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PubMedDate not supplied

Different growth conditions for peripheral blood endothelial progenitors.

PURPOSE: To compare different growth conditions for endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells (PBMNCs). METHODS AND MATERIALS: PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, IGF-I; the same medium with bovine retina-derived extract (RDE); freshly isolated or depleted of adherent cells PBMNCs in HUVEC conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in Ml99 with RDE. PBMNCs were analysed by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. EPCs were seeded alone or with HUVECs on Matrigel to assess in vitro angiogenesis. RESULTS: With growth factors, numerous cell clusters appeared within 1 week. Spindle-shaped and attached cells sprouted, differentiating in endothelial cell (EC)-like cells within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDE, numerous large cell clusters appeared within 1 week, but the number of cells with an EC morphology decreased during culture. FACS confirmed the endothelial phenotype and attached cells were able to migrate in response to VEGF. When nonadherent cells were cultured in HUVEC conditioned medium, they proliferated readily and EPCs were induced while freshly isolated cells neither proliferated nor induced EPCs. FACS analysis of the cocultures showed the presence of double-labeled PBMNCs expressing endothelial antigens. Capillary-like structures were observed on Matrigel only from cocultures and PBMNCs were able to incorporate in these networks. CONCLUSIONS: PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions (growth factors, HUVEC conditioned medium, HUVECs).

Anticoagulants↗
PubMed1996

Selective regulation of RNK-16 cell matrix metalloproteinases by the EP4 subtype of prostaglandin E2 receptor.

Cell surface expression of multiple structurally and functionally distinct prostaglandin E2 (PGE2) receptors (Rs), designated the EP1, EP2, EP3, and EP4 Rs, is a principal determinant of the diverse cellular effects of PGE2. The RNK-16 line of rat large granular lymphocytes, which has served as a model for natural killer cells, coexpresses a mean of 1092 EP3 Rs and EP4 Rs per cell with a mean Kd of 2.7 nM. The presence of the EP3 and EP4 Rs and the absence of the EP1 and EP2 Rs were revealed by inhibition of [3H]PGE2 binding by the EP3/EP1R agonist sulprostone, the EP3/EP2/EP4R agonist M&B 28767, and the EP2/EP4/EP3R agonist misoprostol but not by the EP1R antagonist SC-19220 or the EP2R agonist butaprost. Functional EP4 R expression was confirmed by finding that PGE2 and misoprostol, but not butaprost or sulprostone, evoked increases in the intracellular concentration of cyclic AMP ([cAMP]) in RNK-16 cells. Matrix metalloproteinase (MMP)-1 and -3 were identified by zymography and Western blots as the principal MMPs secreted by RNK-16 cells. Secretion of both MMPs by RNK-16 cells attained a maximal level after 24 h of incubation and was enhanced significantly by 10(-9) to 10(-7) M PGE2, 10(-6) M misoprostol, and 10(-4) M dibutyryl cyclic AMP, but not by the EP3R agonist sulprostone. Thus, the effect of PGE2 on RNK-16 cell MMP secretion is mediated by an EP4 R-dependent mechanism involving increases in [cAMP]i. The migration of RNK-16 cells across micropore filters, without or with a layer of Matrigel, was stimulated chemokinetically by PGE2 and misoprostol. PGE2-elicited chemokinesis of RNK-16 cells across a Matrigel model basement membrane, but not across a microfilter alone, was suppressed by the GM 6001 inhibitor of MMP activities. Stimulation of MMP activities in RNK-16 cells by the EP4R thus facilitates migration of the NK cells across vascular basement membranes.

Alprostadil↗
PubMed2003

Studies on the structure-activity relationship of endostatin: synthesis of human endostatin peptides exhibiting potent antiangiogenic activities.

