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PubMed1998

Inhibition of stromal matrix metalloproteases: effects on breast-tumor promotion by fibroblasts.

Co-injection of fibroblasts with human epithelial breast-tumor MCF7 cells in the presence of Matrigel enhances tumor growth in nude mice. While most of the matrix metalloproteinases (MMPs) have been shown to be produced by stromal cells, tumor cells such as MCF7 cells are unable to produce MMPs. We therefore, hypothesized that the tumor-promoting effect of fibroblasts could be related to their production of MMPs. In order to inhibit stromal proteases, over-production of TIMP-2 was induced in MCF7 cells by in vitro retroviral-mediated gene transfer. TIMP-2-producing MCF7 cells were then co-injected with fibroblasts into nude mice. Alternatively, we evaluated the effect of Batimastat, a synthetic inhibitor of MMPs, on the tumorigenicity of MCF7 cells co-inoculated with fibroblasts into nude mice. Both physiological (TIMP-2) and synthetic (Batimastat) inhibitors of MMPs were able to abolish the tumor-promoting effect of fibroblasts. On the contrary, they failed to modulate the tumorigenicity of MCF7 cells injected alone. Interestingly, Matrigel from which low-molecular-weight proteins or growth factors had been removed failed to favor the tumorigenicity of MCF7 cells inoculated with fibroblasts. These findings emphasize the importance of fibroblasts in cancer progression, and suggest that their role could be related at least in part to production of proteases which can induce the release of factors from the extracellular matrix.

Adenocarcinoma↗

PubMed1999

Enhanced cell motility and invasion of chicken embryo fibroblasts in response to Jun over-expression.

Malignant tumor cells exhibit a number of distinct properties involved not only with deregulated cell proliferation but also enhanced migration and invasion. The Jun oncogene has been well studied in regard to its role in cell proliferation. Many of the target genes deregulated by Jun encode matrix metalloproteases (MMPs) such as MMP1, MMP3 and MMP9. These targets implicate a prominent role for Jun in tumor cell invasion, in addition to its role in growth transformation. To investigate this possibility, we have examined the effect of over-expression of transforming and non-transforming versions of Jun on motility and invasion of chicken embryo fibroblasts (CEFs). We found that over-expression of either form of Jun results in elevated intrinsic cellular motility as well as increased motility in response to several different chemo-attractants, including 3T3-conditioned media, basic fibroblast growth factor, hepatocyte growth factor and Matrigel. The capacity of these cells to invade through Matrigel is also elevated as a result of Jun over-expression. In addition to these effects, CEFs expressing Jun secrete factors that stimulate the motility of a human tongue carcinoma cell line. Our results suggest that Jun plays an important role in the potentiation of cell motility and invasion through multiple mechanisms.

3T3 Cells↗

PubMed1996

Dexamethasone regulation of marrow stromal-derived osteoblastic cells.

The clonal subtypes of cells in the osteogenic family represented by fibroblastoid MBA-15.33, preosteoblast MBA-15.4, and mature osteoblastic MBA-15.6 cells were used to study the effects of glucocorticoid (dexamethasone). The role of dexamethasone was monitored on cell attachment when plated on various protein substrata (BSA, collagen 1, and Matrigel). A 24 h exposure of the cells to 10(-6) M or 10(-7) M dexamethasone differential affects their attachment preference. MBA-15.33 and MBA-15.4 cells increased their attachment capability on collagen 1, while MBA-15.6 cells' attachment was inhibited. Pretreatment with (10(-6) M) dexamethasone caused an increase in attachment on Matrigel by MBA-15.33 cells and to less extent by MBA-15.4 cells. Additionally, measurements of two enzymatic activities were monitored; one is alkaline phosphatase (ALK-P), and the second is neutral endopeptidase (CD10/NEP). MBA-15.33, MBA-15.4, and MBA-15.6 cells were exposed to dexamethasone or to various growth factors (bone morphogenic protein (BMP-2 and BMP-3), TGF beta, and IGF-1). In some experiments, pretreatment of cells by dexamethasone was followed by exposure to the growth factors. The cells' challenged cellular responses were not uniform and revealed a differential pattern when their ALK-P and CD10/NEP enzymatic activities were measured.

