The drug estramustine is an effective drug in the treatment of prostatic cancer. Preclinical and clinical pharmacology have shown that its cytotoxic properties are distinct from its constituent estradiol/nitrogen mustard moieties. In fact, estramustine, as an intact molecule, has an entirely novel mechanism of action unique from other anti-tumorigenic drugs. One long term goal of this grant proposal is to investigate the drug resistant phenotype(s) acquired by human prostatic PC-3 cells grown in the presence of escalated dosages of estramustine. A second major goal is to understand if a causal relationship exists between acquired drug resistance and tumor metastases. Specifically, the following experimental approaches will be adopted: (1) using a PC-3 cell line, estramustine resistance will be induced through sequential selection by drug dose escalation. Established lines of varying resistance will be characterized with respect to clonigenic assays karyotype, drug uptake, P170 phenotype, glutathione S-transferase expression, estramustine-binding-protein content, collateral resistance/sensitivity to other drugs and to estramustine analogues. (2) delineation of the invasive activity of PC-3r/s variants utilizing a 48-well Boyden chamber and a reconstituted basement membrane (Matrigel) as a barrier to tumor cell penetration. (3) analysis of the ability of PC-3r/s variants to penetrate chick heart tissue in vitro. (4) in vivo studies in said mice of the metatastic activity of PC3r/s variants. Particular attention will be given to """"""""bone-targeted"""""""" variants since PC-3 cells were originally derived from a bone metastases and bone-metastases is a common site for tumor cell migration in humans. (5) The PC-3r/s metastases will be put back in culture for recharacterization of their drug resistance, phenotypic properties and invasive activities. These approaches will assist in determining the properties of estramustine resistant cells and establish if the development of drug resistance might be associated with an increased incidence of invasive activity leading to metastases. The in vitro data will provide essential quantitative data which should be in agreement with in vivo qualitative studies. Of further significance, estramustine is proving to be a valuable tool for probing the tumorigenic activities of prostate tumors. Continued progress will consolidate and expand the utility of the drug as an aid to understanding molecular mechanisms regulating metastases.
