Estrogen sulfates: Biological and ultrastructural responses and metabolism in MCF-7 human breast cancer cells

The biological effects and ultrastructural alterations by different estrogen-3-sulfates (E₁-3-S and E₂-3-S) and estradiol-17-sulfate (E₂-17-S) were studied in the MCF-7 mammary cancer cell line in culture. The estrogen-3-sulfates very significantly stimulated the progesterone receptor (PR). The values (in pmoles/mg DNA ± SE) were: control, 0.46 ± 0.09; E₁-3-S, 2.24 ± 0.30, and E₂-3-S, 2.56 ± 0.45. The value of PR after E₂-17-S incubation (0.56 ± 0.24) was similar to the non-treated cells. The PR values obtained by the incubation of unconjugated estrone and estradiol were: 2.63 ± 0.45 and 2.27 ± 0.36, respectively. Analysis of the unconjugated estrogens in the medium indicated significant hydrolysis of estrogen-3-sulfates but not of E₂-17-S. Using [³H]-E₁-3-S, an important transformation was observed inside the cells, a great part being converted to estradiol (>60% in the nuclear fraction). Electron microscopic examination indicated alterations in the secretory system after incubation with estrogen-3-sulfates similar to those obtained with unconjugated estradiol. The effect provoked by E₂-17-S was significantly less than for the other sulfates.As estrogen sulfates are quantitatively the most important form of estrogens in the mammary gland, it is suggested that estrogen-3-sulfates play an important role in the biological responses to estrogens in breast cancer.     

miR-620通过靶向ING4调控乳腺癌MCF-7细胞放射敏感性

目的：探讨miR-620对乳腺癌MCF-7细胞放射敏感性的影响及其机制。方法：收集2017年3月至2018年3月在海南省儋州市人民医院手术切除的21例乳腺癌患者的癌及癌旁组织标本,以及乳腺癌细胞MCF-7、BCaP-37和乳腺上皮细胞HBL-100,采用qPCR法检测癌组织和细胞中miR-620和生长抑制因子4(ING4)mRNA的表达。利用脂质体转染技术,分别将miR-620抑制剂（anti-miR-620）和抑制剂阴性对照（anti-miR-NC）、anti-miR-620和ING4小干扰RNA(si-ING4)、anti-miR-620和小干扰RNA阴性对照序列（si-NC）转染至MCF-7细胞,经放射处理后（依次记为IR+anti-miR-620组、IR+anti-miR-NC组、IR+antimiR-620+si-ING4组、IR+anti-miR-620+si-NC组）,利用克隆形成实验、MTT法和FCM分别检测细胞放射敏感性、细胞增殖活力、细胞周期分布和凋亡率。双荧光素酶报告基因实验和WB法验证miR-620和ING4的靶向关系。结果：与癌旁组织和HBL-100细胞比较,...     

Polynuclear aromatic hydrocarbon carcinogens as antiestrogens in MCF-7 human breast cancer cells: role of the Ah receptor.

Treatment of MCF-7 cells with 1.0 microM 3-methylcholanthrene (MC) caused a decrease in cell proliferation and [3H]thymidine uptake whereas no effects were observed at a lower (0.1 microM) concentration. Co-treatment of the cells with 1 nM 17 beta-estradiol plus 0.1 or 1.0 mu MC resulted in a significant inhibition of 17 beta-estradiol-induced growth and [3H]thymidine uptake. MC also inhibited the 17 beta-estradiol-induced secretion of the 52 kDa protein (procathepsin D) in MCF-7 cells and caused a concentration-dependent decrease in the nuclear estrogen receptor (ER) as determined by either velocity sedimentation analysis or immunoquantitation with human ER antibodies. The effects of several different polynuclear aromatic hydrocarbon (PAH) congeners on the nuclear ER in MCF-7 cells were also determined. Only those congeners which bound to the aryl hydrocarbon (Ah) receptor, namely benzo[a]pyrene, benz[a]anthracene, 7,12-dimethylbenz[a]anthracene and MC, caused a decrease in nuclear ER levels. In contrast, benzo[ghi]perylene, a congener which did not bind to the Ah receptor did not affect nuclear ER levels in MCF-7 cells. Moreover, with some congeners the decrease in nuclear ER levels could be observed without any significant induction of ethoxyresorufin O-deethylase activity, a P4501A1-dependent monooxygenase. These data suggest that the Ah receptor liganded with MC and related PAHs induced a broad spectrum of antiestrogenic responses in MCF-7 cells and complements the results of previous studies which report the antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and other halogenated aromatics which are also Ah receptor agonists.     