Animals↗

PubMed1996

Effects of the carbohydrate-binding protein galectin-3 on the invasiveness of human breast carcinoma cells.

Galectin-3 is a Mr 30,000 protein with carbohydrate-binding specificity for type I and II ABH blood group epitopes and polylactosamine glycans expressed on cell surface and extracellular matrix glycoproteins such as laminin. Cell lines propagated from human normal mammary epithelia and from benign or infiltrating components of primary breast tumours express low levels of galectin-3 in the cytoplasm. However, galectin-3 when added exogenously in solution or when bound within a three-dimensional matrix markedly enhanced the migration of the primary tumour cell lines through a Matrigel barrier. Galectin-3 expression in the cytoplasm and intercellularly on surface membranes was greatly increased in cell lines propagated from malignant ascites and pleural effusions of late stage breast cancer. These cell lines were non-invasive in the Matrigel assay and exogenous galectin-3 had no enhancing effect on invasiveness. These results suggest that galectin-3 could play multiple roles in cell metastasis at an early invasive stage by acting in a paracrine manner to stimulate cell migration through an extracellular matrix, and in later stage cancers in synergy with other mediators of cell-cell aggregation. However, endogenous galectin-3 expression in human breast cancers is not correlated directly with their invasive potential in vitro.

Antigens, Differentiation↗

PubMed1998

Induction of tyrosine aminotransferase of primary cultured rat hepatocytes depends on the organization of microtubules.

We investigated the relationship between the expression of tyrosine aminotransferase (TAT) and cytoskeletal systems of cultured rat hepatocytes by using serum-free culture conditions and changing three factors: 1) the concentration of calcium, 2) the dish-coating material, and 3) the cell-plating density. In hepatocytes in low-calcium medium, induction of TAT by dexamethasone and glucagon was maintained, although cell-cell adhesion was lost. Hepatocytes on Matrigel formed a nonspreading, spherical shape that provided them with the full extent of TAT activity without cell-cell adhesion. Hepatocytes plated on collagen at low cell density spread and changed shape, and the induction of TAT activity was markedly reduced. By using confocal laser-scanning microscopy, we analyzed the three-dimensional organization of cytoplasmic microtubules of hepatocytes maintaining the ability of TAT induction. Hepatocytes plated on collagen at low cell density possessed the radial filamentous structure of cytoplasmic microtubules. When the spherical shape of hepatocytes was maintained by cultivating cells on Matrigel, a ring-like structure of cytoplasmic microtubules beneath the plasma membrane was dominant. Moreover, the induction of TAT activity of hepatocytes in a standard culture system was strongly inhibited by the addition of 1 microM colchicine. These studies suggest that the organization of cytoplasmic microtubules may participate in the shape-related regulation of cell function.

Actins↗

PubMed1998

Virally activated Ras cooperates with integrin to induce tubulogenesis in sinusoidal endothelial cell lines.

Four cell lines, named nonparenchymal 11 (NP11), NP26, NP31, and NP32, were established from sinusoidal endothelial cells (SECs) of rat liver. They still retained expression of receptors for vascular endothelial growth factor (VEGF), Fit-1, and kinase domain-containing receptor (KDR). NP31 and NP32 turned out to be incapable of tubulogenesis in basement membrane matrix (Matrigel), which belongs to endothelial properties, as shown by SECs in primary culture. Expression of temperature-sensitive, virally activated Ras (ts-v-Ras) restored tubulogenic behaviors back to NP31 only at permissive temperature. Matrigel induced long-lasting tyrosine phosphorylation of Shc, with recruitment of Grb-2 and microtubule-associated protein kinase (MAPK) activation in both parental NP31 and NP31 transformed by ts-v-Ras, which was blocked by anti-beta1 integrin antibody. Tubulogenesis was inhibited by adenovirus-mediated expression of dominant-negative Ras in human umbilical vein endothelial cells (HUVECs). PD 098059, a selective inhibitor of MAPK kinase (MEK), nearly perfectly blocked tubulogenesis by ts-v-Ras-expressing NP31 cells at permissive temperature. Furthermore, the botulinum C3 toxin, an inhibitor for Rho, caused fragmentation of branching cords in networks formed by NP31 that expressed ts-v-Ras at permissive temperature. These data suggest that the integrin-mediated Ras signals may be necessary but are not sufficient for tubulogenesis and that an artificial expression of v-Ras might substitute for the second signal required in this system.

