Mechanism Of Dihydroartemisinin Inducing Apoptosis Of MG-63 Cells Via CaMKKβ-AMPK Pathway

Objective: Dihydroartemisinin(DHA)is a main active metabolite of artemisinin derivatives and has been reported to have a broad anti-tumor effect,but the exact mechanism remains unclear.This study focused on the mechanism of DHA-induced apoptosis in MG-63 cells,and the CaMKKβ-AMPK pathway,to provide new ideas for diagnosis and treatment of osteosarcoma.Method 1 MG-63 cells in logarithmic growth phase were cultured in dihydroartemisinin with the concentration of 0,10,20 and 40 μM,respectively.After culturing for 24 hours,the MG-63 cells were treated with Annexin v-fitc / pi by flow cytometry to detect apoptosis.2 MG-63 cells in logarithmic growth phase were cultured in dihydroartemisinin with the concentration of 0,10,20 and 40 μM,respectively.After culturing for 24 hours,protein were extracted,and using Western Blot to detect express level of Caspase-3,calcium / calmodulin-dependent protein kinase kinase beta(CaMKKβ),adenylate-activated protein kinase(AMPK),acetyl-CoA carboxylase(ACC)and phosphorylated AMPK,phosphorylated ACC.3 After BAPTA-AM(intracellular calcium chelator)pretreatment,the above test was repeated.Result Apoptosis test confirmed that dihydroartemisinin can promote the apoptosis of osteosarcoma cells,and positively correlated with the concentration.The results of Western Blot showed that compared with the control group,Caspase-3,CaMKKβ,AMPK,ACC and phosphorylated AMPK and ACC in the experimental group were all increased.The result was positively correlated with the concentration.After BAPTA-AM(intracellular calcium chelator)pretreatment,the above activation is ineffective.Conclusion: Dihydroartemisinin induces the apoptosis of osteosarcoma MG-63 cells through the CaMKKβ-AMPK pathway,and has a significant positive correlation with the concentration.