1Department of Surgery, School of Medicine, Keio University, Shinanomachi 35, Shinjyuku-ku, Tokyo 160-8582 and 3Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
2 To whom correspondence should be addressed. E-mail: m_ueda{at}sc.itc.keio.ac.jp
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Abstract |
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Keywords: angiogenesis/basic fibroblast growth factor/fusion protein/molecular targeting/ribonuclease
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Introduction |
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Various antiangiogenic agents have been identified and some of these are currently under clinical trials (Karp et al., 1995). Most of these inhibitors of angiogenesis, however, pose problems in therapeutic application because of their excessive toxicity and limited efficacy. To resolve these problems, a variety of studies on molecular targeting chemotherapy for neoplasms have been reported, since this might allow higher specificity and reduced systemic toxicity. Several angiogenic factors have been identified and investigated, but vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) have perhaps been the most studied (Li, 2000
). FGF is a potent pleiotropic heparin-binding mitogen for vascular endothelial cells. It acts synergistically with VEGF to stimulate new vessel growth (de Jong et al., 1998
; Seghezzi et al., 1998
; Klint et al., 1999
; Giavazzi et al., 2001
). Therefore, targeting FGF or FGF receptors in order to achieve angiogenesis inhibition can be a strategy for therapy. For example, antiangiogenic platelet factor-4 (PF-4) fragments associate with FGF and impair cell-surface receptor binding to inhibit angiogenesis in vivo (Hagedorn et al., 2001
). Treatment of tumors with three tyrosine kinase receptors, including the FGF receptor, markedly suppressed tumor growth and decreased tumor blood flow (Griffin et al., 2002
).
Recently, we designed a fusion protein, composed of human RNase1, which was cloned from human pancreas and human bFGF and reported that this protein exerted cell type-specific growth inhibition against malignant cells bearing FGF receptors (Futami et al., 1995, 1999
). RNaseFGF fusion proteins are considered to be effectively internalized into the cytosol via the FGF receptors. As a result, they degrade cellular RNA and inhibit protein synthesis.
In a previous study, the generation of an end-to-end fusion protein between RNase1 and bFGF by simple gene linkage was explored. This protein was designed to evade the effect of ribonuclease inhibitor protein (RI) by truncation of seven amino acid residues in the N-terminal sequence of RNase1. Nevertheless, through this method, the protein function could not be demonstrated adequately nor could the stability of the fusion protein be established, because it was difficult to control the configuration between these proteins and choices of the stereostructure were limited. Therefore, we attempted to construct a new concept of fusion protein, by which a domain of the second protein was inserted genetically into the first protein. In this method, the position of the inserted domain could be varied to obtain various protein stereostructures. Thus, to achieve the blocking of RI binding and cellular targeting of RNase simultaneously, bFGF was inserted genetically into Gly89 of RNase1 (RI bindng site of RNase1) to prevent from its stereostructural binding to RI. The resultant protein, CL-RFN89, had abilities both for efficient binding to target cells and for evading RI by masking the RI interaction site with the targeting protein of bFGF. CL-RFN89 retained its activity at >85% even in the presence of a 200-fold molar excess of RI and showed a growth inhibitory effect on mouse B16/BL6 melanoma cells (Tada et al., 2004), which expresses both bFGF and high-affinity FGF receptor (Blanckaert et al., 1993
). Subsequently, tumor angiogenesis that is mediated and promoted by the FGFFGF receptor interaction is assumed to be suppressed by this fusion protein.
In this study, the effect of CL-RFN89 on the antiangiogenic response was investigated, in both in vitro and in vivo angiogenesis models. Furthermore, the possibility of CL-RFN89, reacting with the vascular endothelial cells specifically through FGF receptors was also examined.
