Cranberry phytochemical extracts induce cell cycle arrest and apoptosis in human MCF-7 breast cancer cells

Breast cancer is the most commonly diagnosed cancer in women in the US and is one of the leading causes of death due to cancer. Epidemiological studies have consistently suggested the inverse association between cancer risk and intake of fruits and vegetables. These health benefits have been linked to the additive and synergistic combination of phytochemicals in fruits and vegetables. Cranberries have been shown to possess anti-carcinogenic activities such as inhibition of growth of several cancer cell lines, and inhibition of ornithine decarboxylase (ODC) activity in vitro. However, the molecular mechanisms of the anti-cancer properties of cranberry phytochemical extracts have not been completely understood. Our data showed that cranberry phytochemical extracts significantly inhibited human breast cancer MCF-7 cell proliferation at doses of 5 to 30mg/mL (P<0.05). Apoptotic induction in MCF-7 cells was observed in a dose-dependent manner after exposure to cranberry phytochemical extracts for 4h. Cranberry phytochemical extracts at a dose of 50mg/mL resulted in a 25% higher ratio of apoptotic cells to total cells as compared to the control groups (P<0.05). Cranberry phytochemical extracts at doses from 10 to 50mg/mL significantly arrested MCF-7 cells at G0/G1 phase (P<0.05). A constant increasing pattern of the G1/S index was observed in the cranberry extract treatment group while the G1/S ratio of the control group decreased concomitantly between 10 and 24h treatment. After 24-h exposure to cranberry extracts, the G1/S index of MCF-7 cells was approximately 6 times higher than that of the control group (P<0.05). These results suggest that cranberry phytochemical extracts possess the ability to suppress the proliferation of human breast cancer MCF-7 cells and this suppression is at least partly attributed to both the initiation of apoptosis and the G1 phase arrest.     

Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell

Although the pharmacology and clinical application of water extracts of Ganoderma lucidum have been extensively documented, little is known regarding its alcohol extract. In the present study, the anti-tumor effect of an alcohol extract of Ganoderma lucidum was investigated using MCF-7 cells. We found that the alcohol extract of Ganoderma lucidum inhibited cell proliferation in a dose- and time-dependent manner, which might be mediated through up-regulation of p21/Waf1 and down-regulation of cyclin D1. Furthermore, this compound can directly induce apoptosis in MCF-7 cells, which might be mediated through up-regulation of a pro-apoptotic Bax protein and not by the immune system. Our findings suggest that there are multiple mechanisms underlying the anti-tumor effects of Ganoderma lucidum.     

Rapid onset of nucleolar disintegration preceding cell cycle arrest in roscovitine-induced apoptosis of human MCF-7 breast cancer cells

The aim of our study was to explore the antiproliferative and pro-apoptotic action of roscovitine (ROSC) on human breast cancer MCF-7 cells. We examined the effect of ROSC on cell proliferation, cell cycle progression, nucleolar morphology, posttranslational modifications of histones as well as on induction of apoptosis. The effects of ROSC on the argyrophilic nucleolar organizer regions (AgNORs) and nucleolar RNA of MCF-7 cells were marked: ROSC treatment changed the pattern of AgNORs in a time-dependent manner. The disintegration of nucleoli manifested by increasing number of nucleolar fragments already began at 6 hr posttreatment. This was accompanied by a redistribution of the nucleolin from the nucleolus beginning after 6 hr and preceded a decrease of histone acetylation and phosphorylation. Inhibition of DNA synthesis and accumulation of G(2)/M-arrested cells starting 6 hr posttreatment coincided with a strong increase of the p53 level and with an appearance of a few cells committed to undergo apoptosis. However, all these changes preceded the main wave of apoptosis, which occurred after 24 hr ROSC treatment as assessed by determination of the frequency of Annexin binding, activation of caspases as well as of DNA fragmentation. Onset of PARP-1 cleavage detected by immunoblotting and by immunohistochemistry 6 hr or 9 hr posttreatment, respectively, preceded for a few hours the DNA fragmentation detected in situ by TUNEL assay. Reconstitution of MCF-7 cells with caspase-3 did not change the kinetics of ROSC-induced apoptosis. Our results show that disintegration of nucleoli is an early marker of ROSC-induced changes. Cell cycle arrest precedes the main wave of apoptosis.     

Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells

The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The naturally occurring chemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone. This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage-independent growth, reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2-specific enzymatic activity than either compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs, whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.     

Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin

Quercetin, a widely distributed bioflavonoid, has been shown to induce growth inhibition in certain cancer cell types. In the present study we have pursued the mechanism of growth inhibition in MCF-7 human breast cancer cells. Quercetin treatment resulted in the accumulation of cells specifically at G2/M phase of the cell cycle. Mitotic index measured by MPM2 staining clearly showed that cells were transiently accumulated in M phase, 24 h after treatment. The transient M phase accumulation was accompanied by a transient increase in the levels of cyclin B1 and Cdc2 kinase activity. However, 24 h or longer treatment caused a marked accumulation of cells in G2 instead of M phase. Levels of cyclin B1 and cyclin B1-associated Cdc2 kinase activity were also decreased. We also found that quercetin markedly increased Cdk-inhibitor p21CIP1/WAF1 protein level after treatment for 48 h or longer, and the induction of p21CIP1/WAF1 increased its association with Cdc2-cyclin B1 complex, however, up-regulation of p53 by quercetin was not observed. Quercetin also induced significant apoptosis in MCF-7 cells in addition to cell cycle arrest, and the induction of apoptosis was markedly blocked by antisense p21CIP1/WAF1 expression. The present data, therefore, demonstrate that a flavonoid quercetin induces growth inhibition in the human breast carcinoma cell line MCF-7 through at least two different mechanisms; by inhibiting cell cycle progression through transient M phase accumulation and subsequent G2 arrest, and by inducing apoptosis.     

人参多糖与他莫昔芬联合对人乳腺癌MCF-7细胞凋亡的影响及其机制研究

目的： 探讨人参多糖 (ginseng polysaccharide,GPS)与他莫昔芬 (tamoxifen,TAM)联合对人乳腺癌MCF-7细胞凋亡的影响及其机制。方法：以不同浓度 (0、20、40、80和160 μg/mL)GPS作用于人乳腺癌MCF-7细胞株48 h,以MTT法检测细胞增殖抑制情况,以GPS的最小有作用剂量与不同浓度 (0、0.5、1、2和4 μg/mL)TAM联合处理细胞48 h,以金氏公式筛选出协同抑制作用最明显的联合剂量,以该联合剂量处理MCF-7细胞48 h后,用DAPI染色法、流式细胞术检测细胞凋亡情况;用Giemsa染色检测细胞有丝分裂指数;用Western blot检测细胞中Fas、Caspase-9 以及Parp的表达情况。结果：MTT法检测提示GPS在48 h对MCF-7细胞的最小有作用剂量为40 μg/mL,金氏公式计算结果提示40 μg/mL GPS+1 μg/mL TAM联合用药协同抑制细胞增殖作用最强 (q=1.82);与GPS或TAM单独作用相比,DAPI染色发现联合用药组镜下可见大量凋亡小体;流式细胞仪检测发现,联合用药能够协同增加细胞凋亡率 ( q=2.19,P <0.05);Giemsa染色结果显示联合用药能够协同抑制细胞有丝分裂 (均P<0.05);Western blot检测发现,联合用药能增加细胞中Fas表达,促进Caspase-9以及Parp的活化。结论：GPS与TAM联合能够协同通过Fas信号通路促进人乳腺癌MCF-7细胞发生凋亡。     

