF7细胞的活力明显降低,处于S期细胞的量明显增加,G1和G2/M期细胞明显减少,且细胞凋亡比例明显增加,差异均有统计学显著性(P0.05)。结论:WNT5B在乳腺癌细胞中低表达,过表达WNT5B可使人乳腺癌细胞MCF-7阻滞在S期,抑制细胞增殖,促进细胞凋亡。     

去甲斑蝥素通过半胱氨酸天冬氨酸酶诱导HeLa细胞凋亡

目的：研究去甲斑蝥素（NCTD）通过半胱氨酸天冬氨酸酶（caspase）途径诱导人宫颈癌HeLa细胞凋亡的机制。方法： 采用MTT法、形态学观察、DNA凝胶电泳、乳酸脱氢酶（LDH）检测及Western blot检测法。结果： 去甲斑蝥素能显著诱导HeLa细胞发生凋亡，caspase-家族、caspase-3、-8、-10抑制剂可以部分地抑制NCTD诱导的细胞死亡。细胞凋亡时caspase-3、-8、-9酶活力升高，而且caspase-3的作用底物-caspase-3 激活的DNA酶抑制物（ICAD）蛋白表达下降。结论： 去甲斑蝥素通过激活caspase途径诱导HeLa细胞凋亡。     

siRNA targeting Survivin inhibits growth and induces apoptosis in human renal clear cell carcinoma 786-O cells

We investigated the inhibitory effect of small interfering RNA (siRNA) targeting Survivin gene on cell proliferation and apoptosis in human renal clear cell carcinoma 786-O cells. qRT-PCR, immunocytochemistry, and Western Blot were used to detect Survivin gene expression in 786-O cells. Cell proliferation was determined by BrdU assay and PCNA expression. Cell apoptosis was analyzed through detection of caspase-3 activity, and the effect of Survivin-siRNA on Bcl-2 gene expression was also examined. Forty-eight hours after transfection, Survivin expression was markedly inhibited at the mRNA and protein level. Downregulation of Survivin resulted in a significant inhibition of tumor cell growth. Caspase-3 activity showed that siRNA targeting Survivin gene induced cell apoptosis in 786-O cells. Moreover, Bcl-2 protein expression was markedly inhibited by transfection with siRNA against Survivin. These results indicate that siRNA targeting Survivin gene can downregulate Survivin gene expression in 786-O cells, inhibit growth, and induce apoptosis of renal carcinoma cells.     

BARF1表达下调通过活化caspase依赖的线粒体通路诱导EBV阳性胃癌细胞凋亡

目的:研究BARF1表达下调对EBV阳性胃癌细胞凋亡的影响,以及BARF1基因沉默介导细胞凋亡的分子机制。方法:siRNA和NCsiRNA分别转染NUGC3和SNU719细胞,运用Western blot测定细胞中BARF1、Bcl-2、Bax、细胞色素C、caspase 3和caspase 9的蛋白表达;RT-PCR测定BARF1、Bcl-2和Bax mRNA的表达;台盼蓝染色法测定细胞存活率;Annexin V-FITC/PI染色法和流式细胞仪测定细胞凋亡;细胞凋亡因子抗体芯片分析细胞中凋亡相关蛋白的表达;线粒体膜电位检测试剂盒测定线粒体膜电位;免疫共沉淀检测细胞中Apaf-1和caspase 9的相互作用。结果:与空白对照组和阴性对照组相比,BARF1基因沉默显著诱导NUGC3和SNU719细胞凋亡,而线粒体膜电位显著降低。BARF1沉默基因能促进促凋亡蛋白的表达并抑制抗凋亡蛋白的表达,Bcl-2/Bax比例显著降低;而caspase抑制剂能抑制由BARF1基因沉默介导的细胞凋亡。在siRNA转染的细胞中,caspase 3和caspase 9蛋白发生裂解,细胞色素C的浓度显著高于阴性对照组,Apaf-1蛋白与caspase 9蛋白在细胞质中能够发生相互作用。结论:BARF1基因沉默通过线粒体途径调节Bcl-2和Bax蛋白的表达进而诱导NUGC3和SNU719细胞凋亡,并呈caspase通路依赖关系。     

