The process of malignant progression in human breast cancer

Malignant progression in breast cancer represents the processes through which localized, hormone-dependent tumor cells become resistant to endocrine manipulations and metastasize to sites distant from the primary tumor. By selection in ovariectomized athymic nude mice, we have isolated a variant (MIII) of the hormone-dependent, poorly invasive, human breast cancer cell line MCF-7. MIII cells have lost their absolute requirement for estrogen to form proliferating tumors in nude mice. Furthermore, these tumors are significantly more invasive than the parental MCF-7 cell line. MIII cells retain some responsivity to estrogens and antiestrogens, indicating that they have progressed to a hormone-independent but hormone-esponsive phenotype. In an attempt to determine the nature of this process, we have compared the phenotype of MIII cells with that of other MCF-7 variants. These comparisons strongly suggest that the factors contributing to perturbations in antiestrogen sensitivity, hormone-dependent growth, metastatic potential and tumorigenicity are essentially independent of each other and acquired in a random manner. Loss of estrogen receptor expression and overexpression of EGF receptors tend to occur later in the process of malignant progression.     

PPAPDC1A在体内外对乳腺癌细胞增殖、侵袭和转移能力的影响

目的:通过构建稳定过表达和干扰PPAPDC1A的乳腺癌细胞株,探讨PPAPDC1A对乳腺癌细胞增殖、侵袭和转移能力的影响。方法:利用CCK-8和Transwell实验检测PPAPDC1A稳定过表达和干扰后对乳腺癌细胞体外增殖和侵袭能力的影响。采用裸鼠皮下成瘤实验检测PPAPDC1A对乳腺癌细胞体内增殖和裸鼠致瘤性的作用。利用免疫组织化学染色法检测各组肿瘤组织中Ki-67的表达。通过裸鼠尾静脉注射实验检测PPAPDC1A对乳腺癌MCF-7细胞和MDA-MB-231细胞体内转移能力的影响。结果:成功建立稳定过表达PPAPDC1A的乳腺癌MCF-7细胞株和稳定干扰PPAPDC1A的乳腺癌MDA-MB-231细胞株;CCK-8和Transwell实验结果显示,与MCF-7和MCF-7-Vector细胞株相比,MCF-7-PPAPDC1A细胞株的生长速度显著增快,穿膜细胞数量多（P＜0.05）;与此相反,MDA-MB-231-shPPAPDC1A组细胞的生长速度和穿膜细胞数明显少于MDA-MB-231-shNC和MDA-MB-231 细胞株（P＜0.05）。动物实验结果显示,与MCF-7-Vector组相比,MCF-7-PPAPDC1A组的肿瘤生长速度较快,肿瘤的体积较大,Ki-67的阳性率高,肺转移灶的数目增多（P＜0.05）;与此相反,与MDA-MB-231-shNC组相比MDA-MB-231-shPPAPDC1A组的肿瘤生长速度较慢,肿瘤的体积较小,Ki-67的阳性率低,肺转移灶的数目减少（P＜0.05）。结论:PPAPDC1A对乳腺癌细胞的增殖、侵袭和转移有促进作用。     

Dendritic cells fused with allogeneic breast cancer cell line induce tumor antigen-specific CTL responses against autologous breast cancer cells

Dendritic cell (DC)/tumor cell fusion vaccine has been revealed as a promising tool for the antitumor immunotherapy. Previous research has shown that fusion hybrids of human DCs and autologous tumor cells can induce cytotoxic T lymphocyte (CTL) responses against autologous tumor cells in animal models and human clinical trials. However, a major restriction factor for the clinical use is the difficulty for preparation of sufficient amount of autologous tumor cells especially for the patients with metastasis cancer whose primary tumor lesion is not clear or has been resected. In this study, allogeneic breast cancer cell line MCF-7 cells were electrofused to autologous DCs from patient with breast cancer as a strategy to deliver shared breast cancer antigens to DCs. Fusion cells generated by autologous DCs and allogeneic MCF-7 were able to induce autologous T lymphocytes proliferation, high levels of IFN-gamma production and CTL responses. CTLs induced by DCs/allogeneic MCF-7 fusion cells were able to kill autologous breast cancer cells in an antigen specific and HLA restriction manner. Our study may provide the experiment basis for the use of allogeneic breast cancer cell line in the DC/tumor cell fusion cell vaccination strategy.     

