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Molecular Cancer Research 1:561-568 (2003)
© 2003 American Association for Cancer Research

Angiogenesis, Metastasis, and the Cellular Microenvironment

Amyloid Endostatin Induces Endothelial Cell Detachment by Stimulation of the Plasminogen Activation System1

Arie Reijerkerk1, Laurent O. Mosnier2, Onno Kranenburg1, Bonno N. Bouma2, Peter Carmeliet4, Tom Drixler1,3, Joost C.M. Meijers2,5, Emile E. Voest1 and Martijn F.B.G. Gebbink1

1 Department of Medical Oncology, 2 Thrombosis and Haemostasis Laboratory, Department of Haematology, and 3 Department of Surgery, University Medical Center Utrecht, Utrecht, The Netherlands;
4 Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium; and
5 Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Requests for reprints: M.F.B.G. Gebbink, Department of Medical Oncology—F02.126, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Phone: 31-30-250-6265; Fax: 31-30-252-3741. E-mail: m.gebbink{at}azu.nl


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Endostatin is a fragment of collagen XVIII that acts as an inhibitor of tumor angiogenesis and tumor growth. Anti-tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study, we have investigated the cellular effects of insoluble endostatin. We previously found that insoluble endostatin shows all the hallmarks of amyloid aggregates and potently stimulates tissue plasminogen activator-mediated formation of the serine protease plasmin. We here show that amyloid endostatin induces plasminogen activation by endothelial cells, resulting in vitronectin degradation and plasmin-dependent endothelial cell detachment. Endostatin-mediated stimulation of plasminogen activation, vitronectin degradation, and endothelial cell detachment is inhibited by carboxypeptidase B, indicating an essential role for carboxyl-terminal lysines. Our results suggest that amyloid endostatin may inhibit angiogenesis and tumor growth by stimulating the fibrinolytic system.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Angiogenesis, the formation of new capillaries from pre-existing blood vessels, is important during various pathological processes, including inflammation and tumor growth. Antiangiogenic therapy is being considered as a potentially powerful new therapy for cancer and other angiogenesis-dependent diseases. Endostatin is one of the most potent inhibitors of angiogenesis and can induce tumor regression in mice (1). Clinical trials are currently ongoing (2, 3). Originally, endostatin was purified from conditioned medium of murine hemangioendothelioma (EOMA) cells as a proteolytically cleaved fragment of collagen XVIII. Generation of endostatin can be achieved by cleavage of collagen by cathepsin L (4), matrilysin, (5) or elastase (6).

Endostatin has distinct antiangiogenic and anti-tumoral activities in several animal models (7). Tumor growth was significantly inhibited by i.p. or s.c. injection of endostatin without induction of acquired drug resistance (1, 8). In addition, rat endostatin induced significant inhibition of carcinogen-induced mammary tumor growth (9). Endostatin is also bioactive using delivery approaches such as DNA vaccination and viral expression (10, 11). However, contrary to this, no antiangiogenic effect of endostatin was seen in other studies despite high serum levels (12–15). This paradox has led to discussions about the efficacy of endostatin (16). Until now, this paradox remains unsolved. Possibly, the outcome of endostatin treatment will depend on its structure (see below) and/or the presence of factors provided at the site of action.

A variety of molecular mechanisms have been proposed to underlie endostatin activity. Endostatin blocks vascular endothelial growth factor (VEGF)-mediated signaling through KDR/Flk-1 (17) and induces endothelial cell apoptosis, associated with decreased levels of anti-apoptotic proteins Bcl-2 and BclXL (18). Endostatin can bind tropomyosin, an actin stabilizing protein, and has been suggested to disrupt microfilament integrity ultimately causing endothelial cell apoptosis (19). Direct effects of endostatin on cell adhesion may be caused by suppression of integrin function (20, 21). Other studies have implicated that endostatin acts on the proteolytic system by binding and inhibiting active metalloproteinase (MMP)-2 (22, 23) or down-regulating the urokinase plasminogen activator system (24). However, it is not clear to what extent these activities contribute to the antiangiogenic and anti-tumor effects of endostatin in vivo.

