Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5–7 μM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 μM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17β-estradiol (E₂) or in the presence of a low Tam concentration (1 μM) made the cells even more susceptible to rapid death induction by 5 or 7 μM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X_L and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.     

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.     

Role of the insulin-like growth factor system on an estrogen-dependent cancer phenotype in the MCF-7 human breast cancer cell line

We previously established that exposure of the estrogen receptor (ER) positive MCF-7 human breast cancer cell line to 17-β-estradiol (E₂) results in the post-confluent development of multilayered cellular aggregates (foci) which is consistent with the in vivo cancer phenotype of uncontrolled cellular proliferation. In this investigation, the interaction between the insulin-like growth factor receptor (IGF-IR) and ER-signaling systems in regard to post-confluent focus development was studied. We demonstrated that focus development requires the presence of E₂ and insulin-like growth factor I (IGF-I) or insulin-like growth factor II (IGF-II), as well as intact ER and IGF-IR.

Focus development in MCF-7 cultures, which occurs only after formation of a confluent monolayer, coincides with E₂ regulation of key members of the IGF-signaling system such as IGF-IR, IGF-II, insulin receptor substrate 1 (IRS-1), and insulin-like growth factor binding protein 3 (IGFBP-3), as demonstrated by real-time polymerase chain reaction (PCR). To establish the relevancy of an intact IGF-signaling system for foci formation, we generated stable clones from MCF-7 with IGF-IR suppressed by siRNA. Results from these studies implicate signaling through the IGF-IR to be an integral requirement for E₂-dependent post-confluent proliferation and focus formation. In summary, these studies establish the interactive roles of IGFs and E₂ in the post-confluent development of foci, and will allow subsequent identification of targets for therapeutic intervention in the control and treatment of estrogen-dependent breast cancer.     

17beta-estradiol inhibits forskolin-induced vascular endothelial growth factor promoter in MCF-7 breast adenocarcinoma cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is induced by the cAMP-dependent signalling pathway in several cell types, and by estrogens in some human breast cancer cells. Here, we investigated the cross-talk between estrogens and cAMP/PKA-dependent signalling pathway in human breast cancer MCF-7 cells. The results show that, in the absence of any CRE and ERE, forskolin induces whereas estrogens have no effect on VEGF promoter. Moreover, estrogens, through estrogen receptors, partly inhibit the forskolin-induced VEGF promoter in MCF-7 human breast cancer cells. Therefore, in breast cancers, estrogens could partly inhibit the effect of ligand-activated G protein-coupled receptors on VEGF expression.     

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.     

人重组核苷二磷酸激酶α亚基对MCF-7/S的影响

软琼脂集落形成实验检测rhNDPK -α对乳腺癌细胞株MCF - 7 S集落形成率的影响 ,结果表明 ,MCF - 7 S集落形成率随rhNDPK -α浓度增高下降 ,提示rhNDPK -α可能具有抑制乳腺癌细胞株MCF - 7 S转移作用 ;MTT法检测rhNDPK -α分别联用丝裂霉素C、5 -氟尿嘧啶和千金藤碱对MCF - 7 S的增殖影响 ,结果显示 ,rhNDPK -α分别联用三种化疗物与这三种化疗药物单独作用于MCF - 7 S无明显差异 (P >0 0 5 ) ,提示rhNDPK -α对丝裂霉素C、5 -氟尿嘧啶和千金藤碱没有增敏作用。     

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。     

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.     

Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha. SPHK1 also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK1 stimulated ERK1/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S1P or dihydro-S1P, ligands for the S1P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK1 cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK1 may play an important role in breast cancer progression by regulating tumor cell growth and survival.     

The amino-terminal region of insulin-like growth factor binding protein-3, (1-95)IGFBP-3, induces apoptosis of MCF-7 breast carcinoma cells

In an earlier study, we reported that an N-terminal proteolytic fragment ((1-95)IGFBP-3) corresponding to the first 95 residues of human insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation in a variety of fibroblasts. With a view to investigating its cytostatic capacity in carcinoma cells, we transiently transfected MCF-7 breast adenocarcinoma cells with an expression vector containing (1-95)IGFBP-3 cDNA. The transfected cells secreted a hyper-glycosylated form of (1-95)IGFBP-3. Twenty-four hours after transfection, cell morphology and viability were similar in control and (1-95)IGFBP-3-secreting cells. However, after 48 h, (1-95)IGFBP-3-secreting cells were apoptotic, with marked cytoplasmic vacuolation and increased free histones in the cytoplasm. Culture media conditioned by (1-95)IGFBP-3-secreting cells also induced morphological changes and apoptosis in wild-type MCF-7 cells, indicating that (1-95)IGFBP-3 was responsible for the effects observed. These results provide further evidence that the N-terminal proteolytic fragment of IGFBP-3 has a functional role.     

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.     

Alterations in phosphoinositide metabolism associated with 17 beta-estradiol and growth factor treatment of MCF-7 breast cancer cells

Steady-state levels of phosphatidyl inositol (PtdIns) turnover are examined in MCF-7 human breast cancer cells in response to estradiol treatment. Elevated levels of PtdIns are observed 12-24 h after estradiol treatment, occur at estradiol concentrations as low as 10(-12) M, and are competitively blocked by the antiestrogen LY117018. MCF-7 cells secrete a transforming growth factor (TGF) alpha-like material which can partly replace estradiol in conferring tumorgenicity in nude mice. We show that acute or chronic treatment of MCF-7 cells with TGF alpha results in elevated PtdIns turnover and that chronic treatment increases growth rate. In contrast TGF beta is growth inhibitory and blocks estradiol-induced increases in PtdIns turnover. A phosphatidyl inositol 4,5-bisphosphate specific phospholipase-C activity has been identified and is elevated in association with estradiol treatment. These data are consistent with estradiol-induced autocrine growth factors, including TGF alpha, acting through the PtdIns turnover pathway as part of their mechanism of action.     

