毛蕊异黄酮对人乳腺癌MCF-7细胞作用的体外研究

目的:观察毛蕊异黄酮在体外对雌激素受体阳性细胞人乳腺癌MCF-7细胞的作用及其机制。方法:用不同浓度毛蕊异黄酮作用MCF-7细胞,并设未处理阴性对照组及17-雌二醇(17-estradiol,E2)阳性对照组。MTT法测定细胞增殖抑制的情况,FCM检测细胞凋亡率影响,并通过RT-PCR和免疫细胞化学法分别测定细胞中Bcl-2、Bax的mRNA和蛋白水平变化。结果:低浓度的毛蕊异黄酮(2、4和8μmol/L)促进MCF-7细胞增殖,而高浓度毛蕊异黄酮(16和32μmol/L)抑制MCF-7细胞增殖,并且呈剂量依赖性。同时,低浓度的毛蕊异黄酮能减少MCF-7细胞凋亡率(P<0.05),并且显著降低Bax表达水平,增加Bcl-2表达水平(P<0.05)。结论:毛蕊异黄酮在低浓度时能促进MCF-7细胞增殖,减少细胞凋亡,其机制可能与毛蕊异黄酮的雌激素作用有关。     

Histone acetylation decreased by estradiol in the MCF-7 human mammary cancer cell line

Summary The effect of estradiol (E₂) on the [³H]-acetylation of nuclear histones was studied in the MCF-7 human mammary cancer cell line in culture. Cells (~ 10⁸) were incubated with 8 × 10^–6M [³H]-acetate in the absence (control) or in the presence of estradiol (10^–5–10^–8M). After 20 min incubation, the nuclear histones were extracted and separated by electrophoresis, and each histone band was measured and calculated in DPM/mg protein. It was observed that only the H₂ + H₃ and H₄ histones were associated with the [³H]-acetate. Estradiol (10^–6–10^–8M) provoked a significant inhibition in the incorporation of the acetate. The negative effect, in percentage of the non-treated cell value, was as follows: in E₂ (10^–6M): – 25 ± 10 (SE) for H₂ + H₃ and – 26 ± 5 for H₄; in E₂ (10^–7M): – 35 ± 9 and – 39 ± 10; and in E₂ (10^–8M): – 56 ± 22 and – 30 ± 13 respectively. It is concluded that estradiol has a negative effect in the acetylation of H₂, H₃ and H₄ histones of this mammary cancer cell; no acetylation or effect is observed in H₁ histones. The relationship of this finding to the biological responses of the hormone is to be explored.     

Incorporation of alphafetoprotein by the MCF-7 human breast cancer cell line

The intracellular uptake of alphafetoprotein (AFP) and its fluorescein conjugates, by cultures of the MCF-7 human breast cancer cell line, has been demonstrated using an indirect immuno-peroxidase technique or by direct visualization under fluorescent lighting. The protein was localized in the cytoplasm. Ultrastructural autoradiographs of MCF-7 cells incubated with human 3H-AFP showed protein accumulation in several cytoplasmic organelles, particularly in lipid droplets. Nuclei were free of AFP. A significant species-specificity of AFP internalization was observed in comparative assays with human, mouse, pig and chicken AFP. The incorporation of the protein was prevented by incubation at 0 degrees C or by previous treatment of the cultures with 10nM sodium azide. Attention is paid to the reappearance in a human breast carcinoma cell line of a property associated during ontogenic development with ectodermal derivatives, including the epidermis.     

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。     

Monoclonal antibodies reactive with components in serum-free conditioned medium from a human breast cancer cell line (MCF-7)

Approximately 10,000 primary hybridomas were generated after immunization with either serum-free conditioned medium (SFCM) or extracts from the human breast cancer cell line MCF-7. A total of 11 different monoclonal antibodies (MAbs; 8 generated against SFCM and 3 generated against cell extracts) were selected on the basis of high specificity in cell-binding ELISA for human breast cancer cell lines. The 8 different MAbs obtained by immunization with SFCM all reacted with secreted components in SFCM from MCF-7 cells and 4 of these MAbs reacted with glycolipids extracted from MCF-7 cells. 1 of these MAbs (S2) also recognized a secreted glycoprotein of approximately 77 kilodaltons (kDa). The remaining 4 MAbs did not show specificity solely for carbohydrate determinants. 1 of these MAbs (S7) recognized a secreted protein of approximately 41 kDa. The 3 MAbs raised against cell extracts from breast cancer cells reacted with cytoplasmic antigens in immunofluorescence but also reacted with a secreted component in SFCM from MCF-7 cells. Immunoblotting experiments with proteins from cell extracts and with proteins in SFCM showed that these antibodies all reacted with a protein of a molecular weight of approximately 40 kDa. Our results suggest that this component is cytokeratin 19 or proteolytically processed cytokeratin 19.     

