桃儿七根醇提物对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的:研究桃儿七根醇提物对人乳腺癌MCF-7细胞增殖和凋亡的影响,探讨其抗癌作用的机制。方法:应用MTT法检测桃儿七根醇提物对人乳腺癌MCF-7细胞的生长抑制作用,倒置显徽镜、HE染色及电镜观察细胞形态学改变,流式细胞仪(FCM)分析细胞周期及凋亡率,免疫细胞化学法(ICC)检测细胞增殖、凋亡相关调控蛋白的表达。结果:桃儿七根醇提物可明显抑制MCF-7细胞的增殖,1,2,3 mg．L~(-1)桃儿七根醇提物作用72h后,细胞生长抑制率分别为43．0%,54．9%,78．5%,抑制作用呈时间及浓度依赖性。形态学观察到用药后凋亡细胞典型的形态学特征,伴有多核细胞形成。FCM分析发现桃儿七根醇提物阻断MCF-7细胞生长于细胞周期的G_2/M期,凋亡诱导作用呈时间及浓度依赖性。免疫细胞化学结果显示,2mg·L~(-1)桃儿七根醇提物作用48h后,PCNA,Bcl-2蛋白表达较对照组显著下降;而p21~(WAFI-CIP1)和c-Myc蛋白显著增加(P＜0．01)。结论:桃儿七根醇提物可能通过增加p21~(WAFI-CIP1)蛋白表达及降低PCNA蛋白表达而抑制人乳腺癌MCF-7细胞的增殖,并可能下调Bcl-2蛋白表达和上调c-Myc蛋白表达来促进MCF-7细胞凋亡。     

翡翠贻贝多糖抑制乳腺癌MCF-7细胞增殖的实验研究

目的观察翡翠贻贝多糖（PVPS）对体外培养人乳腺癌MCF-7细胞增殖的抑制作用。方法体外培养的乳腺癌MCF-7细胞经不同浓度翡翠贻贝多糖处理48h后,经CCK-8试剂盒检测分析细胞存活率、用流式细胞术分析各组细胞凋亡率;光镜下观察40mg/L翡翠贻贝多糖处理细胞不同时间后的细胞形态变化。结果翡翠贻贝多糖40mg/L组和80mg/L组的乳腺癌细胞存活率明显低于对照组,而细胞凋亡率则明显高于对照组（P〈0.05或P〈0.01）。光镜下可见肿瘤细胞脱落随作用时间延长而明显。结论翡翠贻贝多糖可抑制体外培养乳腺癌细胞增殖并诱导其凋亡。     

铜绿假单胞茵注射液对人乳腺癌细胞的体外杀伤及促凋亡作用研究

目的研究铜绿假单胞菌（pseudo—monas aeruginosa,PA-MSHA）注射液在体外实验中对人乳腺癌细胞（MCF-7）凋亡和增殖的影响,并探讨其可能的作用机制。方法应用MTT比色法检测铜绿假单胞菌注射液对体外培养的MCF-7细胞增殖抑制作用,应用流式细胞术检测铜绿假单胞菌注射液诱导乳腺癌细胞凋亡,检测其对细胞周期的影响。用Hoechst33258染色法染色,荧光显微镜检测铜绿假单胞菌注射液诱导体外培养的MCF-7肿瘤细胞凋亡的形态学改变。结果铜绿假单胞菌注射液抑制MCK7细胞增殖,呈剂量和时间依赖性;与对照组相比,铜绿假单胞菌注射液组MCF7细胞的凋亡率明显增高（P〈0．01）、G0／G1期细胞比例明显增高、S期细胞所占百分比明显降低（P〈0．05）,染色荧光显微镜检测发现PAMSHA能诱导体外培养的肿瘤细胞呈现明显的凋亡图像。结论铜绿假单胞菌注射液可能通过对细胞周期中G0／G1期阻滞,诱导MCF-7细胞凋亡,从而抑制MCF-7细胞的增殖。     