Role of serum in the prolactin responsiveness of MCF-7 human breast cancer cells in long-term tissue culture

MCF-7 human breast cancer cells, grown in long-term tissue culture, were found to be highly responsive to prolactin in terms of growth even in the presence of serum. Human prolactin, placental lactogen, and growth hormone (50-250 ng/ml) stimulated MCF-7 cells to grow when added to culture medium of cells in the presence of charcoal-stripped serum. Within 3 days of the hormone addition, a 4.4-fold increase in cell number was achieved with human prolactin at 100 ng/ml in the presence of 10% charcoal-stripped serum. Under these same conditions, estradiol-17 beta at 10(-8) M achieved only a 2-fold increase. After 6 days of culture, both estradiol-17 beta and prolactin gave a total 5-fold increase in cell number. No prolactin effect was achieved in the presence of 10% fetal bovine serum. Stripping fetal bovine serum with dextran-coated charcoal removes as much as 85% of the endogenous lactogens. Removal of these hormones is essential for demonstration of subsequent prolactin-induced growth response in MCF-7 cells, since bovine prolactin binds effectively to lactogen receptors on the surface of the cells but does not transmit a growth signal. When added simultaneously with human prolactin, bovine prolactin blocks the growth response to the former hormone. These results clearly demonstrate that, under the proper conditions of culture, the human breast cancer cell line MCF-7 is highly responsive to growth stimulation by homologous lactogenic hormones. This then affords us an excellent model for further studies on the possible role of prolactin in growth and maintenance of human breast cancer.     

RhG-CSF对乳腺癌细胞MCF-7增殖作用的影响

目的:探讨人重组粒细胞集落刺激因子(RhG-CSF)对乳腺癌细胞株MCF-7的增殖作用的影响。方法:体外培养人乳腺癌细胞株MCF-7,将含不同浓度RhG-CSF的培养液与MCF-7细胞共同培养不同时间,采用MTT比色法计算细胞生长增殖率;Anexxin V/PI双染试剂盒检测细胞凋亡率。实验结果用SPSS.v16.0软件分析。结果:不同浓度RhG-CSF处理细胞后,可以显著促进MCF-7细胞株的增殖(P<0.01),且当浓度为10μg/L时增殖作用最强。同时各浓度的RhG-CSF亦可抑制MCF-7细胞凋亡(P<0.01)。结论:RhG-CSF可以显著促进人乳腺癌细胞株MCF-7增殖。当浓度小于10μg/L时,增殖呈浓度依赖性。大于10μg/L时,增殖能力反而逐渐下降。同时RhG-CSF亦可抑制MCF-7细胞凋亡。     

AEG-1基因促进乳腺癌细胞株MCF-7转移

目的 观察AEG-1基因在细胞水平对乳腺癌细胞MCF-7转移的影响。方法 通过将siRNA转染进MCF-7细胞,沉默细胞中AEG-1表达量,以转染阴性siRNA作为对照组。分别采用Transwell小室检测细胞迁移侵袭能力、CCK8实验检测细胞增殖能力。同时通过检测细胞中VEGF的变化及HUVEC细胞体外管腔形成实验考察AEG-1对于血管新生的影响。结果 沉默AEG-1,MCF-7细胞的迁移能力、侵袭能力和增殖能力明显受到抑制。沉默AEG-1,MCF-7细胞的VEGF表达明显降低。上清处理HUVEC细胞,沉默AEG-1组的血管新生能力明显受到抑制。结论 沉默AEG-1基因能显著抑制MCF-7细胞转移的多个层面,包括细胞迁移、侵袭、增殖以及血管新生。表明AEG-1基因在乳腺癌转移过程中起着重要作用,也为将来乳腺癌治疗开拓了新思路。     

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.     