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Materials and methods |
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Dulbecco's modified Eagle's medium (DMEM) and phosphate-buffered saline (PBS) were obtained from Sigma Chemical (St Louis, MO). Fetal bovine serum (FBS) was obtained from JRH Bioscience (Lenexa, KS). Recombinant human fibroblast growth factor-basic was purchased from Pepro Tech EC (London, UK). The EGM bullet-kit (containing endothelial basal medium, hEGF, hydrocortisone, BBE, FBS and GA-1000), trypsin, cell matrix-type I-A, MCDB131 and gel reconstruction buffer were supplied by Asahi Techno Glass (Chiba, Japan). A fluorescein labeling kit was obtained from Roche Diagnostics (Mannheim, Germany). Cell Counting Kit-8, nembutal and diethyl ether were obtained from Wako Pure Chemical Industries (Osaka, Japan). A Millipore chamber (diameter, 14 mm; thickness, 2 mm) and membrane filter (diameter, 13 mm; thickness, 180 µm; filter pore size, 0.45 µm) were obtained from Millipore (Bedford, MA). Six and 24 multi-well plates were purchased from Sumitomo Bakelite (Tokyo, Japan).
Cell lines
A431 cells procured from Riken (Saitama, Japan) were maintained in DMEM containing 10% FBS. Human umbilical vein endothelial cells were procured from Asahi Techno Glass and cultured in EGM containing 2% FBS. All the cells were cultured in an atmosphere of 5% CO2 at 37°C.
Animals
Female 6-week-old BALB/c mice obtained from CLEA Japan (Tokyo, Japan) were used. The animals were housed in an air-conditioned room at 2223°C, with free access to food and water.
Construction of CL-RFN89
The methods of construction and purification of insertional fusion protein, CL-RFN89, were described previously (Tada et al., 2004). To summarize them briefly, recombinant human 4118 cross-linked RNase1(CL-RNase1) and human bFGF (147 amino acid form) were purified from Escherichia coli (Futami et al., 1999
, 2000a
). A cDNA encoding bFGF(19146) (N-terminal 18 residue-truncated form of bFGF) was amplified by polymerase chain reaction. A SacII site was introduced at the position of Gly89 of CL-RNase1 cDNA into a CL-RNase1 expression vector. The resultant plasmids were cleaved with SacII and ligated in frame with the SacII fragment of bFGF(19146) to construct the expression vectors for insertional-fusion proteins (Futami et al., 2000b
). The recombinant proteins were expressed as inclusion bodies in E.coli and then solubilized and refolded (Futami et al., 1995
). The refolded proteins were purified by cation-exchange chromatography. The main peak fractions were collected and concentrated by ultrafiltration.
Binding assay
Fluorescein-labeled CL-RFN89 was synthesized by coupling 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS) to the amino groups of CL-RFN89 using the fluorescein labeling kit, according to the manufacturer's instructions. To explain briefly, 1 mg of CL-RFN89 dissolved in PBS was incubated with 0.14 mg of FLUOS dissolved in 100% DMSO for 2 h at room temperature, with gentle mixing. Unbound FLUOS was removed by Sephadex G-25 gel filtration. The approximate fluorescein to protein ratio was 5:1.
The binding assay was performed to confirm whether the effect of CL-RFN89 was mediated through the FGF receptor. A total of 1 x 105 cells of HUVECs were suspended in phosphate-buffered saline and the cell suspension was mixed with fluorescein-labeled CL-RFN89 to obtain a solution of 2 µM concentration. The cells were washed twice with PBS at 4°C after 15 min of incubation and then examined under a fluorescence microscope (ECLIPSE E1000, Nikon, Tokyo, Japan). An excess amount of human recombinant bFGF was used to investigate the competitive inhibition of FGF receptors by CL-RFN89. Before incubation was started, the cells and the fluorescein-labeled protein complex were treated with 30 µM bFGF. They were also incubated, washed and observed in a similar manner. These experiments were performed in triplicate.
In vitro angiogenesis assay
Tube formation of HUVECs in a type I collagen gel was analyzed as the assay of angiogenesis in vitro. For the preparation of the collagen gel, 8 volumes of Cellmatrix Type I-A (3.0 mg/ml) were mixed with 1 volume of 10x MCDB131 and 1 volume of gel reconstruction buffer (50 mM sodium hydroxide, 260 mM sodium hydrogen carbonate, 200 mM HEPES). A 1 ml volume of the mixture was added at 4°C to each well of the multi-well plate (six wells; 38 mm diameter) and the gel was incubated at 37°C for at least 30 min, to allow for gelation. The gel was covered with 1 ml of a similar collagen gel after HUVECs were plated on the gel at a concentration of 1.5 x 105 cells and incubated in EGM at 37°C for 4 h. The cells were then exposed to various concentrations of reagents added to EGM. After incubation for 48 h, fields of tube-like structures that formed in the gel were observed at random under an optical microscope and the total lengths per field (x40) were measured by computer image analysis (Scion Image, version 0.4.0.2, Scion, Maryland). All the experiments were performed three times and the results were expressed as means and standard deviations.