蒲公英根水提物诱导MDA-MB-231细胞凋亡及其作用机制研究

目的探究蒲公英根水提物对人三阴性乳腺癌细胞MDA-MB-231增殖和凋亡的影响及其作用机制。方法采用Alamar Blue法检测蒲公英根水提物的细胞毒性,以及对MDA-MB-231细胞增殖的抑制作用;通过形态学观察、DAPI染色、流式细胞术,检测蒲公英根水提物对MDA-MB-231细胞凋亡的影响;Western blot分析蒲公英根水提物诱导MDA-MB-231细胞凋亡的作用机制。结果蒲公英各部位提取物无细胞毒性;且7.5、15 g·L~(-1)的蒲公英根水提物明显抑制MDA-MB-231细胞的增殖,细胞出现明显的凋亡特征,流式细胞术进一步证实了蒲公英根水提物能够诱导MDA-MB-231细胞凋亡;蒲公英根水提物呈浓度依赖性降低Bcl-2的蛋白表达,增加Bax、p53、活性c-PARP、caspase-8、caspase-9和caspase-3表达量; caspase-8抑制剂(Z-IETD-FMK)和caspas-9抑制剂(Z-LEHD-FMK)能够逆转蒲公英水提物对MDA-MB-231细胞的增殖抑制活性。结论蒲公英根水提物对人三阴性乳腺癌细胞MDA-MB-231具有明显的抑制作用,对治疗三阴性乳腺癌具有潜在的临床应用价值。     

原花青素通过Caspase途径诱导乳腺癌MCF-7细胞凋亡

目的研究原花青素(Proanthocyanidin/Procyanidin,PC)诱导人乳腺癌细胞凋亡以及凋亡通路,揭示其抗乳腺癌的部分作用机制。方法常规培养细胞,24 h后随机分为阳性对照组、空白对照组和PC组,MTT法检测PC对人乳腺癌细胞增殖的抑制作用;流式细胞术检测凋亡率和细胞周期;ELISA法检测培养上清液中Caspase-9、Caspase-12的含量。结果 PC(10～320μg/mL)6个剂量组可显著抑制MCF-7细胞的增殖,且呈时间和剂量依赖性;在40、80和160μg/mL 3个浓度下体外培养,PC可提高MCF-7细胞的凋亡率,并诱导出现G2-M细胞周期阻滞;PC作用48 h后,Caspase-9、Caspase-12蛋白表达量增加。结论 PC抑制人乳腺癌MCF-7细胞的作用机制与阻滞细胞周期于G2-M期和诱导细胞凋亡有关,其凋亡通路与Caspase途径有关,并涉及Caspase家族的内质网和线粒体两条通路。     

槐米中槲皮素提纯、鉴定以及诱导人乳腺癌MCF-7细胞凋亡作用

目的:研究从槐米中槲皮素(Quercetin,Que)提取、鉴定和对人乳腺癌MCF-7细胞生长的抑制以及诱导凋亡作用.方法:采用碱溶解酸沉淀法提纯槐米中槲皮素,并进行红外光谱分析;采用体外培养人乳腺癌MCF-7细胞,用不同浓度槲皮素处理;采用四甲基偶氮唑蓝(MTT)比色法测定细胞生长抑制率;采用细胞凋亡DNA Lad　der和FITC-Annexin V/PI荧光标记流式细胞仪检测,槲皮素作用后MCF-7细胞凋亡情况.结果:不同浓度槲皮素对人乳腺癌MCF 7细胞有生长抑制作用,并呈浓度和时间依赖性(P＜0.05);10.0 mmol/L槲皮素作用MCF-7细胞2d,DNA电泳出现特征性的凋亡条带;10.0 mmol/L槲皮素处理MCF-7细胞1d、2d和3d,细胞凋亡率分别为(9.2±1.5)％、(30.0±11.8)％和(60.8±10.6)％.结论:用碱溶解酸沉淀法可从槐米中提纯槲皮素,提取的槲皮素对人乳腺癌MCF-7细胞有生长抑制和诱导凋亡作用.     

