Proteomics reveals protein profile changes in doxorubicin--treated MCF-7 human breast cancer cells

MCF-7 cells are extensively used as a cell model to investigate human breast tumors and the cellular mechanism of antitumor drugs such as doxorubicin (DOX), an anthracycline antitumor drug widely used in clinical chemotherapy. To understand the effects of DOX on the protein expression, we perform a comprehensive proteomics to survey global changes in proteins after DOX treatment in MCF-7 cells. Exposure of MCF-7 cells to 0.1 microM DOX for 2 days induced a differentiation-like phenotype with prominent perinuclear autocatalytic vacuoles, abundant filamentous material, and irregular microvilli at the cell surface. In this study, we also present a proteome reference map of MCF-7 cells with 21 identified protein spots via analysis of N-terminal sequencing, mass spectrometry, immunoblot and/or computer matching with protein database. Based on the proteome map, we found that DOX causes a markedly decrease in the levels of three isoforms of heat shock protein 27 (HSP27) whereas the levels of other stress associated proteins including HSP60, calreticulin, and protein disulfide isomerase were not significantly altered in DOX-treated MCF-7 cells. Taken together, we suggest that that action of DOX on breast tumor cells may be partly related to dysregulation of HSP27 expression. Modulation of HSP27 levels may be a clinically useful potential target for design of antitumor drugs and controlling breast tumor growth.     

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.     

Proteasome-mediated degradation of BRCA1 protein in MCF-7 human breast cancer cells

The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. We present several lines of evidence for the mechanism of BRCA1 degradation through the ubiquitin-proteasome pathway by using specific inhibitors of the proteasome in human MCF-7 breast carcinoma cells. The levels of BRCA1 protein were up-regulated by proteasome inhibitors, such as MG-132 and ALLnL, suggesting rapid degradation via the ubiquitin-proteasome pathway. The enhanced loss of BRCA1 protein by taxol, okadaic acid or nocodazole treatment was prevented by the proteasome inhibitors, while inhibition of other proteases was ineffective. Accumulation and proteasomal degradation of BRCA1 protein appear to be restricted entirely to the nucleus. We also detected that high molecular weight BRCA1 protein species appeared after proteasome inhibitor treatments, which indicated that ubiquitinated species were present. Moreover the half-life of BRCA1 protein was significantly increased in response to inhibition of proteasome activity. Our present data demonstrate that BRCA1 protein may be degraded through the ubiquitin-proteasome mediated pathway.     

蝙蝠葛碱在人乳腺癌MCF-7细胞对5氟尿嘧啶敏感性的研究

目的 探讨人乳腺癌MCF-7细胞受蝙蝠葛碱(Dauricine,Dau)作用后,对5氟尿嘧啶(5-FU)敏感性的影响。方法 分别以终浓度为2.5 μg/mL的蝙蝠葛碱、50 μg/mL的5-FU、2.5 μg/mL的蝙蝠葛碱和50 μg/mL的5-FU作用于人乳腺癌细胞MCF-7,运用MTT法检测细胞增殖活性,Transwell实验分析细胞的迁移,DAPI染色检测细胞的凋亡,Western blot法检测cyclinD1和Bcl-2蛋白的表达。结果 MTT实验结果显示联合使用阈下浓度的蝙蝠葛碱增强了5-FU对细胞增殖的抑制;Transwell实验表明联合应用阈下浓度的蝙蝠葛碱进一步加剧了5-FU对细胞迁移的抑制;DAPI染色说明联合应用阈下剂量的蝙蝠葛碱加强了5-FU对细胞凋亡的诱导,Western blot实验表明联合应用阈下浓度的蝙蝠葛碱进一步抑制了cyclinD1和Bcl-2蛋白的表达。结论 蝙蝠葛碱可有效增强人乳腺癌MCF-7细胞对5-FU的敏感性。     

Anti-apoptotic effect of claudin-1 in tamoxifen-treated human breast cancer MCF-7 cells

Background  

Claudin-1 is a membrane protein of tight junctions, and is associated with the development of various cancers. However, the significance of claudin-1 expression in cancer cells is not well understood. Here, we showed for the first time the anti-apoptotic effect of claudin-1 in human breast cancer MCF-7 cells.     

Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells

The expression, DNA binding, and transactivating activity of activator protein 1 (AP-1) was examined in a series of multidrug resistant (MDR) MCF-7 human breast cancer cells that have increasing levels of MDR1 gene expression. We observed an increase in the amount of both c-jun and c-fos mRNA in cells with 12-, 65-, or 200-fold higher resistance to adriamycin when compared to drug-sensitive MCF-7 wild type (WT) cells. Electrophoretic mobility shift assays (EMSA) demonstrated an increase in the DNA binding activity of an AP-1 complex in nuclear extracts from MDR MCF-7 cells when compared to extracts from WT cells. We observed a proportional increase in luciferase expression from a reporter vector containing consensus AP-1 binding sites in transiently transfected MDR cells when compared to WT cells, indicating that AP-1 mediated gene expression is increased in drug-resistant MCF-7 cells. Since the MDR1 promoter contains a putative AP-1 binding site, we used EMSA to examine AP-1 binding activity to an oligonucleotide probe that contained the relevant MDR1 promoter sequences (–123 to –108). Nuclear extracts from resistant MCF-7 cells displayed an increased level of DNA binding of Jun/Jun dimers to the probe, indicating that AP-1 was capable of binding to this promoter site. A luciferase reporter construct containing triplicate copies of the MDR1 promoter sequence was expressed at higher levels in transiently transfected MDR cells when compared to expression in WT cells. Co-transfection of WT cells with a c-jun expression vector and either of the AP-1 luciferase constructs demonstrated that c-jun could activate gene expression from both the consensus and the MDR1 AP-1 sites in a dose dependent manner. In addition, RT-PCR and western blot analysis showed that levels of MDR1 mRNA and Pgp were increased in c-jun transfected WT cells. Taken together, these data indicate that increased AP-1 activity may be an important mediator of MDR by regulating the expression of MDR1.     

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。     

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。     

Title aggregation patterns of argyrophilic nucleolar organizer regions induced by 5-fluorouracil in the nuclei of MCF-7 human breast cancer cells

The effects of tamoxifen and 5-fluorouracil (5-FU) on the patterns of argyrophilic nucleolar organizer regions (AgNORs) in MCF7 human breast cancer cells were studied. Tamoxifen and 5-FU both inhibited the growth of MCF-7 cells by 18% by day 3 of culture, but each had different effects on the AgNORs. Whereas no significant changes were induced by tamoxifen, effects on the AgNORs of MCF-7 cells by 5-FU were dramatic: 5-FU treatment changed the pattern of AgNORs, reducing the number of satellites by aggregation, typically to a single aggregation around nucleoli in a sphenoidal fashion. We named these morphological changes: fluorouracil induced AgNOR aggregations (FAA). Following treatment with 500 ng/ml 5-FU, FAA developed rapidly, AgNORs forming two or three aggregates in 24% (6 h), 24% (12 h), 40% (24 h) and 34% (48 h) of cells, compared to a control rate of 14%. Single large aggregate was rarely found in untreated cultures but after 6, 12, 24 and 48 h treatment with 500 ng/ml 5-FU, AgNORs had formed a single aggregate in 6, 8, 16 and 22% of cells, respectively. FAA were observed at a concentration of 100 ng/ml 5-FU; 48 h treatment resulted in cells in which two or three aggregates were increased by 24% and a single aggregate by 16%. These large single aggregates were larger than nucleoli stained by Papanicolau staining.     

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.     

Estrogens are not required for prolactin induced growth of MCF-7 human breast cancer cells

MCF-7 human breast cancer cells in continuous culture respond to the growth promoting activity of prolactin. Within 3 days of addition to culture medium in the presence of charcoal stripped fetal bovine serum which is depleted of bovine lactogens and estrogens, prolactin at 250 ng/ml promotes a 2- to 3-fold increase in cell number. When phenol red, which is a weak estrogen agonist, is also eliminated from the medium, the cells respond to prolactin to the same extent. When cells are grown for two generations in the presence of lactogen free, phenol red free, charcoal stripped serum, the prolactin induced response is even greater. Human and ovine prolactins are equipotent. The ability of the cells to bind lactogenic hormones remains unaltered by elimination of phenol red from the growth medium. These data indicate that prolactin alone is a mitogen for human breast cancer cells in long-term culture.     

