Experimental Study On The Effect Of MTOR Inhibitor Everolimus Combined With Temozolomide On Autophagy In Malignant Glioma Cell

Objective:We observe the effect of mTOR inhibitor Everolimus(RAD001)on the proliferation and autophagy of U251 cells and investigate the effect of RAD001 combined with Temozolomide(TMZ)on the proliferation and autophagy of U251 cells.We hope that this study Provide a reliable theoretical basis for the treatment of human malignant glioma with RAD001.Methods:The experiment is divided into two parts.In the first part,we investigate the effect of RAD001 on proliferation and autophagy of U251 cells in vitro.First,we use the CCK-8 method to detect the effect of different concentrations of RAD001 on the proliferation inhibition of U251 cells at different time points.Transmission electron microscopy is used to observe the ultrastructure of U251 cells induced by RAD001.Immunofluorescence is used to observe the distribution of GFP-LC3 fusion protein in RAD001-treated human glioma cells.The effect of RAD001 on the protein levels of LC3 B,Beclin-1 and P62 in cells is detected by Western Blot.In the second part,we investigate the effect of RAD001 combined with TMZ on proliferation and autophagy of U251 cells in vitro.We randomly divide U251 cells into the following three groups: blank control group,TMZ single drug group,and RAD001 and TMZ combined group,we use CCK-8 method to understand the cell growth inhibition of each group;Transmission electron microscopy is used to observe the effects of blank control group,TMZ monotherapy and combined treatment The changes of ultrastructure of U251 cells are observed by immunofluorescence staining.Fluorescence distribution of GFP-LC3 fusion protein in blank control group,TMZ single drug and two drugs are observed by immunofluorescence staining.After the combination of two drugs LC3 B,Beclin-1,P62 expression levels.Results:The first part: After U251 cells are treated with different concentrations ofRAD001(0,5,10,20 and 40 ?M)for 12,24 and 48 hours,the results of CCK-8showed that RAD001 could effectively inhibit the proliferation of glioma cells The growth is proliferative and showed a significant dose and time dependence;after 40 ?M RAD001 acting on U251 cells for 48 h,a large number of bilayer membrane structures and autophagosomes containing vacuolar monolayers and autophagosomes containing residual organelles are observed by transmission electron microscopy.After the U251 cells are treated with 20?M and 40?M RAD001 for 48 h,the green fluorescence spots of autophagic protein expression could be observed under fluorescent microscope.At the same time,it is found that the number of fluorescent spots in the high concentration group is significantly lower than that in the concentration group;RAD001(0,5,10,20 and 40?M)for 48 h respectively.Western Blot results show that the expression of LC3-II and Beclin-1 are up-regulated in a dose.The expression level is gradually decreased.The second part: Each dosing group inhibit the growth of U251 cells in a concentration-dependent manner.The inhibition rate of the combined treatment group is much higher than that of the TMZ single drug group,with significant statistical difference(P <0.05).Under the transmission electron microscope,the formation of autophagosomes in the combination group is more obvious than the TMZ single drug group Many observations under fluorescence microscope show no green fluorescence spots in the blank control group,more green fluorescent spots are seen in the TMZ single drug group,and a large number of green fluorescent spots are seen in the combined group;Western Blot results show that the combination group Compare with the TMZ single drug group and the blank control group,the LC3-II/I ratio is significantly higher,the Beclin-1 protein level is significantly increased,and the P62 protein level is significantly decreased.Conclusion:1.RAD001 can inhibit glioma cell growth and proliferation and promote cell autophagy.2.RAD001 combined with TMZ significantly inhibit the proliferation of glioma cells and induc autophagy of cells than TMZ.RAD001 can enhance the drugsensitivity of TMZ to glioma cells,and RAD001 and TMZ have a synergistic effect.