二甲双胍通过激活腺苷酸活化蛋白激酶(AMPK)的抗肿瘤机制

二甲双胍是一种传统的口服降糖药,临床上普遍用于2型糖尿病的治疗。近年来大量流行病学研究报道二甲双胍能够降低2型糖尿病患者的肿瘤发病率,亦有研究发现二甲双胍能在代谢途径、细胞周期、氧化应激、肿瘤干细胞转化等方面通过激活腺苷酸活化蛋白激酶(adenosin emonophosphate-activated protein kinase,AMPK)信号通路,从而抑制肿瘤细胞的生长、增殖以及转化。但二甲双胍通过激活AMPK的抗肿瘤机制仍存在着争议,其确切的作用机制有待进一步深入的研究,同时亟需大规模的临床试验来证实。     

二甲双胍抗肿瘤机制的研究进展

近年来,很多研究发现糖尿病能增加癌症的发生率和死亡率。目前,二甲双胍是临床治疗糖尿病的最常用药物之一。大量研究表明二甲双胍除具有降糖作用外,还有抑制肿瘤细胞生长的作用,因此可以降低2型糖尿病患者恶性肿瘤的发生率和死亡率。二甲双胍能激活腺苷活化蛋白激酶（AMPK）途径、阻滞细胞周期、调节胰岛素/IGF-1轴、调节肿瘤细胞的自噬效应、抑制肿瘤血管生成、激活体内免疫系统、增加化疗药物敏感性及杀伤肿瘤干细胞,从而杀灭肿瘤细胞,抑制肿瘤生长。二甲双胍具有安全、低毒的特性,将其应用于肿瘤的辅助治疗,可能会明显减轻化疗药物的毒副作用,提高患者的耐受性,并有望成为一种新型抗肿瘤药物。目前,二甲双胍的抗肿瘤机制仍处于实验及流行病学研究阶段,并未进入临床实验阶段,但其抗肿瘤作用是确切的。     

二甲双胍对人肝癌细胞HepG2增殖及乙酰辅酶A羧化酶影响的实验研究

目的 探讨二甲双胍在人肝癌细胞HepG2中的抗肿瘤活性及其作用机制。方法 选取二甲双胍不同浓度（0、1、5、10、15 mmol／L）处理HepG2细胞24、48及72 h,用CCK-8法检测其对细胞增殖的影响。设二甲双胍不同浓度（0、5、10、15 mmol／L）处理HepG2细胞72 h,用Western blotting检测腺苷酸活化蛋白激酶（AMPK）、P-AMPK、乙酰辅酶A羧化酶（ACC）、P-ACC蛋白表达,Real-time PCR检测ACC mRNA的表达水平。结果 二甲双胍对HepG2细胞的增殖抑制作用呈时间和浓度依赖性。经不同浓度二甲双胍处理HepG2细胞72 h后,P-AMPK蛋白表达随药物浓度增高而上调,P-ACC蛋白表达随药物浓度增高而上升;与空白对照组（0 mmol／L）中P-AMPK、P-ACC表达比较,10、15 mmol／L组中两者的差异均有统计学意义（P＜0.01）。ACC mRNA表达随二甲双胍浓度的增加呈下降趋势,5、10、15 mmol／L组表达量均较空白对照组显著下降（P＜0.01）。结论 初步实验研究发现,二甲双胍能够抑制人肝癌细胞HepG2的增殖,并从蛋白磷酸化水平和基因水平抑制ACC的活性,其抗肿瘤作用可能与激活AMPK和抑制ACC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。     

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。     

二甲双胍与不同分子亚型乳腺癌的关系

二甲双胍做为糖尿病治疗的一线用药,不仅可以降低血糖,还能促使乳腺癌细胞凋亡。体内外实验证实了二甲双胍对各型乳腺癌的抑制作用,而且二甲双胍还能在新辅助化疗以及放疗中协同治疗乳腺癌。本文将对二甲双胍在乳腺癌治疗中的研究进展做一综述。     

