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Carcinogenesis Advance Access originally published online on September 4, 2008
Carcinogenesis 2008 29(11):2106-2111; doi:10.1093/carcin/bgn206
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Insulin-like growth factor-binding protein-5 (IGFBP-5) acts as a tumor suppressor by inhibiting angiogenesis

Seung Bae Rho*, Seung Myung Dong, Sokbom Kang1, Sang-Soo Seo1, Chong Woo Yoo1, Dong Ock Lee1, Jong Soo Woo and Sang-Yoon Park1

Research Institute, National Cancer Center, 809 Madu 1-dong, Ilsan-gu
1 Center for Uterine Cancer, Research Institute, National Cancer Center, dong, Ilsan-gu, Goyang-si, Gyeonggi-do 411-769, Republic of Korea

* To whom correspondence should be addressed. Tel: +82 31 920 2383; Fax: +82 31 920 2337; Email: sbrho{at}ncc.re.kr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Supplementary material
 References
 
Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family, important for cell growth control, induction of apoptosis and other IGF-stimulated signaling pathways. In this study, we focused on characterizing the specific function of IGFBP-5 as novel antiangiostatic factor. Overexpression of IGFBP-5 suppressed the tube formation as well as the biological functions of angiostatic activity in vivo. This result is due to the reduced expressions of phosphorylated protein kinase B and phosphorylated endothelial NO synthase, which plays important roles in the regulation of angiogenesis when stimulated by vascular endothelial growth factor. Further, IGFBP-5 expression prevented tumor growth and inhibited tumor vascularity in a xenograft model of human ovarian cancer. These results are the first evidence showing that IGFBP-5 plays a role as tumor suppressor by inhibiting angiogenesis.

Abbreviations: Akt, protein kinase B; CAM, chick chorioallantoic membrane; cDNA, complementary DNA; eNOS, endothelial NO synthase; FBS, fetal bovine serum; GFP, green fluorescent protein; HUVEC, human umbilical vein endothelial cell; IGFBP-5, insulin-like growth factor-binding protein; mRNA, messenger RNA; RT–PCR, reverse transcription–polymerase chain reaction; siRNA, small-interfering RNA; VEGF, vascular endothelial growth factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 Supplementary material
 References
 
Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family (1). By binding IGF with high affinity, IGFBPs are able to suppress or enhance the activity of IGFs in a tissue- and cell-dependent manner (1,2). Thus, IGFBPs are involved in various cellular processes including cell growth regulation, induction of apoptosis and other IGF-stimulated signaling pathways in many cell types (3–5).

IGFBP-5 is a soluble protein that comprises 252 amino acid residues. Similar to other IGFBPs, IGFBP-5 has highly conserved cysteine residues within both N- and C-terminal domains that form intradomain disulphide bond (1). IGFBP-5 interacts with several extracellular matrix protein including plasminogen activator inhibitor-1 (6), vitronectin (7), thrombospondin-1 (8) and osteopontin (8) to internalize into the cytosol and further translocates to the nucleus for the action (9,10). The functional role of IGFBP-5 is mainly emphasized on cell growth. Depending on the experimental scheme, IGFBP-5 either induces or suppresses cell proliferation (11–14). Based on these results, the effects of IGFBP-5 on cell survival seem to be dependent on specific soluble or cell-associated ligand interacting with IGFBP-5 (1,2,15).

For tumor therapy, controlling two events are important for deciding the fate of tumor cells during tumor progression. One is inducing an apoptosis and another is inhibiting angiogenesis. Recently, several observations reported that the overexpression of IGFBP-5 induces an apoptosis of some cancerous cells such as squamous cell carcinomas and breast cancer cells (15,16). However, no attempt has been made to study the effect of IGFBP-5 on angiogenesis. Angiogenesis, the sprouting of new blood vessels from pre-existing vasculature, is a crucial process in tumor growth and pathogenesis because it enables the supply of oxygen and nutrients to the growing tumor (17–19). One of the key modulators of blood vessel network during development is vascular endothelial growth factor (VEGF), which can promote the proliferation and invasion in endothelial cells (20). Thus, the activity of endothelial cells plays an essential role for the regulation of various vascular biological functions during tumor progression.

