Recently, numerous studies have shown that endothelin-1 (ET-1) is expressed in ovarian carcinoma and that ET-1 selectively acts as an autocrine or paracrine growth factor through the endothelin A receptor (ETAR), and is involved in cell proliferation, invasiveness, neovascularization, and prevention of apoptosis. Neutral endopeptidase 24.11 (NEP) is a cell surface aminopeptidase with a ubiquitous expression and is capable of degrading a number of bioactive peptides including ET-1. Our previous report showed that stromal NEP expression in ovarian carcinoma was down-regulated as the histologic grade advanced. Here, we confirmed that NEP was expressed in tumor cells as well as stromal tissues in ovarian carcinoma, and investigated the functions of NEP in this carcinoma. We showed that there was a significant decrease in cell proliferation and invasiveness with a reduction in the concentration of ET-1 in the conditioned medium on the NEP overexpression of NEP in ovarian carcinoma cells. In addition, the overexpression of NEP enhanced susceptibility to paclitaxel, resulting in an increased occurrence of apoptotic morphologic change. Furthermore, tumorigenesis was reduced in vivo with the overexpression of NEP, down-regulation of both matrix metalloproteinase-2, and vascular endothelial growth factor expression. This evidence suggests that NEP functionally suppresses the progression of ovarian carcinoma and further study of this enzyme may reveal an effective way to target ET-1 for the treatment of this carcinoma.

Ovarian carcinoma is the leading cause of death from gynecologic malignancy. Owing to ovarian carcinoma frequently remaining clinically silent, the majority of patients with this disease have advanced i.p. metastatic disease at diagnosis (1). However, the regulatory mechanisms responsible for the malignant transformation and progression of this tumor remain unclear. Recently, an extensive characterization of the growth factors and corresponding receptors that regulate the progression of ovarian carcinoma was conducted in an attempt to develop a more effective treatment for this disease.

Endothelin-1 (ET-1) is one such mediator, and is produced by endothelial cells, vascular smooth muscle cells, or tumor cells. ET-1 receptors, ETA and ETB, belong to the family of G-protein-coupled receptors and bind ET-1 with equal affinity (2, 3). Recently, several studies have shown that ET-1 is expressed in ovarian carcinoma and selectively acts as an autocrine or paracrine growth factor through the ETA receptor (ETAR; refs. 4–6). Moreover, in this tumor, ET-1 not only induced cell proliferation but the engagement of ETAR by ET-1 also triggered activation of signal pathways linked to neovascularization, invasiveness, and apoptosis protection (7–9).

Neutral endopeptidase 24.11 (NEP, Neprilysin, EC 3.4.24.11) is a cell surface aminopeptidase which was originally characterized as a T-cell differentiation antigen (CD10/common acute lymphoblastic leukemia antigen; ref. 10). NEP has been reported to be present on epithelial cells of various tissues such as the lung, kidney, intestine, prostate, endometrium, and placenta (11–13), and plays an important role in the maintenance of homeostasis in normal tissues. NEP is capable of efficiently degrading a number of bioactive peptides and cytokines, including ET-1, enkephalins, atrial natriuretic peptides, and bombesin-like peptides (14, 15). Thus, the biological activities of such substrates are regulated by this enzyme by reducing local concentrations available for receptor binding and signal transduction (11).

Recent reports have shown that NEP is involved in neoplastic transformation and tumor progression in certain human malignancies including lung, breast, and prostate carcinomas by inactivating ET-1 or bombesin, which are autocrine growth factors for these tumors (16–19). We previously reported that NEP was also expressed in the stroma of borderline and malignant ovarian tumors and that stromal NEP expression was down-regulated as the histologic grade advanced. This implies that this peptidase plays a role in the biology of neoplastic transformation or tumor progression in ovarian carcinoma through the degradation and modulation of specific peptide substrates such as ET-1.

Therefore, in the present study, we investigated the cellular functions of NEP in ovarian carcinoma cells and examined the effects of NEP on the progression of ovarian carcinoma in vitro and in vivo. A possible role for this enzyme as a suppressor of ovarian carcinoma is proposed.

Cell Culture. We used six human ovarian carcinoma cell lines: NOS2, OVCAR3, RMG-I, RMG-II, SKOV3, and TAOV. SKOV3 cells were generously donated by the memorial Sloan-Kettering Cancer Research Laboratory. RMG-I and RMG-II cells were kindly provided by Dr. Shiro Nozawa (Keio University, Tokyo, Japan). NOS2 and TAOV cells were established in our institute. OVCAR3 cells were purchased from the American Type Culture Collection (Rockville, MD). These cell lines were maintained in RPMI 1640 supplemented with 10% FCS and 100 units/mL of penicillin and streptomycin, and were incubated at 37°C in a humidified atmosphere of 5% CO2.

