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。     

甲异靛诱导白血病细胞HL-60凋亡及机制探讨

背景与目的:甲异靛可有效治疗慢性粒细胞白血病,但其抗白血病的机制尚不清楚。本研究拟观察甲异靛诱导急性白血病细胞株HL-60细胞凋亡的作用及促凋亡机制。方法:用台盼蓝拒染实验观察甲异靛对HL-60细胞增殖抑制作用;琼脂糖凝胶电泳检测DNA条带;荧光显微镜观察细胞形态学的变化;流式细胞术检测细胞凋亡率和Fas蛋白的表达;免疫蛋白印迹法分析caspase-9、caspase-8、caspase-3、PARP、bcl-2、bax和胞浆细胞色素c的含量。结果:甲异靛可抑制HL-60细胞增殖和诱导其凋亡。20μmol/L甲异靛处理HL-60细胞12～48h可明显抑制HL-60细胞增殖;20μmol/L甲异靛作用于HL-60细胞1h,凋亡百分比为(3.70±0.56)%,当延长至3h、6h和12h凋亡细胞比例分别上升至(19.80±1.13)%、(29.20±2.69)%和(47.05±7.70)%,较阴性对照组[(2.65±0.78)%]明显增高(P<0.05)。HL-60细胞经甲异靛处理3h后出现细胞核染色质浓集和DNA梯形条带。甲异靛处理HL-60细胞1h后死亡受体Fas阳性率由(9.35±0.21)%升高到(21.30±1.27)%(P<0.05)。甲异靛可活化HL-60细胞的caspase-8、caspase-9、caspase-3和PARP,降低抗凋亡蛋白bcl-2的表达和上调促凋亡蛋白bax,并升高细胞浆中细胞色素c的浓度。caspase-3抑制剂(z-DEVD-fmk)可以减少甲异靛对HL-60细胞的增殖抑制,降低细胞凋亡率。100μmol/Lz-DEVD-fmk预处理HL-60细胞后再加入20μmol/L甲异靛,5h后凋亡率为(16.5±5.5)%,甲异靛组为(29.8±5.4)%(P<0.05);12h后计数,抑制剂加甲异靛组活细胞高达(3.57±0.18)×105/ml,甲异靛组活细胞仅为(1.80±0.14)×105/ml(P<0.05)。结论:甲异靛可诱导HL-60细胞凋亡,其机制与caspases和bcl-2家族蛋白的调节有关。     

别直参抑制人乳腺癌细胞MCF-7体外增殖

[目的]评价别直参对MCF-7细胞体外增殖的影响,并检测其对凋亡相关蛋白bax/bcl-2表达的影响.[方法]采用MTT比色法检测对MCF-7细胞增殖的作用;流式细胞术检测对MCF-7细胞凋亡的影响;免疫组化法检测bax/bcl-2的表达.[结果]别直参高剂量组作用MCF-7细胞24h后,MTT检测增殖率为89.83％,提示有抑制MCF-7细胞体外增殖的作用(P＜0.05);流式细胞术检测高剂量组细胞凋亡率明显升高(P＜0.05);免疫组化法检测高剂量组能上调MCF-7细胞bax的表达,并下调bcl-2的表达.[结论]别直参高剂量组具有一定的诱导MCF-7细胞凋亡效应作用,其机制可能通过上调bax的表达和下调bcl-2的表达.     

