Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene, MRG

A novel human tumor growth inhibitor was identified by differential cDNA sequencing. The predicted amino acid sequence of this tumor-suppressing factor has a significant sequence homology to mouse mammary-derived growth inhibitor and thus was named mammary-derived growth inhibitor-related gene (MRG). MRG was found to be expressed in normal and benign human breast tissues but not in breast carcinomas. In situ hybridization analysis demonstrated a stage-specific MRG expression as follows. MRG was barely detectable in breast carcinomas, showed partial and weak expression in benign hyperplasia, but was expressed at a high level in normal breast epithelial cells. To determine if MRG can modulate in vivo growth of human breast cancers, we transfected a full-length MRG cDNA into MDA-MB-231 human breast cancer cells and studied the orthotopic growth of MRG transfectants versus control transfectants in the mammary fat pad of athymic nude mice. Overexpression of MRG in human breast cancer cells significantly suppressed cell proliferation in vitro and tumor growth in an orthotopic nude mouse model. These results suggest that MRG has tumor-suppressing activity, and the loss of MRG expression may be involved in the development and progression of breast cancer.     

Interleukin 4 inhibits growth and induces apoptosis in human breast cancer cells

Interleukin-4 (IL-4) is a pleiotropic cytokine produced by mast cells and T lymphocytes that promotes proliferation and immunoglobulin class-switching in B cells. IL-4 receptors (IL-4Rs) are also expressed by nonhematopoietic cells as well as some tumor cells. Unlike its mitogenic effect on B cells, IL-4 inhibits the growth of some cancer cells in vitro. In this study, we show that IL-4R is expressed by breast and ovarian cancer cell lines. Furthermore, anchorage-dependent and -independent growth of breast cancer cell lines MCF-7 and MDA-MB-231 is inhibited by IL-4 treatment, and this effect requires IL-4R. Interestingly, IL-4 only inhibited proliferating breast cancer cells and had no effect on basal, unstimulated growth. We therefore characterized the effect of IL-4 on breast cancer cell growth stimulated by either estradiol or insulin-like growth factor I (IGF-I). In both anchorage-dependent and -independent growth assays, IL-4 inhibited estradiol-stimulated growth. The antiestrogen effect of IL-4 was not due to IL-4 interference with the estrogen receptor, because IL-4 did not interfere with estrogen receptor-mediated reporter gene transactivation. In contrast, IL-4 had no effect on IGF-I-stimulated proliferation. Because IGF-I is known to inhibit programmed cell death, we examined apoptosis as a possible mechanism of IL-4 action. We established that IL-4 induced apoptosis in breast cancer cells by five independent criteria: (a) morphological indicators including pyknotic nuclei and cytoplasmic condensation; (b) DNA fragmentation; (c) the formation of DNA laddering; (d) the cleavage of poly(ADP-ribose) polymerase; and (e) the presence of cells with sub-G1 DNA content. IL-4 increased the percentage of apoptotic cells in MCF-7 and MDA-MB-231 cells 6.0- and 6.7-fold over that of the control, respectively. Finally, the addition of IGF-I reversed IL-4-induced apoptosis, suggesting that the mechanism of IL-4-induced growth inhibition in human breast cancer cells is the induction of programmed cell death.     