In the present work, we evaluated the functional role of IGFBP-5 during angiogenesis. We found that the overexpression of IGFBP-5 prevented the migration and tube formation of endothelial cells both in vitro and in vivo systems by inhibiting expressions of phosphorylated protein kinase B (Akt) and phosphorylated endothelial NO synthase (eNOS), important mediators during VEGF-induced angiogenesis. Moreover, we found that tumor growth and tumor vascularity were decreased in the presence of IGFBP-5 expression in a xenograft model of human ovarian cancer.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 Supplementary material
 References
 
Cell culture and antibodies
SKOV-3 ovarian cancer cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (Walkersville, MD) and cultured on 0.3% gelatin (Sigma, St Louis, MO)-coated dishes using the EGM-2 BulletKit medium (Clonetics). The EGM-2 BulletKit medium consists of a base medium containing 20 µM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Sigma), 5% heat-inactivated fetal calf serum and 1% penicillin–streptomycin (Gibco, New York, NY). HUVECs were used for experiments at passages 3–6. All cells were kept in an atmosphere of 5% CO2 in air at 37°C. The following antibodies were used in this study: anti-Akt (cat. # 9272, Cell Signaling Technology, Danvers, MA), anti-CD31 (PECAM-1) antibody (cat. # AF806, R&D Systems, Minneapolis, MN), anti-eNOS (cat. # 9572, Cell Signaling Technology), anti-GFP (cat. # sc-8334, Santa Cruz Biotechnology, Santa Cruz, CA), anti-IGFBP-5 (cat. # sc-34253, Santa Cruz Biotechnology), anti-phosphorylated Ser473-specific Akt (cat. # 9271, Cell Signaling Technology), anti-phosphorylated Ser1177-specific eNOS (cat. # 9571, Cell Signaling Technology) and anti-β-actin (cat. # sc-47778, Santa Cruz Biotechnology).

Construction of overexpression and small-interfering RNA systems of human IGFBP-5
The complementary DNA (cDNA) encoding full length of human Igfbp5 was obtained from reverse transcription–polymerase chain reaction (RT–PCR). Briefly, total RNA from 293 cells was isolated using a guanidium extraction method (21) and reverse transcribed using moloney-murine leukemia virus reverse transcriptase (Promega, Madison, WI) and random hexamers (Invitrogen, Grand Island, NY). After cDNA synthesis, PCR was performed with a PTC-100 thermal cycler (MJ Research, Watertown, MA) as follows: 30 cycles of 1 min at 94°C, 1 min at 60°C and 2 min at 72°C, followed by a 10 min final extension step at 72°C. Primer sequences used to obtain the full-length human Igfbp5 cDNA sequence were as follows: 5'-CACCTGCTCTACCTGCCAGAA-3' (sense) and 5'-GATGAAATGAGTGGCGTCCT-3' (antisense), resulting in a 1045 bp RT–PCR product. The resulting PCR products were separated on a 1% agarose gel containing ethidium bromide and were visualized by ultraviolet light. For expression of IGFBP-5, full-length cDNA of IGFBP-5 was cloned into pEGFP-C1 vector (Clontech, San Jose, CA) and was digested with BspEI and XhoI, and the subclones were sequenced by the dideoxy-mediated chain termination method using a 310 automatic sequencer (ABI 373, Perkin Elmer, Wellesley, MA). The resulting cloned cDNA sequence was 100% identical to human Igfbp5 (GenBank accession NM_000599 [GenBank] ) and was used for transfection using Lipofectamine (Invitrogen) according to the manufacturer’s protocols.

The small-interfering RNA (siRNA) oligonucleotide sequence targeting Igfbp5 (5'-CGGGAGTCTCTCTCGATCCCTGTCTC-3') corresponded to nucleotides 1123–1106 in the human sequence. The siRNA was synthesized using siRNA construction kit (Ambion, Austin, TX) and was then transfected by oligofectamine (Invitrogen), in accordance with the manufacturer’s protocols. After transfection of overexpressing Igfbp5 cDNA and siRNA of Igfbp5, the messenger RNA (mRNA) and protein expression levels of human IGFBP-5 were compared with mock transfectants (empty vector) by RT–PCR and western blot as described below.