A complicating factor is that endostatin is used in a soluble, as well as in an insoluble form. Whereas both forms have been reported to inhibit tumor growth, the regression of tumors has only been reported with insoluble endostatin (1). Different structural forms of endostatin have distinct bioactivities and may therefore produce their antiangiogenic effects through distinct mechanisms. The structure of soluble, globular endostatin has been elucidated (25, 26). Recently, we found that endostatin is a protein with high propensity to form amyloid fibers through extensive cross-ß sheet formation (27, 28). Moreover, we established that only insoluble endostatin but not soluble endostatin stimulates tissue plasminogen activator (t-PA)-mediated plasminogen activation and induces cell toxicity (27, 28). Here we have studied the effect of insoluble endostatin, to which we refer as amyloid endostatin, on endothelial cell-mediated plasmin formation and cell adhesion.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Amyloid Endostatin Stimulates Plasmin Formation
We previously showed that denatured endostatin forms amyloid fibrils and stimulates t-PA-mediated plasminogen activation in vitro (27). We show here that amyloid endostatin stimulates t-PA-mediated plasminogen activation in a dose-dependent manner (Fig. 1A). Stimulation of t-PA-mediated activation of plasminogen by amyloid endostatin was as potent as stimulation by fibrin fragments, the classical stimulator of t-PA-mediated plasminogen activation (Fig. 1B). No activation of plasminogen was observed in the absence of t-PA and amyloid endostatin alone did not convert the chromogenic substrate (not shown).



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  		FIGURE 1. Amyloid endostatin-stimulated t-PA-mediated plasminogen activation is concentration dependent. Different concentrations of amyloid endostatin (A) and fibrin fragments (B) were added to plasminogen. Plasminogen activation was started by addition of t-PA. At the indicated time points, samples were taken and plasmin activity was analyzed using the chromogenic substrate S-2251. These figures show a representative experiment out of five independent experiments. , 4 µM; , 2 µM; , 1 µM; , 0.5 µM; , 0 µM.

 
Amyloid Endostatin Binds Plasminogen and t-PA
Because endostatin is a stimulator of plasminogen activation by t-PA, we tested whether amyloid endostatin was able to bind plasminogen and t-PA. Both plasminogen and t-PA bound to immobilized amyloid endostatin in a dose-dependent and saturable manner (Fig. 2A). Binding of t-PA to amyloid endostatin [apparent dissociation constant (Kd) = 1.6 ± 0.3 nM] was similar to the binding of t-PA to fibrin degradation products (Kd = 2.3 ± 0.4 nM) (Fig. 2B). The affinity of plasminogen for amyloid endostatin (Kd = 2.7 ± 0.6 nM) was about 10 times higher than the affinity of plasminogen for fibrin fragments (Kd = 28.4 ± 5.8 nM).



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  		FIGURE 2. Amyloid endostatin binds to plasminogen and t-PA. Binding of plasminogen (A) and t-PA (B) to immobilized amyloid endostatin or fibrin fragments was measured by ELISA. Binding was detected using specific antibodies against plasminogen or t-PA followed by peroxidase-conjugated secondary antibodies and substrate addition. •,{blacksquare} EAm; {circ},{square} DESAFIB-X.

 
Carboxypeptidase Inhibits the t-PA-Stimulatory Activity of Amyloid Endostatin
The binding of plasminogen and t-PA to fibrin is mediated by the lysine-binding sites in the kringle domains of plasminogen and t-PA and by carboxyl-terminal lysine residues that are generated in fibrin during plasmin digestion (29). Therefore, we investigated the importance of carboxyl-terminal lysines in amyloid endostatin-mediated plasmin formation. To this end, we used the porcine pancreas carboxypeptidase B (CpB) that removes basic (arginine and lysine) amino acids from proteins. Treatment with CpB reduced amyloid endostatin-stimulated plasminogen activation (Fig. 3). The inhibitory effect of CpB on amyloid endostatin was greatly diminished by the addition of a specific carboxypeptidase inhibitor (CPI), indicating that the effect of CpB is due to carboxypeptidase activity. Binding analyses revealed that CpB treatment of amyloid endostatin abrogated plasminogen binding for more than 90%, but had little or no effect on t-PA binding. In line with this, plasminogen binding to amyloid endostatin was blocked by the lysine analogue {varepsilon}-amino caproic acid (not shown). These results clearly show that carboxyl-terminal lysines are essential for efficient t-PA-mediated plasminogen activation.



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  		FIGURE 3. CpB blocks amyloid endostatin-induced t-PA-mediated plasminogen activation. The formation of plasmin was induced by 4 µM amyloid endostatin. Before the start of the reactions with t-PA, CpB was added for 30 min at 37°C. Carboxypeptidase inhibitor (CPI) was included to demonstrate that the effect of CpB was the result of specific activity. The figure shows a representative experiment out of five independent experiments. , EAm; , EAm + CpB + CPI; , EAm + CpB; , control.

