色胺酮对乳腺癌MCF-7细胞凋亡的诱导作用

目的:探讨色胺酮(Tryptanthrin,Try)对人乳腺癌MCF-7细胞增殖和凋亡的影响。方法:利用MTT方法检测Try(1.56-100μmol/L)对细胞增殖的影响;透射电镜观察细胞的形态学改变;流式细胞术检测细胞周期、凋亡情况及线粒体跨膜电位等指标。结果:MTT结果显示,Try在12.5-100μmol/L浓度范围内能明显抑制MCF-7细胞的增殖,并具有时间和浓度依赖性;透射电镜下可见Try作用48h后,MCF-7细胞有典型的凋亡样改变。Annexin V-FITC与PI双染,流式细胞仪检测结果显示:50、100μmol/LTry作用后,细胞的凋亡情况明显,与对照组相比差异显著;且影响了MCF-7的细胞周期分布,将细胞阻滞于G1期,抑制其DNA的合成;并导致细胞线粒体跨膜电位下降。结论:色胺酮能明显抑制MCF-7细胞增殖并具有诱导细胞发生凋亡的作用。     

二烯丙基二硫诱导人乳腺癌MCF-7细胞凋亡及机制的研究

目的:研究二烯丙基二硫(DADS)诱导人乳腺癌MCF-7细胞凋亡及其分子机制。方法:AO／EB荧光染色法、流式细胞仪检测细胞凋亡率;Western blot法检测DADS对caspase-3剪切片断的影响,及对MAPKs通路相关蛋白,包括p-JNK、JNK表达的影响。结果:DADS对乳腺癌细胞株MCF-7生长具有明显的抑制作用,经AO／EB形态变化分析,可见明显的细胞凋亡特征;DADS处理MCF-7细胞6、12、24、48 h,流式细胞仪检测细胞的凋亡率分别为3．74％、9．22％、20.2％、42％,而对照组细胞的凋亡率仅为3．03％(P<0．05);不同浓度的DADS作用于MCF-7细胞24 h后,Western blot法检测发现caspase-3出现断裂片断,并随着浓度的增加断裂更明显。进一步研究发现,DADS处理MCF-7细胞后,JNK磷酸化水平明显升高。结论:DADS能诱导乳腺癌细胞株MCF-7细胞凋亡,JNK信号通路抑制可能是DADS诱导其调亡的分子机制之一。     

软骨多糖诱导MCF-7乳腺癌细胞凋亡的实验研究

研究软骨多糖诱导MCF-7乳腺癌细胞凋亡及其作用机理。方法:选用MCF-7人类乳腺癌细胞系体外培养,应用MTT法检测细胞生长抑制率,TUNEL法检测细胞凋亡率,HE染色法观察细胞形态学改变,流式细胞仪检测细胞周期的变化,免疫荧光方法检测BCL-2BAD及波形蛋白Vimentin的表达率。结果:软骨多糖对MCF-7细胞体外生长具有明显的抑制作用,且呈时间和浓度依赖性;软骨多糖可诱导MCF-7细胞发生凋亡并伴随有凋亡小体出现等形态学变化;软骨多糖促进BCL-2蛋白的表达水平下降,BAD表达水平上升,及Vimentin的降解。结论:软骨多糖能够在体外诱导MCF-7细胞凋亡,是一种新型的抗乳腺癌活性物质。     

灰树花多糖对乳腺癌细胞MCF-7的凋亡作用

采用MTT法测定不同给药浓度的灰树花多糖(PGF) (1、10、20、50、100和200 μg/mL)在24、48和72 h对乳腺癌细胞(MCF-7)增殖的抑制率,并采用Hoechst染色与流式细胞技术观察20、50和100 μg/mL PGF给药24 h后MCF-7的凋亡情况,同时采用Western blotting对20、50、100 μg/mL PGF给药24 h后MCF-7细胞中Bax、Bcl-2、Pro-Caspase-3以及Cleaved Caspase-3的蛋白表达水平进行检测。研究发现PGF给药24、48和72 h后对MCF-7的增殖均有显著的抑制作用。随着PGF给药浓度增加,MCF-7细胞核裂解增多,细胞凋亡数量增多。PGF 20、50和100 μg/mL给药对MCF-7细胞Bax、Bcl-2、Pro-Caspase-3以及Cleaved Caspase-3的蛋白表达水平可见显著性差异。     