Survivin-2B诱导人乳腺癌细胞凋亡的初步研究*

 目的：探讨存活蛋白2B (survivin-2B)在诱导肿瘤细胞凋亡中的分子机制。方法：将survivin-2B基因插入真核表达载体pCDNA3.1,得到重组载体pCDNA3.1-survivin-2B。将空载体pCDNA3.1及重组载体pCDNA3.1-survivin-2B分别转染人乳腺癌细胞株MCF7,转染后48 h,用annexin V/7-AAD染色法分析细胞凋亡情况,利用碘化丙啶染色法分析转染对细胞周期的影响;同时提取总RNA并反转录成cDNA,进行多重聚合酶链反应。利用GeXP多基因表达分析系统检测21个与肿瘤相关基因的表达情况。结果：过表达survivin-2B基因导致MCF7细胞凋亡与细胞周期阻滞,并引起8个基因表达上调,2个基因表达下调。变化最大的为醛脱氢酶4家族成员A1（ALDH4A1）,表达下降48%;变化最小的为胞质分裂调控蛋白1（PRC1）,上调1.08倍。结论：Survivin-2B能够诱导细胞凋亡与细胞周期G2/M期阻滞,并引起部分肿瘤相关基因的表达变化。     

二甲双胍对人肝癌细胞Bel-7402增殖与凋亡的影响

目的:研究二甲双胍抑制人肝癌细胞Bel-7402增殖及调亡的影响,并初步探讨其机制。方法:体外培养人肝癌细胞株Bel-7402,以不同浓度的二甲双胍(10mM、20mM、40mM)干预细胞48h。采用MTT法检测药物对细胞增殖的影响;采用Hoechst 33258荧光染色法和流式细胞术检测细胞凋亡;Real-time PCR检测caspase-3、bax和bcl-2的mRNA水平表达。结果:与对照组相比,不同浓度的二甲双胍作用于Bel-7402细胞48h后,MTT结果显示对细胞增殖有明显抑制作用(P0.05);Hoechst 33258荧光染色法显示凋亡细胞明显增多;流式细胞术分析显示二甲双胍可明显诱导Bel-7402细胞凋亡,晚期凋亡率分别为3.76%,8.96%和18.67%;caspase-3、bax和bcl-2的mRNA表达水平均上调(P0.05);以上结果均具有浓度依赖性。结论:二甲双胍能抑制Bel-7402细胞增殖并诱导其凋亡,其机制可能与caspase-3、bax和bcl-2的mRNA表达水平改变有关。     

羟基喜树碱通过激活NF-κB诱导人乳腺癌Bcap-37细胞凋亡

 目的: 探讨NF-κB信号转导途径在羟基喜树碱诱导人乳腺癌Bcap-37细胞凋亡中所起的作用。方法: 突变IκB-α-pcDNA3.1(-)质粒转染Bcap-37细胞，并用G418进行阳性克隆选择；采用MTT法测定细胞的增殖活力，琼脂糖凝胶电泳观察细胞凋亡，流式细胞仪进行细胞周期分析，Western blotting法和DIG-EMSA研究细胞凋亡途径。结果: 羟基喜树碱对Bcap-37有明显的生长抑制作用，琼脂糖凝胶电泳检测到典型的DNA梯状条带；羟基喜树碱可通过降解IκBα 蛋白进而激活NF-κB，并使NF-κB发生核移位；羟基喜树碱没有激活突变IκB-α-pcDNA3.1(-)质粒转染细胞的NF-κB，并且转染细胞凋亡受到抑制。结论: 羟基喜树碱对Bcap-37有明显的生长抑制和诱导凋亡作用；NF-κB 信号转导途径在羟基喜树碱诱导人乳腺癌Bcap-37细胞凋亡中起着促进凋亡的作用。     

Paeonol induces apoptosis in human ovarian cancer cells

Paeonol is a broad-spectrum antitumor agent, which is widely used in the treatment of various tumors in Asia. However, the effect of paeonol on ovarian cancer remains unclear. The purpose of this study was to investigate the effect of paeonol on ovarian cancer cells and its possible mechanism. Results measured by MTT (methyl thiazoyltetrazolium) assay showed that cell viability was markedly reduced in a dosage-dependent manner, when treated with paeonol for 24 h. Flow cytometry and Hoechst staining results indicated that the rate of apoptosis in the paeonol pretreatment group was higher than the control group. After co-culture with paeonol, cleaved Caspase 3 protein levels increased while survivin protein levels decreased. In conclusion, our findings indicate that paeonol can induce apoptosis of ovarian cancer cells via activation of Caspase 3 and down-regulation of survivin, and therefore is potentially an effective chemotherapeutic agent for ovarian cancer.     