RhG-CSF对乳腺癌细胞MCF-7增殖作用的影响

目的：探讨人重组粒细胞集落刺激因子（RhG-CSF）对乳腺癌细胞株MCF-7的增殖作用的影响。方法：体外培养人乳腺癌细胞株MCF-7,将含不同浓度RhG-CSF的培养液与MCF-7细胞共同培养不同时间,采用MTT比色法计算细胞生长增殖率;Anexxin V/PI双染试剂盒检测细胞凋亡率。实验结果用SPSS.v16.0软件分析。结果：不同浓度RhG-CSF处理细胞后,可以显著促进MCF-7细胞株的增殖（P〈0.01）,且当浓度为10μg/L时增殖作用最强。同时各浓度的RhG-CSF亦可抑制MCF-7细胞凋亡（P〈0.01）。结论：RhG-CSF可以显著促进人乳腺癌细胞株MCF-7增殖。当浓度小于10μg/L时,增殖呈浓度依赖性。大于10μg/L时,增殖能力反而逐渐下降。同时RhG-CSF亦可抑制MCF-7细胞凋亡。     

人乳腺癌MCF-7细胞中SP亚群细胞的分离及其生物学行为研究

目的:了解肿瘤干细胞的生物学行为及相关的调控机制.方法:采用干细胞对Hoechst33342染料外排的特性进行MCF-7 细胞系肿瘤干细胞的流式分选.并对其增殖、自我更新、分化能力等生物学行为进行研究.还利用流式细胞仪、免疫荧光技术、荧光定量PCR等技术进行了与干细胞生物学行为密切相关的Wnt信号转导通路中相关分子的表达特征研究.结果:MCF-7细胞系中包含SP亚群(side population);且SP细胞具有分化潜能,高增殖能力,及自我更新特性;而且Wnt通路的各种关键分子在该细胞系SP亚群细胞中呈活化状态.结论:MCF-7细胞系的SP亚群可以代表该细胞系的肿瘤干细胞,Wnt通路在该肿瘤细胞系肿瘤干细胞的自我更新等其它生物学行为的调控中起重要作用.     

TrkA-siRNA增强人乳腺癌MCF-7细胞对紫杉醇的敏感性

目的 探讨靶向抑制TrkA基因表达后,人乳腺癌细胞MCF-7对化疗药物紫杉醇敏感性的变化.方法 8 μmol/L紫杉醇作用于乳腺癌MCF-7亲本细胞株和TrkA-siRNA转染细胞株24、48小时后,MTT法检测细胞增殖抑制效应,Western blot检测凋亡蛋白caspase-3的活化.倒置显微镜观察细胞株生长的形态学变化.结果 紫杉醇作用24、48小时后,其对TrkA-siRNA细胞株的生长抑制均高于MCF-7亲本细胞株(P＜0.05),且48小时抑制率高于24小时(P＜0.05).Western blot结果显示,caspase-3蛋白在紫杉醇作用24小时后被激活,其在TrkA-siRNA细胞株中的表达高于MCF-7亲本细胞株(P＜0.05).结论 TrkA-siRNA能增加乳腺癌细胞对化疗药物紫杉醇的敏感性.     

Transfected MCF-7 cells as a model for breast cancer progression

Summary The MCF-7 human breast carcinoma cell line has been used as a recipient for eukaryotic plasmid expression vectors to determine the effects of growth factor and growth factor receptor overexpression on the estrogen-dependent, antiestrogen sensitive and poorly metastatic phenotypes exhibited by this line. Overexpression of some members of the erbB family of ligands and receptors were found to have some effects on these pheno-types. However, only when two members of the fibroblast growth factor family, FGF-1 and FGF-4, were overexpressed was progressivein vivo growth observed is either ovariectomized nude mice without estrogen supplementation or in mice that received tamoxifen treatment. FGF transfected cells also exhibited an increased ability to form micrometastases. The implications of these results with regard to the possible role of the paracrine and autocrine effects of angiogenic growth factor production in breast cancer progression are discussed.     

