Recovery of cellular E-cadherin precedes replenishment of estrogen receptor and estrogen-dependent proliferation of breast cancer cells rescued from a death stimulus

Loss of estrogen-responsiveness and impaired E-cadherin expression/function has been linked to increased metastatic potential of breast cancer cells. In this study, we report that proliferation of breast cancer cells can resume following removal of a toxic stimulus causing severe impairment of cell adhesion and estrogen responsiveness. This type of response was induced by okadaic acid (OA) in MCF-7 cells, and was accompanied by an almost complete block of DNA synthesis, loss of cell-cell contact and cell detachment from culture dishes, loss of estrogen receptor (ER), progesterone receptor (PR) and E-cadherin, whereas only a weak, if any, inhibition of protein synthesis could be observed. These responses were detected in MCF-7 cells after a 1-day treatment with 50 nM OA, and could be reversed if OA-treated cells were recovered in a culture medium devoid of the toxin, so that rescued cells resumed growth 8-12 days after replating. By pulse-chase experiments, we found that protein synthesis was not significantly affected in rescued cells, whose DNA synthesis, instead, was almost completely blocked during the first days of MCF-7 cell rescue from OA treatment. We also analyzed E-cadherin, mitogen activated protein kinase isoforms ERK1 and ERK2, Bcl-2 and BAX proteins during the rescue of MCF-7 cells from OA-induced cell death, and found that their expression followed temporally defined patterns. Cellular levels of E-cadherin returned to control levels within the first days of the rescue, followed by ER, ERK1, and ERK2, and finally by Bcl-2 and BAX proteins. Under our experimental conditions, restoration of cell adhesion did not require a functional ER system, but recovery of a normal ER pool accompanied resumption of estrogen-dependent proliferation of OA-treated MCF-7 cells.     

Rapid induction of histone hyperacetylation and cellular differentiation in human breast tumor cell lines following degradation of histone deacetylase-1

Quinidine inhibits proliferation and promotes cellular differentiation in human breast tumor epithelial cells. Previously we showed quinidine arrested MCF-7 cells in G(1) phase of the cell cycle and led to a G(1) to G(0) transition followed by apoptotic cell death. The present experiments demonstrated that MCF-7, MCF-7ras, T47D, MDA-MB-231, and MDA-MB-435 cells transiently differentiate before undergoing apoptosis in response to quinidine. The cells accumulated lipid droplets, and the cytokeratin 18 cytoskeleton was reorganized. Hyperacetylated histone H4 appeared within 2 h of the addition of quinidine to the medium, and levels were maximal by 24 h. Quinidine-treated MCF-7 cells showed elevated p21(WAF1), hypophosphorylation and suppression of retinoblastoma protein, and down-regulation of cyclin D1, similar to the cell cycle response observed with cells induced to differentiate by histone deacetylase inhibitors, trichostatin A, and trapoxin. Quinidine did not show evidence for direct inhibition of histone deacetylase enzymatic activity in vitro. HDAC1 was undetectable in MCF-7 cells 30 min after addition of quinidine to the growth medium. The proteasome inhibitors MG-132 and lactacystin completely protected HDAC1 from the action of quinidine. We conclude that quinidine is a breast tumor cell differentiating agent that causes the loss of HDAC1 via a proteasomal sensitive mechanism.     

Inhibition of mitogen-activated protein kinase kinase selectively inhibits cell proliferation in human breast cancer cells displaying enhanced insulin-like growth factor I-mediated mitogen-activated protein kinase activation.