无血清悬浮法培养MCF-7细胞系并筛选该细胞系中肿瘤干细胞相关亚群的初步研究

目的：研究无血清培养基悬浮培养MCF-7乳腺癌细胞系,筛选并鉴定MCF-7乳腺癌细胞系中的肿瘤干细胞相关亚群。方法：应用乳腺癌培养基在无血清的条件下悬浮培养MCF-7乳腺癌细胞系。通过无血清培养筛选乳腺癌细胞系肿瘤干细胞相关亚群,将其接种于含血清培养基,观察分化。应用单克隆形成实验、表面标志检测、HOECHST33342染色检测来确定培养出的细胞中肿瘤干细胞的比例及其培养后肿瘤干细胞含量的变化。结果：乳腺癌MCF-7细胞系中有约2.12%的肿瘤细胞在无血清培养基中能够存活、增殖,形成自由漂浮的细胞球。细胞球可连续传代,若重新接种于含血清培养基中可重新贴壁分化,贴壁分化后细胞形态与直接在含血清培养基中培养的MCF-7无明显差别。流式细胞仪表面标志检测,细胞球中约含83.13%表达CD24^-CD44^＋的细胞,HOECHST33342染色提示细胞球中约含10.06%的侧亚群（sidepopulation）细胞。结论：MCF-7细胞系可在含生长因子的无血清培养基中悬浮生长,并维持细胞系。该细胞系中含有比例较低的具有增殖和分化能力的乳腺癌干细胞相关亚群。表面标志以及HOECHST33342检测差异提示细胞球中仅约1/8细胞具有干细胞的功能。     

Differential sensitivity of human mammary epithelial and breast carcinoma cell lines to curcumin

Curcumin has anti-inflammatory, antiproliferative, and antitumor effects. To understand the chemopreventive mechanism of curcumin against human malignancies, the cellular and molecular changes induced by this agent in human mammary epithelial (MCF-10A) and breast carcinoma (MCF- 7/TH) cell lines were investigated. The human multidrug- resistant breast cancer cell line was 3.5 fold more sensitive to curcumin than the mammary epithelial cell line. Even though both cell lines accumulated a similar amount of curcumin, a significantly higher percentage of apoptotic cells was induced in breast cancer cells compared to a very low percentage of apoptosis in mammary epithelial cells. Incubation of breast cancer cells with 20 and 40 microM curcumin for 24 h induced G2 block and sub-G0/G1 cell population, respectively. Curcumin treatment caused a reduction in the expression of Ki67, PCNA, and p53 mRNAs in breast cancer cells. The human mammary epithelial cell line showed a down-regulation of p21 mRNA and an up-regulation of Bax mRNA expression with curcumin treatment. The results suggest that apoptosis is involved in the curcumin-induced inhibition of tumor cell growth, and genes associated with cell proliferation and apoptosis may be playing a role in the chemopreventive action of curcumin.     

利用荧光素酶标记的肿瘤细胞建立活体成像动物模型

目的建立稳定表达荧光素酶的人乳腺癌细胞株并构建适用于小动物活体成像系统观察的裸鼠皮下移植瘤模型。方法采用脂质体将携带荧光素酶基因的质粒转染到人乳腺癌细胞株MCF-7中,G418筛选出稳定表达荧光素酶的单克隆细胞株。扩增后接种于裸鼠,建立裸鼠皮下移植瘤模型,通过活体动物成像系统监测肿瘤的生长过程。结果获得了高水平稳定表达荧光素酶的乳腺癌单克隆细胞株,该单克隆细胞株与母细胞系MCF-7具有相似的生长特性。将稳定表达荧光素酶的克隆接种于裸鼠皮下可成瘤,小动物活体成像系统能准确监测肿瘤细胞在体内的生长过程。结论成功建立了稳定表达荧光素酶的乳腺癌单克隆细胞株。采用活体动物成像系统构建的裸鼠皮下移植瘤模型是拓展肿瘤体内生长、转移及治疗相关研究的理想模型。     

Human breast cancer MCF-7 cell line contains inherently drug-resistant subclones with distinct genotypic and phenotypic features

