A Novel Peptide from Human Apolipoprotein(a) Inhibits Angiogenesis and Tumor Growth by Targeting c-Src Phosphorylation in VEGF-induced Human Umbilical Endothelial Cells

Abstract

Many angiogenesis inhibitors are derived from large plasma proteins. Previous studies showed that the Kringle5-like domain (termed KV) in human Apolipoprotein (a) is a potential antiangiogenic factor. However, its active region and the underling molecular mechanism remain elusive. Here, we identified an 11-amino acid peptide (named KV11) as the key region for the anti-angiogenic function of the KV domain of apolipoprotein (a). We demonstrate that KV11 inhibits angiogenesis in vitro by suppressing human umbilical vein endothelial cell (HUVEC) migration and microtubule formation. KV11 inhibits angiogenesis in chicken chorioallantoic membrane (CAM) assays and mouse corneal micropocket angiogenesis assays in vivo. KV11 peptide shows no effect on tumor cell growth or proliferation, but significantly inhibits tumor growth in SCID mouse xenograft tumor model (p<0.01) by preventing tumor angiogenesis. We elucidate that KV11 peptide suppresses angiogenesis and tumor progression by targeting the c-Src/ERK signaling pathways. Together, these studies provide the first evidence that KV11 from apolipoprotein KV domain has anti-angiogenesis functions and may be an anti-tumor drug candidate.

Introduction

Angiogenesis, the formation of new blood vessels from preexisting ones, is a complex multi-step process, including the destabilization of established vessel, endothelial cell proliferation, migration , and new tube formation. Angiogenesis plays a crucial role in pathological conditions such as tumor progression¹. Both tumor growth and metastasis depend on the expansion of host vasculatures into tumors through angiogenesis². Therefore, targeting tumor angiogenesis is an important therapeutic approach for cancer treatment and therapy.

Vascular endothelial growth factor (VEGF) is an important signaling protein involved in angiogenesis via regulating intracellular signaling networks³. There are numerous preclinical anticancer models through blockading of the VEGF signaling pathways which are among the best known validate approaches for antitumor⁴. Upon stimulation of VEGF, c-Src is generally activated, followed by FAK activation and the down-stream signaling networks. The active complex of c-Src and FAK regulates the small GTPases and in turn mediates the activation of MAPK cascade ⁵. Many angiogenesis inhibitors are endogenous short anti-angiogeneic peptides such as PF-4 peptide, endostatin fragments, and tumstatin peptide ⁶ , suggesting that these peptides may be the core fragments in the anti-angiogenic functions.

Apolipoprotein(a) [apo(a)] contains tandemly repeated Kringle domains followed by a single Kringle 5-like domain (termed KV), and an inactive protease-like domain ⁷ . Previously studies have shown that a truncated recombinant Apo(a) fragment containing the KV domain (86 amino acids) of human Apolipoprotein (a) is potent angiogenesis inhibitors ⁸ , but the key biologic activity region of KV domain and its mechanism involved in angiogenesis are still unknown.

In this study, we identified and characterized a novel 11- amino acid peptide with the sequence of YTMNPRKLFDY (named KV11) from the KV domain. We found that the KV11 peptide inhibited VEGF-induced HUVEC cell migration and tube formation in vitro. In vivo assay showed that KV11 peptide suppressed VEGF-induced angiogenesis in chicken embryo chorioallantoic membrane (CAM) assays and mouse corneal micropocket assays. Furthermore, in mouse xenograft tumor growth assays, we found that the KV11 peptide can significantly suppress the growth of human breast cancer while KV11 had little effect on tumor cell growth per se. Treatment of mouse xenograft tumor models with the KV11 peptide significantly increased the survival of the xenograft tumor mice compared to control group. In addition, we examined the signaling pathway about angiogenesis of the KV11 peptide. Results showed that the KV11 peptide selectively blocked the signaling pathways from the VEGF-stimulated Src-FAK to Cdc42– ERK activation. These interesting results suggest that the KV11 peptide is a key active region of apo (a) KV domain and a potential anti-angiogenesis and anti-tumor therapeutic agent.

Materials and Methods

Reagents and Animals

Medium 199 was purchased from Sigma. Dulbecco's Modified Eagle's medium (DMEM) was from Hyclone Company. VEGF was from R&D System provided by Biological Resources Branch, NCI-Frederick Cancer Research and Development Center. Matrigel was from BD Biosciences. Fertilized chicken eggs were purchased from Shanghai Poultry Breeding Co. Ltd. C57BL/6 mice were from National Rodent Laboratory Animal Resources, Shanghai Branch in China. Severe Combined Immunodeficiency mice (SCID/Ncr) were purchased from National Cancer Institute, USA.