SmacN7诱导乳腺癌细胞MDA-MB-157凋亡的作用及其机制

目的:探讨SmacN7对乳腺癌细胞MDA-MB-157凋亡的作用及机制。方法:将0-20μmol/L的Smac N7应用于乳腺癌细胞MDA-MB-157,用MTS法检测细胞增殖活性,流式细胞仪检测细胞凋亡、细胞周期,hoechst33342染色观察细胞核型变化,JC-1染色检测线粒体膜电位,LDH释放实验检测药物细胞毒性,qPCR检测各基因转录水平,并通过抑瘤实验证实该药抑制乳腺癌增殖的作用。结果:应用Smac N7后,乳腺癌细胞MDA-MB-157增殖抑制率和细胞凋亡率均增加(P<0.01),核型发生显著变化,细胞线粒体膜电位降低,LDH释放量增加,并上调TRAIL、DR4、DR5、p53、PARP-1、Bax、Bid、BAK、caspase-3、caspase-8、caspase-9基因的转录水平(P<0.01),下调Ras、PI3K、AKT、mTOR、Bcl2、Bcl-xL、MCL-1、Survivin、cIAP-1、cIAP-2基因的转录水平(P <0.01)。结论:SmacN7可通过TRAIL介导的死亡受体途径和线粒体介导的内源性凋亡途径诱导乳腺癌细胞MDA...     

SCE诱导MCF-7细胞凋亡及其相关基因表达的研究

目的观察鲨鱼软骨提取物(SCE)对MCF-7细胞凋亡的诱导作用,并进一步研究在此过程中BCL-2和Caspase-3的表达.方法将不同浓度的SCE作用于MCF-7细胞,观察其作用的时间效应及剂量效应;在光镜和电镜下观察其形态变化;用流式细胞术分析细胞DNA含量的改变;用免疫组织化学法观察在此过程中BCL-2和Caspase-3的表达.结果 SCE 能抑制MCF-7细胞生长,在一定剂量和时间范围内引起细胞凋亡,并显示剂量和时间效应, 在此过程中BCL-2蛋白表达减弱.结论 SCE 能明显抑制MCF-7细胞体外生长,并可诱导MCF-7细胞凋亡,其作用机制可能与下调Bcl-2蛋白的表达水平有关.     

miR-200c抑制人乳腺癌MCF-7细胞糖代谢

为探讨miR-200c对人乳腺癌MCF-7 (Michigan cancer foundation-7)细胞糖代谢水平的影响,本研究使用miR-200c mimics转染MCF-7细胞,通过定量即时聚合酶链锁反应(quantitative real time polymerase chain reaction, qRT-PCR)检测各组细胞miR-200c的表达水平;利用细胞计数盒(cell counting kit-8, CCK-8)检测miR-200c mimics转染对MCF-7细胞增殖的影响;通过葡萄糖试剂盒检测各组细胞葡萄糖的消耗,乳酸试剂盒检测各组细胞乳酸的释放量;通过蛋白质印迹法(Western blotting)检测各组细胞糖代谢相关酶己糖激酶(hexokinase 2, HK2)以及肌肉丙酮酸激酶同工酶2 (pyruvate kinase isozyme type M2, PKM2)蛋白表达水平。与miR-NC组相比,miR-200c mimics转染明显上调MCF-7细胞miR-200c m RNA水平;且miR-NC组和MCF-7组miR-200c mRNA水平没有明显差异;细胞计数盒(cell counting kit-8, CCK-8)检测结果显示,与miR-NC组和MCF-7组相比,miR-200c mimics转染不仅明显降低乳腺癌MCF-7细胞的细胞活力,而且显著减少乳腺癌MCF-7细胞葡萄糖的消耗;此外,Western blotting结果显示,miR-200c显著下调MCF-7细胞糖代谢相关酶HK2和PKM2的表达。综上结果表明,miR-200c会抑制人乳腺癌MCF-7细胞糖代谢。本研究成果为乳腺癌防治提供一定的参考价值。     