Cytotoxicity of CL-RFN89 against A431 cells and HUVECs
CL-RFN89 was added to a 96-well plate containing the cells at a density of 104 cells in 100 µl of DMEM per well and then cultured to assess its cytotoxicity against A431 cells and HUVECs. After 48 h of incubation with various concentrations of CL-RFN89, the cell viability was measured with the Cell Counting Kit-8 using the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay. The number of viable cells was estimated and was shown as a ratio compared with an untreated control. Ten wells were used for each cell and the average values were calculated. This experiment was repeated three times.
Mouse dorsal air sac assay
The mouse dorsal air sac assay was used to examine the effect of CL-RFN89 on the angiogenic response triggered by malignant tumor cells, according to a modification of the method described by Tanaka et al. (1989).
Both sides of a Millipore ring were covered with Millipore filters of 0.45-µm pore size and the Millipore chamber was filled with A431 tumor cells (1.5 x 106 cells) in 0.15 ml of PBS, with or without various concentrations of CL-RFN89. The chamber containing the A431 cells was implanted into an air sac formed previously in the dorsum of a 6-week-old female BALB/c mouse by injection of an appropriate volume of air. The control group implanted with the PBS-containing chamber was administered vehicle alone. On day 5, the implanted chambers were removed from the subcutaneous fascia of the treated mice. The angiogenic response was assessed in dissecting microscope photographs, by determining the number of newly formed blood vessels >3 mm in length within the area in direct contact with the chamber. The extent of angiogenesis was scored as 0, 1, 2, 3, 4 or 5, corresponding to the new formation of no, one, two, three, four or five or more blood vessels, respectively. The blood vessels newly formed in response to angiogenic factor(s) released from malignant tumor cells were morphologically distinct from the pre-existing vessels because of their zigzag characteristics, as described previously (Tanaka et al., 1989).
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Results |
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Our first series of experiments were designed to investigate whether the FGFRNase fusion protein, CL-RFN89, reacts with the HUVECs specifically through the FGF receptor. We studied whether CL-RFN89 bound to the HUVECs in the presence of an excess amount of bFGF. When the cells were mixed with fluorescein-labeled CL-RFN89, the appearance of a green fluorescence on the cell surface was detected by fluorescence microscopy. On the other hand, no fluorescence was observed in the presence of an excess amount of bFGF (15 times the amount of CL-RFN89), even when substantial numbers of cells in the same fields were scanned under an optical microscope (Figure 1).
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To examine whether CL-RFN89 inhibits the process of angiogenesis, we employed an in vitro model of angiogenesis in which HUVECs are induced to invade a three-dimensional collagen gel, where they form a network of capillary-like tubes. As shown Figure 2a, complex and branching capillary-tube like structures formed in a random fashion in the control group. In contrast, this dynamic process of in vitro angiogenesis was markedly inhibited in the presence of CL-RFN89.
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Cytotoxicity of CL-RFN89 against A431 cells and HUVECs
To confirm whether A431 cells and HUVECs can coexist with CL-RFN89 in the chamber, we assessed the cytotoxicity of this fusion protein by the use of the WST-8 before conducting the mouse dorsal air sac assay. CL-RFN89 inhibited the growth of HUVECs in a dose-dependent manner (Figure 3). On the other hand, the inhibition rate of the growth of A431 cells by CL-RFN89 was only 1.7% (Figure 3). In addition, no inhibition and proliferation were also observed when A431 cells were added with bFGF at a concentration from 0.1 to 10 µM (data not shown). This implied that CL-RFN89 did not exert any cytotoxicity against A431 cells and that these cells could therefore be applied for the following assay.