 
Amyloid Endostatin Stimulates Plasminogen Activation by Endothelial Cells
We next determined whether amyloid endostatin can stimulate plasminogen activation mediated by endothelial cells, a biological source of tissue-type plasminogen activator. Different concentrations of amyloid endostatin were added to confluent monolayers of endothelial cells in the presence of 0.4 µM plasminogen. After 24 h, the medium was analyzed by Western blotting. Plasminogen cleavage by plasminogen activator results in the formation of plasmin, which migrates at approximately Mr 50,000 in gel electrophoresis. In contrast to soluble endostatin, amyloid endostatin treatment significantly enhanced cell-mediated plasmin formation (Fig. 4A). Cells treated without amyloid endostatin did not convert plasminogen into plasmin. Also in endothelial cell-mediated plasminogen activation induced by amyloid endostatin, we found a similar dependence of amyloid endostatin activity on the presence of carboxyl-terminal lysines. Addition of 50 µg/ml CpB totally abolished plasminogen activation (Fig. 4B). Determination of chromogenic substrate conversion by concentrated medium indicated that plasmin was active (Fig. 4C).



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  		FIGURE 4. Amyloid endostatin stimulates endothelial cell-mediated plasminogen activation. Plasmin formation in culture medium of control cells (C) and cells treated with 30 µM of amyloid endostatin (EAm) or 30 µM of soluble endostatin (Es) was analyzed by SDS-PAGE and Western blotting using anti-human plasminogen antibody. A. Addition of CpB completely blocked amyloid endostatin-induced plasmin formation. B. Plasmin activity was determined in culture medium of amyloid endostatin-treated cells using chromogenic substrate S-2251.

 
Amyloid Endostatin Causes Endothelial Cell Detachment and Extracellular Matrix Degradation
So far, we have shown that amyloid endostatin stimulates plasminogen activation both in vitro and on endothelial cells. Increased plasmin formation has been implicated in endothelial cell detachment (30–33). Therefore, we studied the effects of amyloid endostatin on endothelial cell adhesion in the absence and presence of different plasminogen concentrations (Fig. 5). While amyloid endostatin or plasminogen alone did not induce cell detachment (Fig. 5, A–C), co-treatment of the endothelial cells with plasminogen resulted in dramatic contraction of the cell bodies and complete rounding up of the cells (Fig. 5D). The addition of CpB inhibited amyloid endostatin-mediated plasmin formation (Fig. 4B) and subsequent cell detachment (Fig. 5E). To assess the involvement of t-PA in endostatin-induced plasminogen activation and cell detachment, 20 µM Pefabloc t-PA was used to inhibit t-PA activity. This t-PA inhibiting peptide completely inhibited plasmin-mediated cell detachment, indicating an essential role for t-PA in these processes (Fig. 5F). Treatment with soluble endostatin alone (Fig. 5G) or in combination with plasminogen (Fig. 5H) had no effects.



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  		FIGURE 5. Amyloid endostatin causes endothelial detachment. Bovine pulmonary arterial endothelial cells were cultured to confluency and incubated with 30 µM of amyloid endostatin (EAm) in the presence of different concentrations of plasminogen. After 24 h, cells were washed with PBS, photographed, and analyzed. A. Control-treated endothelial cells. Thirty micromolars EAm (B) or 0.6 µM plasminogen (C) did not induce endothelial cell detachment. D. Cell contraction and rounding up induced by 30 µM EAm and 0.6 µM plasminogen co-treatment. Addition of (E) 50 µg/ml CpB or (F) 20 µM Pefabloc t-PA completely blocked EAm-induced endothelial cell detachment. Soluble endostatin in the absence (G) or presence (H) of plasminogen had no effects. (I) Cell detachment was quantified as described in the "Materials and Methods." Columns, mean; bars, SEM. , 0.6 µM Pg; , 0.3 µM Pg; , 0.15 µM Pg; , 0 µM Pg. (J) Vitronectin (Vn) degradation was analyzed by Western blotting. VnDP, Vn degradation product.

