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Anti-tumor effects and functional evidence of sCA-survivin-siRNA in HCT116 and HT29 solid tumor models.

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posted on 2015-03-04, 04:05 authored by Xin Wu, Hirofumi Yamamoto, Hiroyuki Nakanishi, Yuki Yamamoto, Akira Inoue, Mitsuyoshi Tei, Hajime Hirose, Mamoru Uemura, Junichi Nishimura, Taishi Hata, Ichiro Takemasa, Tsunekazu Mizushima, Sharif Hossain, Toshihiro Akaike, Nariaki Matsuura, Yuichiro Doki, Masaki Mori

(A) For western blot analysis, HCT116 cells were seeded into 6-well plates and transfected with 100 pmol/well of either control or survivin siRNA by Lp (Lipofectamine 2000) or sCA. Actin blots served as loading controls. (B) For proliferation assays, cells were uniformly seeded into 96-well plates (1 × 104 cells/well), and 5 pmol/well siRNA was used. Cell viability was examined at 48 and 72 h by WST-8 assay. Data represent mean ± SEM. *P = 0.0294, **P = 0.0304 (n = 4, Wilcoxon rank test). (C) In vivo tumor growth. Each vehicle, carrying 15 μg of control siRNA or survivin siRNA, was administered by intravenous injection to mice with HCT116 tumors. Data represent the mean ± SEM (n = 10 tumors, Wilcoxon rank test). (D) Immunostaining of survivin in the tumor tissues on day 19. Scale bar, 50 μm. (E) In vivo live imaging of sCA-siRNA (6-FAM labeled) in HT29 tumor by multiphoton microscopy at 90 min. (F) Fluorescent detection of naked-siRNA (6-FAM labeled) or sCA-siRNA (6-FAM labeled) in the HT29 tumor at 4 h. Green: 6-FAM labeled siRNA; Red: microvasculature; Blue: DAPI stained nuclei. Scale bar, 50 μm. (G) Mice were administered with 40 μg of naked-survivin-siRNA or sCA-survivin-siRNA on days 0, 1, and 2. Tumors were removed on day 3, and western blot analysis for survivin was performed. (H) Many tumor cells treated with sCA-survivin-siRNA (6-FAM labeled) had condensed nuclei and were positive for TUNEL assay. Scale bar, 50 μm.