Rictor-dependent AKT activation and inhibition of urothelial carcinoma by rapamycin

Effects of rapamycin on 0.05% BBN-induced urothelial carcinoma. Representative photographs show the gross pictures of bladders (A–D), hematoxylin-eosin staining (E–H), and vascular endothelial growth factor-A staining (I–L), phosphor-AKT (Ser473) staining (M–P) from the control vehicle group (A, E, I), 2 mg/kg/day rapamycin group (B, F, J), BBN-vehicle group (C, G, K), and BBN-rapamycin 2 mg/kg/day group (D, H, L). Original magnification × 400. (Color version of figure is available online.)

Western blot analyses for the effects of rapamycin on the 0.05% BBN-induced urothelial carcinoma (A), the effects of rapamycin, raptor siRNA, and rictor siRNA on T24 cells (B, C). The expressions of phospho-mTOR and cyclin D1 are significantly reduced by 100 ng/mL rapamycin. Rapamycin also increased AKT phosphorylation (B). Raptor siRNA strongly increases AKT phosphorylation, even more than rapamycin treatment. Rictor siRNA, but not raptor siRNA, prevents rapamycin-induced AKT phosphorylation in T24 cells (C). The same blots were stripped and reprobed with actin to confirm equal loading.

Effect of rapamycin on T24 cell proliferation, migration, and invasion. MTT assay in the presence of vehicle and rapamycin at 10 ng/mL and 100 ng/mL for T24 cells (A). Representative photographs show the result of wound scratch assay that the scratch gap dimension with rapamycin treatment for 24 hours was significantly bigger than in control T24 cells (B). The T24 cells spreading along the wound edges were significantly delayed (44.9%) by rapamycin treatment (C). Representative photographs show the nuclei of T24 cells which invaded the filter and attached on the lower surface of the filter, stained with Giemsa stain after treatment of rapamycin 10, 100 ng/ml or vehicle alone for 72 h (D). Invasion capability after rapamycin treatments are expressed as the ratio of cell nuclei numbers of rapamycin to vehicle groups. Rapamycin significantly inhibited the invasion capability of T24 cells in a dose-dependent manner (E). Each value is the mean ± S.E.M of three experiments.

(A) The flow cytometric analysis of T24 cells in the presence of rapamycin 100 ng/mL or vehicle alone. Significant cell arrest occurs at phase G1 with rapamycin. The percentages arrested in phase G1 by rapamycin dosages of 10 and 100 ng/mL were reduced by 12.1% and 12.9%, respectively. (B, C) The expression of VEGF-A in human bladder cancer T24 cells treated with rapamycin or vehicle alone. The concentration of VEGF-A was determined by enzyme-linked immunosorbent assay. Each value is the mean ± S.E.M of three experiments. (B) Treatment with rapamycin 10, 100 and 1000 ng/mL for 24 h significantly inhibits the production of VEGF-A in T24 cells. (C) Rapamycin 100 ng/mL significantly inhibits the production of VEGF-A in T24 cells on a time dependent manner.

Schematic representation of rapamycin's antitumor effect and AKT activation in bladder cancer cells. Inhibition of mTORC1 by rapamycin attenuates the phosphorylation of p70S6K and the expression of cyclin D1, and inhibits T24 cell proliferation and VEGF-A expression. On the other hand, rapamycin treatment leads to rictor-dependent AKT activation. The bold arrow lines indicate the main effects of rapamycin in T24 cells.