细胞周期相关因子与乳腺细胞的癌变及生物学特性的相关性研究

目的：研究正常乳腺上皮细胞与乳腺癌细胞之间以及不同恶性程度的乳腺癌细胞之间的细胞周期相关因子的表达异同。方法：采用Western印迹法检测正常乳腺细胞株AG11132A、ER阳性和乳腺癌细胞株MCF－7及ER阴性的乳腺癌细胞株MDA－MB－231之间细胞周期蛋白D1、E及P21蛋白表达的异同及与其生物学特性的关系。结果：（1）正常乳腺上皮细胞株高表达P21蛋白,低表达周期蛋白E。与正常细胞相比,乳腺癌细胞株MCF－7、MDA－MB－231细胞高表达周期蛋白E,其中表达的周期蛋白E中存在异常的你分子量周期蛋白E成分,而正常乳腺上皮细胞不表达这种异常 的周期蛋白E。（2）在乳腺癌细胞株之间,相对ER阳性的MCF－7细胞,ER阴性的MDA－MB－231细胞则高表达周期蛋白E,基本无表达P21蛋白。结论L（1）正常细胞与这间、相对低恶性程度的民相对高恶性和蔼的癌细胞之间的差别是多环节的、质和量异常导致的结果;（2）周期蛋白E及P21均可反映乳腺癌细胞的增殖活性和恶性程度,可能是乳腺癌的临床预后指标。     

环氧化酶-2抑制剂nimesulide对乳腺癌细胞株增生、凋亡的影响

目的 探讨环氧化酶-2抑制剂nimesulide(NIM)对雌激素受体(ER)阴性(MDA-MB．231)和阳性(MCF-7)人乳腺癌细胞株增生、凋亡的影响。方法 应用MTT比色法分析细胞生长抑制作用，流式细胞技术测定细胞周期分布和凋亡率，透射电镜观察细胞形态与超微结构，AnnexinV法检测细胞的凋亡。结果 NIM以时间、剂量依赖性方式抑制MDA-MB-231(COX-2阳性)、MCF-7(COX-2阴性)细胞生长，阻滞细胞周期于G0／G1期，诱导细胞的凋亡，MDA—MB-231细胞对NIM的作用更为敏感。NIM对COX-2表达阴性的MCF-7细胞同样具有抑制增生、诱导凋亡的作用。结论 NIM对ER阳性和ER阴性乳腺癌细胞均有抑制增生、诱导凋亡的作用。NIM的抗肿瘤作用存在环氧化酶．2依赖性与非依赖性两种途径。     

三阴性乳腺癌中ERβ和NF-κB的表达及其相关性

目的:研究三阴性乳腺癌细胞中雌激素受体β（estrogen receptor β,ERβ）和核因子κB(nuclear factor kappa B,NF-κB)的表达,初步探讨ERβ和NF-κB的关系。方法:Western blot检测MDA-MB-231细胞中ERβ和NF-κB蛋白的表达。免疫共沉淀法检测MDA-MB-231细胞中ERβ和NF-κB蛋白的相互作用。Western blot 检测转染ERβ后的MDA-MB-231细胞中ERβ表达变化和NF-κB蛋白的表达,以及用免疫共沉淀检测转染后ERβ和NF-κB蛋白表达的关系。结果:MDA-MB-231细胞中ERβ和NF-κB蛋白表达之间无明显相关,但转染ERβ后ERβ表达增加,NF-κB表达降低,二者之间表达呈现明显相关性。结论:MDA-MB-231细胞中ERβ抑制NF-κB 蛋白的表达。     

Estrogen receptor (ER) beta1 and ERbetacx/beta2 inhibit ERalpha function differently in breast cancer cell line MCF7

Estrogen receptor (ER) alpha plays an important role in the proliferation and progression of breast cancer. In order to explore the function of wild-type ERbeta (ERbeta1) and its variant form, ERbetacx/beta2, stable transformants of ERalpha-positive breast cancer MCF7 cells with ERbeta1 or ERbetacx/beta2 expression vector were established. Constitutive expression of ERbeta1 or ERbetacx/beta2 reduced the S phase population of the cell cycle in dish culture and the number of colonies in an anchorage-independent assay. DNA-protein complexes of ERE with nuclear extracts from ERbeta1 transformants were observed in the electrophoretic mobility shift assay, while no complex was observed for ERbetacx/beta2 transformants. Reporter gene assay using estrogen-responsive element (ERE)-luciferase showed less responsiveness to estrogen in these transformants compared with parental cells. Endogenous mRNA expression of two known estrogen-responsive genes, cathepsin D and IGFBP4, was weakly induced by estrogen in ERbeta1 and ERbetacx/beta2 transformants compared with parental cells. A comprehensive gene expression analysis using our custom-made cDNA microarray showed that MCF7 and ERbeta1 transformants had a similar gene expression profile, whereas ERbetacx/beta2 showed a distinct profile from others. These results indicate that ERbeta1 and ERbetacx/beta2 inhibit ERalpha function differently in MCF7 cells.     