HAX-1抑制过氧化氢诱发的乳腺癌细胞MCF-7凋亡

目的：应用RNA干扰（RNAi）技术特异地干扰HAX-1在乳腺癌细胞MCF-7中的表达,研究其在过氧化氢诱导的细胞凋亡中的作用。方法：应用载体pSR-GFP／Neo构建针对HAX-1基因的小干扰RNA（siRNA）表达质粒;转染MCF-7细胞,G418筛选稳定细胞系,Western blot鉴定筛选的细胞克隆;用流式细胞仪检测筛选的细胞系在过氧化氢条件下的凋亡率。结果：电泳和测序证实合成的siRNA序列正确并准确克隆到pSR-GFP／Neo载体中;Western blot证实获得MCF-7／HAX-1 siRNA稳定细胞株,其HAX-1蛋白水平降低95％左右;用1mmol／L过氧化氢处理获得的稳定细胞株8h,细胞凋亡率明显高于对照组。结论：HAX-1能够保护乳腺癌细胞MCF-7免于过氧化氢诱导的细胞凋亡。     

川芎嗪逆转人乳腺癌MCF-7/ADM细胞对阿霉素的耐药性

目的 探讨川芎嗪（tetramethylpyrazine,TMP）逆转人乳腺癌MCF-7/ADM细胞对阿霉素（ADM）的耐药性.方法 MTT法测定细胞的药敏性,荧光分光光度法检测细胞内阿霉素浓度的变化,流式细胞术检测耐药细胞凋亡百分率的变化.结果 非细胞毒性剂量（320 mg/L）及低毒剂量（1250 mg/L）川芎嗪均能显著降低MCF-7/ADM的IC50（P＜0.01）,逆转倍数分别为2.13倍和2.82倍;均能显著增加耐药细胞内ADM的浓度（P＜0.01）.320 mg/L川芎嗪能显著增加耐药细胞的凋亡百分率（P＜0.01）.结论 川芎嗪具有部分逆转人乳腺癌MCF-7/ADM细胞对阿霉素的耐药性,其逆转机制与增加细胞内ADM浓度有关.     

两株耐紫杉醇人乳腺癌细胞MCF-7的比较

建立稳定的肿瘤多药耐药(MDR)细胞株是肿瘤MDR机制研究的基础,以MCF-7细胞林为亲本细胞株,采用低浓度加量持续诱导和高浓度短期作用分别建立MCF-7/Taxola和MCF-7/Taxolb细胞模型,并对其耐药谱、动力学周期变化、表形变化、细胞侧群分布、药物蓄积等生物学特性比较评价.结果表明,MCF-7/Taxola和MCF-7/Taxolb细胞的紫杉醇(paclitaxel/Taxol)半数抑制浓度(IC50)分别是亲代MCF-7细胞的525倍和330倍,并且都对多种化疗药物交叉耐药;MCF-7/Taxola细胞S期细胞显著增加,G,期细胞减少;MCF-7/Taxolb细胞各个期变化不大;MCF-7/Taxola细胞P-糖蛋白(P-gP)、肺耐药相关蛋白(LRP)和还原型谷胱甘肽-S转移酶(GSTπ)的表达水平较亲代有显著增加,而MCF-7/Taxolb细胞GSTπ的表达水平较亲代也有显著增加,另外,拓扑异构酶Ⅱ(ToPoⅡ)在两株耐药细胞中表达都明显下降,而两株细胞雌激素受体(ER)、孕激素受体(PR)阳性都表达丢失;光镜下耐药细胞MCF-7/Taxola明显变大并且形态不规则而MCF-7/Taxolb变化不大;电镜下MCF-7/Taxolb表面纤绒毛成小球状隆起和絮状,而MCF-7/Taxolb表面成絮状:MCF-7/Taxola撤药10天后细胞中有紫杉醇蓄积,而MCF-7/Taxolb中没有紫杉醇蓄积.两个模型都具有MDR的基本生物学特性,可用于肿瘤MDR机制的研究,通过两种耐药细胞的比较,推测MCF-7/Taxolb细胞是MCF-7/Taxola细胞的一个亚群.     

