沉默MCM7基因介导AKT信号通路参与黑色素瘤细胞增殖及凋亡的实验

目的 探讨MCM7基因沉默介导AKT信号通路参与人皮肤黑色素瘤细胞的增殖及凋亡。方法 构建MCM7基因和沉默MCM7基因表达的慢病毒RNA（LV-shRNA-MCM7）的表达载体;将A375细胞分为Control组、Empty vector转染组（空载质粒转染组）、siRNA（LV-shRNA-MCM7）转染组、siRNA NC（LV-shRNA-MCM7 negative control）转染组;MTT法检测每组细胞增殖;流式细胞术检测细胞周期、细胞凋亡情况;划痕实验法检测细胞迁移能力;qRT-PCR法测定细胞中MCM7基因、AKT信号通路相关基因、Cyclin D1及凋亡相关基因Bcl-2、Bax、caspase-3的相对表达量;Western blot法测定蛋白相对表达量。结果 沉默MCM7后,黑色素瘤细胞中的MCM7基因、CyclinD1、Bcl-2、AKT信号通路相关基因AKT3的mRNA和蛋白相对表达量显著下调（P<0.05）;凋亡相关基因Bax、caspase-3的mRNA和蛋白表达量显著上调（P<0.05）;siRNA转染组凋亡率显著上升,G₀/G₁期细胞数目明显增多,S期细胞数目明显减少;细胞增殖、迁移能力显著下降（P<0.05）。结论 沉默MCM7基因抑制AKT信号通路的激活,从而抑制A375黑色素瘤细胞增殖、迁移,促进A375黑色素瘤细胞凋亡。     

盐霉素通过靶向调控ALDH影响三阴性乳腺癌细胞MDA-MB- 231增殖和凋亡

目的 探讨盐霉素（salinomycin,Sal）对三阴性乳腺癌细胞MDA-MB-231增殖和凋亡的影响及其作用机制。方法 采用流式细胞术筛选ALDH高表达乳腺癌细胞系;采用MTT法检测不同浓度Sal（1~32 μmol/L）对细胞增殖的影响;采用流式细胞仪检测Sal对细胞凋亡以及肿瘤干细胞表面标志物ALDH表达的影响;采用qRT-PCR、Western blot检测凋亡相关分子Caspase-3、Bax、Bcl-2、PARP mRNA和蛋白表达;TCGA（the Cancer Genome Atlas）数据库下载乳腺癌患者数据集（n=1 218）,采用Pearson法分析ALDH与凋亡相关分子表达的相关性。结果 Sal在24 h、48 h、72 h均可以剂量依赖的方式抑制MDA-MB-231细胞增殖（均P<0.001）;Sal作用MDA-MB-231细胞48 h后,与对照组比较,2 μmol/L组、4 μmol/L组、8 μmol/L组的细胞凋亡率均升高（均P<0.05）;Bcl-2、PARP、Caspase-3 mRNA表达下降（均P<0.05）,Bax mRNA表达升高（均P<0.05）;Bcl-2、Caspase-3 蛋白表达下降（均P<0.05）,PARP、Bax 蛋白表达升高（均P<0.05）;MDA-MB-231乳腺癌干细胞表面标志物ALDH表达下降（均P<0.05）。TCGA数据库分析显示,ALDH与抗凋亡分子Bcl-2、Caspase-3的表达呈正相关（r=0.209,P=0.007;r=0.235,P=0.002）,与Bax、PARP的表达呈负相关（r=-0.326,P＜0.001;r=-0.453,P＜0.001）。结论 Sal可抑制人乳腺癌MDA-MB-231细胞生长,可能通过下调肿瘤干细胞表面标志物ALDH表达实现。     

