Leukemia. associated with activation of -catenin, a putative tumor suppressor in bone and soft tissue sarcoma and an important component of osteogenesis. Our study thereby demonstrates a critical role for GSK-3 in sustaining survival and proliferation of osteosarcoma cells, and identifies this kinase as a potential therapeutic target against osteosarcoma. < 0.01) between cells treated with DMSO and either GSK-3 inhibitor. Open in a separate window Figure 4 Effect of RNA interference on the expression of GSK-3, cell viability, proliferation and apoptosis in osteosarcoma and osteoblast cells(A) Western-blotting analysis compared the level of expression of GSK-3 and GSK-3 between cells treated with non-specific (N) and GSK-3-specific (S) siRNA (20 nmol/L each), respectively. Expression of -actin was monitored as a loading control. (BCD) Relative number of surviving cells, BrdU-positive proliferating cells and TUNEL-positive apoptotic cells were counted and compared between cell types 96 hours after transfection SNX-5422 Mesylate of non-specific and GSK3-specific siRNA. Values shown are the mean SD of six separate experiments. Asterisks denote a statistically-significant difference between cells transfected with non-specific and GSK- 3-specific siRNA. Changes in subcellular localization and activity of -catenin following GSK-3 inhibition Previous studies showed the canonical Wnt/-catenin pathway was inactivated during the development and progression of bone and soft tissue sarcomas including osteosarcoma [26C28]. In contrast to its oncogenic role in many cancer types [29, 30], this observation suggested a tumor suppressor function for the Wnt/-catenin pathway in osteosarcoma [26C28]. Therefore, we focused on -catenin, the downstream effector of the Wnt signaling pathway that is phosphorylated by GSK-3 for ubiquitin-mediated proteasomal degradation [29]. Consistent with the results shown above in Figure ?Figure1,1, -catenin was phosphorylated at the known GSK-3 phospho-acceptor residues (S33, S37 and/or T41) in osteosarcoma cells. Treatment with GSK- 3 inhibitors suppressed the phosphorylation of -catenin and increased its expression (Figure ?(Figure5A).5A). GSK-3 inhibitors also induced the nuclear translocation of -catenin in osteosarcoma cells, whereas the nuclear localization of -catenin was constitutively observed in hFOB1.19 osteoblasts regardless of treatment with DMSO or GSK-3 inhibitor (Figure ?(Figure5B,5B, Supplementary Figure S3). The TOP/FOP flash assay showed a significant increase in T-cell factor-dependent promoter activity in osteosarcoma cells following treatment with GSK-3 inhibitors (Figure ?(Figure5C),5C), reflecting an increase in the co-transcriptional activity of -catenin. Open in a separate window Figure 5 Effect of GSK-3 inhibition on the expression, phosphorylation, subcellular localization and co-transcriptional activity of -catenin in osteosarcoma and osteoblast cells(A) Western-blotting analysis was used to compare the expression and phosphorylation of -catenin between cells treated with DMSO and either GSK-3 inhibitor. Expression of -actin was monitored as a loading control. (B) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of -catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 m. The number shown below each panel indicates the percentage of nuclear -catenin-positive cells among the total number of cells. The SNX-5422 Mesylate bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of -catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear -catenin-positive cells in 3 microscopic fields was evaluated with standard SNX-5422 Mesylate deviation. (C) Relative co-transcriptional activity of -catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and Rabbit Polyclonal to SSTR1 SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3 inhibitors. The influence of -catenin expression on the therapeutic effects of GSK-3 inhibition of osteosarcoma cells was examined by RNA interference of -catenin prior to the treatment of cells with GSK-3 inhibitor. Depletion of -catenin reduced the effects of.