afimoxifene and Urinary-Bladder-Neoplasms

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-β (ERβ) 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 ERβ positive while only a few expressed estrogen receptor-α (ERα). 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 ERα or ERβ 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.

Topics: Apoptosis; Carcinoma, Transitional Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Fulvestrant; Humans; Immediate-Early Proteins; MCF-7 Cells; Poly(ADP-ribose) Polymerases; Raloxifene Hydrochloride; S-Phase Kinase-Associated Proteins; Tamoxifen; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Clinical advances in bladder cancer would require the development of novel animal model systems closely mimicking human disease. We describe a system of conditional gene targeting using the Cre/loxP system that permits temporally controlled mutation of tumor suppressor genes in bladder urothelium.. Mice expressing Cre-ERT, a fusion between Cre-recombinase and a mutated hormone binding domain of the human estrogen receptor ERT, permit temporally and spatially controlled Cre mediated recombination in vivo by the topical application of 4-hydroxy-tamoxifen. Mice expressing Cre-ERT under transcriptional control of the ubiquitously expressed ROSA26 locus R26cre-ERT were crossbred with R26R mice that express the lacZ reporter gene after Cre mediated excision of a neo cassette in all cells of the adult mice. At 7 and 90 days after intravesical application of 1, 2, 5 and 10 mg. 4-hydroxy-tamoxifen the bladder was processed for X-Gal (Life Technologies, Rockville, Maryland) staining.. At doses of 1, 2, 5 and 10 mg. 4-hydroxy-tamoxifen Cre mediated recombination was readily detected in the bladder urothelium in dose dependent fashion. Within the urothelium basal, suprabasal and superficial cells stained. Applying the 10 mg. dose resulted in widespread multifocal staining of the urothelium without recombination in the bladder wall or distant organs.. The R26cre-ERT mouse can be used to induce multifocal somatic mutagenesis in the bladder urothelium in a promoter independent and time controlled manner. This model would enable us to study temporally controlled mutations of bladder cancer related tumor suppressor genes by crossbreeding with mice carrying floxed alleles for Rb, p53 and p16INK4a alone or in combination.

Topics: Animals; beta-Galactosidase; Disease Models, Animal; Dose-Response Relationship, Drug; Estrogen Antagonists; Galactosides; Gene Targeting; Genes, Reporter; Genes, Tumor Suppressor; Indoles; Integrases; Mice; Mice, Transgenic; Mutagenesis, Insertional; Mutation; Proteins; Receptors, Estrogen; Recombination, Genetic; RNA, Untranslated; Tamoxifen; Urinary Bladder; Urinary Bladder Neoplasms; Urothelium; Viral Proteins