异硫氰酸苯乙酯对人乳腺癌MCF-7细胞凋亡作用及机制研究

目的探讨异硫氰酸苯乙酯诱导人乳腺癌MCF-7细胞凋亡的作用及其可能的机制。方法以不同浓度异硫氰酸苯乙酯处理人乳腺癌MCF-7细胞,应用MTS法观察异硫氰酸苯乙酯对MCF-7细胞的抑制率,流式细胞术检测异硫氰酸苯乙酯对细胞凋亡、细胞周期以及活性氧表达的影响。结果将不同浓度的异硫氰酸苯乙酯作用于MCF-7细胞72h后,抑制了MCF-7细胞的增殖,其IC50为15.2μmol.L-1;以10和20μmol.L-1的异硫氰酸苯乙酯作用MCF-7细胞24h后,细胞出现凋亡现象,细胞周期阻滞在G2/M期,并造成细胞氧化损伤。结论异硫氰酸苯乙酯能够促进人乳腺癌MCF-7细胞凋亡,影响细胞周期,其作用机制可能与异硫氰酸苯乙酯可造成肿瘤细胞氧化损伤有关。     

雷公藤甲素对ERα蛋白介导的人乳腺癌MCF-7细胞增殖抑制的影响

目的：研究雷公藤甲素对激素依赖性人乳腺癌细胞MCF-7增殖的抑制作用及可能的机制。方法：MTT法检测雷公藤甲素对MCF-7细胞增殖的抑制作用,Hoechst染色法检测其对细胞凋亡的影响,Western blotting法观察雷公藤甲素对雌激素受体（ER）α蛋白表达的影响。结果：雷公藤甲素对MCF-7细胞增殖有显著的抑制作用,并且呈现良好的时效和量效关系,雷公藤甲素能有效诱导MCF-7细胞凋亡,并且对ERα具有明显的下调作用。结论：雷公藤甲素能抑制人乳腺癌细胞MCF-7增殖并且诱导其凋亡,其诱导MCF-7细胞凋亡的作用机制可能与其下调ERα的表达有关。     

丹参酮ⅡA逆转人ER阳性乳腺癌细胞的恶性表型及其机理

目的 证实丹参酮ⅡA对体外培养的雌激素受体(ER)阳性人乳腺癌细胞(MCF-7)的增殖抑制及恶性表型的逆转作用;探讨其作用的分子机理.方法 用光镜、电镜观察丹参酮ⅡA处理前后细胞形态的改变;细胞增殖试验(MTT法)、集落形成试验检测丹参酮ⅡA对MCF-7细胞生长、增殖的影响;RT-PCR技术半定量检测细胞增殖、分化相关基因mRNA表达的改变;流式细胞仪定量检测细胞增殖、分化相关基因蛋白表达的改变.结果 经一定浓度丹参酮ⅡA处理后MCF-7细胞形态改变,接近正常上皮细胞形态;丹参酮ⅡA对MCF-7细胞增殖抑制作用具有明显的剂量一时问依赖关系;对MCF-7细胞增殖具抑制作用;可上调增殖、分化相关基因ERa、nm23-1、c-fos的表达,下调c-myc的表达.结论 丹参酮ⅡA对MCF-7细胞体外生长、增殖有明显抑制作用;对MCF-7细胞形态具有恶性表型逆转作用;可上调细胞ERa、c-fos、nm23-1和下调c-myc基因表达,可能通过调控细胞增殖和分化相关基因的表达来逆转肿瘤细胞的恶性表型.     

路路通酸对乳腺癌MCF-7细胞和宫颈癌C-33A细胞增殖的影响

目的研究路路通酸(BTA)对乳腺癌MCF-7细胞及宫颈癌C-33A细胞增殖的影响。方法采用MTT法检测不同浓度BTA作用不同时间后对MCF-7细胞、C-33A细胞的增殖抑制作用;流式细胞仪检测BTA处理后细胞周期的变化。结果 BTA作用于乳腺癌MCF-7细胞24、48、72 h后的IC50分别为(37.62±1.72)、(27.32±0.99)、(19.19±0.90)μmol/L。BTA作用于宫颈癌C-33A细胞24、48、72 h后的IC50分别为(34.55±0.88)、(27.20±1.03)、(16.74±0.79)μmol/L,BTA对2种细胞增殖有明显抑制作用,且呈浓度时间依赖性(P<0.05),流式结果显示,BTA将MCF-7细胞阻滞在S期,并诱导其凋亡;BTA将C-33A细胞阻滞在G1-S期。结论BTA对乳腺癌MCF-7细胞和宫颈癌C-33A细胞具有较强的增殖抑制作用,其机制与细胞周期阻滞和诱导细胞凋亡有关。     

