Osteosarcoma is the most prevalent primary malignant bone tumor mainly endangering young adults. could inhibit the proliferation by inhibiting the -catenin signaling and induce apoptosis via upregulation the ratio of Bax/Bcl-2 in human osteosarcoma cells. value of less than 0.05 was considered significant difference. Results PD inhibited the proliferation and viability of osteosarcoma cells The proliferation and viability of 143B and MG63 cells were determined by MTT and crystal violet viability assay, respectively, following treatment with concentrations of 10-6, 10-5, and 10-4 M of PD for 24, 48 and 72 h. We found that PD inhibited the proliferation of these human cells in a dose-dependent manner based on the MTT assay (Figure 2A). In lines with MTT assay, PD also exhibited the inhibitive effects on these osteosarcoma cells in a dose-dependent manner determined by crystal violet viability assay (Figure 2B). Figure 2 Effect of polydatin on proliferation and viability of human osteosarcoma cells. 143B and MG63 osteosarcoma cells BEZ235 (NVP-BEZ235) supplier were treated with polydatin at indicated concentration. A. The proliferation was measured by MTT 24, 48, and 72 h after treatment. B. The … PD induced apoptosis in osteosarcoma cells The apoptosis of MG63 induced by PD was quantified by flow cytometry using Annexin V-FITC/PI staining. As shown in Figure 3, the apoptosis were significantly increased in the groups treated with PD by comparison with control groups, and dose-dependent effects were found. Figure 3 Effect of BEZ235 (NVP-BEZ235) supplier polydatin on apoptosis of human CD86 osteosarcoma cells. 143B osteosarcoma cells were treated BEZ235 (NVP-BEZ235) supplier with 0, 10, 30, and 100 M polydatin for 48 h, apoptosis was detected using annexin V-FITC/PI dual staining by flow cytometry. PD enhanced the activation of caspase-3 in osteosarcoma cells The caspase-cascade system plays a critical role in the initiation, transduction, and amplification of cell apoptosis [18]. To investigate the cleaved effector caspase in the apoptotic pathway, the activation of caspase-3 were measured by the colorimetric assay. As shown in Figure 4, compared with untreated group, exposure of PD can evidently and dose-dependently induce activation of caspase-3 in 143B osteosarcoma cells. Figure 4 Effect of polydatin on caspase-3 activity in human osteosarcoma cells. 143B osteosarcoma cells were treated with 0, 10, 30, and 100 M polydatin for 48 h, caspase-3 activity was measured by the colorimetric assay. Each value is mean … PD up-regulated expression of pro-apoptotic protein (Bax) and down-regulated anti-apoptotic protein (Bcl-2) The Bcl-2 family proteins are important regulators of apoptosis, including both pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2, and the ratio of Bax/Bcl-2 determined the fate of cell [19]. To further study the potential mechanism by which the PD induces apoptosis of human osteosarcoma cells, the effect of PD on the expression of Bcl-2 and Bax in 143B osteosarcoma cells was examined. As shown in Figure 5, PD profoundly up-regulated Bax and down-regulated Bcl-2 in a dose-dependent manner. Figure 5 Effect of polydatin on expression of Bcl-2 and Bax in human osteosarcoma cells. 143B osteosarcoma cells were treated with 0, 10, 30, and 100 M polydatin for 48 h, expression of Bcl-2 and Bax were determined by western blotting. PD suppresses -catenin signal pathway The expression of -catenin is obviously higher than normal tissue [20], subsequently; it can induce over activation of several downstream target genes including andc-Junand Lef1, leading to increased cell survival against apoptosis induced by chemotherapeutic agents and proliferation [33,34]. In the current study, by Quantitative Real-Time PCR and western blotting, we found that PD dose-dependently decreased the expression of -catenin mRNA and protein, which presented a negative association with cell proliferation. In order to identify the activation of -catenin, luciferase reporter assay were used after treatment of PD. The results presented that the reduction of activated -catenin paralleled with the protein and mRNA expression, but inversely related to osteosarcoma cell proliferation. Furthermore, by overexpression of -catenin adenoviruse vector, we found that the inhibitive effect.