槟榔碱对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的：观察槟榔碱对人乳腺癌细胞（MCF-7）增殖和凋亡的影响,并探讨其机制。方法：采用四甲基偶氮唑盐（MTT）法检测不同浓度（0、10、30、50、100、300、500μmol/L）槟榔碱对MCF-7细胞增殖的影响,Hoechst 33342染色和流式细胞术检测细胞凋亡,Western blot法检测Bax,Bcl-2和P53蛋白表达。结果：低浓度（0、10、30、50μmol/L）槟榔碱不影响细胞的增殖和凋亡;而高浓度（100、300、500 μmol/L）槟榔碱呈浓度依赖性抑制MCF-7细胞增殖、诱导MCF-7细胞凋亡、提高P53和Bax蛋白表达、降低Bcl-2蛋白表达。结论：高浓度槟榔碱抑制MCF-7细胞增殖、诱导凋亡,其机制可能与提高P53和Bax蛋白表达,降低Bcl-2蛋白表达有关。     

Methionine cytotoxicity in the human breast cancer cell line MCF-7

Summary Among the first nutrients to be linked to cancer were methyl group containing nutrients including methionine. Methionine and its metabolic derivatives are essential components in several indispensable biological reactions including protein synthesis, polyamine synthesis, and many transmethylation reactions. The purpose of this study was to determine the extent to which methionine excess affects the proliferation and gene expression of the human breast cancer cell line MCF-7. Cells were first grown in control medium; the medium was then replaced with either control or methionine-supplemented treatment media. We found that 5 and 10 g/L methionine significantly suppressed cell growth on day 1, and no further growth was detected after 3 d of treatment. Cell, proliferation in the methionine treated group was significantly lower than that of the control group. Northern analysis revealed that expression of p53 in methionine-treated MCF-7 cells was approximately 70% lower than that of control cells. p53 is a key cell cycle regulatory, protein that has been implicated in tumorigenesis and cancer progression. Alteration of the p53 tumor suppressor gene is the most common genetic change found in a wide variety of malignancies, including cancer. This study shows that excess methionine (5 g/L) inhibited proliferation of MCF-7 breast cancer cells, and down regulation of p53 is correlated with this inhibition. These findings may aid in the development of nutritional strategies for breast cancer therapy.     

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

目的：通过敲低微小RNA （microRNA,miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂（inhibitor）,转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组（Mock组）,阴性对照组（negative control组,NC组）和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。     

In vitro selection of aptamer S1 against MCF-7 human breast cancer cells

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (K_d) value of 29.9 ± 6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.     

Novel thiosemicarbazides induced apoptosis in human MCF-7 breast cancer cells via JNK signaling

Abstract

In this study, novel thiosemicarbazides and 1,3,4-oxadiazoles were synthesized and evaluated for their anticancer effects on human MCF-7 breast cancer cell lines. Among the synthesized derivatives studied, compound 2-(3-(4-chlorophenyl)-3-hydroxybutanoyl)-N-phenylhydrazinecarbothioamide 4c showed the highest cytotoxicity against MCF-7 breast cancer cells as it reduced cell viability to approximately 15% compared to approximately 25% in normal breast epithelial cells. Therefore, we focused on 4c for further investigations. Our data showed that 4c induced apoptosis in MCF-7 cells which was further confirmed by TUNEL assay. Western blotting analysis showed that compound 4c up-regulated the pro-survival proteins Bax, Bad and ERK1/2, while it down-regulated anti-apoptotic proteins Bcl-2, Akt and STAT-3. Additionally, 4c induced phosphorylation of SAPK/JNK in MCF-7 cells. Pretreatment of MCF-7 cells with 10?µM of JNK inhibitor significantly reduced 4c-induced apoptosis. Molecular docking results suggested that compound 4c showed a binding pattern close to the pattern observed in the structure of the lead fragment bound to JNK1. Collectively, the data of current study suggested that the thiosemicarbazide 4c might trigger apoptosis in human MCF-7 cells by targeting JNK signaling.     

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.     

