Hispidulin Enhances Temozolomide(TMZ)-induced Cytotoxicity Gainst Malignant Glioma Cells In Vitro By Inhibiting Autophagy

Objectives:To analyze the effects of Hispidulin,temozolomide and their combination on the activity,apoptosis and autophagy of U87 MG cells,so as to provide theoretical evidence for the treatment of human malignant glioma and explore its feasibility in clinical treatment.Methods:First of all,MTT method was used to detect the effects of different concentration gradients of Hispidulin,TMZ and their combination on the viability of human malignant glioma U87 MG cells,and the optimum concentration of Hispidulin to change the activity of U87 MG cells was selected.Secondly,blank control group,TMZ single drug group and TMZ combined with Hispidulin drug group were set up to observe the apoptosis of U87 MG cells after treatment by Annexin V/PI double staining and TUNEL/DAPI staining,respectively.Then flow cytometry was used to observe the fluorescence labeled autophagosome(AVO)in apoptotic U87 MG cells treated with Hispidulin group,TMZ group and TMZ combined with Hispidulin group by acridine orange staining.Finally,Westernblot was used to detect the expression of pro-apoptotic and anti-apoptotic proteins(Bax,cleaved-capsase-9,cleaved-capsase-3and Bcl-2)and autophagy protein LC3B-II/LC3B-I in U87 MG cells treated with three experimental groups.Results:The results of MTT assay showed that the number of U87 MG cells treated with Hispidulin at high concentration of 100ug/ml(5,10,20,40,100μg/m L)was significantly lower than that of the other four groups(P < 0.05,0,200,400,800μm).At the same time,the cell survival rate of U87 MG cells treated with TMZ(50,100,200,400,800μm)was significantly lower than that of the untreated control group(P <0.01),and the cell survival rate was significantly lower than that of the untreated control group(P < 0.01).Because the number of U87 MG cells treated with Hispidulin of 40ug/m decreased less,and the number of U87 MG cells treated with TMZ of 50μm,100μm and 200μm decreased less than that of U87 MG cells treated with 400,400 and 800μm TMZ,so we chose their combination to evaluate whether Hispidulin has an adjuvant effect to enhance the lethal effect of TMZ on glioma U87 MG cells.After completing the setting of the experimental group,through Annexin V/PI double staining and TUNEL/DAPI staining,it was confirmed that the experimental group of Hispidulin combined with TMZ could promote the increase of apoptosis induced by TMZ in three experimental groups(blank group,TMZ single drug group and combined drug group).In addition,three experimental groups(Hispidulin group,TMZ monotherapy group and combination group)were detected by Westernblot.The TMZ drug group added Hispidulin could up-regulate the expression of pro-apoptotic protein Bax,cleaved-capsase-9 and cleaved-capsase-3induced by TMZ,but reduce the expression of anti-apoptotic protein Bcl-2.Flow cytometry was used to observe that Hispidulin could inhibit autophagy induced by TMZ in the same three experimental groups after staining with acridine orange.It can promote the apoptosis of U87 MG cells,which is characterized by the decrease of the number of autophagosome(AVO)and the decrease of the ratio of autophagy protein LC3B-II/LC3B-I detected by Westernblot.Conclusion:1.Hispidulin has obvious cytotoxic effect on glioma U87 MG cells;2.The killing effect of Hispidulin combined with TMZ on glioma U87 MG cells was significantly higher than that of TMZ alone.Hispidulin could enhance the drug sensitivity and excellent synergistic ability of TMZ to glioma cells.3.Hispidulin enhances TMZ-induced cytotoxicity against malign-ant glioma cells in vitro by inhibiting autophagy.