The aim of the present research was to study the relationship between chemical structure and antiangiogenic activity of endostatin. Four peptides, containing about 40 amino acid residues, designed to cover nearly the whole sequence of endostatin, were synthesized by the solid-phase method. They were termed Fragment I (sequence 6-49), II (sequence 50-92), III (sequence 93-133), and IV (sequence 134-178), with the latter bearing the original disulfide bond Cys135-Cys165. These peptides were tested for their ability to inhibit endothelial cell proliferation, migration, and both in vitro and in vivo angiogenesis assays in matrigel. Fragments I and IV inhibited cell proliferation and cell migration with a potency and an efficacy higher than that of the full length endostatin. Fragment I was also active in inhibiting in vitro the formation of tubules and in vivo the vascularization of the matrigel. Fragments II and III were devoid of antiangiogenic activity. We propose to use the peptides 6-49 and 134-178 as angiogenesis inhibitors in substitution of full length endostatin, in therapeutic applications for cancer, rheumatoid arthritis, and retinopathies.

Amino Acid Sequence↗
PubMed1998

Urokinase induces receptor mediated brain tumor cell migration and invasion.

The plasminogen activation (PA) system plays an important role in tumor invasion by initiating pericellular proteolysis of the extracellular matrix (ECM) and inducing cell migration. Malignant brain tumors overexpress PA members and characteristically invade by migrating on ECM-producing white matter tracts and blood vessel walls. To determine whether urokinase-type plasminogen activator (uPA) and its receptor (uPAR) directly modulate the migration of brain tumor cells, we examined six human brain tumor cell lines, 2 astrocytomas (SW1088, SW1783), 2 medullobastomas (Daoy, D341Med), and 2 glioblastomas (U87MG, U118MG), for their surface uPAR expression, endogenous PA activity, and functional proteolytic activity by an ECM-degradation assay. Migration on Transwell membranes and invasion of Matrigel was then tested by pre-incubating the cells with increasing concentrations of either uPA, the proteolytically inactive amino-terminal fragment (ATF) of uPA, or the uPAR cleaving enzyme, phosphatidylinositol-specific phospholipase C (PI-PLC). All of the cell lines, except D341Med, express surface uPAR protein and uPA activity. High levels of uPAR and uPA activity correlated with cellular degradation of ECM, cell migration, and Matrigel invasion. Cell migration and invasion were enhanced by uPA or ATF in a dose dependent manner, while PI-PLC treatment abolished the uPA effect and inhibited migration and invasion. We conclude that ligation of uPAR by uPA directly induces brain tumor cell migration, independent of uPA-mediated proteolysis; and in concert with ECM degradation, markedly enhances invasion. Conversely, removing membrane bound uPAR from the surface of the cells studied inhibited their ability to migrate and invade even in the presence of proteolytically active uPA.

Brain Neoplasms↗
PubMed1999

SUM-159PT cells: a novel estrogen independent human breast cancer model system.

Breast cancer remains one of the most common malignant diseases in women in North America and Western Europe, yet therapies for the more aggressive estrogen independent tumors are limited and few model systems are available for the study of this type of breast cancer. In these studies, we characterized a novel estrogen independent breast cancer cell line, SUM-159PT. SUM-159PT cells are epithelial in origin, demonstrated by expression of cytokeratin 18. SUM-159PT cells are estrogen independent, demonstrated by lack of estrogen receptor (ER) protein and ER ligand binding studies. Furthermore, SUM-159PT cells injected subcutaneously or orthotopically are tumorigenic in ovariectomized athymic nude mice in the absence of estradiol supplementation. SUM-159PT cells are capable of invading through an 8 microm Matrigel membrane and display a stellate morphology in Matrigel, indicative of a metastatic phenotype. Correlating with this phenotype, we have detected secondary tumors upon inoculation of SUM-159PT cells into the mammary fat pad. To further investigate the metastatic potential of the SUM-159PT cells, we examined the expression of two proteins, vimentin and E-cadherin, implicated in the transition of carcinoma cells to a metastatic phenotype. Western blot and immunohistochemical analysis demonstrated that both SUM-159PT cells and xenografts express vimentin. No expression of E-cadherin was detected in SUM-159PT cells. Our data indicate that despite estrogen independence, SUM-159PT cells are growth inhibited in vitro by compounds such as 1,25(OH)2D3, transforming growth factor beta (TGF-beta), and the phorbol ester TPA. These studies indicate that SUM-159PT cells represent a good model system for the study of late stage estrogen independent, invasive breast cancer.

