反义RNA抑制Ezrin表达对乳腺癌细胞侵袭能力的影响

目的 观察特异性阻断Ezrin的表达对人乳腺癌细胞MDA-MB-231和MCF-7的增殖和侵袭能力的影响.方法 将anti-pCR3.1-Ezrin质粒经脂质体介导,转染人人乳腺癌细胞MDA-MB-231 6、12和24 h,应用Western blot和逆转录-聚合酶链反应(RT-PCR)方法检测Ezrin的表达变化情况;转染质粒24h后,噻唑蓝(MTT)比色法检测抑制Ezrin对MDA-MB-231和MCF-7细胞体外增殖能力的影响,Boyden小室法检测抑制Ezrin对MDA-MB-231和MCF-7细胞体外侵袭能力的影响.结果 转染anti-pCR3.1-Ezrin后,对MDA-MB-231细胞中的Ezrin表达抑制在24 h时达高峰.MTT法比色实验结果显示,MDA-MB-231和MCF-7细胞中转染anti-pCR3.1-Ezrin组、转染空质粒组和对照组的A值分别为0.410±0.018、0.765±0.058、0.795±0.061和0.480±0.021、0.632±0.052、0.648±0.059.转染anti-pCR3.1-Ezrin组细胞的增殖受到明显抑制,抑制率分别为(47.9±3.1)%和(32.0±2.8)%(P<0.05).Boyden小室法检测结果显示,MDA-MB-231和MCF-7细胞中转染anti-pCR3.1-Ezrin组细胞的侵袭能力分别为对照组的(50.5±3.2)%和(74.8±4.6)%(P<0.05).结论 Ezrin在乳腺癌的生长和侵袭过程中发挥重要作用.     

RNA干扰下调midkine基因表达对人乳腺癌细胞粘附和侵袭力的影响

目的 探讨中期因子(midkine,MK)基因siRNA对乳腺癌细胞生物学行为的影响.方法 培养人乳腺癌Bcap-37、LCCI、MCF-7、MDA-MB-231、MDA-MB-435、MDA-MB-468及ZR75-1细胞株,以荧光实时定量PCR方法检测MK基因mRNA表达;筛选出MK表达最高者.采用MK siRNA...     

Vitamin E succinate promotes breast cancer tumor dormancy

BACKGROUND: Vitamin E succinate (VES) is the most potent antitumor analogue of vitamin E. Despite many reports of VES's antitumor activity in vitro, there is little information about its antitumor effects in vivo. MATERIALS AND METHODS: We investigated the effect of VES on the growth of human breast cancer cells in vitro and in vivo. RESULTS: VES decreased cell viability in MDA-MB-231 and MCF-7 human breast cancer cells. Although VES increased apoptosis in MDA-MB-231 cells, it had no effect on apoptosis in MCF-7 cells. The inhibitory effect of VES on cell growth was specific for the intact molecule because a markedly reduced effect was noted when either vitamin E or succinic acid was administered alone. VES inhibited the growth of MDA-MB-231 cells in nude mice. Also, VES was found to inhibit vascular endothelial growth factor (VEGF) gene expression in MDA-MB-231 cells. CONCLUSIONS: VES inhibits the growth of breast cancer cells in vitro and in vivo. This is the first report of VES inhibition of established tumor growth in vivo. The mechanism of VES's in vivo effects may involve inhibition of tumor angiogenesis since VES inhibits VEGF gene expression.     

肿瘤相关钙信号传导蛋白-2基因对人乳腺癌细胞黏附和侵袭力的影响

目的 观察肿瘤相关钙信号传导蛋白-2(TROP-2)基因小干扰RNA (siRNA)对乳腺癌细胞黏附和侵袭力的影响.方法 培养人乳腺癌Bcap-37、LCC1、MCF-7、MDA-MB-231、MDA-MB-435、MDA-MB-468及ZR75-1细胞株,以荧光实时定量聚合酶链反应(PCR)方法检测TROP-2基因mRNA表达;筛选出TROP-2表达最高者.采用TROP4基因siRNA转染乳腺癌细胞,分别以荧光实时定量RT-PCR和免疫荧光方法观察TROP-2基因mRNA和蛋白水平,然后以噻唑蓝(MTT)比色法检测细胞黏附性,以Transwell方法检测癌细胞侵袭能力.结果 人乳腺癌Bcap-37、LCC1、MCF-7、MDA-MB-231、MDA-MB-35、MDA-MB-468及ZR75-1细胞株TROP-2 mRNA分别是1.362±0.057、2.207±0.056、2.997±0.052、0.136±0.045、0.122±0.025、0.194±0.028和2.706±0.039,以MCF-7细胞最高;以TROP-2 siRNA转染乳腺癌MCF-7细胞后,癌细胞TROP-2基因mRNA和蛋白水平明显下降,且呈浓度依赖性;黏附实验结果显示,5、10、20 nmol/L siRNA组黏附率分别为(52.9±2.5)％、(25.6±2.3)％、(12.8±2.2)％(P＜0.01);Transwell实验结果显示,5、10和20 mol/L siRNA组穿过滤膜的细胞分别为78±17、39±15、19±16,而对照组分别为136±25、139±21(P＜0.01).结论 TROP-2基因在乳腺癌细胞黏附和侵袭中发挥着重要作用;以siRNA转染乳腺癌细胞,可抑制乳腺癌细胞黏附和侵袭能力.     