gamma-Glutamyltranspeptidase activity in a human pancreatic cancer cell line (HPC-Y1) in serum-free chemically defined medium

gamma-Glutamyltranspeptidase (gamma-GTP) activity was found in a human pancreatic cancer cell line, HPC-Y1, cultivated in a serum-free chemically defined medium. The gamma-GTP stained in the cytoplasms as fine granules and was produced constantly in a protein-free chemically defined medium. The detergent- or protease-solubilized gamma-GTP of HPC-Y1 cells in serum-free medium was compared with the gamma-GTPs extracted from HPC-Y1 cells in serum-containing medium, human pancreatic carcinoma and normal human pancreas. Their molecular weights, electrophoretic mobilities, affinity to Concanavalin A-Sepharose and isoelectric points were almost identical. No cancer specific properties in the gamma-GTP derived from human pancreatic carcinoma cell line were found by these analyses. However, the serum-free spent medium of HPC-Y1 cells was useful for purifying the gamma-GTP secreted from the human pancreatic carcinoma cell line, since it is not necessary to separate the contaminated serum components that are usually added for cell cultures and the extraction procedures could induce minor structural change and/or artificial modification of gamma-GTP.     

In vitro andin vivo modulation of growth regulation in the human breast cancer cell line MCF-7 by estradiol metabolites

Background  The natural estrogen 17β-estradiol (E₂) functions as a potent tumor promoter during tumorigenic transformation of the mammary gland. From amongst the various pathways of E₂ metabolism upregulation of C16α-hydroxylation of E₂ has been associated with carcinogenesis. In the present studyin vitro andin vivo experiments were performed on estrogen receptor positive human breast cancer MCF-7 cells to examine whether the natural estrogen E₂ and its metabolites 16α-hydroxyestrone (16α-OHEi) and 2-hydroxyestrone (2-OHE₁) function as modulators of tumor cell growth. Methods  An anchorage-independent growth assay was used forin vitro study by counting the number of tri-dimensional colonies formed by MCF-7 cells suspended in 0.33% agar.In vivo experiments examined the effect of implanting metabolite material pellets into female nude mice. Results  In the anchorage-independent growth assay (AIG), continuous 14-day exposure to E₂ and to 16α-OHE₁ at 200 ng/ml induced a 59.4% and a 105.9% increase (P=0.001) respectively in the number of colonies of MCF-7 cells. Identical treatment with 2-OHE₁, however, failed to increase AIG relative to that seen in the solvent treated control cultures. In thein vivo tumorigenicity assay, treatment of nude mice with 1.5 mg E₂ or 16α-OHE₁ resulted in a 335.4% and a 384.1% increase (P<0.0002) in tumor growth, while identical treatment with 2-OHE₁ failed to exhibit any increase relative to the control group. Conclusions  These results suggest that the 16α- and 2-hydroxylated metabolites of E₂ may directly affectin vitro growth of MCF-7 cells via an autocrine mechanism andin vivo growth via paracrine mechanisms. Thus, E2-mediated growth regulation in MCF-7 cells may in part be due to distinct effects of specific E₂ metabolites on the breast cancer cells.     