Tyrosine phosphorylation of an erbB-2 related p90 protein induced by estrogen in human breast epithelial cells

Estrogen induced erbB-2 tyrosine kinase activity in human breast epithelial cells irrespective of estrogen receptor expression. MCF10A is an immortal normal human breast epithelial cell line which does not express estrogen receptor. After treatment of MCF10A cells with estradiol-17beta (E2), a phosphorylated 90 kDa protein which co-immunoprecipitates with p185erbB-2 is detected. The response is transient, detected after 1-5 min exposure to E2, and dose dependent, occurring at 10-10 M E2. A similar response was observed for MCF10A cells transfected with an estrogen receptor, estrogen receptor expressing MCF-7 cells, and estrogen receptor-negative MDA-MB-435 cells but at 10-11 M E2. Overexpression of c-erbB-2 in MCF10A cells prolonged the phosphorylated p90 response to E2.     

微小RNA-148a抑制乳腺癌细胞MDA-MB-231迁移的作用机制

目的:研究微小RNA-148a(miR-148a)在乳腺癌细胞株MDA-MB-231中的表达,探讨上调miR-148a的表达对其迁移的影响及机制.方法:采用实时荧光定量PCR(qRT-PCR)检测正常乳腺上皮细胞MCF-10A和乳腺癌细胞株MDA-MB-231中miR-148a的表达水平.应用脂质体法将miR-148a mimic及阴性对照分别转染MDA-MB-231细胞,qRT-PCR检测转染效率;细胞划痕实验检测转染前后MDA-MB-231细胞迁移能力的变化;Western blot方法检测上皮间质转化(EMT)相关蛋白Slug、Snail和E-cadher-in表达水平的变化.结果:与MCF-10A细胞相比,MDA-MB-231细胞中miR-148a的表达水平明显降低(P<0.05);与阴性对照组相比,miR-148a转染组中miR-148a的表达水平显著升高(P<0.05);过表达miR-148a后,MDA-MB-231细胞迁移能力明显下降,Slug和Snail蛋白表达明显降低,E-cadherin蛋白表达明显增加.结论:miR-148a可通过调控Slug、Snail及E-cadherin蛋白的表达抑制EMT,进而抑制乳腺癌细胞的迁移能力.     

Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene

The breast cancer resistance protein (BCRP) is an ATP-binding cassette half transporter that confers resistance to anticancer drugs such as mitoxantrone, anthracyclines, topotecan, and SN-38. Initial characterization of the BCRP promoter revealed that it is TATA-less with 5 putative Sp1 sites downstream from a putative CpG island and several AP1 sites (K. J. Bailey-Dell et al., Biochim. Biophys. Acta, 1520: 234-241, 2001). Here, we examined the sequence of the 5'-flanking region of the BCRP gene and found a putative estrogen response element (ERE). We showed that estrogen enhanced the expression of BCRP mRNA in the estrogen receptor (ER)-positive T47D:A18 cells and PA-1 cells stably expressing ERalpha. In BCRP promoter-luciferase assays, sequential deletions of the BCRP promoter showed that the region between -243 and -115 is essential for the ER effect. Mutation of the ERE found within this region attenuated the estrogen response, whereas deletion of the site completely abrogated the estrogen effect. Furthermore, electrophoretic mobility shift assays revealed specific binding of ERalpha to the BCRP promoter through the identified ERE. Taken together, we provide evidence herein for a novel ERE in the BCRP promoter.     

