10.1245/ASO.2006.03.038
Annals of Surgical Oncology 13:125-133 (2006)
© 2006 Society of Surgical Oncology
Interferon 
–Inducible Protein 10 Selectively Inhibits Proliferation and Induces Apoptosis in Endothelial Cells
Elizabeth D. Feldman, MD1,
David M. Weinreich, MD1,
Nancy M. Carroll, MD1,
Monika L. Burness, MD1,
Andrew L. Feldman, MD2,
Ewa Turner, BS1,
Hui Xu, BS1 and
H. Richard Alexander, Jr., MD1
1 Surgical Metabolism Section, Surgery Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 2B07, Bethesda, Maryland 20892-1502
2 Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Building 10, Room 2N06, Bethesda, Maryland 20892-1502
Correspondence: Address correspondence and reprint requests to: H. Richard Alexander, Jr., MD; E-mail: richard_alexander{at}nih.gov.
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ABSTRACT
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Background: Interferon 
–inducible protein 10 (IP-10) has antitumor effects in various murine models. The IP-10 receptor has two distinct splice variants, CXCR3A and CXCR3B, that have paradoxical effects after ligand-receptor interaction.
Methods: To characterize the putative antiangiogenic effects of IP-10, we measured proliferation rates and apoptosis in human umbilical vein endothelial cells (HUVECs), fibroblasts, and A375 melanoma or WIDR adenocarcinoma cell lines after exposure to the recombinant protein. CXCR3A (activating) and CXCR3B (inhibitory/proapoptotic) messenger RNA (mRNA) expression levels in fibroblasts, 2 human tumor cell lines, T lymphocytes, and HUVECs of varying cell densities were characterized.
Results: IP-10 resulted in dose-dependent and selective inhibition of proliferation and countered the proliferative effects of vascular endothelial growth factor in HUVECs but did not affect fibroblasts or 2 human tumor cell lines. In addition, IP-10 resulted in potent and selective induction of apoptosis in HUVECS but had no effect on fibroblasts or A375 melanoma. Confluent HUVECs had a predominance of mRNA for the CXCR3B splice variant by reverse transcriptase-polymerase chain reaction, and the ratio of CXCR3B to CXCR3A mRNA was >40 in HUVECs, compared with 
10 in the other cell types. Moreover, CXCR3B mRNA levels were significantly higher in proliferating compared with confluent HUVECs. In vivo, systemic IP-10 administration resulted in slower A375 xenograft growth rates compared with control-treated animals, and immunohistochemical staining showed decreased microvessel density in xenografts of IP-10–treated mice.
Conclusions: IP-10 has antiangiogenic properties and selective effects on endothelial tissue that may be secondary to higher levels of the CXCR3B inhibitory/proapoptotic receptor in that cell type, particularly in its actively proliferating state.
Key Words: Angiogenesis • Interferon –inducible protein 10 • CXCR3 • A375 melanoma • Endothelium
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INTRODUCTION
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Angiogenesis is defined as the growth of neovasculature from preexisting vessels and is an essential biological event encountered in several physiological and pathological processes, including the growth of malignant solid-organ tumors.1 Tumor angiogenesis is characterized by an imbalance that favors overexpression of angiogenic factors and underexpression of angiostatic substances, thus resulting in endothelial cell migration, proliferation, and organization into functional luminal structures.2,3
Investigations of tumor angiogenesis have primarily focused on the role of angiogenic factors, such as vascular endothelial growth factor (VEGF), interleukin (IL)-8, and basic fibroblast growth factor.4–7 More recently, studies have focused on the increasingly important role of endogenous angiostatic factors that may regulate the balance of neovascularization.8 One such putative endogenous angiostatic molecule is the CXC chemokine interferon –inducible protein 10 (IP-10).9 IP-10 is a 10-kDa secreted protein produced by activated monocytes, fibroblasts, endothelial cells, and keratinocytes in response to stimulation with interferon .10,11 It has in vivo antitumor effects in various experimental models1,12–14; these effects have been proposed to be mediated via a T cell–dependent mechanism.15 However, Feldman et al.12 demonstrated significant growth inhibition of A375 human melanoma cells transduced with the human IP-10 gene in a subcutaneous tumor model in nude athymic mice which was associated with a marked reduction in tumor microvessel density.
The IP-10 receptor, CXCR3, has 2 distinct splice variants, CXCR3A and CXCR3B, which have paradoxical effects after ligand-receptor interaction.16 Lasagni et al.16 transfected human microvascular endothelial cell lines (HMVECs) with either variant and noted that overexpression of CXCR3A induced an increase in cell survival, whereas overexpression of CXCR3B dramatically reduced DNA synthesis and upregulated apoptotic cell death. Therefore, CXCR3A may be considered a mediator of cellular activation and endothelial cell angiogenesis, whereas CXCR3B acts as a cellular inhibitor and a promoter of apoptosis.
These experiments were performed to characterize the putative antiangiogenic effects of IP-10. We propose, on the basis of our studies with recombinant IP-10, that tumor growth inhibition by IP-10 is mediated via selective effects of IP-10 on endothelial tissue and that the specificity of these effects may be due to the predominance of the CXCR3B receptor on activated or proliferating endothelium present in the tumor microenvironment.
