三羟异黄酮对MCF-7/HER-2乳腺癌细胞VEGF表达变化的影响

目的观察三羟异黄酮(genistein)对原癌基因HER-2/neu高表达乳腺癌细胞血管内皮生长因子(vascular endothelial growthfactor,VEGF)表达变化的影响,探讨genistein抗HER-2/neu高表达乳腺癌血管生成的分子机制.方法用基因转染技术建立HER-2/neu高表达的乳腺癌MCF-7细胞(命名为MCF-7/HER-2).5×10-5mol/L genistein处理MCF-7/HER-2细胞24、48、72 h后,应用免疫组织化学、Western blot及RT-PCR法检测genistein对MCF-7/HER-2乳腺癌细胞VEGF表达的变化.结果MCF-7/HER-2细胞与MCF-7细胞相比VEGF蛋白和mRNA表达增加,genistein处理MCF-7/HER-2细胞24、48、72 h后,VEGF的mRNA和蛋白表达量均下调,随着处理时间的延长,VEGF mRNA和蛋白表达量下降越明显.结论genistein能在转录和翻译水平下调HER-2/neu高表达乳腺癌细胞VEGF的表达,这可能是genistein抑制HER-2/neu高表达乳腺癌血管生成的机制之一.     

槲皮素对乳腺癌MCF-7细胞热休克蛋白表达的影响

目的:探讨槲皮素对乳腺癌MCF-7细胞株热休克蛋白70(HSP70)、HSP27和HSP90的表达的影响。方法:对体外培养的乳腺癌MCF-7细胞,恒温42℃热处理2h,分别于4、8、12和24h后荧光定量PCR检测HSP70、HSP27和HSP90的基因表达变化并用蛋白质印迹检测蛋白表达量的变化。观察热处理前使用150μmol/L槲皮素对乳腺癌MCF-7细胞HSP70、HSP27和HSP90的基因表达变化、蛋白表达量的影响。结果:42℃热处理后乳腺癌MCF-7细胞HSP70、HSP27和HSP90表达在基因及蛋白水平上均有明显升高,4h达到高峰,8h后开始减少,24h接近正常值。槲皮素能抑制HSP70、HSP27表达,但导致HSP90表达水平先升高,4～8h表达仍处于高峰值,8h后才开始下降。结论:HSP70、HSP27和HSP90在MCF-7细胞内的基因和蛋白表达均在热处理后迅速升高,在热处理前使用槲皮素可以抑制HSP70、HSP27的表达,HSP90水平升高及下降延迟。     

HAP1对乳腺癌细胞MCF-7生物学特性影响的观察

目的:探讨亨廷顿相关蛋白1(HAP1)基因过表达对人乳腺癌细胞株MCF-7增殖、体外迁移侵袭和细胞凋亡的影响及其可能机制.方法:通过转染的方法将逆转录病毒pBabe-puro(嘌呤霉素)HAP1质粒和pBabe-puro质粒导入人乳腺癌细胞系MCF-7,用嘌呤霉素筛选稳定表达两质粒的细胞系,荧光定量PCR和蛋白质印迹法鉴定是否成功构建HAP1过表达细胞系;细胞增殖-毒性检测试剂盒(CCK-8)和克隆形成实验检测细胞的生长增殖,Transwell小室法检测细胞的侵袭和迁移,流式细胞仪检测细胞的凋亡.结果:成功构建稳定表达pBabe-HAP1的MCF-7-pBabe-puro-HAP1细胞模型.CCK-8检测72 h细胞增殖率,MCF-7-pBabe-puro-HAP1为(75.97±6.76)％,明显低于MCF-7-pBabe-puro细胞(93.98±6.63)％(P=0.03)及MCF-7细胞(100.00±0.oo)％,P=0.004;MCF-7-pBabe-puro-HAP1细胞克隆形成率为(22.67±1.26)％,明显低于MCF-7(35.00±0.50)％(P=0.000)和MCF-7-pBabe-puro细胞(33.83±0.76)％,P＝0.000;Transwell小室侵袭和迁移实验表明,MCF-7-pBabe-puro-HAP1组的侵袭(3.33±0.58,P=0.000)和迁移(50.00±3.61,P＜0.01)能力明显降低;流式细胞仪检测细胞凋亡,MCF-7-pBabe-puro-HAP1凋亡率为(8.03±0.15)％,高于MCF-7-pBabe-puro(3.13土0.25)％(P=0.000)和MCF-7细胞(3.33±0.35)％,P=0.000.结论:HAP1基因能够抑制肿瘤细胞增殖和迁移侵袭,并能诱导细胞凋亡,其可能作为一个抑癌基因在肿瘤发生发展中发挥重要作用.     

