| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics, Molecular Targets, and Chemical Biology |
Chain Suppresses Angiogenesis and Tumor Growth
Departments of 1 Dermatology and 2 Medical Pathology and Laboratory Medicine, University of California-Davis Medical Center, Sacramento, California
Requests for reprints: Yoshikazu Takada, Department of Dermatology, University of California-Davis Medical Center, Research III Suite 3300, 4645 Second Avenue, Sacramento, CA 95817. Phone: 916-734-7443; Fax: 916-734-7505; E-mail: ytakada{at}ucdavis.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain has a COOH-terminal globular domain (C, residues 151-411 of the chain, 30 kDa) to which several integrin cell adhesion receptors (e.g., platelet 
IIbß3, endothelial vß3, and leukocyte Mß2) bind. Integrins play a critical role in signal transduction from fibrin(ogen). We found that C and its truncation mutant (designated C399tr), with a deletion of the COOH-terminal 12 residues, induced apoptosis of endothelial cells and blocked tube formation of endothelial cells. DLD-1 human colon cancer cells that secrete C or C399tr grew at similar levels in vitro but grew much slower in vivo than mock-transfected cells. The recombinant purified C399tr fragment markedly suppressed tumor growth, development of intratumoral vasculature, and tumor metastasis in vivo in the highly metastatic Met-1 breast cancer model. The determinant responsible for binding to endothelial cells is cryptic in native fibrinogen but is exposed in C and C399tr. These results suggest that fibrinogen has a novel cryptic determinant, which can exert apoptosis-inducing activity on endothelial cells when exposed, and polypeptides containing this determinant have therapeutic potential. (Cancer Res 2006; 66(19): 9691-7) |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The fibrinogen chain has a conserved globular domain at the COOH terminus (30 kDa, residue 151-411, designated C). According to the crystal structure of the human C domain (8), C has a COOH-terminal fibrin-polymerization domain, a single calcium-binding site and a deep binding pocket. Integrins are a family of cell adhesion receptors that recognize extracellular matrix ligands, including fibrin(ogen) and cell surface ligands (9). Integrins are transmembrane ß heterodimers, and at least 18 and 8ß subunits are known (10). It has been well established that integrins transduce signals inside the cells on ligand binding, and integrin functions are regulated by the signals from inside the cells (9). C is a major integrin-binding site of fibrinogen. Integrin IIbß3 in platelets, vß3 in endothelial cells, and Mß2 in leukocytes recognize C and play an important role in thrombus formation, angiogenesis, and inflammation, respectively. We have published that C and its truncation mutant C399tr (residues 251-411 and 251-399 of the fibrinogen chain, respectively) bind to integrin vß3 in an cation-dependent manner (11, 12). Although these fragments do not have an RGD motif, a prototype integrin recognition sequence, RGD peptide blocked vß3 binding to these fragments (11, 12). Fragment D consists of COOH-terminal portions of , ß, and chains of fibrinogen.
In the present study, we discovered that C and C399tr induced apoptosis of endothelial cells in vitro and blocked tube formation of endothelial cells on Matrigel in vitro, whereas native fibrinogen and fragment D did not induce apoptosis of endothelial cells. Cancer cells that secrete C or C399tr grew much slower in vivo than control mock-transfected cells. Further, the recombinant soluble C399tr suppressed the tumor growth, intratumoral vasculature development, and metastasis in vivo. We provide evidence that the determinant that is involved in endothelial apoptosis is cryptic in fragment D and native fibrinogen but is exposed in C and C399tr. These results suggest that polypeptides that have the apoptosis-inducing determinant have therapeutic potential as an antiangiogenic agent.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C and C399tr fragments were produced in Escherichia coli using the pET21a(+) vector as an insoluble protein and were refolded as described previously (12). Purified proteins have >90% purity in SDS-PAGE. Endotoxin was removed from the purified protein by using the Detoxi-Gel endotoxin removing gel (Pierce Biotechnology, Rockford, IL). We confirmed that the protein sample is endotoxin-free by using an endotoxin detection kit.