Mitogen-activated protein (MAP) kinase mediates cell proliferation, cell differentiation, and cell survival by regulating signaling pathways activated by receptor protein tyrosine kinases (RPTKs), including the insulin-like growth factor 1 receptor (IGF-IR). We analyzed the upstream signaling components of the MAP kinase pathway, including RPTKs, in human breast cancer cell lines and found that some of those components were overexpressed. Importantly, signaling molecules such as IGF-IR, insulin receptor, and insulin receptor substrate 1, leading to the MAP kinase pathway, were found to be concomitantly overexpressed within certain tumor lines, i.e., MCF-7 and T-47D. When compared with the nonmalignant and other breast tumor lines examined, MCF-7 and T-47D cells displayed a more rapid, robust, and sustained MAP kinase activation in response to insulin-like growth factor I (IGF-I) stimulation. By contrast, IGF-I treatment led to a sustained down-regulation of MAP kinase in those lines overexpressing ErbB2-related RPTKs. Interestingly, blocking the MAP kinase pathway with PD098059 had the greatest antiproliferative effect on MCF-7 and T-47D among the normal and tumor lines tested. Furthermore, addition of an IGF-IR blocking antibody to growth medium attenuated the ability of PD098059 to suppress the growth of MCF-7 and T-47D cells. Thus, our study suggests that concomitant overexpression of multiple signaling components of the IGF-IR pathway leads to the amplification of IGF-I-mediated MAP kinase signaling and resultant sensitization to PD098059. The enhanced sensitivity to PD098059 implies an increased requirement for the MAP kinase pathway in those breast cancer cells, making this pathway a potential target in the treatment of selected breast malignancies.     

Rb dephosphorylation and suppression of E2F activity in human breast tumor cells exposed to a pharmacological concentration of estradiol

This report characterizes the influence of a pharmacological concentration of estradiol on growth arrest and cell death in MCF-7 breast tumor cells, with a focus on elements of the Rb-E2F cell-cycle regulatory pathway. Continuous exposure of MCF-7 breast tumor cells to 100 microM estradiol produces a marked reduction in the G1 and S phase populations and a corresponding increase in the G2/M population within 24 h; after 48 h, accumulation of cells in G1 becomes evident while after 72 h the cells appear to be equally distributed between the G1 and G2/M phases. The accumulation of cells in G1 is temporally associated with dephosphorylation of the Rb protein and suppression of E2F activity. Estradiol also produces an initial burst of cell death with loss of approximately 40% of the tumor cell population within 24 h; however, there is no tangible evidence for the occurrence of apoptosis based on terminal transferase end-labeling of DNA, DNA fragmentation analysis by alkaline unwinding, cell-cycle analysis or cell morphology. In addition to the lack of caspase-3 in MCF-7 cells, the absence of apoptosis could be related, at least in part, to the fact that estradiol promotes a rapid reduction in levels of the E2F-1 and Myc proteins. Overall, these studies are consistent with the concept that alterations in the levels and/or activity of the E2F family of proteins as well as proteins interacting with the E2F family may influence the nature of the antiproliferative and cytotoxic responses of the breast tumor cell.     

Cyclic AMP stimulation of transferrin secretion by breast cancer cell grown on extracellular matrix or in two-compartment culture chambers.

Extrahepatic synthesis and secretion of transferrin (Tf), the major iron-carrying protein, have been described in normal and tumoral tissues suggesting a potential role for paracrine or autocrine function. In breast tumor cell MCF-7, we have previously shown a Tf secretion stimulated by estradiol which might confer selective growth advantages of these rapidly proliferating cells. The present work refers to possible additional Tf functions related to differentiation of breast tumor cells. We induced MCF-7 cell differentiation by the cyclic AMP derivative, dibutyryl cAMP (dB cAMP) and studied Tf secretion in different culture conditions after labeling with [35S] methionine. Our results demonstrate that dB cAMP stimulates Tf secretion only in culture environment that permits access to the basolateral surface and caters to the polarity requirements of the cell. These results suggest that Tf may also act as a modulator of cellular differentiation in breast cancer cells.     

Low-frequency low-field magnetic susceptibility of ferritin and hemosiderin

The biologic effects and mechanisms by which bone morphogenetic proteins (BMPs) function in breast cancer cells are not well defined. A member of this family of growth and differentiation factors, BMP-2, inhibited both basal and estradiol-induced growth of MCF-7 breast tumor cells in culture. Flow cytometric analysis showed that in the presence of BMP-2, 62% and 45% of estradiol-stimulated MCF-7 cells progressed to S-phase at 24 h and 48 h, respectively. Estradiol mediates growth of human breast cancer cells by stimulating cyclins and cyclin-dependent kinases (CDKs). BMP-2 significantly increased the level of the cyclin kinase inhibitor, p21, which in turn associated with and inactivated cyclin D1. BMP-2 inhibited estradiol-induced cyclin D1-associated kinase activity. Also estradiol-induced CDK2 activity was inhibited by BMP-2. This inhibition of CDK activity resulted in hypophosphorylation of retinoblastoma protein thus keeping it in its active form. These data provide the first evidence by which BMP-2 inhibits estradiol-induced proliferation of human breast cancer cells.     