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。     

青蒿琥酯对人乳腺癌MCF-7细胞裸鼠移植瘤的作用及IGF—IR的影响

[目的]观察青蒿琥酯对人乳腺癌MCF-7裸鼠移植瘤生长的作用，并探讨其抑制肿瘤的机制。[方法]建立人乳腺癌MCF-7裸鼠移植瘤模型，给予不同剂量青蒿琥酯治疗并观察其对移植瘤的抑制作用；电镜观察移植瘤细胞形态学改变；流式细胞术检测细胞凋亡率和细胞周期的变化：免疫组化检测移植瘤细胞p53、bcl-2、bax和caspase-3的表达情况，分析它们之间的相关性；Westernblot检测IGF—IR蛋白的表达：[结果]给药12d后，低剂量、高剂量青蒿琥酯组、CTX组和联合给药组抑瘤率分别为（24．39±10．20）％、（40．24±7．02）％、（57．01±5．84）％和（68．29±5．1）％；青蒿琥酯使肿瘤细胞阻滞于G0／G1期而使S期细胞减少；在青蒿琥酯组中，bcl-2的蛋白表达量明显下降，bax、caspase-3蛋白表达上调，p53蛋白表达量与对照组比较无差异：bcl-2／bax及bcl-2／caspase-3表达均呈负相关．IGF-IR蛋白表达下调。[结论]青蒿琥酯可显著抑制乳腺癌MCF-7细胞裸鼠移植瘤的生长，其机制可能与其影响细胞凋亡相关蛋白、诱导凋广、抑制细胞增殖有关．     

褐藻糖胶对人乳腺癌MCF-7细胞增殖与凋亡的影响

目的：研究褐藻糖胶（fucoidan）对体外培养的人乳腺癌MCF-7细胞增殖及凋亡的影响，并探讨其凋亡机制。方法：应用MTT法检测细胞的增殖；Hoechst33258染色、琼脂糖凝胶电泳法观察细胞的凋亡；RT-PCR和Western印迹法分别检测细胞中bcl-2和bax的mRNA及其蛋白的表达。结果：不同浓度的褐藻糖胶均能抑制MCF-7细胞的增殖（P〈0．01），并随其浓度增加，抑制率逐渐增大；褐藻糖胶诱导MCF-7细胞凋亡数增加，且可见明显的、凋亡特有的DNA梯形条带；在褐藻糖胶存在下。bcl-2基因的mRNA和蛋白表达减少，bax基因的mRNA和蛋白表达增加，bcl-2／bax比值下降（P〈0．05）。结论：褐藻糖胶可抑制MCF-7细胞增殖，且诱导其凋亡，其凋亡机制可能与下调bcl-2和上调bax基因的表达有关。     

SCD1抑制剂对乳腺癌细胞增殖凋亡的影响及其机制

目的 检测硬脂酰辅酶A去饱和酶1（stearoyl-CoA desaturase 1, SCD1）在乳腺癌组织中的表达,分析抑制SCD1的活性对乳腺癌MCF-7细胞增殖凋亡的影响及其机制。方法 采用免疫组织化学法检测乳腺癌及癌旁正常组织中SCD1蛋白的表达。应用MTS法测定SCD1抑制剂MF-438对MCF-7细胞增殖的抑制率。应用Hoechst33342染色荧光显微镜观察细胞形态并计算凋亡指数,PI染色流式细胞术检测细胞凋亡率。蛋白质印迹法检测凋亡相关蛋白Bcl-2和Bax的表达。结果 乳腺癌组织中SCD1阳性表达率显著高于正常乳腺组织（P<0.05）,并且三阴性乳腺癌中的SCD1表达水平低于其他亚型（P<0.05）。MF-438在0.1~100 μmol/L浓度范围内,对MCF-7细胞显示出显著的剂量依赖性的增殖抑制作用,并在低血清条件下更为敏感。MCF-7细胞在5 μmol/L MF-438作用48 h后,表现出典型的细胞凋亡特征性变化,细胞凋亡指数及细胞凋亡率显著高于对照组（P<0.05）;同时,MF-438下调MCF-7细胞抑凋亡蛋白Bcl-2的表达,并上调促凋亡蛋白Bax的表达。结论 抑制SCD1能抑制乳腺癌细胞增殖并诱导凋亡,对SCD1的深入研究将有望为乳腺癌的分子靶向治疗提供一个新的靶标。     