人参皂甙Rg3对乳腺癌MCF-7线粒体凋亡途径的影响

目的探讨20(R)–人参皂甙Rg3(SPG-Rg3)对人乳腺癌MCF-7细胞的诱导凋亡作用及其可能机制。方法人乳腺癌细胞MCF-7细胞株分为空白对照组、实验对照组及SPG-Rg3多个浓度组。利用MTT法观察人参皂甙Rg3对MCF-7细胞生长的抑制作用,并计算出IC-50,进一步确定其有效浓度;流式细胞术检测人参皂甙Rg3作用后MCF-7细胞周期的变化;利用AnnexinV-EGFP/PI双染法,检测人参皂甙Rg3诱导MCF-7细胞凋亡情况;免疫细胞化学染色检测MCF-7细胞凋亡与Fas、FasL蛋白表达的关系。结果 SPG-Rg3作用48h的IC-50为244.54μg/mL。流式细胞仪检测Rg3使MCF-7的S期细胞比率明显增加(P<0.01),凋亡率明显增加(P<0.01)。免疫细胞化学显示Rg3能使MCF-7细胞胞浆内细胞色素C增加(P<0.01)。结论 SPG-Rg3能诱导乳腺癌MCF-7细胞凋亡,其机制可能与诱导线粒体释放细胞色素C有关。     

Anastatica hierochuntica (L.) methanolic and aqueous extracts exert antiproliferative effects through the induction of apoptosis in MCF-7 breast cancer cells

Breast cancer therapy using anticancer bioactive compounds derived from natural products as adjuvant treatment has gained recognition due to expensive and toxic conventional chemotherapeutic drugs. The whole plant of Anastatica hierochuntica (L.) (A. hierochuntica) has been investigated for its pharmacologically important anticancer properties but without categorizing the biological activities of the plant parts. We assessed the anticancer potential of different parts of A. hierochuntica (seeds, stems and leaves) and explored their mechanisms of action using the human breast cancer cell line, MCF-7. Currently, we investigated the antiproliferative effects of methanolic (MSD, MST, ML) and aqueous (ASD, AST, AL) extracts of A. hierochuntica plant parts on the MCF-7 cells using cell viability assays. Flow cytometry, Western Blot, DNA fragmentation, and gene expression assays were performed to evaluate apoptosis and cell cycle regulatory proteins. The results indicate that the methanolic and aqueous extracts decreased MCF-7 cell viability in a dose-dependent manner. The induction of apoptosis was observed in all the methanolic and aqueous-treated MCF-7 cells. The cell death process was confirmed by the visualization of DNA fragmentation and cleavage of the intrinsic apoptotic pathways, caspase-9 and caspase-3, the key enzyme causing apoptosis hallmarks. In addition, the most pro-apoptotic extracts, ASD and ML, up-regulated the expression of pro-apoptotic Bax, tumor suppressor TP53 genes and the cyclin inhibitor CDKN1A gene. In conclusion, of the aqueous and methanolic extracts of A. hierochuntica plant parts exerting antiproliferative effects through the induction of apoptosis in breast cancer MCF-7 cells, ASD and ML extracts were the most promising natural-based drugs for the treatment of breast cancer.     