Synthesis of recombinant soluble vß3. We used recombinant soluble vß3 (constructs were provided by T. Springer, Center for Blood Research, Boston, MA; ref. 13) for binding assays. We generated Chinese hamster ovary (CHO) cells that stably express soluble vß3 and purified the secreted protein in serum-free medium by Ni-NTA affinity chromatography using a 6-His tag at the COOH terminus of the protein.
Site-directed mutagenesis. Site-directed mutagenesis was carried out using a modified Quick Change method (14). The presence of the mutations was verified by DNA sequencing. We generated the C399tr mutant, in which residues 400 to 411 of C are deleted by the insertion of the stop codon at position 400 (12).
Analysis of apoptosis. Apoptosis was measured by using the Annexin V-FITC Apoptosis Detection kit (BD PharMingen, San Diego, CA). Cells were seeded in wells of six-well tissue culture plates (1 x 106 per well) in the presence of fibrinogen or its fragments (10-20 µg/mL) and cultured in 5% CO2 incubator at 37°C for 24 hours. Cells were harvested by incubating with trypsin-EDTA (5 minutes at 37°C). After a trypsin inhibitor was added, cells were washed with PBS, and Annexin V-FITC (5 µL) and propidium iodide (PI) solutions (5 µL in 100 µL) were then added. After 15 minutes of incubation at room temperature, Annexin V-FITC bound to cells was analyzed by flow cytometry.
We measured caspase-3/caspase-7 activities by using Apo-One homogeneous caspase-3/caspase-7 assay (Promega, Madison WI). Briefly, cells plated in wells of 96-well tissue culture plates at a density of 2.0 x 104 per well were lysed with an equal volume of lysis buffer containing the caspase substrate, Z-DEVD-R100, and incubated at room temperature for 1 hour. The fluorescence from each well was measured at an excitation wavelength of 498 nm and an emission wavelength of 521 nm using a Wallac 1420 multilabel counter (PerkinElmer, Wellesley, MA).
Cell proliferation. Cells were plated in wells of 96-well tissue culture plates (5 x 104 per well) and cultured in the presence of 10 to 20 µg/mL fibrinogen or its fragments. Cell growth was measured by the 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. CellTiter 96 aqueous one solution (20 µL) cell proliferation assay kit (Promega) was added to 100 µL medium in the 96-well culture plate and incubated at 37°C in a humidified chamber for 2 to 3 hours. The absorbance was read at 490 nm using the Bio-Tek fluorescent microplate reader (Bio-Tek Instruments, Inc., Randburg, South Africa). Each sample was measured in triplicate.
Tube formation on Matrigel. Wells of 96-well FluoroNunc black plates were coated with 50 µL Matrigel basement membrane matrix (BD Bioscience, San Jose, CA) and allowed to gel at 37°C for 30 minutes. Endothelial cells growing on the tissue culture plastic were trypsinized, washed, and added to the matrix-coated wells at 2 x 104 per well. Unless otherwise noted, plating was done in fully supplemented growth medium: DMEM plus 10% FCS for CPAE and M-131 plus microvascular growth supplement (Cascade Biologics, Portland, OR) for human microvascular endothelial cells (HMVEC). Cells were incubated for 24 hours in the presence of fibrinogen fragments. To enhance images, calcein AM (Invitrogen, Carlsbad, CA) was added to the medium (10 µg/mL) and incubated for 15 minutes at 37°C. Digital images were taken by using a fluorescent microscope and analyzed by using ImageJ.