The resistance of cancer cells to multiple chemotherapeutic agents poses a major problem in the successful treatment of breast cancer. Whether drug resistance is due to changes induced in the drug-exposed tumor cells or represents the selective growth of one or more drug-resistant clones present in the initial tumor remains controversial. Here we provide evidence that the development of multidrug resistance in a human breast cancer cell line (MCF-7) is a result of propagation of an inherently resistant subclone. The drug-resistant MCF-7 (MCF-7/DOX) cells exhibited several phenotypic and genotypic features that were notably distinct from those observed in the parental drug-sensitive (MCF-7/WT) cells. The most striking change was the presence of a full-length functional caspase-3 in MCF-7/DOX cells that was missing in the parental MCF-7/WT cells due to a deletion mutation in the caspase-3 gene. A drug-resistant MCF-7 cell subline (MCF-7/WT/DOX) was established by exposing the MCF-7/WT cells directly to a high dose of doxorubicin and used for determining the phenotypic and genotypic alterations associated with drug resistance. The MCF-7/WT/DOX cells exhibited alterations identical to those of the MCF-7/DOX cells but which were strikingly distinct from the parental MCF-7/WT cell line. These results suggest that drug resistance is an inherent property of some cancer cells that are present in the initial tumor burden and exhibit distinct phenotypic/genotypic alterations.     

ＲｈｏＡ小干扰ＲＮＡ抑制乳腺癌细胞株ＭＣＦ-７及其裸鼠皮下移植瘤的实验研究

目的　观察ＲｈｏＡ小干扰ＲＮＡ（ｓｉＲｈｏＡ）对乳腺癌细胞株ＭＣＦ-７增殖、迁移、周期和凋亡的影响以及对裸鼠移植瘤生长的影响。方法　ｓｉＲｈｏＡＬｉｐｏｆｅｃｔａｍｉｎｅ２０００介导下转染乳腺癌细胞ＭＣＦ-７,转染４８ｈ后,采用Ｗｅｓｔｅｒｎｂｌｏｔ技术检测ＲｈｏＡ蛋白的表达,ＭＴＴ实验检测ｓｉＲｈｏＡ转染细胞的增殖变化,损伤刮擦实验检测ｓｉＲｈｏＡ转染细胞的迁移能力,流式细胞仪检测ｓｉＲｈｏＡ转染细胞的周期和凋亡,裸鼠移植瘤实验检测ｓｉＲｈｏＡ对肿瘤生长的影响。结果　成功转染ｓｉＲｈｏＡ的肿瘤细胞,Ｗｅｓｔｅｒｎｂｌｏｔ显示ＲｈｏＡ蛋白表达在ＭＣＦ-７细胞中明显下降;ｓｉＲｈｏＡ对ＭＣＦ-７细胞的增殖、迁移均有显著的抑制作用并能促进肿瘤细胞凋亡及细胞周期中Ｓ期细胞减少,Ｇ１／Ｇ０期细胞增加;裸鼠移植瘤内重复注射ｓｉＲｈｏＡ后肿瘤生长明显减缓。结论　ｓｉＲｈｏＡ能够明显抑制ＲｈｏＡ基因在乳腺癌细胞ＭＣＦ-７中的表达,部分逆转ＭＣＦ-７的恶性生物学行为并抑制裸鼠移植瘤的生长。     

Suppression of tumorigenesis by the breast cancer cell line MCF-7 following transfer of a normal human chromosome 11.

Breast cancer development is associated with several genetic abnormalities. Loss of heterozygosity in the short arm of chromosome 11 has been observed in 30% of tumors. We found homozygosity at five chromosome 11 polymorphic loci in genomic DNA of the MCF-7 breast carcinoma cell line, suggesting a possible loss of one chromosome 11. We have studied the transformed and tumorigenic phenotypes of MCF-7 cells following introduction of a normal human chromosome 11 via microcell fusion. MCF-7/H11 cell hybrids, containing chromosome 11, showed in vitro characteristics similar to the parental cell line. However, tumorigenicity in athymic mice was completely suppressed. Since tumor formation by MCF-7 cells is estrogen dependent, we have analysed the expression of the estrogen receptor and of the estrogen-activated gene pS2. No difference was detected between the parental MCF-7 cells and the derived chromosome 11 cell hybrids, indicating that the mechanism of MCF-7 tumor suppression by chromosome 11-associated functions does not directly involve the estrogen/estrogen receptor molecular pathway.     