青蒿琥酯抑制MCF-7细胞的机制探讨

目的探讨青蒿琥酯(Artesunate,ART)对乳腺癌细胞MCF-7抑制作用及其机制。方法ART处理细胞3天后,采用MTT比色法检测细胞增殖功能,观察细胞形态、结构的改变,免疫细胞化学检测bax、nm23、PCNA、VEGF、bcl-2蛋白的表达情况。结果ART对乳腺癌细胞MCF-7的抑制作用呈明显浓度依赖性,可导致细胞形态、结构的改变;免疫细胞化学结果显示20μmol/L青蒿琥酯作用两种细胞72小时后,可上调bax、nm23,下调PCNA、VEGF蛋白的表达,bcl-2蛋白表达无明显变化。结论ART有抑制MCF-7细胞增殖的作用,其机制可能与上调bax、nm23,下调PCNA、VEGF蛋白的表达有关。     

LBH589或联合多烯紫杉醇对人卵巢癌OVCAR-3细胞增殖和凋亡的研究

  目的  探讨新型组蛋白去乙酰化酶抑制剂LBH589或联合多烯紫杉醇（DTX）对人卵巢癌OVCAR-3细胞增殖和凋亡的影响。  方法  采用不同浓度LBH589、DTX或两者联合处理OVCAR-3细胞, 用四甲基偶氮唑蓝（MTT）法检测细胞增殖活力, 台盼兰、吖啶橙溴化乙啶（AO/EB）双染色法检测细胞凋亡; Western blot检测聚腺苷二磷酸核糖聚合酶（PARP）、caspase-3、caspase-7、bcl-2、bax蛋白水平。  结果  LBH589、DTX均能抑制OVCAR-3细胞增殖, 诱导凋亡, 小剂量LBH589联合DTX作用更强。经Calcusyn软件分析证明两者联合具有明显的协同作用。Western blot检测发现caspsae-3、PARP-85kD剪切蛋白增加, bax表达增加, bcl-2表达减少。caspase-7无明显变化。  结论  LBH589、DTX能抑制OVCAR-3细胞增殖, 诱导凋亡, 两者联合有明显的协同作用。      

丹酚酸乙对乳腺癌MCF-7细胞的生长抑制及诱导凋亡研究

[目的]观察丹酚酸乙对乳腺癌MCF-7细胞的生长抑制及诱导凋亡作用。[方法]MTT法观察丹酚酸乙对体外培养MCF-7细胞的增殖抑制作用;Hoechst33258荧光染色法观察细胞凋亡的形态学变化;流式细胞仪检测丹酚酸乙作用于MCF-7细胞48h后细胞凋亡率和细胞周期的变化;Western Blot检测caspase-3蛋白表达的变化。[结果]丹酚酸乙能明显抑制MCF-7细胞的生长,呈剂量和时间依赖性;荧光染色结果显示,丹酚酸乙与MCF-7细胞共培养24、48和72h后,细胞呈现明显的核固缩、碎裂以及凋亡小体形成等细胞凋亡现象;流式细胞仪检测结果显示,不同浓度的丹酚酸乙处理MCF-7细胞48h后,细胞凋亡率和S期细胞的比例逐渐升高;Western Blot显示,随着丹酚酸乙浓度的增加,MCF-7细胞caspase-3蛋白表达水平逐渐升高。[结论]丹酚酸乙对MCF-7细胞具有明显的生长抑制和促凋亡作用,这种作用可能与上调caspase-3表达以及阻滞细胞于S期有关。     