Animals↗
PubMed2000

Contortrostatin, a dimeric disintegrin from Agkistrodon contortrix contortrix, inhibits breast cancer progression.

We report the results of a multidisciplinary study on the inhibitory effect of a snake venom disintegrin, contortrostatin, a 13.5 kDa homodimeric protein isolated from Agkistrodon contortrix contortrix (southern copperhead) venom, on breast cancer progression. We demonstrate that contortrostatin binds to integrins and blocks the adhesion of human breast cancer cells (MDA-MB-435) to extracellular matrix (ECM) proteins including fibronectin and vitronectin, but it has no effect on adhesion of the cells to laminin and Matrigel. Contortrostatin also prevents invasion of MDA-MB-435 cells through an artificial Matrigel basement membrane. Daily local injection of contortrostatin (5 microg per mouse per day) into MDA-MB-435 tumor masses in an orthotopic xenograft nude mouse model inhibits growth of the tumor by 74% (p = 0.0164). More importantly, it reduces the number of pulmonary macro-metastasis of the breast cancer by 68% (p < 0.001), and micro-metastasis by 62.4% (p < 0.001). Contortrostatin is not cytotoxic to cancer cells, and does not inhibit proliferation of the breast cancer cells in vitro. However, contortrostatin inhibits angiogenesis induced by the breast cancer, as shown by immunohistochemical quantitation of the vascular endothelial cells in tumor tissue removed from the nude mice. We have identified alpha(v)beta3, an important integrin mediating cell motility and tumor invasion, as one of the binding sites of contortrostatin on MDA-MB-435 cells. We conclude that contortrostatin blocks alpha(v)beta3, and perhaps other integrins, and thus inhibits in vivo progression.

Agkistrodon↗
PubMed1998

Hepatocyte growth factor stimulates the invasion of gallbladder carcinoma cell lines in vitro.

Human gallbladder cancer is highly malignant and its prognosis is usually poor depending on the extent of surrounding tissue invasion. We examined in vitro the invasive activity of four gallbladder cancer cell lines (GB-d1, GB-h3, GB-d2 and FU-GBC-1) in the absence or presence of hepatocyte growth factor (HGF). In type 1 collagen gel culture, HGF stimulated cell proliferation and induced an invasive phenotype of arborizing structures in GB-d1, GB-h3 and GB-d2. In a Matrigel invasion assay, invasion was also induced in three of these cell lines by HGF but not in FU-GBC-1. Cellular motility was, however, stimulated by HGF in all of the four cell lines to various extents. Zymography for proteolytic enzymes demonstrated high levels of type IV collagenase and urokinase-type plasminogen activator (u-PA) activity in GB-d1, GB-h3 and GB-d2 even in the absence of HGF. In the presence of HGF, the 72 kDa type IV collagenase (MMP-2) activity of GB-h3 and u-PA activities of GB-d1, GB-h3 and GB-d2 were enhanced. In contrast, the MMPs and PAs activities of FU-GBC-1 were faint irrespective of the addition of HGF. A Western blot analysis demonstrated higher levels of 190 kDa c-MET product (HGF receptor) of GB-d1, GB-h3 and GB-d2 than that of FU-GBC-1. The invasion in the Matrigel assay stimulated by HGF was inhibited by protease inhibitors, aprotinin and FOY-305, as well as by anti-HGF antibody. These results thus suggest that, in addition to the importance of the proteolytic activity, the cellular motility induced via the HGF/HGF-receptor system is essential for the invasive progression of gallbladder carcinoma cells.

Adenocarcinoma↗
PubMed1998

Effect of MRC-5 fibroblast conditioned medium on breast cancer cell motility and invasion in vitro.