Mice were maintained according to the NIH standards established in the “Guidelines for the Care and Use of Experimental Animals,” and all of the experimental protocols were approved by the Animal Investigation Committee of Texas A&M University.

Peptides Sequence Analysis and Synthesis

Using the cysteine in disulfide bonds for cleavage sites (not including cysteine), human Apolipoprotein (a) KV domain was mainly separated into four peptides: P1, MFGNGKGYRGKKATTVTGTP; P2, QEWAAQEPHRHSTFIPGTNKNAGLEKNY; P3, RNPDGDINGPW; and P4, YTMNPRKLFDY (Figure 1A). These four peptides were analyzed by DNAStar software package and BioEdit software.

Figure 1. Peptide analyses in KV domain of Apolipoprotein (a) and P1–P3 proliferation and migration assay.

(A) The Kringle domain (KV) of human apolipoprotein (a). Since the cysteine residues (bigger white circle) of disulfide bonds are the cleavage sites, the KV domain of Apo (a) mainly contain four short peptides: P1, ⁵-MFGNGKGYRGKKATTVTGTP-²⁴; P2, ²⁶-QEWAAQEP-HRHSTFIPGTNKNAGL EKNY-⁵³; P3, ⁵⁵-RNPDGDINGPW-⁶⁵ (grey); and P4, ⁶⁷-YTMNPRKLFDY-⁷⁷. (Note, the numbers are the amino acid position in the whole KV domain, and the first cysteine from N-terminus was number 4). (B) Bioinformatics analysis of the four peptides by the Protean Program in DNAStar software. The antigenic index (Jameson-Wolf), surface probability (Emini), and hydrophilicity (Kyte & Dolittle) were analyzed. The high antigenic index regions are from amino acids 5–15, 50–60, 65–75. These regions are from the anterior segment of P1, large part of P3, and almost the whole P4, respectively. P4 also has high mean hydrophilicity region. (C–E left panel), P1–P3 peptide has little effect on HUVEC proliferation. HUVECs were plated at 5000 cells/well of 96-well plate. KV11 peptide with different concentrations was added into the cell culture and cells were then measured using the CellTiter96 AQueous One solution cell proliferation assay kit (Promega). (C–E right panel), migration assay of P1–P3 migration. HUVECs were plated, scraped, and incubated in medium with 4 ng/ml VEGF in the presence or absence of various concentrations of P1–P3 peptide. Quantitative measurement of cell migration were shown. Data are expressed as the migrated cell number in the VEGF-treated cultures. The data shown are representative of three independent experiments. * p < 0. 05 versus VEGF-treated control.

Using ExPASy (http://www.expasy.ch/tools /protparam.html) ⁹ , We analyzed the half –lifes of P1–P4 , results(P4,i.eKV11 , 8 hours; P1 , 30 hours; P2,0.8 hours; P3, 1 hour) showed all of them are more than that of recombinant human VEGF-165 (approximately 30 min¹⁰).

The solid-phase synthesis was performed by H.D Biosciences Co. Ltd in Shanghai, P.R China. The purified peptide was characterized by means of amino acid analysis, analytical HPLC, and mass spectrometry with the purity more than 95%.

Cell Culture

Human umbilical vascular endothelial cells (HUVEC) were kindly gifted from Dr. Xinli Wang (Cardiothoracic Surgery Division of the Michael E. DeBakey Department of Surgery at Baylor College of Medicine Hospital) and cultured in M199 containing 100 mg/L heparin, 15 mMol/L HEPES, and 20% fetal bovine serum (FBS) ¹¹. Human breast cancer cell MCF-7 were obtained from the American Type Tissue Collection and cultured in DMEM supplemented with 10% fetal bovine serum, 0.1 mg/mL streptomycin and 25 units/mL penicillin¹² .

Cell Proliferation Assays

Proliferation studies were carried out using the CellTiter96 AQueous One solution cell proliferation assay (Promega) as previously described ¹³. Briefly, cells were plated at about 5000 HUVECs or MCF-7cells /well in 96-well plate and allowed to adhere to the plate with different concentration peptides (P1–P4, P4 named KV11 here). The cells were incubated for 48–72 hours and then the AQueous One solution was added to the samples and measured at 490 nm.

