Implication of the Akt2/survivin pathway as a critical target in paclitaxel treatment in human ovarian cancer cells.
OBJECTIVE
Although multiple mechanisms have been implicated in paclitaxel (PTX)-induced resistance in ovarian cancer, recent evidence has suggested that Akt2 has an important role in the protection of cells from paclitaxel-induced apoptosis. In the present study, we investigated the role of the Akt2/survivin pathway in paclitaxel-induced resistance by a modified method to generate an effective shRNA vector.


METHODS
We applied RNAi-mediated silencing techniques to investigate the mechanism of the Akt2/survivin pathway on PTX-induced resistance in ovarian cancer cells (A2780 and SKOV3). The expression of Akt2 and survivin mRNA and related protein levels were evaluated with semiquantitative real-time RT-PCR and western blot analysis, respectively. Inhibition of cell proliferation was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, and the induction of apoptosis was examined through flow cytometry (FACS) and Hoechst staining.


RESULTS
Akt2 down-regulation sensitized ovarian cancer cells to paclitaxel-induced apoptosis, and inhibited survivin expression. We further demonstrated that suppressing the inhibition of survivin expression can induce the drug-resistance to paclitaxel. We introduced a modified vector to generate shRNA to induce RNA interference, which contained three U6 promoters to express different shRNAs; it severely reduced Akt2 gene expression and showed good specificity.


CONCLUSIONS
Our findings will aid in understanding the molecular mechanism of paclitaxel-induced resistance in ovarian cancer and facilitate the development of novel anti-neoplastic strategies.
Weng
Danhui
D
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China.
Song
Xiaohong
X
Xing
Hui
H
Ma
Xiaoli
X
Xia
Xi
X
Weng
Yanjie
Y
Zhou
Jianfeng
J
Xu
Gang
G
Meng
Li
L
Zhu
Tao
T
Wang
Shixuan
S
Ma
Ding
D
D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002469:Cell Separation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D000077022:Survivin; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D000077022:Survivin; D013778:Tetrazolium Salts; D013844:Thiazoles; C494921:AKT2 protein, human; D051057:Proto-Oncogene Proteins c-akt; C022616:thiazolyl blue; D017239:Paclitaxel