Antiprogestins are a promising new class of mammary tumor inhibitors with a unique mechanism of action which does not seem to involve a classical progesterone antagonism. The objective of this application is to confirm an additive antitumor activity and to clarify the intracellular mechanism of action of the antiprogestin mifepristone, and the antiestrogen tamoxifen, in inhibition of growth of established tumors (chemotherapy) and in prevention or delay of tumor development (chemoprevention/adjuvant setting) in clinically relevant breast cancer models. The focus of the mechanistic studies is to explore the role of (and the specific pathways for) induction of apoptosis in breast cancer cells as a treatment response. The importance of (and the effect of treatments on) estrogen (ER) and progesterone (PR) receptors as well as the potential additive or synergistic interaction between mifepristone and tamoxifen will also be explored. The research design includes the use of the ER(+) and PR(+) MCF-7 and the ER(-) and PR(-) MDA-231 human breast cancer cell lines grown in culture (in vitro studies) or inoculated into female nude mice supplemented with 17BETA-estradiol (in vivo studies). Companion experiments utilizing the MXT [hormone-dependent ER(+) and PR(+)] and MXT-OVEX [hormone-independent ER(-) and PR(-)] homograft mammary carcinoma models of the mouse are also included to compare the antitumor effects in a normal (nonimmunologically suppressed) animal with those induced in nude mice. The following cellular parameters and their time-dependent sequence will be assessed: morphological features (e.g., mitotic and apoptotic indices), in vitro cell viability (cytotoxicity), Cdk1 protein concentration, DNA fragmentation, PKA and PKC isoform activity and subcellular distribution, as well as the changes in the expression of bax, bcl2 and TGFBETA isoforms. The expression of other known """"""""cell death genes"""""""" or """"""""cell cycle determinants"""""""" will also be included whenever they seem appropriate. These various outcome measures will be evaluated using immunocytochemistry, pulsed field gel electrophoresis, Western blots, Northern analysis, specific ELISA, enzyme kinetics and saturation lignad binding assays. The results generated from the described studies will confirm an additive antitumor activity of mifepristone and tamoxifen. This activity is mainly the result of a steroid receptor-mediated interaction with the cell suicide mechanism which involves distinct changes in gene expression (an increase in the bax/bcl2 ratio) in distinct subpopulations (different receptor status) of breast cancer cells. These changes initiate a direct and/or an indirect cell death command via distinct PKC or PKA second messenger signaling pathways and/or via expression or specific TGF-b isoforms. Induction of apoptosis in two distinct subpopulations of breast cancer cells and/or through multiple reinforcing molecular biochemical mechanisms would provide an excellent rationale for a combination therapy. The expected additive effect may delay malignant progression, reduce metastatic spread and increase survival of breast cancer patients. The studies could also impact the future clinical development of """"""""antiprogestins"""""""" for this indication.
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