Gold nanoparticles induce apoptosis in MCF-7 human breast cancer cells

Background: Gold nanoparticles have recently been investigated with respect to biocompatibility accordingto their interactions with cells. The purpose of this study was to examine cytotoxicity and apoptosis induction bywell-characterized gold nanoparticles in human breast epithelial MCF-7 cells. Methods: Apoptosis was assessedby TUNEL, cytotoxicity by MTT assay and caspase 3, 9, p53, Bax and Bcl expression by real-time PCR assays.Results: Gold nanoparticles at up to 200 μg/mL for 24 hours exerted concentration-dependent cytotoxicity andsignificant upregulation of mRNA expression of p53, bax, caspase-3 & caspase-9, whereas expression of antiapoptoticbcl-2 was down-regulated. Conclusion: To the best of our knowledge this is the first report showingthat gold nanoparticles induce apoptosis in MCF-7cells via p53, bax/bcl-2 and caspase pathways.     

Reversine induces cell cycle arrest,polyploidy, and apoptosis in human breast cancer cells

Background

Reversine, a small synthetic purine analogue, has been reported to be effective in tumor suppression. In the present study, we demonstrated an antitumor activity of reversine that could suppress cellular proliferation and induce cell cycle arrest and apoptosis in human breast cancer cell lines.

Methods

To evaluate whether reversine could suppress cell growth of MCF-7 and MDA-MB-231 cells and induce cell death, the cell viability, cell cycle, and apoptosis were determined in this study.

Results

Reversine treatment in human breast cancer cells reduced cell viability in a dose-dependent manner. Cell cycle accumulation at the G₂/M phase in reversine-treated cells was also determined. Moreover, polyploidy was also found in reversine-treated cells. Apoptosis in reversine-treated cells was exhibited with PARP cleavage and caspase-3 and caspase-8 activation, but not caspase-9 activation, indicating that caspase-dependent apoptosis mediated by an extrinsic pathway took place in reversine-treated cells. Furthermore, reversine attenuated cell death in cells pretreated with a pan-caspase inhibitor before reversine treatment.

Conclusions

In the present study, we demonstrated that reversine contributes to growth inhibition in human breast cancer cells through cell cycle arrest, polyploidy, and/or apoptosis induction. The apoptosis mediated by reversine was induced by the mitochondria-independent pathway. Therefore, the potential role of reversine as a novel therapeutic agent for the treatment of breast cancer is worthy of further investigation.     

Tautomycetin诱导乳腺癌耐药细胞MCF-7/ADR的凋亡及其机制

目的:研究tautomycetin对乳腺癌耐药细胞MCF-7/ADR增殖及凋亡的影响及其机制.方法:MTr法检测tautomycetin对MCF-7/ADR细胞增殖的影响,流式细胞术检测MCF-7/ADR细胞的凋亡,Western blotting法检测tautomycetin对MCF-7/ADR细胞caspase相关蛋白、Bcl-2、Cyto-C、P53蛋白表达和Akt磷酸化的影响.结果:Tautomycetin可剂量(0.01～100μmol/L)依赖性地抑制MCF-7/ADR细胞的增殖(P＜0.05),IC50值为(1.26±0.12)μmol/L;与对照组相比,tautomycetin (1 μmol/L)可诱导MCF-7/ADR细胞凋亡,早期凋亡比例由(0.67±0.18)％升高至(17.2±3.8)％,晚期凋亡比例由(0.96±0.23)％升高至(28.4±5.7)％(P＜0.05).Tautomycetin可活化MCF-7/ADR细胞中caspase-7和caspase-9,降低Bcl-2蛋白的表达,促进线粒体释放Cyto-C,降低p-Akt的水平,但对caspase-8和P53的表达没有影响.结论:Tautomycetin可阻断Akt活化,以P53非依赖的方式通过Cyto-C介导的通路诱导MCF-7/ADR细胞凋亡.     