5,4'-二-正辛烷氧基-7-二氟亚甲基异黄酮体外抗人类乳腺癌细胞作用

目的 探讨金雀异黄素(Gen)衍生物5,4'-二-正辛烷氧基-7-二氟亚甲基异黄酮(DOdFMG)体外抗人类乳腺癌细胞的作用.方法 平皿克隆形成法检测DOdFMG对人类乳腺癌MCF-7细胞抑制生长和增生作用,琼脂糖凝胶电泳法观测DOdFMG诱导MCF-7细胞凋亡作用,Western blotting 法测定DOdFMG对MCF-7细胞PTEN、pAkt、caspase-3蛋白的表达及活性.结果 DOdFMG抑制MCF-7细胞生长,呈剂量依赖性,比Gen具有更强的抗肿瘤活性,DOdFMG能诱导MCF-7细胞凋亡,DOdFMG处理的MCF-7细胞PTEN蛋白及caspase-3蛋白表达上调,pAkt蛋白表达下调,呈时间剂量依赖性.结论 DOdFMG具有抗人类乳腺癌MCF-7细胞的作用,其机制可能与抑制PKB/Akt通路的信号传导、激活PTEN及caspase-3有关,是一种新型治疗乳腺癌的候选药物.     

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.     

bcl-2/bcl-xL反义寡核苷酸对乳腺癌细胞增殖和凋亡的影响

目的　观察靶向bcl 2 /bcl xL基因的反义寡核苷酸 (ASODN )对乳腺癌细胞株MCF 7增殖和凋亡的影响。方法　将bcl 2 /bcl xLASODN及其对照序列通过脂质体转染MCF 7细胞。采用MTT法测定MCF 7细胞增殖 ,荧光显微镜定量检测MCF 7细胞凋亡率。结果　与对照序列相比 ,bcl 2 /bcl xLASODN能抑制MCF 7增殖和诱导其凋亡 (P <0 .0 1)。结论　bcl 2 /bcl xLASODN在乳腺癌治疗方面作为 1种新的化合物值得进一步研究     

Apoptosis-mediated inhibition of human breast cancer cell proliferation by lemon citrus extract

Dietary phytochemicals have a variety of antitumor properties. In the present study, the antitumor activity of methanolic extract of lemon fruit (lemon extract; LE) (LE) on the MCF-7 breast cancer cell line was investigated in vitro. Apoptotic cell death was analyzed using the TUNEL assay. In addition, the apoptosis mediated by LE extract in the MCF-7 cells was associated with the increased expression of the tumor suppressor p53 and caspase-3. Additionally, the expression of a pro-apoptotic gene, bax, was increased, and the expression of an anti-apoptotic gene, bcl-2, was decreased by LE extract treatment, resulting in a shift in the Bax:Bcl-2 ratio to one that favored apoptosis. The expression of a major apoptotic gene, caspase-3, was increased by LE extract treatment. In light of the above results, we concluded that LE extract can induce the apoptosis of MCF-7 breast cancer cells via Bax-related caspase-3 activation. This study provides experimental data that are relevant to the possible future clinical use of LE to treat breast cancer.     

Bisphosphonates induce apoptosis in human breast cancer cell lines

Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50 values of 15, 20 and 3 microM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 microM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells.     

红松松塔提取物多聚苯丙素-多糖复合物诱导人乳腺癌MCF-7细胞凋亡

目的 研究红松松塔提取物多聚苯丙素-多糖复合物(Polyphenylpropenoid-polysaccharide complex,PPC)诱导人乳腺癌MCF-7细胞凋亡的机制。方法 用体外培养的MCF-7细胞与不同浓度PPC作用不同时间,应用MTT法、荧光染色法、凋亡检测试剂盒、DNA凝胶电泳及Western blot检测PPC对MCF-7细胞增殖的影响和诱导凋亡的作用。结果 PPC可诱导MCF-7细胞发生凋亡。细胞凋亡时Caspase-3和Caspase-9的酶活力升高,Caspase-3前体酶蛋白表达下降;Caspase-3抑制剂(z-DEVE-fmk)可部分抑制PPC诱导的细胞凋亡,并抑制Caspase-3前体酶的活化。结论 PPC通过激活Caspase家族诱导MCF-7细胞凋亡。     

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF-7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.