间充质干细胞对乳腺癌MCF-7细胞侵袭力的研究

目的探讨人胎盘羊膜来源的间充质干细胞(h AMSCs)向乳腺肿瘤相关成纤维细胞(CAFs)转化后,对乳腺癌细胞上皮间质转化(EMT)的影响。方法选取2017年7月至2018年7月间牡丹江医学院附属红旗医院收住的80例产妇的足月剖腹产胎儿羊膜。MCF-7单独培养或Transwell小室共培养人乳腺癌细胞系MCF-7和MSCs共培养组即试验组,MCF-7细胞MSC培养基组为对照组,MCF-7常规培养组为对照组2。采用RT-qPCR检测共培养后h AM-MSCs向CAFs转化及CAFs相关特征标志物平滑肌肌动蛋白-α(α-SMA)和腱生蛋白-c(tenascin-c)的基因表达。对共培养后的肿瘤细胞进行细胞EMT相关标志及肿瘤特性检测。结果与对照组比较,共培养试验组MCF-7细胞发生上皮间质转化。与MSCs共培养能明显促进乳腺癌细胞的侵袭和迁移。结论人胎盘羊膜来源的间充质干细胞可诱导乳腺癌细胞上皮间质转化。     

Two new estrogen-supersensitive variants of the MCF-7 human breast cancer cell line

Two new estrogen-sensitive variants of MCF-7 human breast cancer cells, CG-4 and CG-5, are described in this report. These cells were derived from a casual contamination by MCF-7 cells of primary cultures from a human adenocarcinoma of the breast and a pleural effusion of a patient with advanced breast cancer, respectively. Careful characterization of these variants revealed chromosomal properties highly similar to and alloenzyme phenotypes identical to those of MCF-7 cells which were simultaneously cultured in the laboratory. MCF-7, CG-4 and CG-5 cells were tested for estrogen responsiveness under identical growth conditions, that is in the presence of culture medium supplemented with 5% charcoal-treated serum. While the number of MCF-7 cells increases by about 40% over the controls in the presence of physiological concentrations of estradiol, the number of CG-4 cells doubles and the number of CG-5 cells triples. All these cell lines have approximately the same number of estrogen, androgen, glucocorticoid, and progesterone receptor sites/cell. Since several difficulties arise in demonstrating the estrogen responsive growth stimulation of currently available human breast cancer cells, these two new variants, characterized by a high and reproducible estrogen responsiveness, afford a new model for studying the mechanisms by which estrogen regulates cell proliferation. The problems related to the careful characterization of every established cell line are discussed.     

LOXL2促进乳腺癌MCF-7细胞侵袭研究

目的:研究赖氨酰氧化酶样蛋白-2(lysyl oxidase like-2 protein,LOXL2)对乳腺癌MCF-7细胞侵袭转移的影响。方法:利用脂质体法将构建好的Flag-LOXL2质粒转染到乳腺癌MCF-7细胞中,应用Western blot检测转染效率,Transwell侵袭实验检测乳腺癌MCF-7细胞的体外侵袭力,并进一步使用Western blot检测LOXL2蛋白对金属蛋白酶MMP2/MMP9表达的影响。结果:在乳腺癌MCF-7细胞中瞬时转染FlagLOXL2,LOXL2蛋白表达明显增加,并促进MCF-7细胞的侵袭能力,上调金属蛋白酶MMP2和MMP9蛋白的表达。结论:LOXL2促进MCF-7细胞的侵袭能力,并影响金属蛋白酶MMP2以及MMP9的表达。     

塞来昔布预防MCF-7细胞获得性耐药发生

目的:观察环氧合酶-2 (cyclooxygenase-2, Cox-2) 抑制剂塞来昔布(celecoxib)对乳腺癌细胞株MCF-7获得性耐药的预防作用,并对其机制进行初步探讨.方法:采用小剂量多柔比星 (doxorubicin, Doxo) 对MCF-7细胞进行诱导,建立获得性耐药诱导模型,同时以此模型,联合应用塞来昔布进行耐药预防研究.MTT法检测多柔比星对MCF-7细胞的生长抑制效应及半数抑制浓度(half inhibition concentration,IC50)值; RT-PCR方法检测MDR1、MRP1、GST及TopoⅡ等多药耐药相关基因的变化;Western印迹法检测蛋白的变化; FCM法检测细胞膜P-糖蛋白(P-glycoprotein,P-gp)的表达及功能. 结果:多柔比星(0.05 μg/mL)作用7 d,MCF-7细胞内MDR1及MRP1在mRNA及蛋白水平均上调,而GST及TopoⅡ未见明显变化;联合塞来昔布(10 和20 μmol/L)进行干预,可有效抑制多柔比星诱导的MDR1及MRP1在mRNA及蛋白水平的上调,细胞膜P-gp功能均降低.多柔比星与塞来昔布(10 μmol/L)联合作用组,MCF-7细胞对多柔比星的IC50值为(0.38±0.04) μg/mL与多柔比星单药组的 (0.67±0.03) μg/mL相比,差异有统计学意义(P<0.01). 结论:选择性Cox-2抑制剂塞来昔布可有效预防MCF-7细胞获得性耐药的发生, 且具有明显的化疗增敏作用,其初步作用机制部分涉及塞来昔布抑制多柔比星引起的P-gp及MRP1的上调.     

