Effects Of Bisphosphonates On Growth And Expression Of OPG/RANKL In Osteosarcoma MG-63 Cells In Vitro

ObjectiveTo study the effects of bisphosphonates on proliferation and apoptosis in MG-63 cells in vitro. In this study, the effects of proliferation and apoptosis were examined by N-BPs treatment on Osteosarcoma cell line. Further, the expression of RANKL and OPG were assessed for the purpose of benefiting the development of novel strategies to a target molecular treatment on Osteosarcoma. To investigate anti-tumor effects of N-BPs on MG-63 cells in vitro.Methods1. Different concentration of bisphosphonates and the different time of cultivate were used to treat MG-63 cell. The proliferation inhibition was analysed by alive cell count, MTT assay was used to measure the direct anti-tumor effect of bisphosphonates against MG-63 cell line with inhibition concentration 50% (IC50) values.2. Morphological studies included transmission electron microscope observation and Hoechst 33258 fluorescent staining of Osteosarcoma cells affected by bisphosphonates to detect early and late apoptotic cells.3. Flow cytometer(FCM)with PI and Annexin V-FITC staining were applied to determine cell cycle of MG-63 after medical intervention.4. RT-PCR and western blotting as the expression of OPG and RANKL.Results1. MTT assay showed that Zoledronic acid induced a distinct dose- and time-dependent diminution of cell viability (IC₅₀:52.37±1.0μmol/L for 72 h). 2. The MG-63 cells apoposis was comfirmed by type cell morphology DNA content, Annexin V-FITC /PI and Hoechst33258 labeling method. Furthermore, N-BPs induced apoptosis of MG-63 cells were verified by electron microscopy.3. Flow cytometry analysis showed that N-BPs induced cell apoptosis was through arrest of cell cycle in G₁ phase. Cell cycle analysis showed that the proportion of S phase in cells of intervention groups increased while that of G₂/M phase decreased markedly, which demonstrated cells were blocked in G₁ phase.4. Densitometry analysis showed that zoledronic acid induced 1.8-fold increases in OPG expression in MG-63 cells. There was no significant change in the expression of RANKL in the MG-63 cells. Furthermore, Western blotting demonstrated that N-BPs up-regulated OPG expression.ConclusionsThese results demonstrate that bisphosphonates inhibits MG-63 cell line growth directly, and induces cell apoptosis, as well as up-regulates osteoprotegerin expression.