Connective tissue growth factor induces apoptosis in human breast cancer cell line MCF-7

Connective tissue growth factor (CTGF) is a member of an emerging CCN gene family that is implicated in various diseases associated with fibro-proliferative disorder including scleroderma and atherosclerosis. The function of CTGF in human cancer is largely unknown. We now show that CTGF induces apoptosis in the human breast cancer cell line MCF-7. CTGF mRNA was completely absent in MCF-7 but strongly induced by treatment with transforming growth factor beta (TGF-beta). TGF-beta by itself induced apoptosis in MCF-7, and this effect was reversed by co-treatment with CTGF antisense oligonucleotide. Overexpression of CTGF gene in transiently transfected MCF-7 cells significantly augmented apoptosis. Moreover, recombinant CTGF protein significantly enhanced apoptosis in MCF-7 cells as evaluated by DNA fragmentation, Tdt-mediated dUTP biotin nick end-labeling staining, flow cytometry analysis, and nuclear staining using Hoechst 33258. Finally, recombinant CTGF showed no effect on Bax protein expression but significantly reduced Bcl2 protein expression. Taken together, these results suggest that CTGF is a major inducer of apoptosis in the human breast cancer cell line MCF-7 and that TGF-beta-induced apoptosis in MCF-7 cells is mediated, in part, by CTGF.     

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Influenza virus neuraminidase activates latent transforming growth factor beta.

Transforming growth factor beta (TGF-beta) is a family of proteins secreted by virtually all cells in a biologically inactive form. TGF-beta levels increase during many pathophysiological situations, including viral infection. The mechanism for increased TGF-beta activity during viral infection is not understood. We observed an increase in active TGF-beta levels within 1 day in mice infected with influenza virus. Further studies showed that the neuraminidase glycoprotein of influenza A and B viruses directly activates latent TGF-beta in vitro. There are sufficient levels of TGF-beta activated by virus to induce apoptosis in cells. In addition, influenza virus-induced apoptosis is partially inhibited by TGF-beta-specific antibodies. These novel findings suggest a potential role for activation of TGF-beta during the host response to influenza virus infection, specifically apoptosis. This is the first report showing direct activation of latent TGF-beta by a viral protein.     

Chemotherapy cytotoxicity of human MCF-7 and MDA-MB 231 breast cancer cells is altered by osteoblast-derived growth factors

One-third of women with breast cancer will develop bone metastases and eventually die from disease progression at these sites. Therefore, we analyzed the ability of human MG-63 osteoblast-like cells (MG-63 cells), MG-63 conditioned media (MG-63 CM), insulin-like growth factor I (IGF-I), and transforming growth factor beta 1 (TGF-beta1) to alter the effects of adriamycin on cell cycle and apoptosis of estrogen receptor negative (ER-) MDA-MB-231 and positive (ER+) MCF-7 breast cancer cells, using cell count, trypan blue exclusion, flow cytometry, detection of DNA fragmentation by simple agarose gel, and the terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method for apoptosis (TUNEL assay). Adriamycin arrested MCF-7 and MDA-MB-231 cells at G2/M phase in the cell cycle and inhibited cell growth. In addition, adriamycin arrested the MCF-7 cells at G1/G0 phase and induced apoptosis of MDA-MB-231 cells. Exogenous IGF-I partially neutralized the adriamycin cytotoxicity/cytostasis of cancer cells. MG-63 CM and TGF-beta1 partially neutralized the adriamycin cytotoxicity of MDA-MB-231 cells but enhanced adriamycin blockade of MCF-7 cells at G1/G0 phase. MG-63 osteoblast-like cells inhibited growth of MCF-7 cells while promoting growth and rescued MDA-MB-231 cells from adriamycin apoptosis in a collagen co-culture system. These data suggest that osteoblast-derived growth factors can alter the chemotherapy response of breast cancer cells. Conceivably, host tissue (bone)-tumor cell interactions can modify the clinical response to chemotherapy in patients with advanced breast cancer.     

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.     

Insulin-like growth factor binding protein-related protein 1 inhibits proliferation of MCF-7 breast cancer cells via a senescence-like mechanism.