Spatial organization of three-dimensional cocultures of adriamycin-sensitive and -resistant human breast cancer MCF-7 cells

Genetic and cellular heterogeneity is one of mechanisms involved in increasing tumour aggressiveness during neoplastic progression. Development of drug-resistant tumour cell subpopulations is a major problem in clinical oncology. Multi-drug resistant tumour cells survive when exposed to cytotoxic agents. Here, we studied in a three-dimensional (3D) coculture system, called "ex vivo nodules", how drug-resistant and sensitive tumour cells settle down in a 3D space. For this, we cocultured adriamycin-sensitive (MCF-7S) and -resistant (MCF-7R) human breast cancer cells in long term nodules. We showed that both types of cells are able to grow separately or in coculture until five weeks in spheroidal forms. MCF-7R cells did not loose their multi-drug resistance when cultured in nodules as measured by RT-PCR. Curiously, the exterior aspects of mixed (MCF-7S/ MCF-7R) nodules and MCF-7R nodules were similar whereas MCF-7S nodules were completely different. Nevertheless, morphologically these three nodule types were distinct, in particular in their density. Immunostaining showed that in mixed nodules, MCF-7R cells were arranged at the periphery, whereas the MCF-7S cells are in the central part of the nodules.Even if the mechanism of this arrangement remained unclear, this work shows that three-dimensional cell culture is well adapted to the study of the relationships between adhesion mechanisms and drug-resistance.     

Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells

Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 μg/mL of oleuropein or 50 μg/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G₁ to S phase transition manifested by the increase of cell number in G₀/G₁ phase.     

Growth,morphological and biochemical changes in oxa-spermine derivative-treated MCF-7 human breast cancer cells

The growth inhibitory properties of two oxa-spermine derivatives named compound 1 and compound 2, representatives of a novel type of polyamine derivatives, were studied. Dose-response growth inhibitory curves obtained after 48h drug exposure demonstrated the much higher cytotoxic activity of compound 1 towards MCF-7 human breast cancer cells. Further experiments with compound 1 showed that this oxa-spermine derivative exhibited considerable cytotoxicity with IC(50) values of 3.74 microM and 2.93 microM after 24h and 48h drug exposure respectively. In MCF-7 cells, after 8h drug (10 microM) exposure it caused shrinkage, chromatin condensation and nuclear fragmentation. However, no clear DNA laddering was detected in treated cells. Drug treatment provoked an increase in polyamine oxidase (PAO) activity. This enzyme is able to produce cytotoxic H(2)O(2) and 3-acetamidopropanal, catalyzing the oxidative deamination of N(1)-acetylated derivatives of spermine and spermidine to spermidine and putrescine respectively. Taken together these data demonstrate that the novel oxa-polyamine derivative compound 1 has considerable cytotoxic activity towards MCF-7 cells and indicate that an induction of PAO may be involved in its cytotoxic and apoptotic effects.     

Estrogenic effects of two derivatives of icariin on human breast cancer MCF-7 cells

The aims of the present study were to determine the estrogenic activities of icariin (ICA) and its derivatives and their structure–estrogenic activity relationship. Therefore, icaritin (ICT) and desmethylicaritin (DICT) were derived from ICA. The estrogenic activities of ICA, ICT and DICT were examined by cell proliferation and progestogen receptor mRNA expression of estrogen-receptor-positive MCF-7 cells. Current studies exhibited that ICT and DICT both markedly enhanced the proliferation of MCF-7 cells; as compared to estradiol (100%), their relative proliferative effects (RPE) were 90% and 94%, respectively. Cell proliferation induced by ICT and DICT was completely antagonized by ICI182,780. ICT and DICT increased progestogen receptor (PR) at mRNA levels at 48 h after treatment, although the effects were not as prominent as 17β-estradiol (E₂). These phenomena were not observed with ICA. Results demonstrate that ICT and DICT (nonconjugated forms) possess estrogen-like activity; however, ICA appears to have no estrogenicity in the MCF-7 cell line model in vitro.     

The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.     

Two-signal electrochemical method for evaluation suppression and proliferation of MCF-7 cells based on intracellular purine

Two electrochemical signals ascribed to xanthine/guanine and hypanthine/adenine in MCF-7 cells were detected at 0.726 and 1.053 V, respectively. Based on the intensity of signals, the genistein-induced proliferation and suppression of MCF-7 cells could be evaluated. The results showed that with the increase of genistein dose at the range of 10⁻⁹ to 10⁻⁶ M, the two electrochemical signals of MCF-7 cell suspension increased due to the proliferation, whereas the tendency at the high dosage range of more than 10⁻⁵ M was decreased. The proliferation and cytotoxicity obtained by the electrochemical method were in agreement with those obtained by cell counting and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] method. Thus, the two-signal electrochemical method is an effective way to evaluate the effect of drugs on cell activity based on purine metabolism.     

The influence of lead and arsenite on the inhibition of human breast cancer MCF-7 cell proliferation by American ginseng root (Panax quinquefolius L.)