Inhibition of endothelial cell proliferation and tumor angiogenesis by up-regulating NDRG2 expression in breast cancer cells

The N-myc downstream-regulated gene 2 (NDRG2) is involved in tumor cell differentiation and apoptosis, but its function in tumor angiogenesis remains to be established. Here, we employed adenovirus overexpressing NDRG2 (Ad-NDRG2) to efficiently up-regulate target gene expression in the NDRG2-low-expressing, breast cancer cell line MCF-7. Moreover, VEGF secretion was decreased in MCF-7 cells infected by Ad-NDRG2, and medium conditioned by these infected cells could significantly inhibit the proliferation, tube formation and invasion of human umbilical vein endothelial cells (HUVECs). Further study indicated that the angiogenesis promoting factors VEGF and HIF-1α were down-regulated, whereas the angiogenesis suppressing factors p53 and VHL were up-regulated in MCF-7 cells infected by Ad-NDRG2. Finally, in a nude mouse model, intratumoral injections of Ad-NDRG2 every 3 days for 20 days significantly inhibited the growth and angiogenesis of xenografted MCF-7 tumors. In summary, these data indicate that NDRG2 may be involved in angiogenesis by impacting the expression of angiogenesis related factors. Thus, specific overexpression of NDRG2 by adenovirus represents a promising approach for the treatment of tumor angiogenesis.     

上皮细胞黏附分子增强细胞间紧密连接促进乳腺癌细胞耐药

乳腺癌是致死率很高的恶性肿瘤,由ABCG2 (ATP-binding cassette G2)介导的多药耐药(multidrug resistance,MDR)是导致其化疗失败的重要原因,探讨ABCG2介导的耐药机制并探寻其关键分子是当前亟待解决的难题。上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)参与多种肿瘤耐药,且与乳腺癌MDR密切相关,但它在ABCG2介导的乳腺癌耐药中的作用尚未阐明。本研究目的在于探究EpCAM对于ABCG2介导的乳腺癌细胞的多药耐药的调节作用及其机制。CCK8细胞毒性结果证实,相对于人乳腺癌药物敏感株MCF-7,耐药株MCF-7/MX对米托蒽醌(mitoxantrone,MX)的耐药性显著增强;Western 印迹结果显示,与MCF-7相比,MCF-7/MX细胞中ABCG2高表达,EpCAM表达上调。siRNA法敲低MCF-7/MX细胞中EpCAM可下调其ABCG2表达,并恢复对MX的敏感性。倒置显微镜观察细胞形态,发现敲低EpCAM可减少MCF-7/MX细胞间连接。免疫荧光双染法观察到EpCAM与密封蛋白1(claudin 1)在MCF-7/MX细胞共定位;进一步Western 印迹结果表明,敲低EpCAM减少MCF-7/MX细胞中密封蛋白1表达。综上所述,EpCAM可能通过与密封蛋白1相互作用,增强细胞间紧密连接,促进ABCG2介导的乳腺癌多药耐药。     

Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.     

Elevation of Soluble Guanylate Cyclase Suppresses Proliferation and Survival of Human Breast Cancer Cells

Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.     