MiR-218-5p通过靶向抑制TDP1表达促进鱼藤酮诱导的胃癌细胞凋亡

目的 探讨过表达miR-218-5p和抑制TDP1的表达对鱼藤酮诱导损伤胃癌细胞凋亡的影响，阐明其可能的作用机制。方法 采用RT-PCR检测人正常胃黏膜上皮细胞和四种胃癌细胞中miR-218-5p及TDP1表达水平，并分析其相关性。双荧光素酶报告基因验证miR-218-5p对TDP1的靶向调控作用。采用1.0 μmol/L鱼藤酮诱导胃癌细胞损伤，流式细胞术检测细胞周期及凋亡率。Western blot检测细胞线粒体中TDP1水平及细胞Bax、Cyt-c蛋白的表达。结果 miR-218-5p在胃癌细胞中低表达（P<0.05），TDP1高表达（P<0.01），两者表达呈负相关（R²=0.9580, P=0.0212）。与对照组比较，损伤组SGC-7901细胞发生G1期阻滞，凋亡率升高（P<0.01）。与损伤组比较，miR-218-5pmimic组SGC-7901细胞G1期阻滞加剧，细胞凋亡率进一步升高（P<0.01），Bax及Cyt-c表达上调（P<0.01），而线粒体中TDP1蛋白水平降低（P<0.01）；TDP1过表达组细胞G1期阻滞得到缓解，凋亡率降低（P<0.01），线粒体中TDP1蛋白水平升高（P<0.01），Bax及Cyt-c表达降低（P<0.01）。结论 miR-218-5p可靶向抑制TDP1表达，诱导胃癌细胞凋亡，其作用机制可能与抑制线粒体DNA损伤修复及功能维持、激活线粒体内源性凋亡途径有关。     

miR-204对乳腺癌MCF-7细胞增殖及凋亡的影响

目的 探讨miR-204在人乳腺癌组织及MCF-7细胞中的表达水平及其对乳腺癌细胞增殖及凋亡的影响。方法 提取癌症和肿瘤基因组图谱（the cancer genome atlas,TCGA）数据库中浸润性乳腺癌的miR-204相关数据进行汇总,转染miR-204过表达病毒,筛选稳定转染细胞株并通过RT-qPCR检测转染率,MTT法检测过表达miR-204后乳腺癌MCF-7细胞的增殖情况,流式细胞仪检测细胞凋亡情况。 结果 miR-204在浸润性乳腺癌组织中的表达水平较癌旁组织明显下调（P＜0.001）,且与淋巴结转移和远处转移相关（P＜0.05）。过表达miR-204能够抑制乳腺癌MCF-7细胞的增殖能力（P＜0.05）;miR-204上调后,细胞凋亡率达到（34.7±1.9）%,细胞凋亡能力明显增强（P＜0.001）。结论 miR-204可抑制乳腺癌细胞增殖并介导细胞凋亡。     

辛伐他汀对人乳腺癌细胞MCF-7的生长抑制作用及机制研究

目的 探讨辛伐他汀（SVA）体外对乳腺癌细胞MCF-7的生长抑制作用及其可能的机制。 方法 选用人乳腺癌MCF-7细胞进行体外培养,采用四甲基偶氮唑盐法检测不同浓度SVA（0、3.25、6.25、12.5、25、50和100 μmol／L）处理MCF-7细胞24、48、72 h后的增殖抑制作用,荧光染色法观察SVA（0、2和4 μmol／L）处理MCF-7细胞48 h后的凋亡形态学变化,流式细胞仪测定SVA（0和4 μmol／L）处理MCF-7细胞48 h后的细胞周期变化,免疫印迹法分析SVA（0、2、4和8 μmol／L）处理MCF-7细胞72 h后细胞内Bcl-2和Bax蛋白水平的表达。结果 不同浓度SVA处理不同时间后,MCF-7细胞的增殖明显受到抑制,且增殖抑制作用呈时间和浓度依赖性;荧光显微镜观察发现SVA能够诱导MCF-7细胞出现核固缩、染色质凝集等凋亡形态学改变。流式细胞仪检测细胞周期显示,SVA阻滞MCF-7细胞于G0／G1期。免疫印迹 结果 显示,SVA处理组的Bcl-2蛋白表达水平低于对照组,Bax蛋白表达水平明显高于对照组,且随SVA浓度的增高,Bcl-2蛋白水平逐渐降低,Bax蛋白水平逐渐升高。结论 SVA对人乳腺癌MCF-7细胞具有增殖抑制作用,且呈浓度和时间依赖性,其抗肿瘤机制可能与诱导细胞周期阻滞、下调Bcl-2蛋白及上调Bax蛋白表达有关。     

