Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Jul 15.
Published in final edited form as: Cancer Res. 2010 Jun 22;70(14):5880–5890. doi: 10.1158/0008-5472.CAN-09-4341

Identification of a metalloprotease-chemokine signaling system in the ovarian cancer microenvironment: implications for anti-angiogenic therapy

Anika Agarwal 1,2, Sarah L Tressel 1, Rajani Kaimal 1, Marianthi Balla 1, Francis H Lam 1, Lidija Covic 1,2,3, Athan Kuliopulos 1,2,3
PMCID: PMC2917243  NIHMSID: NIHMS210306  PMID: 20570895

Abstract

Ovarian cancer is lethal gynecologic malignancy that may benefit from new therapies that block key paracrine pathways involved in tumor-stromal interactions and tumor vascularity. It was recently shown that matrix metalloprotease-1 (MMP-1) activation of the G protein-coupled receptor protease-activated receptor-1 (PAR1) is an important stimulator of angiogenesis and metastasis in peritoneal mouse models of ovarian cancer. In the present study we tested the hypothesis that MMP1-PAR1 promotes angiogenesis through its paracrine control of angiogenic chemokine receptors. We found that MMP1-PAR1 activation induces secretion of several angiogenic factors from ovarian carcinoma cells, most prominently IL-8, GRO-α, and MCP-1. The secreted IL-8 and GRO-α acts on endothelial CXCR1/2 receptors in a paracrine manner to cause robust endothelial cell proliferation, tube formation and migration. A cell penetrating pepducin, X1/2pal-i3, that targets the conserved third intracellular loop of both CXCR1 and CXCR2 receptors significantly inhibited endothelial cell proliferation, tube formation, angiogenesis and ovarian tumor growth in mice. Matrigel plugs mixed with MMP1-stimulated, OVCAR-4 conditioned media showed a dramatic 33-fold increase in blood vessel formation in mice. The X1/2pal-i3 pepducin completely inhibited the MMP1-dependent angiogenesis as compared to a negative control pepducin or vehicle. Conversely, a VEGF-directed antibody, Avastin, suppressed angiogenesis in mice, but as expected, was unable to inhibit IL-8 and GRO-α dependent endothelial tube formation in vitro. These studies identify a critical MMP1-PAR1-CXCR1/2 paracrine pathway that might be therapeutically targeted for ovarian cancer treatment.

Keywords: Ovarian cancer, angiogenesis, PAR1, MMP-1, IL-8, GRO-α, X1/2pal-i3 pepducin

Introduction

Chemokines have traditionally been viewed as attractants for inflammatory leukocytes, however, accumulating evidence suggests that they play important roles in modulating the tumor-stromal microenvironment (1–3). Ovarian cancer is a lethal disease that recruits a network of chemokines, proteases, and their receptors to grow and metastasize with no anatomical barriers to prevent peritoneal dissemination (4). Inflammatory chemokines such as CXCL8 (IL-8), CXCL6 (IL-6), CXCL1 (GRO-α) and CCL2 (MCP-1) are highly upregulated in epithelial ovarian cancers (5–7) and are postulated to play pivotal functions in tumor growth, inflammation, angiogenesis and metastasis (2, 6, 8).

IL-8 and GRO-α, the ligands for the CXCR1 and CXCR2 chemokine receptors, are produced by the ovulating follicle to attract leukocytes and assist in ovulation (9), and IL-8 and IL-8 antibodies have been detected in the serum of ovarian cancer patients (10). Upregulated IL-8 expression has been shown to be a marker of poor prognosis in breast cancer (2, 11) and more recently has been associated with poor clinical outcome and worse survival in ovarian cancer patients (12). IL-8 is a strong stimulator of capillary tube formation via both CXCR1 and CXCR2 receptors (13) and co-culturing of cancer cells with stromal fibroblasts causes an induction in IL-8, GRO-α and GRO-β oncogenes (14). Moreover, GRO-α is expressed at higher levels in the tissue and serum of ovarian cancer patients as compared to normal women (15).

Recent work has shown that thrombin activation of PAR1 causes induction of GRO-α in breast carcinoma and melanoma (16). PARs are a unique class of G protein-coupled receptors that are activated by proteolytic cleavage of their extracellular domains (17). PAR1 has been identified as an oncogene and a potent invasogenic receptor for breast, ovarian, melanoma and prostate carcinoma cells (18–24). We recently found that matrix metalloprotease-1 (MMP-1), also functions as a protease agonist of PAR1 cleaving the receptor (22, 25) to generate PAR1-dependent migration, invasion and angiogenesis in breast and ovarian tumors (22, 23, 26, 27). Furthermore, we found that fluids from patients with ovarian cancer harbor high levels of MMP-1 that correlates with malignancy. Inhibition of PAR1 results in pronounced reduction of stromal infiltration and angiogenesis in mouse models of breast and ovarian cancer. However, little is known regarding the mechanism of the MMP1-PAR1 signaling system in paracrine communication to the endothelial cells and angiogenesis.

Here, we tested the hypothesis that MMP-1 activated PAR1 is a critical regulator of cancer-stromal communication and angiogenesis in ovarian carcinomas through paracrine control of chemokine production and endothelial CXCR1/2 receptors. To block these angiogenic chemokine receptors, we used cell-penetrating pepducins based on the intracellular loops of CXCR1 and CXCR2 (28). Pepducins are lipidated peptides that act on the inside surface of the cell membrane and block signaling between their cognate receptor and its G protein effectors. The first (i1) and third (i3) loops of CXCR1 and CXCR2 receptors are identical and pepducins based on these loops are potent antagonists of both receptors. Pepducins have have been extensively studied in the context of PAR and chemokine receptor signaling and have been shown to possess drug-like properties through pharmacologic and efficacy studies in various animal models of angiogenesis, cancer, thrombosis and inflammation (22, 23, 25–30).

We found that MMP1-PAR1 stimulation of ovarian carcinoma cells leads to secretion of angiogenic chemokines, in particular IL-8 and GRO-α. Media from MMP1-stimulated ovarian carcinoma cells produced a florid angiogenesis both in vitro and in mice. The X1/2pal-i3 pepducin completely inhibited the MMP-1 effects in the angiogenesis models indicating that the MMP1-PAR1-CXCR1/2 paracrine system may be an attractive new target to block angiogenesis in ovarian cancer.