Senescence evasion by MCF-7 human breast tumor-initiating cells

Introduction  

A subpopulation of cancer cells, tumor-initiating cells, is believed to be the driving force behind tumorigenesis and resistance to radiation and chemotherapy. The persistence of tumor-initiating cells may depend on altered regulation of DNA damage and checkpoint proteins, as well as a reduced propensity to undergo apoptosis or senescence.     

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。     

Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids

The interaction between the estrogen receptor and a variety of flavonoids was studied in the presence or absence of estradiol using a stably-transfected human breast cancer cell line (MVLN). On the other hand, flavonoids were evaluated for their effects on proliferation in estrogen-dependent (MCF-7) and independent (MDA-MB231) human breast cancer cells. We established a relationship structure-activity and determined regions and/or substituents essential for estrogenic or antiestrogenic activities. In contrast, we did not find the same relationship for cell proliferation. Among all flavonoids used, only 7-methoxyflavanone and 7,8-dihydroxyflavone at high concentrations (50 μM) possess antiestrogenic and antiproliferative activities. These results suggest that two hydroxyls (in positions 7 and 8) or 7-methoxy substituents are essential for the antiestrogenic activity of flavonoids. However, it seems that flavonoids at high concentrations exert their antiproliferative activity through other estrogen receptor-independent mechanisms.     

Modulation of 5-FU metabolism in human MCF-7 breast carcinoma cells

Summary We have previously demonstrated a highly significant relationship (P<0.0001) between the incorporation of 5-fluorouracil (5-FU) into total cellular RNA and loss of clonogenic survival of the human MCF-7 breast carcinoma cell line. The present studies explore the applicability of this relationship to MCF-7 cells exposed to 5-FU and modulting agents such as PALA, MTX, and MMPR. PALA treatment produces a minimal increase in the absolute amount of 5-FU incorporated into total cellular RNA, but it results in a three-fold enhancement of the [³H]FU/³²P ratio, which measures 5-FU misincorporation into newly synthesized RNA. MTX and MMPR increase intracellular PRPP levels up to four-fold; nevertheless these agents result in only minimal increases in absolute (5-FU) RNA formation. In contrast, the relative incorporation of 5-FU into newly synthesized RNA of MTX-or MMPR-treated cells is increased 2.5-fold. The combination of PALA/MMPR results in a two-fold absolute increase in (5-FU) RNA formation and a nine-fold enhancement of the [³H]FU/³²P ratio. Combinations of modulating agents with 5-FU result in more than additive decreases in MCF-7 clonogenic survival. The relationship between 5-FU incorporation into RNA and loss of clonogenic survival was highly significant (P<0.0002) when corrected for newly synthesized RNA, while the correlation with absolute amounts of (5-FU) RNA formation was less significant (P<0.05). These studies demonstrate that the relationship previously established between (5-FU)RNA formation and loss of clonogenic survival should be corrected for the amount of newly synthesized RNA when 5-FU is combined with modulating agents that alter rates of RNA synthesis.     

Down-regulation of P-glycoprotein expression in MDR breast cancer cell MCF-7/ADR by honokiol

P-glycoprotein accounts for the most intrinsic and acquired cancer multidrug resistance. To inhibit the expression of P-glycoprotein is one of the effective ways to reverse cancer drug resistance. Honokiol, a naturally occurring compound, has been demonstrated to combat cancer through mechanisms including inhibition of angiogenesis and induction of apoptosis. Here, we show that honokiol down-regulated the expression of P-glycoprotein at mRNA and protein levels in MCF-7/ADR, a human breast MDR cancer cell line. The down-regulation of P-glycoprotein was accompanied with a partial recovery of the intracellular drug accumulation, and of the sensitivities toward adriamycin. This study reveals a novel function of honokiol as an anti-cancer agent.     

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.