上调SCD1对乳腺癌细胞增殖凋亡的影响及与其相关通路的调控机制

目的:探究上调硬脂酰辅酶A去饱和酶1（SCD1）对乳腺癌细胞增殖凋亡的影响及与其相关通路的调控机制。方法:将细胞分为三组,即对照组、阴性对照组和SCD1组。检测和比较每组的转染效率、细胞活力、增殖情况以及凋亡率。qPCR检测mRNA水平,Western blot检测蛋白表达水平。结果:SCD1组细胞的SCD1 mRNA和蛋白的表达水平显著高于对照组和阴性对照组。SCD1组的细胞活力和克隆形成数目显著高于对照组和阴性对照组。SCD1组的细胞凋亡率显著低于对照组和阴性对照组。转染SCD1后,细胞表达的Cyclin D1、PCNA和Bcl-2 mRNA水平显著升高,Caspase3、Bax mRNA水平显著降低。乳腺癌MCF-7细胞SCD1过表达后,Wnt、β-catenin mRNA和蛋白的表达水平均显著升高。结论:上调SCD1的水平可通过促进Wnt/β-catenin通路促进乳腺癌细胞的增殖并抑制其凋亡。     

桔梗皂苷D对人移行细胞癌5637细胞株的诱导凋亡作用及其分子机制

目的 探讨桔梗皂苷D对人移行细胞癌5637细胞诱导凋亡作用及分子机制。方法 HE染色和透射电子显微镜观察细胞的形态学变化;流式细胞学技术检测细胞凋亡率的变化;Western blot法检测凋亡通路相关蛋白的表达变化;PCR法检测p53、Bcl-2、Bax mRNA表达;免疫组织化学法检测凋亡相关蛋白表达的影响。结果 通过HE染色和电子显微镜观察桔梗皂苷D组5637细胞,可见凋亡细胞,凋亡指数明显高于对照组（P<0.01）;与对照组细胞相比,桔梗皂苷D组5637细胞Survivin、Livin表达量明显下降（P<0.05）;Caspase-9、Caspase-8、Caspase-3、Cyt-c表达量与对照组比较,明显增加（P<0.05）;p53、Bax mRNA水平明显升高,Bcl-2 mRNA表达下降（P<0.01）; Bax、Caspase-9、Caspase-8以及Caspase-3表达较对照组增多,而Bcl-2表达则明显下降（P<0.01）。结论 桔梗皂苷D能诱导人移行细胞癌5637细胞凋亡,其作用机制可能与上调Caspase-9、Caspase-8、Caspase-3、Cyt-c、p53和下调Bcl-2表达、激活线粒体途径和死亡受体途径有关。     

自噬基因Beclin1在细针穿刺乳腺病变中的表达及其与Bcl-2和p53的相关性

目的 检测细胞自噬基因Beclin1在细针穿刺乳腺良恶性病变中的表达及其与Bcl-2、p53的关系,探讨其在乳腺癌发生发展中的作用及其机制。方法 采用RT-PCR和免疫组织化学检测乳腺良恶性病变中Beclin1、Bcl-2、p53mRNA及蛋白表达水平。结果 Beclin1 mRNA在乳腺癌中的表达明显低于在乳腺良性病变中的表达（P<0.05）,Bcl-2、p53 mRNA在乳腺癌中的表达高于在乳腺良性病变中的表达（P<0.05）。Beclin1蛋白在乳腺癌中的阳性表达率明显低于在乳腺良性病变中的阳性表达率（P<0.05）;Bcl-2、p53蛋白在乳腺癌中的阳性表达率明显高于在乳腺良性病变中的阳性表达率（P<0.05）;在乳腺癌中Beclin1蛋白表达与Bcl-2和p53蛋白表达之间存在负相关（P<0.05）;而Bcl-2和p53蛋白的表达无相关性（P>0.05）。结论 Beclin1在乳腺癌组织中表达下调,而Bcl-2、p53在乳腺癌中均有高表达。Beclin1蛋白与Bcl-2、p53蛋白在乳腺癌组织中的表达存在负相关。     

