A Study On Autophagic Activity Of Typical Human Glioblastoma Cells Induced By Ardipusilloside â… 

Objective:To study the effect of the ardipusilloside I(ADS I) on autophagic cell death in the typical human glioma cells U87 MG, U251 MG, U373 MG and SHG-44 MG. Methods:The cultured U87 MG, U251 MG, U373 MG and SHG-44 MG cells were randomly divided into control group and ADS I treatment groups. MTT assay was performed to measure the effect of the different concentrations of ADS on proliferation of U87 MG, U251 MG, U373 MG and SHG-44 MG cells. Cell autophagosome was observed by TEM. The expression of Beclin-1 and LC-3, autophagic related protein, was analyzed by immunocytochemistry and western blot. Flow cytometry analysis tested the effects of ADS Ⅰon cell cycle and apoptosis in U373 MG cells. At last, CQ-induced reduction of autophagic cell death after ADS Ⅰtreatment was measured in U373 MG cells. Results:MTT detection showed that ADS I obviously inhibited the typical glioma U87 MG, U251 MG, U373 MGand SHG-44 MG cell growth in a dose and time-dependent manner. The typical autophagosome characteristics, including nucleation and autophagosome formation, were observed by TEM, Immunocytochemistry results showed that the ratio of Beclin 1 and LC3-positive cells significantly increased in all the four cell lines 24 h later after ADS I treatment in a dose-dependent manner. In addition, the expression of all of three autophagy-related proteins(LC3-I, LC3-II and Beclin-1) was up-regulated in all the cells following an increase in the concentration of ADS-I in the cultures by western blot analysis. FCM showed that 3.8 mg/L ADSⅠsignificantly increased the proportion of cells in G2/M phase from 6.86% to 27.31%; moreover, addition of 5 μM CQ significantly reduced ADS-I induced cell death by flow cytometric analysis. Conclusions:ADS I inhibited the cell viability of the typical glioma cells U87 MG, U251 MG, U373 MG and SHG-44 MG in a dose-dependent manner, increased the level of all of three autophagy-related proteins(LC3-I, LC3-II and Beclin-1), and induced autophagic cell death. All of these supply the important evidence in vitro for the anti-glioma activity of ADS I.