Materials and Methods

Pepducins

The CXCR1/2 pepducins X1/2pal-i3 (C15H31CONH-RTLFKAHMGQKHRAMR-NH2), X1/2LCA-i1 (lithocholic-CONH-YSRVGRSVTD-NH2), and the PAR1-based negative control pepducin P1pal-19E (C15H31CONH-RCESSSAEANRSKKERELF-NH2) were synthesized by standard fmoc solid phase methods as before (29, 30). Palmitic acid and lithocholic acid were dissolved in 50% N-methyl pyrolidone/50% methylene chloride and coupled overnight to the deprotected N-terminal amine of the peptide. After cleavage from the resin, palmitoylated peptides were purified to >95% purity by C18 or C4 reverse phase liquid chromatography and dissolved in DMSO as before (28).

Reagents

Quantikine ELISAs for human IL-8, GRO-α and VEGF-A-165, were obtained from R&D Systems and used as recommended by the manufacturer. Recombinant human IL-8 and GRO-α were commercially obtained from PeproTech. Pure proMMP-1 was obtained from EMD BioSciences, FN-439 (MMP Inh-1) from Calbiochem and human α-thrombin was from Haematologic Technologies and 3-(4,5-dimethylthiazole-2-yl)-2,5-dipheyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich.

Angiogenesis array

The human Angiogenesis antibody array (AAH-ANG-1) was bought from RayBiotech. The array membranes were incubated with either MMP-1 or buffer-stimulated OVCAR-4 media and used as recommended by the manufacturer. Results were calibrated using Image J by comparing signal intensities in the membranes after simultaneous exposure to X-ray film. Stock solutions of 800 nM proMMP-1 were activated with 2 mM APMA in 50 mM Tris, pH 7.7, 5 mM CaCl2, 0.2 M NaCl, 50 μM ZnCl2 at 37°C for 30 min and then transferred to ice. The APMA was then removed by overnight dialysis in 10 kDa MWCO Mini Slide-A-Lyzers (Pierce) at 4°C as previously described (23).

Cell culture

OVCAR-4, OVCAR-3, IGROV-1 cells were obtained from the NCI (Frederick, Maryland) and were grown in RPMI with 10% FBS. Cells were serum starved for 7 h in RPMI with 0.1% BSA then stimulated with MMP-1 or vehicle and conditioned media (CM) was collected 18 h later. OVCAR-4 PAR1 shRNAi cells were stable cell lines prepared by transfecting OVCAR-4 cells with PAR1 shRNAi from Sigma using OligofectAMINE and selecting clones with puromycin (0.5μg/ml). HUVEC cells were bought from Cambrex (Lonza) and cultured in EBM2 media with Bullet kit and 10% FBS. HUVEC cells were serum starved in EBM2 media with 0.5% BSA.

Flow cytometry

A rabbit polyclonal antibody specific to PAR1 was purified by peptide affinity chromatography as previously described (20). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody was purchased from Zymed. Flow cytometry was performed on ovarian carcinoma cells as before (23). CXCR1 (IL8-RA) and CXCR2 (IL8-RB) chemokine receptor antibodies were bought from R&D Systems.

Tube formation assay

MatTek plates were chilled to 4°C and coated with 100 μl Matrigel per well. 35,000 freshly passaged HUVEC cells (P2–5) in EBM2 media with 0.5% BSA were plated on matrigel-coated MatTek plates and stimulated with either IL-8, GRO-α, or MMP1-stimulated OVCAR-4 CM. MMP1-stimulated CM was quenched with FN-439 before adding the CM to the HUVEC cells. Endothelial tube morphogenesis was carried out in the presence or absence of X1/2pal-i3, X1/2LCA-i1, P1pal-19E pepducins (300 nM), Avastin (0.25 mg/ml), or vehicle (0.2% DMSO). Endothelial tube formation was observed after 18 h and endothelial tubes were photographed under phase contrast microscopy using an inverted Olympus microscope. Quantification of the digital images was performed in a blinded manner by counting total tubes per five 40x fields and quantified for tube length and branch complexity using NIH ImageJ software. Tube formation was expressed as fold change or percentage over the controls.

MTT assay

HUVEC cells were plated in 96 well plates and subjected to various treatment conditions or vehicle (0.2% DMSO) for 72 h. MTT reagent was added at a concentration of 0.5 mg/ml and allowed to incubate at 37°C for 5 h. The resulting formazan crystals were dissolved with 100% DMSO and absorbance was measured on a SPECTRmax 340 microplate reader (Molecular Devices).

Matrigel plug assays and ovarian cancer xenografts in mice

All animal experiments were conducted in full compliance with Tufts Medical Center Institutional Animal Care and Use Committee. Female NCR Nu/Nu mice (5–7 weeks) were purchased from Taconic farms. Mice were injected subcutaneously into their flanks with 300 μl matrigel plugs consisting of equal volumes of CM and matrigel. Animals were subsequently treated with either X1/2pal-i3, X1/2 LCA-i3, P1pal-19E pepducin (5 mg/kg/d × 7 d), Avastin (5 mg/kg) or vehicle (15% DMSO) in 100 μL volumes. At the end of 7 days, mice were euthanized and plugs were excised and fixed in 10% formalin/PBS. For ovarian cancer xenografts, OVCAR-4 cells were injected subcutaneously into each flank of female NCR Nu/Nu mice. The mice were treated 24 h later with either X1/2pal-i3 or vehicle (15% DMSO) until day 30. Tumor volume was calculated using the formula, V = length × diameter2/2. On day 30, mice were euthanised and tumors were harvested for assessment of angiogenesis.

