Genistein-Induced Apoptosis of Prostate Cancer Cells is Preceded by a Specific Decrease in Focal Adhesion Kinase Activity

Abstract

Genistein (5,7,4'-trihydroxyisoflavone), an isoflavinoid found in soy beans, has been identified as potentially causal for the low incidence of metastatic prostate cancer (PCa) in certain countries. Although genistein-induced PCa cell adhesion has been identified as a possible causative mechanism, direct growth inhibition by genistein has been reported and also could be causal. If in vivo growth inhibition was significant, then growth inhibition should occur at concentrations attained with dietary consumption, the mechanism of growth inhibition should be relevant to PCa, and genistein (a broad-spectrum in vitro protein-tyrosine kinase inhibitor) should have relatively specific kinase inhibitory effects in vivo. These considerations were investigated by measuring growth inhibitory activity in a variety of PCa cell lines. Growth inhibitory effects were shown not to occur with concentrations below the low micromolar range (i.e., 3 logs above that attained in serum). In-depth mechanistic studies with the PC3-M metastatic variant cell line demonstrated that growth inhibition was independent of genistein's estrogenic effects. Genistein was shown to decrease the viability of nonadherent cells, suggesting a lack of dependence on cell adhesion for growth inhibition. However, important molecular and kinetic differences between genistein's effects on growth in adherent versus nonadherent cells were identified. Specific suppression of focal adhesion kinase activity (without global decreases in phosphotyrosine) was shown to precede induction of apoptosis, which was responsible for growth inhibition in adherent cells. These findings do not support an in vivo growth inhibitory role by genistein consumed in quantities associated with a soy-based diet. They do, however, identify genistein as a potential therapeutic agent for PCa and as a tool with which to study the control of apoptosis in PCa.

Prostate cancer chemoprevention agents exhibit selective activity against early stage prostate cancer cells

“…33,34 The PC3-M cell line is a well-characterized variant of the PC3 cell line and represents a more rapidly growing metastatic variant of the parental cells. 12,13,18 While widely reported in the literature, the LNCaP cell line was not subjected to phenotypic analysis in this study. This is because the current focus was not upon identi®cation of androgen receptor binding agents and there was concern that tamoxifen may exhibit promiscuous receptor activation, as has been reported with a variety of agents.…”

“…PC3-M cells are a metastatic variant of PC3 cells whose origin and characteristics have been described previously. 12,13 Normal prostate epithelial cells (NPECs) were obtained from Clonetics (Palo Alto, CA). Human papillomavirus (HPV) transformed normal epithelial and primary cancer cells were obtained from prostate glands as described.…”

“…13,17 Brie¯y, between 1200 and 2000 cells in a ®nal volume of 100 ml were added to each well of a 96-well plate and allowed to adhere overnight. Each of 10 different drugs, in 10 different serial dilution concentrations, were then added in a volume of 100 ml to each well.…”

“…After incubating for 3 days, thymidine incorporation was measured as described. 13,17 Cells were harvested onto glass ®ber ®lters (Packard, Meriden, CT) with a Packard cell harvester and counted in a Matrix 9600 Microplate counter (Packard). All microtiter assays in this study were run in replicates of six and were repeated at least once at a separate time.…”

“…13,18 To determine if cell growth was associated with metastatic potential, the ability of cells to adhere to a solid matrix was measured. As can be seen in Figure 1, the highly metastatic PC3-M cell variant ®rst attached more rapidly than all other cell types, then entered a phase of spontaneous cell detachment.…”