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MATERIALS AND METHODS
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Cell Culture
Human umbilical vein endothelial cells (HUVECs; Clonetics, San Diego, CA) were maintained at 37° C in a 5% carbon dioxide incubator in basal endothelial cell media (EGM-2; Clonetics) enriched with fetal bovine serum, hydrocortisone, human recombinant basic fibroblast growth factor, insulin-like growth factor 1, ascorbic acid, epidermal growth factor, GA-1000 (gentamicin/amphotericin), and heparin (Clonetics). They were passaged for 2 generations for the experiments described below. Human fibroblasts (donated by Dr. J. Wunderlich, Surgery Branch, National Cancer Institute) were maintained at 37° C in a 5% carbon dioxide incubator in RPMI complete media (Life Technologies, Carlsbad, CA) enriched with fetal bovine serum (Gemini Bio-products, Woodland, CA), L-glutamine, and penicillin-streptomycin (Life Technologies). A375 human melanoma cells (American Type Culture Collection, Frederick, MD) were maintained at 37° C in a 5% carbon dioxide incubator in Dulbecco’s modified Eagle medium media (Dulbecco’s modified Eagle medium enriched with 10% non–heat-inactivated fetal bovine serum; American Type Culture Collection). WIDR, a human colon cancer cell line, was maintained at 37° C in a 5% carbon dioxide incubator in RPMI complete media.
Proliferation Assays
Various cell types were plated at a density of 1000 cells per well in 96-well plates treated with nonpyrogenic polystyrene (Corning Incorporated, Corning, NY) and incubated at 37° C in complete medium for 24 hours. Cell types included HUVECs, human fibroblasts, A375 melanoma, and WIDR adenocarcinoma. After 24 hours, each well was aspirated, and cells were treated with the appropriate complete media alone or IP-10 1, 5, or 10 µg/mL in complete media. Proliferation was analyzed at 30 minutes and 24, 48, and 72 hours by tetrazolium salt assay (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. Six samples in each group were tested. The absorbance of the samples was measured against a background control, basal medium, by using a Multiskan MCC/340 plate reader (Titertek, Huntsville, AL) at 450 nm with a reference wavelength of 650 nm. The degree of proliferation was expressed as the absorbance at 450 nm.
Inhibition of VEGF Stimulation With Exposure to IP-10
HUVECs were plated at a density of 1000 cells per well in 96-well plates (Corning Incorporated) and incubated at 37° C in complete medium for 24 hours. The medium was then aspirated, and new complete media were added that contained no additional additives, IP-10 10 µg/mL, VEGF .1 µg/mL (R&D Systems, Minneapolis, MN), or IP-10 10 µg/mL plus VEGF .1 µg/mL, all in complete media. Proliferation was analyzed at 30 minutes and 24, 48, and 72 hours by tetrazolium salt assay (Boehringer Mannheim) according to the manufacturer’s instructions. Six samples in each group were tested. The absorbance of the samples was measured against a background control, basal medium, by using a Multiskan MCC/340 plate reader (Titertek) at 450 nm with a reference wavelength of 650 nm. The degree of proliferation was expressed as the absorbance at 450 nm.
Deoxyuride-5'-Triphosphate Biotin Nick End Labeling Assays for Apoptosis: HUVECs
A375 human melanoma cells and fibroblasts were plated in six-well plates treated with nonpyrogenic polystyrene (Corning Incorporated) at a density of 200,000 cells per well at 37° C in complete medium for 24 hours. The medium was aspirated, and fresh media were applied that contained IP-1001 or 10 µg/mL in complete media. Cells were trypsinized at 24 or 48 hours, washed in phosphate-buffered saline (PBS), fixed for 1 hour with 1% paraformaldehyde, permeabilized with 70% ethanol at –20° C for several days, and subjected to analysis for the presence of apoptosis by using the APO-BRDU kit (BD Biosciences Pharmingen, San Diego, CA) according to the manufacturer’s instructions via flow cytometry within 2 hours of staining. The assay was run on a single laser flow cytometer (FACScan; Becton Dickinson Immunocytometry Systems, San Jose, CA) at 488 nm and analyzed with the CellQuest program (Becton Dickinson Immunocytometry Systems). The gate for detection of apoptosis was set with reference to the positive and negative controls provided by the manufacturer.
Reverse Transcriptase-Polymerase Chain Reaction for CXCR3 Messenger RNA Expression
HUVECs were plated at known cell densities from 50,000 to 800,000 cells per well in 6-well plates (Corning Incorporated) in complete media for 48 hours at 37° C in 5% carbon dioxide. Total RNA was harvested from HUVECs, human fibroblasts, A375, WIDR, and IL-2–stimulated T lymphocytes (provided by Dr. R. Morgan, Surgery Branch, National Cancer Institute) by using the Qiagen RNAeasy mini kit (Qiagen, Hilden, Germany), and 1 µg of total RNA was transcribed to complementary DNA by using Superscript II reverse transcriptase (Life Technologies).
