Figure 1.
Synthesis of MSX-122 and in vitro functional assays.
(A) Scheme of MSX-122 preparation. (B) Representative immunofluorescence images show competition-binding assay using the biotinylated CXCR4 antagonist TN14003. MDA-MB-231 cells on an 8-well slide chamber were treated with TN14003 or MSX-122 at various concentrations for 15 minutes at room temperature. The cells were subsequently fixed and incubated with biotin-labeled TN14003 (0.05 µg/ml). After washing, cells were incubated with streptavidin-rhodamine. Red color represents CXCR4. Nuclei were counterstained with cytox blue. (C) RT-PCR results of CXCR4 and CXCR7 reveal that MDA-MB-231 cells do not express CXCR7. (D) Scheme of fluorine-18 labeling of MSX-122 is shown. To determine binding of MSX-122 to a CXCL12 binding site on CXCR4, we tested whether preincubation with CXCL12 (11, 33, 100, and 400 nM) before adding [18F]MSX-122F blocked binding to CXCR4 in the CXCR4-positive metastatic SCCHN cells. (E) A low energy predicted binding pose for MSX-122 (magenta) in the CXCR4 X-ray structure (grey) is illustrated by two different visualizations: ribbon (left) and protein surface (right) representations. The complex was constructed by extracting the small molecule antagonist IT1t from the X-ray structure (pdb code 3ODU; [33]) followed by docking MSX-122 into the corresponding binding site.
Figure 2.
MSX-122 blocks invasion and angiogenesis in vitro.
(A) Micrographs of Matrigel invasion assay induced by CXCR4/CXCL12-mediated interaction using MDA-MB-231 cells in the presence of anti-CXCR4 compounds. (B) Representative micrographs of the endothelial tubular network formation in the presence of anti-CXCR4 compounds. HUVECs formed excellent tubular networks in the presence of CXCL12 (#1), but poor tubular networks when CXCL12 was blocked by prior treatment with 100 nM of AMD3100 (#2) or MSX-122 (#3). The results are summarized in a graph in the right panel.
Figure 3.
Comparison of inhibition of cAMP modulation by MSX-122 and AMD3100.
(A) With 15 min pre-treatment with MSX-122 or AMD3100 at various concentrations, the effect of 150 ng/ml of CXCL12 on cAMP reduction was measured by the TR-FRET based LANCE assay kit using U87CD4CXCR4 cells. (B) RT-PCR analysis of CXCR4 and CXCR7 mRNA in wild-type MDA-MB-435 cells. CXCR4-overexpressing MDA-MB-435 cells were generated by transfection using a CXCR4 expressing plasmid (Missouri S&T cDNA resource center) and Lipofectamine2000. (C) No effect of MSX-122 on cAMP reduction induced by 150 ng/ml of CXCL12 in CXCR4-negative, wild-type MDA-MB-435 cells. (D) The effect of MSX-122 on cAMP reduction induced by 150 ng/ml of CXCL12 in CXCR4-overexpressing MDA-MB-435 cells.
Figure 4.
MSX-122 attenuates colonic damage in mice with experimental colitis.
(A) Colonic sections were stained with H&E. (B) Histological scores were determined by a blinded examination of the sections. n = 16 per group. *, p<0.05 between DSS and DSS + MSX-122 treated mice. (C) MPO activity was measured in the colons of control, DSS-, and DSS/MSX-122-treated mice. MPO activity was expressed as unit of MPO per mg colonic lysate. n = 12. *, p<0.05. (D) The presence of CD4+ lymphocytes was detemined by immunohistochemically using anti-CD4 antibodies. CD4+ lymphocytes are depicted in the brown color and nuclei were counter-stained in the blue color. Quantification of CD4+ lymphocyte per high powered field is shown. n = 4–5. *, p<0.01. (E) Representatives of immnohistological staining of CXCR4-positive cells are shown. CXCR4 protein is depicted by the brown color, whereas nuclei were counter-stained in blue. 1, control; 2, 7 days DSS; 3, 7 days DSS + 3 days or recovery; 4, 7 days DSS/MSX-122; and 5, 7 days DSS/MSX-122 + 3 days MSX-122. (F) Quantitative analysis of CXCR4-positive immune cells in the colon is shown. *, p<0.01. MSX-122 blocks migration of CXCR4 positive cells. (G) Expression of cytokine mRNAs was determined by qRT-PCR. Expression levels of cytokines were normalized to 18S. n = 8. *, p<0.05. (H) MSX-122 attenuates TNF-α secretion induced by invasive E.coli. J774A.1 macrophages in triplicates were infected with overnight cultures of commensal E.coli EFC-1, CD-isolated E.coli LF82 or 13I at MOI 10∶1 for 3 h. Following 3 h infection, macrophage culture was washed and incubated for additional 21 h in a growth medium containing antibiotics and MSX-122. The amounts of TNF-α in the supernatant were quantified by ELISA. Representative data from three independent experiments, each performed in triplicate, are shown. *, p<0.05.
Figure 5.
MSX-122 inhibits inflammation induced by carrageenan and lung fibrosis induced by bleomycin.
(A) Suppression effect of anti-CXCR4 compounds on carrageenan-induced mouse paw edema. Acute paw inflammation was induced by subcutaneous injection of 50 µL of λ-carrageenan in one hind paw. The mice in the treatment group were all administered CXCR4 antagonists at 10 mg/kg i.p., while control animals received corresponding i.p. injections of vehicle. Left panels show the control mice with left paw induced inflammation by carrageenan; right panels show the treated mice with left paw induced inflammation by carrageenan with about 55% suppression. (B) MSX-122 inhibits bleomycin-induced pulmonary fibrosis. A total of 20 mice, 10 in each group, received 10 mg/kg, i.p. of MSX-122ms or saline one day before bleomycin treatment and daily for 20 days. Representative H&E stainings of saline treated control (left) or MSX-122ms treated (right) lung tissues on day 20 after bleomycin treatment.
Figure 6.
MSX-122 blocks metastasis in an experimental animal model of breast cancer metastasis.
(A) Representative photographs of 5 lungs were shown from each group. (B) Histological quantization shows a decrease in tumor area in the treated group. Quantitative real-time RT-PCR of human CXCR4 confirms that animals injected with MDA-MB-231 cells and treated with vehicle develop lung metastasis. On the other hand, animals injected with MDA-MB-231 cells and treated with MSX-122 (4 mg/kg, i.p., daily) develop significantly less metastasis.
Figure 7.
MSX-122 blocks metastasis in two more animal models for metastasis.
(A) Non-invasive [18F]FDG-PET imaging of lung metastasis in head and neck cancer animal models. Three randomly selected mice revealed lung metastases in all mice in the control group; significantly less metastasis was found in the MSX-122 (10 mg/kg i.p., daily) treated group. Left panels show the axial images of three subjects from the control group (bottom) and three from the treated group (top). Right panels show the corresponding coronal images of the same mice. White arrows indicate metastasized SCCHN tumor cells. (B) The inhibition effect of MSX-122 (10 mg/kg i.p., daily) in a uveal melanoma micrometastasis animal model. Micrometastatic clones in liver are shown by small white arrows in the images. MSX-122 significantly decreased the numbers of hepatic micrometastases.