Confocal microscopy of matrigel plugs and ovarian cancer xenografts

Subcutaneously implanted matrigel plugs or tumors were removed, cleaned, and fixed overnight with 10% formalin. Plugs were blocked for 1 h with 5% goat serum in Tris buffered saline containing 0.3% Triton-X 100 (TBST) and incubated overnight at 4°C with primary antibody CD31 (Millipore) diluted 1:1000 in TBST. After several washes with TBST, plugs or tumors were then incubated with fluorescently tagged secondary antibody Cy3-anti-hamster (Jackson Immunolabs) diluted in TBST for 4 h. Plugs and tumors were washed and post-fixed with 4% paraformaldehyde for 10 min. Whole mounted matrigel plugs and tumors were imaged using a Leica TCS SP2 confocal microscope. Confocal images were constructed into 3D projections of Z-stacks. Quantification of images was performed using NIH ImageJ.

Statistical analysis

All in vivo and in vitro data are presented as mean ± SD or mean ± SE. Comparisons were made with Wilcoxon-Rank Sum Student’s t test following ANOVA analyses. Statistical significance was defined as * P<0.05, ** P<0.005.

Results and Discussion

MMP-1 induces chemokine production from ovarian cancer cells in a PAR1-dependent manner

MMP-1 activation of PAR1 has recently been implicated in tumor angiogenesis of breast and ovarian cancers (22, 23, 27) but the mechanism of action of PAR1-dependent tumor-endothelial cell communication is not well understood. Therefore, we first characterized the profile of angiogenic factors that resulted from stimulation of PAR1 in ovarian cancer cells (Fig. 1A). We exposed a high PAR1-expressing ovarian carcinoma cell line, OVCAR-4, to MMP-1 and found that several angiogenic factors were secreted into the conditioned media (CM). As shown in Fig. 1A, the CXCR1/2 chemokines IL-8 and GRO (α/β/γ), and the CCR2 chemokine MCP-1 were the most highly upregulated angiogenic/inflammatory factors with a 4–5.5 fold increase above baseline (P<0.005). Thrombin, a PAR1 agonist, is known to upregulate VEGF-A in chick allantoic membrane and human vascular smooth muscle cells (31, 32), however we noted no significant change in VEGF-A levels (the 2 major isoforms 165 and 121) following MMP-1 stimulation and a slight increase in VEGF-D (Fig. 1A). Other chemotactic and angiogenic factors such as angiogenin were increased by 1.5–3 fold following MMP-1 stimulation of OVCAR-4 cells (Fig. 1A). We focused on the CXCR1/2 chemokine receptors and their two major agonists IL-8 and Gro-α because we have recently developed the first dual antagonist pepducins targeted against both receptors (28).

Figure 1.

Figure 1

Open in a new tab

MMP-1-PAR1 stimulates secretion of CXCR1/2 chemokines from ovarian carcinoma cells.

A, OVCAR-4 cells were stimulated with either 1 nM MMP-1 or PBS buffer in RPMI with 0.1% BSA and conditioned media (CM) was collected after 18 h. The angiogenesis array membranes were incubated with CM from the OVCAR-4 cells and fold change of the spot intensities was quantified using NIH ImageJ software.

B, Relative surface PAR1 expression was measured by flow cytometry as before (20) in OVCAR-4, IGROV-1, OVCAR-3 and OVCAR-4/PAR1 shRNAi ovarian carcinoma cell lines.

C–E, ELISA analysis was used to measure IL-8 (C), GRO-α (D), and VEGF-A-165 (E) levels in CM from various ovarian carcinoma cell lines that were stimulated with 1 nM MMP-1 in the presence or absence of the PAR1 antagonist pepducin P1pal-7 (3 μM), the small molecule PAR1 antagonist RWJ-56110 (5 μM), or buffer control as indicated. Data (mean ± SE) are from 2–4 experiments performed in duplicate.

To confirm the findings of the cytokine array we tested whether MMP-1 stimulated IL-8 and GRO-α secretion in several ovarian cancer cell lines expressing varying levels of PAR1. PAR1 surface expression was quantified on the OVCAR-4 (high), IGROV-1 (medium) and OVCAR-3 (low) ovarian cancer cells by FACS using a PAR1-specific antibody (Fig. 1B). In addition, we performed stable knockdown of PAR1 in the high PAR1 expressing OVCAR-4 using shRNAi (Fig. 1B, Supplementary Fig. S1). ELISA analysis validated that MMP-1 treatment caused increased secretion of IL-8 from PAR1-expressing OVCAR-4 and IGROV-1 cells (P<0.005) but had no effect in the low PAR1-expressing cell line, OVCAR-3, or following gene silencing of PAR1 in OVCAR-4 (Fig. 1C). A similar pattern in GRO-α secretion was confirmed by a GRO-α ELISA for OVCAR-4 and IGROV-1 cells whereas the low PAR1 expressing OVCAR-3 and OVCAR-4/PAR1-shRNA cells did not show an increase in GRO-α when stimulated with MMP-1 (Fig. 1D).

We also blocked the effect of PAR1 in the ovarian cancer cells with a PAR1 small molecule antagonist RWJ-56110 (33) and a PAR1 antagonist pepducin, P1pal-7 (23, 27, 29, 30). Pepducins are lipidated peptides based on the intracellular loops of the G-protein coupled receptor that have been previously validated in animal models of thrombosis, sepsis and cancer (23, 27, 29, 30, 34–36). We observed that IL-8 induction by MMP-1 and thrombin was inhibited by RWJ-56110 or P1pal-7 (Fig. 1C, Supplementary Fig. S2A) confirming that the protease-induced production of IL-8 was PAR1-dependent. The PAR1 inhibitors RWJ-56110 and P1pal-7 were also able to inhibit the GRO-α increases induced by MMP1 and thrombin (Fig. 1D, Fig 2A, Supplementary Fig. S2B). By comparison, VEGF-A-165 levels (Fig. 1E) remained essentially unchanged (0.9–1.15 fold) following MMP-1 stimulation in the tested ovarian cancer cell lines. As was observed with MMP-1, thrombin stimulation of PAR1 caused the secretion of IL-8 and GRO-α, and gave only a slight induction in VEGF-A-165 in OVCAR-4 and OVCAR-3 cells (Supplementary Fig. S2C).

Figure 2.