乳腺癌阿霉素耐药细胞株MCF-7/ADM的上皮间质化现象及机制

目的:建立乳腺癌阿霉素耐药细胞株,命名为MCF-7/ADM,探讨耐药细胞的上皮间质化(EMT)现象及机制,为耐药乳腺癌的治疗奠定基础.方法:中等浓度间歇法建立乳腺癌阿霉素耐药细胞株MCF-7/ADM.采用MTT和Transwell实验分别检测耐药对细胞增殖、迁移与转移能力的影响;荧光定量PCR检测MCF-7/ADM细胞EMT相关分子标志物E-钙黏蛋白及间质细胞分子标记N-钙黏蛋白、波形蛋白、纤维连接蛋白等的表达.ELISA检测TGF-β的表达,Western blot检测TGF-β及Smad2/3在蛋白水平的表达.结果:乳腺癌阿霉素耐药细胞MCF-7/ADM的耐药指数为32.2,对5-氟脲嘧啶、顺铂、环磷酰胺产生交叉耐药性.与MCF-7细胞相比,MCF-7/ADM细胞迁移和转移能力明显增强,E-cadherin表达降低而N-cadher-in表达显著增高,细胞上清中TGF-β1表达增加,细胞内TGF-β及磷酸化Smad2/3在蛋白水平表达升高.结论:乳腺癌阿霉素耐药细胞MCF-7/ADM发生EMT现象,可能与经典TGF-β通路激活有关.     

乳腺癌干细胞相关亚群细胞周期分析

目的：流式细胞仪分选乳腺癌细胞系MCF-7的肿瘤干细胞相关SP亚群（side population，SP），并检测SP和非SP细胞周期。方法：MCF-7细胞悬液经Hoeehst33342染色，流式细胞仪分选SP和非SP后，乙醇固定，PI染色，流式细胞仪检测细胞周期。结果：SP细胞在MCF-7细胞中占4％，Vempamil阻断后比例减为0．5％。SP细胞中S／G2／M期细胞最低，仅占7．9％，而非SP细胞增殖期比例相对高，S／G2／M期细胞约占34．2％。未分选细胞中S／G2／M期细胞比例居中，为22．9％。结论：人乳腺癌细胞系MCF-7含SP亚群，比例约为4％，且SP细胞多处于静止期，具有一般干细胞的特性。     

乳腺癌干细胞相关亚群细胞周期分析

目的:流式细胞仪分选乳腺癌细胞系MCF-7的肿瘤干细胞相关SP亚群(side population,SP),并检测SP和非SP细胞周期。方法:MCF-7细胞悬液经Hoechst33342染色,流式细胞仪分选SP和非SP后,乙醇固定,PI染色,流式细胞仪检测细胞周期。结果:SP细胞在MCF-7细胞中占4%,Verapamil阻断后比例减为0·5%。SP细胞中S/G2/M期细胞最低,仅占7·9%,而非SP细胞增殖期比例相对高,S/G2/M期细胞约占34·2%。未分选细胞中S/G2/M期细胞比例居中,为22·9%。结论:人乳腺癌细胞系MCF-7含SP亚群,比例约为4%,且SP细胞多处于静止期,具有一般干细胞的特性。     

MDA-7 results in downregulation of AKT concomitant with apoptosis and cell cycle arrest in breast cancer cells

The melanoma differentiation-associated gene-7 (mda-7) is a known mediator of apoptosis in cancer cells but not in normal cells. We hypothesized that MDA-7 interferes with the prosurvival signaling pathways that are commonly altered in cancer cells to induce growth arrest and apoptosis. We also identified the cell signaling pathways that are antagonized by MDA-7 leading to apoptosis. Using an adenoviral expression system, mda-7 was introduced into the breast cancer cell lines SKBr3, MCF-7 and MDA-MB-468, each with a different estrogen receptor (ER) and HER-2 receptor status. Downstream targets of MDA-7 were assessed by reverse phase protein array analysis, western blot analysis and immunofluorescence confocal microscopy. Our results show that MDA-7-induced apoptosis was mediated by caspases in all cell lines tested. However, MDA-7 modulates additional pathways in SKBr3 (HER-2 positive) and MCF-7 (ER positive) cells including downregulation of AKT-GSK3β and upregulation of cyclin-dependent kinase inhibitors in the nucleus. This leads to cell cycle arrest in addition to apoptosis. In conclusion, MDA-7 abrogates tumor-promoting pathways including the activation of caspase-dependent signaling pathways ultimately leading to apoptosis. In addition, depending on the phenotype of the breast cancer cell, MDA-7 modulates cell cycle regulating pathways to mediate cell cycle arrest.     