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Implantation of the chambers containing only PBS (negative control) was associated with minimal angiogenesis (Figure 4). The mean angiogenesis index was only 0.5 ± 1.22 (N = 6) (Figure 5), indicating that the experimental manipulation and subsequent healing process did not induce any significant angiogenic response. On the other hand, A431 cells separated by a semipermeable filter (positive control) triggered the neovascularization process, with an angiogenesis index of 4.4 ± 0.55 (N = 5). CL-RFN89 significantly suppressed the A431 cell-induced angiogenesis when the concentration of the chamber was kept at 1 and 2 µM. The mean angiogenesis index was 0.8 ± 1.30 (N = 5) for both concentrations. As a control, the assay was used with RNase1 alone, bFGF alone and the buffer of same constituents with CL-RFN89, none of which proved to inhibit the angiogenesis (data not shown).
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Discussion |
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In an attempt to make it safer for clinical use, we and others have introduced the use of mammalian enzymes such as ribonucleases (RNases) instead of bacterial toxins (Rybak et al., 1991; Jinno et al., 1996
; Psarras et al., 1998
). RNases constitute a large superfamily of proteins crossing over many species. Neither human RNases nor bovine pancreatic RNase A are themselves cytotoxic. However, when injected into Xenopus oocytes, they abolished protein synthesis at concentrations comparable to that of the toxin ricin by direct degradation of the cellular RNA (Rybak et al., 1991
; Saxena et al., 1991
).
Mammalian RNases are non-toxic to cultured cells and can be substituted for plant or bacterial toxins as an immunotoxin (Rybak et al., 1995). However, it must enter the cell and reach the cytosol, where RNA degradation would ultimately lead to a cell death (Kim et al., 1995
; Wu et al., 1995
). In the cytosol, RNase encounters the ribonuclease inhibitor protein (RI). RI constitutes >0.01% of the total protein in the cytosol (Bretscher et al., 2000
) and it inactivates RNase by forming a tight complex that prevents RNA substrates from entering the active sites of the enzyme (Kobe and Deisenhofer, 1996
). With the exception of bovine seminal RNase, which has been reported to be resistant to RI independently (Murthy et al., 1996
), it is important for RNase to possess the ability to evade RI in order to exert the desired cytotoxicity and several studies have been performed in this connection. It was shown that truncation of seven amino acid residues in the N-terminal sequence of RNase1 resulted in a more pronounced reduction in the affinity to human placental RI; however, it also significantly reduced the activity of the enzyme. A version of this 17 des RNase has 466-fold lower affinity to placental RI, while its ribonucleolytic activity is 8.6 times lower than that of RNase1 (Futami et al., 1995
) and it has no ability of cellular targeting. To maintain the activity of the enzyme, simultaneously reducing the affinity to RI and to achieve cellular targeting, bFGF was inserted genetically into Gly89 of RNase1 to prevent its stereostructural binding to RI. CL-RFN89 retained their activity at >85% even in the presence of 200-fold molar excess amount of RI and showed cell type-specific growth inhibition against malignant cells bearing FGF receptors (Tada et al., 2004
).
Our results for a binding assay using fluorescein-labeled CL-RFN89 showed that this protein could adhere to the cell surface of HUVECs. In the presence of an excess amount of unlabeled bFGF, however, no adhesion or uptake of CL-RFN89 to HUVECs occurred. This means that the excess bFGF occupies the FGF receptors and competitively prevent CL-RFN89 from binding to the cells, which suggests that the binding of the protein to HUVECs is FGF receptor dependent. This selective inhibition of CL-RFN89 for the HUVECs was also demonstrated in an in vitro cytotoxicity assay. CL-RFN89 inhibited the cellular growth of HUVECs, which express the FGF receptor (Chen et al., 2001), in a dose-dependent manner. On the other hand, the growth of A431 human epidermal carcinoma cells remained intact despite the presence of CL-RFN89 at concentrations of up to 10 µM. Although it was previously reported that bFGF accelerated the growth of A431 cells in vitro (Sugimoto and Nishino, 1996
), our result can be explained by a report showing that A431 cells express barely detectable levels of FGF receptor, by the analysis of [125I]bFGF binding to A431 cells (Muggeridge et al., 1992
) and by a report showing that 16-mer oligopeptide, with conformation similar to the putative receptor-binding domain of bFGF had no ability to bind to A431 cells (Kono et al., 2003
). Furthermore, proliferation of A431 cells was not accelerated by bFGF at concentrations from 0.1 to 10 µM (data not shown).