ADP Ribose Transferases↗

PubMed1996

Different metastatic potentials of ras- and src-transformed BALB/c 3T3 A31 variant cells.

The metastatic phenotype of tumor cells is thought to be induced by an aberrant signaling cascade or cascades that are different from those required for tumorigenicity. Oncogene-transfected cells with different tumorigenicities and metastatic potentials have been used to identify such pathways and responsible molecules. However, oncogenes that can induce tumorigenicity in recipient cells also frequently induce the metastatic phenotype at the same time. The difficulty in obtaining cell lines that are tumorigenic but not metastatic has hampered such studies. In this report, we transfected the activated c-Ha-ras oncogene into BALB/c 3T3 A31 variant cells and found that the transfectants were tumorigenic but they did not form metastatic lung modules in the experimental metastasis assay. The phenotype was very stable and was maintained during cultivation. On the other hand, the metastatic potentials of either the transfected cells or the original variant cells could be induced by transfection of the v-src oncogene. The src transfectants formed extensive nodules in lung when injected into the tail veins of congeneric mice. The cell motility of the metastatic src transfectants on Matrigel-coated dishes was greater than that of the ras transfectants. The src transfectants were also invasive in Matrigel when analyzed on a filter. These variant cells transformed by the ras and src oncogenes will be a useful system for identifying the signaling cascades responsible for the metastatic potential of tumors.

3T3 Cells↗

PubMed1999

Dynamics of prostate cancer cell invasion studied in vitro by NMR microscopy.

Understanding the dynamics and pathogenesis of invasion is vital for developing strategies to prevent cancer metastasis. Conventional invasion assays provide information for a single time point. NMR microscopic imaging as used in the current study to measure cell invasion in vitro provides a nondestructive method for scoring cell invasion thus offering a unique possibility to study this process dynamically. An additional advantage is that cells can be retrieved for metabolic and physiological characterization. Two prostate cancer cell lines, DU-145 and Mat-Ly-Lu, preselected for differences in invasive behavior, were studied. Cells were seeded in 12-mm culture plate inserts containing a 15-microm-thick porous membrane with 3.0 microm pore size that was coated with a 100 microm Matrigel layer. Cell invasion in the Matrigel layer was obtained from the profile of intracellular water measured with diffusion-weighted 1D imaging. Additional experiments were also performed with confocal microscopy to validate the NMR results. Significant differences were detected between the invasive behavior of DU-145 and Mat-Ly-Lu cells. The obtained results show that NMR microscopy can be used to dynamically study invasion by cancer cells. The noninvasive nature of NMR microscopy permits determination of cell migration dynamically for any given sample, which is especially important if cell availability is limited to the unique sample, such as for biopsy specimens. Magn Reson Med 42:277-282, 1999.

Animals↗

PubMed2000

Urokinase-type plasminogen activator and its receptor in colorectal cancer: independent prognostic factors of metastasis and cancer-specific survival and potential therapeutic targets.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plasminogen (Plg), and plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) have been observed in many cancers and may contribute to progression and metastasis. In our study, we examined the expression of the 5 proteins by immunohistochemistry in 59 consecutive primary colorectal cancers (CRC) and correlated the protein expression with patient outcome. In addition, we determined the effect of down-regulation of uPAR on the invasive/metastatic capability of CRC cells, by measuring antisense-uPAR transfected HCT116 and control cell lines, in terms of uPAR expression, uPA-binding activity, invasiveness through Matrigel in vitro and metastasis after cecal orthotopic implantation in nude mice in vivo. We found that higher expression of uPA or uPAR in primary tumor tissues was positively correlated with distant metastasis of CRC (Mann-Whitney, p < 0.02) and negatively correlated with both patient overall survival (OS) and cancer-specific survival (CSS; Cox model, p < 0.04). The prognostic value of uPA and uPAR for both OS and CSS was independent of other variables (multivariate Cox model, p < 0. 007). Antisense-uPAR transfected HCT116 cells, which expressed significantly lower levels of total cellular and cell surface uPAR proteins and uPA-binding activity compared with either wild-type or cells transfected with vector alone (Bonferroni, p < 0.05/3), consistently showed decreased invasiveness through Matrigel (Bonferroni, p < 0.05/3) and decreased metastasis formation in nude mice (Fisher, p < 0.05). Our data suggest that uPAR and uPA are independent prognostic factors in CRC; anti-uPAR treatment, which affects both uPAR and uPA levels, may have potential for new treatment of the disease.