Figure 2

Open in a new tab

CXCR1/2 chemokines produced by MMP-1 stimulation of OVCAR-4 cells lead to tube formation of endothelial cells. OVCAR-4 cells (0.2 million) were plated in each well of a 6 well plate, serum starved and then treated overnight with either PBS buffer or 1 nM MMP-1 in the presence or absence of 0.2% DMSO vehicle, P1pal-7 (5 μM), or RWJ-56110 (5 μM). CM was collected and the residual MMP-1 activity was quenched with FN439 (5 μM). CM was then added to HUVECs (30,000) on matrigel-coated 12 well MatTek plates and incubated overnight at 37°C.

A, Tube formation of HUVECs was visualized at 40x magnification.

B, Tube formation in A was quantified using ImageJ software in a blinded manner. As indicated, FN-439 (5 μM) was also added to the OVCAR4 cells prior to MMP-1 treatment. Experiments (mean ± SD) shown are representative of 2 or more experiments performed in triplicate.

MMP1-PAR1 stimulation of ovarian carcinoma cells triggers endothelial tube formation

We next investigated the angiogenic potential of IL-8 and GRO-α induction by the MMP1-PAR1 signaling system in endothelial cells. Media from OVCAR-4 cells stimulated with either MMP-1 or vehicle was added to freshly cultured human umbilical vein endothelial cells (HUVEC) seeded on matrigel and incubated overnight. As MMP-1 can stimulate endothelial cells directly (37, 38), we quenched any residual MMP-1 activity with the MMP-1 inhibitor FN-439 before adding the CM to the HUVECs. Consistent with our hypothesis, MMP-1 CM containing elevated levels of IL-8 and GRO-α and basal levels of VEGF enhanced tube formation and branching complexity of the endothelial cells by 4-fold (Fig. 2A–B). Further, the MMP-1 paracrine effect on tube formation could be significantly attenuated by pretreating OVCAR-4 cells with the MMP-1 inhibitor FN-439, or the PAR1 antagonists RWJ-56110 or P1pal-7 (Fig. 2A–B). Taken together, these findings suggest that the MMP1-induced paracrine communication between ovarian carcinoma cells and endothelial cells was mediated through PAR1.

A cell penetrating CXCR1/2 pepducin blocks IL-8 and GRO-α stimulated endothelial cell proliferation

The chemokine receptors, CXCR1 and CXCR2 are expressed on the endothelium and are important for endothelial survival, proliferation and angiogenesis (13, 39). We confirmed the presence of CXCR1/2 chemokine receptors on our endothelial cells using flow cytometry (Fig. 3A). IL-8 and GRO-α were highly potent inducers of endothelial cell proliferation with EC50 values of 200 pM and 600 pM respectively (Fig. 3B). The IL-8 and GRO-α effects on endothelial cell proliferation were completely blocked by the X1/2pal-i3 pepducin (Fig. 3B), a palmitoylated pepducin based on the third intracellular loop of the CXCR1/2 chemokine receptors (28).

Figure 3.

Figure 3

Open in a new tab

A cell penetrating CXCR1/2 pepducin blocks IL-8 and GRO-α induced proliferation of human endothelial cells (HUVECs).

A. HUVECs express CXCR1 and CXCR2 chemokine receptors. Surface expression of CXCR1 and CXCR2 using monoclonal antibodies as compared to secondary antibody alone (2°) was measured by flow cytometry.

B. HUVECs were plated on a 96-well plate (10,000/well) in EBM2 media with the Bullet kit. The next day IL-8 or GRO-α (0.006–60 nM) was added after 5 h of serum starvation with X1/2pal-i3 (300 nM) or 0.2% DMSO vehicle (buffer). MTT was added to the cells after 72 h of growth, and cell proliferation quantified as previously described (27).

C. Media from MMP1-stimulated ovarian carcinoma cells induces proliferation of HUVECs. HUVECs were plated in 24 wells and treated with either EBM2/0.5% BSA (–) or MMP1-stimulated OVCAR-4 (high PAR1) CM or OVCAR-3 (low PAR1) CM. Proliferation was measured by 3[H]-thymidine incorporation into HUVECs after 72 h.

D, MMP1-stimulated OVCAR-4 CM was added to HUVECs in a 96 well plate in the presence or absence of CXCR1/2 pepducin X1/2pal-i3 (300 nM) and proliferation was measured by MTT incorporation 72 h after treatment as described above and expressed as fold change above unstimulated controls. Mean ± SE (n=3–6).

We then tested whether the media from the MMP-1 stimulated, OVCAR-4 cells could cause endothelial cell proliferation. As expected, the MMP1-OVCAR-4 media induced mitogenesis and proliferation by 3.5–4.5 fold (Fig. 3C). The MMP-1 mitogenic effect was not observed in the low-PAR1 expressing OVCAR-3 cells (Fig. 3C). The MMP1-induced proliferation was inhibited by addition of X1/2pal-i3 to the HUVECs (Fig. 3D) again demonstrating that the observed paracrine effect between the ovarian cancer cells and the endothelial cells was dependent on the CXCR1/2 receptors. Exogenous IL-8 and GRO-α also stimulated migration of endothelial cells in a wound healing migration assay which was blocked by X1/2pal-i3 (Supplementary Fig. S3).

The X1/2pal-i3 pepducin blocks IL-8 and GRO-α stimulated endothelial cell tube formation

As MMP-1 caused a 4-fold increase in both IL-8 and GRO-α secretion from carcinoma cells, we tested the ability of these chemokines to induce endothelial cell tube formation on matrigel coated wells. Exogenous IL-8 and GRO-α caused a 23-fold increase in branch point complexity and a 13-fold increase in tube length over unstimulated controls (Fig. 4A). This effect was significantly inhibited by 300 nM X1/2pal-i3 but not by Avastin (Fig. 4A) suggesting that the effect was mediated through the CXCR1/2 receptors. In the reciprocal experiment, exogenous VEGF stimulated HUVEC tube formation which was significantly inhibited by Avastin but not by X1/2pal-i3 (Supplementary Fig. S4). To validate the inhibitory effects of the CXCR1/2 pepducins on endothelial tube formation, we used neutralizing antibodies to the IL-8 receptors CXCR1 and CXCR2. The CXCR1 and CXCR2 neutralizing antibodies significantly (P<0.05) suppressed IL-8 induced branch point complexity by 68% and tube length by 58% as compared to control IgG (Supplementary Fig. S5), quantitatively similar to the inhibition observed with the X1/2pal-i3 pepducin.