慢病毒介导高表达OTUD7B对乳腺癌MCF-7细胞生物学行为影响

目的 去泛素化酶OTUD7B与肿瘤的发生、发展密切相关.为了明确OTUD7B在乳腺癌中所发挥的作用,实验利用慢病毒构建高表达OTUD7B载体感染MCF-7乳腺癌细胞后对其生物学行为的影响.方法 构建带有绿色荧光蛋白标签的人OTUD7B表达质粒的慢病毒(pEGFP-hOTUD7B)及对照(pEGFP-CI)感染MCF-7乳腺癌细胞;于荧光倒置显微镜下观察病毒转染效果及应用蛋白质印迹法及免疫组化法检测OTUD7B的表达水平;MTS法检测实验组(pEGFP-hOTUD7B)、阴性对照组(pEGFP-CI)和正常对照组对MCF-7乳腺癌细胞增殖能力的影响;应用细胞划痕实验检测细胞迁移能力;流式细胞仪检测细胞凋亡.结果 感染病毒后于荧光倒置显微镜下观察病毒感染效率,可见病毒感染成功.应用蛋白质印迹法检测病毒感染率并找出最适病毒感染复数(multiplicity of infection,MOI),当MOI=30时,实验组、阴性对照组和正常对照组灰度值分别为3.81±0.08、2.12±0.078和2.05±0.15,差异有统计学意义,F=402.03,P<0.001.应用免疫组化法可见感染OTUD7B表达水平.MTS法结果显示,实验组、阴性对照组和正常对照组细胞24 h A值分别为0.36±0.08、0.56±0.25和0.69±0.17,F=11.819,P<0.001;48 h A值分别为0.65±0.17、1.45±0.48和1.82±0.63,F=23.752,P<0.001;在72 h A值分别为0.73±0.21、1.58±0.63和1.99±0.27,F=35.563,P<0.001.细胞划痕试验显示,24 h后实验组组迁移率为(7.7±0.91)%,阴性对照组和正常对照组迁移率分别为(13.4±1.52)%和(12.1±1.32)%,F=49.36,P<0.001,48 h后实验组迁移率为(12.4±1.29)%,阴性对照组及正常对照组迁移率分别为(32.9±1.71)%和(31.8±1.59)%,F=504.50,P<0.001.流式细胞仪检测细胞凋亡结果提示,实验组与阴性对照组及正常对照组相比明显使细胞阻滞在G0/G1期,促进其凋亡,FG0/G1期=425.102,FS期=135.063,均P<0.001.结论 成功构建了能够高表达OTUD7B的慢病毒载体,明显抑制了MCF-7乳腺癌细胞增殖、迁移,并促进了其凋亡.     

二甲双胍与乳腺癌关系的研究进展

糖尿病增加乳腺癌的发病风险, 并影响乳腺癌患者的预后。二甲双胍作为一种降糖药除了能降低血糖外, 尚有抑制多种癌症的作用, 并对乳腺癌有独特的作用, 体内和体外实验均被证实能抑制乳腺癌细胞, 甚至对表皮生长因子受体-2(HER-2)阳性且曲妥株单抗耐药的乳腺癌细胞、乳腺癌干细胞、三阴性乳腺癌细胞也能发挥抗肿瘤作用。二甲双胍尚能减低糖尿病患者的乳腺癌发病风险、降低乳腺癌组织学级别、增加雌激素受体(ER)及孕激素受体(PR)表达, 对乳腺癌新辅助化疗也有一定的作用。本文将从基础及临床研究方面阐明二甲双胍对乳腺癌的抑制机制。      

二甲双胍治疗乳腺癌的分子机制与临床研究进展

乳腺癌已成为严重影响妇女身心健康甚至危及生命的最常见的恶性肿瘤。糖尿病增加了乳腺癌的发病率,并对乳腺癌的预后产生重要的影响。二甲双胍作为Ⅱ型糖尿病一线的降糖药,不仅能够降低血糖,增强胰岛素的敏感性,还能够促进乳腺癌细胞的凋亡,降低乳腺癌患者死亡率。在体内体外的实验中均已证实二甲双胍能诱导细胞周期停滞,抑制乳腺癌细胞的增殖,促进其凋亡,甚至对ErbB2过度表达的乳腺癌细胞、乳腺癌干细胞、三阴性乳腺癌也能发挥抗肿瘤作用。二甲双胍还能预防线粒体功能障碍,下调丙酮酸激酶表达,抑制Ki-67表达,起到抑制乳腺癌细胞的作用。这对乳腺癌新辅助化疗及放射治疗起到一定的协同作用。因此,本文拟对二甲双胍治疗乳腺癌的分子机制与临床研究进行综述。     