13-MTD通过激活MAPK途径和抑制Akt存活途径诱导乳腺癌MCF-7细胞凋亡

目的： 探讨侧链饱和脂肪酸13-甲基十四烷酸(13-MTD)诱导人乳腺癌MCF-7细胞凋亡的作用机制。方法：140 mg/L 13-MTD处理体外培养的人乳腺癌MCF-7细胞和人乳腺正常细胞,采用流式细胞仪检测技术观察13-MTD对人乳腺癌MCF-7细胞凋亡的影响,免疫印迹法检测13-MTD处理后细胞内c-Jun氨基末端激酶(JNK）,p38, Fas 相关死亡结构域蛋白(FADD)和丝氨酸/苏氨酸蛋白激酶(Akt)等蛋白磷酸化变化。结果：流式细胞仪实验结果显示13-MTD能有效地诱导人乳腺癌MCF-7细胞凋亡,但不引起正常人乳腺上皮细胞凋亡。免疫印迹检测显示经13-MTD处理后的人乳腺癌MCF-7细胞JNK和p38磷酸化蛋白明显增加,Akt磷酸化蛋白明显减少。结论：13-MTD是一个新的安全高效抗肿瘤药物,其作用机制可能是通过激活MAPK途径和抑制Akt存活途径来诱导肿瘤细胞凋亡。     

Immunohistochemical analysis of pro- and active-caspase 3 in laryngeal squamous cell carcinoma

Active caspase 3 is considered to be the main executioner caspase in apoptotic process. The mechanisms of apoptosis in laryngeal squamous cell carcinoma (LSCC) have been investigated by examining the expression profiles of pro-caspase 3 and active-caspase 3. The correlation between the two forms of caspase 3 and the p53 status was also determined. LSCCs (n=65) were studied using immunohistochemistry with antibodies to pro-caspase 3, active-caspase 3 and p53. The expression of pro-caspase 3 was absent or weak in 16 (24.6%), moderate in 21 (32.3%) and strong in 28 (43.1%) cases. Survival curves for different levels of pro-caspase 3 differed, but the differences were not statistically significant. An apoptotic index (AI) was determined by quantifying the active-caspase 3-positive cells. The AI ranged from 0.2% to 9.4% and did not differ among the different levels of pro-caspase 3 expression. Even in cases in which the expression of pro-caspase 3 was considered negative, caspase 3-positive apoptotic cells were found. The AIs were significantly higher in supraglottic tumours compared with glottic counterparts (P=0.008) and were higher in poorly differentiated tumours compared with well-differentiated and moderately differentiated LSCC (P=0.06). No correlation between AI and p53 expression was found, although pro-caspase 3 expression trended to be higher in the p53-positive group of LSCC. Our results suggest that the expression of pro-caspase 3, a key executioner caspase in apoptosis, is downregulated in a proportion of LSCC, but this is not associated with decreased apoptotic activity, measured by active-caspase 3 labelling.     

肝细胞黏附分子诱导肾癌786-0细胞系凋亡

目的 探讨肝细胞黏附分子(HEPACAM)基因转染肾癌细胞对其凋亡的影响.方法 将携带HEPACAM基因的重组质粒转染786-0细胞,RT-PCR鉴定HEPACAM基因在786-0中的表达;Hoechst染色结合荧光显微镜观察细胞形态改变;FCM检测细胞周期及细胞凋亡情况;Western印迹法检测BAX、BCL-2、C...     

槲皮素诱导MCF-7细胞凋亡及其与Fas/FasL通路的相关性研究

目的:了解槲皮素诱导人乳腺癌MCF-7细胞凋亡的作用及其与Fas/Fas L通路的相关性。方法:建立槲皮素诱导的MCF-7细胞凋亡模型。采用透射电镜观察细胞核形态变化,Annexin V-FITC/PI和JC-1荧光标记流式细胞术检测细胞凋亡率、线粒体膜电位(Δψm)及Fas L中和抗体对细胞凋亡的阻断作用。采用免疫荧光法和流式细胞术检测细胞Fas/Fas L的表达。流式细胞术观察p38 MAPK抑制剂SB203580对细胞Fas/Fas L表达的影响。Western blot检测p38 MAPK和p-p38 MAPK蛋白水平的变化。结果:80.0μmol/L槲皮素处理MCF-7细胞48h,透射电镜可见染色质浓缩及边缘化现象;处理24 h、48 h和72 h,Δψm分别下降17.4%、44.3%和68.9%,细胞凋亡率分别为(10.2±3.3)%、(28.9±7.5)%和(39.2±8.9)%。Fas L中和抗体预处理细胞后,24 h、48 h和72 h的细胞凋亡率则分别为(8.2±2.8)%、(19.2±5.3)%和(22.5±6.9)%,细胞凋亡阻断率分别为19.6%、33.6%和42.6%。Fas/Fas L表达率随处理时间增加而升高,SB203580可显著抑制细胞Fas/Fas L的表达率。p38 MAPK的蛋白水平变化不大,而p-p38 MAPK的蛋白水平在48 h和72 h显著增高。结论:槲皮素可上调MCF-7细胞的Fas/Fas L表达,并经膜Fas/Fas L途径诱导MCF-7细胞凋亡,p-p38 MAPK可能是上调Fas/Fas L表达的重要信号分子。     