RT–PCR analysis
For detecting expression of human Igfbp5, mRNA transcripts were detected by RT–PCR as described above. Glyceraldehyde-3-phosphate dehydrogenase mRNA transcripts were used in equal amounts alongside the experimental mRNA as an internal control. Primer sequences for human glyceraldehyde-3-phosphate dehydrogenase were as follows: 5'-ATGACCACAGTCCATGCCATCA-3' (sense) and 5'-CCTGCTTCACCACCTTCTTG-3' (antisense), yielding a 271 bp RT–PCR product.

Western blot analysis
Cells were harvested, washed in phosphate-buffered saline, centrifuged and resuspended in cell lysis solution (50 mM Tris, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin and 200 µg/ml phenylmethylsulfonyl fluoride). The cell lysates were subsequently resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto Immobilon P membranes (Millipore Corporation, Billerica, MA) and immunoblotted with relevant antibodies described above. Immunoreactive bands were visualized using an ECL system (GE Healthcare, Buckinghamshire, UK).

[3H]thymidine uptake assay
Cells were seeded at a density of 1 x 104 cells per well in Dulbecco’s modified Eagle’s medium containing 5% heat-inactivated fetal calf serum and 1% penicillin–streptomycin in the gelatinized plates on day 0. After 18 h, cells were incubated for 6 h in M199 containing 1% FBS and then stimulated with VEGF (10 ng/ml, R&D Systems) for 24 h in M199 containing 1% FBS. [3H]thymidine (0.5 µCi/ml, Amersharm, Arlington, IL) was then added 4 h prior to the assay. High-molecular mass [3H] radioactivity was precipitated using 5% trichloroacetic acid at 4°C for 1 h. Cells were solubilized in 0.2 N NaOH and 0.1% sodium dodecyl sulfate and [3H]thymidine uptake was evaluated with a liquid scintillation counter (Beckman Coulter, Fullerton, CA). Three independent experiments were conducted in triplicate; values shown represent means ± SDs of total c.p.m.

Migration and invasion assay
Migration and invasion were assayed using Transwells (Corning Costar, 8 µm, Rochester, NY) as described previously (22). For migration assays, the lower surface of a filter was coated with 10 µg of gelatin. M199 containing 1% FBS with VEGF (10 ng/ml) was placed in the lower wells. Cells were fixed and stained with hematoxylin and eosin (Sigma). Non-migrating cells on the upper filter surface were removed by wiping with a cotton swab. The numbers of cells that migrated to the lower side of the filter were counted under a light microscope and the mean values of eight fields were determined. For the invasion assays, the lower and upper surfaces of a filter were coated with 10 µg of gelatin and 10 µg of Matrigel (BD Biosciences, San Jose, CA), respectively. The fixation and quantification methods used were the same as described for the migration assay. Three independent experiments were conducted in triplicate and the values shown represent means ± SDs.

Expression and purification of recombinant IGFBP-5
cDNA encoding the full length of IGFBP-5 was isolated by RT–PCR described above and subcloned into pET28a (EMD Chemicals, Gibbstown, NJ) using EcoRI and XhoI. Then construct was expressed in Escherichia coli strain BL21 (DE3) grown in Luria–Bertani medium supplemented with kanamycin (75 µg/ml). Culture was grown at 37°C to an A600 = 0.4–0.5, transferred at 30°C and were induced by the addition of 0.4 mM isopropyl-1-thio-β-D-galactopyranoside for 4 h. The induced cells were then harvested and resuspended in 20 mM Tris–HCl (pH 8.0) containing 10% glycerol, 50 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid and 1 mM dithiothreitol. Cells were lyzed by ultrasonication and were centrifuged for 15 min to remove cell debris. The supernatant was filtered and loaded onto a nickel affinity column matrix (Invitrogen) and incubated at 4°C for 2 h. The slurry was pelleted by centrifugation and washed with washing buffer (20 mM Na2HPO4, pH 6.0, and 500 mM NaCl) three times. The pellet of the gel matrix was resuspended in elution buffer (20 mM Na2HPO4, pH 6.0, and 500 mM NaCl containing 400 mM imidazole) and incubated at 4°C for 20 min to elute the bound His fusion proteins. The His-tag was cleaved off with thrombin, and each protein was further purified using high-performance liquid chromatography (Waters 600, Waters, Milford, MA). Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin (Sigma) as a standard.

