Distinctive alterations of invasiveness, drug resistance and cell–cell organization in 3D-cultures of MCF-7, a human breast cancer cell line, and its multidrug resistant variant

Growth of human tumor cells as three-dimensional (3D) multicellular spheroids modifies their invasive properties. Here we study the differences in the biological features of MCF-7, a human breast cancer cell line, and its multidrug resistant variant (MDR-MCF-7) cultured as spheroids or as monolayers. Three-dimensional culture decreased the proliferative rate of both cell lines, reduced the drug sensitivity of MCF-7 cells and did not affect the resistance of MDR-MCF-7 cells. Transmission electron microscopic studies and intercellular junctions labeling showed that MCF-7 spheroids had a junctional system involving E-cadherin, tight-junctions and desmosomes. In MDR-MCF-7 cell spheroids, cell cohesion was mostly due to membrane interdigitations. MDR-MCF-7 cells, but not their parental counterpart, displayed a higher invasive potential when cultured as spheroids, as shown in the Boyden chamber assay. 3D-induced invasiveness was correlated with serine protease and plasminogen activator (PA) secretion. MCF-7 cells did not show any tendency to invade, whatever the mode of culture. These results show that 3D-cultures as spheroids distinctively altered structural features of parental and MDR-MCF-7 cells. In MCF-7 cells, 3D-culture increased cell–cell contacts and drug resistance; in MDR-MCF-7 cells, it induced invasive properties. This revised version was published online in July 2006 with corrections to the Cover Date.     

IGFBP7对人乳腺癌MCF-7细胞增殖的影响及其机制

目的: 探究胰岛素样生长因子结合蛋白7(IGFBP7)对人乳腺癌MCF-7细胞增殖的影响及其分子生物学机制。方法: 将质粒pCMV6-IGFBP7转染MCF-7细胞,构建稳定表达IGFBP7的MCF-7细胞系;采用Western blotting检测IGFBP7在MCF-7细胞稳定转染子的表达;采用软琼脂培养克隆形成实验检测IGFBP7对MCF-7细胞克隆形成能力的影响;采用流式细胞术检测IGFBP7对MCF-7细胞周期的影响;采用Western blotting检测IGFBP7对MCF-7细胞细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、细胞周期素D1(cyclin D1)、细胞周期素依赖性激酶4(CDK4)、cyclin E、CDK2、p21^CIP1/WAF1、p27^KIP1、p53、视网膜母细胞瘤蛋白(Rb)和p-Rb蛋白含量的影响。结果: (1)只有稳定转染质粒pCMV6-IGFBP7的MCF-7细胞表达IGFBP7。(2)IGFBP7能够显著降低MCF-7细胞的克隆形成率(P<0.01),阻止细胞从G₁期进入S 期,使其停滞于G₁期(P<0.01)。(3)IGFBP7能够显著抑制ERK1/2的磷酸化(P<0.01)。(4)IGFBP7能够下调cyclin D1和cyclin E蛋白表达(P<0.01),上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达(P<0.01),抑制Rb的磷酸化(P<0.01)。(5)MEK1/2阻断剂PD98059可部分模拟IGFBP7的肿瘤抑制效应。结论: (1) IGFBP7可通过下调cyclin D1和cyclin E蛋白表达,上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达,以及抑制Rb磷酸化发挥抗肿瘤作用;(2) IGFBP7对cyclin D1和p27^KIP1的调节可能与其抑制ERK1/2信号通路有关。     

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。     

细胞内GSH耗竭逆转人乳腺癌耐药细胞株MCF-7/ADM对阿霉素的耐药性

目的：探讨癌细胞内GSH浓度在肿瘤细胞耐药机制中的作用。方法：以马来酸二乙酯（DEM）处理人乳腺癌耐药细胞株MCF-7／ADM3小时,以消耗其细胞内还原型谷胱甘肽（GSH）,MTT法检测DEM处理前后MCF-7／ADM细胞对ADM耐药程度的变化,并采用荧光分光光度法同步检测其细胞内GSH的消耗程度,分析细胞内GSH浓度的变化与MCF-7／ADM细胞对ADM耐药程度变化间的相关关系。结果：0．1umol／L DEM使细胞内GSH浓度减少37．4％（P〈0．01）;ADM亦使细胞内GSH含量呈ADM浓度依赖性的下降;DEM与ADM合用时,GSH浓度显著下降,其下降程度大于DEM和ADM单独应用之和。随着细胞内GSH数量的减少．MCF-7／ADM细胞对ADM的敏感性增强、耐药性下降（P〈0.01）。结论：DEM耗竭细胞内GSH的作用与ADM有协同效应,耗蝎细胞内GSH可部分逆转人乳腺癌MCF-7／ADM细胞对阿霉素的耐药性。     

