Figure 2. ET-1 production, autocrine regulation, and proliferative effect on bladder cancer cell lines.

(A) Bladder cancer cell lines secrete ET-1 in culture medium. UMUC3, T24, and T24T cell lines were plated in 24-well plates in CGM and SFM. Cells were harvested, and conditioned media (CM) were collected at the indicated time points. ET-1 level was determined in CM by an ELISA kit as per the manufacturer’s instructions. ET-1 values were normalized to protein contents of the cell lysates as determined by BCA. Bars represent mean ± SEM of results of 3 experiments, each done in duplicate. *P < 0.05, ANOVA, comparing ET-1 levels at the indicated time points under similar experimental conditions; **P < 0.05 comparing growth in SFM and CGM in the same cell line. (B) Autocrine regulation of ET-1 production by ET_AR. Bladder cancer cells were grown to 75% confluence before treatment with ZD4054 (1 μmol/l), BQ788 (1 μmol/l), and A-182086 (10 μmol/l). CM was collected at 72 hours, and ET-1 was measured by ELISA. Bars represent mean ± SEM of results of 3 experiments each done in duplicate. *P < 0.05, Student’s t test, comparing treated cell lines with control untreated cells in SFM. (C) ET-1 promotes bladder cancer cell proliferation. ET-1 promotes proliferation of UMUC3, T24, and T24T cells in a concentration-dependent fashion. Cell lines were plated in 96-well plates (1,000 cells/100 μl/well) in CGM for 6 hours, before being stimulated with the indicated concentrations of ET-1 in growth medium with 2% FBS for 72 hours. DNA incorporation was measured by CyQuant assay as per the manufacturer’s instructions. *P < 0.05, ANOVA.