Chick chorioallantoic membrane assay
The chick chorioallantoic membrane (CAM) assay was performed with minor modifications (23). Fertilized chick embryos were preincubated at 37°C with 70% humidity in order to conduct the chorioallantoic membrane assay. After 3 days, a square window was opened after the removal of 2–3 ml of albumin to detach the developing CAM from the shell. A 1.5 x 1.5 cm window in the shell was made to expose the CAM. Clear tape was used to seal the windows that were formed and the eggs were incubated for 60 h. On day 8, CAMs were implanted, under sterile conditions within a laminar flow hood, with sterilized Thermanox discs. Thermanox discs were loaded with 100 ng of mock, IGFBP-5 or siIGFBP-5 recombinant protein, respectively. The CAM was examined daily until day 12 and photographed in ovo with an Axioskope2 plus microscope (Zeiss, Switzerland) equipped with color charge-coupled device camera (ProgResC14, Jenoptik, Jena, Germany). Recombinant human VEGF (100 ng/disk), incorporated into Thermanox discs, induced branching of blood vessels. Two independent, blinded investigators performed the count of blood vessels for each group. Three independent experiments were conducted in triplicate and the values shown represent means ± SDs.

Mouse tumor model
To determine the effect of IGFBP-5 in a murine tumor models, specific pathogen-free nu/nu mice (5-week-old female) were purchased from Charles River Laboratories (Wilmington, MA). To establish tumors in mice, 2 x 106 of SKOV-3 tumor cells were subcutaneously injected into the middorsal region. One hundred percent of mice were allowed to grow for 12 days. Then, 10 animals from each group were subcutaneously injected with 100 µg of mock or IGFBP-5 recombinant protein, respectively. An intratumoral injection of recombinant proteins was done thrice, once every 3 days. Tumor growth was determined by caliper measurements every 3 days. Tumor volume was calculated by the following formula: tumor volume (mm3) = (a x b2)/2, where a = length in mm and b = width in mm. Tumor volume and body weight was measured every other day. Mice were killed on day 24 after final injection. Tumors were then excised and prepared for immunohistochemistry.

Immunohistochemistry
Growing tumors were fixed in 10% neutral-buffered formalin (Sigma), embedded in Tissue-Tek OCT (Sakura, Tokyo, Japan) and frozen at –80°C. Sections measuring 5 µm were cut by using a Leica CM1800 cryostat (Leica, Wetzla, Germany) and air dried at room temperature. Slides were then stained with hematoxylin and eosin (Sigma) according to the manufacturer’s protocol. For immunohistochemistry, sections were treated with a saturating concentration of the biotin-conjugated anti-human CD31 (PECAM-1) antibody. For visualization of antibody, staining was conducted using EnVision+ System-HRP (DAB)TM according to the manufacturer’s protocol (Dako, Copenhagen, Denmark). Slides were then counter stained in Mayer’s hematoxylin (Sigma). Preparations were examined and photographed on an Axiophot 2 apparatus (Zeiss).

