We recently reported that this lectin surfactant protein D (SP-D) suppresses epidermal growth factor receptor (EGFR) signaling by interfering with ligand binding to EGFR through an interaction between the carbohydrate-recognition domain name (CRD) of SP-D and A549 human lung adenocarcinoma cells were serum-starved overnight and incubated with 1 m gefitinib for 2 h at 37 C. the indicated antibodies. The display the densitometric evaluation, and data are presented as mean S.D. (same experiment as was performed using CHOK1 cells Vcam1 stably expressing human EGFR. display densitometric analyses, and data are presented as mean S.D. (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01 (compared with control). SP-A suppresses the proliferation, migration, and invasion of A549 cells Next, we examined the effects of SP-A around the proliferation of lung cancer cells. A549 cells were incubated with 10 g/ml SP-A, and the cell proliferation was assayed after 24, 48, and 72 h. As shown in Fig. 2SP-A suppressed the proliferation of A549 cells. Dose dependence was also confirmed (Fig. 2A549 cells were plated in a 96-well plate (1 103 cells/well), maintained in DMEM with 10% (v/v) FCS, and incubated with 10 g/ml SP-A at 37 C. The cell proliferation was assayed after 24, 48, and 72 h using the WST-1 reagent. The absorbance at 440 nm was measured on the plate reader. A549 cells were incubated with various concentrations of SP-A, and the cell proliferation was assayed after 72 h. The data shown are presented as mean S.D. (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01 (compared with the SGI-1776 kinase activity assay control). A549 cells were incubated with the indicated concentrations of gefitinib with or without 20 g/ml SP-A. Cell proliferation was assessed after 48 h using the WST-1 reagent. The data shown are presented as mean S.D. (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01 (compared with the control). A549 cells were seeded into the upper insert of a transwell double chamber in DMEM with 0.1% (v/v) BSA and EGF (10 ng/ml) with or without SP-A (10 g/ml). DMEM with 10% (v/v) FCS was added to the bottom wells as a chemoattractant. A control insert was used for migration assay (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01. A549 cells were seeded into the upper insert of a transwell double chamber using DMEM with 0.1% (v/v) BSA and EGF (10 ng/ml), with or without SP-A (20 g/ml) or gefitinib (10 m). DMEM with 10% (v/v) FCS was then added to the bottom wells as a chemoattractant. A control insert was used for the migration assay (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01. A549 cells were applied into each well of ibidi chambers. After incubation for 24 h, the culture inserts were removed, and the dishes were filled with a serum-free medium. EGF (100 ng/ml) and SP-A (20 g/ml) were added to the medium, and the cells were incubated for 24 h. The migrated cells were measured under a microscope. The data shown are the mean S.D. (test or Welch correction was used for statistical comparisons. *, 0.05; **, 0.01 (compared with EGF-treated control cells). We then SGI-1776 kinase activity assay evaluated the effects of SP-A around the migration and invasion of A549 cells. When SP-A was added, the number of EGF-induced migration and invasion cells was significantly decreased (Fig. 2dose-dependent suppression of EGF binding by SP-A. Binding of EGF to the cells was evaluated SGI-1776 kinase activity assay using a -counter as described under Experimental procedures. The data are expressed as relative values with the binding in the absence of SP-A being 100%. Experiments were performed in duplicate and were repeated three times. The data are representative of three impartial experiments. Open in a separate window Physique 4. SP-A does not influence cell-surface expression of EGFR in A549 cells. A549 cells were serum-starved overnight. The next day, cells were incubated with 20 g/ml SP-A for 2 h, washed, and incubated with 0.5 mg/ml Sulfo-NHS-LC-biotin for 30 min at 4 C. Whole-cell lysates were immunoprecipitated with the monoclonal anti-EGFR antibody (clone Ab-11) or control IgG. Samples were separated by SDS-PAGE, transferred onto PVDF membranes, and probed with HRP-conjugated SGI-1776 kinase activity assay streptavidin (and whole-cell lysate of A549 cells was immunoprecipitated (and experimental paradigm described in was performed in H441 cells (and sEGFR was produced in Flp-In CHOK1 cells and purified as described under Experimental procedures. 0.5 g of proteins with or without PNGase F treatment were subjected to SDS-PAGE, which was followed by Coomassie Brilliant Blue R-250 staining (indicated concentrations of SP-A were incubated with sEGFR (100 ng/well).