Issue Archive

[Inhibition of Beclin 1 enhances apoptosis by H2O2 in glioma U251 cells.] [Article in Chinese]

KONG Xiao-Xia, ZHANG Hong-Yu^*, CHEN Zhao-Qin, FAN Xiao-Fang, GONG Yong-Sheng

Institute of Hypoxia Medical Research, Teaching Center of Functional Experiment； Pharmacy School, Wenzhou Medical College, Wenzhou 325035, China

Abstract

Oxidative stress could induce apoptosis and autophagy process simultaneously, but the role of autophagy is still not clear. Beclin 1, a key gene regulating the preautophagosome formation, is involved in the injury induced by oxidative stress. To observe the role of autophagy in H2O2-induced injury of U251 cells, the recombinant plasmid Psilencer3.1-siRNA-Beclin 1 was transfected into U251 cells by eukaryotic cell transfection technique. Plasmid vector and cell culture medium were used as negative and control groups respectively. The cells were collected 24 h later, and the cell total protein was extracted to detect Beclin 1, Bcl-2 and Bax protein expressions by Western blot. After the Beclin 1-siRNA cells were treated with 1 mmol/L H2O2, the autophagic vacuoles in the cells were stained with monodansylcadaverine (MDC), and the cell apoptotic ratio was determined with PI/Annexin V-FITC staining by flow cytometry analysis. The results showed that the synthetic siRNA decreased the expression of Beclin 1 protein significantly, but had no obvious effect on the levels of Bcl-2 and Bax protein expressions. Compared with those in the control group, the autophagic vacuoles, the level of LC3-II protein expression and the percentage of apoptotic cells increased (P < 0.05) in 1 mmol/L H2O2 group. In Beclin 1- siRNA + H2O2 group, autophagic vacuoles and the levels of LC3-II protein expression decreased obviously, the percentage of apoptot ic cells increased significantly compared with that in 1 mmol/L H2O2 group (P < 0.05). H2O2 and autophagy inhibitor 3-methyladenine (3-MA) combination also increased the percentage of apoptotic cells obviously (P < 0.05). These results revealed that the transfection of Psilencer3.1-siRNA-Beclin 1 effectively inhibited the expression of Beclin 1 protein expression, degraded the autophagy level and increased the apoptotic rate in U251 cells under oxidative stress, which was coincident with the effect of autophagy inhibitor 3-MA. This study suggests that autophagy is a cell protective role in oxidative stress process, and the inhibition of autophagy may enhance apoptosis.

Key words: RNA interference; Beclin 1; autophagy; apoptosis

Received: 2010-12-11　　Accepted: 2011-04-08

Corresponding author: 张宏宇　　E-mail: st.hyz@hotmail.com

Citing This Article：

KONG Xiao-Xia, ZHANG Hong-Yu, CHEN Zhao-Qin, FAN Xiao-Fang, GONG Yong-Sheng. [Inhibition of Beclin 1 enhances apoptosis by H2O2 in glioma U251 cells.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (3): 238-244 (in Chinese with English abstract).