Evidence of a genomic biomarker in normal human epithelial mammary cell line, MCF-10A, that is absent in the human breast cancer cell line, MCF-7

This study investigated the use of DNA amplification fingerprinting (DAF) to identify biomarkers useful in the elucidating genetic factors that lead to carcinogenesis. The DNA amplification fingerprinting (DAF) technique was used to generate fingerprint profiles of a normal human mammary epithelial cell line (MCF-10A) and a human breast cancer cell line (MCF-7). When compared with one another, a polymorphic biomarker gene (262 base pairs (bps)) was identified in MCF-10A but was not present in MCF-7. This gene was cloned from the genomic DNA of the MCF-10A cell line, and subjected to Genbank database analysis. The analysis of the nucleotide sequence polymorphic marker (Genbank account: AC079630) shows that this biomarker has 100% homology with the nucleotide sequence of human chromosome 12 BAC RP11-476D10 (bps 19612-19353). The nucleotide sequence was used for possible protein translation product and the result obtained indicated that the gene codes for hypothetical protein XF2620. In order to evaluate the effects that the 262 bps biomarker would have on the morphology of MCF-7 cells, it was transfected into MCF-7 cells. There were observable changes in the morphology of the transfected cells. These changes included an increase in cell elongation and a decrease in cell aggregation.     

表没食子儿茶素没食子酸酯对人乳腺癌MCF-7细胞及其血管内皮细胞生长因子-C表达的影响

目的:探讨体外应用表没食子儿茶素没食子酸酯(EGCG)对人乳腺癌MCF-7细胞增殖、侵袭及其对血管内皮细胞生长因子-C (VEGF-C)表达的影响.方法:体外培养MCF-7细胞,设立对照组及EGCG实验组,应用MTT法及Transwell侵袭实验观察不同浓度EGCG对MCF-7细胞增殖及侵袭的影响;应用免疫细胞化学显色...     

阿霉素诱导人乳腺癌细胞系耐药后的细胞行为变化

目的研究和完善乳腺癌细胞多药耐药机制及多药耐药后肿瘤细胞的性质、行为改变。方法分别对野生组(MCF-7)、阿霉素诱导的耐药组(MCF-7/ADM)、撤药60d组(MCF-7/撤药60d)进行细胞学分析,观察细胞增殖速度及群体增殖时间,SP免疫组化法检测细胞表型变化。结果MCF-7/ADM与MCF-7细胞增殖速度无明显差异,撤药组随着撤药时间的延长,细胞的增殖速度加快,群体倍增时间缩短;MCF-7/ADM、MCF-7/撤药60d细胞的分化程度低于MCF-7组;MCF-7/ADM的耐药相关标记蛋白Pgp、LRP、GST-π、HER-2表达较MCF-7明显增加,而雌激素受体(ER)、TOPO-Ⅱ转为阴性表达,孕激素受体(PR)随撤药时间的延长表达逐步减低。结论应用阿霉素诱导的MCF-7具有明显的耐药细胞性状,耐药细胞具有去分化的能力;耐药细胞的遗传和生化特性发生了变化,可用于肿瘤多药耐药机制的研究。     

载有槲皮素的PEG-PE胶束对乳腺癌细胞耐药性的逆转效应

目的探索载有槲皮素的PEG-PE聚合物胶束(M-Q)能否在体外逆转耐阿霉素人乳腺癌细胞(MCF-7 ADRr)的耐药性。方法将PEG-PE聚合物与槲皮素混合制备脂膜,加0.9%氯化钠注射液形成胶束,用动态光散射粒径仪表征其尺寸分布,并将其和阿霉素与MCF-7 ADRr细胞共孵育72 h,用MTS方法检测对肿瘤细胞的杀伤率。结果PEG-PE聚合物胶束能有效运载槲皮素,其包封率高达74%以上,平均粒径为11.11 nm左右,与游离槲皮素相比,M-Q能在体外更大程度地逆转MCF-7 ADRr细胞对于阿霉素的耐药性(P0.05),且PEG-PE胶束自身对靶细胞无明显杀伤作用。结论载有槲皮素的PEG-PE胶束在逆转肿瘤耐药性方面具有应用潜能。     

