PURPOSE: 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. CONCLUSION: 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.
PURPOSE: 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. CONCLUSION: 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.
Authors: Niamh H Foley; Isabella M Bray; Amanda Tivnan; Kenneth Bryan; Derek M Murphy; Patrick G Buckley; Jacqueline Ryan; Anne O'Meara; Maureen O'Sullivan; Raymond L Stallings Journal: Mol Cancer Date: 2010-04-21 Impact factor: 27.401
Authors: N Mori; S Kyo; M Nakamura; M Hashimoto; Y Maida; Y Mizumoto; M Takakura; S Ohno; T Kiyono; M Inoue Journal: Br J Cancer Date: 2010-07-27 Impact factor: 7.640
Authors: Jung Hye Choi; Jim Jinn-Chyuan Sheu; Bin Guan; Natini Jinawath; Paul Markowski; Tian-Li Wang; Ie-Ming Shih Journal: Cancer Res Date: 2009-02-03 Impact factor: 12.701