Bcl-2 small hairpin RNAs enhance radiation-induced apoptosis in A549 cells.
Bcl-2, a prominent member of the family of proteins, is responsible for dys-regulation of apoptosis and resistance to chemotherapy and radiotherapy. This study investigated whether small hairpin RNA (shRNA) targeting Bcl-2 could render A549 cells more susceptible to gamma radiation-induced apoptosis. Recombinant Bcl-2 shRNAs expression vector were transfected into A549 cells with Lipofectamine 2000. Transfected cells were screened in 800 mg/ml G418 screening medium, and after stable transfection, silencing was examined. Expression of the Bcl-2 protein was assayed using Western blot in A549 cells. Inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by morphological observation and flow cytometry. Expression levels of Bcl-2 protein from A549 cells decreased after stable transfection with Bcl-2 shRNAs. No differences in Bcl-2 protein levels between control shRNA group and untreated cells were noted. After stable transfection with Bcl-2 shRNAs the viability of cells was less than after stable transfection with those with control shRNAs and untransfected A549, respectively (P<0.05). Control shRNA had no significant effect on growth of cells. Radiation significantly inhibited the growth of cells stably transfected with Bcl-2 shRNA (P<0.05). No difference in survival between the cells with control shRNA and untransfected cells was noted. Using Giemsa staining, cells stably transfected with Bcl-2 shRNA combined with radiation at 48 h displayed changes of apoptosis. After treatment with radiation apoptotic rates of the A549 cells stably transfected with Bcl-2 shRNA significantly increased (P<0.05), compared with the cells with control shRNA and untransfected cells. shRNAs against the Bcl-2 mRNA increases radiation-induced apoptosis in A549 cells.
Institute of Haematology, Medical College of Jinan University, No. 601 West Huangpudadao, Guangzhou 510632, China. thedm@jnu.edu.cn
Dong-Mei
DM
He
Yuan
Y
Zhang
Ge-Xiu
GX
Liu
D017209:Apoptosis / Q000528:radiation effects*; D045744:Cell Line, Tumor; D005720:Gamma Rays / Q000009:adverse effects*; D020868:Gene Silencing; D006801:Humans; D009690:Nucleic Acid Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D022661:RNA, Untranslated / Q000235:genetics*; D014162:Transfection
D019253:Proto-Oncogene Proteins c-bcl-2; D022661:RNA, Untranslated