mda-7 (IL-24) Inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling

Despite therapeutic interventions including surgery, chemotherapy and radiotherapy, glioblastoma multiforme (GBM) has a very poor prognosis and novel therapies are required. MDA-7 (IL-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound anti-proliferative and cytotoxic effects in a variety of tumor cells, but not in non-transformed cells. The present studies examined the combined impact of Ad.mda-7 and ionizing radiation on the proliferation and survival of GBM cells. Ad.mda-7 reduced the proliferation of rodent and human glioma cells in MTT assays and in colony formation assays. The anti-proliferative effects of Admda-7 were enhanced by radiation in a greater than additive fashion. In vitro, this cellular change correlated with enhanced cell numbers in G1/G0 and G2/M phases of the cell cycle, implying Ad.mda-7 radiosensitizes tumor cells in a cell cycle-independent manner. The radiosensitizing effects were not observed in cultures of non-transformed primary astrocytes. The enhanced reduction in growth correlated with increased necrosis and DNA degradation. Ad.mda-7 enhanced p38 and ERK1/2 activity but did not alter JNK or Akt activity. Irradiation of cells expressing MDA-7 suppressed ERK1/2 activity and dramatically enhanced JNK1/2 activity without altering either Akt or p38 activity. Inhibition of JNK1/2, but not p38, signaling abolished the radiosensitizing properties of MDA-7. Inhibition of neither ERK1/2 nor PI3K signaling enhanced the anti-proliferative effects of Ad.mda-7, whereas combined inhibition of both pathways enhanced cell killing, suggesting that ERK and PI3K signaling can be protective against MDA-7 lethality.     

Ligands for peroxisome proliferator-activated receptorgamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice

Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel and promising approach to cancer therapy. All-trans-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers. The NHR, peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in breast cancer cells. Activation of PPARgamma through a synthetic ligand, troglitazone (TGZ), and other PPARgamma-activators cause inhibition of proliferation and lipid accumulation in cultured breast cancer cells. TGZ (10(-5) M, 4 days) reversibly inhibits clonal growth of MCF7 breast cancer cells and the combination of TGZ (10(-5) M) and ATRA (10(-6) M, 4 days) synergistically and irreversibly inhibits growth and induces apoptosis of MCF7 cells, associated with a dramatic decrease of their bcl-2 protein levels. Similar effects are noted with in vitro cultured breast cancer tissues from patients, but not with normal breast epithelial cells. The observed apoptosis mediated by TGZ and ATRA may be related to the striking down-regulation of bcl-2, because forced over-expression of bcl-2 in MCF7 cells cultured with TGZ and ATRA blocks their cell death. TGZ significantly inhibits MCF7 tumor growth in triple immunodeficient mice. Combined administration of TGZ and ATRA causes prominent apoptosis and fibrosis of these tumors without toxic effects on the mice. Taken together, this combination may provide a novel, nontoxic and selective therapy for human breast cancers.     

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.     

Transforming growth factor beta 1 can induce estrogen-independent tumorigenicity of human breast cancer cells in athymic mice

We have examined the effect of transforming growth factor beta 1 (TGF-beta 1) overexpression in human breast cancer cell tumorigenicity in athymic mice. Estrogen-dependent MCF-7 cells were stably transfected with pSVTGF beta 1. A clone was isolated which overexpressed TGF-beta 1 mRNA and secreted > 10-fold more TGF-beta activity into the tissue culture medium. Similar to the parent line, the MCF-7/TGF-beta 1 cells were relatively insensitive to exogenous TGF-beta 1 and exhibited low levels of TGF-beta receptors. Clonogenicity in soft agarose, doubling time, morphology, and sensitivity to 17 beta-estradiol and the antiestrogen tamoxifen were not altered in the transfected cells. Inoculation s.c. of MCF-7/TGF-beta 1 cells in ovariectomized nude mice resulted in 100% tumor formation which was totally abrogated by i.p. administration of the neutralizing anti-TGF-beta 2G7 IgG2B. The parent cells formed tumors only after estrogen supplementation. By immunohistochemistry, higher levels of TGF-beta 1 protein were detected in MCF-7/TGF-beta 1 tumors than in estrogen-induced parent MCF-7 tumors. Administration of 1 microgram TGF-beta 1 i.p. daily for 3 weeks after tumor cell inoculation transiently supported estrogen-independent growth of parent MCF-7 tumors in castrated nude mice. These data indicate that overexpression of TGF-beta 1 in human breast cancer cells can contribute to their escape from hormone dependence.     