Isolation of protein kinase C-alpha-regulated cDNAs associated with breast tumor aggressiveness by differential mRNA display

Elevated levels of protein kinase C (PKC) are associated with increased metastatic capacity in both human breast cancer cells and breast tumors. MCF-7 breast cancer cells stably transfected with PKC-alpha were recently shown to display a more aggressive phenotype and increased tumorigenicity in nude mice. To identify genes involved in the progression to the aggressive phenotype, mRNA differential display was performed to isolate cDNAs that are differentially expressed between the parental, non-metastatic MCF-7 cell line and the metastatic derivative MCF-7-PKC-alpha cell line. One cDNA was identified which was upregulated and four cDNAs were downregulated in MCF-7-PKC-alpha cells. The upregulated cDNA may be a differentiation-specific gene as it is 100% homologous to a putative glialblastoma cell differentiation-related protein, GBDR1. DNA sequence analysis and flow cytometry revealed that three of the downregulated cDNAs correspond to histone 3.B, and integrins alpha3 and alpha6. The fourth downregulated cDNA clone, G2Q, is a novel sequence. G2Q is expressed in normal breast and bronchial tissue, but is downregulated in a variety of tumor cell lines and in aggressive primary and secondary breast tumors, suggesting that G2Q may be a useful prognostic indicator of tumor aggressiveness. Further, downregulation of G2Q expression in the non-metastatic MCF-7 cells by antisense oligonucleotides resulted in increased in vitro invasive capacity of these cells in a Matrigel matrice. This study provides the basis for identifying new genes involved in breast tumor progression and the role that PKC plays in the pathogenesis of this cancer.     

Establishment of Paclitaxel-resistant Breast Cancer Cell Line and Nude Mice Models,and Underlying Multidrug Resistance Mechanisms in Vitro and in Vivo

Background: Breast cancer is a common malignant tumor which affects health of women and multidrugresistance (MDR) is one of the main factors leading to failure of chemotherapy. This study was conducted toestablish paclitaxel-resistant breast cancer cell line and nude mice models to explore underlying mechanisms ofMDR. Methods: The breast cancer drug-sensitive cell line MCF-7 (MCF-7/S) was exposed in stepwise escalatingpaclitaxel (TAX) to induce a resistant cell line MCF-7/TAX. Cell sensitivity to drugs and growth curves weremeasured by MTT assay. Changes of cell morphology and ultrastructure were examined by optical and electronmicroscopy. The cell cycle distribution was determined by flow cytometry. Furthermore, expression of proteinsrelated to breast cancer occurrence and MDR was tested by immunocytochemistry. In Vivo, nude mice wereinjected with MCF-7/S and MCF-7/TAX cells and weights and tumor sizes were observed after paclitaxeltreatment. In addition, proteins involved breast cancer and MDR were detected by immunohistochemistry.Results: Compared to MCF-7/S, MCF-7/TAX cells had a higher resistance to paclitaxel, cross-resistance andprolonged doubling time. Moreover, MCF-7/TAX showed obvious alterations of ultrastructure. Estrogen receptor(ER) expression was low in drug resistant cells and tumors while expression of human epidermal growth factorreceptor 2 (HER2) and Ki-67 was up-regulated. P-glycoprotein (P-gp), lung resistance-related protein (LRP)and glutathione-S-transferase-π (GST-π) involved in the MDR phenotype of resistant cells and tumors were alloverexpressed. Conclusion: The underlying MDR mechanism of breast cancer may involve increased expressionof P-gp, LRP and GST-π.     

Stable knockdown of estrogen receptor alpha by vector-based RNA interference suppresses proliferation and enhances apoptosis in breast cancer cells

Breast cancer, the most common malignancy in women, has a known association with the steroid hormone estrogen. Estrogen receptor alpha (ERalpha) plays an important role in the clinical care of breast cancer patients, both as a prognostic factor and as a therapeutic target. Here, we show that a small interfering RNA (siRNA) against ERalpha downregulates ERalpha expression in human MCF-7 and Bcap-37 breast cancer cells, causing a significant decrease in breast cancer cell proliferation. Tumor cells lacking ERalpha expression grew at a much slower rate than did control cells in vitro. Moreover, ERalpha knockdown in breast cancer cells resulted in decreased, even completely abrogated tumor growth in BALB/c nude mice, providing direct evidence for an essential role of ERalpha in breast cancer growth. Our results suggest siRNA-mediated gene silencing of ERalpha may impair tumorigenicity, and even suppress the tumor growth.     