尿多酸肽对乳腺癌细胞凋亡的影响

目的研究肿瘤细胞分化诱导剂尿多酸肽（CDA—Ⅱ）对乳腺癌细胞凋亡的影响。方法将CDA—Ⅱ与不同生物学特性的乳腺癌细胞株MCF-7和MDA-MB-231进行体外培养，同时以维甲酸为阳性对照，观察CDA—Ⅱ对乳腺癌细胞生长曲线、细胞凋亡及形态学等方面的影响。结果CDA—Ⅱ可减缓两株乳腺癌细胞的生长和增殖能力，诱导乳腺癌细胞凋亡。结论CDA—Ⅱ可抑制MCF-7和MDA-MB-231两株乳腺癌细胞的生长和增殖能力，诱导乳腺癌细胞凋亡．本研究为CDA-Ⅱ治疗乳腺痛提供了实验依据。     

多种人乳腺癌细胞株CCL5表达和其侵袭能力关系的初步研究

目的探讨四种人乳腺癌细胞株（MCF-7，SK—BR-3，T-47D和MDA—MB-231）之间，CCL5表达与其侵袭能力间是否有相关性。方法使用RT—PCR方法和采用Cell Invasion Assay Kit分别测定四种人乳腺癌细胞株CCL5表达及其侵袭能力。结果CCL5基因在四种细胞株中均有表达，其中MCF-7细胞株中该基因表达量相对较高，MDA—MB-231和T47-D细胞株次之，SK—BR-3细胞株表达量相对较低。MDA—MB-231细胞株侵袭能力最强，SK—BR-3细胞株次之，MCF-7、T-47-D细胞株侵袭能力弱。结论在四种人乳腺癌细胞株之间，尚未发现CCL5 mRNA表达量与其侵袭能力间有明显的线性相关性。该研究为进一步探讨CCL5和乳腺癌的关系提供新方向。     

人平衡型核苷载体在乳腺癌细胞中的表达情况及其对5-FU药效的影响

目的研究乳腺癌细胞株MDA-MB-231、MDA-MB-468、SK-BR-3、MCF-7中人平衡型核苷载体（hENTS）的表达及其对5氟尿嘧啶（5-Fu）细胞毒性的影响。方法MDA-MB-231、MDA—MB-468、SK-BR-3、MCF-7细胞常规培养于含10％小牛血清的高糖DMEM培养液中。逆转录．聚合酶链反应（RT-PCR）检测4种细胞株中hENTlmRNA及hENT2mRNA的表达。每种细胞分别在含有一定浓度序列（1．28×10^4ng／L～2．00×10^8ng／L）5-FU的培养液中培养48h。MTT法检测4种细胞株增殖,并计算其5-FU的半数抑制浓度（IC50）。结果①bENT1及hENT2mRNA在MDA-MB-231、MDA-MB-468、SK-BR-3细胞中的表达均较其在MCF-7细胞中（P伊〈0．05）,但其表达在MDA-MB-231、MDA-MB-468、SK-BR-3细胞中差异无统计学意义（P〉0．05）。hENT2mRNA表达在4种细胞中均较hENT1 mRNA表达低,差异有统计学意义（P〈0．05）。②MTT结果显示,5-FU的IC50在MDA-MB-231、MDA-MB-468、SK-BR-3细胞株中均较在MCF-7细胞株中低（P〈0．05）,在MDA-MB-231、MDA-MB-468、SK-BR-3细胞株之间差异无统计学意义（P〉0．05）。③在5-FU的IC50 较低的乳腺癌细胞株（MDA-MB-231、MDA—MB-468、SK-BR-3）中高表达hENTs,而在5-Fu的Ic50较高的乳腺癌细胞株（MCF-7）中,低表达hENTs或无表达。结论在乳腺癌细胞株中,细胞膜上hENTs的表达情况能显著影响5-FU的作用效果,其结果提示,在临床上可能需要依据hENTs的表达情况来选择核苷类抗癌药物。     