 
The observed detachment of endothelial cells could be mediated by extracellular matrix degradation. Vitronectin (serum spreading factor) is an important component of the extracellular matrix involved in cell attachment and cell spreading. Vitronectin can be degraded by plasmin (34). Vitronectin degradation was studied in lysates of amyloid endostatin-treated endothelial cells by Western blotting. Whereas only small amounts of vitronectin degradation products were generated in the presence of plasminogen alone, co-treatment with amyloid endostatin potently increased vitronectin degradation (Fig. 5J). CpB, which blocked plasmin formation (Fig. 4B), also abrogated vitronectin degradation.

Inhibitory Activity of Amyloid Endostatin on Subcutaneous Tumor Growth Is Reverted by CpB
To determine the possible role for carboxyl-terminal lysine residues in the antitumoral effect of amyloid endostatin in vivo, we treated mice with amyloid endostatin in the absence or presence of CpB. Treatment was started directly after tumor cell injection and stopped on day 13. Amyloid endostatin-treated mice showed approximately 50% reduced growth of a s.c. colon carcinoma (P = 0.046 treatment versus control), while the presence of CpB completely abolished this reduction (Fig. 6). The anti-tumor effect of CpB alone was not significant (P = 0.229 CpB versus control).



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  		FIGURE 6. Inhibition of tumor growth by amyloid endostatin is abolished by CpB treatment. Mice (n = 4/group) were inoculated subcutaneously with 1 x 106 C26 colon carcinoma cells and treated daily with amyloid endostatin or control solvent in the presence or absence of CpB as indicated. The inhibitory effect of amyloid endostatin on tumor growth was completely blocked by continuous administration of CpB. The experiment shown is a representative of three independent experiments.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The formation of plasmin by activation of its zymogen plasminogen is associated with degradation of the extracellular matrix as it occurs in the dissolution of blood clots, tissue remodelling, invasive growth of cancer cells, and angiogenesis (35–38). Plasmin mediates proteolysis of the extracellular matrix by degrading fibrin and other matrix molecules. In addition, plasmin mediates proteolysis indirectly by the activation of metalloproteinases, which further degrade the extracellular matrix. During angiogenesis, extracellular matrix components regulate cell proliferation, migration, and survival through interactions with adhesion molecules on the cell surface. Important adhesion molecules include the receptors for fibrin and vitronectin, the integrins {alpha}vß3 and {alpha}vß5 (39). Plasmin has been implicated as an important modulator of the interactions between cells and the extracellular matrix (31, 40). Plasmin may also liberate cytokines, such as transforming growth factor ß (41), from inactive precursors and could play a role in the formation of plasminogen breakdown products with antiangiogenic properties, including angiostatin and kringle 5 (42). Thus, plasmin is a key enzyme in the angiogenic process.

Here, we have demonstrated that amyloid endostatin, a cofactor for t-PA-mediated plasminogen activation, induces endothelial cell-mediated plasmin formation resulting in vitronectin degradation, cell remodelling, and detachment.

Numerous studies revealed high expression of t-PA in several human tumors (43–47). Therapeutic administration of amyloid endostatin may result in stimulation of t-PA activity that is produced in the tumor. In light of this, it is interesting to note that t-PA expression by endothelial cells is induced by angiogenic factors, including basic fibroblast growth factor (bFGF) and VEGF (48). Overstimulation of the plasminogen activation system may result in excessive matrix degradation. Induction of plasminogen activation has led to endothelial cell detachment (30), inhibition of cell adhesion (31), endothelial cell destruction (32), or regression of capillary tubes (33). Vitronectin plays an important role in endothelial cell survival (49). We found that vitronectin, a main adhesive matrix protein present in serum and deposited in the extracellular matrix of cultured endothelial cells, is degraded on treatment with amyloid endostatin. Its breakdown may cause cell detachment and subsequent apoptosis.

With the discovery of t-PA as a general cross-ß sheet-binding protein (27), we have identified a molecule that may contribute to the cellular effects induced by amyloid proteins like endostatin. Indeed, a specific inhibitor of t-PA activity completely blocked plasmin-mediated changes of endothelial cell morphology induced by amyloid endostatin.

We noted that several other antiangiogenic peptides, such as amyloid endostatin, can stimulate t-PA-mediated plasminogen activation. Stimulatory activity toward plasminogen activation and antiangiogenic activity has been described for a cleaved or denatured conformation of antithrombin, aaATIII (50, 51), prothrombin fragments (52, 53), thrombospondin (54–57), maspin (58, 59), and amphoterin (60, 61). This could implicate that a common antiangiogenic pathway may exist that is induced by t-PA binding proteins. Clinical studies underscore that high t-PA levels are associated with good prognosis in cancer patients (62–64).