Activation of caspase-3 during tube formation in HMVEC. Sixteen-well chamber slides were coated with Matrigel (50 µL/well) at 37°C. HMVECs (4 x 104 per well) were added to the well and incubated for 8 hours in the presence of fibrinogen or its fragments at 30 µg/mL in the medium (100 µL/well). Cells were fixed with 2% paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were then treated with bovine serum albumin (BSA) and then incubated with Alexa 488–labeled antibody specific to cleaved caspase-3 (green; Cell Signaling, Danvers, MA) overnight at 4°C. Nuclei were stained with 4',6-diamidino-2-phenylindole (blue). Cells were observed under fluorescent microscope.
Generation of DLD-1 colon carcinoma cells that secrete C or C399tr. We engineered DLD-1 human colon carcinoma cells to secrete C or C399tr by transfecting the pSecTag construct encoding C or C399tr. Control DLD-1 cells were transfected with vector only. These cells were selected for zeocin resistance (designated C-DLD-1, C399tr-DLD-1, and mock-DLD-1, respectively). We inserted the 6-His and S tags in the KpnI/BamHI site in pSecTag vector (between the IgG
secretory signal and the NH2 terminus of the C or C399tr). The expression of C or C399tr in DLD-1 cells was tested by staining with FITC-labeled anti-6-His tag antibody. Briefly, we incubated the cells with Brefeldin A (Sigma-Aldrich, St. Louis, MO) that blocks transport of proteins from the endoplasmic reticulum to the Golgi apparatus at 10 µg/mL medium for 8 to 16 hours. Cells were washed with PBS and fixed with 3.7% formaldehyde for 20 minutes at room temperature. Cells were incubated with FITC-labeled anti-S tag antibody and observed under fluorescent microscope. We found that most of the C or C399tr DLD-1 cells were positive, but nontransfected or mock-transfected DLD-1 cells were not (data not shown). These cells were s.c. injected into severe combined immunodeficient (SCID) mice (one million cells per mouse) without further cloning or enrichment. The tumor growth was monitored using caliper, and tumor volume (v) was calculated as v = 0.4 (a x b2), where a is a maximum tumor diameter and b is a diameter at 90° to a.
Intravital microscopy of Met-1 cells. Thirteen nude mice were used in this study with eight mice serving as experimental mice treated via i.p. injection of C399tr and five mice serving as nontreated controls. Each mouse was s.c. transplanted with a small piece of the Met-1 metastatic mammary tumor, either on one side or both sides of the trunk, following a procedure that has been approved by the University of California, Davis Animal Use and Care Committee. All experimental mice were injected with C399tr (150 µg/d/mouse) everyday, except weekends for a total of 4 weeks. Starting from the day after tumor transplantation, the size of all tumors, in both the experimental mice and control mice, was measured via caliper 5 days weekly, Monday to Friday, but not on weekends. Individual growth curves were plotted using the measurements (see Fig. 4A). At the end of 4 weeks (28 days) post-tumor transplantation, all the animals were sacrificed. The lungs of all the experimental animals as well as the controls were scored for pulmonary metastasis via x10 magnification gross analysis.
|
C399tr lies in the 0.6 to 0.7 range, whereas the nontreated control lies in the 0.3 to 0.4 range. |
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C induced, but native fibrinogen or fragment D did not induce, apoptosis of endothelial cells. We determined the effect of fibrinogen, fragment D, and C on the proliferation of CPAE cells. The isolated recombinant C effectively blocked proliferation of CPAE (Fig. 1A and B
) and BAE (data not shown) cells in tissue culture at concentrations of <10 µg/mL, whereas native fibrinogen and fragment D did not have a noticeable effect at the same concentrations. We showed that the effect of C is due to induction of apoptosis, as evidenced by the detection of apoptotic cells by fluorescence-labeled Annexin V (Fig. 1C) and presence of activated caspase-3/caspase-7 (Fig. 1D) in C-treated cells.