Plasmid Construction and Transfection. Full-length cDNA for NEP was kindly provided by Dr. David M. Nanus (Cornell University, New York). The eukaryotic expression vector pcDNA 3.1(+) (Invitrogen, San Diego, CA) was used to drive the expression of the inserted NEP cDNA. Transfections were carried out using Lipofectamine according to the instructions of the manufacturer (Invitrogen). SKOV3 cells were transfected with pcDNA3.1(+), SKpcDNA, or pcDNA3.1(+) with NEP cDNA inserted. Stable transfectants were selected by growth in media supplemented with 400 μg/mL of G 418 (Sigma-Aldrich, St. Louis, MO). Several hundred clones resistant to G 418 were obtained and two independent monoclonal cells (SKNEP3 and SKNEP16) from these transfectants were used in the subsequent experiments.

Enzyme Activity Assay. NEP enzyme activity was measured spectrophotometrically using succinyl-Ala-Ala-Phe-p-nitroanilide (Suc-Ala-Ala-Phe-pNA; Bachem Bioscience, Inc., PA) as a substrate. Whole cell suspensions were prepared in test tubes, then washed with PBS. Thereafter, 5 × 104 cells were resuspended in 200 μL of PBS contained in each well of 96-well microtiter plates. Then the substrate and aminopeptidase M (0.5 units/mL) were added (final concentration, 0.9 mmol/L Suc-Ala-Ala-Phe-pNA), and incubated at 37°C for 60 minutes. NEP enzyme activity was measured at 405 nm with a microplate reader (Labsystems, Multiskan Bichromatic, Helsinki, Finland) every 10 minutes during incubation at 37°C. We used the NEP inhibitor phosphoramidon (Peptide Institute, Osaka, Japan) as a specific inhibitor of NEP enzyme activity.

Flow Cytometric Analysis. Fluorescence-activated cell sorting (FACS) was done to quantify the expression of NEP on the cell surface of various clones of the NEP transfectant. Then, the cells were incubated with phycoerythrin-conjugated monoclonal antibody specific for NEP (PharMingen, San Diego, CA) for 30 minutes at 4°C and washed thrice with PBS. FACS data were acquired on a FACS Calibur (Becton Dickinson, San Jose, CA), and analyzed using CELL Quest software (Becton Dickinson).

Immunohistochemical Staining of Ovarian Carcinoma Tissues. Tissue samples of ovarian carcinoma were obtained from patients who were surgically treated at Nagoya University Hospital with informed consent. All samples were fixed in 10% formalin and embedded in paraffin, and sections were cut at a thickness of 4 μm. For heat-induced epitope retrieval, deparaffinized sections in 0.01 mol/L citrate buffer were treated thrice at 90°C for 5 minutes at 750 W using a microwave oven. Immunohistochemical staining for NEP was done using the avidin-biotin immunoperoxidase technique (Histofine SAB-PO kit, Nichirei, Tokyo, Japan) according to the instructions of the manufacturer as previously described (20). As a primary antibody for NEP staining, anti-NEP/CD10 monoclonal antibody (NCL-CD10-270, Novocastra, United Kingdom) was used at a dilution of 1:100. For the negative control, mouse immunoglobulin G replaced the primary antibody. We used placental chorion as a positive control for NEP expression.

Western Blot Analysis. Immunoblot analysis of NEP, ETAR, and endothelin converting enzyme-1 (ECE-1) was done as described previously (21). Briefly, cells were grown to 70% to 80% subconfluence and treated with lysis buffer containing 1% Triton-X in PBS and protease inhibitor cocktail tablets (Roche, Barcelona, Spain). Total cell lysate (20 μg) was electrophoresed on a 10% (NEP) or 15% (ETAR and ECE-1) SDS polyacryl-amide gel and transferred electrophoretically to an Immobilon membrane (Millipore, Bedford, MA). After blocking in blocking solution (5% nonfat dry milk/0.1% Tween 20/PBS), the membranes were incubated with a recommended dilution of primary antibodies overnight. We used the following antibodies: anti-NEP/CD10 monoclonal antibody (NCL-CD10-270, Novocastra), anti-ETAR polyclonal antibody (R19, Santa Cruz Biotechnogy, Inc.), and anti-ECE-1 polyclonal antibody (Zymed Laboratories, Inc.) The primary antibodies were washed in 0.1% Tween 20/PBS, and then incubated with horseradish peroxidase–conjugated secondary antibody. Proteins were visualized using enhanced chemiluminescence (ECL) reagent (Amersham Bioscience, Buckinghamshire, United Kingdom) followed by exposure to X-ray film.