We used Transwell chambers to study separately cellular motility and invasion. In order to assess the cellular motility, polycarbonate microporous filters were coated with extracellular matrix proteins which adsorbed on the filters without clogging the pores. To investigate the invasive behavior of tumor cells, filters were covered with a layer of Matrigel which clogged the pores. The motility and the invasion of breast cancer cell lines (MDA-MB-231, MCF-7/6 and MCF-7/AZ cells) were assessed quantitatively in different culture media: defined (serum-free), serum-containing and normal human fibroblast MRC-5 conditioned media. In serum-containing medium, tumor cells migrated and invaded through the coated and covered filters. Their motility and invasion potentials were considerably lower in defined medium, whereas medium conditioned by MRC-5 fibroblasts stimulated both motility and invasion but not growth. The MRC-5 conditioned medium induced also the spreading of clusters of MCF-7/6 cells grown on Matrigel-coated plates.

Adenocarcinoma↗
PubMed1998

Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green alga, Spirulina platensis.

We have investigated the effect of calcium spirulan (Ca-SP) isolated from a blue-green alga, Spirulina platensis, which is a sulfated polysaccharide chelating calcium and mainly composed of rhamnose, on invasion of B16-BL6 melanoma, Colon 26 M3.1 carcinoma and HT-1080 fibrosarcoma cells through reconstituted basement membrane (Matrigel). Ca-SP significantly inhibited the invasion of these tumor cells through Matrigel/fibronectin-coated filters. Ca-SP also inhibited the haptotactic migration of tumor cells to laminin, but it had no effect on that to fibronectin. Ca-SP prevented the adhesion of B16-BL6 cells to Matrigel and laminin substrates but did not affect the adhesion to fibronectin. The pretreatment of tumor cells with Ca-SP inhibited the adhesion to laminin, while the pretreatment of laminin substrates did not. Ca-SP had no effect on the production and activation of type IV collagenase in gelatin zymography. In contrast, Ca-SP significantly inhibited degradation of heparan sulfate by purified heparanase. The experimental lung metastasis was significantly reduced by co-injection of B16-BL6 cells with Ca-SP. Seven intermittent i.v. injections of 100 microg of Ca-SP caused a marked decrease of lung tumor colonization of B16-BL6 cells in a spontaneous lung metastasis model. These results suggest that Ca-SP, a novel sulfated polysaccharide, could reduce the lung metastasis of B16-BL6 melanoma cells, by inhibiting the tumor invasion of basement membrane probably through the prevention of the adhesion and migration of tumor cells to laminin substrate and of the heparanase activity.

Animals↗
PubMed1999

Inhibition by vasoactive intestinal polypeptide (VIP) of angiogenesis induced by murine Colon 26-L5 carcinoma cells metastasized in liver.

We investigated the effect of VIP on the liver metastases and angiogenesis by Colon 26-L5 carcinoma cells in mice. Daily systemic administration of VIP, beginning 3 days after tumor inoculation into a portal vein of mice, inhibited significantly the development of their liver metastases. Immunohistochemical staining for factor VIII-related antigen in the sections of liver metastases showed that the systemic administration of VIP caused significant prevention of angiogenesis within tumor masses. VIP (10-(10) to 10(-6) M) inhibited the invasion of reconstituted basement membrane (Matrigel) by hepatic sinusoidal endothelial (HSE) cells in a concentration-dependent manner in a Transwell chamber assay in vitro and achieved approximately 50% reduction of control at 10(-6) M. VIP (10(-6) M) also significantly suppressed the haptotactic migration of HSE cells to fibronectin, laminin or type I collagen substrates with a similar inhibition rate to the invasion assay. Exposure of VIP to HSE cells induced accumulation of intracellular cAMP in a concentration-dependent manner. The inhibitory effect of VIP (10(-6) M) on HSE cell migration was significantly abrogated in the presence of 3 x 10(-6) M H-89, a cAMP-dependent protein kinase inhibitor. VIP (10(-6) M) inhibited the morphogenesis of HSE cells into capillary-like structures on Matrigel-coated wells. VIP did not affect the proliferation of HSE cells and the production of gelatinases in HSE cells in vitro at the concentrations used in the invasion assay. These observations suggest that the anti-metastatic effect of VIP on liver metastases by Colon 26-L5 carcinoma cells in mice is partly due to the prevention of tumor angiogenesis probably through suppression of the motility of endothelial cells.

Animals↗
PubMed1999

Chemoattraction of femoral CD34+ progenitor cells by tumor-derived vascular endothelial cell growth factor.