Adult↗

PubMed2000

Down-regulation of N-acetylglucosaminyltransferase V by tumorigenesis- or metastasis-suppressor gene and its relation to metastatic potential of human hepatocarcinoma cells.

The effects of transfection of the metastasis suppressor gene nm23-H1 and cell-cycle related tumor-suppressor gene p16 on the activity of N-acetylglucosaminyltransferase V (GnT-V) and their relations to cancer metastatic potential were investigated. After transfection of nm23-H1 into 7721 human hepatocarcinoma cells and A549 human lung cancer cells, the activities of GnT-V were decreased by 28%-42% in the cells. In contrast, when p16 was transfected into these two cell lines, the decrease of GnT-V activity was only observed in A549 cells. This was probably to be due to the obvious expression of p16 gene in parental 7721 cells and the deletion of p16 in A549 cells. The decrease of GnT-V mRNA was only observed in nm23-H1-transfected cells, but not in p16-transfected A549 cells, suggesting that these two genes regulated GnT-V via different mechanisms. Horseradish peroxidase (HRP)-lectin staining showed that the 7721 cells transfected with nm23-H1 or the A549 cells transfected with p16 displayed a decreased intensity with HRP-leucoagglutinating phytohemagglutinin and increased intensity with HRP-concanavalin A, indicating the decline of beta1,6 N-acetylglucosamine branching structure on the asparagine-linked glycans of cell-surface and intracellular glycoproteins. The nm23-H1 transfected 7721 cells also displayed some changes in metastasis-related phenotypes, including the increase in cell adhesion to fibronectin (Fn), the decline in cell adhesion to laminin (Ln), and the decreased cell migration and invasion through matrigel. Transfection of antisense GnT-V cDNA into 7721 cells resulted in a decrease of GnT-V activity, an increase of cell adhesion to Fn or Ln, and a decrease in cell migration and invasion through matrigel. These phenotypes bore similarity to those of the 7721 cells transfected with nm23-H1. Our findings indicate that the down-regulation of GnT-V by nm23-H1 contributes to the alterations in metastasis-related phenotypes, and is an important molecular mechanism of metastasis suppression mediated by nm23-H1.

Adenocarcinoma↗

PubMed2000

Antiangiogenesis efficacy of nitric oxide donors.

Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis-mediated disorders. Because nitric oxide (NO) is known to play a key role in various vascular diseases, the present study was undertaken to determine the role of NO in angiogenesis-mediated processes using the NO donor, S-nitroso N-acetyl penicillamine (SNAP) and S-nitroso N-acetyl glutathione (SNAG). The antiangiogenic efficacy of these NO donors was examined using in vivo and in vitro model systems. The in vitro studies demonstrated the ability of SNAP to inhibit cytokine fibroblast growth factor (FGF2)-stimulated tube formation and serum-induced cell proliferation. The inhibitory effect on cell proliferation by SNAP concentrations above the millimolar range was associated with significant shifts in the concentration of NO metabolites. Furthermore, using the mouse Matrigel implant model and the chick chorioallantoic membrane (CAM) models, SNAP demonstrated maximal inhibitory efficacy (85-95% inhibition) of cytokine (FGF2)-induced neovascularization in both in vivo models. SNAP and SNAG resulted in 85% inhibition of FGF2-induced neovascularization in the mouse Matrigel model when given at 5 mg/kg/day infusion in minipumps during 14 days and 87% inhibition of angiogenesis induced by FGF2 in the CAM when administered a single dose of 50 microg. Thus, NO donors might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or neovascular, ocular, and inflammatory diseases.

Angiogenesis Inhibitors↗

PubMed2000

PKC and ERK1/2 regulate amylase promoter activity during differentiation of a salivary gland cell line.