透骨草提取物通过Bcl-2和Bax蛋白诱导人乳腺癌细胞株MCF-7凋亡

目的探讨Bax和Bcl-2蛋白在透骨草提取物诱导人乳腺癌细胞株MCF-7细胞凋亡过程中的作用,为透骨草提取物治疗乳腺癌提供实验依据。方法采用吖啶橙染色方法透骨草提取物对MCF-7细胞形态的影响;采用流式细胞术检测透骨草提取物对MCF-7细胞凋亡率的影响;采用蛋白印迹法检测MCF-7细胞Bcl-2和Bax蛋白的表达。结果 30.5μg/ml透骨草提取物可诱导MCF-7细胞胞体皱缩,胞核固缩、呈新月状且边集,出现凋亡小体。30.5μg/ml透骨草提取物处理的MCF-7细胞凋亡率（22.3±1.2）%明显高于对照组（3.2±1.0）%（P〈0.05）。30.5μg/ml透骨草提取物可使MCF-7细胞Bcl-2蛋白表达明显低于对照组（P〈0.05）,而Bax蛋白表达明显高于对照组（P〈0.05）。结论透骨草提取物对MCF-7细胞有诱导凋亡作用,其作用机制可能与下调Bcl-2蛋白表达、上调Bax蛋白表达有关。     

三氧化二砷诱导乳腺癌细胞凋亡的早期事件——凋亡性容量下降

目的探讨凋亡性容量下降(apoptotic volume decrease,AVD)是否是三氧化二砷(arsenic trioxide,As2O3)诱导乳腺癌MCF-7细胞凋亡的早期事件。方法噻唑兰(MTT)法检测As2O3对MCF-7细胞存活率的影响;流式细胞技术检测As2O3能否引起MCF-7细胞凋亡;时滞显微摄影技术检测细胞容量;Caspase-3试剂盒检测凋亡信号活性。结果 MTT实验表明As2O3使MCF-7细胞存活率下降并呈现剂量依赖的量效关系;流式细胞测定结果证实As2O3对乳腺癌细胞的致凋亡作用;As2O3作用MCF-7细胞在2h内细胞出现AVD,但Caspase-3的活性无明显变化,直到作用MCF-7细胞6h后Caspase-3活性显著升高。结论 AVD是As2O3诱导乳腺癌细胞凋亡的早期事件。     

保护性自噬对顺铂诱导人乳腺癌 MCF-7细胞凋亡的抑制作用探讨

目的：探讨顺铂对乳腺癌 MCF-7细胞自噬的影响及自噬在顺铂诱导凋亡中的作用。方法顺铂处理乳腺癌 MCF-7细胞,MTT 检测细胞增殖的能力,Hoechst 33342染分析细胞的凋亡,吖啶橙染色分析细胞的自噬,Western blot 分析自噬蛋白 LC3Ⅰ／Ⅱ和 p62的表达和凋亡蛋白多聚 ADP-核糖聚合酶 PARP 表达。结果顺铂呈时间和剂量依赖性抑制乳腺癌 MCF-7细胞的增殖,并且凋亡细胞数量随顺铂浓度的递增而增加;同时顺铂能诱导微管相关蛋白轻链3-Ⅱ（LC3Ⅱ）蛋白的增加,p62蛋白的减少以及酸性自噬溶酶体的增加,顺铂联合氯喹明显增加了 LC3Ⅱ和 p62的蛋白的表达;与单药顺铂相比,自噬抑制剂氯喹明显降低细胞存活率（89．17％±2．56％）vs （74．63％±1．51％）,（P ＜0．05）,而且 PARP 蛋白发生了明显的裂解（P ＜0．05）。结论顺铂诱导乳腺癌 MCF-7细胞保护性自噬,抑制自噬可以增加顺铂诱导乳腺癌 MCF-7细胞凋亡,自噬抑制剂联合顺铂为乳腺癌提供了新的治疗策略。     

Growth inhibition, G1-arrest, and apoptosis in MCF-7 human breast cancer cells by novel highly lipophilic 5-fluorouracil derivatives