KDR靶向RNA干扰对MCF一7细胞凋亡的影响

目的：研究KDR靶向RNA干扰对MCF-7细胞凋亡的影响,探讨其可能的机制。方法：采用阳离子脂质体Lipofecta．mine2000TM作为转染试剂将人KDR基因的siRNA转染人类乳腺细胞株MCF-7,诱RNAi,采用Hoechst33258染色和半定量RT—PCR检测Caspase-3、survivin的mRNA表达及细胞凋亡变化;比色法检测Caspase．3的活性;利用免疫组织化学方法检测survivin的表达,并用图像分析仪分析蛋白表达强度。结果：靶向KDR的siRNA转染MCF-7后,Caspase-3的mRNA表达上调,survivin基因mRNA及蛋白表达水平下调（P〈0．05）。结论：KDRsiRNA通过减少乳腺癌细胞survivin的表达,增加Caspase．3表达来促进肿瘤细胞凋亡,发挥其抗肿瘤作用。     

Oligonucleosomal DNA Fragmentation in MCF-7 Cells Undergoing Palmitate-Induced Apoptosis

Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.     

Quercetin Suppresses Twist to Induce Apoptosis in MCF-7 Breast Cancer Cells

Quercetin is a dietary flavonoid which exerts anti-oxidant, anti-inflammatory and anti-cancer properties. In this study, we investigated the anti-proliferative effect of quercetin in two breast cancer cell lines (MCF-7 and MDA-MB-231), which differed in hormone receptor. IC₅₀ value (37μM) of quercetin showed significant cytotoxicity in MCF-7 cells, which was not observed in MDA-MB-231 cells even at 100μM of quercetin treatment. To study the response of cancer cells to quercetin, with respect to different hormone receptors, both the cell lines were treated with a fixed concentration (40μM) of quercetin. MCF-7 cells on quercetin treatment showed more apoptotic cells with G1 phase arrest. In addition, quercetin effectively suppressed the expression of CyclinD1, p21, Twist and phospho p38MAPK, which was not observed in MDA-MB-231 cells. To analyse the molecular mechanism of quercetin in exerting an apoptotic effect in MCF-7 cells, Twist was over-expressed and the molecular changes were observed after quercetin administration. Quercetin effectively regulated the expression of Twist, in turn p16 and p21 which induced apoptosis in MCF-7 cells. In conclusion, quercetin induces apoptosis in breast cancer cells through suppression of Twist via p38MAPK pathway.     

Docosahexaenoic Acid Induces Apoptosis in MCF-7 Cells In Vitro and In Vivo via Reactive Oxygen Species Formation and Caspase 8 Activation

Background

The present study sought to further investigate the in vitro and in vivo anticancer effects of a representative omega-3 fatty acid, docosahexaenoic acid (DHA), with a focus on assessing the induction of oxidative stress and apoptosis as an important mechanism for its anticancer actions.

Methodology/Principal Findings

In vitro studies showed that DHA strongly reduces the viability and DNA synthesis of MCF-7 human breast cancer cells in culture, and also promotes cell death via apoptosis. Mechanistically, accumulation of reactive oxygen species and activation of caspase 8 contribute critically to the induction of apoptotic cell death. Co-presence of antioxidants or selective inhibition or knockdown of caspase 8 each effectively abrogates the cytotoxic effect of DHA. Using athymic nude mice as an in vivo model, we found that feeding animals the 5% fish oil-supplemented diet for 6 weeks significantly reduces the growth of MCF-7 human breast cancer cells in vivo through inhibition of cancer cell proliferation as well as promotion of cell death. Using 3-nitrotyrosine as a parameter, we confirmed that the fish oil-supplemented diet significantly increases oxidative stress in tumor cells in vivo. Analysis of fatty acid content in plasma and tissues showed that feeding animals a 5% fish oil diet increases the levels of DHA and eicosapentaenoic acid in both normal and tumorous mammary tissues by 329% and 300%, respectively.

Conclusions/Significance

DHA can strongly induce apoptosis in human MCF-7 breast cancer cells both in vitro and in vivo. The induction of apoptosis in these cells is selectively mediated via caspase 8 activation. These observations call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of human breast cancer.     