基因转染LL-37/hCAP-18对人乳腺癌细胞凋亡的影响

目的：探讨基因转染人源抗菌肽LL-37/hCAP-18对人乳腺癌细胞MCF-7增殖、凋亡的影响。方法：将含人LL-37/hCAP-18的真核表达质粒瞬时转染人MCF-7细胞,48h后,MTT比色检测MCF-7细胞的增殖,流式细胞术检测MCF-7细胞周期及凋亡,细胞免疫组化染色检测MCF-7细胞中Bax、Bcl-2的表达。结果：重组基因瞬时转染MCF-7细胞48h后,与各对照组相比,肿瘤细胞的生长增殖受到显著抑制（P〈0.05）;转染重组基因组肿瘤细胞凋亡率显著高于各对照组（P〈0.05）,但细胞周期无明显变化;与对照组相比,转染重组基因pEGFP-c1-LL-37的MCF-7细胞中Bcl-2表达显著下降（P〈0.05）,Bax由阴性表达转为表达明显升高。结论：LL-37/hCAP-18可以通过上调促凋亡因子Bax、下调抗凋亡因子Bcl-2诱导肿瘤细胞凋亡,从而抑制乳腺癌细胞MCF-7的增殖。     

新藤黄酸抑制人乳腺癌细胞株MCF-7增殖的实验研究

目的 探讨新藤黄酸对人乳腺癌细胞株MCF-7的增殖抑制情况及其作用机制。方法 0.5～20μg／ml新藤黄酸处理MCF-7细胞72h,四甲基偶氮唑盐（MTT）法检测MCF 7细胞增殖抑制率;流式细胞术Annexin V-FITC/PI双染色检测细胞凋亡率;线粒体膜电位检测试剂盒（JC-1）检测线粒体跨膜电位变化;Western blotting检测Fas、FasL、caspase-3、caspase-8、caspase-9、Bax和Bcl-2蛋白表达水平。结果 0.5～20μg／ml新藤黄酸均能够抑制MCF-7细胞增殖,且增殖抑制作用呈浓度依赖,半数抑制浓度（IC₅₀）为1763 μg／ml。0.5～3.0μg／ml新藤黄酸即可诱导MCF-7细胞凋亡,凋亡作用呈时间和浓度依赖。0.5μg／ml新藤黄酸处理MCF 7细胞48h后早期凋亡率为3.7％,总凋亡率为7.2％,72h早期凋亡率为6.7％,总凋亡率为13.7％;3μg／ml新藤黄酸48h后早期凋亡率为69.5％,总凋亡率为71.7％,72h早期凋亡率为76.9％,总凋亡率为81.5％。0.5、1.0、1.5 μg／ml新藤黄酸导致线粒体跨膜电位下降的细胞比例升高,促凋亡相关蛋白 FasL、caspase-3、caspase-8、caspase-9表达水平呈浓度依赖性上升,Fas和Bax蛋白表达水平变化不大,抗凋亡蛋白Bcl-2表达水平则呈浓度依赖性下降。结论 新藤黄酸通过诱导人乳腺癌细胞株MCF-7凋亡,抑制细胞增殖,其诱导凋亡的分子机制与死亡受体及线粒体凋亡途径密切相关。     