Trichostatin A and 5 Aza-2′ deoxycytidine decrease estrogen receptor mRNA stability in ER positive MCF7 cells through modulation of HuR

Trichostatin A (TSA) and 5-Aza 2'deoxycytidine (AZA), two well characterized pharmacologic inhibitors of histone deacetylation and DNA methylation, affect estrogen receptor alpha (ER) levels differently in ER-positive versus ER-negative breast cancer cell lines. Whereas pharmacologic inhibition of these epigenetic mechanisms results in re-expression and increased estrogen receptor alpha (ER) levels in ER-negative cells, treatment in ER-positive MCF7 cells results in decreased ER mRNA and protein levels. This decrease is dependent upon protein interaction with the ER 3'UTR. Actinomycin D studies showed a 37.5% reduction in ER mRNA stability from 4 to 1.5 h in AZA/TSA treated MCF7 cell lines; an effect not seen in 231ER + cells transfected with the ER coding region but lacking incorporation of the 3'UTR. AZA/TSA do not appear to directly interact with the 3'UTR but rather decrease stability through altered subcellular localization of the RNA binding protein, HuR. siRNA inhibition of HuR expression reduces both the steady-state and stability of ER mRNA, suggesting that HuR plays a critical role in the control of ER mRNA stability. Our data suggest that epigenetic modulators can alter stability through modulation of HuR subcellular distribution. Taken together, these data provide a novel anti-estrogenic mechanism for AZA and TSA in ER positive human breast cancer cells.     

人乳腺癌细胞株WAF1/CIP1/p21基因的表达及其与细胞生物学特性的关系

目的研究人乳腺癌细胞株ＷＡＦ１／ＣＩＰ１基因的ＤＮＡ状况、ｍＲＮＡ和蛋白的表达水平及其意义。方法应用细胞培养、分子生物学Ｓｏｕｔｈｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交以及免疫组化染色等技术，检测人乳腺癌表达野生型ｐ５３（ｗｔｐ５３）的ＭＣＦ７细胞和表达突变型ｐ５３（ｍｔｐ５３）的ＭＤＡＭＢ２３１细胞中ＷＡＦ１／ＣＩＰ１基因ＤＮＡ状况、ｍＲＮＡ和蛋白质的表达水平，研究其与ｍｄｍ２、ｐ５３蛋白的表达和细胞生物学特性的关系。结果比较ＭＣＦ７细胞与ＭＤＡＭＢ２３１细胞：（１）两者ＷＡＦ１／ＣＩＰ１基因ＤＮＡ状况无明显差异，前者ｍＲＮＡ和蛋白质的表达水平明显高于后者（Ｐ＜０．０５）；（２）两者ｐ５３蛋白的性质和分布不同，前者ｍｄｍ２蛋白的表达水平明显高于后者（Ｐ＜０．０５）；（３）前者生物学特性好于后者。结论人乳腺癌细胞株ＷＡＦ１／ＣＩＰ１基因ｍＲＮＡ和蛋白质的表达水平与ｐ５３基因表型和细胞生物学特性有关。     

EGFRvIII‐induced estrogen‐independence,tamoxifen‐resistance phenotype correlates with PgR expression and modulation of apoptotic molecules in breast cancer

The tumor‐specific, ligand‐independent, constitutively active epidermal growth factor receptor (EGFR) variant, EGFRvIII, remains understudied in breast cancer. Here, we report that expression of EGFRvIII in the ErbB‐2‐overexpressing, estrogen‐dependent MDA‐MB‐361 breast cancer cell line resulted in significant estrogen‐independent tumor growth in ovariectomized, athymic nude mice in comparison to MDA‐MB‐361/wt cells. MDA‐MB‐361/vIII breast cancer cells maintained estrogen‐induced tumor growth, but were tamoxifen‐resistant in the presence of estrogen, while MDA‐MB‐361/wt cells had a significant reduction in tumor growth in the presence of estrogen and tamoxifen. Tamoxifen alone did not have a significant effect on EGFRvIII‐mediated estrogen‐independent tumor growth. Constitutive signaling from the EGFRvIII receptor resulted in an increased activation of both the Akt and MAPK pathways. Compared to estrogen‐dependent, tamoxifen‐sensitive MCF‐7/vIII breast cancer cells, which had unchanged levels of ERα, but an increase in progesterone receptor (PgR) in comparison to MCF‐7/wt cells, MDA‐MB‐361/vIII cells had a reduction in ERα expression as well as a more pronounced reduction in PgR compared with MDA‐MB‐361/wt cells. EGFRvIII expression was also significantly associated with an absence of PgR protein in invasive human breast cancer specimens. Alterations of proapoptotic proteins and antiapoptotic proteins were observed in EGFRvIII transfectants. In conclusion, constitutive signaling through EGFRvIII and its downstream effector proteins crosstalks with the ERα pathway, resulting in loss of PgR expression and alterations in the apoptotic pathway, which may result in the estrogen‐independent, tamoxifen‐resistant phenotype conferred to EGFRvIII‐expressing breast cancer cells. © 2009 UICC     

Inhibition of breast cancer growth by suramin.

