Inhibition of APN/CD13 leads to suppressed progressive potential in ovarian carcinoma cells

Abstract

Background: Aminopeptidase N (APN/CD13), a 150-kDa metalloprotease, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression of several human malignancies. In the current study, we investigated the role of APN/CD13 in ovarian carcinoma (OVCA) progression.

Methods: We first examined the expression of APN/CD13 at the protein level in a variety of OVCA cell lines and tissues. We subsequently investigated whether there was a correlation between APN/CD13 expression and invasive potential of various OVCA cell lines. Moreover, we investigated the function of APN/CD13 in OVCA cells using bestatin, an APN/CD13 inhibitor, or transfection of siRNA for APN/CD13.

Results: We confirmed that APN/CD13 was expressed in OVCA tissues and cell lines to various extents. There was a positive correlation between APN/CD13 expression and migratory potential in various OVCA cell lines with accordingly enhanced secretion of endogenous MMP-2. Subsequently, we found a significant decrease in the proliferative and migratory abilities of OVCA cells after the addition of bestatin or the inhibition of APN/CD13 expression by siRNA. Furthermore, in an animal model, daily intraperitoneal administration of bestatin after inoculation of OVCA cells resulted in a decrease of peritoneal dissemination and in prolonged survival of nude mice.

Conclusion: The current data indicate the possible involvement of APN/CD13 in the development of OVCA, and suggest that clinical use of bestatin may contribute to better prognosis for ovarian carcinoma patients.

Figure 1

APN/CD13 expression in OVCA tissues A, B, C, D and E; Immunohistological staining of APN/CD13 in surgically resected OVCA tissues of 14 patients representing four different histological types using APN/CD13 specific Ab. A-D; APN/CD13 was expressed intensely in tumor cells but only slightly in stromal cells. A; clear cell carcinoma (case 7), B; endometrioid adenocarcinoma (case 8), C; mucinous cystadenocarcinoma (case 5), D; serous cystadenocarcinoma (case 12), E; serous cystadenocarcinoma (case 2), APN/CD13 was intensely expressed in stromal cells but was almost undetectable in tumor cells. F; APN/CD13-negative control of serous cystadenocarcinoma. Black arrows indicate tumor cells expressing APN/CD13; green arrows indicate stromal cells expressing APN/CD13.

Figure 2

APN/CD13 expression in various OVCA cell lines, as estimated by FACS analysis A; ES-2 cells, B; HEY cells, C; SKOV-3 cells, D; TAOV cells, E; NOS-4 cells, F; NOS-2 cells and G; HRA cells.

Figure 3

Differential effects of bestatin on cell proliferation of ES-2 and HRA cells. A; Decrease of APN/CD13 enzyme activity by bestatin in a concentration-dependent manner in ES-2 cells, which expressed APN/CD13. More than 100 μg/ml of bestatin significantly inhibited APN/CD13 enzyme activity in ES-2 cells (p < 0.01). B; Cytotoxicity of bestatin was evaluated by trypan blue dye-exclusion test for APN/CD13-positive ES-2 cells and APN/CD13-negative HRA cells. C; Bestatin dose-dependently suppressed cell proliferation of ES-2 cells, which strongly expressed APN/CD13. More than 100 μg/ml of bestatin significantly inhibited the proliferation of ES-2 cells (p < 0.01). D; Bestatin exerted no significant effect on the proliferation of HRA cells, which expressed a low level of APN/CD13.

Figure 4

Bestatin inhibited the cell motility of APN/CD13-expressing OVCA cells in the Transwell migration assay in a concentration-dependent manner. A: SKOV-3 cells; *; p < 0.01, **; p < 0.001, B; ES-2 cells; *; p < 0.001, **; p < 0.0001.

Figure 5

Suppression of APN/CD13 expression by siRNA induced a marked decrease in migratory potential of ES-2 cells. A; Western blot analysis showed a decrease in APN/CD13 expression after siRNA transfection. B, C; Giemsa staining showing the migration of ES-2 cells transfected with non-specific control siRNA (B; si-cont) or siRNA specific for APN/CD13 (C; si-CD13), respectively. D; The level of migration of ES-2 cells transfected with si-CD13 relative to the control was 33%. Data are expressed as the mean ± SD *; p < 0.01.

Figure 6

Decrease of MMP-2 and VEGF expression caused by RNA interference of APN/CD13 in ES-2 cells using MMP-2 and VEGF ELISA kit. The MMP-2 and VEGD expression in the conditioned medium of si-CD13-transfected ES-2 cells was significantly lower than that of si-cont-transfected cells. Data are expressed as the mean ± SD, A; *p < 0.001, B; *p < 0.05.

Figure 7

Inhibition of proliferative potential by the transfection of siRNA for APN/CD13 in ES-2 cells in a 4-day modified MTT assay. Analysis of the proliferation rate was performed as described in "MATERIALS AND METHODS". si-CD13-transfected cells showed almost no growth, whereas the si-cont-transfected cells grew similarly to the parental ES-2 cells. Closed circles; si-cont-transfected cells, gray squares; si-CD13-transfected cells. Data are expressed as the mean ± SD of four independent experiments. *; p < 0.01, **; p < 0.001.

Figure 8

Anti-metastatic effect of bestatin treatment in SKOV-3 cells. A representative example of nude mice 30 days after i.p. inoculation of 1.0 × 10⁷ of SKOV-3 cells with or without subsequent daily treatment with bestatin, showing a mouse treated with PBS alone (a; left) or with bestatin (20 mg/kg, daily)(b; right). More apparent symptoms of carcinomatous peritonitis were observed in mice treated with PBS alone than in those treated with bestatin. In this experiment, each group consisted of seven mice.