The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells

BACKGROUND: Topotecan, which is a Camptothecin derivative, shows a large spectrum in anti-tumor activity. Topotecan exerts its cytotoxic effect on tumor cells mainly by inhibition of topoisomerase I activity resulting in double-strand DNA breaks. In our study, we investigated the combined cytotoxic action of Topotecan and Quercetin in MCF-7 and MDA-MB 231 human breast cancer cells. To examine the possible relation between the cytotoxic activity of Topotecan and oxidative stress, we measured ROS and nitrite levels in both human breast cell lines. MATERIALS AND METHODS: MCF-7 and MDA-MB 231 cells were exposed to Topotecan, Quercetin, or a combination of both agents for 24 h at 37 degrees C. The viability of the cells was measured using the colorimetric MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. We determined reactive oxygen species and nitrite levels as indicators of oxidative stress in both cell lines with and without Topotecan and/or Quercetin incubations using fluorometric dichlorofluorescin diacetate (DCFH-DA) and diaminonaphtalene (DAN) assay. RESULTS: The IC(50) concentration of Topotecan was 100 ng/ml in MCF-7 cell line and 160 ng/ml in MDA-MB231 cell line. Treatment with Quercetin enhanced cytotoxicity of Topotecan as 1.4-fold in MCF-7 and 1.3-fold in MDA-MB-231 cell line. A significant increment on ROS and nitrite levels was found in MCF-7 and MDA-MB-231 cells following Topotecan incubation. CONCLUSIONS: Our results suggest that Topotecan has cytotoxic activity against both of the breast cancer cell lines in vitro. A combination with Quercetin increases efficacy of Topotecan in the treatment of breast cancers. Our results indicate that increased oxidative stress plays a role in the cytotoxic action of Topotecan.     

A biochemical mechanism for resistance of intervertebral discs to metastatic cancer: Fas ligand produced by disc cells induces apoptotic cell death of cancer cells

Metastatic spinal cancer is characterized by the maintenance of normal disc structure until the vertebral body is severely destroyed by cancer cells. Anatomic features of the discs have been thought to be the main factor which confer the discs their resistance to metastatic cancer. However, little is known about the biochemical mechanism to prevent or attenuate the local infiltration of cancer cells into the discs. The purpose of this study was to investigate whether Fas ligand (FasL) produced by disc cells can kill Fas-bearing breast cancer cells by Fas and FasL interaction. Two human breast cancer cells (MCF-7 and MDA-MB-231) were obtained and cultured (1 × 10⁶ cells/well), and the expression of Fas was investigated by western blot analysis. Annulus fibrosus cells were isolated and cultured, and the presence of FasL was quantified in the supernatants of three different numbers of annulus fibrosus cells (1×, 2×, and 4 × 10⁶ cells/well) by ELISA assay. The MCF-7 and MDA-MB-231 cancer cells were cultured with supernatants of annulus fibrosus cells for 48 h. As controls, MCF-7 and MDA-MB-231 cancer cells were also cultured by themselves for 48 h. Finally, we determined and quantified the apoptosis rates of MCF-7 and MDA-MB-231 cancer cells by Annexin V–FITC and PI and TUNEL at 48 h, respectively. The expression of Fas was identified in MCF-7 and MDA-MB-231 cancer cells. The mean concentrations of FasL in supernatants of annulus fibrosus cells (1×, 2×, and 4 × 10⁶ cells/well) were 10.8, 29.6, and 56.4 pg/mL, respectively. After treatment with the supernatant of three different numbers of annulus fibrosus cells, the mean apoptosis rate of MCF-7 cancer cells was increased (2.8%, P < 0.01; 6.7%, P < 0.001; 31.0%, P < 0.001) in a dose-dependent manner of FasL compared to that of control (1.1%). The mean apoptosis rate of MDA-MB-231 cancer cells was also increased (5.7%, P < 0.01; 11.1%, P < 0.001; 25.3%, P < 0.001) in a dose-dependent manner of FasL compared to that of control (2.1%). TUNEL also demonstrated direct evidence of apoptoses of MCF-7 and MDA-MB-231 cancer cells. Our results demonstrate that Fas-bearing cancer cells undergo apoptosis by FasL produced by disc cells, which may be considered as a potential biochemical explanation for the disc’s resistance to metastatic cancer. This is the EuroSpine Winning Paper 2007 on Basic Science.     

