Effects of mTOR inhibitor everolimus (RAD001) on bladder cancer cells

Abstract

Purpose: We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder cancer (BC) cells in vitro and in vivo.

Experimental design: The effect of RAD001 on the growth of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 BC cells were assessed by crystal violet and [(3)H]thymidine incorporation assays. Flow cytometric cell-cycle analyses were done to measure the apoptotic cell fraction. Protein synthesis was measured using tritium-labeled leucine incorporation assays. The effects of RAD001 on the mTOR pathway were analyzed by Western blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, and UM-UC-9 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with RAD001 or placebo. Tumors were harvested for immunohistochemical analysis.

Results: In vitro, RAD001 transiently inhibited BC cell growth in a dose-dependent manner. This effect was augmented by re-treatment of cells after 3 days. UM-UC-14 cells were the most sensitive to RAD001, whereas UM-UC-9 cells were the least sensitive. After re-treatment with RAD001, only sensitive cell lines showed G(1)-phase arrest, with no evidence of apoptosis. RAD001 significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of protein synthesis through the S6K and 4EBP1 pathways seems to be the main mechanism for the RAD001-induced growth inhibition. However, inhibition of angiogenesis was the predominant mechanism of the effect of RAD001 on UM-UC-9 cells.

Conclusions: The mTOR inhibitor RAD001 inhibits growth of BC cells in vitro. RAD001 is effective in treating BC tumors in an in vivo nude mouse model despite the heterogeneity of in vitro responses.

Fig. 1

The inhibitory effects of RAD001 on bladder cancer cell growth and proliferation determined by crystal violet assays (A and B) and [³H]Thymidine incorporation assays (C) respectively. UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 cells were treated with A, RAD001 and incubated for 6 days. B, Cells were treated with RAD001 initially at day 0 and then retreated at day 3 with a dose equivalent to the IC₅₀ for each cell line and incubated for 5 days. C, RAD001 and incubated for 48 h.

Fig. 1

The inhibitory effects of RAD001 on bladder cancer cell growth and proliferation determined by crystal violet assays (A and B) and [³H]Thymidine incorporation assays (C) respectively. UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 cells were treated with A, RAD001 and incubated for 6 days. B, Cells were treated with RAD001 initially at day 0 and then retreated at day 3 with a dose equivalent to the IC₅₀ for each cell line and incubated for 5 days. C, RAD001 and incubated for 48 h.

Fig. 1

The inhibitory effects of RAD001 on bladder cancer cell growth and proliferation determined by crystal violet assays (A and B) and [³H]Thymidine incorporation assays (C) respectively. UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 cells were treated with A, RAD001 and incubated for 6 days. B, Cells were treated with RAD001 initially at day 0 and then retreated at day 3 with a dose equivalent to the IC₅₀ for each cell line and incubated for 5 days. C, RAD001 and incubated for 48 h.

Fig. 2

Flow cytometry analysis of bladder cancer cell lines. A, Cell cycle analysis was performed at 48 h for control- and RAD001-treated (0.5 nM) UM-UC-6 cells, control- and RAD001-treated (10 nM) UM-UC-9 cells, UM-UC-6 cells treated with RAD001 (0.5 nM) every 3 days, and UM-UC-9 cells treated with RAD001 (10 nM) every 3 days. Bar charts comparing the differences in the percentage of G1 phase cells between controls and RAD001-treated cells are shown. B, Propidium iodide exclusion assays were performed 24 h after UM-UC-6 cells and UM-UC-9 cells were treated with RAD001. The flow cytometric profiles showing the percentage of cells undergoing nonapoptotic cell death (G) are shown.

Fig. 2

Flow cytometry analysis of bladder cancer cell lines. A, Cell cycle analysis was performed at 48 h for control- and RAD001-treated (0.5 nM) UM-UC-6 cells, control- and RAD001-treated (10 nM) UM-UC-9 cells, UM-UC-6 cells treated with RAD001 (0.5 nM) every 3 days, and UM-UC-9 cells treated with RAD001 (10 nM) every 3 days. Bar charts comparing the differences in the percentage of G1 phase cells between controls and RAD001-treated cells are shown. B, Propidium iodide exclusion assays were performed 24 h after UM-UC-6 cells and UM-UC-9 cells were treated with RAD001. The flow cytometric profiles showing the percentage of cells undergoing nonapoptotic cell death (G) are shown.

Fig. 3

Western blot analysis of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 cells 24 h after treatment with RAD001.

Fig. 4

In vivo growth-inhibitory effect of prolonged treatment of subcutaneous tumor– bearing nude mice. The graphs show tumor size with time after RAD001 or placebotreatment, in mice with UM-UC-3–, UM-UC-6– and UM-UC-9-derived tumors respectively.

Fig. 5

Immunohistochemical staining of representative tumor tissue sections harvested after prolonged treatment with either a placebo or RAD001. Phospho-mTOR, phosphop70S6K and phospho-AKT are shown at 400X magnification, and CD31 is shown at 100X magnification.