Antiestrogenic action of 3-hydroxytamoxifen in the human breast cancer cell line MCF-7

The antiestrogenic action of 3-hydroxytamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1-(3-hydroxyphenyl)-2 -phenylbut-1-ene] was characterized in vitro and compared with that of tamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene]. The relative binding affinities of 3-hydroxytamoxifen to estrogen receptor were 3.3% in cytosol of MCF-7 cells and 1.5% in human mammary carcinoma cytosol compared to values of 0.2 and 0.3% for tamoxifen (the affinity of 17 beta-estradiol considered to be 100%). The concentration of 3-hydroxytamoxifen necessary to suppress the 17 beta-estradiol-induced growth stimulation of MCF-7 cells was about tenfold lower than that for tamoxifen. The induction of progesterone receptor in MCF-7 cells by 17 beta-estradiol was inhibited by 3-hydroxytamoxifen. In the absence of 17 beta-estradiol, 3-hydroxytamoxifen gave rise to a moderate increase in the progesterone receptor levels, which demonstrates the partially estrogenic character of hydroxytamoxifen.     

雌二醇联合睾丸酮对乳腺癌MCF-7细胞增殖和凋亡的影响

目的:探讨雌二醇联合睾丸酮对乳腺癌MCF-7细胞增殖和凋亡的影响及其可能机制。方法:将10-10mol/L雌二醇和10-5、10-7、10-9和10-11mol/L睾丸酮单独或联合作用于乳腺癌MCF-7细胞24、48和72h,MTT法检测细胞的生长情况,FCM检测细胞周期和细胞凋亡的情况,以及cyclinD1和雄激素受体(androgen receptor,AR)蛋白的表达情况。结果:雌二醇可促进MCF-7细胞的增殖。高浓度(10-5mol/L)睾丸酮可抑制细胞增殖,并抑制雌二醇促细胞增殖的作用;低浓度(10-9mol/L)睾丸酮可促进MCF-7细胞增殖,并增强雌二醇的促细胞增殖作用。雌二醇联合10-5mol/L睾丸酮作用48h后促使细胞周期由G1期进入S期,细胞凋亡率上升,cyclinD1蛋白的表达上调,AR蛋白的表达未见明显变化。结论:雌二醇联合高浓度睾丸酮可抑制乳腺癌细胞的增殖和促进细胞凋亡,可能与上调细胞cyclinD1蛋白的表达有关。     

Inhibition of estradiol uptake and transforming growth factor alpha secretion in human breast cancer cell line MCF-7 by an alkyl-lysophospholipid

We investigated the effect of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), an alkyl-lysophospholipid, on the uptake of estrogen, the secretion of transforming growth factor (TGF) alpha and the content of progesterone receptors (PRs) in the hormone-dependent breast cancer cell line, MCF-7. The uptake of labeled estradiol by MCF-7 was dose dependently decreased by 12 h pretreatment with 10-25 micrograms/ml ET-18-OCH3, and this suppression occurred prior to the onset of the inhibitory action of ET-18-OCH3 on MCF-7 growth. Scatchard analysis demonstrated that ET-18-OCH3 reduced the number of estrogen receptors in MCF-7 without affecting their affinity. Both the secretion of TGF-alpha from MCF-7 into the conditioned medium and the PR content of MCF-7 were decreased by 48 h treatment with 10 micrograms/ml ET-18-OCH3. The estradiol uptake, the TGF-alpha secretion, and the PR content were not affected by platelet-activating factor, lyso-PAF, and palmitoyl-lysophosphatidylcholine, all at 10 micrograms/ml. These results suggest that the reduction of estrogen receptor level induced by ET-18-OCH3 resulted in decreases in both the secretion of TGF-alpha and the content of PR in MCF-7, and these effects are specific to ET-18-OCH3. We concluded that these effects of ET-18-OCH3 may lead, at least partly, to its antitumor action in hormone-dependent breast cancer cell lines.     