Cooperative effect of gefitinib and fumitremorgin c on cell growth and chemosensitivity in estrogen receptor alpha negative fulvestrant-resistant MCF-7 cells

The selective ER downregulator, fulvestrant, is currently approved as a second line endocrine therapy after onset of resistance to prior antiestrogen therapy in postmenopausal breast cancer patients. Resistance to antihormonal therapies is common and, therefore, we anticipate that fulvestrant-resistance will occur as well. The current study was undertaken to investigate the underlying molecular changes after fulvestrant-resistance and find new therapeutic targets and agents for fulvestrant-resistant breast cancer cells. We developed a unique fulvestrant-resistant cell line (MCF-7/F), derived from MCF-7 estrogen receptor alpha (ERalpha)-positive human breast cancer cells, by culturing them in 1 microM fulvestrant containing medium for approximately 18 months. MCF-7/F cells became irreversibly ERalpha negative as withdrawal of fulvestrant did not alter the ERalpha-negative phenotype, determined by real-time PCR, Western blot analysis, and ERE-luciferase transfection assays. MCF-7/F cells grew in a hormone-independent manner. Interestingly, MCF-7/F cells overexpressed both epidermal growth factor receptor (EGFR) and breast cancer resistant protein (BCRP). Gefitinib, a specific EGFR tyrosine kinase inhibitor, preferentially inhibited the growth of MCF-7/F cells relative to MCF-7 cells by inhibiting both MAPK44/42 and Akt phosphorylation. MCF-7/F cells became less sensitive to chemotherapeutic agents such as mitoxantrone. Moreover, fumitremorgin C, a specific BCRP inhibitor, significantly increased the efficacy of mitoxantrone in MCF-7/F cells. Gefitinib increased the inhibitory effect of mitoxantrone on cell growth. Similarly, fumitremorgin C increased the inhibitory effect of gefitinib on cell growth, suggesting that there is a bidirectional crosstalk between EGFR and BCRP. More importantly, these results provide a molecular basis for using gefitinib, BCRP inhibitors, and chemotherapeutic agents as combination therapy approaches in fulvestrant-resistant breast cancer.     

乳腺癌CYP1B1的表达对紫杉醇耐药性的影响

  目的  探讨乳腺癌细胞CYP1B1表达与紫杉醇耐药产生的相关性。   方法  以乳腺癌细胞系MCF-7为研究对象, 经TCDD诱导MCF-7表达CYP1B1, 观察CYP1B1表达后对紫杉醇药物的细胞毒效应的影响。将紫杉醇联合CYP1B1的拮抗剂ANF使用, 观察ANF是否可逆转CYP1B1对紫杉醇的耐药作用。采用RT-PCR测定诱导后CYP1B1 mRNA水平, 采用细胞免疫化学染色和蛋白印迹技术测定诱导后CYP1B1蛋白水平, MTS比色法测定紫杉醇对细胞增殖的抑制强度。   结果  TCDD可诱导MCF-7细胞表达CYP1B1, 其mRNA和蛋白表达水平与TCDD浓度具有明显的剂量依赖性(P < 0.05);而单独紫杉醇不能诱导MCF-7细胞表达CYP1B1。当MCF-7细胞经TCDD诱导表达CYP1B1后, 紫杉醇浓度在0.01~0.1μg/mL时, 紫杉醇对MCF-7细胞的抑制率与对照组相比明显下降(P <0.05)。使用CYP1B1的特异性拮抗剂ANF后, MCF-7细胞对紫杉醇的敏感性升高, 细胞抑制率与对照组比较无显著性差异(P>0.05)。   结论   CYP1B1表达后可导致细胞产生对紫杉醇耐药。      

Growth inhibition of human breast cancer cells in vitro with an antibody against the type I somatomedin receptor