cyclin G2在乳腺病变组织中表达及其对MCF-7细胞的作用

目的 研究细胞周期蛋白G2(cyclin G2)在乳腺病变中的表达及意义,探讨cyclin G2对乳腺癌MCF-7细胞凋亡的影响.方法 用免疫组化SP法检测cyclin G2在人乳腺不同病变中的蛋白表达,并进行显微图像分析;利用阳离子介导的脂质体法转染pDsRed2-cyclin G2融合基因至人乳腺癌细胞系MCF-7细胞中,流式细胞术检测MCF-7细胞的凋亡率.结果 (1)cyclin G2在乳腺增生症中的阳性表达率及表达强度与正常乳腺组织相当,而在导管原位癌(ductal carcinoma in situ,DCIS)以及浸润性导管癌(invasive ductal carcinoma,IDC)中表达逐渐下降,差异有统计学意义(P<0.05).(2)转染了重组质粒pDsRed2-cyclin G2的MCF-7细胞凋亡率明显增高(P<0.01).结论 cyclin G2蛋白表达随着乳腺疾病恶性程度增加而降低,外源性转染cyclin G2至MCF-7细胞中可促进其凋亡,抑制细胞增殖.     

人喉癌长春新碱耐药细胞株的建立及其生物学特性

目的:建立人喉癌长春新碱多药耐药细胞系。方法:以药物浓度梯度递增法诱导筛选人喉癌Hep-2的多药耐药细胞株Hep-2/v,比较两组细胞的形态和倍增时间;MTT法确定细胞的IC50及其耐药指数;流式细胞仪检测两组细胞的细胞周期分布以及细胞内的罗丹明聚集情况;实时定量RT-PCR法检测MDR1基因的mRNA表达,Western blot法检测相应的蛋白表达情况。结果:建成的Hep-2/v耐药细胞株,其耐药性能稳定,耐药指数为45,并与顺铂及5-氟尿嘧啶有不同程度的交叉耐药性;Hep-2/v的体外群体细胞倍增时间较亲代细胞延长13.58小时;细胞周期分析结果显示其G0/G1期细胞升高,而S期细胞则明显降低(P0.05);罗丹明染色显示,Hep-2细胞内的罗丹明较Hep-2/v明显升高(P0.01);Hep-2/v细胞MDR1表达在基因及蛋白水平明显高于Hep-2细胞(P0.01)。结论:Hep-2/v细胞株具有明确及稳定的多药耐药性,是研究多药耐药机制及筛选逆转剂的良好模型。     

蛴螬提取物对MCF-7人乳腺癌细胞株凋亡的影响

目的：研究蛴螬提取物对MCF-7人乳腺癌细胞株凋亡通路的影响，为天然药物蛴螬的开发提供实验和理论依据。方法:应用MTT法检测蛴螬提取物对MCF-7人乳腺癌细胞株的抗增殖作用及细胞毒活性；通过倒置显微镜、HE染色、吖碇橙（AO）/溴化乙啶（EB）荧光染色方法观察肿瘤细胞的形态学改变；应用流式细胞仪检测细胞凋亡率的变化；应用免疫细胞化学技术（S-P法）检测用药前后凋亡通路中Bcl-2、Fas、caspase-9、caspase-3的表达改变，探讨蛴螬提取物对凋亡通路的影响。结果:(1)用MTT法检测结果表明，蛴螬提取物在体外对MCF-7人乳腺癌细胞株有明显的抑制增殖作用，实验组与对照组相比其生长抑制率有显著差异（P<0.01），而且这种抑制作用呈现浓度和时间的依赖性；(2)倒置显微镜观察表明，实验组可见胞核固缩、碎裂、凋亡小体形成等凋亡形态学变化；(3)HE染色表明实验组胞核浓缩呈蓝黑色、胞浆呈淡红色、核染色质浓缩、呈碎块状，有凋亡小体形成；(4)经AO／EB荧光染色观察结果表明，实验组出现凋亡细胞；(5)流式细胞仪结果表明实验组凋亡率明显增加，呈时间依赖性；(6)免疫细胞化学染色结果表明，实验组 Bcl-2表达下调，Fas、caspase-3、caspase-9表达均上调，与对照组比差异显著（P<0.01）。结论:①蛴螬提取物在体外显著抑制MCF-7人乳腺癌细胞株增殖；②蛴螬提取物对MCF-7人乳腺癌细胞株凋亡通路的影响机制可能是通过下调Bcl-2，上调Fas、caspase-3 、caspase-9的蛋白表达而起作用，是经由细胞凋亡的线粒体途径和死亡受体途径来完成凋亡的启动和执行。     