HUVEC Wound-Healing/ Migration Assay

To determine the effect of P1–P4 peptides on HUVEC migration toward VEGF, wound-healing assays were performed using HUVEC cells. Cells were allowed to grow to confluency on six-well plates and washed twice with PBS. Monolayer cells were wounded by scratching with 1 ml-pipette tip and washed three times with PBS. Fresh medium was added with 4 ng/ml of VEGF and different concentration of peptides. This was allowed to incubate for 24 hours and pictures were taken using a Nikon digital camera ¹³.

HUVEC Boydem Chamber Migration Assay

As described previously ¹⁴, Boydem chamber migration assay assays were performed. 24-well transwell (Corning Incorporated) migration chambers having an 8-µm pore size were used. The transwells were coated with 0.1% gelatin for 30min in 37°C and then washed twice with PBS. The bottom chambers were filled with 600 µl medium supplemented with 10ng/ml VEGF. The top chamber was seeded with approximately 4 × 10⁴ HUVE cells/well in 100 µl containing different concentrations of KV11 (P4) peptide. Cells were allowed to migrate for 4 hours at 37°C. After incubation, cells on the top surface of the membrane were gentlely scraped with cotton swabs. Cells on the bottom side of the membrane were fixed with 4% paraformaldehyde for 20min, washed three times with PBS, and then stained with hematoxlin and eosin. The cells were then destained in PBS, and the membrane was left to air dry at room temperature. Migrated cells were counted using inverted microscope. Three independent areas per filter were counted, and the mean number of migrated cells was calculated.

HUVEC Matrigel Tubule Formation Assay

Matrigel (BD Biosciences) was thawed overnight on ice. Each well of 24-well plates was coated at 4°C with 100µl matrigel and incubated at 37 °C for 1 hour. HUVECs were harvested and approximately 4 × 10⁴ cells in 1 ml medium with various concentrations of KV11 peptide were seeded into the wells. After 12–16 hours, microtube formation was assessed with an inverted photomicroscope and the images were photographed using Nikon digital camera.

Tubular structures were quantitated by manual counting of low power fields and percentage of inhibition was expressed using untreated wells as 100% ¹⁵.

Chicken Embryo Chorioallantoic Membrane (CAM) Assays

According to previous method ¹⁶, embryonic eggs were incubated in 38.5–39°C with the relative humidity at 65–70%. Five days later, a 1–2 cm² window was opened. The shell membrane was removed to expose the CAM. As the carrier, 6 mm-diameter Whatman filter disk which absorbed KV11 peptide solution was put on the CAM. Only PBS in the carrier was the control group. Then the window was sealed with plastic tape and eggs were incubated again. All the above procedures were done under aseptic conditions. Four days later, the CAM microvessels were observed under stereomicroscope and the neovascularization was quantified as described ¹⁷. Assays for each test sample were carried out three times and each experiment contained 10–20 eggs per data point.

Mouse Corneal Micropocket Assay and Histological Examination

The mouse corneal assay was performed according to procedures previously described ¹⁸ . Briefly, corneal micropockets were created with a modified von Graefe cataract knife in one eye of each 5 to 6-week C57BL/6 mice. A micropellet (0.35×0.35 mm) of sucrose octasulfate-aluminum complex (SIGMA-Aldrich, Inc) coated with hydron polyhydroxyethylmethacrylate [polyHEMA, Aldrich] containing saline, 160ng VEGF or 160ng VEGF plus 1µg KV11 peptide was implanted into each corneal pocket, positioned about 1 mm from the corneal limbus. After implantation, chlortetracycline hydrochloride ophthalmic ointment was applied to each operated eye to prevent infection. Data and photos were obtained on day 7 after pellet implantation. The area of neovascular response, vessel length and clock hours of new blood vessel of 10–11 mice in each group were calculated according to the formula Area(mm²)= 0.2×3.14×VL(mm)×CN(mm) , here CN is the clock hours of neovascularization, where 1 clock hour equals 30 degrees of arc, (VL) = the maximal vessel length extending from the limbal vasculature toward the pellet)¹⁹.

And then , mouse corneas were dissected and fixed in 10% formaldehyde. The tissues were sectioned in 3 µm slices, and subjected to hematoxylin and eosin (H&E) staining according to previous method²⁰ .

Xenograft Tumor Growth Assay in SCID mice

5-week-old SCID mice were weighed, coded, and divided into experimental groups at random (n = 6). 8×10⁶ MCF-7 cells in 100 µl medium were injected subcutaneously into the right sides of the dorsal area as described ²¹ . Peptides was intralesionally injected ²² at the dose of 50 µg peptide/100 µl /mouse/day. Control groups were injected only with PBS. The growth of the tumor xenograft was evaluated in a pilot study by determining the tumor volume using digital caliper every 3 days. Tumor volume was calculated using the relationship A×B²×0.52 ²³, where A is the longest diameter and B the shortest. Mice were continually observed until death or were killed when moribund.