降糖药物二甲双胍在癌症防治方面的新用途

二甲双胍是世界上应用最广范的抗糖尿病药物,而现在越来越多的证据证明这种药物具有潜在的抗癌功能。流行病学调查显示,使用二甲双胍治疗的糖尿病癌症患者发病率会降低。而且,二甲双胍能活化细胞能量感受器腺苷酸活化蛋白激酶(AMPK)途径,后者可以作为治疗癌症的最新靶点。最后,多项研究指出,二甲双胍能直接抑制癌细胞生长,具有抗肿瘤功能。     

Effects of Rapamycin on Cell Apoptosis in MCF-7 Human Breast Cancer Cells

Background: Rapamycin is an effective anti-angiogenic drug. However, the mode of its action remainsunclear. Therefore, in this study, we aimed to elucidate the antitumor mechanism of rapamycin, hypotheticallyvia apoptotic promotion, using MCF-7 breast cancer cells. Materials and Methods: MCF-7 cells were platedat a density of 15105 cells/well in 6-well plates. After 24h, cells were treated with a series of concentrations ofrapamycin while only adding DMEM medium with PEG for the control regiment and grown at 37oC, 5% CO2and 95% air for 72h. Trypan blue was used to determine the cell viability and proliferation. Untreated andrapamycin-treated MCF-7 cells were also examined for morphological changes with an inverted-phase contrastmicroscope. Alteration in cell morphology was ascertained, along with a stage in the cell cycle and proliferation.In addition, cytotoxicity testing was performed using normal mouse breast mammary pads. Results: Our resultsclearly showed that rapamycin exhibited inhibitory activity on MCF-7 cell lines. The IC50 value of rapamycin onthe MCF-7 cells was determined as 0.4μg/ml (p<0.05). Direct observation by inverted microscopy demonstratedthat the MCF-7 cells treated with rapamycin showed characteristic features of apoptosis including cell shrinkage,vascularization and autophagy. Cells underwent early apoptosis up to 24% after 72h. Analysis of the cell cycleshowed an increase in the G0G1 phase cell population and a corresponding decrease in the S and G2M phasepopulations, from 81.5% to 91.3% and 17.3% to 7.9%, respectively. Conclusions: This study demonstrated thatrapamycin may potentially act as an anti-cancer agent via the inhibition of growth with some morphologicalchanges of the MCF-7 cancer cells, arrest cell cycle progression at G0/G1 phase and induction of apoptosis inlate stage of apoptosis. Further studies are needed to further characterize the mode of action of rapamycin asan anti-cancer agent.     

Effects of Metformin on Cell Kinetic Parameters of MCF-7 Breast Cancer Cells in Vitro

In this study, the antiproliferative effects of the metformin was evaluated on MCF-7 Cells (human breastadenocarcinoma cell line). For this purpose cell kinetic parameters including cell proliferation assay, mitoticindex and labelling index analysis were used. 30 μM, 65 μM and 130 μM Metformin doses were applied to cellsfor 24, 48 and 72 hours. The results showed that there was a significant decrease in cell proliferation, mitoticindex and labelling index for all experimental groups (p<0.05) for all applications.     