表柔比星抑制乳腺癌细胞c-FLIP表达的时间窗和量效关系

目的 探讨表柔比星抑制乳腺癌细胞中c-FLIP表达的时间窗和量效关系.方法 将MCF7及MDA-MB-231乳腺癌细胞株分成两组:处理组中加入终浓度分别为4.0,2.0,1.0,0.5,0.25 mg/L的表柔比星,对照组加入等量生理盐水.各组分别作用24,48,72 h后采用RT-PCR技术检测两组细胞株中c-FLIP的表达,流式细胞仪检测细胞凋亡百分率.结果 随着表柔比星浓度的增加和作用时间的延长,c-FLIP的表达整体呈下降趋势;随着表柔比星浓度的升高及作用时间的延长,乳腺癌细胞凋亡率逐渐增加,呈剂量和时间依赖性.其中以浓度为2 mg/L作用72 h凋亡率最高.结论 表柔比星化疗诱导乳腺癌细胞的凋亡可能系通过抑制c-FLIP的表达,且其效果以浓度为2 mg/L作用72 h时为最佳.     

转染ERα基因对MDA-MB-231乳腺癌细胞白细胞介素-8表达的影响

目的 通过转染GFP-C1-ERα质粒建立稳定表达ERα的MDA-MB-23l细胞株,观察ERα对该细胞株白细胞介素(IL)-8表达的影响.方法 pEGFP-C1-ERα质粒经酶切和测序后转染MDA-MB-231细胞,使用G418筛选出稳定表达的克隆并鉴定.使用荧光逆转录-聚合酶链反应(RT-PCR)分别测定稳定转染ERα的MDA-MB-231细胞、MDA-MB-231细胞及MCF-7细胞的IL-8mRNA的表达,使用酶联免疫吸附试验(ELISA)法测定细胞上清液IL-8的浓度.结果 成功建立ERα阳性表达的MDA-MB-231细胞株,转染ERα的细胞株IL-8 mRNA的合成(105±16)ng/L明显低于MDA-MB-231细胞(195±32)ng/L(P<0.05),但仍然高于MCF-7细胞(32±4)ng/L(P<0.05),转染后细胞上清液IL-8浓度较未转染细胞明显降低,分别为(3499±312)ng/L和(6788±427)ng/L(P<0.05),但仍然高于MCF-7细胞(1846±44)ng/L(P<0.05).结论 稳定转染ERα基因抑制了MDA-MB-231细胞的IL-8的合成和分泌.     

畸胎瘤细胞源性生长因子对雌激素受体阴性乳腺癌细胞雌激素依赖性的影响

目的分析乳腺癌细胞雌激素依赖性与畸胎瘤细胞源性生长因子（PC—cell derived growth factor,PCDGF）表达之间的关系,探讨雌激素受体（ER）阴性乳腺癌患者接受内分泌治疗的可能性。方法荧光定量聚合酶链反应检测乳腺癌细胞系MCF-7、MDA-MB-231、T47D、MDA-MB-435s中PCDGF的表达,CCK-8法检测不同浓度雌激素培养条件下细胞的存活情况。以ER阳性乳腺癌细胞系MCF-7为对照,用RNA干扰技术抑制ER阴性乳腺癌细胞系MDA-MB-231中PCDGF基因的表达,观察抑制前后雌激素依赖性的变化。结果PCDGF在4株乳腺癌细胞系中均有不同程度的表达,其中在MDA-MB-435s中表达量最高,PCDGF高表达的乳腺癌细胞系的雌激素依赖性较高,PCDGFshRNA可以有效抑制乳腺癌细胞系MCF-7和MDA-MB-213中PCDGF的表达（抑制率分别为81．1％和86．7％）。MDA-MB-231细胞系雌激素依赖性增加的程度高于MCF-7细胞系（P〈0．05）。结论PCDGF高表达于ER阴性乳腺癌细胞,抑制其表达可能会更好的逆转内分泌治疗的耐药性。     