In this study we evaluate the antitumour activity, the cell cycle arrest and apoptotic properties of novel lipophilic benzene-fused seven-membered 5-fluorouracil (5-FU) analogs in comparison to 5-FU on MCF-7 human breast cancer cells. The lipophilicities of ESB-786B, ESB-252A and ESB-928A were predicted by using the CDR option of the PALLAS 2.0 program. Cytotoxic assays were evaluated in MCF-7 cells treated with the sulforhodamine B colorimetric method. Cell cycle perturbations were studied by flow cytometry. Apoptosis was determined by both DNA fragmentation and annexin V-FITC and propidium iodide staining. The novel derivatives were more lipophilic than 5-FU and induced a marked growth inhibition, in a dose-dependent manner. After treatment with IC(50) value (ranged from 2.5 to 22 microM) for each compound, light microscopy observation showed modifications in the morphology of MCF-7 cells. In addition, the 5-FU analogs arrest cells in the G(0)/G(1) phase of the cell cycle whereas 5-FU induced arrest in S-phase. Moreover, induction of apoptosis was demonstrated by the annexin-V-based assay and confirmed using DNA fragmentation analysis on MCF-7 cells, a cell line in which the induction of DNA laddering is very difficult. The novel benzannelated seven-membered 5-FU analogs can be considered as specific apoptotic inducers. These experimental findings provide support for the use of these novel compounds as new weapons in the fight against breast cancer.     

Effect of shikonin on human breast cancer cells proliferation and apoptosis in vitro

Shikonin, isolated from the plant Lithospermum erythrorhizon Sieb. Et Zucc, has been reported to induce apoptosis in several tumor cells. However, such effect of shikonin on human breast cancer cells has not been reported. Thus, in the present study, whether shikonin could induce MCF-7 human breast cancer cell apoptosis was investigated. The results showed that shikonin (2.5-80 microM) induced MCF-7 cell death in a time- and dose-dependent manner, as measured by MTT assay. The IC(50) of a 24 h, 48 h and 72 h time course for MCF-7 cells was 7.4+/-0.4, 6.3+/-0.6 and 3.9+/-0.5 microM, respectively. Cellular morphology observation showed that MCF-7 cells underwent marked apoptotic morphological changes upon treatment with 10 microM shikonin compared with the untreated control. Flow cytometric analysis of shikonin-treated MCF-7 cells showed that the ratio of the apoptotic DNA fragmentation increased in a dose-dependent manner. The present study demonstrated for the first time that the cytotoxic effect of shikonin on MCF-7 cells underwent apoptosis process.     

Adenovirus-mediated p53 gene therapy reverses resistance of breast cancer cells to adriamycin

The aim of this study was to determine whether adenovirus-mediated p53 gene (Ad-p53) transfection can enhance adriamycin cytotoxicity and reverse adriamycin resistance in human breast cancer cells and explore its effect on the expression of MDR1 gene and permeability-glycoprotein (P-gp). Human breast cancer cell lines, MCF-7 and MCF-7/ADR, were used in in-vitro studies. After infection with Ad-p53, the cytotoxicity of adriamycin was evaluated using the Cell Counting Kit-8 assay. The expression of MDR1 mRNA was detected by quantitative real-time PCR. The expression of P-gp was analyzed using western blotting. In in-vivo studies, MCF-7/ADR tumor cells were inoculated subcutaneously in athymic nude mice. After 14 days of inoculation, tumor size was measured. Apoptosis and expression of P-gp in the tumor tissue were analyzed by fluorescence activated cell sorting and western blotting. After transfection with a multiplicity of infection of 50 for Ad-p53, chemosensitivity of MCF-7/ADR cells increased by 18.1 times (P=0.001), and 50% inhibitory concentration (IC50) of adriamycin decreased from 4.54 ± 0.91 to 0.26 ± 0.11 mg/l. Real-time PCR showed that MDR1 mRNA decreased from 1.32 to 0.85 (P=0.001). Western blotting analysis showed that P-gp also decreased. In in-vivo studies, Ad-p53 combined with adriamycin dramatically inhibited the growth of subcutaneous xenograft of MCF-7/ADR. The fluorescence activated cell sorting assay showed that there were more apoptotic cells in tumor tissues treated with Ad-p53 and adriamycin. The expression of P-gp was significantly decreased in tumor tissues. This study suggests that Ad-p53 can reverse MCF-7/MDR cell resistance to adriamycin. The reversal effect was associated with inhibition of P-gp expression and induction of apoptosis.