The angiostatic activity of interferon-inducible protein-10/CXCL10 in human melanoma depends on binding to CXCR3 but not to glycosaminoglycan

Abstract

Human interferon-inducible protein 10 (IP-10; HGMW-approved gene symbol CXCL10) is an ELR(-) CXC chemokine that contains binding domains for both the chemokine receptor CXCR3 and glycosaminoglycans. IP-10 has been recently demonstrated to be a potent angiostatic protein in vivo. Whether IP-10 exerts its angiostatic function through binding to CXCR3, glycosaminoglycans, or both, is not clear. To clarify this issue, we created expression constructs for mutants of IP-10 that exhibit partial (IP-10C) or total (IP-10C22) loss of binding to CXCR3 or loss of binding to glycosaminoglycans (IP-10H and IP-10C22H). The A375 human melanoma cell line was transfected with these expression vectors, and stable clones were selected and inoculated subcutaneously into nude mice. As expected, tumor cells secreting wild-type IP-10 showed remarkable reduction in tumor growth compared to control vector-transfected tumor cells. Surprisingly, mutation of IP-10 resulting in partial loss of receptor binding (IP-10C), or loss of GAG binding (IP-10H), did not significantly alter the ability to inhibit tumor growth. This tumor growth inhibition was associated with a reduction in microvessel density, leading to the observed increase in both tumor cell apoptosis and necrosis. In contrast, expression of the IP-10C22 mutant failed to inhibit melanoma tumor growth. These data suggest that CXCR3 receptor binding, but not glycosaminoglycan binding, is essential for the tumor angiostatic activity of IP-10. We conclude that the arginine 22 amino acid residue of IP-10 is essential for both CXCR3 binding and angiostasis.

FIG. 1

Mutagenesis and expression of IP-10 protein. (A) Protein sequence of human IP-10 wild type and C and H mutants used in this study. (B) The cDNAs of IP-10 (wild type or mutant) were cloned into the pIRES2-EGFP vector and the empty vector served as control. (C) The secretion of human IP-10 proteins by A375 human melanoma cells transfected with IP-10 expression vectors was evaluated by Western blot with anti-IP-10 antibody (top) and ELISA (bottom). (D) The nontransfected (top) or IP-10-transfected cells (bottom) were identified by EGFP expression using fluorescence flow cytometry. (E) In vitro proliferation of the transfected A375 cells was examined by cell number assays in serum-free medium over 5 days by hemocytometer counting. Data shown are from two separate experiments performed in duplicate each time.

FIG. 2

Competitive CXCR3 receptor binding assay. (A) Glycosaminoglycan-deficient Chinese hamster ovary cells (pgsA-745) were stably transfected with empty expression vector (lane 1, bottom) or with the human CXCR3 expression vector (lane 2, bottom), and the CXCR3 expression in the cell lysate was verified by Western blot with anti-CXCR3 antibody. Binding of 100 pM ¹²⁵I-labeled IP-10 to the cells without (lane 1, top) or with CXCR3 expression (lane 2, top) was measured as described under Materials and Methods. (B) The percentage of cold IP-10 competition binding with specific ¹²⁵I-labeled IP-10 binding was calculated after subtraction of nonspecific binding. The input of ¹²⁵I-labeled IP-10 is 100 pM and the increasing concentrations of unlabeled IP-10 range from 0 to 100 nM. Each point represents the mean of three experimental. The inset is the transformed data of ¹²⁵I-labeled binding to a one-binding-site model of CXCR3. (C) The GAG-deficient CHO cells expressing CXCR3 were used to test the competition of the ¹²⁵I-labeled IP-10 binding to CXCR3 receptor with unlabeled wild-type or mutant IP-10 in increasing concentrations of up to 1000-fold. Data shown are representative of three independent experiments.

FIG. 3

GAG-mutant IP-10 binding assay. (A) Defined GAG-chemokine binding motifs and proposed GAG-IP-10 binding motif. (B) Cleared culture medium supernatants from A375 cells stably expressing IP-10 proteins or mock-transfected A375 cells were incubated with heparin – Sepharose in 0.9% NaCl at 4°C and rotated for 4 h, and the heparin – Sepharose was washed with 0.5 M NaCl. The washed heparin beads (lower band) and cell supernatants (upper band), which had been concentrated with Microcon YM-3 (cutoff M_r 3000) membranes, were resolved on a 16% SDS – PAGE reducing gel and transferred to nitrocellulose. The membrane was blotted with anti-human IP-10 antibody and visualized by enhanced chemiluminescence assay as described under Materials and Methods.

FIG. 4

In vivogrowth of the transfected human A375 cells. (A) Human A375 melanoma cells (1 × 10⁶) stably producing IP-10 proteins were injected subcutaneously into nude mice and tumors were measured in two dimensions using digital calipers at the indicated time. (B) The tumor volume was calculated according to [17] and data are expressed as mean mm³ ± standard deviation (SD) of five mice per group per experiment. The entire experiment was performed twice. Comparisons between groups were performed using Student’s t test. Two-tailed P values ≤0.01 were considered significant. (C) The IP-10 expression levels in tumor xenografts and serum were determined by ELISA at termination of experiments. IP-10 protein levels are expressed as mean ng ± SD per milligram tissue protein or per milliliter serum from five mice per group.

FIG. 5

Histopathological alterations of the transfected melanoma tumors. (A) H&E-stained sections of control tumors showed mitotic activity and (B) occasionally the tumor cells invaded the muscles; (C) the IP-10-transfected melanomas showed central necrosis and a pyknotic ring of tumor cells around the necrotic zone. (D) High magnification of box in C showing pyknotic nuclei. Green, yellow, and black arrows indicate viable, apoptotic or pyknotic, and dead cells, respectively.

FIG. 6

Vessel and microvessel distributions of the transfected melanoma tumors. To evaluate the endothelial cell content of tumors, the fixed, embedded tumor tissues were sectioned and stained for immunoreactive CD31 in control tumors (A) and (B) and IP-10-transfected melanoma tumors (C) and (D). (E) Microvessel density was digitally measured from five random fields using the Image-Pro Plus program (Media Cybernetics, LP).

FIG. 7

Binding analysis of double mutant IP-10 protein. (A) Protein sequence of human wild-type IP-10 (line 1); IP-10C22, an arginine 22 to alanine substitution at the N loop of IP-10C (line 2); and an additional double mutant, IP-10C22H. (B) The glycosaminoglycan-deficient Chinese hamster ovary cells expressing CXCR3 were used to evaluate the competition of ¹²⁵I-labeled IP-10 binding to CXCR3 receptor with excess unlabeled wild-type or mutant IP-10 over increasing concentrations (up to 1000-fold). (C) Heparin-binding assay for wild-type or mutant IP-10 was performed under physiological conditions as described in the legend to Fig. 3.

FIG. 8

In vivo growth of the transfected human A375 cells with double mutant IP-10. (A) Human A375 melanoma cells (1 × 10⁶) stably producing mutant IP-10 proteins were inoculated subcutaneously into nude mice (five mice per group) and tumors were measured in two dimensions using digital calipers at the indicated time. The entire experiment was performed twice with similar results each time. The tumor volume was calculated according to [17] and data from one of the repeat experiments are expressed as mean mm³ ± SD. Comparisons between groups were performed using the Student t test. Two-tailed P values ≤0.01 were considered significant. (B) The IP-10 expression levels in tumor tissues and mouse serum were determined by ELISA at the termination of the experiments.