长链非编码RNA RP1-85F18.6在乳腺癌细胞中的表达及其对增殖和细胞周期的影响

背景与目的:近年来,大量研究表明长链非编码RNA(lncRNA)在肿瘤发生、发展中起着重要作用。lncRNA RP1-85F18.6是新近发现的非编码RNA,其在结肠癌组织和细胞系中高表达,并可促进肿瘤细胞增殖和侵袭,抑制凋亡及调控细胞周期。然而,目前尚无lncRNA RP1-85F18.6在乳腺癌中的研究报道,因此,本研究初步探讨lncRNA RP1-85F18.6在乳腺癌细胞中的表达及其对增殖和细胞周期的影响。方法:qRT-PCR法检测lncRNA RP1-85F18.6在乳腺癌细胞系MDA-MB-231、MBA-MD-468、MCF-7 及正常乳腺细胞系MCF-10A中的表达。将MDA-MB-231细胞分别转染RP1-85F18.6沉默序列(沉默组)与阴性对照序列(阴性对照组),将无处理的MDA-MB-231细胞作为空白对照组,在各组细胞中采用MTT法测定增殖能力,PI染色流式细胞仪检测法测定细胞周期,Western blot法测定Ki-67、增殖细胞核抗原(PCNA)、cyclin A1和p21~(C1P1)蛋白的表达。结果:乳腺癌细胞系MDA-MB-231、MBA-MD-468及MCF-7中lncRNA RP1-85F18.6相对表达量均高于正常乳腺上皮细胞系MCF-10A(均P0.05)。沉默组在转染后24、48、72、96 h的OD_(490) _(nm)值均明显低于空白对照组(均P0.05)。与空白对照组比较,沉默组的G_1/G_0期细胞比例无明显变化(P0.05),但S期细胞比例明显升高、G_2/M期细胞比例明显降低(均P0.05);沉默组Ki67﹑PCNA及cyclin A1蛋白表达量明显下调,而p21~(C1P1)蛋白表达量明显上调(均P0.05)。阴性对照组与空白对照组间以上指标差异均无统计学意义(均P0.05)。结论:lncRNA RP1-85F18.6在乳腺癌细胞系中高表达,其可能通过调节增殖相关蛋白及细胞周期蛋白的表达而参与乳腺癌的发生与发展,对lncRNA RP1-85F18.6及其相关靶基因的干预可能为乳腺癌的治疗提供新的途径。     

miR-26b的表达及其与Foxf2相互作用在乳腺癌细胞生物学行为中的作用

目的:探讨miR-26b在乳腺癌细胞中的表达及其对乳腺癌细胞生物学行为的的影响。方法:比较正常乳腺细胞系MCF-10A及乳腺癌细胞系MCF-7中miR-26b的表达差异。以无处理的MCF-7细胞为空白对照,分别检测MCF-7细胞转染miR-26b模拟物(miR-26b组)、空质粒(阴性对照组)后的miR-26b表达与增殖、迁移、侵袭能力,以及Foxf2的mRNA与蛋白表达的变化。用双荧光素酶报告系统检测miR-26b对MCF-7细胞中Foxf2转录活性的影响。结果:miR-26b在MCF-7细胞中的表达水平明显低于MCF-10A细胞(P0.05)。与空白对照组和阴性对照组比较,miR-26b组miR-26b mRNA表达水平明显升高、细胞增殖迁移、侵袭能力明显降低,Foxf2的mRNA和蛋白表达量均明显下调(P0.05)。转染miR-26b模拟物后,MCF-7细胞中Foxf2-3'UTR的转录活性明显抑制(P0.05)。结论:miR-26b在乳腺癌细胞中表达降低、增加其表达能抑制乳腺癌细胞的恶性生物学行为,机制可能与其下调Foxf2的表达有关。     

基质金属蛋白酶-2和纤维粘连蛋白在乳腺癌细胞系的定量表达

目的分析乳腺癌细胞系的基质金属蛋白酶-2(MMP-2)和纤维粘连蛋白(FN)mRNA表达及蛋白表达与乳腺癌转移的关系，阐明MMP-2和FN在乳腺癌转移中的作用。方法采用荧光定量逆转录-聚合酶链反应(RT—PCR)检测乳腺癌细胞系的MMP-2和FN mRNA表达量；采用Western blot方法检测细胞系的MMP-2和FN蛋白表达量。结果MMP-2和FN的mRNA表达量在乳腺癌高转移细胞系MDA-MB-231、MDA-MB-435中表达下调，而在低转移细胞系MDA-453、T47D、SK-BR-3和不转移细胞系MCF-7、ZR-75-30表达上调。MMP-2和FN蛋白表达在高转移细胞系上调，而在低转移细胞系和不转移细胞系中下调。结论MMP-2和FN mRNA表达和蛋白表达与乳腺癌转移相关，MMP-2和FN mRNA水平和蛋白水平的表达存在负反馈调节。