| Background: Bone osteosarcoma is a kind of highly malignant tumor;which has long been identified as the most representative sarcoma on account of the following characteristics: high incidence of early metastasis,high recurrence rate and poor survival rate.it has been found that cantharidin,an active component of cantharides,has a therapeutic effect on a variety of malignant tumors.Compare with cantharidin,sodium sulfate maintain the specific anticancer activity of cantharidin,and the toxicity and irritation is significantly lower.Due to the low molecular weight,highly permeability,sodium cantharidinte is widely used in clinical.Methods: This study will choose the human osteosarcoma cell line MG-63 as the object,to observe the inhibitory effect of sodium cantharidinate on MG-63 cells by flow cytometry.The apoptosis and cell cycle of MG-63 treated by sodium cantharidinate will be considered.The anti-osteosarcoma mechanism of sodium cantharidinate will provide the basis for clinical study of sodium cantharidate for treatment of osteosarcoma tumor.Results: It showed that the proliferation of MG-63 cells was significantly inhibited by sodium cantharidinate.Sodium cantharidinate could significantly promote the apoptosis of MG-63 cells by activation of Caspase-3 pathway.We found that sodium cantharidinate could induce Caspase-3 activation in a concentration dependent manner,leading to cell apoptosis.When Caspase-3 gene was silenced in MG-63 cells,the apoptosis of MG-63 cells induced by sodium cantharidinate was inhibited.Next,we study the role of autophagy pathway in LC3.The results showed that after LC3 gene silencing,the ability of sodium cantharidate induced apoptosis in MG-63 cells decreased significantly.The decline in the proportion is beyond 50%,indicating that MG-63 cell apoptosis is mediated by caspase-3 induced by LC3 downstream autophagy pathway.The apoptosis of MG-63 cells induced by sodium chloride is caused by the activation of LC3 signaling protein,which is caused by autophagy,which leads to the activation of the Caspase-3 gene,which leads to the apoptosis of MG-63 cells.Cell cycle detection showed that sodium cantharidate inhibited the expression of Cyclin D1 and Cdk-4,Cdk-6 kinase in MG-63 cells,which led to the deposition of cells in G0/G1 phase and the cell cycle arrest.In the following experiments,we observed that sodium cantharidate specifically stimulated the phosphorylation of AKT and the activation of AKT signaling pathway.The activation of JNK,P38 and m TOR was not affected by sodium cantharidate.PI3K/AKT inhibitor and p53 gene silencing on MG-63 cells significantly reduced the MG-63 cell cycle arrest caused by sodium cantharidinate.PI3K/AKT inhibitor down-regulated the activation of AKT signaling stimulated by sodium cantharidinate obviously,further inhibited the expression of p53 and p21,so that the cell cycle protein kinase Cdk-4 Cyclin D1 and corresponding Cdk-6 expression returned to normal,eventually leading to cell smoothly through the G0/G1 phase into S phase.The silence of p53 could not affect the activation of PI3K/AKT signaling pathway induced by sodium cantharidinate.However,the expression of p21 was significantly decreased in p53 silenced MG-63 cells,and then inhibited the cell cycle and protein kinase activity.Conclusion: we demonstrated that MG-63 cell apoptosis is induced by sodium cantharidate,by trigger cell autophagy,LC3 protein activation,followed by activation of apoptosis gene Caspase-3,eventually lead to apoptosis of MG-63 cells.At the same time,sodium cantharidinate activated MG-63 cells PI3K/AKT phosphorylation,which lead to the activation of p53/p21 and inhibit the expression of Cyclin D1 and Cdk-4,Cdk-6 kinase,leading to cell accumulation in G0/G1 phase,causing cell cycle arrest.The completion of this thesis will open up a new way for the treatment of osteosarcoma,and provide the theoretical and experimental basis for the development of antitumor drugs. |
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