Histology and Immunohistochemistry

Tumors were removed and fixed and embedded with paraffin. Specific blood vessel staining was performed on the 5-µm sections with CD31 antibody ¹². Images were taken with ZEISS Axioskop 40 photo microscope. The number of blood vessels was counted.

Western Blot and Immunoprecipitation

HUVEC lysates were sampled with lysis buffer (50mM Tris-HCl, pH7.4, 1% Nonidet P-40, 150mM NaCl, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride, 1µg/mM aprotinin, 1µg/mL leupeptin, 1mM sodium orthovanadate and 1mM NaF). Either c-Src or FAK was precipitated from the cell lysates (500µg of total proteins) with the appropriate antibodies for 3 hour at 4°C, and followed by collecting the precipitated complex with protein A-Sepharose beads for 3 hours at 4°C. The precipitates were washed three times with ice-cold lysis buffer and resolved by 8% SDS-PAGE. Protein levels were assessed by immunoblotting with the specific antibodies and detected by the chemiluminiscence solution.

Statistical Analysis

Results are expressed as the mean of at least triplicate determinations and statistical comparisons are based on Student’s t test or analysis of variance. A probability value of 0.05 was considered to be significant.

Results

Identification of KV11 peptide as a potential inhibitor of angiogenesis from the KV domain of Apolipoprotein (Apo) (a)

Previous data had shown that a truncated recombinant Apo (a) fragment containing the KV domain (86 amino acids) of human Apolipoprotein (a) were potent angiogenesis inhibitor^{7, 8}. However, the functional element in the KV domain of Apolipoprotein (a) is still not clear and their potential role in tumor angiogenesis is not fully understood. Structural analyses revealed that the KV domain of human Apolipoprotein (a) mainly contains four peptides, named P1 to P4 (Figure 1A). The 11 amino acid peptide of P4 was shown and named KV11 (Figure 1A). Using DNAStar and BioEdit software, we analyzed the antigenic index (Jameson-Wolf), surface probability (Emini), and mean hydrophilicity (Kyte & Dolittle) of these four peptides. As shown in Figure 1B, the high antigenic regions (above zero-reticle) were found mainly in the whole P4, anterior parts of P1, P3, and the posterior part of P2. Furthermore, we found that only P4 has high surface probability and high mean hydrophilicity regions. Similar results were obtained using other methods, such as Eisenberg analysis, suggesting that P4 (KV11) is a potentially active peptide with high antigenic index, surface probability, and mean hydrophilicity compared to other three peptides of the KV domain.

To identify the key element in KV domain of Apo (a) protein in anti-angiogenesis, we examined the potential effects of the four peptides (P1–P4) on cell proliferation and wound-healing/migration. As shown in Figure 1C–1E left panel and Figure2A, all of P1–P4 did not inhibit HUVEC proliferation. In wound-healing/migration, little effect was observed with other P1–P2peptides (Figure 1C–1D right panel), The result also showed that KV11 peptide (P4) can inhibit HUVEC migration at the concentration of 15µM (Figure2C right panel) but P3 have no effect on it at this concentration (Figure1E right panel) and KV11 showed more significant inhibition on migration than P3. The inhibitory effect of KV11 peptide on HUVEC migration is dose-dependent from the range of 7.5– 120 µM of peptide concentrations and the IC50 for this assay is about 120µM (Figure 2C).

Figure 2. Inhibition of HUVEC migration, and tube formation by KV11 peptide in vitro.

(A) KV11 peptide has little effect on HUVEC proliferation. The assay was performed as Figure1C–1E. (B) Inhibition of HUVC Boyden chamber migration by KV11 peptide. 4 × 10⁴ cells were placed into the top chamber of Boyden chamber (0.8µm) coated with gelatin. VEGF was added to the bottom chamber and incubated at 37°C for 4 hours. HUVECs were then fixed and stained with H&E and counted under the microscope. ** p < 0.01. (C) KV11 peptide inhibits wound-healing migration. HUVECs were plated, scraped, and incubated in medium with 4ng/ml VEGF in the presence or absence of various concentrations of KV11 peptide. The HUVECs migrated into the scraped area in the presence of VEGF. KV11 peptide significantly inhibited the migration of HUVEC to the wounded area. Representative photomicrographs of cells treated with VEGF (left), and cells treated with VEGF and KV11 peptide (right) were shown. Dotted lines indicate the area occupied by the initial scraping. Bars=200µm. The left panel is the quantitative measurement of cell migration. Data are expressed as the migrated cell number in the VEGF-treated cultures. The data shown are representative of three independent experiments. * p < 0. 05; and ** p < 0.01 versus VEGF-treated control. (D) KV11 inhibited HUVEC tubule formation. Representative photomicrographs of tubule formation in the control and KV11-treated groups were shown. About 4 × 10⁴ cells were plated in 24-well plates previously coated with matrigel, and incubated for 16 hours at 37°C in the absence or presence of KV11 peptide (right). Bars =200µm. Tubular structures were quantitated by mannual counting under low power fields. “Percentage (%) of control” is the mean number of tubules expressed as a proportion of that in the control group. ** p<0.01 and *** p<0.001.