Wnt信号通路在姜黄素诱导乳腺癌MCF-7细胞凋亡中作用机制

目的 探讨Wnt通路在姜黄素诱导人乳腺癌MCF-7细胞凋亡中的作用机制.方法 将MCF-7细胞培养液随机分为5组,分别加入15、30、60、120μmol/L的姜黄素,对照组不加,培养12,24,48 h后采用CCK-8法检测细胞增殖能力.分A、B两组(剂量组、时间组)采用Annexin V-FITC/PI双染流式细胞术(FCM)观测细胞凋亡及细胞周期分布;Western Blotting法检测A、B两组的Wnt通路相关蛋白,并进行相应的相关性分析探讨Wnt通路与乳腺癌MCF-7细胞凋亡的关系.结果 CCK-8结果显示,随培养时间延长,姜黄素各浓度组MCF-7细胞增殖抑制率逐渐增高(均P<0.05);在同一时间点,随着姜黄素浓度升高,细胞增殖抑制率逐渐增高(均P<0.05).FCM染色结果显示,随着姜黄素浓度增加、作用时间延长MCF-7细胞凋亡指数逐渐上升(均P<0.05),且G1/S期细胞增加越多(P<0.05).Western Blotting实验结果显示MCF-7细胞中Wnt1、β-catenin的表达下调程度呈现剂量时间依赖性(P<0.05).相关性分析显示细胞抑制率、G0/G1期细胞百分比、细胞凋亡率均与Wnt通路相关蛋白Wnt1、β-catenin的表达呈现较明显的相关性(P<0.05).结论 姜黄素的抗癌活性与抗增殖能力具有明显的剂量时间依赖性,且Wnt通路在姜黄素诱导乳腺癌MCF-7细胞抑制、凋亡过程中发挥了重要作用.     

PXD101对人乳腺癌细胞MCF-7增殖及凋亡影响的机制探讨

  目的  探讨组蛋白去乙酰化酶抑制剂PXD101对人乳腺癌细胞MCF-7增殖、细胞周期及凋亡的影响及分子机制研究。  方法  应用不同浓度PXD101处理培养的乳腺癌细胞株MCF-7, 通过赛唑蓝比色(MTT)法和平板克隆形成实验检测药物对细胞增殖的影响; HoechSt33342荧光染色法观察细胞形态变化; 流式细胞仪PI染色法检测细胞周期变化以及AnnexinV-FITC/PI双染法检测细胞凋亡情况; Westen blot检测p21、CyclinB1、PARP、Bcl-2以及Bax的蛋白表达。  结果  PXD101以剂量时间依赖性抑制MCF-7细胞的增殖; 荧光显微镜观察发现细胞核碎裂, 出现凋亡小体; 0、0.1、1、10μmol/L PXD101作用24 h后, G₂/M期细胞比例增加, 分别为(12.66±1.55)%、(20.63±1.32)%、(23.20±1.82)%、(32.19±2.37)%(P < 0.05), 凋亡细胞也增加(P < 0.05);p21表达增多, CyclinBl表达减少, PARP剪切明显增加, Bcl-2表达减少, Bax表达增加。  结论  PXD101在体外条件下能够明显抑制乳腺癌MCF-7细胞的增殖, 诱导细胞周期阻滞及凋亡, 并呈剂量依赖性。      

Ectopic expression of cyclin D1 amplifies a retinoic acid-induced mitochondrial death pathway in breast cancer cells.

All-trans retinoic acid inhibits growth associated with downregulation of cyclin D1 and can cause low level apoptosis in estrogen receptor positive breast cancer cell lines. The cyclin D1 gene is amplified and/or the protein overexpressed in about one-third of breast cancers. Constitutive expression of cyclin D1 in estrogen receptor positive MCF-7 and ZR-75 breast cancer cells (MCF-7(cycD1) and ZR-75(cycD1)) Increased the fraction of cells in S phase and reduced the G1 accumulation following retinoic acid treatment compared with control cells. However, culture of MCF-7(cycD1) with 1 microM all-trans retinoic acid resulted in about threefold greater growth inhibition compared with vector-transfected cells. Hoechst staining of DNA and in situ DNA end-labeling analysis indicated that MCF-7(cycD1) and ZR-75(cycD1) cultures contained 4-6-fold more retinoic acid-induced apoptotic nuclei as vector-transfected cells. Retinoic acid treatment of vector-transfected clones resulted in Bax protein activation as assessed by exposure of the NH(2)-terminus of Bax but the proportion of cells containing activated Bax was increased in cyclin D-expressing cells treated with retinoic acid. The latter cells also displayed both immunocytochemical and biochemical evidence of translocation of cytochrome c into the cytosol following RA-treatment. Retinoic acid markedly decreased the Bcl-2 levels in MCF-7 and ZR-75 cells. Accordingly, coexpression of Bcl-2 and cyclin D1 rendered the cells resistant to retinoic acid-induced apoptosis. We conclude that constitutive expression of cyclin D1 sensitizes ER-positive breast cancer cells to a retinoic acid-induced mitochondrial death pathway involving Bax activation, cytochrome c release and caspase-9 cleavage.     