4-氨基吡啶与多西紫杉醇抗人乳腺癌细胞MCF-7 作用及相互关系探讨*

目的:通过研究钾离子通道阻断剂4- 氨基吡啶（4-AP ）对肿瘤化疗药物多西紫杉醇（docetaxel ,DOC ）的抗人乳腺癌细胞MCF-7 作用的影响,探讨4-AP 能否增强DOC 的作用。方法:用MTT 比色法分析DOC 、4-AP 以及两药联合应用对人乳腺癌细胞MCF-7 增殖的影响;用流式细胞术PI 单染法及Annexin V-FITC/PI双染法检测上述两种药物对人乳腺癌细胞MCF-7 的细胞周期及早期凋亡的影响。结果:DOC 能明显抑制人乳腺癌细胞株MCF-7的增殖,并呈剂量和时间依赖性。4-AP 对MCF-7 细胞增殖亦具有一定的抑制作用,在用药后24h、48h 及72h 的抑制率分别为11.9%±1.7% 、42.1%±3.2% 、44.2%±1.6% 。且5mmol/L 4-AP 可使DOC 的作用增强,使25μ mol/L DOC 对人乳腺癌细胞株MCF-7 最大杀伤作用时间从72h 提前至24h。5 μ mol/L DOC 能够使MCF-7 G2/M期细胞比率明显增加（53.58%±1.44% 与对照组8.83%±0.44% 相比,P<0.01）,使G0/G1 期细胞减少（11.48%±0.14% 与对照组63.89%±0.98% 相比,P<0.01）,说明DOC 主要在G2/M期阻滞MCF-7 细胞的增殖。5mmol/L 4-AP 作用于MCF-7 使其G0/G1 期细胞比率增加,G2/M期细胞明显减少,甚至消失（0.42%±0.17% 与对照组8.83%±0.44% 相比,P<0.05）。 而两药联用可见4-AP 使MCF-7 G2/M期细胞比率有所减少,而G0/G1 期细胞比率有所增加。DOC 单独作用于人乳腺癌细胞株MCF-7 细胞24h 后,能明显增加晚期凋亡和死亡率（由6.97%±0.75% 增加到20.77%±0.75% ,P<0.05）;而两药联合时,与对照组相比,早期凋亡比例由4.60%±0.91% 增加到12.20%±0.82%（P<0.05）,晚期凋亡和死亡细胞比例由6.97%±0.75% 增加到58.42%±0.31%（P<0.05）,提示4-AP（5mmol/L ）能明显增加DOC 诱导的MCF-7 早期凋亡。结论:DOC 和4-AP 分别在G2/M期和G0/G1 期抑制MCF-7 细胞增殖,4-AP 通过促进DOC 诱导细胞凋亡发挥抑制MCF-7 细胞增殖的作用。      

辛基酚对MCF-7乳腺癌细胞周期及周期蛋白表达的影响

目的观察辛基酚对MCF-7乳腺癌细胞周期及周期蛋白表达的影响。方法以MTT试验、流式细胞分析、免疫细胞化学和RT-PCR等方法观察辛基酚对MCF-7乳腺癌细胞增殖、细胞周期相和细胞凋亡、CDK2和CDK4 mRNA表达、Cyclin D1 mRNA及蛋白表达的影响。结果4、8、16μmol/LOP作用MCF-7乳腺癌细胞72h时,细胞增殖率分别为107.31%、168.06%、62.00%,G0/G1期细胞分别为(52.46±6.67)%、(50.19±7.39)%、(67.31±5.47)%,对照组(55.27±7.53)%,细胞凋亡率分别为(4.84±1.12)%、(6.48±1.36)%、(19.26±3.57)%,对照组(5.56±1.125)%,16μmol/LOP减少了细胞中CDK2、CDK4、Cyclin D1 mRNA及Cyclin D1的表达。结论4、8μmol/LOP促进MCF-7乳腺癌细胞增殖,16μmol/LOP则抑制细胞增殖,促进细胞凋亡,这可能与OP影响细胞中CDK2、CDK4、Cyc-lin D1 mRNA及Cyclin D1表达有关。     

白藜芦醇抑制MCF-7乳腺癌细胞增殖的机制研究

研究白藜芦醇对MCF-7乳腺癌细胞抑制效应及其作用机制。方法：以人MCF-7乳腺癌细胞株为研究对象,利用MTT方法研究白藜芦醇抑制MCF-7乳腺癌细胞的生物学效应;观察在ERK1/2抑制剂PD98059预处理情况下,白藜芦醇抑制MCF-7乳腺癌细胞增殖效应的改变;利用免疫印迹方法观察白藜芦醇对MCF-7乳腺癌细胞中ERK1/2与AKT信号分子的蛋白表达。结果：白藜芦醇能够明显降低MCF-7乳腺癌细胞增殖能力,该作用呈一定的浓度依赖性关系。在ERK1/2抑制剂PD98059预处理情况下,白藜芦醇对MCF-7乳腺癌细胞增殖抑制效应能明显抑制,PD98059可明显减轻该效应。同时,白藜芦醇明显增加p-ERK1/2蛋白表达,降低p-AKT表达水平,但对ERK1/2与AKT蛋白表达无改变。结论：白藜芦醇能够有效抑制MCF-7乳腺癌细胞增殖,该效应与白藜芦醇对ERK1/2及AKT信号途径的调节有关。     