The endothelial cell proliferation is very important and migration is critical step during the angiogenesis. According to above result, we select P4 (KV11) to study its further activity of angiogenesis inhibition. And then , we tested how KV11 affects cell migration using the modified Boyden chamber assays. HUVECs were placed in the upper chamber of modified Boyden chamber containing membrane coated with 0.1% gelatin and 8 ng/ml of human VEGF was used as the chemo-attractant for HUVEC migration assays. The addition of 10 µM KV11 peptide to the cells dramatically arrested cell migration from the upper chamber to the lower one in a dose-dependent manner (Figure 2B).

Effect of KV11 peptide on microvascular endothelial cell tubule formation

The complex process of angiogenesis involves the ordered assembly and alignment of endothelial cells as well as cell migration. In vitro, endothelial cells can spontaneously align and form a three-dimensional tubular capillary-like network on matrigel²⁴. To examine the effect of KV11 peptide on human vascular endothelial cell tubule formation, we examined the tubule formation ability of HUVECs using 24-well plate coated with matrigel for 12 hours with or without KV11 peptide. As shown in Figure 2D, addition of 100µM KV11 peptide to the culture medium dramatically inhibited the ability of HUVEC cells to form tubular structures in matrigel as compared in absence of KV11 (Figure 2D). The number of microvascular tubule structures in the matrigel in the presence of KV11 peptide was quantitatively analyzed and KV11 peptide significantly inhibited HUVEC tubule formation on matrigel in a dose-dependent manner (Figure 2D right panel).

KV11 peptide inhibits angiogenesis in vivo

To study the in vivo antiangiogenic activity, we examined the effect of KV11 peptide on angiogenesis using the chorioallantoic membrane (CAM) assays. As shown in Figure 3, KV11 peptide inhibited new embryonic blood vessel growth (Figure 3) at the concentration of 0–1600µM per disk. Within the avascular areas (the circle area of 15 mm diameter with about 176 mm² around the filter paper dick), the number of microvessels (pointed with black arrows) of KV11 peptide-treated group was much less than that of untreated group (Figure 3A and 3B). The number of newly-formed blood vessels was significantly blocked in a dose-dependent manner over the range of 0–1500µM KV11 peptide per disk (Figure 3C) without inflammation. These data suggest that KV11 peptide suppresses angiogenesis in chicken embryos.

Figure 3. Inhibition of angiogenesis by KV11 peptide in the chicken embryo chorioallantoic membrane (CAM) assays.

(A) The control CAM assay with a filter paper (white disk) containing PBS alone. (B) A representative picture of KV11 peptide-treated CAM, showing inhibition of new blood vessel growth. (C) KV11 inhibited the number of microvessels within a defined area surrounding the implanted disk. **, p<0.01 and ***, p<0.001. Bars =3mm. As the carrier, Whatman filter papers (6 mm) with different amounts of KV11 peptide were implanted on the CAMs of 5-day-old embryos. After 4 days, the formation of avascular areas was analyzed under the stereomicroscope.

To further investigate the anti-angiogenic activity of KV11 peptide in vivo, the inhibitory effect of KV11 on VEGF-induced corneal neovascularization was examined in the mouse corneal micropocket angiogenesis assays (Figure 4). Micropellets coated with the slow release polymer-hydron containing VEGF were implanted into the avascular corneas of C57BL/6 mice. New blood vessels stimulated by 160 ng VEGF was detected on day 7 after implantation (Figure 4C). The KV11 peptide significantly inhibited VEGF-induced angiogenesis (Figure 4D). The density of corneal vessels of the mouse group treated with VEGF and KV11 peptide was markedly suppressed comparing with the group treated with VEGF only (Figure 4C and 4D). The vessel length, clock hours and vessel area were all inhibited by 50–70% in the corneas of 10 mice examined in the absence or presence of KV11 peptide (Figure 4E–4G).