Recombinant PML adenovirus suppresses growth and tumorigenicity of human breast cancer cells by inducing G1 cell cycle arrest and apoptosis

Our previous studies demonstrated that the promyelocytic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing effect in breast cancer cells were examined. We showed that Ad-PML effectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post-infection and a detectable level remained at day 16. Ad-PML significantly suppressed the growth rate, clonogenicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the effect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a significant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphorylated 48 h post-infection. These findings indicate that PML exerts its growth suppressing effects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppressing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy.     

Effect of NCAM-transfection on growth and invasion of a human cancer cell line

A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.     

Risk and preventive factors for cot death in The Netherlands, a low-incidence country

BACKGROUND: The p53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-type p53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-type p53 tumor suppressor gene. METHODS: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/beta-gal. Cell proliferation was monitored using a 3H-thymidine incorporation assay, Western blot analysis for p53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis. p53 gene therapy was tested in vivo in a subcutaneous tumor model. RESULTS: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (beta-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the beta-gal gene. Adenovirus-mediated p53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. CONCLUSIONS: Introduction of the wild-type p53 gene using an adenoviral vector in pancreatic cancer with p53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediated p53 tumor suppressor gene therapy of human pancreatic cancer.     

Dietary fat and breast cancer metastasis by human tumor xenografts

Human breast cancer cell lines growing as xenografts in athymic nude mice have been used to examine the effects of dietary fat and fatty acids on tumor progression. The estrogen independent MDA-MB-435 cell line has the advantage that it metastasizes consistently to the lungs and forms quantifiable secondary nodules when injected into the mammary fat pads. With these breast cancer cells, the stimulating effects of polyunsaturated omega-6 fatty acids on both primary tumor growth and metastasis were demonstrated; in contrast, the long-chain omega-3 fatty acids were inhibitory. The model can also be adapted to examine dietary fatty acids, and inhibitors of their metabolism, as experimental adjuvant therapy after surgical excision of the primary tumors. Unfortunately, estrogen dependent human breast cancer cells do not metastasize, or do so rarely, in nude mice; in consequence, it is not possible to use the model to study estrogen-fatty acid interactions on the metastatic process. In addition to metastasis from a primary location, intravenous injection of MDA-MB-435 cells into the nude mouse host, particularly when combined with studies using Matrigel-based in vitro invasion assays, permits further dissection of the steps in the metastatic cascade which are influenced by dietary fatty acids. The results obtained by these several approaches have demonstrated distinct roles for the cyclooxygenase and lipoxygenase-mediated products of omega-6 fatty acid metabolism, and suggest new approaches to experimental breast cancer therapy.     

H-cadherin expression inhibits in vitro invasiveness and tumor formation in vivo

H-cadherin is a newly characterized cadherin molecule whose expression is decreased in a variety of human carcinoma cells, suggesting that it may play a role in maintaining normal cellular phenotype. To investigate how re-expression of H-cadherin could influence the malignant phenotype of human breast carcinoma cells in vivo, we transfected both control and H-cadherin expression vectors into human breast cancer cells (MDAMB435), which do not express H-cadherin constitutively. We found that invasiveness of these cells could be prevented by transfection with H-cadherin. We also compared the ability of control- and H-cadherin-transfected cells to induce subcutaneous tumors after injection into mammary fat pads of nude mice. Our results show that H-cadherin transfection produced a marked inhibition of tumor growth and modified the morphology of tumor cells: tumors from mice injected with control cells were significantly larger and contained larger cells having a higher degree of pleomorphism than those of tumors generated from carcinoma cells expressing H-cadherin. Altogether, these results indicate that H-cadherin expression antagonizes tumor growth in nude mice, presumably by enhancing cell-cell association in a tissue environment. These findings strongly suggest that H-cadherin could provide a possible target for corrective gene therapy against breast cancer.     

Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-based in vitro assays

In vitro analyses of basement membrane invasiveness employing Matrigel (a murine tumor extract rich in basement membrane components) have been performed on human breast cancer model systems. Constitutive invasiveness of different human breast cancer (HBC) cell lines has been examined as well as regulation by steroid hormones, growth factors, and oncogenes. Carcinoma cells exhibiting a mesenchymal-like phenotype (vimentin expression, lack of cell border associated uvomorulin) show dramatically increased motility, invasiveness, and metastatic potential in nude mice. These findings support the hypothesis that epithelial to mesenchymal transition (EMT)-like events may be instrumental in the metastatic progression of human breast cancer. The MCF-7 subline MCF-7ADR appears to have undergone such a transition. The importance of such a transition may be reflected in the emergence of vimentin expression as an indicator of poor prognosis in HBC. Matrix degradation and laminin recognition are highlighted as potential targets for antimetastatic therapy, and analyses of laminin attachment and the matrix metalloproteinase (MMP) family in HBC cell lines are summarized. Matrigel-based assays have proved useful in the study of the molecular mechanisms of basement membrane invasiveness, their regulation in HBC cells, and their potential as targets for antimetastatic therapy.     

Inhibitory effects of ginsenoside Rh2 on tumor growth in nude mice bearing human ovarian cancer cells

Ginsenoside Rh2 (Rh2), isolated from an ethanol extract of the processed root of Panax ginseng CA Meyer, inhibits the growth of B16 melanoma cells. This study was designed to evaluate the ability of Rh2 to inhibit growth of human ovarian cancer cells (HRA) in vitro and in nude mouse. Rh2 inhibited proliferations of various established human ovarian cancer cell lines in a dose-dependent manner between 10 and 60 microM in vitro and induced apoptosis at around the IC50 dose. When HRA cells were inoculated s.c. into the right flank of nude mice, all mice formed a palpable tumor within 14 days. Although i.p. administration of Rh2 alone hardly inhibited the tumor growth, when Rh2 was combined with cis-diamminedichloroplatinum(II) (CDDP) the tumor growth was significantly inhibited, compared to treatment with CDDP alone. When mice were treated p.o. with Rh2 daily (but not weekly), the tumor growth was significantly (P<0.01) inhibited, compared to CDDP treatment alone. When Rh2 was combined with CDDP, the degree of tumor growth retardation was not potentiated. The survival time was significantly (P<0.05) longer than that of medium alone-treated controls or the group treated with CDDP alone. Then, we examined whether p.o. administration of Rh2 has a dose-dependent inhibitory effect on the tumor growth. I.p. and weekly administration of CDDP had more potent antitumor activity in the order of 1 mg/kg, 2 mg/kg and 4 mg/kg, whereas p.o. and daily administration of Rh, (0.4 to 1.6 mg/kg) not only had antitumor activity comparable to that of 4 mg/kg CDDP, but also resulted in a significant increase of the survival. Doses of Rh2 used in this study did not result in any adverse side-effects as confirmed by monitoring hematocrit values and body weight, unlike 4 mg/kg CDDP, which had severe side-effects. It is noteworthy that p.o. but not i.p. treatment with Rh2 resulted in induction of apoptotic cells in the tumor in addition to augmentation of the natural killer activity in spleen cells from tumor-hearing nude mice. Thus, particularly in view of the toxicity of CDDP, Rh2 alone would seem to warrant further evaluation for treatment of recurrent or refractory ovarian tumor.     