|
C also blocked tube formation from HMVEC in Matrigel (Fig. 2A and B
), although it did not induce apoptosis in these cells grown in tissue culture flasks (data not shown). C induced caspase-3 activation in 8 hours in these cells in Matrigel (Fig. 2C). These results suggest that the human endothelial cells used are resistant to C-induced apoptosis in tissue culture but become sensitive to C during tube formation. In contrast, C did not affect the proliferation of CHO cells, HaCaT cells (transformed keratinocytes), primary human keratinocytes, or DLD-1 human colon carcinoma cells (data not shown). These results suggest that the C-induced apoptosis is specific to endothelial cells among the several cell types tested.
|
C or C399tr grew slower than nonsecreting control cells in SCID mice. We decided to test whether C and C399tr affect angiogenesis in vivo by using tumor xenograft models. It has been reported that platelets play a role in tumor growth (3, 4, 16, 17). Because platelet integrins IIbß3 recognizes the COOH-terminal AGDV motif of C, the binding of C in vivo to this integrin potentially influences tumor growth in vivo. We thus generated the C399tr mutant, in which the COOH-terminal 12 residues, including the AGDV motif, are deleted. We found that C399tr induced apoptosis of BAE and CPAE cells (data not shown), blocked tube formation of HMVEC in Matrigel (Fig. 2A and B), and activated caspase-3 in Matrigel in these cells (Fig. 2C) to the extent similar to that of C. These results suggest that the COOH-terminal 12 residues of C are not critical for inducing endothelial apoptosis in vitro.
We first engineered DLD-1 cells to secrete C or C399tr by transfecting them with a pSecTag construct encoding C or C399tr (designated C-DLD-1 and C399tr-DLD-1, respectively). Control DLD-1 cells were transfected with the vector only (designated mock-DLD-1). We found that mock-DLD-1, C-DLD-1, and C399tr-DLD-1 cells grew at similar rates in tissue culture (Fig. 3A
), suggesting that the secretion of C or C399tr has a minor effect on the growth of DLD-1 cells in vitro. We s.c. injected the transfectants into SCID mice (one million cells per mouse) without further cloning or enrichment. We found that both C-DLD-1 and C399tr-DLD-1 cells grew much slower than mock-DLD-1 cells in SCID mice (Fig. 3B and C), suggesting that the secretion of C and C399tr affects tumor growth in vivo indirectly through blocking angiogenesis.
|
C399tr blocked the tumor growth, intratumoral vascularization, and metastasis of the highly metastatic Met-1 breast cancer in nude mice. We studied whether the recombinant C399tr protein affects the tumor growth, intratumoral microcirculation, and metastasis in a xenograft model with the use of a breast cancer cell line, Met-1 (15, 18, 19), containing the polyoma virus middle T transgene, which forms highly vascularized tumors and metastasizes effectively in nude mice (15, 20). Histology and computer-assisted intravital microscopy showed that Met-1 exhibited high microvessel density and tortuosity. These indices of microvascular complexity correlated with the high Met-1 metastatic rate (15, 20). C399tr (i.p. injection of 150 µg protein/mouse, once daily, 4-5 days weekly) markedly reduced the growth of the Met-1 tumor (Fig. 4A
), suggesting that C399tr effectively blocked tumor growth. Intravital microscopic study (Fig. 4B) shows that the tumors from the C399tr-injected mice are sparsely vascularized. The intratumoral microvessels in the C399tr-injected mice are underdeveloped and nontortuous, whereas those in the tumors in the non-C399tr-injected control mice were well developed, densely distributed, and tortuous. These results suggest that C399tr directly affects angiogenic development and the development of the intratumoral microvasculature. Similar to previous studies, all of the control mice that were not treated with C399tr (n = 5) had massive pulmonary metastasis (100%). In contrast, mice injected with C399tr (n = 8) had much less metastasis: five of eight mice had no detectable metastasis, two had confined pulmonary metastasis, and one had discoloration (suspected metastasis) but was confirmed not to have any metastatic lesions based on histopathology. Occurrence of pulmonary metastasis in the treated mice differed significantly (P < 0.05) from the nontreated controls. These results suggest that C399tr effectively blocked angiogenesis, leading to suppressed growth and diminished metastasis.
