Shirasuna KInvolvement of proteasomes in migration matrix metalloproteinase-9 production of oral squamous cell carcinoma. a selective IKK inhibitor, blocks IB phosphorylation, and prevents NF-B p65 nuclear translocation, and its prodrug is Quinagolide hydrochloride under assessment in a clinical trial for inflammation-related cardiovascular diseases and rheumatoid arthritis [19, 20]. In this study, we examined the potential for the C13orf18 clinical evaluation of IMD-0560 for the treatment of bone invasion by OSCC cells. RESULTS IMD-0560 inhibits TNF-induced p65 phosphorylation and IB degradation in human and mouse OSCC cells Pretreatment with IMD-0560 inhibited TNF-induced Quinagolide hydrochloride p65 phosphorylation (Ser-536) and IB degradation in a dose-dependent manner in SCCVII, HSC-2, and Ca9-22 cells Quinagolide hydrochloride (Figure ?(Figure1A).1A). We used IMD-0560 at 1 M for SCCVII and 10 M for HSC-2 and Ca9-22 cells following experiments, respectively. TNF induced the translocation of p65 from the cytoplasm to the nucleus, and IMD-0560 significantly blocked this translocation in HSC-2, Ca9-22, and SCCVII cells (Figure ?(Figure1B).1B). Pretreatment with IMD-0560 inhibited both IB degradation and p65 phosphorylation induced by TNF (Figure ?(Figure1C).1C). IMD-0560 also suppressed TNF-induced transcriptional activity (Figure ?(Figure1D).1D). These results strongly indicate that IND-0560 inhibits TNF-induced NF-B activation in OSCC cells. Open in a separate window Figure 1 IMD-0560 inhibits TNF-induced p65 phosphorylation and IB degradation in OSCC cells(A) SCCVII, HSC-2 and Ca9-22 cells were pretreated with various concentrations of IMD-0560 for 120 min and then treated with TNF (10 ng/ml) for 15 min. p65 phosphorylation and IB degradation were examined via Western blot. -actin was used as a loading control. Similar results were obtained in three independent experiments. (B) SCCVII, HSC-2 and Ca9-22 cells were pretreated with or without IMD-0560 (1 or 10 M) for 120 min and then further treated with or without TNF (10 ng/ml) for 30 min. Then, the cells were fixed and incubated in an anti-p65 antibody, followed by incubation in Alexa Fluor 430-conjugated anti-rabbit IgG. The subcellular localization of Alexa Fluor 430-labeled p65 was determined via fluorescence microscopy (magnification 200x). Bar = 50 m. Similar results were obtained in three independent experiments. (C) SCCVII and HSC-2 cells were pretreated or without IMD-0560 (1 or 10 M) for 120 min and then treated with TNF (10 ng/ml) for the indicated periods. p65 phosphorylation and IB degradation were examined via Western blot. -actin was used as a loading control. Similar results were obtained in three independent experiments. (D) SCCVII cells were transiently transfected with a PBIIx reporter, pretreated with or without IMD-0560 (1 M) for 120 min and then treated with or without TNF (10 ng/ml) for 8 hrs. The cells were assessed for luciferase activity after 8 hrs. The data are expressed as the mean SD (n=3). *and mRNA levels were analyzed via real-time PCR. The data represent the mean SD of the expression levels of relative to (n=3). *(Supplementary Figure S2). Open in a separate window Figure 4 Early treatment with IMD-0560 reduced bone invasion by inhibiting osteoclastogenesisTwenty-eight days after tumor inoculation, the tissues were fixed in 3.7% formaldehyde, decalcified in 10% EDTA, sectioned in the coronal axis and stained with H&E (upper panels), TRAP (middle panels) or RANKL (lower panels). Mice treated with CMC alone served as controls. NT: no tumor inoculation, C: control, E3: mice treated with IMD-0560 at 3 mg/kg, E5: mice treated with IMD-0560 at 5 mg/kg. (A) Upper panels: Original magnification 40x. Bar=100 m. Middle panels: Original magnification 400x. Bar=100 m. Lower panels: Some specimens from each group were processed for immunohistochemical staining with an anti-RANKL antibody. Original magnification 200x. T: tumor. Bar=100 m. Mice treated with CMC alone served as controls. (B) In each specimen, 5 tumor fields were randomly selected, and the number of TRAP+ MNCs was counted. The data are expressed as the mean SD of the number of TRAP+ MNCs/bone surface (mm2)/section (n=10). *(magnification 400x). Bar=100 m. (F) A representative photograph of MMP-9 staining of a tumor from the control and IMD-0560-treated groups (magnification 400x). Bar=100 m. (F) A representative photograph of MMP-9 staining.