羟喜树碱通过诱导自噬抗宫颈癌的机制研究

目的探讨抗肿瘤药物羟喜树碱(HCPT)对宫颈癌HeLa细胞作用的机制。方法 CCK8检测Hela细胞增殖,Western blot检测自噬和凋亡相关蛋白的表达,GFP-LC3 shRNA转染和Hoechst染色后,荧光显微镜下观察细胞自噬特异小体量的改变以及凋亡形态学的变化。结果 HCPT抑制Hela细胞生长呈浓度依赖性(P<0.05),IC503μmol/L HCPT作用Hela细胞后,自噬相关蛋白Beclin1、p62的表达以及LC3-Ⅱ/LC3-Ⅰ比值发生改变,差异有显著性意义(P均<0.05);凋亡相关蛋白Bax、cleaved caspase-3、Bcl-2表达也发生明显改变(P均<0.05);荧光显微镜下观察Hela细胞带有GFP-LC3的自噬体增加,凋亡细胞也增多(P均<0.05)。结论 HCPT能够诱导HeLa细胞中自噬相关基因Beclin1、p62以及LC3的表达增强,从而激活细胞自噬发生;且HCPT能够激活宫颈癌HeLa细胞发生自噬,进而诱导细胞凋亡来达到抗肿瘤目的。     

20(S)-Protopanaxadiol Induces Human Breast Cancer MCF-7 Apoptosis through a Caspase-Mediated Pathway

20(S)-Protopanaxadiol (PPD), a ginsenoside isolated from Pananx quinquefolium L., has been shown toinhibit growth and proliferation in several cancer cell lines. The aim of this study was to evaluate its anticanceractivity in human breast cancer cells. MCF-7 cells were incubated with different concentrations of 20(S)-PPDand cytotoxicity was evaluated by MTT assay. Occurrence of apoptosis was detected by DAPI and AnnexinV-FITC/PI double staining. Mitochondrial membrane potential was measured with Rhodamine 123. The Bcl-2and Bax expression were determined by Western blot analysis. Caspase activity was measured by colorimetricassay. 20(S)-PPD dose-dependently inhibited cell proliferation in MCF-7 cells, with an IC50 value of 33.3 μM at24h. MCF-7 cells treated with 20(S)-PPD presented typical apoptosis, as observed by morphological analysisin cell stained with DAPI. The percentages of annexin V-FITC positive cells were 8.92%, 17.8%, 24.5% and30.5% in MCF-7 cells treated with 0, 15, 30 and 60μM of 20(S)-PPD, respectively. Moreover, 20(S)-PPD couldinduce mitochondrial membrane potential loss, up-regulate Bax expression and down-regulate Bcl-2 expression.These events paralleled activation of caspase-9, -3 and PARP cleavage. Apoptosis induced by 20(S)-PPD wasblocked by z-VAD-fmk, a pan-caspase inhibitor, suggesting induction of caspase-mediated apoptotic cell death.In conclusion, the 20(S)-PPD investigated is able to inhibit cell proliferation and to induce cancer cell death bya caspase-mediated apoptosis pathway.     

Id-1 regulates Bcl-2 and Bax expression through p53 and NF-κB in MCF-7 breast cancer cells