Elevated insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) mRNA in senescent human mammary epithelial cells suggested that the IGFBP-3 gene product may inhibit cell proliferation. To test this hypothesis, we used a retroviral vector to express IGFBP-rP1 cDNA in the IGFBP-rP1-deficient MCF-7 breast cancer cell line. Compared with control vector-transduced cells, cumulative cell numbers for IGFBP-rP1-transduced polyclonal or clonal cell cultures were reduced by 39 and 74%, respectively, after 1 week. Medium conditioned by IGFBP-rP1-producing cultures reduced cumulative cell numbers in parental MCF-7 cultures by 20% compared with medium from cultures of a control vector-transduced cell line. Nuclear fragmentation analysis and cell proliferation assays completed in the presence of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone excluded apoptosis as the responsible mechanism. The percentage of cells containing senescence-associated beta-galactosidase activity was doubled compared with control cell cultures. Flow cytometry analysis indicated that twice as many noncycling cells were present in the IGFBP-rP1-transduced MCF-7 cell cultures compared with controls. These findings indicate that IGFBP-rP1 is an inhibitor of MCF-7 breast cancer cell proliferation and may act via a cellular senescence-like mechanism.     

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

Placental transforming growth factor-beta is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression

The p53 tumor suppressor gene and members of the transforming growth factor-beta (TGF-beta) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-beta superfamily, designated placental TGF-beta (PTGF-beta), that is up-regulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-beta is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-beta promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-beta promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-beta overexpression from a recombinant adenoviral vector (AdPTGF-beta) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50-60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-beta overexpression was seen to induce both G(1) cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-beta superfamily and implicate PTGF-beta as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.     

Endogenous aromatization of testosterone results in growth stimulation of the human MCF-7 breast cancer cell line

Estrogens produced within breast tumors may play a pivotal role in growth stimulation of the breast cancer cells. However, it is elusive whether the epithelial breast cancer cells themselves synthesize estrogens, or whether the surrounding tumor stromal cells synthesize and supply the cancer cells with estrogen. The aromatase enzyme catalyzes the estrogen production, aromatizing circulating androgens into estrogens. The aim of this study was to investigate aromatase expression and function in a model system of human breast cancer, using the estrogen responsive human MCF-7 breast cancer cell line. Cells were cultured in a low estrogen milieu and treated with estrogens, aromatizable androgens or non-aromatizable androgens. Cell proliferation, expression of estrogen-regulated proteins and aromatase activity were investigated. The MCF-7 cell line was observed to express sufficient aromatase enzyme activity in order to aromatize the androgen testosterone, resulting in a significant cell growth stimulation. The testosterone-mediated growth effect was completely inhibited by the aromatase inhibitors letrozole and 4-hydroxy-androstenedione. Expression studies of estrogen-regulated proteins confirmed that testosterone was aromatized to estrogen in the MCF-7 cells. Thus, the results indicate that epithelial breast cancer cells possess the ability to aromatize circulating androgens to estrogens.     

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines

"Loss of function" alterations in growth inhibitory signal transduction pathways are common in cancer cells. In this study, we show that growth arrest (GA) treatments--serum and growth factor withdrawal and growth inhibitory IL-6 family cytokines (Interleukin-6 and Oncostatin M (OSM))--increase STAT3 phosphorylation (pSTAT3), increase CCAAT enhancer binding protein delta (C/EBPdelta) gene expression and induce GA of primary, finite-lifespan human mammary epithelial cells (HMECs), and immortalized breast cell lines (MCF-10A and MCF-12A). In contrast, serum and growth factor withdrawal from human breast cancer cell lines (MCF-7, SK-BR-3, T-47D, and MDA-MB-231) for up to 48 h induced a relatively modest increase in pSTAT3 levels and C/EBPdelta gene expression and resulted in varying levels of GA. In most breast cancer cell lines, IL-6 family cytokine treatment increased pSTAT3 levels and C/EBPdelta gene expression, however, growth inhibition was cell line dependent. In addition to "loss of function" alterations in growth inhibitory pathways, breast cancer cell lines also exhibit "gain of function" alterations in growth signaling pathways. The Akt growth/ survival pathway is constitutively activated in T-47D and MCF-7 breast cancer cells. The Akt inhibitor LY 294,002 significantly enhanced T-47D growth inhibition by serum and growth factor withdrawal or IL-6 family cytokine treatment. Finally, we show that activation of the pSTAT3/C/EBPdelta growth control pathway is independent of estrogen receptor status. These results demonstrate that "loss of function" alterations in the pSTAT3/C/EBPdelta growth inhibitory signal transduction pathway are relatively common in human breast cancer cell lines. Defective activation of the pSTAT3/ C/EBPdelta growth inhibitory signal transduction pathway, in conjunction with constitutive activation of the Akt growth stimulatory pathway, may play a synergistic role in the etiology or progression of breast cancer.     

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.     