Figure 4. CXCR1/2 i1 and i3-loop pepducins inhibit endothelial tube formation.

Figure 4

Open in a new tab

A, IL-8 and GRO-α induced HUVEC tube formation is blocked by the CXCR1/2 pepducin X1/2pal-i3. HUVECs were added to matrigel-coated MatTek wells and stimulated with IL-8 (60 nM) plus GRO-α (60 nM) or IL-8 alone (30nM) in the presence of X1/2pal-i3 (300 nM), Avastin (0.25 mg/ml) and/or PBS buffer control (–) and incubated overnight. Tube formation was quantified by ImageJ software in a blinded manner to assess tube length and branch point complexity.

B, OVCAR-4 cells were plated in each well of a 6-well plate, serum starved and then treated overnight with 1 nM MMP-1. Stimulated CM was collected and residual MMP-1 activity quenched with FN-439 before adding to the HUVEC cells. Tube formation was assessed after 24 h in presence of 300 nM CXCR1/2 pepducins X1/2pal-i3, X1/2LCA-i1, or control PAR1 i3 pepducin P1pal-19E or 0.2% DMSO vehicle (–). Tube formation was quantified by ImageJ software in a blinded manner. Experiments (mean ± SE) are representative of 2 or more experiments done in triplicate.

We further tested whether X1/2pal-i3 was able to block endothelial cell tube formation induced by the media from MMP-1 stimulated ovarian carcinoma cells. We used two different CXCR1/2 pepducins—X1/2pal-i3 and X1/2LCA-i1 (a lithocholic-tagged i1 loop of CXCR1/2), and a negative control pepducin P1pal-19E based on the i3 loop of PAR1 (29) which has a similar structure to X1/2pal-i3. Both X1/2pal-i3 and X1/2LCA-i1 significantly inhibited endothelial cell tube formation and branch complexity induced by the media from MMP-1 stimulated ovarian carcinoma cells, whereas P1pal-19E had no effect (Fig. 4B). These data provide further evidence that the CXCR1/2 pepducins were specifically blocking the CXCR1/2 receptors to inhibit tube formation.

A CXCR1/2 pepducin suppresses angiogenesis in mice

Next, we tested whether the X1/2pal-i3 pepducin would inhibit blood vessel formation in mouse models of angiogenesis. As stimulation of ovarian carcinoma cells produced a marked increase in secretion of both IL-8 and GRO-α, we first spiked unstimulated ovarian carcinoma cell media with IL-8 plus GRO-α versus buffer alone and added the mixtures to matrigel plugs which were injected into the flanks of nude mice. Mice receiving the IL-8/GRO-α matrigel plugs were divided into 3 treatment groups. The mice were injected subcutaneously with either vehicle, X1/2pal-i3 or Avastin. On day 7, the matrigel plugs were harvested and stained for blood vessel formation. Unstimulated conditioned media (buffer-treated) from OVCAR-4 cells containing basal levels of VEGF, IL-8 and GRO-α was unable to form blood vessels. Addition of IL-8/GRO-α to the unstimulated ovarian carcinoma cell media caused a dramatic increase (P=0.0016) in angiogenesis within the plugs as compared to media with buffer alone, which was completely blocked by treatment with X1/2pal-i3 (P=0.0075) (Fig. 5A). Treatment with Avastin also significantly inhibited angiogenesis, suggesting that the basal levels of VEGF (~200 pg/ml) in the OVCAR-4 CM were contributing to the IL-8/GROα-driven angiogenesis in vivo. These basal levels of VEGF, however, were not sufficient to stimulate angiogenesis in the mice as the unstimulated CM from the OVCAR-4 cells did not support angiogenesis without supplementation with IL-8 and GRO-α.

Figure 5. X1/2pal-i3 blocks chemokine-induced angiogenesis in vivo.

Figure 5

Open in a new tab

OVCAR-4 cells were serum starved and treated with PBS buffer (A), or 1 nM MMP-1 (B) for 18 h. CM was then collected, mixed with equal parts matrigel and HUVECs (10,000), and injected as a plug into each flank of NCR Nu/Nu female mice

A. CM from unstimulated OVCAR-4 cells was spiked with IL-8 (60 nM) plus GRO-α (60 nM), mixed with matrigel and injected into the flanks of mice. Mice with were then treated with either X1/2pal-i3 (5 mg/kg per day for 7 days), Avastin (5 mg/kg) or vehicle (15% DMSO) as indicated. After 7 days the plugs were harvested, and stained for blood vessel formation with a CD31 (PECAM) antibody and analyzed by confocal microscopy in a blinded manner.

B. MMP-1 stimulated OVCAR-4 CM was concentrated 6-fold and mixed with matrigel and HUVECs and injected into the flanks of NCR Nu/Nu mice. The mice received either X1/2pal-i3 (5 mg/kg/d × 7 days), Avastin (5 mg/kg), P1pal-19E (5 mg/kg/d × 7 days) or vehicle (15% DMSO). After 7 days, plugs were harvested, stained and subjected to confocal microscopy. Data are plotted as mean ± SE.

Mice were then injected with matrigel mixed with media from MMP1-stimulated OVCAR-4 cells. These mice were divided into the following treatment groups: X1/2pal-i3 (5 mg/kg/day × 7 days), Avastin (5 mg/kg) or negative control i3 loop pepducin P1pal-19E (5 mg/kg/day × 7 days). Mice injected with MMP1-stimulated OVCAR-4 CM showed a significant 33-fold increase in blood vessel formation as assessed by confocal microscopy (Fig. 5B). As observed above, Avastin inhibited the MMP1-induced angiogenesis. Treatment of the mice with the CXCR1/2 antagonist pepducin X1/2pal-i3 gave a striking reduction (P=0.002) in blood vessel formation (Fig. 5B, Supplementary Fig. S6). However, the negative control i3 loop pepducin, P1pal-19E, had no effect in the mouse model providing further evidence that the observed angiogenesis was mediated by the CXCR1/2 receptors.