In vitro Cell Proliferation Assay. SKpcDNA, SKNEP3, and SKNEP16 cells were plated in triplicate at a density of 1,500 cells per 200 μL of culture medium containing 10% FCS in 96-well plates and cultured for 1 to 4 days. Cell viability was assayed every 24 hours using a modified tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with a CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) according to the instructions of the manufacturer. Absorbance was measured at 492 nm with a microplate reader (Labsystems, Multiskan Bichromatic). Furthermore, we assessed the effect of recombinant ET-1 (Calbiochem) and/or TAK-044, an ETAR antagonist (Takeda Chemical Industries, Osaka, Japan), on cell proliferation. SKpcDNA and SKNEP3 cells were plated in triplicate at a density of 2,000 cells per 200 μL of culture medium containing 10% FCS in 96-well plates and incubated for 24 hours; after which the medium was replaced with a serum-free medium (RPMI 1640) for serum starvation. After 12 hours of incubation, the medium was replaced again with fresh serum-free medium with or without 100 nmol/L of recombinant ET-1, 100 nmol/L of TAK-044, or 10 μmol/L of phosphoramidon. After an additional 36 hours, cell viability was assayed as described above. Experiments were done in triplicate.

In vitro Invasion Assay. Cell invasion was evaluated using 24-well Matrigel invasion chambers (Becton Dickinson Labware). SKOV3 parental and vector or NEP-transfected cells were suspended in the upper chamber at a final concentration of 75 × 104 /mL in 200 μL of RPMI 1640 supplemented with or without 100 nmol/L of ET-1 or 10 μmol/L of phosphoramidon. The lower chamber contained 700 μL of RPMI 1640 supplemented with chemoattractants. After 16 hours of incubation, the tumor cells remaining on the upper surface of the filters were removed by wiping with cotton swabs, and the cells on the lower surface were stained with May-Grünwald Giemsa reagent. The number of cells on the lower surface of the filters was counted under a microscope at a magnification of 200. Four individual experiments were done in triplicate.

In vitro Migration Assay. Cell migration was assayed in 24-well Transwell cell culture chambers (Costar). Cells were suspended in the upper chamber at a final concentration of 50 × 104 /mL in 200 μL of RPMI 1640 supplemented with 0.1% bovine serum albumin. The subsequent procedures were the same as those used for the invasion assay except that the incubation time was 12 hours.

Quantification of Endothelin-1 by ELISA. SKpcDNA, SKNEP3, and SKNEP16 cells were seeded into 24-well culture dishes and incubated in the culture medium. After become confluent, the cells were washed with a serum-free medium and incubated for 24 hours in the presence or absence of phosphoramidon (10 μmol/L; Peptide Institute). After the incubation, the culture supernatants were tested using ET-1 ELISA kits (G-T, Minneapolis, MN) according to the directions of the manufacturer. Experiments were done in triplicate.

Measurement of Matrix Metalloproteinase-2 Protein by ELISA. SKpcDNA, SKNEP3, and SKNEP16 cells were seeded into 24-well culture dishes, and incubated in the culture medium. Once confluent, the cells were washed with a serum-free medium, and incubated for 24 hours in the presence or absence of recombinant ET-1 (100 μmol/L). After the incubation, the culture supernatants were tested using matrix metalloproteinase (MMP)-2 ELISA kits (R & D Systems, Minneapolis, MN) according to the directions of the manufacturer. Experiments were done in triplicate.

Gelatin Zymography. The activities of MMPs in the conditioned medium of SKpcDNA, SKNEP3, and SKNEP16 cells were assayed by zymography as described previously (22).

Paclitaxel Chemosensitivity Assay. SKpcDNA and SKNEP3 cells were seeded in triplicate in 96-well plates at a density of 5,000 cells in 200 μL of culture medium containing 10% FCS. After incubation for 24 hours at 37°C, the medium was replaced with fresh medium with or without various concentrations of paclitaxel (Bristol Myers, Tokyo, Japan). After 36 hours, cell viability was measured as described above. Data were expressed as a percentage of the control (without paclitaxel).