In this study, CL-RFN89 was shown to exhibit antiangiogenic activity both in vitro and in vivo. Cytotoxicity assay showed that CL-RFN89 at a concentration of 10 µM caused 46% inhibition of the growth of HUVECs. It was thought that CL-RFN89 inhibited the proliferative phase of the cells. In the presence of CL-RFN89 at a concentration of 2 µM in the 3D culture system, the length of the tubular structures formed in the culture was decreased to 43% of that in the control group. The later stages of angiogenesis involve morphological alterations and functional maturation of endothelial cells, which result in lumen formation (Risau, 1997). These processes are controlled by the interaction of growth factors and extracellular matrix components (Folkman and Klagsbrun, 1987
; Madri and Marx, 1992
). By using the 3D culture system as an experimental model, we examined whether CL-RFN89 inhibited the remodeling phase of angiogenesis, corresponding to the late phase (Madri and Marx, 1992
; Marx et al., 1994
; Prols et al., 1998
). It was revealed that a specific expression pattern of transmembrane receptors, including the FGF receptor and induction of a new set of differentiation-specific genes, occurred during the course of angiogenesis (Marx et al., 1994
; Sankar et al., 1996
). CL-RFN89 had the ability to inhibit the process of angiogenesis at this point in vitro. In addition, CL-RFN89 was also shown to inhibit angiogenesis by the mouse dorsal air sac assay. Neovascularization of the mouse subcutaneous tissues, which was triggered by A431 cells, was effectively inhibited by the administration of CL-RFN89. This result was not caused by the growth suppression of A431 cells in the membrane chamber, because the growth of these cells was shown to be unaffected by CL-RFN89 in the cytotoxicity assay. It was assumed that CL-RFN89 infiltrated the surrounding tissues through the membrane filter and inhibited vascularization by the cells, bearing FGF receptors, such as the endothelial cells, the smooth muscle cells and the others responsible for the neovascularization. These results show that CL-RFN89 is a potent inhibitor of angiogenesis.
Recent studies have examined the role of FGF receptors in tumor growth and several tumor cell lines that express FGF receptors on their cell surface. Amplification of the FGFR1 and FGFR2 genes in breast tumor tissues from over 350 patients was examined and it was found to occur in 10% of the patients (Adnane et al., 1991
). Screening of breast tumor cell lines or tissues for FGFR4 revealed overexpression of the FGFR4 mRNA (Lehtola et al., 1992
; Ron et al., 1993
; Penault-Llorca et al., 1995
), suggesting that FGFR4 may have a role in the development of breast cancer in humans. FGF has also been implicated as an autocrine growth factor for melanomas (Halaban et al., 1987
), gliomas, meningiomas (Ueba et al., 1994
) and pancreatic ductal adenocarcinomas (Kornmann et al., 1998
). This expression of FGFR may provide a mechanism for targeting tumor cells. It was observed that CL-RFN89 exerted efficient growth inhibition of mouse metastatic melanoma B16/BL6 cells in vitro (Tada et al., 2004
). An in vivo study using tumor cell lines expressing FGFR is now under way in order to demonstrate the therapeutic potential of CL-RFN89. It is expected that CL-RFN89 would exhibit anti-tumor activity both by a direct effect (inhibition of tumor cell growth) and by an indirect effect (inhibition of tumor angiogenesis). It may be possible to devise a new strategy by utilizing the synergism between the direct and indirect anti-tumor activities of this compound.
In summary, our results provided evidence that the bFGFRNase fusion protein could be efficiently targeted via the FGF receptors in HUVECs and that this protein inhibited proliferation and tube formation of the cells in vitro. In addition, this protein also effectively inhibited tumor angiogenesis in vivo. These experiments have demonstrated that tumor angiogenesis can be targeted by CL-RFN89 via FGF receptors and that this protein holds promises as an antiangiogenic agent that exerts specificity for the cells bearing the FGF receptors.
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Acknowledgements |
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Received December 7, 2004; revised April 21, 2005; accepted May 18, 2005.
Edited by Dario Neri
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