Prostate cancer is the second leading cause of cancer death in the United States and, thus far, there has been no effective therapy for the treatment of hormonerefractory disease. Recently, the androgen receptor (AR) has been shown to play a critical role in the development and progression of the disease. In this report, we showed that knocking down the AR protein level by a small interfering RNA (siRNA) approach resulted in a significant apoptotic cell death as evidenced by an increased annexin V binding, reduced mitochondrial potential, caspase-3/6 activation, and DFF45 and poly(ADP-ribose) polymerase cleavage.

Despite the specificity inferred by its name, glycogen synthase kinase (GSK)-3beta is an important kinase with a plethora of significant cellular targets, including cytoskeletal proteins and transcription factors, and its activity is regulated by phosphorylation on tyrosine/serine residues. As part of our efforts to dissect the molecular basis responsible for androgen-independent progression of prostate cancer, we investigated the role of GSK-3beta in androgen-stimulated gene expression in human prostate cancer cells. Pretreatment of prostate cancer cells harboring wild-type or mutant androgen receptor with the GSK-3beta inhibitors, lithium chloride (LiCl), RO318220, or GF109203X, inhibited R1881-stimulated androgen-responsive reporter activity in a dose- and time-dependent manner. In addition, the expression of two endogenous androgen-stimulated gene products, prostate-specific antigen and matrix metalloproteinase-2, was suppressed by the GSK-3beta inhibitors in those cells. Most importantly, knocking down GSK-3beta expression via a small interference RNA-mediated gene silencing approach also reduced R1881-stimulated gene expression, demonstrating the specificity of GSK-3beta involvement. Moreover, R1881 treatment of the cells increased phosphorylation status of GSK-3beta on tyrosine residue Y(216) but not on serine residue S(9). Pretreatment of the cells with phosphatidylinositol 3-kinase inhibitor LY294002 or wortmannin, which blocks androgen action in cells, abolished R1881-induced GSK-3beta Y(216) phosphorylation. However, the phosphatidylinositol 3kinase or GSK-3beta inhibitors did not block R1881-induced nuclear translocation of androgen receptor. Finally, knocking down the expression of Akt or beta-catenin, the two GSK-3beta-related signaling molecules, via siRNA-mediated gene silencing did not significant affect R1881-stimulated gene expression. These findings suggest that GSK-3beta activity is required for androgen-stimulated gene expression in prostate cancer cells.

Prostate growth and differentiation is androgen dependent, and increased expression of matrix metalloproteinase 2 (MMP-2) has been found in more aggressive prostate cancers. As part of our efforts to elucidate the mechanisms responsible for prostate cancer progression, we evaluated the MMP-2 expression after androgen stimulation in human prostate cancer LNCaP and LAPC-4 cells, which express a functional androgen receptor. Treatment of the cells with a synthetic androgen R1881 resulted in an increase of pro-MMP-2 expression assessed by Western blot and gelatinolytic zymography in both cell lines. R1881-stimulated pro-MMP-2 expression occurred in a dose-dependent manner, which was completely abrogated in the presence of the nonsteroid androgen antagonist bicalutamide. In accordance with the protein expression, MMP-2 promoter activity was also increased by R1881 in a cell-based luciferase reporter assay. However, R1881 treatment did not significantly affect either the pro-MMP-9 expression or its promoter activity. Although we observed an appearance of active form of MMP-2, its activator MT1-MMP was not changed after R1881 treatment. Pretreatment of the cells with inhibitors of RNA transcription, actinomycin D, or protein translation, cycloheximide, significantly suppressed R1881-induced pro-MMP-2 expression in LNCaP cells, indicating that androgen stimulates pro-MMP-2 gene expression. In addition, phosphatidylinositol 3'-kinase inhibitor, LY294002 or wortmannin, strongly inhibited R1881-induced pro-MMP-2 expression. Finally, R1881-enhanced LNCaP cell migration was clearly suppressed by LY294002 or the MMP-2 inhibitor OA-Hy in an in vitro migration assay. In conclusion, our data demonstrated that androgen stimulates pro-MMP-2 expression in LNCaP cells via phosphatidylinositol 3'-kinase-dependent androgen receptor transactivation.