X1/2pal-i3 blocks angiogenesis and inhibits tumor growth in ovarian cancer xenografts

Lastly, we examined the effect of CXCR1/2 receptor blockade on ovarian cancer tumor growth and related these effects on tumor angiogenesis. OVCAR-4 ovarian carcinoma cells were subcutaneously injected into the flanks of female NCR Nu/Nu mice. The mice were then randomly divided into 2 treatment groups: X1/2pal-i3 or vehicle, and tumor volume measured. After 30 days the mice were euthanized and tumors assessed for angiogenesis. As shown in Fig. 6A–B, mice treated with the CXCR1/2 pepducin showed a significant decrease in tumor volume on day 23 (P=0.01) and at the 30 day endpoint (P=0.02). The X1/2pal-i3 treated tumors also demonstrated a highly significant (P<0.0001) 5-fold decrease in angiogenesis (Fig. 6C) confirming an important role for the CXCR1/2 receptors in ovarian tumor growth and angiogenesis.

Figure 6.

Figure 6

Open in a new tab

X1/2pal-i3 inhibits tumor progression and angiogenesis of ovarian cancer xenografts. OVCAR-4 cells (3 × 106) were injected subcutaneously into each flank of female NCR Nu/Nu mice. Mice (n=8) were treated 24 h later with either X1/2pal-i3 (5 mg/kg/d × 6/7 days) or vehicle (15% DMSO) until day 30 and tumor volume was measured with calipers. On day 30, mice were euthanised and tumors were harvested, fixed in formalin and stained with CD31 and subjected to confocal microscopy to assess angiogenesis. Data are plotted as mean ± SE.

Targeting MMP1-chemokine communication in ovarian cancer angiogenesis

Angiogenesis inhibitors, as exemplified by the VEGF-antibody Avastin, have recently emerged as potential treatments for recurrent ovarian and primary peritoneal cancer (40, 41). Nonetheless, anti-VEGF therapy has not universally been able to translate response rates into cure rates in patients (42), necessitating the need to identify new pathways involved in tumor-stromal interactions. However, there is limited information regarding the involvement of other tumor-produced angiogenic factors and their cognate receptors in ovarian cancer angiogenesis. In this study, we found that the metalloprotease MMP-1, a poor prognostic factor for ovarian cancer (43, 44), mediates tumor-stromal communication by stimulating release of the angiogenic chemokines IL-8 and GRO-α via the PAR1 receptor on the ovarian carcinoma cells. The secreted GRO-α and IL-8 chemokines then act in a paracrine manner to promote endothelial cell proliferation, migration, tube formation and angiogenesis. A cell-penetrating X1/2pal-i3 pepducin directed against both the CXCR1 and CXCR2 receptors was able to block the MMP1-induced effects of IL-8 and GRO-α on the endothelial cells and inhibited tumor progression and angiogenesis in ovarian cancer xenografts. These findings are consistent with our previous observations that upstream blockade of MMP1-PAR1 also significantly inhibited angiogenesis in mouse models of ovarian peritoneal carcinomatosis (23).

Pro-angiogenic factors like IL-8 and Gro-α are upregulated by MAPK activation of the NF-kB and AP-1 transcription factors in many cell types including inflammatory cells, endothelium and carcinoma cells (45). Although little is known regarding PAR1-dependent MAPK signaling pathways in ovarian carcinoma cells, both thrombin and MMP-1 have been shown to upregulate IL-8 gene expression in melanoma cells presumably through PAR1 (21, 46). We found that MMP-1 activation of PAR1 did not significantly stimulate VEGF-A production above basal levels from the ovarian carcinoma cells. Despite this, Avastin had a partial inhibitory effect on the IL-8/GROα-induced angiogenesis in mice. This inhibition may be due to the synergistic effects of VEGF-A on IL-8 in endothelial cells. Indeed, recent studies showed that IL-8 can induce the physical interaction of CXCR1/2 with VEGFR2 and cause subsequent activation of RhoA (47). RhoA is critical for endothelial angiogenesis (48) suggesting that there may be a critical relationship between VEGF and IL-8 in promoting angiogenesis. Other reports showed that shRNAi knock-down of GRO-α impaired tumor growth and angiogenesis in melanoma (16) and silencing IL-8 expression with lipocalated RNAi decreased ovarian tumor growth (12).

Novel treatment strategies in ovarian cancer have combined standard chemotherapeutics with angiogenesis-inhibiting agents to prolong survival as maintenance therapy (49) and as adjuvants in metronomic chemotherapy (50). However, the rich peritoneal milieu that promotes growth and angiogenesis for the spread of ovarian cancer provides multiple communication pathways that may need to be interrupted to prevent metastasis. As exemplified by the inhibitory effects of the CXCR1/2 pepducins, this study presents a potential orthogonal strategy to target ovarian cancer angiogenesis and progression.

Supplementary Material

1
2

Acknowledgments

We thank Ya Li, Ina Klebba and Vandana Iyer for expert advice regarding experiments, Claudia Derian and Patricia Andrade-Gordon of Johnson & Johnson Pharmaceuticals for providing RWJ-56110, Sheida Sharifi for histopathology analysis and Chi Luo for help with developing OVCAR-4 PAR1shRNAi clones.

Grant Support: The Liz Tilberis award from OCRF (A. Agarwal); Cancer in Aid grant from Tufts Medical School (F. Lam); NIH grants CA 104406 (L. Covic), and CA122992, HL64701, HL57905 (A. Kuliopulos).

Footnotes

Disclosure of Potential Conflicts of Interest: Tufts Medical Center has out-licensed the pepducins used in this study. L. Covic and A. Kuliopulos are Scientific Founders and consultants for Anchor (Ascent) Therapeutics. The other authors disclosed no potential conflicts of interest.