Figure 4. Inhibition of angiogenesis by KV11 peptide using the mouse corneal micropocket model.

(A–D) Micropellets containing 160 ng of VEGF were implanted into corneal micropockets of C57BL/6 mice as described in materials and methods. The effects of KV11 on angiogenesis in vivo were examined using slow-releasing polymer containing saline alone (A), KV11 peptide alone (B), 160 ng VEGF alone (C), or 160 ng VEGF and 1 µg KV11 peptide (D). Corneal neovascularization was measured and photographed with a stereomicrocsrope on day 7 after implantation. Positions of pellets were pointed with arrows. Bars =500µm. (E–G) KV11 inhibited vessel length, clock hours of circumferential neovascularization, and the area of neovascularization, respectively. The bio-microscopic assessment was conducted by two independent observers. Results are given as mean ± SEM. ***, p<0.001. (H–J) Histological analyses of mouse corneal assays in the absence or presence of KV11. Mouse corneas under different treatment (saline, VEGF or VEGF with KV11 peptide) were harvested and stained with hematoxylin and eosin (H&E). KV11 inhibited multiple lumen-like formations (arrows, some of them containing red blood cells) (J) compared to VEGF-treated group (I). Bars =10 µm.

To further examine the effect of KV11 peptide on cornea angiogenesis, we performed histological analyses of corneas treated with saline, VEGF, and VEGF together with KV11 peptide, respectively (Figure 4H–4J). VEGF (160 ng in the pellet) dramatically induced corneal neovascularization as observed by the large numbers of lumen-like formations in the section (Figure 4I) as compared to corneal section treated with saline only (Figure 4H). However, in the presence of KV11 peptide (1 µg in the pellet), VEGF-induced neovascularization was significantly reduced and diminished compared to the VEGF-treated control (Figure 4J). The number of multiple lumen-like formations (blue arrows) observed in the corneas of VEGF and KV11 peptide-treated group were significantly less than that of VEGF-treated group while more than that of saline-treated group (Figure 4H–4J), suggesting that KV11 peptide significantly inhibit VEGF-induced angiogenesis in corneal assay. In all treated mice over the course of experiment, no inflammation was found and no weight loss or unusual behavior was detected, suggesting that KV11 peptide had no toxicity at our experimental dose (1 µg in pellet treatment).

KV11 peptide inhibits tumor angiogenesis and suppresses tumor growth in SCID mouse model with little toxicity

To understand whether KV11 peptide directly affect tumor cell growth, we performed cell proliferation assay using breast MCF-7cancer cell line. As shown in Figure 5A, KV11 peptide at 100 µM did not affect tumor cell growth in vitro using MTS cell proliferation assays (P>0.05, Figure 5A), suggesting that KV11 had no direct effect on tumor cell proliferation. Given that tumor growth is angiogenesis-dependent (Norrby K., 2006) and that suppression of angiogenesis can inhibit tumor growth, we further examined the anti-angiogenic activity and tumor growth of KV11 peptide using SCID mouse model. MCF-7 cells were injected subcutaneously (s.c.) into SCID mice and the growth of tumor xenograft was evaluated with the treatment of KV11 peptide or PBS as control. As shown in Figure 5B and 5D, after a 100-day treatment with KV11 peptide, the mean volume and tumor size of KV11 peptide-treated group were much less than that of PBS-treated group in SCID mouse tumor model (about 70% tumor size reduction in KV11 treated group was observed, P<0.01) . To examine the inhibitory effect of KV11 on tumor angiogenesis, we stained the 5-µm tumor sections with CD31 antibody. The average vessel number in KV11-treated group was dramatically less than that in tumors of control group (Figure 5C), indicating that KV11 significantly inhibited tumor angiogenesis and prevented prostate tumor growth.

Figure 5. KV11 peptide inhibits tumor growth in vivo.

(A) KV11 has little effect on MCF-7 cell growth in vitro up to 100 µM of peptide concentrations using Cell Titer96 AQueous One solution cell proliferation assay. (B) KV11 dramatically inhibited tumor growth in xenographic tumor mouse model using SCID mice. Typical example of tumor-bearing mice of the PBS control (upper) or KV11-treated groups (bottom) on day 100. Arrows pointed to the tumor and bars equal 5 mm. (C) Effects of KV11 on tumor angiogenesis in xenograft mouse tumor model. Tumors were fixed and embedded with paraffin. The 5-µm sections were stained with CD31 antibody. The average vessel number in tumors of control group was significantly more than that of KV treatment group. (D) Quantitative analysis of tumor volumes between PBS-treated and KV11- treated tumor models. The tumor volume data are presented as mean ± SD tumor volumes in KV11 treated or control groups (**, P<0.01). (E) Survival rate of mice bearing tumors in control group and in KV11-treated group. Mice (6 per group) were monitored until they died or moribund. Median survival rate of mice in the control group was 110 days (black squares).