Although increasing evidence supports the protective role of inhibitor of differentiation and DNA binding-1 (Id-1) against anticancer drug-induced apoptosis, the underlying molecular mechanisms seem to vary depending on the tumor system. Here, we examined the direct role of Id-1 in MCF-7 breast cancer cells by ectopically overexpressing Id-1 under serum-free condition, where the endogenous Id-1 expression was suppressed. Id-1 expression resulted in increased number of viable cells, reduced Bax expression, enhanced Bcl-2 expression, but no change in Bcl-xL expression. The expression of nuclear factor-κB (NF-κB) was augmented, while those of p53 and IκB were reduced. Such changes in p53 and NF-κB pathways were also functional, as assessed by real-time polymerase chain reactions and reporter assays of their known downstream targets, p21 and Il-6, as well as Bax and Bcl-2 genes. Finally, Id-1 played a protective role against taxol-induced apoptosis in breast cancer cells as assessed by MTT assay and apoptotic cell count upon taxol treatment (0–200 nM). Reduced Bax expression and enhanced Bcl-2 expression by Id-1 were also noted in the presence of taxol. Taken together, we present a molecular mechanism where Id-1 regulates p53 and NF-κB pathways, which in turn regulates Bax and Bcl-2 genes, thus providing a survival advantage under exogenous stress such as serum-free or taxol treatment in MCF-7 breast cancer cells. In this regard, inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of breast cancer progression and anti-cancer drug resistance. Hwan Kim and Heekyoung Chung equally contributed to this work.     

Estrogen increases intracellular p26Bcl-2 to p21Bax ratios and inhibits taxol-induced apoptosis of human breast cancer MCF-7 cells

Recent studies have demonstrated that following estrogen ablation, estrogen responsive breast cancer cells undergo apoptosis. In addition, estrogen receptor (ER) expression has been strongly correlated with the expression of the bcl-2 gene product, p26Bcl-2 protein, which is known to inhibit apoptosis. In the present studies, we investigated whether estrogen affects the intracellular levels of p26Bcl-2 and thereby modulates taxol-induced apoptosis of estrogen responsive human breast cancer MCF-7 cells. Transfer of MCF-7 cells to a culture-medium without estrogens reduced their intracellular p26Bcl-2 levels by 50%. Inclusion of 0.1 M estradiol in the medium produced approximately a four-fold increase in p26Bcl-2, but not p29Bcl-xL or p21Bax levels; the expression of the c-myc and mdr-1 genes remained unchanged. Estradiol-induced four-fold increase in the ratio of the p26Bcl-2 to p21Bax levels caused a significant decline in the lethal, kilobase size DNA fragments of apoptosis, which had resulted when MCF-7 cells were cultured in a medium without estrogen. In addition, in MCF-7 cells, estradiol-induced increase in the intracellular p26Bcl-2 to p21Bax ratios was associated with a significant reduction in the large-sized DNA fragmentation induced by treatment with taxol. The increased ratios also protected MCF-7 cells against taxol-mediated cytotoxicity as assessed by the MTT assay. These results suggest that by modulating p26Bcl-2 levels, estrogens may affect the antitumor activity of taxol and potentially of other anti-breast cancer drugs against estrogen responsive human breast cancer cells.     

Radiosensitizing effects of arsenic trioxide on MCF-7 human breast cancer cells exposed to 89strontium chloride

The aim of this study was to investigate the radiosensitizing effects of arsenic trioxide (As2O3) on MCF-7 human breast cancer cells irradiated with 89strontium chloride (89SrCl2). The 50% inhibitory concentration (IC50) was calculated from results of an MTT assay. The concentration of As2O3 less than 20% IC50 was selected for subsequent experiments. Cells were treated with As2O3 and 89SrCl2. Morphological changes of cells were observed under an inverted microscope. The radiosensitivity enhancing ratio (SER) was computed based on a clone formation assay. Cell cycle distribution and apoptosis were measured by flow cytometry (FCM). Expression of Bcl-2 and Bax at both the mRNA and protein levels was assessed by RT-PCR and western blotting. The IC50 of As2O3 at 24?h was 11.7?μM. Doses of As2O3 (1 and 2?μM) were used in combin-ation treatments and SER values were 1.25 and 1.79, respectively. As2O3 significantly suppressed cell growth, caused G2/M arrest, enhanced cell death and apoptosis induced by 89SrCl2 and decreased expression of the Bcl-2 gene. Since expression of Bax was unchanged following treatment, As2O3 effectively reduced the Bcl-2/Bax ratio. As2O3 (1-2?μM) enhances the cytotoxic effects of 89SrCl2 on the MCF-7 human breast cancer cell line by inducing G2?phase delay and promoting apoptosis through the reduction of the Bcl-2/Bax ratio.