Biosynthesis of alpha fetoprotein by MCF-7 human breast cancer cells

Direct evidence was obtained for de novo synthesis of AFP by MCF-7 human breast cancer cells per se. Synthesis was demonstrated by L-14C-leucine and L-35S-methionine incorporation into immunochemically isolated AFP, and confirmed by radioimmunodiffusion and radioimmunoelectrophoresis. This information indicates that AFP synthesis is associated with normal and neoplastic cells of several different histotypes, and suggests that AFP detected and measured previously in primary human breast cancer tissue cytosol (Sarcione et al., 1983) also resulted from in situ biosynthesis by breast cancer cells per se rather than uptake of exogenous AFP originating from extracellular sources. Evidence that AFP obtained after treatment of 14C-leucine radiolabelled MCF-7 breast cancer cell protein with 0.4 M KCl contained 2.6 times more radioactivity than did AFP obtained before such salt treatment is interpreted as indicating that two different molecular species of de novo synthesized AFP existed in breast cancer cells: (1) larger amount of non-immunoreactive AFP which became immunoreactive and measurable after KCl treatment, and (2) smaller amounts of free immunoreactive AFP. 14C-radiolabelled AFP obtained before and after treatment of cell protein with 0.4 M KCl codiffused, comigrated with alpha 1 electrophoretic mobility and gave an identical radioimmunologic reaction both with each other and with added carrier human cord serum AFP. Furthermore, preliminary studies indicated that radiolabelled non-immunoreactive AFP could be separated from lower-molecular-weight free AFP by chromatography on Sephadex G-200. Taken together, these findings suggest that synthesized free AFP was bound as a non-immunoreactive high-molecular-weight macromolecular complex rather than being covalently linked. Our current working hypothesis is that most of the de novo synthesized endogenous AFP in MCF-7 human breast cancer cells was rapidly and reversibly bound by hydrophobic bonding to a specific cytoplasmic AFP-receptor.     

Serum regulation of the estrogen responsiveness of the human breast cancer cell line MCF-7

Initially after receiving MCF-7 cells, we were able to confirm their estrogen responsiveness. We observed significant increases in thymidine incorporation, in thymidine kinase activity, and in cell numbers in response to 10(-8) M estradiol. Subsequently, however, the cells failed to show a response to estradiol. A growth response to estradiol could be restored by increasing the serum concentration in the medium. Cells grown in 15% serum (calf or human) responded to estradiol with increased rates of growth and thymidine incorporation and increased activities of thymidine kinase and DNA polymerase. We suggest that there is present in serum a "factor" which can influence the expression of a growth response to estradiol.     

The role of peroxiredoxin II in radiation-resistant MCF-7 breast cancer cells

Although several signaling pathways have been suggested to be involved in the cellular response to ionizing radiation, the molecular basis of tumor resistance to radiation remains elusive. We have developed a unique model system based upon the MCF-7 human breast cancer cell line that became resistant to radiation treatment (MCF+FIR30) after exposure to chronic ionizing radiation. By proteomics analysis, we found that peroxiredoxin II (PrxII), a member of a family of peroxidases, is up-regulated in the radiation-derived MCF+FIR3 cells but not in the MCF+FIS4 cells that are relatively sensitive to radiation. Both MCF+FIR3 and MCF+FIS4 cell lines are from MCF+FIR30 populations. Furthermore, the resistance to ionizing radiation can be partially reversed by silencing the expression of PrxII by PrxII/small interfering RNA treatment of MCF+FIR3 resistant cells, suggesting that PrxII is not the sole factor responsible for the resistant phenotype. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing MCF+FIS4 cells when compared with vector control cells. The up-regulation of the PrxII protein in radioresistant cancer cells suggested that human peroxiredoxin plays an important role in eliminating the generation of reactive oxygen species by ionizing radiation. The present finding, together with the observation that PrxII is also up-regulated in response to ionizing radiation in other cell systems, strengthens the hypothesis that the PrxII antioxidant protein is involved in the cellular response to ionizing radiation and functions to reduce the intracellular reactive oxygen species levels, resulting in increased resistance of breast cancer cells to ionizing radiation.     