References

  • 1.Balkwill F. Cancer and the chemokine network. Nat Rev Cancer. 2004;4:540–50. doi: 10.1038/nrc1388. [DOI] [PubMed] [Google Scholar]
  • 2.Matsuo Y, Ochi N, Sawai H, et al. CXCL8/IL-8 and CXCL12/SDF-1alpha co-operatively promote invasiveness and angiogenesis in pancreatic cancer. Int J Cancer. 2009;124:853–61. doi: 10.1002/ijc.24040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.DeNardo DG, Johansson M, Coussens LM. Immune cells as mediators of solid tumor metastasis. Cancer Metastasis Rev. 2008;27:11–8. doi: 10.1007/s10555-007-9100-0. [DOI] [PubMed] [Google Scholar]
  • 4.Bast RC, Jr, Hennessy B, Mills GB. The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer. 2009;9:415–28. doi: 10.1038/nrc2644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Malik S, Balkwill F. Epithelial ovarian cancer: a cytokine propelled disease? Br J Cancer. 1991;64:617–20. doi: 10.1038/bjc.1991.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Milliken D, Scotton C, Raju S, Balkwill F, Wilson J. Analysis of chemokines and chemokine receptor expression in ovarian cancer ascites. Clin Cancer Res. 2002;8:1108–14. [PubMed] [Google Scholar]
  • 7.Negus RP, Stamp GW, Relf MG, et al. The detection and localization of monocyte chemoattractant protein-1 (MCP-1) in human ovarian cancer. J Clin Invest. 1995;95:2391–6. doi: 10.1172/JCI117933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420:860–7. doi: 10.1038/nature01322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Karstrom-Encrantz L, Runesson E, Bostrom EK, Brannstrom M. Selective presence of the chemokine growth-regulated oncogene alpha (GROalpha) in the human follicle and secretion from cultured granulosa-lutein cells at ovulation. Mol Hum Reprod. 1998;4:1077–83. doi: 10.1093/molehr/4.11.1077. [DOI] [PubMed] [Google Scholar]
  • 10.Lokshin AE, Winans M, Landsittel D, et al. Circulating IL-8 and anti-IL-8 autoantibody in patients with ovarian cancer. Gynecol Oncol. 2006;102:244–51. doi: 10.1016/j.ygyno.2005.12.011. [DOI] [PubMed] [Google Scholar]
  • 11.Lerebours F, Vacher S, Andrieu C, et al. NF-kappa B genes have a major role in inflammatory breast cancer. BMC Cancer. 2008;8:41. doi: 10.1186/1471-2407-8-41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Merritt WM, Lin YG, Spannuth WA, et al. Effect of interleukin-8 gene silencing with liposome-encapsulated small interfering RNA on ovarian cancer cell growth. J Natl Cancer Inst. 2008;100:359–72. doi: 10.1093/jnci/djn024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Li A, Dubey S, Varney ML, Dave BJ, Singh RK. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol. 2003;170:3369–76. doi: 10.4049/jimmunol.170.6.3369. [DOI] [PubMed] [Google Scholar]
  • 14.Sato N, Maehara N, Goggins M. Gene expression profiling of tumor-stromal interactions between pancreatic cancer cells and stromal fibroblasts. Cancer Res. 2004;64:6950–6. doi: 10.1158/0008-5472.CAN-04-0677. [DOI] [PubMed] [Google Scholar]
  • 15.Yang G, Rosen DG, Zhang Z, et al. The chemokine growth-regulated oncogene 1 (Gro-1) links RAS signaling to the senescence of stromal fibroblasts and ovarian tumorigenesis. Proc Natl Acad Sci U S A. 2006;103:16472–7. doi: 10.1073/pnas.0605752103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Caunt M, Hu L, Tang T, Brooks PC, Ibrahim S, Karpatkin S. Growth-regulated oncogene is pivotal in thrombin-induced angiogenesis. Cancer Res. 2006;66:4125–32. doi: 10.1158/0008-5472.CAN-05-2570. [DOI] [PubMed] [Google Scholar]
  • 17.Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000;407:258–64. doi: 10.1038/35025229. [DOI] [PubMed] [Google Scholar]
  • 18.Even-Ram S, Uziely B, Cohen P, et al. Thrombin receptor overexpression in malignant and physiological invasion processes. Nat Med. 1998;4:909–14. doi: 10.1038/nm0898-909. [DOI] [PubMed] [Google Scholar]
  • 19.Nierodzik ML, Chen K, Takeshita K, et al. Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. Blood. 1998;92:3694–700. [PubMed] [Google Scholar]
  • 20.Kamath L, Meydani A, Foss F, Kuliopulos A. Signaling from protease-activated receptor-1 inhibits migration and invasion of breast cancer cells. Cancer Res. 2001;61:5933–40. [PubMed] [Google Scholar]
  • 21.Tellez C, Bar-Eli M. Role and regulation of the thrombin receptor (PAR-1) in human melanoma. Oncogene. 2003;22:3130–7. doi: 10.1038/sj.onc.1206453. [DOI] [PubMed] [Google Scholar]
  • 22.Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, Kuliopulos A. PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell. 2005;120:303–13. doi: 10.1016/j.cell.2004.12.018. [DOI] [PubMed] [Google Scholar]
  • 23.Agarwal A, Covic L, Sevigny LM, et al. Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer. Mol Cancer Ther. 2008;7:2746–57. doi: 10.1158/1535-7163.MCT-08-0177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Arora P, Cuevas BD, Russo A, Johnson GL, Trejo J. Persistent transactivation of EGFR and ErbB2/HER2 by protease-activated receptor-1 promotes breast carcinoma cell invasion. Oncogene. 2008;27:4434–45. doi: 10.1038/onc.2008.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Trivedi V, Boire A, Tchernychev B, et al. Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site. Cell. 2009;137:332–43. doi: 10.1016/j.cell.2009.02.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Nguyen N, Kuliopulos A, Graham RA, Covic L. Tumor-derived Cyr61(CCN1) promotes stromal matrix metalloproteinase-1 production and protease-activated receptor 1-dependent migration of breast cancer cells. Cancer Res. 2006;66:2658–65. doi: 10.1158/0008-5472.CAN-05-2082. [DOI] [PubMed] [Google Scholar]