In this study, the polyanionic compound suramin was shown to be a potent in vitro growth inhibitor of both hormone-insensitive, estrogen receptor-negative human breast cancer cells (MDA MB231 and SK-BR-3) and hormone-responsive, estrogen receptor-positive human breast cancer cells (ZR 75-1, T47D, and MCF7). The inhibitory effect of suramin was dose dependent, with a median effective dose varying from 7 microM for MDA MB231 cells to 50 microM for MCF7 cells. This result indicated that estrogen receptor-negative cells were more sensitive to the drug. In MCF7 cells, not only did suramin block the mitogenic action of growth factors such as epidermal growth factor (EGF) and insulin-like growth factors I and II (IGF-I and IGF-II, respectively), but it also totally abolished the increase in cell proliferation induced by the steroid hormone 17 beta-estradiol (E2). Maximal inhibition was obtained after 5 days of suramin treatment, and inhibition either was partially reversed by E2, IGF-I, and IGF-II or was not reversible by EGF following removal of drug. In addition, suramin significantly decreased synthesis and secretion of the lysosomal enzyme cathepsin D, which was shown to be associated with a high risk of breast tumor metastasis. These results therefore suggest that, because of its effects on growth and cathepsin D secretion, suramin might be a helpful additional therapeutic tool for breast cancer patients, especially for patients with estrogen receptor-negative tumors which are insensitive to antihormonal strategies.     

Binding analysis of the estrogen receptor to its specific DNA target site in human breast cancer

The estrogen receptor (ER) is a nuclear protein with a hormone- and a DNA-binding domain. We examined the DNA binding of ER in MCF-7 cells and 79 primary breast cancers by gel mobility shift assay using as a probe the estrogen response element (ERE). The mobility shift assay showed saturable, specific binding of ER to ERE in crude, high molar extracts containing greater than or equal to 4 mg/ml protein. Nonspecific binding was reduced by increasing concentrations of poly(deoxyinosidylate.deoxycytidylate) and shortening of the ERE probe from 35 to 15 base pairs. In the presence of Mg2+ the ER-ERE complex formation was hormone dependent at 22 degrees C but not at 37 degrees C. In the absence of Mg2+ estradiol was not necessary for ER-ERE complex formation. Correlation of the mobility shift assay with the hormone-binding (E2) assay showed agreement in 55 of the 79 tumors. Both assays were positive (E2 +/ERE+) in 35 cases and both were negative (E2-/ERE-) in 20 cases. In 11 tumors the hormone-binding assay was positive and the mobility shift assay negative (E2+/ERE-), suggesting an alteration of the DNA-binding domain. In 13 cancers the hormone-binding assay was negative and the mobility shift assay positive (E2-/ERE+) suggesting an alteration of the hormone-binding domain. By performing both hormone- and DNA-binding assays of ER and the hormone-binding assay of progesterone receptor (PR), we found the following subgroups of breast cancer: (a) E2+/ERE+/PR+, (b) E2+/ERE+/PR-, (c) E2+/ERE-/PR+, (d) E2+/ERE-/PR-, (e) E2-/ERE+/PR+, (f) E2-/ERE+/PR-, (g) E2-/ERE-/PR-. The simultaneous determination of 17 beta-estradiol and ERE binding may provide a better definition of the ER status of individual tumors and prove useful in refining endocrine therapy of patients with breast cancer.     

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts

The importance of epithelial‐stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA‐MB231) basal cell lines, with fibroblasts isolated from breast benign‐disease adjacent tissues (NAF) or with carcinoma‐associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA‐MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA‐MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA‐MB231 by a decrease in the expression of genes induced by TGFβ1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. © 2009 UICC     

Inhibition of estrogen-dependent breast cell responses with phenylacetate

The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4-chlorophenylacetate (4-CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4-CPA were estrogen receptor (ER) positive (T47-D, MCF-7 and ZR-75-1) whereas those that were little affected by these compounds were ER-negative (MDA-MB-157, MDA-MB-231 and SK-Br-3). Dose response studies indicated that 4-CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3-4 times that of PA. These findings suggest that there is "cross-talk" between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen-dependent events. This hypothesis was directly tested in vitro using ER+ MCF-7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF-7-D1). Our experiments with MCF-7-D1 cells indicated that PA and 4-CPA inhibited basal and estrogen-induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen-dependent cyclin D1 gene activation. Using a reporter gene construct (ERE(V)-tk-Luc) containing a canonical estrogen response element that was transiently transfected into MCF-7 and MDA-MB-231 cells, we have also demonstrated inhibition of promoter activity by PA and 4-CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.