Patients and animals with GM-CSF-producing tumors have an increased number of mobilized CD34+ progenitor cells within their peripheral blood and tumor tissue. These CD34+ cells are inhibitory to the activity of intratumoral T-cells. The present study used the murine Lewis lung carcinoma (LLC) model to assess mechanisms that could lead to the accumulation of CD34+ cells within the tumor tissue. In vitro analyses showed that LLC tumor explants released chemoattractants for normal femoral CD34+ cells. The LLC tumor cells contributed to the production of this activity since CD34+ cell chemoattractants were also released by cultured LLC cells. Antibody neutralization studies showed that most, although not all, of the chemotactic activity that was produced by LLC cells could be attributed to VEGF. In vivo studies with fluorescent-tagged CD34+ cells showed their accumulation within the tumor tissue, but not within the lungs, spleen or bone marrow, suggesting a selective accumulation within the tumor. Whether or not VEGF could chemoattract CD34+ cells in vivo was measured with a VEGF-containing Matrigel plug assay. Infusion of fluorescent-tagged CD34+ cells into mice after the plugs became vascularized revealed the accumulation of fluorescent-tagged cells within the plugs. However, these CD34+ cells failed to accumulate within the VEGF-containing Matrigel plugs when they were infused together with neutralizing anti-VEGF antibody. Through a combination of in vitro and in vivo analyses, the LLC cells were shown to be capable of chemoattracting CD34+ cells, with most of the tumor-derived chemotactic activity being due to tumor release of VEGF.

Animals↗
PubMed1999

Inhibition of B16BL6 melanoma invasion by tyrosine and phenylalanine deprivation is associated with decreased secretion of plasminogen activators and increased plasminogen activator inhibitors.

We previously found that dietary tyrosine (Tyr) and phenylalanine (Phe) limitation significantly decreased the metastatic phenotype of B16BL6 melanoma cells in vivo and decreased the in vitro invasion of these cells. To more specifically characterize the effects of Tyr and Phe deprivation we examined the three steps involved in invasion: attachment to host cells and components, elaboration of proteases that degrade basement membranes, and migration of invading tumor cells. Here we report that B16BL6 melanoma cell invasion through growth factor reduced (GFR) Matrigel is significantly decreased by Tyr and Phe deprivation. Tyr and Phe deprivation in vitro decreased the attachment of B16BL6 melanoma cells to GFR Matrigel, heparin sulfate proteoglycans (HSPG), neonatal murine epidermal (NME) cells and the extracellular matrix (ECM) from these cells. These cells also exhibited a decrease in chemotactic response to fetal bovine serum (FBS). Deprivation of these two amino acids decreased the secretion of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) while plasminogen activator inhibitor (PAI)-1 and -2 were increased in these cells. These observations suggest that Tyr and Phe deprivation decreases the in vitro chemotactic and invasive ability of B16BL6 melanoma cells by decreasing attachment and secreted PA activity and by increasing secreted PAIs in these cells.

Animals↗
PubMed1999

Biological properties associated with the enhanced lung-colonizing potential in a B16 murine melanoma line grown in a medium conditioned by syngeneic Corynebacterium parvum-elicited macrophages.

A previous study by our laboratory showed that the peritoneal murine Corynebacterium parnum-elicited macrophages released into their growth medium an activity which enhanced the ability of B16-F10 melanoma cells to form experimental metastases in the lung of syngeneic mice. In the present study, we used a clone of B16-F10 line (F10-M3 cells) to investigate whether the increase in lung-colonizing potential due to the pro-clonogenic activity released by C. parvum-elicited macrophages was associated with biological properties characteristic of a metastatic phenotype. We have found that the pulmonary retention, growth rate in lung parenchyma, invasiveness through Matrigel, adhesiveness to IL-1-activated endothelium and MHC class I expression were increased in F10-M3 cells stimulated by the macrophage pro-clonogenic activity. By using an in vitro experimental protocol, the enhancement of lung-colonizing potential in the stimulated melanoma cells turned out to be a transient phenomenon as was the increase of invasiveness through Matrigel and the higher expression of MHC class I antigens. In conclusion, the melanoma cells stimulated by the pro-clonogenic activity released by C. parvum-elicited macrophages showed changes in biological parameters which are relevant to metastatic diffusion. These changes appeared as a temporary phenomenon which sustains the view that the metastatic phenotype represents a transient biological character influenced by host factors.