Aromatase expression and its localization in human breast cancer

Aromatization or in situ estrogen production by aromatase has been considered to play an important role in the development of human breast carcinoma. In the human breast, aromatase overexpression is observed in the stromal or interstitial cells of the carcinoma, especially at the sites of frank invasion and/or adipose tissue. Transplantation experiments in the nude mouse employing MCF-7 and/or SF-TY human fibroblast cell lines revealed that aromatase activity and expression were much higher in the tumour with MCF-7 and SF-TY than that with MCF-7 alone. Aromatase overexpression in human breast carcinoma tissue is considered to occur as a result of carcinoma-stromal cell interactions, i.e. paracrine communication between stromal and carcinoma cells. Aromatase overexpression is correlated with the malignant phenotype in the human breast, but not with stage, age, clinical stages, clinical course, or proliferative activity of breast carcinoma. Aromatase overexpression may be correlated with development, rather than the biological behaviour of breast malignancy. Aromatase overexpression is not necessarily correlated with expression of 17beta-hydroxysteroid dehydrogenase type 1, which converts estrone to estradiol and estrogen receptor. Different mechanisms may be involved in the regulation of expression of these two important estrogen-metabolizing enzymes and estrogen receptor in human breast cancer. Aromatase overexpression in intratumoral stromal cells was much more frequently detected in male breast cancer than in female counterparts, which confers a growth advantage on cancer cells in a male hormonal environment with low serum estrogen levels.     

Breast cancer increases initiation of angiogenesis without accelerating neovessel growth rate

BACKGROUND: Recurrence and mortality rates in patients with breast cancer correlate with the degree of tumor angiogenesis (angiogenic index). We have developed a novel angiogenesis model by using disks of fresh human placental vein that initiate an angiogenic response and exhibit linear radial capillary growth in culture. We hypothesized that the addition of human breast cancer cells to this human placental vein angiogenesis model would increase the incidence of angiogenesis and accelerate the rate of neovessel growth compared with vein disk cultured without tumor cells. METHODS: To test this hypothesis, vein explants from seven human placentas were incorporated into clots of 0.3% fibrin in Medium 199 and fetal bovine serum with or without 1.5 x 10(5) T-47D (n = 6 placentas) or MCF-7 (n = 1 placenta) breast cancer cells. Statistical differences between the experimental (with breast cancer cells) and control (no added cells) cultures were determined by repeated measures ANOVA. RESULTS: The proportion of disks exhibiting neovessel growth (initiation) by day 12 was significantly increased in the presence of T-47D cells (p < 0.05 at day 12, p < 0.001 at day 15). No statistical difference was seen in rates of neovessel growth (millimeters per day). Similar results were seen with MCF-7 cells. CONCLUSIONS: Tumor enhancement of angiogenesis may occur by increased initiation of the angiogenic response. Subsequent vessel growth rates may be tumor independent. We predict that effective antiangiogenic therapies will block a tumor's ability to augment angiogenesis initiation rather than subsequent neovessel growth.     

Inhibition of human squamous cell carcinoma growth in vivo by epidermal growth factor receptor antisense RNA transcribed from the U6 promoter

BACKGROUND: Squamous cell carcinomas of the head and neck (SCCHN), unlike normal mucosal squamous epithelial cells, overexpress epidermal growth factor receptor (EGFR) messenger RNA and protein. EGFR protein is required to sustain the proliferation of SCCHN cells in vitro. To determine whether EGFR expression contributes to tumor growth, we investigated the effect of suppressing EGFR expression in tumor xenografts through in situ expression of antisense oligonucleotides. METHODS: Intratumoral cationic liposome-mediated gene transfer was used to deliver plasmids capable of expressing sense or antisense EGFR sequences into human head and neck tumors, which were grown as subcutaneous xenografts in nude mice. The oligonucleotides were expressed under the control of the U6 RNA promoter. RESULTS: Direct inoculation of the EGFR antisense (but not the corresponding sense) plasmid construct into established SCCHN xenografts resulted in inhibition of tumor growth, suppression of EGFR protein expression, and an increased rate of apoptosis (programmed cell death). Sustained antitumor effects were observed for up to 2 weeks after the treatments were discontinued. CONCLUSION: These results suggest that interference with EGFR expression, using an antisense-based gene therapy approach, may be an effective means of treating EGFR-overexpressing tumors, including SCCHN.