Figure 1.

DKK-1 is highly expressed in osteolytic prostate cancer cells and inhibits Wnt3a-induced osteoblastogenesis in C2C12 cells. (a) Total mRNA and secreted protein levels of DKK-1 were measured by qRT-PCR analysis and ELISA respectively in prostate cancer cell lines. (b) Supernatants of prostate cancer cell lines MDA-PCa-2b and PC3 where harvested after 48 h. C2C12 cells underwent differentiation in the presence of Wnt3a media (10%), 5% FCS DMEM/F-12 (75%) and prostate cancer supernatant (15%) for 72 h. Ten percent L-cell media were used in the control conditions. The mRNA levels of the osteoblastic marker ALP were assessed by qRT-PCR. (c) C2C12 cells were transfected with the TCF/LEF Wnt promoter and treated in the presence of Wnt3a medium with PC3 supernatant and 1 μg/ml anti-DKK-1 or 1 μg/ml IgG goat for 24 h before lysis and assay. Activation of Wnt signaling was detected by measuring luciferase activity. ALP mRNA expression levels by qRT-PCR and ALP activity (arbitrary units) by enzymatic assay were assessed following the same experimental conditions as listed in (b). For prostate cancer cell lines and C2C12 experiments, mRNA expression data shown are normalized to beta-actin and murine beta-actin, respectively. Results are shown as the mean±S.D. (*P<0.05; **P<0.01, ***P<0.001) and N=3