Differential effect of glucose deprivation on MAPK activation in drug sensitive human breast carcinoma MCF-7 and multidrug resistant MCF-7/ADR cells

We have investigated the effect of glucose deprivation treatment on the activation of mitogen activated protein kinases (MAPKs) in the drug-sensitive human breast carcinoma cells (MCF-7) and its drug resistant variant (MCF-7/ADR) cells. Western blots and in-gel kinase assays showed that glucose free medium was a strong stimulus for the activation of MAPK in MCF-7/ADR cells. No activation was seen in MCF-7 cells. MAPK was activated within 3 min of being in glucose free medium and it remained activated for over 1 h in MCF-7/ADR cells. After being returned to complete medium, 1 h was required for the MAPK to become deactivated. To investigate whether alternative sources of ATP could inhibit glucose deprivation induced MAPK activation, we added glutamine and glutamate to glucose deprived medium. The addition of glutamine did not reverse glucose deprivation induced MAPK activation in MCF-7/ADR cells. The addition of glutamate, however, decreased the MAPK activation and the length of time of activation. We observed an increase greater than three fold in MEK, Raf, Ras, and PKC activity with glucose deprivation in MCF-7/ADR cells. This suggests that glucose deprivation-induced MAPK activation is mediated through this signal transduction pathway.     

eIF-4E腺病毒载体构建及对乳腺癌MCF-7细胞转移能力的影响

摘要 目的：构建乳腺癌真核细胞起始因子一4E(eukaryotic initiation factor 4E,eIF-4E)基因重组腺病毒载体,并观察其对乳腺癌细胞MCF-7转移能力的影响。方法：应用基因重组技术将eIF-4E基因构建于腺病毒载体pAD-X,转染293包装细胞得到高滴度重组腺病毒,并用real-time PCR进行eIF-4E基因表达的验证。将重组腺病毒pAD-eIF-4E感染MCF-7细胞后,应用transwell小室法观察其对细胞侵袭和运动能力的影响。结果：酶切结果与预期相符,real-time PCR可检测到转染后MCF-7细胞有eIF-4E基因表达。且病毒转染后transwell小室可见,MCF-7细胞的侵袭和运动能力均受到显著的抑制（均为p<0.01）。结论：重组eIF-4E腺病毒载体正确构建,其对乳腺癌细胞MCF-7的侵袭和运动都有抑制作用。     

白细胞介素-2受体在乳腺癌MCF-7细胞的表达

目的 :观察乳腺癌MCF 7细胞上白细胞介素 2受体 (IL 2R)α、β和γ链的表达、IL 2对MCF 7细胞增殖的作用及雌激素对三条链表达的影响。方法 :使用特异性IL 2R多克隆抗体以免疫细胞化学方法和流式免疫荧光法检测MCF 7细胞上IL 2R的表达 ,以MTT法及3 H TdR掺入法检测细胞增殖情况。结果 :MCF 7细胞上存在IL 2Rα、β、γ的免疫阳性物质 ,其中IL 2Rγ的表达要强于IL 2Rα、β的表达 ;10 -6mol/L浓度的雌二醇可促进IL 2Rα、β的阳性细胞数及IL 2Rγ的免疫阳性物质的含量 ;IL 2在 10 0U/ml至 10 0 0U/ml的浓度范围内可显著促进MCF 7细胞的增殖。结论 :MCF 7细胞上存在IL 2R且其表达受雌二醇的调节 ,IL 2可能通过IL 2R影响MCF 7细胞的增殖     

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.     

The potentiation of estrogen on insulin-like growth factor I action in MCF-7 human breast cancer cells includes cell cycle components

To gain insight into the mechanisms involved in the cross-talk between IGF-1 receptor (IGF-1R) and estrogen receptor signaling pathways, we used MCF-7-derived cells (SX13), which exhibit a 50% reduction in IGF-1R expression. Growth of NEO cells (control MCF-7 cells) was stimulated by both IGF-1 and estradiol (E2), and the addition of both mitogens resulted in a synergistic response. Estrogen enhanced IGF-1R signaling in NEO cells, but this effect was markedly diminished in SX13 cells. Estrogen was also able to potentiate the IGF-1 effect on the expression of cyclin D1 and cyclin E and on the phosphorylation of retinoblastoma protein in control but not in SX13 cells. IGF-1 increased the protein level of p21 and the luciferase activity of the p21 promoter, whereas it only reduced the protein level of p27 without affecting p27 promoter activity. Estrogen did not affect the p21 inhibitor, but it decreased the protein level of p27 and the p27 promoter luciferase activity. These effects of both mitogens were also observed at the level of association of both cyclin-dependent kinase inhibitors with CDK2 suggesting that IGF-1 and E2 affect the activity of both p21 and p27. Taken together, these data suggest that in MCF-7 cells, estrogen potentiates the IGF-1 effect on IGF-1R signaling as well as on the cell cycle components. Moreover, IGF-1 and E2 regulate the expression of p21 and p27 and their association with CDK2 differently.     

Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Abstract

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6?months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2?days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.