American ginseng root (Panax quinquefolius) has a number of purported therapeutic effects, including inhibition of cancer cell proliferation. The ability of environmentally relevant heavy metals to alter ginseng effects on cancer cell growth was the subject of this study. A water extract of American ginseng root was applied alone or in combination with physiologically relevant doses of either lead (Pb) or arsenite to MCF-7 breast cancer cells in vitro and effects on cell proliferation were determined. Ginseng alone produced a significant dose-dependent inhibition of MCF-7 cell proliferation starting at 0.5 mg ml(-1). Treatment of MCF-7 cells with 2.5 microM arsenite significantly decreased MCF-7 cell proliferation (p < 0.01). When cells were treated with arsenite (1.25 or 2.5 microM) in combination with ginseng extract (0.5 mg ml(-1)), there was an apparent synergistic inhibition of cell proliferation. Treatment of MCF-7 breast cancer cells with 50 microM Pb significantly decreased cell proliferation relative to control (p < 0.01), and concomitant ginseng and Pb treatment did not lead to a further decrease. These results suggest that contaminant heavy metals, some of which have been detected in ginseng root extracts or commercial ginseng preparations, may alter the biological activity of ginseng.     

Molecular changes associated with the acquisition of oestrogen hypersensitivity in MCF-7 breast cancer cells on long-term oestrogen deprivation

The growth dependence of many breast cancers on oestrogen has been exploited therapeutically by oestrogen deprivation, but almost all patients eventually develop resistance largely by unknown mechanisms. Wild-type (WT) MCF-7 cells were cultured in oestrogen-deficient medium for 90 weeks in order to establish a long-term oestrogen-deprived MCF-7 (LTED) which eventually became independent of exogenous oestrogen for growth. After 15 weeks of quiescence (LTED-Q), basal growth rate increased in parallel with increasing oestrogen sensitivity. While 10⁻⁹ M oestradiol (E2) maximally stimulated WT growth, the hypersensitive LTED (LTED-H) were maximally growth stimulated by 10⁻¹³ M E2. By week 50, hypersensitivity was apparently lost and the cells became oestrogen independent (LTED-I), although the pure antioestrogen ICI182780 still inhibited cell growth and reversed the inhibitory effect of 10⁻⁹ M E2 at 10⁻¹² to 10⁻⁷ M. Tamoxifen (10⁻⁷ to 10⁻⁶ M) had a partial agonist effect on WT, but had no stimulatory effect on LTED. Whilst LTED cells have a low progesterone receptor (PgR) expression in all phases, oestrogen receptor (ER) a expression was, on average, elevated five- and seven-fold in LTED-H and LTED-I, respectively, and serine118 was phosphorylated. ERβ expression was up-regulated and the levels of insulin receptor substrate 1 (IRS-1) remained low throughout all phases. The levels of RIP140 mRNA appeared to decrease to approximately 50% of the WT message in LTED-Q and remained constant into the hypersensitive phase. No significant changes were observed in the expression of SUG-1, TIF-1 and SMRT in LTED. The overall changes in nuclear receptor interacting proteins do not appear to be involved in the hypersensitivity. Thus, the resistance of these human breast cancer cells to oestrogen-deprivation appears to be due to acquired hypersensitivity which may be explained in part by increased levels of and phosphorylated ER.     

Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

Recently, salidroside (p-hydroxyphenethyl-β-d-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) were sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.     

MiR-155 promotes proliferation of human breast cancer MCF-7 cells through targeting tumor protein 53-induced nuclear protein 1

Background

MiR-155 has emerged as an “oncomiR”, which is the most significantly up-regulated miRNA in breast cancer. However, the mechanisms of miR-155 functions as an oncomiR are mainly unknown. In this study, the aims were to investigate the effects of miR-155 on cell proliferation, cell cycle, and cell apoptosis of ERalpha (+) breast cancer cells and to verify whether TP53INP1 (tumor protein 53-induced nuclear protein 1) is a target of miR-155, and tried to explore the mechanisms of miR-155 in this process.

Results

The expression of miR-155 is significantly higher in MCF-7 cells compared with MDA-MB-231 cells. Ectopic expression of TP53INP1 inhibits growth of MCF-7 cells by inducing cell apoptosis and inhibiting cell cycle progression. Overexpression of miR-155 increases cell proliferation and suppress cell apoptosis, whereas abrogating expression of miR-155 suppress cell proliferation and promotes cell apoptosis of MCF-7 cells. In addition, miR-155 negatively regulates TP53INP1 mRNA expression and the protein expression of TP53INP1, cleaved-caspase-3, -8, -9, and p21, and luciferase reporter reveals that TP53INP1 is targeted by miR-155.

Conclusions

TP53INP1 is the direct target of miR-155. MiR-155, which is overexpressed in MCF-7 cells, contributes to proliferation of MCF-7 cells possibly through down-regulating target TP53INP1.