毛蕊异黄酮抑制SIRT1促乳腺癌细胞MCF-7凋亡

目的:探讨毛蕊异黄酮促乳腺癌细胞MCF-7凋亡的机制。方法:MTT检测低、中、高(10μM,50μM,100μM)剂量的毛蕊异黄酮对细胞活力的影响;Tunel检测毛蕊异黄酮对细胞凋亡的影响;Western blot检测SIRT1,p53和cleaved caspase-3的蛋白表达;Real-time PCR检测caspase-3 mRNA的表达。结果:毛蕊异黄酮能够剂量依赖性地降低细胞活力,100μM剂量组的毛蕊异黄酮显著地促进肿瘤细胞凋亡并降低SIRT1,增加p53和cleaved caspase-3的蛋白表达。SIRT1抑制剂烟酰胺(Nicotinamide,NAM,300μM)组与毛蕊异黄酮处理组相比显著地抑制SIRT1的蛋白表达,p53和cleaved caspase-3蛋白表达水平进一步增加;SRT1720(SIRT1特异性激动剂)与毛蕊异黄酮共孵育组逆转SIRT1蛋白表达,降低p53和cleaved caspase-3的蛋白水平。结论:毛蕊异黄酮促进肿瘤细胞MCF-7的凋亡,部分可能是通过降低SIRT1的表达水平,从而增加p53和cleaved caspase-3的蛋白表达促进细胞凋亡。     

SNX-2112, a Novel Hsp90 Inhibitor,Induces G2/M Cell Cycle Arrest and Apoptosis in MCF-7 Cells

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor with anticancer properties currently in clinical trials. This study investigated the effects of SNX-2112 on inhibition of cell growth, the cell cycle, and apoptosis in MCF-7 human breast cancer cells, in addition to the various molecular mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis suggest that SNX-2112 inhibits cell growth in a time- and dose-dependent manner more potently than 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), a traditional Hsp90 inhibitor, probably as a result of cell-cycle arrest at the G2/M phase and the induction of apoptosis. Downregulation of Bcl-2 and Bcl-xL, upregulation of Bax, cleavage of caspase-9 and poly (ADP-ribose) polymerase (PARP), and degradation of the breast cancer-related Hsp90 client proteins human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, and nuclear factor kappa-B kinase (IKK) were observed in SNX-2112 treated cells by Western blot assay. These findings suggest that the molecular mechanisms of cell-growth inhibition by SNX-2112 involve activation of the mitochondrial apoptotic pathway and the degradation of breast cancer-related proteins.     

Metformin Induces Apoptosis and Cell Cycle Arrest Mediated by Oxidative Stress,AMPK and FOXO3a in MCF-7 Breast Cancer Cells

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration- dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G₀-G₁ phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H₂O₂ increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.     

全反式维甲酸联合辛二酰苯胺异羟肟酸诱导MCF-7 细胞凋亡

目的:探讨全反式维甲酸(ATRA)和辛二酰苯胺异羟肟酸(SAHA)单独及联合应用对乳腺癌MCF-7细胞凋亡的影响。方法:培养人乳腺癌MCF-7细胞,待细胞进入对数生长期,加入一定浓度的ATRA和(或)SAHA,采用流式细胞术检测细胞的凋亡情况。结果:ATRA和SAHA单药及联合应用早期凋亡率均高于对照组,但24 h时测得联合组早期凋亡率低于单药组,48 h、72 h联合组早期凋亡率高于SAHA组却低于ATRA组,差异均有统计学意义(P0.05);联合组死亡细胞和晚期凋亡细胞率之和高于单药组及对照组,差异有统计学意义(P0.05)。结论:ATRA和SAHA单独或联合作用于乳腺癌MCF-7细胞均可促进细胞凋亡,两药联合应用时诱导细胞凋亡作用更强。     

Oxygen Toxicity Induces Apoptosis in Neuronal Cells

1. A high oxygen atmosphere induced apoptosis in cultured neuronal cells including PC12 cells and rat embryonic cortical, hippocampal, and basal forebrain neurons associated with DNA fragmentation and nuclear condensation.2. The sensitivity of CNS neurons to a high-oxygen atmosphere was the following order; cortex > basal forebrain > hippocampus.3. Cycloheximide and actinomycin-D inhibited the apoptosis, indicating that it depends on new macromolecular synthesis. In contrast, cultured postnatal CNS neurons were resistant to oxidative stress.4. Neurotrophic factors such as nerve growth factor (NGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) blocked the apoptosis induced by a high-oxygen atmosphere.