Bcl-2 Small Interfering RNA Sensitizes Cisplatin-resistant Human Lung Adenocarcinoma A549/DDP Cell to Cisplatin and Diallyl Disulfide  

作　　者：Zexiang HUANG Xiaoyong LEI Miao ZHONG Bingyang ZHU Shengsong TANG Duanfang LIAO 

机构地区：[1]Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China

出　　处：《Acta Biochimica et Biophysica Sinica》2007年第11期835-843,共9页生物化学与生物物理学报（英文版）

基　　金：This work was supported by the grants from the National Natural Science Foundation of China （No. 30300426） and the Youth Foundation of Hunan Province Education Department （No. 03B034）

摘　　要：Bcl-2 is overexpressed in a variety of human tumors and is involved in tumorigenesis and chemoresistance. In this study, we investigated the inhibitory effect of the hairpin Bcl-2 small interfering （si）RNA on the expression of the Bcl-2 gene in the cisplatin （DDP）-resistant human lung adenocarcinoma cell line A549/DDP, and the effect of Bcl-2 siRNA on drug sensitization in A549/DDP cells. Bcl-2 siRNA and negative siRNA plasmids were constructed and stably transfected into A549/DDP cells. Reverse transcription- polymerase chain reaction, immunofluorescence microscopy and Western blot analysis were used to detect the target gene expression. Spontaneous cell apoptosis was detected by acridine orange and ethidium bromide staining. Drug sensitivity of the cells to DDP and diallyl disulfide （DADS） was analyzed by 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） assay and flow cytometry. Expression levels of Bcl-2 mRNA and protein in siRNA stable transfectants were clearly reduced compared with negative siRNA transfectants and untreated cells. MTT results indicated that Bcl-2 transfectants had a higher cell inhibition rate after treatment with 0.2-200 μg/ml DDP or 50-200 μM DADS. Flow cytometry revealed increased apoptosis in Bcl-2 siRNA cells. After the addition of 20 μg/ml DDP or 100 μM DADS, siRNA targeting of the Bcl-2 gene specifically down-regulated gene expression in A549/DDP cells, increased spontaneous apoptosis, and sensitized cells to DDP and DADS.Bcl-2 is overexpressed in a variety of human tumors and is involved in tumorigenesis and chemoresistance. In this study, we investigated the inhibitory effect of the hairpin Bcl-2 small interfering （si）RNA on the expression of the Bcl-2 gene in the cisplatin （DDP）-resistant human lung adenocarcinoma cell line A549/DDP, and the effect of Bcl-2 siRNA on drug sensitization in A549/DDP cells. Bcl-2 siRNA and negative siRNA plasmids were constructed and stably transfected into A549/DDP cells. Reverse transcription- polymerase chain reaction, immunofluorescence microscopy and Western blot analysis were used to detect the target gene expression. Spontaneous cell apoptosis was detected by acridine orange and ethidium bromide staining. Drug sensitivity of the cells to DDP and diallyl disulfide （DADS） was analyzed by 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） assay and flow cytometry. Expression levels of Bcl-2 mRNA and protein in siRNA stable transfectants were clearly reduced compared with negative siRNA transfectants and untreated cells. MTT results indicated that Bcl-2 transfectants had a higher cell inhibition rate after treatment with 0.2-200 μg/ml DDP or 50-200 μM DADS. Flow cytometry revealed increased apoptosis in Bcl-2 siRNA cells. After the addition of 20 μg/ml DDP or 100 μM DADS, siRNA targeting of the Bcl-2 gene specifically down-regulated gene expression in A549/DDP cells, increased spontaneous apoptosis, and sensitized cells to DDP and DADS.

关 键 词：small interfering RNA ADENOCARCINOMA BCL-2 A549/DDP APOPTOSIS 

分 类 号：R730[医药卫生—肿瘤]