The addition of transforming growth factor alpha (TGFalpha) to a human submandibular gland cell line (HSG) cultured on basement membrane extract Matrigel, synergistically activates the acinar cell-specific salivary amylase promoter. Signaling through beta1 integrins and increased phosphorylation of ERK1/2 are involved in the increased promoter activity. Phorbol-12-myristate-13-acetate (PMA) and thapsigargin increase amylase promoter activity, suggesting that phorbol ester and calcium-dependent protein kinase C (PKC) pathways are also involved. The combination of specific inhibitors of PKC and MEK1 inhibits the amylase promoter. Inhibitors of the calcium-dependent PKC isoforms alpha, beta, and gamma decrease the promoter activity; however, PKCbeta is not detectable in HSG cells. TGFalpha alters the cellular localization of PKCalpha but not -gamma, suggesting PKCalpha is involved in TGFalpha upregulation of the amylase promoter. Furthermore, rottlerin, a PKCdelta-specific inhibitor, increases the promoter activity, suggesting PKC isoforms differentially regulate the amylase promoter. In conclusion, beta1-integrin and TGFalpha signaling pathways regulate the amylase promoter activity in HSG cells. In response to Matrigel and TGFalpha, the activation of both PKCalpha and phosphorylation of ERK1/2 results in synergistic activation of the amylase promoter. Published 2000 Wiley-Liss, Inc.

Amylases↗

PubMed2000

Antiphospholipid antibodies affect trophoblast gonadotropin secretion and invasiveness by binding directly and through adhered beta2-glycoprotein I.

OBJECTIVE: To investigate the in vitro ability of antiphospholipid antibodies (aPL) to bind human trophoblast cells and to affect gonadotropin secretion and invasiveness. METHODS: Antiphospholipid antibody IgG from women with recurrent miscarriages, beta2-glycoprotein I (beta2GPI)-independent IgG aPL human monoclonal antibody (mAb) (519), and IgM anti-beta2GPI human mAb (TMIG2) were investigated for their binding to trophoblasts cultured for various amounts of time, their ability to affect invasiveness of Matrigel-coated filters, and their release of human chorionic gonadotropin (hCG). RESULTS: Polyclonal IgG aPL, as well as mAb 519 and TMIG2, bound to trophoblasts, the highest binding being found when cells displayed the greatest amount of syncytium formation. TM1G2 binding was found to be betaGPI dependent. Both polyclonal and monoclonal aPL, but not the controls, significantly reduced hCG release and Matrigel invasiveness. CONCLUSION: These findings suggest that aPL recognition of both anionic PL and adhered beta2GPI on trophoblast cell structures might represent a potential pathogenetic mechanism for defective placentation in women with the antiphospholipid syndrome.

Anions↗

PubMed1994

Maturation of undifferentiated lung epithelial cells into type II cells in vitro: a temporal process that parallels cell differentiation in vivo.

BACKGROUND: Formation of alveolar-like structures (ALS) by mature fetal rabbit type II pneumocytes (day 29 gestation) and long-term differentiation on Engelbreth-Holms-Swarm mouse tumor extract or EHS gel (Matrigel) were reported by our group (Blau et al., 1988. J. Cell Physiol., 136:203-214). We now describe structural organization and differentiation of immature lung epithelial cells, isolated at day 22 gestation, into mature type II cells in vitro. METHODS: Peripheral pulmonary tissue was pooled and undifferentiated epithelial cells isolated for primary culture on Matrigel. Cells were examined 12-16 h after plating and on days 1, 3, 5, and 7 of culture and assessed by phase contrast and by transmission electron microscopy after fixation in situ. RESULTS: Cells formed ALS 12-16 h after plating. Spherule diameter increased about four to eight times from day 1-7 in culture. There was rapid transformation of tall columnar cells to cuboidal, normal polarization of cells with respect to cell-free lumen of ALS, progressive reduction of glycogen zones, apparent gradual increase of cell organelles such as Golgi apparatus, rough endoplasmic reticulum and mitochondria, and apparent extrusion of lipidic figures into the lumen. These morphologic transformations in vitro temporally paralleled cell differentiation in vivo. The relative increase of 14C-acetate precursor into phosphatidylcholine in contrast to cardiolipin was consistent with these transformations. CONCLUSIONS: Under the conditions of our culture system, maturation of undifferentiated pulmonary epithelial cells is reproduced in vitro along the same time course and according to the same developmental sequence of fetal lungs in vivo.

