The Effect And Mechanism Of Adiponectin On Proliferation Inhibition And Apoptosis Induction Of Osteosarcoma Cell Through MTOR Signaling Pathway

Part Ⅰ Effect of adiponection on proliferation and apoptosis of osteosarcoma cellsAbstractObjective:To investigate effects of adiponectin on proliferation and apoptosis of osteosarcoma cells and possible mechanisms.Methods:Western-blot (WB) was applied to measure levels of adiponectin receptor (AdipoR1, AdipoR2) in three human osteosarcoma cell lines (MG-63, Saos-2, U2OS) and human osteoblast cell line hFOB1.19. After treatment of adiponectin in different concentrations on those osteosarcoma cells, CCK-8was adopted to measure cell proliferation and to screen out adiponectin-sensitive cell line. Experiments on proliferation and apoptosis were performed on the adiponectin-sensitive cells:cell cycle variation was measured by flow cytometry; cell apoptosis was measured by co-staining of TUNEL and DAPI; Cyclin D1and Caspase-3were measured by WB as cell cycle and apoptosis markers respectively.Results:①WB results showed specific expression of AdipoRl and AdipoR2in all three osteosarcoma cell lines (MG-63, Saos-2, U2OS) and osteoblast hFOB1.19. Level of AdipoRl was significantly higher in all three osteosarcoma cell lines than hFOB1.19. Saos-2had the highest level of AdipoRl and AdipoR2when compared to the other three cell lines.②CCK-8showed that adiponectin had effects of dose-dependent inhibition on proliferation of the three osteosarcoma cell lines. However, the effect of inhibition on proliferation reached to the plateau over20ug/ml, indicating the concentration around20ug/ml was the peak point. MG-63was the most adiponectin-sensitive as displayed the highest inhibition ratio when compared to the other two cell lines.③Flow cytometry results showed significant reduced ratio of S phase in osteosarcoma cells treated with adiponectin (lOug/ml) when compared to untreated group. Ratio of G0/G1increased dramatically, whereas ratio of G2and S phase decreased greatly in osteosarcoma cells treated with adiponectin (20ug/ml) when compared to untreated group and lOug/ml group.④Co-staining results showed increased apoptosis in osteosarcoma cells treated with lOug/ml adiponectin (6.52±0.878%) and20ug/ml adiponectin (8.24±0.11%), when compared to untreated group (2.57±0.44%).⑤WB results showed level of Cyclin D1in osteosarcoma cell lines treated with adiponectin (lOug/ml,0.995±0.113;20ug/ml,0.904±0.095) decreased significantly when compared to untreated group (1.276±0.081). Level of Caspase-3was raised in adiponectin group (10ug/ml,1.027±0.083;20ug/ml,1.069±0.142) compared to untreated group (0.794±0.033). Conclusion:AdipoRl and AdipoR2were specifically expressed in human osteosarcoma cell lines and osteoblast cells, and levels of adiponectin receptors can be varied in different types of osteosarcoma cell lines. Adiponectin has dose-dependent effects of inhibition on proliferation and promotion on apoptosis of osteosarcoma cell lines, possibly via regulation of Cyclin D1and Caspase-3. Part Ⅱ Adiponection exerts effects in osteosarcoma cells via mTOR signaling pathwayAbstractObjective:To investigate mechanisms of effect of adiponectin on osteosarcoma cells via PI3K/Akt/mTOR axis.Methods:Adiponectin-sensitive osteosarcoma cell line was treated with adiponectin at different concentration and duration. Downstream molecules of mTOR pathway (p70S6K1, p-p70S6Kl1Thr389,4EBP1, p-4EBPlThr37/46) and upstream molecules (Akt, p-AktSer473) were measured via WB. Furthermore, after mTOR in MG-63was enhanced or blocked by Rhebl-overexpression via lentivirus transfection or exert of PI3K/Akt antagonist LY294002respectively, adiponectin was given and levels of Cyclin D1and Caspase-3were measured via WB.Results:mTOR in MG-63was inhibited by adiponectin in dose and time-dependent way, as levels of p-p70S6K1Thr389, p-4EBP1Thr37/46and p-AktSer473in a resembled way. The down-regulation of Cyclin D1expression and up-regulation of Caspase-3expression by adiponectin were less remarkable after mTOR was universally activated by lentiviral transfection overexpressing Rheb1, indicating the role of adiponectin on proliferation inhibition and apoptosis induction of osteosarcoma cells were impaired when mTOR was activated. Moreover, adiponectin had a synergistic effect on proliferation inhibition and apoptosis induction of MG-63osteosarcoma cell line with PI3K/Akt/mTOR specific inhibitor—LY294002.Conclusion:Adiponectin can suppress expression of Cyclin D1but enhance expression of Caspase-3by imparing phosphorylation of Akt and following activation of mTOR signaling pathway. Conclusively, adiponection exerts inhibition effect on osteosarcomas by suppressing proliferation and promoting apoptosis of osteosarcoma cells.