Figure 1.
WNT1-inducible signaling pathway protein 1 (WISP-1) induces the migration activity of human oral squamous cell carcinoma (OSCC) cells.
(A) Cells were incubated with various concentrations of WISP-1, and the in vitro migration activity was measured with the Transwell assay after 24 h (n = 5). (B) SCC4 cells were incubated with WISP-1 for 24 h, and the wound-scratching assay was performed (n = 4). (C) SCC4 cells were incubated with various concentrations of WISP-1, and the invasion activity was measured with the Transwell assay after 24 h (n = 5). Results are expressed as the mean ± standard error of the mean (SEM); *, p < 0.05 compared with the control; #, p < 0.05 compared with the WISP-1–treated group.
Figure 2.
WISP-1 increases cell migration and intercellular adhesion molecule-1 (ICAM-1) expression through the αvβ3 integrin receptor.
(A&B) SCC4 cells were incubated with WISP-1 for 24 h, and ICAM-1 expression was examined by western blotting and quantitative real-time polymerase chain reaction (qPCR) (n = 5). (C) Cells were transfected with ICAM-1 small interfering RNA (siRNA) for 24 h, followed by stimulation with WISP-1. The in vitro migration activity was measured with the Transwell assay (n = 4). (D&E) The protein levels and migratory activity of WISP-1 and ICAM-1 in SCC4/control short hairpin RNA (shRNA) and SCC4/WISP-1 shRNA cells were examined by western blotting and the Transwell assay (n = 4). (F–H) Cells were pretreated with αvβ3 monoclonal antibody (mAb) (10 µg/mL) for 30 min followed by stimulation with WISP-1. The in vitro migration activity and ICAM-1 expression were measured with the Transwell assay, qPCR, and western blotting (n = 5). Results are expressed as the mean ± SEM; *, p < 0.05 compared with the control; #, p < 0.05 compared with the WISP-1–treated group.
Figure 3.
Apoptosis signal-regulating kinase 1 (ASK1) is involved in WISP-1–induced migration and ICAM-1 expression.
(A–D) Cells were pretreated for 30 min with thioredoxin (200 ng/mL) or transfected with ASK1 shRNA for 24 h and stimulated with WISP-1. The in vitro migration and ICAM-1 expression were measured by the Transwell assay, qPCR, and western blotting (n = 5). (E) SCC4 cells were incubated with WISP-1 for the indicated time intervals, and ASK1 phosphorylation was examined by western blotting (n = 4). (F) SCC4 cells were pretreated for 30 min with αvβ3 mAb and stimulated with WISP-1 for 15 min; ASK1 phosphorylation was determined by western blotting (n = 4). Results are expressed as the mean ± SEM; *, p < 0.05 compared with the control; #, p < 0.05 compared with the WISP-1–treated group.
Figure 4.
WISP-1 increases cell motility and ICAM-1 expression through the c-Jun N-terminal protein kinase (JNK) and p38 pathways.
(A–C) Cells were pretreated for 30 min with SB203580 (10 µM) and SP600125 (10 µM) or transfected with dominant negative (DN) mutants of p38 and JNK for 24 h followed by stimulation with WISP-1. The in vitro migration and ICAM-1 expression were measured by the Transwell assay, qPCR, and western blotting (n = 5). (D) SCC4 cells were incubated with WISP-1 for the indicated time intervals, and p38 and JNK phosphorylation was examined by western blotting (n = 4). (E) SCC4 cells were pretreated for 30 min with αvβ3 mAb or thioredoxin for 30 min followed by stimulation with WISP-1 for 60 min, and JNK and p38 phosphorylation was determined by western blotting (n = 5). Results are expressed as the mean ± SEM; *, p < 0.05 compared with the control; #, p < 0.05 compared with the WISP-1–treated group.
Figure 5.
Activator protein 1 (AP-1) is involved in WISP-1–mediated migration in human OSCC cells.
(A–C) Cells were pretreated for 30 min with curcumin (10 μM) and tanshinone (10 µM) or transfected for 24 h with c-Jun siRNA followed by stimulation with WISP-1 for 24 h. The in vitro migration and ICAM-1 expression were measured by the Transwell assay, qPCR, and western blotting (n = 5). (D) SCC4 cells were incubated with WISP-1 for the indicated time intervals, and c-Jun phosphorylation was examined by western blotting (n = 4). (E–G) SCC4 cells were pretreated for 30 min with αvβ3 mAb, thioredoxin, SB203580, or SP600125 for 30 min followed by stimulation with WISP-1 for 120 min. The c-Jun phosphorylation, c-Jun binding to the AP-1 element, and c-Jun translocation into the nucleus was determined by western blotting, chromatin immunoprecipitation, and immunofluorocytochemistry (n = 5). Results are expressed as the mean ± SEM; *, p < 0.05 compared with the control; #, p < 0.05 compared with the WISP-1–treated group.
Figure 6.
WISP-1 and ICAM-1 expression correlates with the tumor stage of patients with OSCC.
Immunohistochemistry of WISP-1 (A&B) and ICAM-1 (A&C) expression in normal and OSCC tissue. The correlation and quantitative data are shown in (D).