Animals↗
PubMed2000

Different levels of TGFbeta, IL-10, IFNgamma and gelatinase A occur in experimental white and black metastases induced by bryostatin 1 or by phorbol ester-treated BL6T murine melanoma cells.

Bryostatin 1 and phorbol esters reduce the intracellular melanin level in high metastatic overexpressing nPKCdelta BL6 (BL6T) cells, thereby inducing white experimental metastasis in syngeneic mice. We evaluate here the possible differences between white and black metastases induced by both treatments on the proliferative and metastatic potential as well as on the expression of some cytokines involved in the metastatic process such as TGFbeta, IL-10 and IFNgamma. The level of expression of gelatinase A is also considered. White and black metastases induced after the injection of bryostatin 1- or phorbol ester-treated cells into the tail vein of syngenic mice were isolated and analysed for the levels of LDH usually used as markers of cytotoxicity, for the levels of cytokines and gelatinase A or dissociated and cultured in vitro for a few passages. The cultured cells were analysed in vitro for the proliferative capacity and the melanin synthesis. The same cells were also re-injected into syngeneic mice and the number of experimental metastases were counted after 17 days or injected with matrigel in order to quantify the proliferative capacity in vivo. The results show only one significant difference between bryostatin I and phorbol ester, namely the cells obtained from white bryostatin 1-treated cells return to a black phenotype after a few passages in culture. This suggests that PKC mediates many of the biological effects of bryostatin 1 but that its effect is lost in vitro. On the other hand, white and black metastases (at least for metastases induced by BL6T cells treated with phorbol ester) do appear significantly different. In vivo white metastases show lower levels of LDH, lower levels of proliferative capacity into matrigel, higher levels of TGFbeta and IFNgamma and, when re-injected into syngeneic mice, give big black metastases. Therefore, in murine melanoma cells, the treatment with bryostatin I induces the appearance of a white population expressing different levels of TGFbeta, IFNgamma, IL-10 and gelatinase A. Such a white population is more difficult to diagnose and is capable of turning into a more aggressive phenotype under suitable environmental conditions.

Animals↗
PubMed2000

MMP-9 secretion and MMP-2 activation distinguish invasive and metastatic sublines of a mouse mammary carcinoma system showing epithelial-mesenchymal transition traits.

We have investigated the gelatinase profiles and invasiveness of clonal tumour sublines derived from a spontaneously arising mammary tumour in a Balb/cfC3H mouse. The 67NR. 66c14 and 4T1.2 sublines have low, intermediate and high metastatic potential respectively. In Boyden chamber studies, Matrigel invasion was seen to be progressively higher in the more metastatic lines 4T1.2>66c14>67NR, consistent with MMP-2 activation potential, MMP-9 secretion, and migration over either type I or IV collagen, which were low in both 67NR and 66c14 cells compared to 4T1.2 cells. These attributes are consistent with those seen in human breast cancer cell lines which appear to have undergone an epithelial-mesenchymal transition (EMT) as indicated by vimentin expression. We were, however, surprised to find vimentin expression, MT1-MMP expression and stellate Matrigel outgrowth in the non-invasive, non-metastatic 67NR cells. indicating that they had undergone an EMT despite not being invasive. We conclude that the EMT is manifested to differing degrees in these three clonal cell lines, and that the 67NR cells have either undergone a partial EMT or have since lost certain important attributes of the EMT-derived phenotype. This model should prove useful in further characterizing the regulation of MTI-MMP mediated MMP-2 activation and delineating the EMT in breast cancer progression.

Animals↗
PubMed2001

Glioma cell invasion: regulation of metalloproteinase activity by TGF-beta.

Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that selectively degrade components of the extracellular matrix. MMPs are implicated in tumor cell invasion because they mediate the breakdown of the basal membrane. In addition, they seem to be important for the creation and maintenance of a microenvironment that facilitates tumor cell survival. Among the essential characteristics of human malignant gliomas are infiltrative growth, angiogenesis and suppression of antitumor immune surveillance. Transforming growth factor-beta (TGF-beta) is intimately involved in the regulation of these processes. We have previously demonstrated that TGF-beta promotes the migration of LN- 18 and LN-229 glioma cells via a process that may involve the upregulation of alphaVbeta3 integrin expression. Furthermore, we have defined a novel pathway for hepatocyte growth factor (HGF)-induced glioma cell migration and invasion which requires the induction of TGF-beta2 expression. Here, we demonstrate that TGF-beta2 induces MMP-2 expression and suppresses tissue inhibitor of metalloproteinases (TIMP)-2 expression and that concentration-dependently promotes the invasion of U87MG and LN-229 glioma cells in a matrigel invasion assay. Similarly, ectopic expression of the anti-apoptotic BCL-x, protein leads to enhanced matrigel invasion by LN-18 and LN-229 glioma cells. We outline the possible interrelations of TGF-beta, proteins of the BCL-2 family, integrins and metalloprotease activity. By virtue of its promotion of glioma invasion and its growth regulatory and immunomodulatory properties. TGF-beta continues to be one of the most promising targets for the experimental therapy of human malignant glioma.

3T3 Cells↗
PubMed2002

Influence of polyamines on in vitro and in vivo features of aggressive and metastatic behavior by human breast cancer cells.

Increased cellular activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) synthesis, is an independent adverse prognostic factor for overall survival in human breast cancer, thus suggesting an important role for PA in tumor progression. The experiments presented here were designed to investigate the role of PA in invasion and metastasis, using the highly aggressive MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. Administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, significantly reduced, in a dose-dependent manner, the invasiveness in matrigel of both MDA-MB-435 and MDA-MB-231 cells by approximately 70%. DFMO treatment also inhibited (P < 0.0001) 'stellate' colony formation (an indicator of aggressive phenotype) by MDA-MB-435 cells plated in the matrigel outgrowth assay. Administration of DFMO (2% in drinking water) reduced the growth rate of both cell lines implanted orthotopically in nude mice. To evaluate metastasis while minimizing effects on proliferation, DFMO-treated mice were sacrificed later to allow their tumors to reach the same size of the tumors in the control mice. The most striking finding was that DFMO, while ineffective in reducing local invasion, nearly totally abolished (P = 0.0152) pulmonary metastasis in mice bearing MDA-MB-435 xenografts. These results support a role of PA in promoting breast cancer aggressiveness, particularly with regard to the development of distant metastasis. Furthermore, the data suggest that PA involvement is distal to local invasion in the metastatic cascade.

Animals↗
PubMed2001

TGF beta is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells.

New vessels form de novo (vasculogenesis) or from pre-existing vessels (angiogenesis) in a process that involves the interaction of endothelial cells (EC) and pericytes/smooth muscle cells (SMC). One basic component of this interaction is the endothelial-induced recruitment, proliferation and subsequent differentiation of pericytes and SMC. We have previously demonstrated that TGF beta induces the differentiation of C3H/10T1/2 (10T1/2) mesenchymal cells toward a SMC/pericyte lineage. The current study tests the hypothesis that TGF beta not only induces SMC differentiation but stabilizes capillary-like structures in a three-dimensional (3D) model of in vitro angiogenesis. 10T1/2 and EC in Matrigel were used to establish cocultures that form cord structures that are reminiscent of new capillaries in vivo. Cord formation is initiated within 2-3 h after plating and continues through 18 h after plating. In longer cocultures the cord structures disassemble and form aggregates. 10T1/2 expression of proteins associated with the SMC/pericyte lineage, such as smooth muscle alpha-actin (SMA) and NG2 proteoglycan, are upregulated in these 3D cocultures. Application of neutralizing reagents specific for TGF beta blocks cord formation and inhibits expression of SMA and NG2 in the 10T1/2 cells. We conclude that TGF beta mediates 10T1/2 differentiation to SMC/pericytes in the 3D cocultures and that association with differentiated mural cells is required for formation of capillary-like structures in Matrigel.

Animals↗