Statistical analysis
Student’s t-test was used to compare mean values for independent variables. Significant differences at 95% confidence interval (P < 0.05) are depicted with an asterisk (*) on each graph.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 Supplementary material
 References
 
Overexpression of IGFBP-5 inhibits VEGF-induced proliferation, invasion and tube formation
To evaluate the function of IGFBP-5 during VEGF-induced angiogenesis, we first attempted to construct overexpression and siRNA system of IGFBP-5 in HUVECs. To test whether siRNA transfection efficiently blocks endogenous IGFBP-5, we measured both mRNA and protein expression levels of IGFBP-5 overexpression transfectants [green fluorescent protein (GFP)–IGFBP-5 in Figure 1A)] as well as siRNA and overexpression cotransfectants of IGFBP-5 (GFP–IGFBP-5 + siRNA). As shown in Figure 1A, the mRNA and protein expression levels of IGFBP-5 in siRNA and overexpression cotransfectants (GFP–IGFBP-5 + siRNA) were significantly decreased comparing with overexpression transfectants (GFP–IGFBP-5). Endogenous level of IGFBP-5 expression was detected as low level in HUVECs (Figure 1A).


Figure 1
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  		Fig. 1. IGFBP-5 inhibits VEGF-induced proliferation, invasion and tube formation of HUVEC in vitro. (A) The construction of overexpression and siRNA system of IGFBP-5 in HUVECs. HUVECs were transfected with GFP-fused Igfbp5 cDNA alone (GFP–IGFBP-5) or cotransfected GFP-fused Igfbp5 cDNA with siRNA (GFP–IGFBP-5 + siRNA). After transfection, the cells were visualized under fluorescence microscope and analyzed the mRNA or protein expression levels by RT–PCRs and immunoblots. Size bar, 20 µm. (B) The effect of human IGFBP-5 on the proliferation of HUVECs. Each transfectants was cultured in the presence of VEGF for 3 days. The c.p.m. value of [3H]thymidine was determined with a liquid scintillation counter. Data are shown as means ± SDs. *P < 0.05. (C) The effect of human IGFBP-5 on the invasion of HUVECs. Each transfectants was tested using Matrigel-coated Transwell for the invasion assays followed by stimulation with or without VEGF for 48 h. Numbers of invaded cells were counted under a light microscope and mean values were determined. Data are shown as means ± SDs. *P < 0.05. (D) The effect of human IGFBP-5 on the tube formation of HUVECs. Each transfectants was seeded on growth factor-reduced Matrigel and then treated with or without VEGF for 48 h. The formation of tubular structures was detected by an inverted microscope. Tube lengths were quantified and are expressed as the means ± SDs. *P < 0.05.

 
Next, we overexpressed IGFBP-5 in HUVECs and determined whether IGFBP-5 had angiostatic activity. First, we tested their effects on VEGF-induced proliferation of endothelial cells by measuring [3H]thymidine incorporation (24). The VEGF-induced DNA synthesis was observed in both untransfected HUVECs and mock (vector only) transfectants comparing with those of unstimulated cells (Figure 1B). As shown in Figure 1B, the overexpression of IGFBP-5 markedly inhibited cell proliferation comparing with those of mock transfectant and untransfectant. Also, the inhibition of IGFBP-5 by siRNA (siIGFBP-5) reversed the inhibitory effect of IGFBP-5 on HUVEC cell proliferation (Figure 1B). This inhibitory effect was not due to the cell cytotoxicity of IGFBP-5 in endothelial cells since IGFBP-5 had no effect on the viability of HUVECs in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (data not shown). These results indicate that IGFBP-5 specifically inhibits VEGF-induced endothelial cell proliferation.

To further investigate whether ectopic expression of IGFBP-5 modulates the effects of VEGF on endothelial cell invasion, we performed Transwell invasion assays. VEGF enhanced the invasion of untransfected HUVECs and of mock transfectant comparing with that of unstimulated cells as expected (Figure 1C). Through the assays, we found that the overexpression of IGFBP-5 significantly reduced VEGF-stimulated invasion, but siIGFBP-5 did not (Figure 1C). Therefore, ectopic expression of IGFBP-5 inhibits key events during the VEGF-stimulated angiogenic process, such as the proliferation and invasion of endothelial cells in vitro.

To confirm the antiangiogenic effects of IGFBP-5 on VEGF-induced angiogenesis, we performed endothelial tube formation assay. Untransfectant or mock transfectant incubated with VEGF formed an organized network of endothelial cells on Matrigel (Figure 1D). In contrast, ectopic expression of IGFBP-5 markedly inhibited VEGF-stimulated tube formation (Figure 1D). The inhibitory effect of IGFBP-5 on VEGF-stimulated tube formation was completely recovered by IGFBP-5 siRNA transfectant (siIGFBP-5).