敲除人乳腺癌MCF-7细胞中PTEN基因对JNK通路活性的影响

背景与目的:PTEN与多种肿瘤发生发展密切相关,大量研究表明PTEN基因通过直接或间接作用整合复杂的信号网络系统,并影响靶分子及其下游信号级联反应.本研究探讨敲除MCF-7细胞中PTEN基因对JNK通路活性的影响.方法:PTEN反义寡核苷酸转染后,激光共聚焦显微镜检测MCF-7细胞内PTEN蛋白表达:流式细胞术、四甲基偶氮唑蓝(MTT)及Western blot法分别检测SP600125诱导的细胞早期凋亡和细胞周期改变、细胞增殖抑制、细胞中JNK及其下游底物ATF-2、C-Jun的磷酸化水平.结果:FTEN反义寡核苷酸有效封闭MCF-7细胞中PTEN蛋白表达:SP600125(10 μmol/L)诱导已敲除PTEN的MCF-7细胞发生早期凋亡.凋亡率达(32.4±2.4)%,细胞发生G1期阻滞、细胞增殖明显受抑制,与SP600125组、反义组细胞相比差异有显著性(P<0.05);反义+SP600125组MCF-7细胞中磷酸化JNK及其下游底物ATF-2及C-Jun磷酸化水平下调.结论:MCF-7细胞中JNK通路激活与PTEN表达水平相关,PTEN缺失使MCF-7细胞中JNK通路活化,细胞对JNK相关抑制剂的敏感性增加.     

PCDGF shRNA对乳腺癌细胞系MCF-7增殖凋亡和VEGF表达的影响

背景与目的：畸胎瘤细胞源性生长因子（PC cell-derived growth factor,PCDGF）是调节乳腺癌细胞增殖和凋亡的重要因子,然而其作用机制目前尚不清楚。本研究通过构建并体外转染针对PCDGF的shRNA,分析PCDGF对乳腺癌细胞系MCF-7增殖、凋亡及细胞中血管内皮细胞生长因子（vascular endothelial growth factor,VEGF）表达的影响。方法：RT-PCR法检测PCDGF在4种乳腺癌细胞系中的表达,构建含2条shRNA的RNAi质粒,脂质体转染质粒于MCF-7细胞中,MTT法检测转染前后细胞增殖变化,ELISA法检测VEGF表达。结果：被检测的4种乳腺癌细胞中均有不同程度的PCDGFmRNA表达,转染shRNA质粒对MCF-7细胞PCDGF mRNA表达抑制率为59．8％;转染质粒后MCF-7细胞增殖受到抑制,实验组早期细胞凋亡率为30．41％,晚期细胞凋亡率为35．38％;阴性对照组早期细胞凋亡率为3．69％,晚期细胞凋亡率为15．39％。同阴性对照质粒相比,转染阳性质粒的MCF-7细胞VEGF的表达抑制率为47．2％。结论：PCDGF调节MCF-7细胞的增殖、凋亡和VEGF的表达。     

洛伐他汀对MCF-7细胞核转录因子和细胞周期调控蛋白表达的影响

目的 探讨胆固醇合成抑制剂洛伐他汀(LOV)对MCF-7乳腺癌细胞核转录因子(IκBα)和细胞周期调控蛋白表达的影响。方法 MCF-7乳腺癌细胞经4μmoL／L、8μmol／L和16μmol／L终浓度的LOV处理后,流式细胞仪观察细胞周期分布状况,Western blot分析胞浆中IκBα、CDK4、p16、pRb的蛋白表达及核内IκBα的蛋白表达。结果 不同剂量的LOV处理48～72h能显著阻滞MCF-7细胞细胞周期于G0／Gl期,且具有一定的剂量-效应和时间依赖关系;同剂量的LOV能显著下调胞浆内IκBα、CDK4和pRb蛋白表达,上调核内IκBα蛋白表达,但对p16蛋白表达无明显影响。结论 洛伐他汀可能通过影响IκBα和细胞周期调控蛋白的表达,诱导MCF-7细胞周期受阻于G0／G1期。     