In fibrin, the generation of carboxyl-terminal lysines is key to the efficient activation of plasminogen, as they form high-affinity binding sites for plasminogen and, less so, t-PA. The importance of carboxyl-terminal lysines in endostatin-mediated plasminogen activation and subsequent detachment of endothelial cells was shown since the addition of CpB totally abrogated endostatin activity. Physiologically, plasminogen activation induced by partially degraded fibrin is regulated by thrombin-activable fibrinolysis inhibitor (TAFI), a CpB-type enzyme present in blood (65, 66). We found that stimulation of t-PA-mediated plasminogen activation by endostatin was similarly inhibited by activated TAFI (not shown). Thus, TAFI could function as a regulator of the anti-tumoral activity of endostatin.

In line with our in vitro data, co-treatment of mice with endostatin and CpB abolished the inhibitory effect of endostatin. We cannot exclude that other CpB sensitive pathways involved in endostatin bioactivity may be affected, but these results strongly suggest that the plasminogen activation system plays an important role in endostatin function.

Our suggestion that increased plasminogen activation might be responsible for at least part of the endostatin effect on tumor growth is supported by data of others. Tumor vascularization and tumor invasion is prevented in the absence of PAI-1 when increased levels of plasmin are likely to be formed (67, 68). In addition, high concentrations of t-PA are generated and PAP levels are elevated in patients with peripheral tumors that regress in response to tumor necrosis factor {alpha} (TNF{alpha}) (69, 70).

Taken together, overstimulation of t-PA by agents such as amyloid endostatin may result in excessive matrix degradation, thereby preventing angiogenesis and tumor growth (71). This is a novel pathway to intervene in tumor growth and warrants further study.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Proteins and Reagents
The cDNA for murine endostatin (kindly provided by Dr. Fukai, Boston, MA) was amplified by PCR and cloned into the prokaryotic expression vector pET15b (Novagen, WI). Recombinant murine endostatin was produced by Escherichia coli as described (1) and resuspended in PBS. Soluble Pichia pastoris-produced endostatin was provided by Entremed, Inc. (Rockville, MD). Plasminogen was obtained from Sigma Chemical Co. (St. Louis, MO) or purified from plasma as described (72); t-PA and plasmin substrate S-2251 were obtained from Chromogenix AB (Mölndal, Sweden); Pefabloc t-PA was obtained from Pentapharm AG (Basel, Switzerland); porcine pancreas CpB was obtained from Boehringer Mannheim (Mannheim, Germany); and potato carboxypeptidase inhibitor and rabbit polyclonal antibody against vitronectin were from Calbiochem (La Jolla, CA). Anti-plasminogen monoclonal antibody and anti-t-PA polyclonal antibodies were obtained from American Diagnostica (Greenwich, CT).

Preparation of Yeast-Produced Amyloid Endostatin
P. pastoris-produced endostatin (Entremed) was denatured by dialyses against 8 M urea, 10 mM Tris (pH 7.5), and 10 mM ß-mercaptoethanol. The denaturing buffer was subsequently removed by extensive dialysis against H2O. Amyloid endostatin became visible as a white precipitate.

Binding Experiments
Binding of plasminogen and t-PA was carried out in 96-well microtiter plates coated overnight at room temperature with 50 µl amyloid endostatin (20 µg/ml) or fibrin fragments (DESAFIB-X, Chromogenix, 20 µg/ml) in coating buffer [15 mM Na2CO3, 35 mM NaHCO3, 0.02% NaN3 (pH 9.6)]. The wells were blocked with 3% BSA in PBS for 1 h and washed three times with PBS containing 0.3% BSA. Where indicated, the wells were treated with porcine pancreas CpB (50 µg/ml) for 1 h at 37°C in PBS containing 3% BSA and washed three times with PBS/BSA. Plasminogen and t-PA, at various concentrations, were allowed to bind for 1 h. After three washes, bound plasminogen or t-PA were detected with specific anti-plasminogen and anti-t-PA antibodies followed by peroxidase-conjugated secondary antibodies. Peroxidase activity was measured using ortho-phenylenediamine as substrate. The reaction was stopped by the addition of 1 M H2SO4 and absorbance was measured at 490 nm.