ß-Actin was used as a housekeeping gene to standardize quantification of CXCR3A and CXCR3B copy number and to account for varying amounts of initial complementary DNA in the reaction. All primers and probes were obtained from Biosource International (Camarillo, CA). Polymerase chain reaction amplification of ß-actin was performed with the primers 5'-GCGAGAAGATGAC CCAGATC-3'(forward primer) and 5'-CCAGTGGTACGGCCA GAGG-3' (reverse primer) by using the Platinum Taq DNA polymerase kit (Life Technologies). The quantity and quality of the amplified products were determined by spectrophotometry and gel electrophoresis, and then progressive 10-fold dilutions were conducted to generate the standards for use in an ABI Prism 7700 Sequence Detector (Applied Biosciences). The probe sequence was 5'-TCAAGATCA TTGCTCCTCCTGAGCGC-3'.
Standards were generated for both CXCR3A and CXCR3B by using the same conditions as described previously. The primers and probe sequences for CXCR3A were 5'-CAGGTGCCCTCTTC AACATCA-3' (forward primer), 5'-ATGTTCAGGTAGCGG TCAAAGC-3' (reverse primer), and 5'-CCCTCCTG CTGGCCT GCATCA-3' (probe). The primers and probe sequences for CXCR3B were 5'-TGCCAGGC CTTTACACAGC-3' (forward primer), 5'-TCGGCG TCATTTAGCACTTG-3' (reverse primer), and 5'-CCCGTTCCCGCCCTCACAGG-3' (probe). Gene copy numbers of CXCR3A and CXCR3B were assessed in each cell line and expressed as a ratio to 1 x 105 copies of ß-actin.
In Vivo Studies
Animal experiments were conducted according to protocols approved by the National Institutes of Health Animal Care and Use Committee. Twenty 7-to 8-week-old female nude athymic mice (Taconic, Germantown, NY, and Frederick Cancer Research Center, Frederick, MD) were inoculated with 2 x 106 A375 human melanoma cells in 100 µL of PBS subcutaneously in the right flank. Subjects were then divided into 2 groups and randomized to receive peritumoral subcutaneous injections with either recombinant IP-10 10 µg/100 µL (R&D Systems) or carrier, .1% bovine serum albumin. IP-10 was received in lyophilized form and was reconstituted by using .1% bovine serum albumin. The peritumoral injections were initiated on the same day as tumor inoculation. Tumors were measured in two dimensions by using calipers at regular intervals by a blinded observer (M.B.), and tumor areas were calculated as the product of the greatest perpendicular diameters.
Immunohistochemistry
Tumors from animals treated with IP-10 or carrier were harvested immediately after mice were sacrificed after cessation of the subcutaneous injections, as described previously. Tumors were snap-frozen in liquid nitrogen and stored at –80° C. Frozen sections (7 µm) were cut on a cryostat and stained with hematoxylin and eosin or antibodies specific for CD31 (Pharmingen, San Diego, CA). For immunostaining, sections were fixed in acetone for 10 minutes. After endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes, sections were incubated for 1 hour in a blocking solution that contained 10% normal goat serum. Sections were incubated with primary antibody at 4° C overnight at a 1/50 dilution. Slides were then washed three times in PBS, incubated in biotinylated species-specific appropriate secondary antibody for 1 hour, and exposed to avidinbiotin-peroxidase complex (Vector Laboratories, Inc., Burlingame, CA). Sections were reacted with .06% 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and counterstained with hematoxylin.
Hematoxylin and eosin–stained and immunostained sections were analyzed by a pathologist (A.L. Feldman) who was blinded to the identity of the groups. Only good-quality sections with uniform, well-demarcated staining and low background were analyzed. Microvascular density of two tumors from each group was assessed by using a scoring system based on the number of CD31-positive cells per high-power field (x400).
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RESULTS
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Proliferation
We first evaluated the effect of IP-10 on the proliferation rates of HUVECs, fibroblasts, and two human tumor cell lines, A375 and WIDR. IP-10 had a dose-dependent selective inhibition of the proliferation of HUVECs that was evident 24 hours after exposure to the protein (Fig. 1A
). Application of IP-10 decreased the HUVEC proliferation rate by 30% and 47% at doses of 5 and 10 µg/mL, respectively, at 72 hours. However, treatment with IP-10 1, 5, or 10 µg/mL did not affect the proliferation rates of fibroblasts, A375 human melanoma, or WIDR human colon carcinoma at any given time point (Fig. 1B–D). Exposure to VEGF .1 µg/mL resulted in augmented HUVEC proliferation as compared with untreated control HU-VECs (Fig. 2). IP-10 inhibited the proliferative effects of VEGF in HUVECs such that proliferation was equivalent to that in untreated HUVECs.
Received for publication March 4, 2005.
Accepted for publication August 2, 2005.
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ABSTRACT
INTRODUCTION
). CD31 staining demonstrated larger vessels and individual endothelial cells. The scores for the representative sections of the 2 tumors of treated mice were 4 and 2.8 microvessels per high-power field, compared with 2 control mice that had scores of 10.6 and 11.7. 
chemokine induced by interferon 
