The vitamin D analogue BXL-628 inhibits growth factor-stimulated proliferation and invasion of DU145 prostate cancer cells

Abstract

Purpose: Suppression of the invasive phenotype is essential in developing new therapeutic tools to treat advanced prostate cancer (PC) indicating that androgen-independent prostate cancer (AI-PC) is characterized by increased metastatic potential. In the present study, we have investigated the effect of the nonhypercalcemic vitamin D analogue BXL-628 on proliferation and invasive properties of the human PC cell line DU145. In particular, the effect of the analogue was tested following stimulation with a potent growth factor, keratinocyte growth factor (KGF), which stimulates both proliferation and invasion of these cells. We have also evaluated the effect of the analogue on KGF stimulation of PI3K/AKT signaling pathway.

Methods: Cell proliferation was determined by cell counting. Invasion through Matrigel was evaluated using Boyden chambers. PI3K activity was measured by immunokinase assay and AKT phosphorylation was evaluated by western blot analysis. Keratinocyte growth factor receptor (KGFR) autotransphosphorylation was evaluated by western blot after immunoprecipitation of the receptor.

Results: BXL-628 is able to inhibit both proliferation and invasion of DU145 cells in basal conditions and in response to KGF. Following stimulation with KGF, the inhibition is due to suppression of KGFR autotransphosphorylation and downstream PI3K/AKT activation, both achieved following a brief (5 min) incubation with the analogue. This effect on KGFR autophosphorylation was still present when cells were treated with the alpha-amanitin, an inhibitor of RNA transcription, indicating a rapid, nongenomic effect.

Conclusions: Our results demonstrate that the vitamin D analogue BXL-628 is able to suppress KGF-induced proliferation and invasion of AI-PC cells in vitro, prospecting a possible use of the drug, which is currently in phase II clinical studies for benign prostatic hyperplasia, in the treatment of advanced prostate cancer.

Fig. 1

Effect of BXL-628 on basal (inset) and KGF-stimulated proliferation of DU145 cells, determined by cell counting. Cells were treated for 48 h with increased concentrations of the analogue with or without fixed concentrations of KGF (10 ng/ml). Each experimental point was determined in triplicate and experiments were performed at least three times. Results are expressed as the percentage of growth compared with their relative controls

Fig. 2

Effect of BXL-628 (1×10⁻⁸ M) on Matrigel invasion of DU145 cells (panel a) and PC3 cells (panel b) in basal conditions and following stimulation with KGF (10 ng/ml). Matrigel invasion was evaluated by using Boyden chambers. Number of cells migrated was evaluated in at least ten fields for each experimental point and averaged. Data are mean ± SEM of the percentage of cell migrated with respect to control of four different experiments

Fig. 3

Effect of BXL-628 (1×10⁻⁸ M) on KGF (10 ng/ml)-induced autotransphosphorylation of its receptor. Cells were pre-treated (panel b) or not (panel a) for 4 h with α-amanitin (4 μg/ml, 4 h). After stimulations, cell lysates were immunoprecipitated using anti-KGFR antibody, run onto SDS-PAGE and analyzed first for expression of phosphorylation using anti-phosphotyrosine (PY20) antibody (upper blots) and, after stripping and re-probing, for receptor expression using anti-KGFR and, for the experiment with α-amanitin, for actin expression. Representative of two similar experiments

Fig. 4

Effect of the phosphatidylinositol-3 kinase inhibitor, LY294002, on proliferation of DU145 cells, determined by cell counting. Cells were treated for 48 h with fixed concentration of LY294002 (10 nM) with or without KGF (10 ng/ml). Each experimental point was determined in triplicate and experiments were performed at least three times. Results are expressed as the percentage of growth compared with their relative controls

Fig. 5

Effect of BXL-628 (1×10⁻⁸ M) on KGF (10 ng/ml)-mediated PI3K activation. After stimulation, cell lysates were immunoprecipitated using an anti-phosphotyrosine (PY20) antibody, followed by immunokinase assay in the presence of [γ-³²P]ATP (for details, see Materials and methods). Products of the reaction are evaluated by thin-layer chromatography followed by autoradiography. Upper panels show representative experiments, where spots correspond to the PI3-kinase catalytic product [³²P]phosphatidylinositol phosphate (PIP), while lower panels show mean ± SEM quantification (arbitrary units) of the band for the indicated number of experiments

Fig. 6

Effect of BXL-628 (1×10⁻⁸ M) on KGF (10 ng/ml)-mediated phosphorylation of the PI3K downstream effector AKT. After stimulation, equal amount of total cell lysates was subjected to SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-phosphoserine AKT antibodies (upper panels) followed by stripping and re-probing with anti-AKT antibodies (lower panels). Representative of two similar experiments