Study On Synergistic Inhibition Of Dihydroartemisinin And Afatinib On Glioma Cells

Glioma is a primary brain tumor which is caused by carcinogenesis of glial cell in brain and spinal cord.For a long time,the treatment of gliomas has been a problem obsessing neurosurgeons.Features of glioma include fast progression,poor prognosis,and strong invasion to nearby healthy brain tissue.Even if the glioma sufferers receive the treatment plan of surgery radiotherapy and chemotherapy,the prognosis effect is still unsatisfactory.Afatinib is an irreversible and effective dual inhibitor of human epidermal growth factor receptor 2(HER2)and epidermal growth factor receptor(EGFR)tyrosine kinase,which was approved to enter the Chinese market in 2017.Dihydroartemisinin(DHA)is a derivative of artemisinin.In recent years,more and more reports have shown that DHA has a good anticancer effect and can induce sensitization to chemoradiotherapy.However,the research on the treatment of glioma with the combination of DHA and afatinib has not yet been reported.To this end,we attempt to study the inhibitory effect of DHA and afatinib on glioma cells,focus on analyzing the effect of its combination,and initially explore the possible functional mechanism to provide a new idea for the treatment for glioma.First of all,we tested the inhibition of glioma cell line U87-MG by single drug and drug combination through CCK-8 method to detect whether the drug combination has a good synergistic effect,and then determine the drug concentration for the subsequent experiments.Changes of morphology and state of the cells were observed under inverted microscope,and then the apoptosis and cycle of U87-MG cells after adminisration were detected by flow cytometry.Next,the fluctuations of migration ability and invasion ability of U87-MG cells were detected by Transwell method.Finally,the expression level of caspase3 and AKT protein in U87-MG cells after administration was detected by Western Blot technique,and the medication mechanism was preliminarily explored.Through in vivo experiments of subcutaneous transplantation tumor in nude mice,we randomLy divided the nude mice inoculated with U87-MG transplantation tumor into 4 groups:control group,afatinib group,DHA group,and drug combination group.The control group was administered with solvent;afatinib group:10mg/kg/d;DHA group:25mg/kg/d;drug combination group:afatinib 10mg/kg/d;DHA 25mg/kg/d;afatinib administered intragastrically,every seven days for one treatment course,of which five days for administration and two days for rest;DHA was administered intraperitoneally,of which five days for administration and two days for rest.The tumor volume was measured every two days.The tumor was removed and weighed three weeks later to learn the inhibitory effect of the combination therapy on the transplantation tumor in nude mice.The results showed that CCK-8 method detected the inhibitory effect on U87-MG cells from both afatinib and DHA.The inhibitory effect of altidine combined with DHA at 2μM,4μM on the growth of U87-MG cells markedly enhanced,indicating significant synergy;the combination of the two drugs had no significant synergistic effect on the U87-MG cells cycle,but synergistically inhibited the invasion and migration of U87-MG cells,and had an obvious synergistic effect on the apoptosis of U87-MG cells.In addition,drug combination can induce apoptosis by activating Caspase3 in U87-MG cells and inhibiting the activation of AKT.Also,we found in vivo experiment that the drug combination can inhibit the growth of subcutaneous transplantation tumor of U87-MG cells in nude mice.The results of this study demonstrated that the combination of afatinib and DHA could significantly synergistically inhibit U87-MG cells in vivo and in vitro.This study laid a foundation for further study on the mechanism of the combination of afatinib and DHA,and also provided a new idea for the treatment of glioma.