Figure 1.
Oxyclozanide binds S100A9 protein.
A: General formulae of synthesized salicylic and benzoic amides. B: Structure of Oxyclozanide (OX). C-D: Inhibition of S100A9 binding to immobilized RAGE (C) or TLR4 (D) by OX. 50 nM S100A9 was injected over RAGE or TLR4±0.4–200 µM OX in the presence of 1 mM CaCl2, 20 µM ZnCl2 and 1% DMSO. Binding was expressed as % inhibition of S100A9 response without OX and IC50 calculated after curve fit to a sigmoidal dose-response model. IC50 values of ∼1.3 and 1.6 µM were obtained for OX inhibition of S100A9 binding to RAGE and TLR4. E: Effect of Zn++ on direct binding of OX to amine coupled S100A9. 3.125–100 µM OX was injected (2 min at 30 µL/min) over S100A9 (density ∼2.6 kRU) in HBS-P with 1 mM Ca++ ±20 µM Zn++. Responses at late association phase are plotted versus OX concentration and curves fit to a 1∶1 model. Zn++ specific binding was obtained by subtraction of responses with Ca++ alone.
Figure 2.
OX inhibits EL4 lymphoma growth in vivo.
A: Anti-tumor effect of OX in EL4 tumors inoculated (s.c.) into wild type mice. OX was administrated p.o. at 30 mg/kg/day 7 days/week from day 1 throughout the experiment. Each data point represents mean ± SEM (n = 10; *p<0.05, Mann Whitney U). Control animals received only water. B: Tumor weights (*p<0.05; Mann Whitney U).
Figure 3.
A: Effect of Zn++ on direct binding of OX to amine coupled S100A4. 3.125–100 µM OX was injected (2 min at 30 µL/min) over S100A4 (density ∼2.4 kRU) in HBS-P with 1 mM Ca++ ±20 µM Zn++. Responses at late association phase are plotted versus OX concentration. Zn++ specific binding was obtained by subtraction of responses with Ca++ alone and curves fit to a 1∶1 model. B-C: Inhibition of S100A4 binding to immobilized RAGE (B) and TLR4 (C) by OX. 100 nM S100A4 was injected over RAGE or TLR4±0.4–200 µM OX in the presence of 1 mM Ca, 20 µM Zn and 1% DMSO. Binding was expressed as % inhibition of response with S100A4 in the absence of OX. IC50 values (inserted) were calculated after curve fit to a sigmoidal dose-response model.
Figure 4.
S100A4 and S100A9 can form heterodimers.
A: Binding of S100 proteins to immobilized S100A4 and S100A9. S100A1, A4, A7, A9 and A13 (∼1.3 µg/mL) were injected (2 min at 30 µL/min) over S100A4 and S100A9 (density ∼2.5 kRU) in the presence of 1 mM Ca++ and 20 µM Zn++. Responses were calculated at late association phase. B: Formation of homo- and hetero-complexes of S100A4 and S100A9 is Zn++ dependent. S100A4 and S100A9 were injected over immobilized S100A4 or S100A9 at ∼1.3 µg/mL HBS-P containing 1 mM Ca++ ±20 µM Zn++. Responses at late association phase were calculated. C: HEK293T cells were transfected either with S100A4 or S100A9 cDNA construct alone or the two together, as indicated. After 24 hrs of culture some of the transfected cells were exposed to the membrane permeable cross-linker DSS. Thereafter cell lysates were prepared, equal amounts (30 µg) of protein loaded on an SDS-PAGE gel and western blots were performed using either anti-S100A4 or anti-S100A9 antibodies as indicated. Representative results from one out of two experiments performed are shown.
Figure 5.
Binding of homo- and hetero-complexes of S100A4 and S100A9 to RAGE are displaced by OX and heparan sulfate (HS).
A: Binding of homo- and hetero-complexes of S100A4 and S100A9 to immobilized RAGE and TLR4. S100A4, S100A9 and a 1∶1 mixture of S100A4/9 were injected (2 min at 30 µL/min) over RAGE (left panel) or TLR4 (right panel). B: Inhibition of ∼1.3 µg/mL S100A4, S100A9 and the 1∶1 mixture with 0.195–100 µM OX in the presence of 1 mM Ca++, 20 µM Zn++ and 1% DMSO. Samples were injected (2 min at 30 µL/min) over immobilized RAGE. C: Responses at late dissociation phase (expressed as % of signal in the absence of OX) were plotted against concentration of competitor and IC50 calculated by fit of curves to a sigmoidal dose-response or a one-site competition model. IC50 values of 1.4, 52 and 17 µM were calculated for S100A9, S100A4 and S100A4/S100A9. D: Corresponding experiment with 0.98–500 ng HS/mL as competitor. Conditions were identical except that DMSO was omitted from the sample buffer. HS inhibited binding of S100A9, S100A4 and S100A4/S100A9 to RAGE with 50% at 22, 36 and 60 ng/mL.
Figure 6.
Analysis of S100A9 and S100A4 expression in vivo.
FACS sorting of spleen cells from C57BL/6 animals. Panel A: Left: The gate used for defining CD11b+ cells is shown. Right: The CD11b+Ly6G+C+/CD11b+Ly6C++ populations used for comparison and defined as indicated. Purity is shown to the far right. Panel B: Quantitative real time RT-PCR analysis of S100A9, S100A8 and S100A4 RNA expression from indicated cell populations (>90% pure by FACS analysis) from the spleen and BM of C57BL/6, animals. The mean expression from 4 separate experiments is shown (*** = p<0.001; Students unpaired t test). C. FACS Aria sorted cells were used for Western blotting. Antibodies to S100A4, S100A9 and β-tubulin were used to reveal respective protein. Panel D. Indicates the gating strategy on S100A4 GFP mice on C57BL/6 background. E. Quantitative RT-PCR analysis of S100A4 and S100A9 expression from THP-1 cells cultivated alone or for 48 h with LPS or S100A9, as indicated. Two independent experiments with triplicate cultures were performed.
Figure 7.
Analysis of S100A9 and S100A4 expression in vivo during disease.
Panel A and B; Quantitative real time RT-PCR analysis (see Materials and Methods). Spleen cells, from control C57BL/6 animals, from mice inoculated with 50,000 EL4 lymphoma cells or spleen cells from EAE induced animals with MOG-peptide and with Pertussis toxin were FACS-sorted and used to analyze RNA expression of S100A4 (Panel A) and S100A9 (Panel B) (*** = p<0.001; Students unpaired t test). Panel C; Immunohistochemical analysis show cells expressing S100A4 (green), S100A9 (red) in brain sections from mice with MOG induced EAE. The labeled cells are localized close to and around a vessel. An overlay of the two first pictures including nuclear staining of DAPI (blue) is shown in the last picture. Scale bar represents 100 µm. Panel D; Expression of S100A4 (green) and S100A9 (red) respectively in sections from an EL-4 tumor. An overlay of the two first pictures including nuclear staining with DAPI (blue) is shown in the last picture. The overlay shows that S100A4 and S100A9 is expressed in different cells, no overlap can be detected. Scale bar represents 50 µm.