Raloxifene Inhibits Growth of RT4 Urothelial Carcinoma Cells via Estrogen Receptor-Dependent Induction of Apoptosis and Inhibition of Proliferation

Bladder cancer is the fifth most common type of cancer in the USA, with over 70,000 new cases diagnosed each year. Treatment often involves invasive surgical therapies, as chemotherapy alone is often ineffective and associated with high recurrence rates. Identification of estrogen receptor-beta (ER beta) in up to 75 % of urinary tumors raises the question of whether this receptor could be targeted to effectively treat bladder cancer. In this study, a panel of five bladder cancer cell lines representing a variety of disease stage and grades were treated with the antiestrogens 4-hydroxytamoxifen, raloxifene, or the pure antagonist ICI 182,780. All cell lines were ER beta positive while only a few expressed estrogen receptor-alpha (ER alpha). Notably, all but the TCCSUP cell line were growth inhibited 20-100 % by at least two antiestrogens. Using RT4 cells, we demonstrate that growth inhibition by raloxifene is ER dependent and either ER alpha or ER beta can mediate this response. Activation of caspase-3 and its effector poly-ADP ribose polymerase (PARP) demonstrate that raloxifene-induced growth inhibition is in part the result of increased apoptosis; this PARP cleavage was ER dependent. Moreover, changes in the expression of cell cycle genes indicate that cell proliferation is also affected. Specifically, raloxifene treatment results in the stabilization of p27 protein, likely via the downregulation of S-phase kinase-associated protein (SKP2). Expression of the negative cell cycle regulator B-cell translocation gene (BTG2) is also increased, while cyclin D1 transcription is reduced. These results indicate that antiestrogens may be useful therapeutics in the treatment of bladder cancer by targeting ER and inhibiting growth via multiple mechanisms.