血管内皮生长因子受体2为靶的反义寡核苷酸筛选及其对乳腺癌MCF-7细胞的抗肿瘤作用

目的筛选出可与血管内皮生长因子受体2（KDR）mRNA高效、特异结合的反义寡核苷酸。探讨其体外抗肿瘤作用。方法先用寡核苷酸库杂交和计算机预测初筛反义寡核苷酸;经寡核苷酸芯片杂交验证,挑选与KDRmRNA有强杂交信号的反义寡核苷酸,用MTT法、逆转录聚合酶链反应（RT-PCR）和免疫印迹（Western blotting）探讨反义寡核苷酸对乳腺癌细胞系MCF-7细胞增殖和KDR表达的抑制作用。结果在寡核苷酸库杂交筛选出的13条反义寡核苷酸中,有8条（8／13,61.5％）在芯片杂交中显示较强杂交信号;而在计算机预测设计的17条探针中,只有1条显示较强信号。合成杂交信号较强的9条硫代反义寡核苷酸,均能有效抑制MCF-7细胞增殖,并呈剂量依赖性。其中抑制率最高的2条反义寡核苷酸为asON4和asON7,由寡核苷酸库杂交联合芯片杂交筛出,在0.8μmol／L时抑制率分别为51．6％和62．2％;同时,这2条反义寡核苷酸在mRNA水平和蛋白质水平抑制了KDR基因表达,并呈剂量相关性。结论寡核苷酸库杂交筛选和寡核苷酸芯片杂交筛选的结果有较好一致性,将寡核苷酸库和寡核苷酸芯片联用是一种较好筛选反义寡核苷酸方法。KDR反义寡核苷酸有明显的抗肿瘤作用。     

去磷酸化RB蛋白表达对阿霉素诱导MCF-7/S细胞凋亡的影响

目的检测经阿霉素(ADR)处理的人乳腺癌MCF-7/S细胞的凋亡率(AR)及去磷酸化RB蛋白表达的变化,以探讨ADR诱导细胞凋亡的可能机理。方法将体外培养的MCF-7/S细胞分为实验组(以不同浓度ADR处理细胞)及对照组(以等体积的生理盐水处理细胞);应用MTT比色法检测ADR对MCF-7/S细胞的抑制率(IR);应用流式细胞术检测实验组和对照组细胞的AR;采用S-P免疫组化染色法检测ADR作用后去磷酸化RB蛋白表达的变化。结果ADR抑制MCF-7/S细胞增殖,呈剂量依赖性,IC50为0.128mg/L;0.25、2、5μg/mlADR处理的MCF-7/S细胞的AR分别为0.171、0.184、0.259,而对照组MCF-7/S细胞的AR为0.045,两者比较,有非常显著性差异(P<0.01);5μg/mlADR实验组MCF-7/S细胞的去磷酸化RB蛋白的表达量为986.8±207.4,而对照组为131.7±31.9,两者比较,有非常显著性差异(P<0.01);5μg/mlADR实验组MCF-7/S细胞的AR与其去磷酸化RB蛋白的表达量呈正相关(γ=0.998,P=0.037)。结论ADR能抑制MCF-7/S细胞增殖并诱导其凋亡,其机理可能与去磷酸化RB蛋白表达水平上调有关。     