In addition, the survival rate of tumor-bearing mice began to decrease after 100 days and about 80% mice in control group were dead or moribund after 140 days due to tumor progression and metastasis (Figure 5E). However, in KV11-treated group, no mouse was dead or moribund after 140 days and no significant body weight loss was found comparing to the control group without tumors (data not shown), suggesting that KV11 can inhibit tumor growth and potential metastasis in SCID mouse model with little side-effect.

KV11 peptide inhibits VEGF-induced c-Src phosphorylation

To examine the molecular mechanisms of KV11 in anti-angiogenesis, we examined how KV11 affect VEGF-activated cell signaling pathways in HUVEC cells. Since c-Src phosphorylation and c-Src-mediated FAK phosporylation are essential for the VEGF signaling pathways and in angiogenesis⁵, we first examined the effects of KV11 peptide on VEGF-activated phosphorylation of c-Src and FAK. As shown in Figure 6A, addition of KV11 peptide significantly inhibited VEGF (4 nM)-stimulated phosphorylation and activation of FAK and c-Src in the signaling complex in a dose-dependent manner (Figure 6A). However, the effects of KV11 on the phosphorylation of c-Src/FAK complex are VEGF-specific since KV11 has little inhibitory effect on integrin-dependent FAK autophosphorylation (FAK phosphorylation at Y397) (Figure 6B).

Figure 6. KV11 peptide inhibits VEGF activation of c-Src, FAK, PAK, and ERK in HUVEC.

(A) KV11 inhibited VEGF-induced phosphorylation of c-Src, which resulted in the inhibition of c-Src-mediated FAK phosphorylation. Cells treated with the indicated for 5min and then sampled for the immunoprecipitation against FAK or c-Src. (B) KV11 did not affect FAK autophosphorylation in VEGF-activated HUVEC. To examine FAK autophosphorylation, FAK phosphorylation at Y397 was detected using anti-pFAK (Y397) antibody. (C) KV11 did not affect VEGF-induced activation of Rho GTPases. For Rac1 or Cdc42, GST-PBD (PAK-binding domain) was used for the pull-down assay. For RhoA, GST-Rhotekin was pulled down. (D) KV11 reduced VEGF-induced αPAK1 activation but not MLK3. To examine PAK activation, phosphorylated PAK was detected using p-αPAK1 antibody. (E) KV11 blocked VEGF activation of ERK. HUVEC was treated with the indicated for 20min. In case of a positive sample for the inhibition of ERK phosphorylation, cells were pretreated with 10µM of PD98059 for 30min and then stimulated with VEGF for another 20min. (F) KV11 suppresses VEGF-induced ERK phosphorylation. Cells were pretreated with KV11 for 1hr and then stimulated with VEGF for the indicated time points.

KV11 peptide downregulates PAK1 but not Rho GTPases

Rho GTPases are implicated in cell migration, and also regulated by FAK/c-Src complex⁵. Thus, we next examined whether KV11-inhibited c-Src/FAK activation affects Rho GTPases such as Rac1, Cdc42, and RhoA. As shown in figure 6C, KV11 did not significantly affect VEGF-induced Rho GTPases.

Since PAK (p21-activated protein kinase)/MLK (mixed lineage kinase) family is thought to be required for cell migration, and are linked to c-Src/FAK signaling during angiogenesis⁵, we further examined the effect of KV11 on the phosphorylation of PAK/MLK in HUVEC. In VEGF-stimulated HUVEC, KV11 peptide significantly inhibited the phosphorylation of PAK1 (Figure 6D). To examine whether KV11-inhibition of PAK1 was selective, the phosphorylation of MLK3, another member of PAK/MLK family, was examined. As shown in figure 6D, KV11 did not affect MLK3, suggesting that KV11 selectively regulate the VEGF-Src-FAK-PAK in cell migration.

