Figure 6. ZEB1 is required for chemo-resistance to PTX in vivo.

(A) Generation of NOS3TR cells that constitutively expressed control shRNA (shCtrl) or ZEB1 shRNA (shZEB1) by retrovirus infection (immunoblot). The morphology of shCtrl and shZEB1 cells. shZEB1 showed an epithelial cell-like morphology with clear cell-cell adhesion. (B) The injection of either shCtrl or shZEB1 cells into the murine peritoneum (IP administration: 7.0 × 10⁶ cells / 500 μL). (C) Decreased intraperitoneal dissemination in the shZEB1 cell-inoculated mice, compared with the control mice (upper panels). Suppressed expressions of ZEB1 observed in the peritoneal metastatic lesions in the shZEB1 cell-injected mice (lower panels). (D) Survival curves of the parental, shCtrl or shZEB1 cell- inoculated mice. The mice injected with shZEB1 cells survived for longer than the shCtrl-injected mice (P = 0.04). (E) The in vivo PTX-sensitivity model. Mice were intraperitoneally injected with either shCtrl or shZEB1 cells, and were treated with or without 20 mg/kg of PTX once a week for three weeks. Mice were sacrificed 4 weeks after tumor cell injection and the total volume of disseminated tumors of each mouse was measured. (F) Total weight of intraperitoneal disseminated tumors in shCtrl and shZEB1 cell-inoculated mice in the presence or absence of PTX treatment. Asterisks show significance (P < 0.05). (G) TUNEL assay of tumor tissues from PTX-treated mice. A number of shZEB1 cells were apoptotic, whereas only a small fraction of shCtrl cells were positive for apoptosis.