Endothelial cell-binding sites of C are cryptic in native fibrinogen. Because C and C399tr induced, but native fibrinogen and fragment D did not induce, apoptosis in endothelial cells, fibrinogen and its fragments may have different abilities to bind to endothelial cells. We determined whether FITC-labeled fibrinogen and its fragments bind to endothelial cells using flow cytometry. We found that C and C399tr bound to CPAE cells, but native fibrinogen and fragment D did not (Fig. 5A
). This observation suggests that the determinant(s) in fibrinogen and fragment D that are involved in apoptosis of endothelial cells are cryptic but become exposed in C and C399tr. Integrin vß3 is a major integrin in CPAE and BAE cells (21). We found that the C bind to vß3 on the cell surface, but native fibrinogen and fragment D did not (Fig. 5B). This result suggests that the binding site for vß3 is cryptic in fibrinogen and fragment D but is exposed in C and C399tr and that it is likely that vß3 is one of the C/C399tr-binding proteins on endothelial cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C and C399tr induced apoptosis of endothelial cells in vitro. Importantly, DLD-1 colon cancer cells that secrete these fibrinogen fragments grow slower than mock-transfected cells in SCID mice. Further, we showed that purified C399tr protein suppressed the tumor growth, development of intratumoral vasculature, and metastasis in vivo in the Met-1 model. Because C and C399tr induced, but native fibrinogen or fragment D did not induce, apoptosis of endothelial cells, it is expected that the determinant involved in endothelial cell apoptosis is cryptic. We provided evidence that this is really the case. These results suggest that fibrinogen has a novel cryptic determinant, which can exert apoptosis-inducing activity on endothelial cells when exposed and that C399tr has a therapeutic potential as an antiangiogenic agent.
FDPs are implicated in tissue injury associated with the adult respiratory distress syndrome, disseminated intravascular coagulation, and septic shock (2), in which plasma concentrations of FDP are markedly elevated (3, 4). Activation of fibrinolysis and the resulting generation of FDPs contribute to lung vascular injury (5). Fragment D detaches BAE monolayers from the substratum (6) and increases CPAE monolayer permeability to 125I-albumin (7). Apparently, the cryptic determinant identified in the present study plays a role in vascular injury in such pathologic situations. It has also been reported that FDP-induced cell death of giant trophoblasts leads to embryonic lethality in thrombomodulin-deficient mice (22) and that low-molecular weight FDP suppressed the proliferation of cultured globular mesangial cells (23). It is interesting to study whether C is involved in the antiproliferative effects induced by FDP in nonendothelial cells in future studies.
Integrins are candidate receptors involved in C-induced endothelial apoptosis because several integrins bind to C and because there is evidence that ligand binding to integrins can induce apoptosis. Indeed, snake venom echistatin induces apoptosis through intracellular signaling on binding to integrin vß3 (24). In addition, the binding of several ligands to integrins activates mitogen-activated protein kinases (stress-activated c-Jun NH2-terminal kinase and p38) that are related to apoptosis (25, 26), in addition to extracellular signal-regulated kinase1/2 that is related to cell proliferation. By engineering DLD-1 cells to secrete C or C399tr, we showed that the deletion of the COOH-terminal domain did not affect the ability of C to block the growth of DLD-1 cells in vivo. This suggests that the binding of platelet integrin IIbß3 to the COOH-terminal AGDV motif is not important for the tumor-suppressive effect of C or C399tr. It has been reported that fibrin(ogen) is a physiologically relevant ligand for Mß2, and integrin engagement of fibrin(ogen) is critical to leukocyte function in vivo (27). Because the Mß2-binding site (e.g., the NRLSIGE sequence, residue 390-396 of C) is exposed in C399tr (28), we do not rule out the possibility that leukocytes are involved in suppressing tumor growth by C399tr in vivo. Because C does not expose the Mß2-binding site (28), it is unlikely that C requires leukocyte binding for blocking tumor growth in vivo.