DKK-1通过上调nm23表达抑制乳腺癌细胞迁移

Dickkopf-1(DKK-1)作为Wnt/β-连环蛋白(Wnt/β-catenin)经典信号传导通路的拮抗剂而受到关注.为了进一步阐明DKK-1在乳腺癌细胞迁移中的作用及其分子机制,应用我们建立的乳腺癌细胞MCF-7高转移倾向亚克隆LM-MCF-7细胞株,比较了DKK-1在不同转移能力的乳腺癌细胞株中表达水平及其与细胞迁移能力的关系.结果显示,DKK-1在LM-MCF-7细胞中表达明显下调;"伤口愈合"实验结果表明,在MCF-7细胞中,RNA干扰DKK-1可导致细胞迁移能力增强;相反,在LM-MCF-7细胞中过表达DKK-1则可抑制细胞的迁移.进一步研究结果显示,DKK-1为肿瘤转移抑制因子nm23的上游激活因子.因此,我们的研究结果表明,DKK-1表达水平下调导致nm23表达水平下调,解除了对乳腺癌细胞迁移的抑制作用,是LM-MCF-7乳腺癌细胞具有高迁移能力的原因之一;反之,与LM-MCF-7相比,DKK-1在MCF-7细胞中高表达,其通过上调nm23可抑制乳腺癌细胞迁移.这一发现对进一步揭示乳腺癌细胞转移的分子机制具有的重要意义.     

Cyclin A1 Modulates the Expression of Vascular Endothelial Growth Factor and Promotes Hormone-Dependent Growth and Angiogenesis of Breast Cancer

Alterations in cellular pathways related to both endocrine and vascular endothelial growth factors (VEGF) may contribute to breast cancer progression. Inhibition of the elevated levels of these pathways is associated with clinical benefits. However, molecular mechanisms by which endocrine-related pathways and VEGF signalling cooperatively promote breast cancer progression remain poorly understood. In the present study, we show that the A-type cyclin, cyclin A1, known for its important role in the initiation of leukemia and prostate cancer metastasis, is highly expressed in primary breast cancer specimens and metastatic lesions, in contrasting to its barely detectable expression in normal human breast tissues. There is a statistically significant correlation between cyclin A1 and VEGF expression in breast cancer specimens from two patient cohorts (p<0.01). Induction of cyclin A1 overexpression in breast cancer cell line MCF-7 results in an enhanced invasiveness and a concomitant increase in VEGF expression. In addition, there is a formation of protein–protein complexes between cyclin A1 and estrogen receptor ER-α cyclin A1 overexpression increases ER-α expression in MCF-7 and T47D cells. In mouse tumor xenograft models in which mice were implanted with MCF-7 cells that overexpressed cyclin A1 or control vector, cyclin A1 overexpression results in an increase in tumor growth and angiogenesis, which is coincident with an enhanced expression of VEGF, VEGFR1 and ER-α Our findings unravel a novel role for cyclin A1 in growth and progression of breast cancer, and suggest that multiple cellular pathways, including cell cycle regulators, angiogenesis and estrogen receptor signalling, may cooperatively contribute to breast cancer progression.     

Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.     

EGF Induces Cell Motility and Multi-Drug Resistance Gene Expression in Breast Cancer Cells

The epidermal growth factor receptor (EGFR) is important for normal development, differentiation, and cell proliferation. Deregulation of EGFR has been observed in breast cancer. EGFR and signal pathways activated by these receptors have been associated with an advanced tumor stage and a poor clinical prognosis in breast cancer, however, the precise mechanisms responsible for this process are still not known. Here we show that treatment of MCF-7 breast cancer cells with EGF activated Akt and ERK, induced morphological changes, and increased cell motility. In addition, the constitutive expression of Raf-1 and the use of a MEK inhibitor demonstrated the participation of the Raf/MEK/ERK pathway in these processes. Importantly we detected that EGF induced MRP-1, 3, 5 and 7 gene expression and an increase in MRP1 promoter activity. In conclusion, treatment of MCF-7 breast cancer cells with EGF, in the absence of other growth factors, resulted in activation of EGFR signal transduction pathways; which were related with cell motility and drug resistance.     