Apogossypolone抑制乳腺癌MCF-7细胞增殖及诱导细胞凋亡

目的:研究Apogossypolone(ApoG2)对人乳腺癌细胞系MCF-7体外的增殖抑制和诱导凋亡作用.方法:采用MTT法和平板克隆形成实验检测ApoG2作用于MCF-7细胞后,对其体外增殖和细胞克隆形成能力的影响;Hoechst 33258荧光染色法观察ApoG2(20μmol/L)作用48 h后,细胞凋亡的形态学变化;FCM检测ApoG2作用于MCF-7细胞后的细胞凋亡率以及细胞周期的变化.结果:ApoG2药物在浓度为5μmol/L、作用14 d后就能对MCF-7细胞增殖起到明显的抑制作用,且呈剂量和时间依赖性;平板克隆实验表明,经不同浓度(5、10和20 μmol/L)ApoG2作用14 d后,对MCF-7细胞克隆形成能力呈现明显的抑制作用,且同样呈剂量和时间依赖性.Hoechst 33258荧光染色结果显示,ApoG2与MCF-7细胞共培养72 h后,细胞呈现明显的核固缩和碎裂、染色质凝集和凋亡小体形成等细胞凋亡现象.FCM检测结果显示,20 μmol/LApoG2作用72 h后,细胞凋亡率较对照组升高,而且S期和G2/M期细胞比例较对照组升高(P＜0.05).结论:ApoG2能够抑制人乳腺癌细胞系MCF-7的增殖和克隆形成,阻滞细胞周期,诱导细胞凋亡.     

电化学疗法对乳腺癌MCF-7细胞凋亡及细胞内钙离子浓度的影响

目的探讨电化学疗法(ECT)对乳腺癌MCF-7细胞内钙离子浓度及细胞凋亡、坏死和细胞生长抑制的影响。方法将MCF-7细胞分为对照组及实验组,对照组为0 C(C:库仑),实验组按电量不同分为4组:3 C、5 C、10 C及20 C组。每组重复6孔,用不同电量处理MCF-7细胞后培养6 h和24 h,通过MTT法、GENMED细胞钙离子浓度比色法、流式细胞仪和激光共聚焦分别检测细胞生长抑制率、细胞内Ca2+浓度、观察凋亡和坏死情况。组间均数比较采用方差分析,应用SNK方法进行两两比较,应用5×2析因分析探讨电量与时间对细胞的影响。结果不同电量干预MCF-7细胞后培养6 h及24 h,细胞生长抑制率(6 h:F=508.43,P=0.000;24 h:F=232.76,P=0.000)、坏死率(6 h:F=2282.07,P=0.000;24 h:F=2644.60,P=0.000)、细胞内Ca2+浓度(6 h:F=1416.06,P=0.000;24 h:F=2394.26,P=0.000)随电量增加而增加,电量及培养时间之间具有交互作用(F=288.93,P=0.000;F=2305.39,P=0.000;F=1651.96,P=0.000)。流式细胞仪检测细胞凋亡率结果显示,细胞凋亡率在3、5、10 C组明显增高,而20 C组细胞凋亡率降低,电量及培养时间有交互作用(F=51.20,P=0.000)。结论 ECT可抑制乳腺癌MCF-7细胞株的生长并诱导其凋亡,并引起细胞内Ca2+浓度升高。低剂量ECT主要诱导细胞凋亡,高剂量ECT主要引起细胞坏死。     

Regulation of estrogen sulfotransferase by estrogen in MCF-7 human mammary cancer cells

Summary The importance of the steroid hormone microenvironment within cells is now recognised in studies on endocrine-related neoplasms such as breast cancer. This focuses attention on ezymes which control the intracellular levels of estradiol-17 (E₂). One such enzyme, estrogen sulfotransferase, which converts E₂ to inactive E₂-3 sulfate, has now been shown to be regulated by estrogen in MCF-7 human mammary cancer cells. Hydroxysteroid sulfotransferase, which sulfurylates the adrenal-derived estrogen 5-androstene-3,17-diol, is also under estrogen control. Evidence is provided which shows that one function of these enzymes may involve elimination of estrogen from the cell following processing of the ligand-charged estrogen receptor (ER).     

甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究

目的：研究甲异靛对人乳腺癌MCF-7细胞增殖抑制和诱导凋亡作用。方法：采用MTT法检测甲异靛对MCF-7乳腺癌细胞的增殖抑制;流式细胞仪测定细胞凋亡率;光学显微镜观察细胞形态的变化;westernblot法测定caspase-3、PARP及bcl-2表达。结果：甲异靛能明显抑制MCF-7乳腺癌细胞增殖。甲异靛诱导MCF-7细胞凋亡,呈现典型细胞凋亡的形态变化,并呈时间和剂量依赖性,凋亡率最高可达（68.40±4.87）%,在细胞凋亡过程中出现PARP分子断裂和bcl-2下调,未检测到caspase-3。结论：甲异靛能诱导MCF-7乳腺癌细胞凋亡抑制细胞增殖,其发生机制可能与下调bcl-2有关。     

甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究

目的:研究甲异靛对人乳腺癌MCF-7细胞增殖抑制和诱导凋亡作用。方法:采用MTT法检测甲异靛对MCF-7乳腺癌细胞的增殖抑制;流式细胞仪测定细胞凋亡率;光学显微镜观察细胞形态的变化;westernblot法测定caspase-3、PARP及bcl-2表达。结果:甲异靛能明显抑制MCF-7乳腺癌细胞增殖。甲异靛诱导MCF-7细胞凋亡,呈现典型细胞凋亡的形态变化,并呈时间和剂量依赖性,凋亡率最高可达(68.40±4.87)%,在细胞凋亡过程中出现PARP分子断裂和bcl-2下调,未检测到caspase-3。结论:甲异靛能诱导MCF-7乳腺癌细胞凋亡抑制细胞增殖,其发生机制可能与下调bcl-2有关。     

Effect of hyperosmolality on alkaline phosphatase and stress-response protein 27 of MCF-7 breast cancer cells

Summary MCF-7, a continuous cell line derived from a human breast carcinoma, exhibits very low alkaline phosphatase (ALP) activity. The enzyme is heat-stable and is inhibited by L-phenylalanine and L-phenylalanylgly-cylglycine, but not by L-homoarginine, 1-bromotetramisole, or levamisole. These data indicate that MCF-7 produces term-placental ALP, the oncodevelopmental enzyme form inappropriately expressed by a variety of human tumors. In contrast to human cancer cells that produce this enzyme monophenotypically, ALP activity of MCF-7 cells is not significantly increased by glucocorticoids or sodium butyrate. By comparison, exposure to hyperosmolality causes a striking increase in enzyme activity. Cycloheximide blocks this effect. The results obtained with cell-free assays were confirmed by cytochemical and immunocytochemical assays on whole cells. Because some of the agents tested in the enzyme modulation experiments affect cell proliferation, their possible effect on two stress-response proteins (srp 27 and srp 72) was also examined; specific immunocytochemical assays were used. These tests revealed that neither protein is affected by glucocorticoids; that sodium butyrate has no effect on srp 27, but alters the intracellular distribution of srp 72; and that hyperosmolality, while not significantly affecting srp 72, causes an increase in srp 27.     

Colocalization of estrogen and progesterone receptors with an estrogenregulated heat shock protein in paraffin sections of human breast and endometrial cancer tissue

Summary We have studied by immunocytochemistry and monoclonal antibodies the presence and localization of estrogen receptors, progesterone receptors, and a 24-kD estrogen-regulated heat shock protein in biopsies from breast and endometrial cancer patients. Three different tissue processing protocols were used to colocalize the antigens in the same tissue sections: a) frozen sections, b) formalin fixation with routine paraffin embedding, and c) picric acid-formaldehyde (PAF) fixation with a rapid embedding in paraffin. Frozen sections showed good receptor staining but poor 24-kD protein immunoreactivity, while routine paraffin sections (with or without DNase pretreatment) were inadequate to reveal the nuclear receptor proteins at the same level seen in frozen sections. On the other hand, all three proteins could be detected satisfactorily in PAF-fixed paraffin-embedded tissue. Using this procedure we were able to visualize 24-kD protein and estrogen receptor or progesterone receptor in individual cells in paraffin sections. The study revealed that in all of the estrogen receptor positive breast and endometrial tumor samples, almost 90% of the cells expressing the cytoplasmic 24-kD protein contained estrogen receptor in the cell nucleus. In contrast, 24-kD immunoreactive cells did not express progesterone receptors in almost 40% of the progesterone receptor positive tumor samples.     