Acetates↗

PubMed2002

Interleukin-15 inhibits sodium nitroprusside-induced apoptosis of synovial fibroblasts and vascular endothelial cells.

OBJECTIVE: One of the pathologic hallmarks of juvenile rheumatoid arthritis (JRA) is a tumor-like expansion of inflamed synovial tissue, or pannus, which causes much of the joint damage in this disease. The expansion of pannus is supported by extensive formation of new blood vessels. We have previously shown that revascularization of minced JRA synovial tissues engrafted into SCID mice correlated with the intensity of inflammatory activity in the tissues and with interleukin-15 (IL-15) expression. Since synovial vascular endothelial cells (VECs) expressed IL-15 receptors, the present study was undertaken to investigate the hypothesis that IL-15 might play a role in neovascularization of the pannus. METHODS: To evaluate IL-15 for possible angiogenic activity, we assessed the ability of recombinant human IL-15 (rHuIL-15) to induce VEC growth directly and to stimulate synovial cells to produce endothelial growth factors. Since IL-15 had been shown to inhibit apoptosis of certain immune cells, we were also interested in whether it might have similar effects on VECs. Apoptosis was induced by addition of sodium nitroprusside (SNP) at 1-2 mM to >80% confluent primary VECs, and numbers of apoptotic cells were determined by annexin V assay. RESULTS: Addition of rHuIL-15 at 10-100 ng/ml to primary synovial fibroblast cultures failed to up-regulate expression of vascular endothelial growth factor and angiopoietin 1 by these cells. Although rHuIL-15 failed to induce a mitogenic response of VECs, it promoted survival of these cells on Matrigel. Preincubation of VECs with rHuIL-15 at 50 ng/ml significantly reduced the proportion of VECs undergoing apoptosis. CONCLUSION: IL-15 promotes survival of VECs on Matrigel and inhibits SNP-induced apoptosis of endothelial cells. We hypothesize that this mechanism may be relevant to the stabilization of newly formed vascular structures in JRA synovium.

Apoptosis↗

PubMed2005

Invasiveness of fibroblast-like synoviocytes is an individual patient characteristic associated with the rate of joint destruction in patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by inflammation and destruction of synovial joints. Fibroblast-like synoviocytes (FLS) harvested from synovial tissue of patients with RA can invade normal human cartilage in severe combined immunodeficient (SCID) mice and Matrigel basement membrane matrix in vitro. This study was undertaken to investigate the association of these in vitro characteristics with disease characteristics in patients with RA. METHODS: Synovial tissue samples from 72 RA and 49 osteoarthritis (OA) patients were obtained. Samples of different joints were collected from 7 patients with RA. The FLS invasiveness in Matrigel was studied, and the intraindividual and interindividual differences were compared. From the patients with FLS who exhibited the most extreme differences in in vitro ingrowth (most and least invasive FLS), radiographs of the hands and feet were collected and scored according to the Sharp/van der Heijde method to determine the relationship between in vitro invasion data and estimated yearly joint damage progression. RESULTS: FLS from patients with RA were more invasive than FLS from patients with OA (P < 0.001). The mean intraindividual variation in FLS invasion was much less than the mean interindividual variation (mean +/- SD 1,067 +/- 926 and 3,845 +/- 2,367 for intraindividual and interindividual variation, respectively; P = 0.035), which shows that the level of FLS invasion is a patient characteristic. The mean +/- SEM Sharp score on radiographs of the hands or feet divided by the disease duration was 4.4 +/- 1.1 units per year of disease duration in patients with the least invasive FLS (n = 9), which was much lower compared with the 21.8 +/- 3.1 units per year of disease duration in patients with the most invasive FLS (n = 9) (P < 0.001). CONCLUSION: The ex vivo invasive behavior of FLS from RA patients is associated with the rate of joint destruction and is a patient characteristic, given the much smaller intraindividual than interindividual FLS variation.