  • 27.Yang E, Boire A, Agarwal A, et al. Blockade of PAR1 signaling with cell-penetrating pepducins inhibits Akt survival pathways in breast cancer cells and suppresses tumor survival and metastasis. Cancer Res. 2009;69:6223–31. doi: 10.1158/0008-5472.CAN-09-0187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kaneider NC, Agarwal A, Leger AJ, Kuliopulos A. Reversing systemic inflammatory response syndrome with chemokine receptor pepducins. Nat Med. 2005;11:661–5. doi: 10.1038/nm1245. [DOI] [PubMed] [Google Scholar]
  • 29.Covic L, Gresser AL, Talavera J, Swift S, Kuliopulos A. Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides. Proc Natl Acad Sci U S A. 2002;99:643–8. doi: 10.1073/pnas.022460899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Covic L, Misra M, Badar J, Singh C, Kuliopulos A. Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation. Nat Med. 2002;8:1161–5. doi: 10.1038/nm760. [DOI] [PubMed] [Google Scholar]
  • 31.Caunt M, Huang YQ, Brooks PC, Karpatkin S. Thrombin induces neoangiogenesis in the chick chorioallantoic membrane. J Thromb Haemost. 2003;1:2097–102. doi: 10.1046/j.1538-7836.2003.00426.x. [DOI] [PubMed] [Google Scholar]
  • 32.Arisato T, Sarker KP, Kawahara K, et al. The agonist of the protease-activated receptor-1 (PAR) but not PAR3 mimics thrombin-induced vascular endothelial growth factor release in human smooth muscle cells. Cell Mol Life Sci. 2003;60:1716–24. doi: 10.1007/s00018-003-3140-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Andrade-Gordon P, Maryanoff BE, Derian CK, et al. Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor. Proc Natl Acad Sci U S A. 1999;96:12257–62. doi: 10.1073/pnas.96.22.12257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Kaneider NC, Leger AJ, Agarwal A, et al. ‘Role reversal’ for the receptor PAR1 in sepsis-induced vascular damage. Nat Immunol. 2007;8:1303–12. doi: 10.1038/ni1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Covic L, Singh C, Smith H, Kuliopulos A. Role of the PAR4 thrombin receptor in stabilizing platelet-platelet aggregates as revealed by a patient with Hermansky-Pudlak syndrome. Thromb Haemost. 2002;87:722–7. [PubMed] [Google Scholar]
  • 36.Leger AJ, Jacques SL, Badar J, et al. Blocking the protease-activated receptor 1–4 heterodimer in platelet-mediated thrombosis. Circulation. 2006;113:1244–54. doi: 10.1161/CIRCULATIONAHA.105.587758. [DOI] [PubMed] [Google Scholar]
  • 37.Goerge T, Barg A, Schnaeker EM, et al. Tumor-derived matrix metalloproteinase-1 targets endothelial proteinase-activated receptor 1 promoting endothelial cell activation. Cancer Res. 2006;66:7766–74. doi: 10.1158/0008-5472.CAN-05-3897. [DOI] [PubMed] [Google Scholar]
  • 38.Eck SM, Hoopes PJ, Petrella BL, Coon CI, Brinckerhoff CE. Matrix metalloproteinase-1 promotes breast cancer angiogenesis and osteolysis in a novel in vivo model. Breast Cancer Res Treat. 2009;116:79–90. doi: 10.1007/s10549-008-0085-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Li A, Varney ML, Valasek J, Godfrey M, Dave BJ, Singh RK. Autocrine role of interleukin-8 in induction of endothelial cell proliferation, survival, migration and MMP-2 production and angiogenesis. Angiogenesis. 2005;8:63–71. doi: 10.1007/s10456-005-5208-4. [DOI] [PubMed] [Google Scholar]
  • 40.Burger RA. Experience with bevacizumab in the management of epithelial ovarian cancer. J Clin Oncol. 2007;25:2902–8. doi: 10.1200/JCO.2007.12.1509. [DOI] [PubMed] [Google Scholar]
  • 41.Kumaran GC, Jayson GC, Clamp AR. Antiangiogenic drugs in ovarian cancer. Br J Cancer. 2009;100:1–7. doi: 10.1038/sj.bjc.6604767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Miller K, Wang M, Gralow J, et al. Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med. 2007;357:2666–76. doi: 10.1056/NEJMoa072113. [DOI] [PubMed] [Google Scholar]
  • 43.Sood AK, Seftor EA, Fletcher MS, et al. Molecular determinants of ovarian cancer plasticity. Am J Pathol. 2001;158:1279–88. doi: 10.1016/S0002-9440(10)64079-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Wang L, Madigan MC, Chen H, et al. Expression of urokinase plasminogen activator and its receptor in advanced epithelial ovarian cancer patients. Gynecol Oncol. 2009;114:265–72. doi: 10.1016/j.ygyno.2009.04.031. [DOI] [PubMed] [Google Scholar]
  • 45.Hoffmann E, Dittrich-Breiholz O, Holtmann H, Kracht M. Multiple control of interleukin-8 gene expression. J Leukoc Biol. 2002;72:847–55. [PubMed] [Google Scholar]
  • 46.Blackburn JS, Liu I, Coon CI, Brinckerhoff CE. A matrix metalloproteinase-1/protease activated receptor-1 signaling axis promotes melanoma invasion and metastasis. Oncogene. 2009 doi: 10.1038/onc.2009.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Petreaca ML, Yao M, Liu Y, Defea K, Martins-Green M. Transactivation of vascular endothelial growth factor receptor-2 by interleukin-8 (IL-8/CXCL8) is required for IL-8/CXCL8-induced endothelial permeability. Mol Biol Cell. 2007;18:5014–23. doi: 10.1091/mbc.E07-01-0004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Hoang MV, Whelan MC, Senger DR. Rho activity critically and selectively regulates endothelial cell organization during angiogenesis. Proc Natl Acad Sci U S A. 2004;101:1874–9. doi: 10.1073/pnas.0308525100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Mabuchi S, Terai Y, Morishige K, et al. Maintenance treatment with bevacizumab prolongs survival in an in vivo ovarian cancer model. Clin Cancer Res. 2008;14:7781–9. doi: 10.1158/1078-0432.CCR-08-0243. [DOI] [PubMed] [Google Scholar]
  • 50.Kerbel RS, Kamen BA. The anti-angiogenic basis of metronomic chemotherapy. Nat Rev Cancer. 2004;4:423–36. doi: 10.1038/nrc1369. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS210306-supplement-1.pdf (2.8MB, pdf)
2
NIHMS210306-supplement-2.doc (34.5KB, doc)

RESOURCES