Dichloromethane fraction of Melissa officinalis induces apoptosis by activation of intrinsic and extrinsic pathways in human leukemia cell lines

Abstract

Various components from medicinal plants are currently used in cancer therapy because of their apoptosis-inducing effects. The present study has aimed to investigate the growth inhibitory and apoptotic effects of Melissa officinalis on tumor cells. We prepared different fractions of this plant to investigate their inhibitory effects on two leukemia cell lines, Jurkat and K562. Fractions with the highest inhibitory effects were examined for induction of apoptosis by the annexin V/propidium iodide assay and cell cycle changes by flow cytometry. Real-time polymerase chain reaction evaluated the changes in expression of apoptosis-related genes. Among different fractions, dichloromethane and n-hexane dose-dependent showed the strongest inhibitory effects on both K562 and Jurkat cells. The dichloromethane fraction significantly induced apoptosis at concentration of 50?µg/ml on Jurkat (85.66?±?4.9%) and K562 cells (65.04?±?0.93%) at 24?h after treatment (p?<?0.002). According to cell cycle analysis, more than 70% of the cells accumulated in the sub-G1 phase when cultured in the presence of the dichloromethane fraction. This fraction up-regulated Fas and Bax mRNA expression as well as the Bax/Bcl-2 ratio according to cell type, showing its effect on the activation of both extrinsic and intrinsic pathways of apoptosis. The expression of apoptosis-related genes did not significantly change following treatment with the n-hexane fraction. These data indicated that the dichloromethane fraction of M. officinalis had the ability to induce apoptosis and change apoptosis-related gene expression in leukemia cells.     

Caprine arthritis-encephalitis virus induces apoptosis in infected cells in vitro through the intrinsic pathway

Caprine arthritis-encephalitis virus (CAEV) is a lentivirus that causes natural inflammatory disease in goats, with chronic lesions in several different organs. CAEV infection of in vitro cultured cells is accompanied by apoptosis, but the involvement of the intrinsic and extrinsic pathways has not previously been elucidated. We have studied the activation of caspases-3, -8 and -9 by fluorescent assays in various goat cells infected in vitro by CAEV, and the effects of transfected dominant negative variants of theses caspases, to show that CAEV-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of mitochondrial pathway.     

MDA-7/IL-24对乳腺癌MDA-MB-231细胞生长抑制和凋亡的影响

 摘要：目的 探讨IL-24基因转染乳腺癌MDA-MB-231细胞的增殖抑制和促凋亡作用。方法 利用脂质体将穿梭质粒pDC316-hIL-24-EGFP瞬时转染至乳腺癌MDA-MB-231细胞,通过RT-PCR检测转染后hIL-24基因mRNA的转录,Western blot检测转染后IL-24和Caspase-3蛋白的表达,MTT比色法测定细胞增殖的抑制, 流式细胞仪检测细胞的凋亡和细胞周期的变化,Hoechst 33258染色检测细胞的凋亡情况。结果IL-24基因可在MDA-MB-231细胞中成功转录及表达。IL-24可上调MDA-MB-231细胞中Caspase-3蛋白表达。IL-24基因的表达使得乳腺癌MDA-MB-231细胞出现增殖抑制。流式细胞仪检测显示MDA-MB-231细胞DNA 合成受到抑制,细胞周期主要抑制在G2/M期。Hoechst 33258染色显示IL-24基因转染后MDA-MB-231细胞出现凋亡。结论IL-24基因转染乳腺癌MDA-MB-231细胞后能够抑制细胞增殖并促进其凋亡,其机制之一可能是上调Caspase-3的表达。     

Norcantharidin induces human melanoma A375-S2 cell apoptosis through mitochondrial and caspase pathways

Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris. To examine the pathway of NCTD-induced A375-S2 cell death, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, photomicroscopical observation, DNA agarose gel electrophoresis, caspase activity assay and Western blot analysis were carried out. A375-S2 cells treated with NCTD exhibited several typical characteristics of apoptosis. The inhibitory effect of NCTD on human melanoma, A375-S2 cells, was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-9. The activities of caspase-3 and -9 were significantly increased after treatment with NCTD at different time. The expression of inhibitor of caspase-activated DNase was decreased in a time-dependent manner, simultaneously, the ratio of Bcl-2/Bax or Bcl-xL/Bax was decreased and the expression ratio of proteins could be reversed by caspase-3 inhibitor. The expression of cytochrome c in cytosol was increased after NCTD treatment and caspase- 3 inhibitor had no significant effect on the up-regulation of cytochrom c. These results suggest that NCTD induced A375-S2 cell apoptosis and the activation of caspase and mitochondrial pathway were involved in the process of NCTD-induced A375-S2 cell apoptosis.