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Archive > Volume 20 Issue 4 > 2013,20(4):419-424. DOI:10.3872/j.issn.1007-385X.2013.04.007 Prev Next
Basic Research
Inhibitory effect of FIM-A, a mTOR inhibitor, on the proliferation and apoptosis of human MG-63 osteosarcoma cell line and its mechanism
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Abstract:
Objective: To investigate the effect of phosphorus sirolimus derivatives FIM-A, a new mammalian mammalia target of rapamycin (mTOR) inhibitor, on the proliferation and apoptosis of human MG-63 osteosarcoma cell line. Methods: Human MG-63 osteosarcoma cells and hF-OB1.19 osteoblasts were cultured in vitro and incubated with different concentrations of FIM-A (1×10-9-1×10-5 mol/L) for 24 hours. CCK-8 assay was used to evaluate the cell proliferation. The cell cycle and apoptosis were analyzed using flow cytometry. ELISA was used to detect the secretions of vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α). The expressions of mTOR, p70S6 kinase protein (p70s6k) and 4E-binding protein 1 (4E-BP1) mRNA and protein were detected by RT-PCR and Western blotting, respectively. Results: The expressions of mTOR, p70s6k and 4E-BP1 mRNA in MG-63 osteosarcoma cells were significantly higher than that in the hF-OB1.19 osteoblasts (P<0.05). The proliferation of the MG-63 osteosarcoma cells were significantly inhibited after FIM-A treatment. The proliferation inhibition rate of MG-63 cells was significantly higher than that of the negative control group after the treatment of 1×10-7 mol/L FIM-A (\[37.64±2.07\]% vs 0, P<0.05), and the cell proliferation inhibition rate increased along with FIM-A concentrations in a dose-dependent manner (r=0940, P<0.01). After the treatment of 1×10-6 mol/L FIM-A for 24 hours, the proportion of MG-63 cells in G0/G1 phase was significantly increased compared with the control group (\[56.4±3.2\]% vs \[43.4±6.9\]%, P<005). No obvious changes were found in the apoptotic rate of MG-63 cells compared with the control group. The expression levels of HIF-1 α and VEGF in MG-63 cells were significantly lower than those of the control group after the treatment of different concentrations of FIM-A for 24 hours (P<005), and as concentrations increased, the level of HIF-1 α (r=0.988, P<001) and VEGF (r=0.998, P<0.01) decreased gradually in a dose-dependent manner. Meanwhile, the inhibitory effect of FIM-A on phosphorylations of p-mTOR (r=-0.919, P<0.01), p-p70s6k (r=-0.843, P<0.01) and p-4EBP1 (r=-0.818, P<0.01) proteins in MG-63 cells was also in a dose-dependent manner. Conclusion: FIM-A can significantly inhibit human MG-63 osteosarcoma cells and induce G0/G1 phase cell cycle arrest, and its anti-tumor effect was probably through the intervention of mTOR pathway.
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Project supported by the Major Science and Technology Foundation of Fujian Province (No. 2011YZ0002-1)
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