Arthritis, Rheumatoid↗

PubMed2002

A lymph node metastatic mouse model reveals alterations of metastasis-related gene expression in metastatic human oral carcinoma sublines selected from a poorly metastatic parental cell line.

BACKGROUND: Greater than 40% of patients with squamous cell carcinoma (SCC) of the oral cavity have lymph node metastasis at the time of diagnosis and a 5-year survival rate of less than 50%. Changes in gene expression that regulate metastasis of SCC to lymph nodes have not been identified. METHODS: To study metastasis of oral SCC, highly metastatic oral SCC cell lines from a poorly metastatic oral SCC cell line were established by in vivo selection using a lymph node metastatic mouse model. The metastatic potential of the cells was studied using Matrigel invasion and cell surface protein adhesion assays. mRNA and protein encoded from metastasis-related genes in the metastatic derivatives and in their parental cells were examined using Northern blot analysis, immunoblotting, rapid analysis of gene expression, and a cDNA microarray technique. RESULTS: The in vivo selected metastatic cells showed much higher Matrigel invasion capability than the parental cells. They also showed alterations in their adhesion properties to three cell surface proteins. Comparison of metastatic and nonmetastatic cells revealed several significant alterations in the expression of metastasis-related genes, including up-regulation of the urokinase-type plasminogen activator receptor, integrin beta1, membrane type 1-matrix metalloproteinase, and down-regulation of protease-activated receptor-1. CONCLUSIONS: To the authors' knowledge, the current study is the first to report on gene expression analysis using a lymph node metastatic mouse model of human oral SCC. The data suggest that certain alterations of metastasis-related gene expression favor invasion of oral SCC and that cell surface proteins may play major roles in the metastasis of oral SCC to the lymph nodes.

Animals↗

PubMed2006

ACTIBIND, an actin-binding fungal T2-RNase with antiangiogenic and anticarcinogenic characteristics.

BACKGROUND: ACTIBIND is an Aspergillus niger extracellular ribonuclease (T2-ribonuclease [RNase]) that possesses actin-binding activity. In plants, ACTIBIND inhibits the elongation and alters the orientation of pollen tubes by interfering with the intracellular actin network. The question rose whether ACTIBIND can also affect mammalian cancer development. METHODS: Cell colony formation was performed in human colon (HT-29, Caco-2, RSB), breast (ZR-75-1), and ovarian (2780) cancer cells in the presence or absence of 1 muM ACTIBIND. In HT-29 and ZR-75-1 cells, the effect of ACTIBIND on cell migration was studied by microscopic observations and by invasion assay through Matrigel. Tube formation was assessed in human umbilical vein endothelial cells (HUVEC) in the presence of angiogenin or basic fibroblast growth factor (bFGF) (1 microg/mL each) following overnight incubation with 1 or 10 microM ACTIBIND. In an athymic mouse xenograft model, HT-29 cells were injected subcutaneously, followed by subcutaneous (0.4-8 mg/mouse/injection) or intraperitoneal (0.001-1 mg/mouse/injection) injections of ACTIBIND. In a rat dimethylhydrazine (DMH)-colorectal carcinogenesis model, ACTIBIND was released directly into the colon via osmotic micropumps (250 microg/rat/day) or given orally via microcapsules (1.6 mg/rat/day). Aberrant crypt foci, tumors in the distal colon, and tumor blood vessels were examined. RESULTS: ACTIBIND had an anticlonogenic effect unrelated to its ribonuclease activity. It also inhibited angiogenin-induced HUVEC tube formation in a dose-responsive manner. ACTIBIND was found to bind actin in vitro. It also bound to cancer cell surfaces, leading to disruption of the internal actin network and inhibiting cell motility and invasiveness through Matrigel-coated filters. In mice, ACTIBIND inhibited HT-29 xenograft tumor development, given either as a subcutaneous or intraperitoneal treatment. In rats, ACTIBIND exerted preventive and therapeutic effects on developing colonic tumors induced by DMH. It also reduced the degree of tumor observation. CONCLUSIONS: This study indicated that ACTIBIND is an effective antiangiogenic and anticarcinogenic factor.

Angiogenesis Inhibitors↗