PS341对他莫昔芬诱导乳腺癌MCF-7细胞凋亡影响及其机制的探讨

目的:研究蛋白酶体抑制剂PS341是否可增强他莫昔芬(TAM)诱导的乳腺癌细胞凋亡,并探讨可能的机制。方法:采用MTT方法测定细胞活力,采用流式细胞仪检测细胞凋亡,采用蛋白质印迹法检测Caspase-8的活化,PARP的裂解及死亡受体Fas蛋白的表达。结果:TAM以时间及浓度依赖的方式抑制乳腺癌MCF-7细胞增殖,24及48h的IC50分别为25和6μmol/L,25μmol/L的TAM处理24h可诱导21.9%的MCF-7细胞发生凋亡,10nmol/L的PS341预处理后给予25μmol/L的TAM处理24h细胞凋亡增至34.6%,PS341与TAM均可轻微上调Fas的表达水平,两药联合应用后可显著上调Fas的表达水平,并明显增强线粒体外凋亡通路的活性。结论:PS341与TAM联合应用可通过上调Fas的表达水平,增强TAM诱导的乳腺癌MCF-7细胞凋亡。     

瘦素对人类乳腺癌细胞MCF-7增殖及凋亡的影响

 目的 观察不同浓度瘦素对人类乳腺癌细胞MCF-7增殖及凋亡的影响,探讨瘦素在乳腺癌发生及发展中的作用。方法 采用四甲基偶氮唑蓝（MTT）比色法检测不同浓度瘦素对体外培养的MCF-7细胞生长的增殖作用,流式细胞术分析细胞周期的分布, Annexin V-FITC／PI染色法检测细胞凋亡。结果 MTT比色法结果显示：不同浓度瘦素作用24、48、72 h能够促进MCF-7细胞增殖,浓度与时间不存在交互作用（F＝0.919,P＝0.523）,浓度及时间各自的主效应均差异有统计学意义（F＝12.699,P＝0.000; F＝647.881,P＝0.000）。多重比较结果显示：200　ng／ml及400　ng／ml瘦素组与对照组相比差异有统计学意义（P＝0.007;P＝0.000）,不同时间段之间均差异有统计学意义（P＝0.000）。流式细胞术分析显示100、400 ng／ml瘦素作用48 h后,G0／G1期细胞比例下降14.42 %（F＝10.464,P＝0.044）,S期比例分别上升7.57 %、22.19 %（F＝47.361,P＝0.005）,G2／M期变化差异无统计学意义（F＝1.77,P＝0.311）。未发现瘦素对MCF-7细胞早期凋亡的抑制作用。结论 瘦素能够促进乳腺癌细胞MCF-7的增殖并改变生长周期,但不会抑制凋亡,提示瘦素可能在乳腺癌的发展中发挥促进作用。     

Overexpression of bax sensitizes human breast cancer MCF-7 cells to radiation-induced apoptosis

