Abstract
Estrogen receptor beta (ERβ) plays a role in prostate carcinogenesis. In this study, we investigated the effects of ERβ gene silencing in PC3 androgen-independent prostate cancer cells. PC3 cells were transfected with vector alone, scrambled shRNA vector, vector encoding ERβ-targeting shRNA (shERβ), or shERβ followed by addition of PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor (shERβ+PD98059). Cyclin D1, Bcl-2, matrix metalloproteinase (MMP)2, and phosphorylated (p-) extracellular signal-regulated kinase (ERK1/2) expression was detected by western blotting. While ERK1/2 expression was comparable in all cells, p-ERK1/2 expression was highest in shERβ cells, and lowest in shERβ+PD98059 cells. Bcl-2, cyclin D1, and MMP2 expression was highest and lowest in shERβ and shERβ+PD98059 cells, respectively. Flow cytometry analysis showed that ERβ silencing promoted cell proliferation by decreasing the percentage of cells in G0/G1. Analysis of colony formation, migration, and invasion capacities, measured using soft agar colony-formation, wound-healing, and transwell invasion assays, respectively, showed that ERβ silencing augments cell proliferation, migration, and invasion, and that this increase is reversed by PD98059 treatment. A tumor xenograft model in nude mice was used to assess the effect of ERβ silencing on the biological behavior of PC3 cells. Colony formation assays and tumor transplantation data indicated that ERβ silencing promotes tumor formation. Immunohistochemical analysis of tumors showed that vascular endothelial growth factor (VEGF) and p-ERK1/2 expression, but not that of total ERK1/2, was increased upon ERβ silencing. In conclusion, out data demonstrate that ERβ gene silencing enhances malignant biological behaviors of PC3 cells by activating the ERK1/2 signaling pathway.