Bone marrow stromal cell-derived growth inhibitor inhibits growth and migration of breast cancer cells via induction of cell cycle arrest and apoptosis

Genes encoding growth-inhibitory proteins are postulated to be candidate tumor suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumors. Here we report the cloning and functional characterization of a novel human bone marrow stromal cell (BMSC)-derived growth inhibitor (BDGI) by large scale random sequencing of a human BMSC cDNA library. Human BDGI cDNA encodes a 477-amino acid residue protein that shares high homology with rat and mouse pregnancy-induced growth inhibitors. The C-terminal of BDGI is identical to a novel human pregnancy-induced growth inhibitor, OKL38. BDGI is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved family of BDGI-like proteins. BDGI overexpression inhibits the proliferation, decreases anchorage-dependent growth, and reduces migration of MCF-7 human breast cancer cells, whereas down-regulation of BDGI expression promotes the proliferation of MCF-7 and HeLa cervix epitheloid carcinoma cells. Interestingly, the inhibitory effect of BDGI on MCF-7 cells is more potent than that of OKL38. We demonstrate that BDGI induces cell cycle arrest in S phase and subsequent apoptosis of MCF-7 cells, which is likely to account for the antiproliferative effects of BDGI. This process may involve up-regulation of p27Kip1 and down-regulation of cyclin A, Bcl-2, and Bcl-xL. The inhibitory effect of BDGI on cell proliferation and the induction of apoptosis were also observed in A549 lung cancer cells but not HeLa cells. These results indicate that BDGI might be a growth inhibitor for human tumor cells, especially breast cancer cells, possibly contributing to the development of new therapeutic strategies for breast cancer.     

Ebp1, an ErbB-3 binding protein, interacts with Rb and affects Rb transcriptional regulation

Ebp1, an ErbB-3 binding protein, inhibits the proliferation and induces the differentiation of human breast cancer cells. The mechanisms of these effects are unknown. Rb, the product of the retinoblastoma gene, is an important modulator of cell cycle progression and cellular differentiation. We report that Rb is a binding target for Ebp1. Ebp1 was localized to both the nucleus and the cytoplasm of logarithmically growing AU565 breast cancer cells and HeLa cells as determined by confocal immunofluorescent microscopy. Ebp1 was present in Rb immunoprecipitates derived from AU565 breast cancer cells. GST-Rb also bound endogenous Ebp1. Using GST-Ebp1 constructs, we determined that the 72 C-terminal amino acids of Ebp1 were sufficient to bind Rb. Dephosphorylation of Ebp1 enhanced the interaction of Ebp1 with Rb. The overexpression of Ebp1 in MCF-7 and AU565 (Rb(+)) cells inhibited the activity of the E2F1 regulated cyclin-E promoter. Ebp1 bound E2F1 indirectly via Rb in lysates of MCF-7 cells. The interaction of Ebp1 with Rb may prove to be an important mechanism of Ebp1 induced changes in cell proliferation and differentiation.     

p53-mediated up-regulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells

Induction of CD95 (Fas/APO-1) and CD95 ligand during chemotherapeutic treatment may contribute to the death by apoptosis of some tumor cells. In this study, we have analyzed the role of the CD95 system in genotoxic drug-induced death of human breast tumor cells. Incubation of the breast tumor cell lines MCF-7 and EVSA-T with doxorubicin or methotrexate caused apoptosis after 48 h of treatment. These drugs induced a marked increase in the level of CD95 mRNA and protein in wild-type p53-expressing MCF-7 cells. On the contrary, the breast cancer cell line EVSA-T that expresses high levels of an inactive form of p53, did not up-regulate CD95 upon drug treatment. Elevation of CD95 expression by DNA-damaging drugs was notably blocked in MCF-7 cells expressing the human papillomavirus type 16 E6 protein (E6 cells) which prevented p53 accumulation upon DNA damage. However, E6 cells were still killed by the drugs. Furthermore, the genotoxic drugs did not induce the expression of CD95 ligand in MCF-7 cells at doses that caused apoptosis in these breast tumor cells. Moreover, drug-induced apoptosis of breast tumor cells was not prevented in the presence of either a CD95 antagonistic antibody or a CD95 ligand blocking antibody. We also observed a strong synergism between lower doses of DNA-damaging drugs and CD95 agonistic antibody in the induction of apoptosis in MCF-7 cells. In summary, our data indicate that drug-induced apoptosis of breast tumor cells occurs by a CD95/CD95L-independent mechanism although by elevating the tumor suppressor proteins p53 and CD95, genotoxic drugs may sensitize breast tumor cells to CD95-mediated apoptosis.