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy and ionizing radiation (IR). bax, which forms a heterodimer with bcl-2 and accelerates apoptosis, is not, or only weakly, expressed in most human breast cancer cells, and weak bax expression is considered to be related to the resistance of breast cancer cells to apoptosis. bax expression vector was introduced to human breast cancer MCF-7 cells, which exhibit weak expression of bax, to demonstrate its role of modulating radiation-induced apoptosis. bax overexpression in MCF-7 cells by stable transfection does not affect viability by itself, but each stable transfectant was more sensitive to IR than the parental MCF-7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax. IR upregulated p53 and p21^WAFI about 5- to 10-fold and downregulated bcl-2 and bcl-XL by 80–90% at 6 hr in both parent and bax stably transfected MCF-7 cells to the same degree. FACS analysis and DNA electrophoresis revealed that this sensitization was due to apoptosis. We suggest that exogenous bax expression might be one of the factors determining cellular radiosensitivity in MCF-7 breast cancer cells and may have therapeutic applications for enhancing radiation sensitivity in breast cancer cells. © 1996 Wiley-Liss, Inc.     

Resveratrol inhibits cell proliferation and induces apoptosis of human breast carcinoma MCF-7 cells

Resveratrol, which is found in grapes and wine, has been reported to have a variety of important pharmacological effects including anti-inflammatory, anti-platelet, and anti-carcinogenetic properties. In this study, using the human breast cancer cell line MCF-7, we have analyzed a possible mechanism by which resveratrol could interfere with cell cycle control and induce cell death. Resveratrol treatment of MCF-7 cells resulted in a dose-dependent inhibition of the cell growth and the cells accumulated at the S phase transition of the cell cycle at low concentrations, but high concentrations do not induce S phase accumulation. The anti-proliferative effects of resveratrol were associated with a marked inhibition of cyclin D and cyclin-dependent kinase (Cdk) 4 proteins, and induction of p53 and Cdk inhibitor p21WAF1/CIP. Growth suppression by resveratrol was also due to apoptosis, as seen by the appearance of a sub-G1 fraction and chromatin condensation. In addition, the apoptotic process involves activation of caspase-9, a decrease of Bcl-2 as well as Bcl-XL levels, and an increase of Bax levels.     

Acacetin-induced cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells

In this study, we examined acacetin (5,7-dihydroxy-4'-methoxyflavone), a flavonoid compound, for its effect on proliferation in human non-small cell lung cancer A549 cells. The results first reported that acacetin not only inhibited A549 cell proliferation but also induced apoptosis and blocked cell cycle progression in the G1 phase. ELISA assay demonstrated that acacetin significantly increased the expression of p53 and p21/WAF1 protein, which caused cell cycle arrest. An enhancement in Fas and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by acacetin. Taken together, p53 and Fas/FasL apoptotic system may participate in the antiproliferative activity of acacetin in A549 cells.     

致康胶囊对人乳腺癌MCF-7细胞增殖及凋亡的影响

目的:探讨致康胶囊对人乳腺癌细胞系MCF-7增殖及凋亡的影响。方法:采用0（对照组）、0.5、1.0、2.0 mg/ml 致康胶囊培养液分别处理乳腺癌细胞MCF-7,分别于培养后24、48和72 h计数MCF-7细胞数。采用MTT法检测致康胶囊对乳腺癌MCF-7细胞生长增殖的影响;采用流式细胞仪检测细胞周期;PI 单染流式细胞仪检测致康胶囊对MCF-7细胞凋亡的影响。结果:不同浓度致康胶囊能有效地抑制MCF-7细胞增殖,且随浓度增加和作用时间延长,细胞的增殖抑制率增加。0.5、1.0、2.0 mg/ml的致康胶囊作用MCF-7细胞72 h后,G0/G1期细胞所占细胞周期的比例分别为19.33%±10.38%、14.12%±5.37%和26.84%±2.13%;而对照组G0/G1期比例为51.83%±1.90%。0.5、1.0、2.0 mg/ml致康胶囊处理48 h后细胞的凋亡率分别为2.09%±0.74%、3.84%±0.78%和 5.35%±0.83%。结论:致康胶囊能抑制MCF-7细胞的体外增殖,且抑制作用表现为时效和量效关系;并能通过阻滞细胞周期于G2/M期,促进乳腺癌细胞凋亡。