Increasing the cytotoxicity of doxorubicin in breast cancer MCF-7 cells with multidrug resistance using a mesoporous silica nanoparticle drug delivery system

Resistance to cytotoxic chemotherapy is the main cause of therapeutic failure and death in women with breast cancer. Overexpression of various members of the superfamily of adenosine triphosphate binding cassette (ABC)-transporters has been shown to be associated with multidrug resistance (MDR) phenotype in breast cancer cells. MDR1 protein promotes the intracellular efflux of drugs. A novel approach to address cancer drug resistance is to take advantage of the ability of nanocarriers to sidestep drug resistance mechanisms by endosomal delivery of chemotherapeutic agents. Doxorubicin (DOX) is an anthracycline antibiotic commonly used in breast cancer chemotherapy and a substrate for ABC-mediated drug efflux. In the present study, we developed breast cancer MCF-7 cells with overexpression of MDR1 and designed mesoporous silica nanoparticles (MSNs) which were used as a drug delivery system. We tested the efficacy of DOX in the breast cancer cell line MCF-7/MDR1 and in a MCF-7/MDR1 xenograft nude mouse model using the MSNs drug delivery system. Our data show that drug resistance in the human breast cancer cell line MCF-7/MDR1 can be overcome by treatment with DOX encapsulated within mesoporous silica nanoparticles.     

乳腺癌细胞株MCF-7/ADM中肿瘤干细胞的研究

目的： 通过对阿霉素耐药乳腺癌细胞系MCF-7/ADM中乳腺癌干细胞（breast cancer stem cells,BCSCs）成分分析，观察化疗耐药处理的MCF-7乳腺癌细胞株是否可高效富集乳腺癌干细胞，为研究乳腺癌的发病机制提供思路。方法: MTT法分别测定阿霉素耐药乳腺癌细胞系MCF-7/ADM及其亲本细胞株MCF-7对阿霉素的IC50，计算其耐药倍数。通过细胞侧群（side population,SP）分析、球囊培养、流式细胞仪检测MCF-7/ADM及MCF-7中CD+44CD-24细胞比例三方面鉴定MCF-7/ADM和MCF-7中乳腺癌干细胞比例。结果: 阿霉素耐药乳腺癌细胞系MCF-7/ADM相对于MCF-7对阿霉素的耐药倍数为37.1；MCF-7/ADM中SP细胞比例为（9.60±0.66）%，MCF-7细胞的SP细胞比例为（0.39±0.11）%；两者球囊形成率分别为 （10.27±0.64）%和（1.03±0.15）%；两者的CD+44CD-24细胞比例分别为（64.87±3.87）%和（3.70±0.53）%,差异显著（P<0.05）。结论: 阿霉素耐药乳腺癌细胞系MCF-7/ADM中乳腺癌干细胞比例明显高于MCF-7细胞。化疗耐药处理的MCF-7乳腺癌细胞株可高效富集乳腺癌干细胞，这对于乳腺癌发病机制的研究具有重要意义。     

HER2介导乳腺癌细胞多药耐药的作用及机制

目的筛选HER2高表达乳腺癌细胞化疗耐受的药物种类,探讨HER2介导的乳腺癌多药耐药的机制。方法构建HER2稳定高表达的乳腺癌细胞MCF-7/HER2模型;MTT法检测该细胞对多种临床常用的抗乳腺癌药物的敏感性;Hochest33258染色观察药物诱导的MCF-7/HER2的凋亡率,并采用聚合酶链式反应(RT-PCR)检测细胞中bcl-2和survivin基因的mRNA表达。结果MCF-7/HER2细胞对Taxol,MMC,5-FU,VP-16及TSPA的耐药指数分别为对照细胞的74,22,2.5,3.5和2.8倍,出现了明显的药物抗性(P<0.05);而对CDDP,ADM,VBL,VCR,NBV和MTX等的耐药指数与对照组相比,差异无统计学意义(P>0.05);由Taxol,MMC,5-FU,VP-16诱导的MCF-7/HER2细胞凋亡率明显低于对照细胞;MCF-7/HER2细胞survivin基因表达明显高于对照组,而bcl-2基因表达与对照组比较,差异无统计学意义。结论HER2可介导乳腺癌细胞对Taxol,MMC,5-FU,VP-16和TSPA等的多药抗性,这种多药抗性的产生可能与HER2上调survivin表达所致的凋亡抗性有关。     

Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cells

A direct correlation exists between increased choline kinase (Chk) expression, and the resulting increase of phosphocholine levels, and histological tumor grade. To better understand the function of Chk and choline phospholipid metabolism in breast cancer we have stably overexpressed one of the two isoforms of Chk‐α known to be upregulated in malignant cells, in non‐invasive MCF‐7 human breast cancer cells. Dynamic tracking of cell invasion and cell metabolism were studied with a magnetic resonance (MR) compatible cell perfusion assay. The MR based invasion assay demonstrated that MCF‐7 cells overexpressing Chk‐α (MCF‐7‐Chk) exhibited an increase of invasion relative to control MCF‐7 cells (0.84 vs 0.3). Proton MR spectroscopy studies showed significantly higher phosphocholine and elevated triglyceride signals in Chk overexpressing clones compared to control cells. A test of drug resistance in MCF‐7‐Chk cells revealed that these cells had an increased resistance to 5‐fluorouracil and higher expression of thymidylate synthase compared to control MCF‐7 cells. To further characterize increased drug resistance in these cells, we performed rhodamine‐123 efflux studies to evaluate drug efflux pumps. MCF‐7‐Chk cells effluxed twice as much rhodamine‐123 compared to MCF‐7 cells. Chk‐α overexpression resulted in MCF‐7 human breast cancer cells acquiring an increasingly aggressive phenotype, supporting the role of Chk‐α in mediating invasion and drug resistance, and the use of phosphocholine as a biomarker of aggressive breast cancers. Copyright © 2010 John Wiley & Sons, Ltd.     

转染肿瘤坏死因子-α及MDR1反义RNA逆转乳腺癌多药耐药的研究

目的：转染肿瘤坏死因子-α(TNF-α)cDNA和多药耐药基因(MDR1)的反义RNA到乳腺癌耐药细胞株MCF-7/ADR中进行表达，并观察它们在乳腺癌耐药逆转中的作用。 方法：应用RT-PCR和DNA重组技术构建反义绿色荧光蛋白pEGFP-MDR1融合蛋白表达载体和红色荧光蛋白pDsRed2-TNF-α融合蛋白表达载体，分别和同时导入乳腺癌耐药细胞株MCF-7/ADR中进行表达，检测转染前后细胞的生长曲线、细胞凋亡程度、MDR1-mRNA和P糖蛋白（P-gp）表达情况及对ADR敏感性的变化。 结果：转染后的细胞生长明显减慢，凋亡率显著增加，MDR1-mRNA和P糖蛋白（P-gp）表达明显降低，对ADR的耐药性明显下降，敏感性增加。 结论：联合运用不同的逆转耐药机制，将TNF-α cDNA及MDR1反义RNA分别或同时导入乳腺癌耐药细胞中，能有效达到逆转耐药的目的。     

转染肿瘤坏死因子-α及MDR1反义RNA逆转乳腺癌多药耐药的研究

目的:转染肿瘤坏死因子-α(TNF-α)cDNA和多药耐药基因(MDR1)的反义RNA到乳腺癌耐药细胞株MCF-7/ADR中进行表达,并观察它们在乳腺癌耐药逆转中的作用.方法:应用RT-PCR和DNA重组技术构建反义绿色荧光蛋白pEGFP-MDR1融合蛋白表达载体和红色荧光蛋白pDsRed2-TNF-α融合蛋白表达载体,分别和同时导入乳腺癌耐药细胞株MCF-7/ADR中进行表达,检测转染前后细胞的生长曲线、细胞凋亡程度、MDR1-mRNA和P糖蛋白(P-gp)表达情况及对ADR敏感性的变化.结果:转染后的细胞生长明显减慢,凋亡率显著增加,MDR1-mRNA和P糖蛋白(P-gp)表达明显降低,对ADR的耐药性明显下降,敏感性增加.结论:联合运用不同的逆转耐药机制,将TNF-αcDNA及MDR1反义RNA分别或同时导入乳腺癌耐药细胞中,能有效达到逆转耐药的目的.     

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。