CAM assay was performed to test for antiangiostatic activity of IGFBP-5 on in vivo angiogenesis. As shown in Figure 2, recombinant human IGFBP-5 clearly inhibited VEGF-stimulated angiogenesis ~75% in CAMs. Again, antiangiostatic activity of IGFBP-5 was completely abolished by siIGFBP-5 treatment (Figure 2). These observations strongly demonstrated that IGFBP-5 effectively suppressed the formation of blood vessels in vitro and in vivo.


Figure 2
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  		Fig. 2. Angiostatic activity of IGFBP-5 in the CAM assay. (A) Digital images of in vivo angiogenesis assay in CAM. The open box indicate the point at which the mock, IGFBP-5 or siIGFBP-5-treated coverslips were loaded onto the CAM of the developing eggs. (B) A quantitative evaluation of the antiangiostatic activity. The data are presented as the mean number of blood vessel branches ±SD from 6–9 embryos in each sample. *P < 0.05.

 
IGFBP-5 suppresses the phosphorylation of Akt and eNOS
To define the mechanism how IGFBP-5 inhibits angiogenesis, we investigated whether IGFBP-5 effects on the phosphorylation of Akt and eNOS, two key molecules during VEGF-stimulated angiogenesis (25). As presented in Figure 3, VEGF-stimulated phosphorylated Akt (Ser473) and eNOS (Ser1177) were dramatically reduced by the IGFBP-5.


Figure 3
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  		Fig. 3. IGFBP-5 interferes with VEGF-induced phosphorylation of Akt and eNOS in HUVECs. HUVECs were stimulated for 20 min with VEGF. Then, phosphorylated Akt and eNOS were visualized by immunoblotting with antibodies that were specific for phosphorylated proteins. Detection of the total Akt and eNOS served as a loading control.

 
IGFBP-5 suppresses tumor growth by antiangiogenic activity in vivo
To explore the possibility that IGFBP-5 has direct antitumor activity, we tested the effects of IGFBP-5 on tumor cell growth in vivo. Briefly, SKOV-3 ovarian cancer cells were subcutaneously injected into nude mice in group of 10. One hundred percent of mice were allowed to grow for 12 days. Each group was subcutaneously injected with mock or IGFBP-5 recombinant protein, respectively. Tumor growth and morphology were analyzed over 24 days at 3 days interval. We allowed the tumors to grow until they reached a mean volume of 100 mm3. As shown in Figure 4A, the volume of IGFBP-5-treated tumors was 70% smaller than those from mock mice. Moreover, immunohistologic staining of endothelial cells in the IGFBP-5-treated mice showed an 82% decrease in the number of blood vessels stained with anti-CD31 (PECAM-1) (Figure 4B). These results indicated that IGFBP-5 overexpression is capable of suppressing tumor growth by inhibiting angiogenesis in vivo.


Figure 4
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  		Fig. 4. IGFBP-5 suppresses tumor growth by inhibiting angiogenesis in vivo. (A) Intratumoral injection of IGFBP-5 inhibits SKOV-3 ovarian cancer cell growth as xenografts in nude mice (n = 10). Mean tumor volume was determined by caliper measurements (upper panel). Bars, SD. *P < 0.05. (B) Frozen sections of the tumors were stained for endothelial cells using an anti-CD31 (PECAM-1) antibody. *P < 0.05.

 
We measured the endogenous IGFBP-5 expression in SKOV-3 cells and MDAH2774 cells (another ovarian cancer cell line). As shown in supplementary Figure 1 (available at Carcinogenesis Online), the endogenous IGFBP-5 expression in two ovarian cancer cells is relatively lower than those of normal cells such as HEK293 cells (human normal kidney epithelial cell line) and MRC-5 cells (human normal lung fibroblast line). We also did mouse tumor model using MDAH2774 cells. Results are very similar to the observation seen in mouse tumor model using SKOV-3 cells (supplementary Figure 2 is available at Carcinogenesis Online). Thus, results are very consistent with other ovarian cancer cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 Supplementary material
 References
 
Many studies on the biological functions of IGF in mammals have been published so far. IGFs are regulated by at least six members of the IGFBP family. As such, members of IGFBP family play a major role in the development and regulation of many tissues. Especially, increasing evidence supports the view that IGFBPs could be suppressors in malignant tumors (15,16,26–29).