Inhibition of VEGF-induced ERK phosphorylation by KV11 peptide

To further examine how KV11 peptide regulates the VEGF-induced signaling pathways, we analyzed the effect of KV11 peptide on VEGF-stimulated MAPK phosphorylation in HUVEC. As shown in Figure 6E, KV11 peptide inhibited VEGF-induced ERK phosphorylation in a dose-dependent manner while a phosphorylation of JNK and p38 was not changed in the presence of KV11 peptide (Figure 6E). To further confirm the effect of KV11 peptide on ERK phosphorylation, we pre-treated the cells with 10 µM KV11 peptide and assayed for the activation of ERK. As shown in figure 6F, a pretreatment of HUVEC with 10µM KV11 peptide completely inhibited VEGF-induced ERK phosphorylation.

Discussion

There are more than 80 amino acids in the KV domain of Apo (a) protein with four different fragments and three disulfide bonds. Although the domain has been shown to inhibit angiogenesis, it is unclear which fragment of the KV domain is the key active region and the underlying mechanism of anti-angiogenesis. In this study, we have identified and characterized a 11- amino acid peptide (KV11) with potent anti-angiogenic activity using both in vitro and in vivo assays, such as CAM and mouse corneal micropocket models²⁵. Furthermore, we demonstrated that KV11 inhibited tumor growth by blocking tumor angiogenesis in a xenographic tumor model using SCID mice with little toxicity. And finally, we showed that KV11 inhibited the VEGF-mediated c-Src phosphorylation and signaling pathways downstream of c-Src including FAK, PAK, and ERK in angiogenesis.

Angiogenesis is a complex multi-step process that involves cell proliferation, migration, and finally tube formation. Peptides or compounds that inhibit any of the multi-step processes will lead to the disruption of angiogenesis and can serve as potential candidates for therapeutic intervention of angiogenesis. It has been shown that suppression at any one of the steps in angiogenesis will inhibit the formation of new vessels and arrest tumor growth ²⁶. In this study, we demonstrate that KV11 peptide specifically suppressed HUVEC cell migration and tube formation but has little effect on cell proliferation, suggesting that KV11 peptide directly affect endothelial cells and act on the cell migration steps of the angiogenesis processes.

Since the growth of tumor is dependent on tumor angiogenesis and angiogenesis inhibition is a novel therapeutic modality towards controlling solid tumors ²⁷, we demonstrate that KV11 peptide significantly inhibits tumor growth in SCID mice with MCF-7 tumor cells but the peptide has little effect on the growth of MCF-7 cell in vitro even at high concentration (100 µM, Figure 5A). On the other hand, KV11 peptide inhibited HUVEC cell migration, and tube formation at low concentrations (15 µM in Wound-Healing/ Migration migration assay, 10 µM in Boyden chamber migration assay, and 1 µM in tube formation assays, respectively) (Figure 2). These results suggest that KV11 peptide inhibits tumor growth in vivo not directly through inhibiting tumor cell proliferation per se but through inhibiting tumor angiogenesis. Previous data showed that one of the widely-used anti-tumor drugs for clinic treatment of many malignancies, Cisplatin, can dramatically make tumor-bearing mice lost body weight and lower survival rate ²⁸. In our experiments, we found that KV11 peptide-treated mice did not show any body weight loss and their survival rate was much higher than that of the control groups, suggesting that KV11 peptide has lower side effect and little toxicity compared to traditional anti-tumor drugs.

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis, such as tumor growth and ischemic diseases ²⁹. In VEGF-stimulated intracellular signaling events, c-Src phosphorylation of FAK, and activation of Rho GTPases (RhoA, Rac and Cdc42) and their down-stream signaling pathways control cell migration⁵. In addition, it has been reported that PAK activation is required for the activation of ERK via Raf to MEK at different stimulation conditions⁵. Although it is not clear how KV11 selectively regulates c-Src-PAK-ERK in angiogenic endothelial cells, previous reports have suggested that the regulation of c-Src-mediated ERK phosphorylation as well as its localization control the cell dynamics, including cell adhesion and migration ^{5, 30}.

In conclusion, our results showed that a short 11-amino acid peptide (KV11) from Apo (a) is the potential biological active region of KV domain. We highlighted the roles of KV11 peptide in the inhibition of tumor growth through regulating cell migration, and tumor angiogenesis by selectively blocking VEGF-induced c-Src signaling pathway. In VEGF signaling pathway , ligands bind in an overlapping pattern to three cell surface receptor tyrosine kinases (RTKs), known as VEGF receptor-1, -2 and -3 (VEGFR1–3)31. VEGF receptor-2 is the primary receptor in VEGF signaling pathway that regulates angiogenesis 12. The exact receptor (s), the acting site (outside or inside the cells) through which KV11 inhibit angiogenesis and the activitiy comparing of KV11 and parent Kringle5 domain are under active investigation.