We provided evidence that the binding site for vß3, a major integrin in CPAE and BAE cells (21), is cryptic in fibrinogen and fragment D but is exposed in C and C399tr and that it is likely that vß3 is one of the C/C399tr-binding proteins on endothelial cells. However, anti-vß3, anti-ß1, and anti-ß3 antibodies did not block the binding of C to CPAE cells (data not shown). This is possibly because C or C399tr is too small for antibodies to induce steric hindrance. In addition, antagonists to vß3 (e.g., anti-vß3 monoclonal antibodies) could not be used to test whether vß3 is critical for C-induced apoptosis because antagonists to vß3 by themselves induce the apoptosis of endothelial cells (29). It is thus necessary to clarify whether integrins are involved in the C-induced apoptotic process using integrin antagonists that do not induce apoptotic effects on endothelial cells in future studies.
|
Acknowledgments |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Lan Yu for the help in animal studies.
|
Footnotes |
|---|
Received 5/ 9/06. Revised 6/30/06. Accepted 7/31/06.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain of human fibrinogen. Structure 1997;5:125–38.[Medline]vß3. J Biol Chem 2000;275:16891–8.vß3 to the fibrinogen and E chain C-terminal domains. Biochemistry 1999;38:5872–7.[CrossRef][Medline] subunit, and ligand binding. Biochemistry 2002;41:4339–47.[CrossRef][Medline]vß3-mediated activation of apoptosis. Exp Cell Res 1999;251:33–45.[CrossRef][Medline]v integrin, p38 mitogen-activated protein kinase, and urokinase plasminogen activator are functionally linked in invasive breast cancer cells. J Biol Chem 2001;276:47901–5.2ß1 mediates isoform-specific activation of p38 and upregulation of collagen gene transcription by a mechanism involving the 2 cytoplasmic tail. J Cell Biol 1999;147:401–16.Mß2/Mac-1 is critical for host inflammatory response in vivo. J Clin Invest 2004;113:1596–606.[CrossRef][Medline]Mß2 in the C-domain of fibrinogen. Biochemistry 2002;41:12942–51.[CrossRef][Medline]vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994;79:1157–64.[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  M. Guo, D. Daines, J. Tang, Q. Shen, R. M. Perrin, Y. Takada, S. Y. Yuan, and M. H. Wu Fibrinogen-{gamma} C-Terminal Fragments Induce Endothelial Barrier Dysfunction and Microvascular Leak via Integrin-Mediated and RhoA-Dependent Mechanism Arterioscler. Thromb. Vasc. Biol., March 1, 2009; 29(3): 394 - 400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Parmar, L. R. Berry, M. Post, and A. K. C. Chan Effect of covalent antithrombin-heparin complex on developmental mechanisms in the lung Am J Physiol Lung Cell Mol Physiol, March 1, 2009; 296(3): L394 - L403. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Springer, J. Zhu, and T. Xiao Structural basis for distinctive recognition of fibrinogen {gamma}C peptide by the platelet integrin {alpha}IIb{beta}3 J. Cell Biol., August 25, 2008; 182(4): 791 - 800. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mori, C.-Y. Wu, S. Yamaji, J. Saegusa, B. Shi, Z. Ma, Y. Kuwabara, K. S. Lam, R. R. Isseroff, Y. K. Takada, et al. Direct Binding of Integrin {alpha}v{beta}3 to FGF1 Plays a Role in FGF1 Signaling J. Biol. Chem., June 27, 2008; 283(26): 18066 - 18075. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, 10 µg/mL; 
, 1 µg/mL) for the indicated time and caspase-3/caspase-7 activities in the cell lysates were measured. The data suggest that 