Synergistic Effects of Exemestane and Aspirin on MCF-7 Human Breast Cancer Cells

Objective: The purpose of this study is to investigate the combined effects of exemestane and aspirin on MCF-7 human breast cancer cells. Methods: Antiproliferative effects of exemestane and aspirin, alone and in combination, on growth of MCF-7 human breast cancer cells were assessed using the MTT assay. Synergistic interaction between the two drugs was evaluated in vitro using the combination index (CI) method. The cell cycle distribution was analyzed by flow cytometry and Western blotting was used to investigate the expression of cyclooxygenase-1, cyclooxygenase-2 and Bcl-2. Results: MTT assays indicated that combination treatment obviously decreased the viability of MCF-7 human breast cancer cells compared to individual drug treatment (CI<1). In addition, the combination of exemestane and aspirin exhibited a synergistic inhibition of cell proliferation, significantly arrested the cell cycle in the G0/G1 phase and produced a stronger inhibitory effect on COX-1 and Bcl-2 expression than control or individual drug treatment. Conclusion: These results indicate that the combination of exemestane and aspirin might become a useful method to the treatment of hormonedependent breast cancer. The combination of the two inhibitors significantly increased the response as compared to single agent treatment, suggesting that combination treatment could become a highly effective approach for breast cancer.     

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.     

Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells

Purpose

Isocryptotanshinone (ICTS) is a natural bioactive product that is isolated from the roots of the widely used medical herb Salvia miltiorrhiza. However, few reports exist on the mechanisms underlying the therapeutic effects of ICTS. Here, we report that ICTS has anticancer activity and describe the mechanism underlying this effect.

Methods

The antiproliferative effect of ICTS was determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and clonogenic assays. The effect of ICTS on the cell cycle was measured using flow cytometry. Apoptosis was determined by Hoechst 33342 staining, DNA fragmentation assays, and Western blotting for apoptotic proteins. Finally, the effect of ICTS on mitogen-activated protein kinases (MAPKs) was determined by Western blotting.

Results

ICTS significantly inhibited proliferation of MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 human liver cancer cells, and A549 human lung cancer cells in vitro. Among the tested cell lines, MCF-7 cells showed the highest sensitivity to ICTS. ICTS significantly inhibited colony formation by MCF-7 cells. Furthermore, exposure of MCF-7 cells to ICTS induced cell cycle arrest at the G1 phase and decreased mitochondrial membrane potential. Hoechst 33342 staining and Western blot analysis for apoptotic proteins suggested that ICTS induced apoptosis in MCF-7 cells. In addition, ICTS activated MAPK signaling in MCF-7 cells by inducing time- and concentration-dependent phosphorylation of JNK, ERK, and p38 MAPK.

Conclusion

Our results suggest that ICTS inhibited MCF-7 cell proliferation by inducing apoptosis and activating MAPK signaling pathways.     

Oxidants-Antioxidants Profile in the Breast Cancer Cell Line MCF-7

Reactive oxygen species (ROS) have various biological effects and they are non-linear in characteristic. In high oxidativestress, they may cause cytotoxicity, inhibit cell proliferation, and induce cell death in the form of apoptosis/necrosis;while in low or medium oxidative stress, ROS may cause DNA damage, cell mutation, inflammation, cell proliferation,and eventually they may induce carcinogenesis. Antioxidants are compounds with the ability to reduce ROS. Cell lineMCF-7 is one of the breast cancer cell lines that is known to have small amount of antioxidant MnSOD compared to theother cell lines. Low antioxidant MnSOD level in breast cancer cell line MCF-7 leads to low concentration of hydrogenperoxide, because antioxidant MnSOD will convert radical superoxide to hydrogen peroxide. The aim of this researchwas to analyze oxidants and antioxidants profile in breast cancer cell line MCF-7 and their relationship with cell number.Observations were conducted for 5 days. The cell number was counted with tryphan blue method and haematometer.The concentration of radical superoxide was measured with DHE staining using LSCM tipe Olympus Fluoview FV1000-Ver 1.7. MnSOD activity, hydrogen peroxide concentration, and catalase activity were measured with ELISA.The results showed that the longer of observation, the greater concentration of oxidants and MnSOD activity, but therewas no change in catalase activity. Conclusion the increase in cancer cells number is influenced by radical superoxide.