Multidrug-resistant MCF-7 breast cancer cells contain deficient intracellular calcium pools

Emergence of resistance to antineoplastic drugs poses a major impediment to the successful treatment of breast cancer. We previously reported that human breast carcinoma MCF-7 cells selected for resistance against doxoru-bicin (MCF-7/DOX cells) expressed high levels of tissue-type transglutaminase (tTGase), a calcium-dependent protein cross-linking enzyme that plays a role in apoptosis. The purpose of this study was to determine the mechanisms by which MCF-7/DOX cells survive and proliferate despite high levels of tTGase expression. Our results demonstrate that the MCF-7/DOX cells contain deficient intracellular calcium pools, which may explain their ability to survive and tolerate the high levels of tTGase expression. Treatment with thapsigargin failed to induce any significant killing of MCF-7/DOX cells. Similar treatment of the drug-sensitive MCF-7 wild-type (MCF-7/WT) cells, however, induced significant apoptosis. Treatment with the ionophore A23187, on the other hand, killed a large percentage of both the MCF-7/DOX and the MCF-7/WT cells. We also established a revertant cell line, MCF-7/RT, from MCF-7/DOX cells to rule out the involvement of P-glycoprotein (P-gp) in these phenomena. Unlike the MCF-7/DOX cells, the MCF-7/RT cells showed no detectable P-gp expression; the MCF-7/RT cells, however, continued to express high levels of tTGase. Moreover, like MCF-7/DOX cells, the MCF-7/RT cells were highly resistant to thapsigargin-induced apoptosis but were sensitive to the ionophore A23187-induced apoptosis. These results suggest that the resistance of MCF7/DOX cells to thapsigargin is linked to their defective intracellular Ca²⁺ stores, a notion that was directly confirmed by single-cell spectrofluorometric analysis.     

紫花牡荆素抑制乳腺癌MCF-7细胞增殖与侵袭能力的研究

目的观察紫花牡荆素对乳腺癌MCF-7细胞株增殖与侵袭能力的影响并探讨其分子机制。方法应用四甲基偶氮唑蓝（MTT）比色法与侵袭实验检测紫花牡荆素对MCF-7细胞增殖与侵袭能力的影响,应用反转录PCR、Western blot法、Tunel法检测紫花牡荆素对基因表达、蛋白表达、细胞凋亡的影响。结果不同浓度紫花牡荆素均抑制MCF-7细胞增殖水平,同未加药对照组比较差异均有统计学意义（P〈0．05）,5、10、20μmol／L紫花牡荆素作用后,MCF-7细胞迁移数与未处理组相比分别降低20.3％、44．4％和50．3％（P〈0．05）。以10μmol／L紫花牡荆素处理后,凋亡细胞数增多,可以上调Bax与Caspase-3蛋白的表达水平,下调基质金属蛋白酶（MMP）-2与MMP-9的mRNA和蛋白表达水平。结论紫花牡荆素对于乳腺癌细胞恶性增殖与侵袭能力具有显著抑制作用。     

Specific cytoplasmic alpha-fetoprotein binding protein in MCF-7 human breast cancer cells and primary breast cancer tissue

Summary Direct evidence was obtained for the existence of a specific high affinity alpha-fetoprotein (AFP)-binding protein in the cytosol of both MCF-7 human breast cancer cultured cells and primary breast cancer tissue from postmenopausal women using a nitrocellulose blotting assay. Scatchard analysis of the binding data for MCF-7 cells at 37° C revealed the presence of a single class of AFP binding sites with an apparent K_d of 4.5 × 10^–8 M, and 75,000 binding sites per cell. All 9 primary breast cancer cytosols obtained from postmenopausal women also contained measureable levels of this specific AFP-binding protein. The number of AFP molecules specifically bound varied considerably between patients and ranged from 29–250 fmol per mg cytosol protein. Levels of AFP-binding protein levels and estrogen receptor measured in these same breast cancer cytosols showed a positive statistical correlation (r = 0.85). Taken together, the present evidence for the existence of a specific cytoplasmic AFP-binding protein in MCF-7 cells and previously reported evidence forde novo synthesis of free immunoreactive and bound nonimmunoreactive forms of cytoplasmic AFP by MCF-7 cells is consistent with the conclusion that most of the endogenous AFP synthesized in breast cancer cells is rapidly bound to specific cytoplasmic AFP-receptors, and that binding of AFP to these receptors masks its immunoreactivity. The association of AFP synthesis with rapidly growing fetal liver and adult regenerating liver, germ-cell tumors, immature uterus, and breast cancer cells suggests that a positive correlation exists between cytoplasmic AFP-receptor levels and the proliferative capacity of malignant breast tumors, and therefore such measurements may provide useful therapeutic and/or prognostic information in individual patients.