Measurement of Plasmin Activity
The reactions were performed at 37°C in HBS buffer [20 mM HEPES, 4 mM KCl, 137 mM NaCl, 3 mM CaCl2, 0.1% BSA (pH 7.4)] containing 50 µg/ml of plasminogen with various concentrations of amyloid endostatin, DESAFIB-X, or with a control sample. The reactions were started by the addition of t-PA at a final concentration of 30 units/ml. At several time points, 20 µl samples were taken and the reaction was stopped with 20 µl buffer containing 150 mM {varepsilon}ACA and 150 mM EDTA. Plasmin activity was determined in 96-well plates after the addition of 20 µl chromogenic substrate S-2251 at a final concentration of 1.6 mM. Increase in absorbance was measured at 405 nm for 10 min. When applicable, test samples were preincubated for 30 min with 25 µg/ml CpB and/or 50 µg/ml potato carboxypeptidase inhibitor.

Plasminogen activation by endothelial cells was determined by SDS-PAGE analyses and Western blotting. Conditioned medium was concentrated five times with nanosep 10K Omega (Pall Life Science, Portsmouth, UK). Plasmin activity was analyzed by adding 10 µl of concentrated conditioned medium to S-2251 (1.25 mM) and subsequent reading at 405 nm.

Cell Culture
Bovine pulmonary arterial endothelial cells (BPAEC) (CCL-209) were obtained from the American Type Culture Collection (Rockville, MD) and cultured in DMEM (Gibco BRL, Invitrogen Corporation, UK) with the supplement of 10% FCS and antibiotics. BPAEC were seeded onto 48-well culture plates (Costar Inc., NY) and grown to confluency in culture medium. The cells were washed two times with PBS and incubated with yeast-produced amyloid endostatin in human endothelial-SFM basal growth medium (Gibco BRL, Invitrogen Corporation, UK) supplemented with antibiotics.

Assay of Endothelial Cell Detachment
BPAEC were incubated with amyloid endostatin in the presence or absence of plasminogen. Where indicated, 50 µg/ml CpB was used for complete blockage of plasminogen activation, and 20 µM Pefabloc t-PA was used to inhibit t-PA activity. After 24 h, the cells were washed with PBS and photographed using phase-contrast microscopy. The supernatant was stored for further analyses. Cell detachment was analyzed by encircling the non-cell area in four different photographs using Adobe Photoshop version 6.0 (Adobe Systems Inc., CA) and subsequently quantified by calculating the percentage coverage of the selected area using Optimas 6.0 software (DVS, Breda, The Netherlands).

Tumor Experiments
Male 6- to 8-week-old BALB/c mice (General Animal Laboratory, University Medical Center Utrecht, Utrecht, the Netherlands) were used. All mice were fed a diet of animal chow and water ad libitum. Experiments were performed according to the guidelines of the Utrecht Animal Experimental Committee, University Medical Center (Utrecht, the Netherlands). Mice were inoculated subcutaneously with 1 x 106 C26 colon carcinoma cells in 200 µl PBS. E. coli-derived amyloid endostatin (20 mg/kg/day) or control solvent was given by daily s.c. injection, starting just after tumor cell injection and stopped at the end of the experiment. This results in approximately 50% inhibition of tumor growth in this model. CpB was given continuously using a mini-osmotic pump (Alzet pump, Alza, Palo Alto, CA, type 2002 [14 days]) containing 200 µl CpB at a concentration of 5 mg/ml. The pump was implanted subcutaneously in the dorsal skin fold. In mice bearing s.c. tumors, absence of contact between tumor deposit and pump was assured by implanting the pump in the contralateral side. Animals received an initial s.c. bolus injection of 40 µg CpB in 100 µl PBS at the time of tumor cell injection. Tumor diameters were determined on day 13 by a caliper, and tumor volume was calculated with the formula width2 x length x 0.52. Significance of differences in tumor growth among groups was determined by the unpaired Student's t test. P < 0.05 was considered to be statistically significant. Values represent mean number ± SEM.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Colinda Aarsman and Maria de Mol for their excellent technical help.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Dutch Cancer Society (M.F.B.G.G.); the Fischer Stichting (A.R.); The Netherlands Heart Foundation; and Crucell N.V., Leiden, The Netherlands. Note: J.C.M.M. is an established investigator of The Netherlands Heart Foundation. A.R. and L.O.M. contributed equally to this work. Back

Received February 14, 2003; revised April 16, 2003; accepted April 22, 2003.


   References
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 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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