In this study, we first focused on characterizing the specific function of IGFBP-5 as a novel antiangiogenic protein. Endothelial cell proliferation and migration are initial steps critical to the angiogenesis process. IGFBP-5 significantly inhibited endothelial cell proliferation and migration. In addition, IGFBP-5 directly inhibited the tube formation of endothelial cells on Matrigel and reduced the expression of phosphorylated Akt and phosphorylated eNOS in HUVECs. Phosphorylated Akt and eNOS play a significant role in angiogenesis when stimulated by VEGF (25). The observations from our study show that IGFBP-5 had little to no effect on unphosphorylated Akt and eNOS. These data indicate that IGFBP-5 has a direct inhibitive effect on angiogenesis in vitro. We also observed that exogenous IGFBP-5 has a direct inhibitive effect on endothelial cell migration and tube formation using a CAM model.

Previous observation indicated that IGF-1 and VEGF increased the IGFBP-5 expression in large vessel endothelial cells (30). During all the experiments, we have not used exogenous source of IGF-1. Although we detected the low level of IGF-1 expression in endogenous level of HUVECs (data not shown), we believed that this level of expression does not affect on IGFBP-5 expression. Supporting this notion, only IGF-1 (100 ng/ml)-treated HUVECs showed the high expression of IGFBP-5 (supplementary Figure 3 is available at Carcinogenesis Online). Thus, this suppressive effect of IGFBP-5 on HUVECs might be IGF-1 independent. Similar observation was seen in recent article, which described that IGFBP-5 has a suppressive effect on head and neck squamous cell carcinoma (15). To measure the effect of VEGF on IGFBP-5 expression, we treated VEGF on HUVECs and measured the expression pattern of IGFBP-5 expression. Similar to previous observation (30), VEGF increased the level of IGFBP-5 expression in HUVECs by dose-dependent manner (supplementary Figure 4 is available at Carcinogenesis Online). During all the experiments, we used exogenous source of VEGF at the concentration of 10 ng/ml and this dose of VEGF slightly increased IGFBP-5 expression in HUVECs.

Recently, many molecules that inhibit tumor angiogenesis have been identified and characterized. These include antagonists of angiogenic growth factors, receptors, integrin–adhesion molecules and matrix proteinases, some of which are currently subjects of clinical trials (31,32). However, one of the major challenges in terms of designing such therapies is that there are numerous direct and indirect mechanisms by which tumors can induce new blood vessel growth. It is therefore reasonable to assume that successful antiangiogenic protocols must address each of the possible mechanisms by which tumors can induce new blood vessel growth. These new therapeutic agents could prospectively be added to chemotherapy or radiotherapy regimens or could also be used in combination with immunotherapy or vaccine therapy.

With our findings, it is clear that IGFBP-5 is a vital component in the inhibition of oncogenesis. We provide the first evidence to our knowledge that IGFBP-5 exerts a direct antiangiogenic effect on endothelial cells. We further postulate that IGFBP-5 provides a new mode for angiogenic regulation and a novel therapeutic target for the angiogenesis-related vascular diseases such as cancer. Further study of IGFBP-5 and its inhibiting characteristics may create effective future cancer therapies.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
Supplementary material
 References
 
National Cancer Center (NCC-0810410-1); Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A040004).


   Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Supplementary material
References
 
Supplementary Figures 1–4 can be found at http://carcin.oxfordjournals.org/


   Acknowledgments
 
We thank Dr S.A.Martinis (Department of Biochemistry, University of Illinois at Urbana-Champaign) and Richard Yoo (University of Washington) for critical reading of the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Supplementary material
 References
 

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Received July 2, 2008; revised August 12, 2008; accepted August 23, 2008.


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