Reconstitution of TGF-β sensitivity in the VACO-411 human colon carcinoma line by somatic cell fusion with MCF-7

We characterized the mechanism of transforming growth factor beta (TGF-beta) resistance in the VACO-411 human colon carcinoma line. VACO-411 is unique for several reasons, including having a novel mutator phenotype and wild-type p53. Like many colon tumors, VACO-411 is not growth inhibited by TGF-beta. However, VACO-411 represents a subset of colon tumors that are resistant to TGF-beta-mediated growth inhibition, despite the expression of functional TGF-beta receptors. VACO-411 expresses cell surface TGF-beta receptor types I and II, and the coding regions of these receptors are wild type. To further characterize the nature of the VACO-411 defect, we fused VACO-411 with the human breast carcinoma line MCF-7. MCF-7 is also resistant to TGF-beta-mediated growth inhibition. However, unlike VACO-411, MCF-7 lacks cell surface expression of TGF-beta receptor type II, but does contain an intact postreceptor signaling pathway, as shown by regeneration of TGF-beta sensitivity following wild-type TGF-beta receptor type II transfection. In contrast to parental VACO-411 and MCF-7, the morphologically distinct cell hybrids were growth inhibited by TGF-beta. Therefore, the TGF-beta defect in VACO-411 is a postreceptor, loss-of-function mutation which can be genetically complemented. The data suggest that the VACO-411 defect in TGF-beta signaling will be able to be further complemented by microcell-mediated chromosome transfer.     

Purification and characterization of an alpha 1-antichymotrypsin-like 66 kDa protein from the human breast cancer cell line, MCF-7.

The synthesis of a 66 kDa protein immunoreactive with antibodies to human alpha 1-antichymotrypsin (alpha 1-ACT) is induced by estradiol (E2) in the human breast cancer cell line MCF-7. We have purified this alpha 1-ACT-like 66 kDa protein from medium conditioned by MCF-7 cells, performed a comparative physico-chemical characterization with serum alpha 1-ACT, and analysed its presumed positive regulatory effect on growth of MCF-7 cells. The 66 kDa protein is a functional antiproteinase which is antigenically identical to serum alpha 1-ACT. The 66 kDa protein does however deviate from serum alpha 1-ACT with respect to mol. wt. and pattern of microheterogeneity, the molecular mechanism for this is probably an incomplete glycoprotein processing in the MCF-7 cells. The results of our growth experiments suggest that the 66 kDa protein is a minor positive growth regulatory factor, which may contribute to breast carcinoma cell proliferation in a cooperative manner.     

A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo

MCF-7 human breast cancer cells provide a useful in vitro model system to study hormone-responsive breast cancer as they contain receptors for estrogen and progesterone, and estrogen both induces the synthesis of specific proteins in these cells and increases their rate of proliferation. An MCF-7 cell line which was selected for resistance to adriamycin (MCF-7/AdrR) exhibits the phenotype of multidrug resistance (MDR), and displays multiple biochemical changes. MDR in MCF-7/AdrR is also associated with a loss of mitogenic response to estrogen and the development of cross-resistance to the antiestrogen 4-hydroxytamoxifen. In addition, while the parental MCF-7 cell line responds to estrogen with increased levels of progesterone receptors and the secretion of specific proteins, these estrogen responses are lost in MCF-7/AdrR. Furthermore, while the formation of tumors in nude mice by wild-type MCF-7 cells is dependent upon the presence of estrogen, MCF-7/AdrR cells form tumors in the absence of exogenous estrogen administration. These changes in hormonal sensitivity and estrogen-independent tumorigenicity of the multidrug-resistant MCF-7 cell line are associated with a loss of the estrogen receptor and a concomitant increase in the level of receptors for epidermal growth factor. Thus, in MCF-7/AdrR cells, the development of MDR is associated with alterations in the expression of both cytosolic and membrane receptors, resulting in resistance to hormonal agents and the expression of hormone-independent tumor formation.     

Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene

The effect of isoflavones on the growth of the human breast carcinoma cell lines, MDA-468 (estrogen receptor negative), and MCF-7 and MCF-7-D-40 (estrogen receptor positive), has been examined. Genistein is a potent inhibitor of the growth of each cell line (IC50 values from 6.5 to 12.0 micrograms/ml), whereas biochanin A and daidzein are weaker growth inhibitors (IC50 values from 20 to 34 micrograms/ml). The isoflavone beta-glucosides, genistin and daidzin, have little effect on growth (IC50 values greater than 100 micrograms/ml). The presence of the estrogen receptor is not required for the isoflavones to inhibit tumor cell growth (MDA-468 vs MCF-7 cells). In addition, the effects of genistein and biochanin A are not attenuated by overexpression of the multi-drug resistance gene product (MCF-7-D40 vs MCF-7 cells).