Estrone sulfate promotes human breast cancer cell replication and nuclear uptake of estradiol in MCF-7 cell cultures

Estradiol levels in breast tumors from post-menopausal women are similar to those in pre-menopausal women even though plasma estrogens are much lower after the menopause. In situ estrogen production by the tumor provides a potential means of maintaining high estradiol levels in post-menopausal breast cancer tissue. The estrone sulfatase pathway has been proposed as the mediator of in situ estrogen production. A number of studies suggest that estrone sulfate may be converted into estradiol in breast tumors via the catalytic activity of estrone sulfatase and 17β-hydroxysteroid dehydrogenase. However, these studies used pharmacologic levels of estrogen sulfates and have not shown that physiologic levels can support biologic effects. Accordingly, the present study examined the dose relationship of estrone sulfate to a variety of biologic endpoints in MCF-7 breast cancer cells in culture. These cells converted physiologic concentrations of estrone sulfate to quantities of free estradiol capable of stimulating cell growth. Under these conditions, the nuclear steroids observed were free estrone and estradiol. Increase in cell number after 6 days of exposure to steroid required 100 nM estrone sulfate. However, S-phase, a more sensitive measure of cell proliferation, was stimulated by 0.1 nM estrone sulfate, a clearly physiologic concentration. Stimulation of estrogen-dependent protein markers such as pS2 and progesterone receptor required much higher concentrations of estrone sulfate. These effects were mediated through the estrogen receptor since the pure anti-estrogen, ICI 164384, blocked all effects produced by estrone sulfate. While it has been suggested that anti-estrogens may partly exert their effects by inhibition of sulfatase and 17β-hydroxysteroid dehydrogenase, this did not occur under our experimental conditions. These data provide evidence of the relevance of the estrone sulfatase pathway since biologic effects can be demonstrated in response to physiologic concentrations of estrone sulfate.     

米非司酮逆转人乳腺癌细胞MCF-7/ADR耐多柔比星机制的探讨

目的 探讨米非司酮对乳腺癌耐药细胞株MCF-7/ADR的逆转耐药作用.方法 以亲本乳腺癌细胞MCF-7和耐多柔比星(阿霉素)乳腺癌细胞MCF-7/ADR为研究对象,分别应用MIF进行干预后,流式细胞仪检测MIF作用前后瘤细胞P-gp的表达、细胞内阿霉素蓄积量的变化以及细胞周期的分布.结果 (1)10 μmol/L MIF作用72小时后,MCF-7/ADR细胞P-gp表达率[(23.21±1.80)％]明显高于MCF-7细胞[(19.37±2.37)％,P＜0.05].(2)5 μmol/L ADR处理后,MCF-7/ADR细胞内ADR蓄积量为(47.13±4.11)％,低于MCF-7细胞[(60.24±2.61)％,P＜0.05].(3) 10 μmol/L MIF联合5 μmol/L ADR处理细胞,MCF-7/ADR和MCF-7细胞内ADR的蓄积量分别为(82.72±2.42)％及(88.63±2.75)％ (P ＞0.05);但均较单用ADR时升高(均P＜0.01).(4) MIF作用前,MCF-7/ADR细胞G0/G1期比例[(77.21±3.10)％]高于MCF-7细胞G0/G1期比例[(59.05±2.16)％,P＜0.05];MCF-7/ADR细胞S期比例明显低于MCF-7细胞(P＜0.05).经10 μmol/L MIF作用后,MCF-7细胞G0/G1期比例(75.28±2.53)％较MIF作用前明显升高(P＜0.05);S期比例则较MIF作用前显著降低(P＜0.05);MCF-7/ADR细胞G0/G1期比例和S期比例分别为(80.13±2.72)％及(13.52±1.03)％,与MIF作用前比较差异均无统计学意义(均P＞0.05);两种瘤细胞的G2/M期比例与MIF作用无关(P＞0.05).结论 (1)MIF可以逆转MCF-7/ADR的耐药性,其作用机制与降低细胞P-gp含量、增加细胞内ADR蓄积量有关.(2) 10 μmol/L浓度的MIF对MCF-7/ADR细胞周期分布影响不大.     

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.