GSDME通过调控细胞焦亡影响乳腺癌MCF-7细胞对紫杉醇的敏感性

[摘要] 目的：探讨GSDME是否通过调控细胞焦亡影响乳腺癌MCF-7 细胞对化疗药物紫杉醇（paclitaxel,PTX）的敏感性。方法：利用RNA干扰技术敲降GSDME在MCF-7 细胞中的表达,采用CCK-8 法、流式细胞术、乳酸脱氢酶（lactate dehydrogenase,LDH）释放实验及Wb技术分别检测GSDME低表达前后,PTX对细胞增殖、焦亡、LDH释放、GSDME-N端蛋白及cleaved-caspase-3 蛋白表达水平的变化情况。结果：与对照组比较,PTX处理组细胞的焦亡率、LDH释放量、GSDME-N端蛋白及cleaved-caspase-3 蛋白的表达水平均显著升高（均P<0.01）;与si-NC组比较,敲低GSDME可使si-GSDME组细胞对PTX的敏感性降低,其细胞焦亡率、LDH释放量及GSDME-N端蛋白表达水平均显著下降（均P<0.01）。结论：敲减MCF-7 细胞中GSDME的表达量可显著抑制细胞焦亡并降低细胞对PTX的敏感性。     

Progression in MCF-7 breast cancer cell tumorigenicity: compared effect of FGF-3 and FGF-4

The transforming properties of fibroblast growth factor 3 (FGF-3) were investigated in MCF7 breast cancer cells and compared to those of FGF-4, a known oncogenic product. The short form of fgf-3 and the fgf-4 sequences were each introduced with retroviral vectors and the proteins were only detected in the cytoplasm of the infected cells, as expected. In vitro, cells producing FGF-3 (MCF7.fgf-3) and FGF-4 (MCF7.fgf-4) displayed an amount of estrogen receptors decreased to around 45% of the control value. However, MCF7.fgf-3 cell proliferation remained responsive to estradiol supply. The sensitivity of the MCF7.fgf-4 cells, if existant, was masked by the important mitogenic action exerted by FGF-4. In vivo, the MCF7.fgf-3 and MCF7.fgf-4 cells gave rise to tumors under conditions in which the control cells were not tumorigenic. Supplementing the mice with estrogen had the paradoxical effect of totally suppressing the start of the FGF-3 as well as the FGF-4 tumors. Tumorigenicity in the presence of matrigel was similar for MCF7.fgf-3 and control cells and was increased by estrogen supplementation. Once started, the MCF7.fgf-4 tumors grew with a characteristic high rate. Remarkably, FGF-4 but not FGF-3, stimulated the secretion of vascular endothelial growth factor (VEGF₆₅) without altering the steady-state level of its mRNA, suggesting a possible regulation of VEGF synthesis at the translational level in MCF7 cells. The increased VEGF secretion is probably involved in the more aggressive phenotype of the MCF7.fgf-4 cells while a decreased dependence upon micro-environmental factors might be part of the increased tumorigenic potential of the MCF7.fgf-3 cells.     

Anti-estrogenic effect of unliganded progesterone receptor is estrogen-selective in breast cancer cells MCF-7

We have reported that the ectopic expression of progesterone receptor (PR) in MCF-7 cells disrupted the effects of estradiol-17beta (E2), and this anti-estrogenic effect of PR was associated with heightened E2 metabolism and inhibition of estrogen receptor (ER) binding to estrogen response element (ERE). This study determined if the transfected PR was also able to abolish the effect of other estrogens including estrone (E1), estriol (E3) and estradiol-17alpha. The study revealed that the transfected PR in MCF-7 cells abolished the growth-stimulatory effects of E1 and E2, but had minimal effect on the effects of E3 and estradiol-17alpha. This observation is associated with a faster metabolic inactivation of E1 and E2 than that of E3 and estradiol-17alpha. The conditioned media collected after 72h of E3 treatment from PR-transfected MCF-7 cells retained full estrogenic potential whereas E2-treated conditioned media after 72h have lost all of the estrogenic activity. On the other hand, PR was able to inhibit ER-ERE binding induced by all the estrogens. This suggests that the estrogen-selective anti-estrogenic effect of transfected PR was due to the difference in rate of metabolic inactivation of different estrogens in MCF-7 cells. It is plausible that inhibition of ER-ERE interaction by PR resulted in faster estrogen-ER dissociation, and the dissociated E1 and E2 were metabolically inactivated but enzymes for E3 and estradiol-17alpha inactivation were lacking in MCF-7 cells. The findings suggest an interesting mechanism by which the disruption of ER-ERE interaction heightens the inactivation of estrogens in breast cancer cells.