Inhibition of proliferation, invasion, and migration of prostate cancer cells by downregulating elongation factor-1alpha expression

Abstract

Overexpression of elongation factor-1alpha (EF-1alpha) has been reported to contribute to the development and progression of various cancers. However, its role in prostate cancer (PCa) still remains poorly understood. In the present study, we investigate the influence of EF-1alpha in Du145, a high-grade metastatic PCa cell line, and demonstrate that EF-1alpha plays an essential role in cellular properties associated with tumor progression, namely cell proliferation, invasion, and migration. In this study, EF-1alpha expression in human PCa cell line Du145 was reduced by RNA interference (RNAi) technology, and the proliferation, invasion, and migration of EF-1alpha-reduced Du145 cells were examined. We also detected an EF-1alpha expression pattern in 20 pairs of primary PCa samples and their corresponding normal tissues. Expression of EF-1alpha was detectable in four PCa cell lines (22RV1, LnCap, Du145, and PC3), indicating its possible role in pathogenesis of PCa. RNAi-mediated knockdown of EF-1alpha expression in Du145 cells, which expressed the highest level of EF-1alpha among four PCa cell lines, led to a decrease in proliferation. Similarly, suppression of EF-1alpha inhibited Du145 cell migration and invasion through a basement membrane substitute. Furthermore, we found that the normal prostate tissues showed a relatively low level of EF-1alpha expression, whereas PCa tissues demonstrated significantly higher expression levels of EF-1alpha (P < 0.001). Taken together, these findings support the hypothesis that EF-1alpha affects multiple processes involved in tumor progression, and identify EF-1alpha as a potential therapeutic target.

Figure 1

EF-1α expression in PCa cell lines 22RV1, LnCap, Du145, and PC3. Total RNA was prepared from the indicated cell lines, reverse-transcribed, and subjected to QRT-PCR using oligonucleotide primers specific for EF-1±α. Expression was normalized using β-actin expression as an internal standard.

Figure 2

Stable suppression of EF-1α by siRNA in Du145 cells. (A) Du145 cells were trans-fected with either siRNA-C or siRNA-E. Cells were then lysed and subjected to Western blot analysis with either anti-EF-1α or anti-β-actin antibodies. (B) Densitometric analysis of EF-1α protein in cells of nontransfected group, siRNA-C–transfected group, and siRNA-E–transfected group.

Figure 3

Du145 cells were cultured in 6-well chamber slides at a concentration of 1×10⁴ and transfected with either siRNA-C or siRNA-E; parental cells were simultaneously maintained; 72 h after transfection, the cells were fixed in cold methanol. Expression of EF-1α was detected in intact cells (see Materials and Methods).

Figure 4

EF-1α enhances proliferation of Du145 cells. Equal numbers of indicated cells were plated in triplicate, incubated under standard culture conditions, following the MTT assays (A) and the cell counting assay (B) as described in Materials and Methods. Values shown are X&macr; ± s.

Figure 5

EF-1α is important for migration and invasion of Du145 cells. (A) The indicated cells were detached from culture plates, washed, and plated in the upper chamber of Transwell inserts as described in Materials and Methods. After 20 h, migrated cells were stained and counted in five random high power fields. (B) The indicated cells were plated in Matrigel-coated Transwell chambers as described in Materials and Methods; 16 h later, invading cells were stained and counted in five random high power fields. Values shown are X̄ ± s. **P < 0.001, comparison of the number of siRNA-E–transfected Du145 cells/field with that of nontransfected and siRNA-C–transfected Du145 cells/field.

Figure 6

Immunohistochemical staining of EF-1α in clinical PCa tissues (A) and matched normal prostate tissue (B) (400×).