Insulin and insulin-like growth factors (IGFs) stimulate the growth of human breast cancer cells in vitro. The type I somatomedin receptor (SR), expressed in these cells, may mediate the mitogenic effects of these peptides. We have examined the effect of type I SR blockade on human breast cancer growth with a monoclonal antibody (alpha-IR3) that blocks the receptor binding domain. alpha-IR3 inhibited binding of 125I-IGF-I in all breast cancer cell lines tested. Binding affinity of alpha-IR3 was 2 to 5 times higher than that of IGF-I in MDA-231 (Kd 2.1 nM) and MCF-7 cells (Kd 0.6 nM), respectively. In the presence of 10% calf serum, the antibody inhibited anchorage-independent growth of six of seven breast cancer cell lines. This inhibition was reversible with excess IGF-I. In serum-free medium, alpha-IR3 blocked IGF-I-stimulated DNA synthesis in four of four breast cancer cell lines (MCF-7, ZR75-1, MDA-231, and HS578T). However, the antibody did not inhibit basal growth of any of the breast cancer cell lines in serum-free conditions. In three estrogen receptor-positive, estrogen-responsive breast cancer cell lines (MCF-7, ZR75-1, and T47D), type I SR blockade with alpha-IR3 failed to block estrogen-stimulated DNA synthesis or cell proliferation, indicating that secreted IGF activity is not the sole mediator of the growth effects of estrogen. In conclusion, antibody-mediated type I SR blockade does not inhibit basal growth of breast cancer cells under serum-free conditions, arguing against a critical autocrine role of endogenously secreted IGF activity in vitro. However, type I SR blockade inhibits breast cancer cell growth in the presence of serum, suggesting that serum IGFs might be critical endocrine or paracrine regulators of human breast cancer.     

Function analysis of estrogenically regulated protein tyrosine phosphatase gamma (PTPgamma) in human breast cancer cell line MCF-7

Protein tyrosine phosphatase gamma (PTPgamma) is a member of the receptor-like family of tyrosine phosphatases and has been implicated as a tumor suppressor gene in kidney and lung cancers. Based on our previous findings, we hypothesize that PTPgamma is a potential estrogen-regulated tumor suppressor gene in human breast cancer. To examine the effects of PTPgamma on growth of MCF-7 human breast cancer cells and compare the estrogenic responses of human breast cells with different PTPgamma expression levels, we established several stably transfected MCF-7 cell lines expressing different levels of PTPgamma, which were confirmed by RT-PCR and immunostaining. In our work, we used the antisense construct to breakdown endogenous PTPgamma level in MCF-7 cells. The results from doubling time assay suggested that PTPgamma is capable of inhibiting MCF-7 breast cancer cell growth. We further demonstrated that PTPgamma is able to inhibit anchorage-independent growth of breast cancer cells in soft agar and reduce the estrogenic responses of MCF-7 cell proliferation to estradiol-17beta (E(2)) and zeranol (Z, a nonsteroidal growth promoter with estrogenic activity). Our data suggest that PTPgamma may function as an important modulator in regulating the process of tumorigenesis in human breast.     

Oncoprotein expression and morphological phenotypes of human breast epithelial cells transformed by the c-Ha-ras oncogene

Activated oncogenes have been detected in a variety of malignant tumors and altered expressions of certain genes are known to play a functional role in the cancer process. The chemical carcinogen, BP, and the insertion of c-Ha-ras, induced characteristics of transformed phenotypes in a suitable human breast epithelial cell line. Carcinogen-treated and Ha-ras-transfected cells showed a progression of changes in the morphology, anchorage independent growth, invasiveness and tumorigenicity in SCID mice. Tumor growth occurs after a series of molecular events that parallel morphological changes. The aim of this work was to determine the neoplastic phenotypes following treatment with benzo(a)pyrene (BP) and transfection with c-Ha-ras oncogene changes and PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in MCF-10F cells, a spontaneously immortalized human breast epithelial cell line. Protein expression was determined by immunofluorescent staining coupled with confocal microscopy. An increased oncoprotein expression in comparison to MCF-10F cells was observed in PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in breast epithelial cells transformed with a chemical carcinogen and/or oncogene transfected that are not present in the MCF-10F. This in vitro cancer model can be used as a valuable model in the study of breast carcinogenesis.