In vivo Experiments. Female nude mice (BALB/c), 5 weeks of age, were obtained from Japan SLC (Nagoya, Japan). The treatment protocol followed the guidelines of animal experimentation adopted by Nagoya University. Mice were given injections s.c. on the bilateral dorsal flank with 1.0 × 106 viable SKpcDNA or SKNEP3 cells resuspended in 100 μL of PBS. After 7 days, established tumors were detectable. Tumor volume was measured with calipers and calculated using the formula π / 6 × (larger diameter) × (smaller diameter)2. One experiment was terminated after 40 days to allow the harvesting of tumor xenografts for immunohistochemical analysis. Each group consisted of six mice. At the end of the experiment, mice underwent euthanasia with diethyl ether. Tumor tissue was excised, fixed in 4% paraformaldehyde, and embedded in paraffin sections (5 mm). Furthermore, SKpcDNA and SKNEP3 cells (1 × 107 cells /0.5 mL of medium/mouse) were injected i.p. to examine their peritoneal metastatic potential. Mice were sacrificed 30 days after the injection of carcinoma cells, and the i.p. dissemination was evaluated. In addition, the total weight of disseminated tumors was compared between the two groups. Each group consisted of six mice.

Statistical Analysis. For the data from in vitro and in vivo experiments, statistical comparisons among groups were done using the Student's t test and ANOVA with Bonferroni corrections. Differences between groups were considered statistically significant at P < 0.05. Data are expressed as the mean ± SD.

Neutral Endopeptidase 24.11 Expression in Ovarian Carcinoma Tissues and Cell Lines. We first confirmed expression of NEP in surgically resected ovarian carcinoma tissues. NEP staining was found in tumor cells as well as stromal tissues (Fig. 1A-C) although NEP was mainly expressed in stromal tissues as shown in our previous study (20). We examined 55 ovarian cancer tissue samples including all histologic types of ovarian carcinoma. Of these samples, seven (12.7%) were positive for NEP immunohistochemical staining in tumor cells, 29 (52.7%) were positive in stromal cells but not in tumor cells, and 19 (34.5%) were negative in both tumor and stromal cells. The intensity of immunohistochemical staining varied among the tissues. No expression of NEP was found in ovarian surface epithelial cells. However, NEP was partially expressed in follicular granulosa cells or the corpus luteum (data not shown).

Figure 2 shows the extent of NEP expression in ovarian carcinoma cell lines as assessed by Western blot analysis. NEP was expressed in RMG-I, RMG-II, and SKOV3 cells but not in the other cell lines. However, no apparent correlation between the expression profile of NEP and proliferative or invasive potential was found in the current study (data not shown).

Overexpression of Neutral Endopeptidase 24.11 in Ovarian Carcinoma Cells. To investigate the functional effect of NEP on ovarian carcinoma cells, we transfected NEP cDNA into ovarian carcinoma SKOV3 cells. A previous report showed that SKOV3 cells overexpress ETAR and secrete high levels of ET-1 (5). Whereas parental SKOV3 cells expressed a limited amount of NEP, NEP-transfected monoclonal cells (SKNEP3 and SKNEP16) expressed a remarkably high level of NEP as determined by FACS analysis (Fig. 3A). To confirm the enzyme activity of NEP overexpressed by the transfection, we also checked for aminopeptidase activity. The NEP activity of SKNEP3 and SKNEP16 was ∼30 times higher than that of the parental SKOV3 cells and SKpcDNA cells in terms of absorbance (P < 0.001; Fig. 3B).

Functional Analysis of Neutral Endopeptidase 24.11 Using Neutral Endopeptidase 24.11–Overexpressing Ovarian Carcinoma Cells. We first investigated the effect of NEP on cell proliferation using SKpcDNA, SKNEP3, and SKNEP16 cells as described in MATERIALS AND METHODS. As shown in Fig. 4A, cell proliferation rates were lower for SKNEP3 and SKNEP16 cells than for SKpcDNA cells (P < 0.05). Next, we assessed the effect of recombinant ET-1 on cell proliferation in SKpcDNA and SKNEP3 cells in the presence or absence of an ETAR antagonist, TAK-044, and an NEP inhibitor, phosphoramidon. As shown in Fig. 4B, 100 nmol/L of recombinant ET-1 significantly stimulated the proliferation of SKpcDNA cells (P < 0.005) and this effect was almost completely inhibited by the addition of TAK-044 (100 nmol/L). In contrast, the addition of recombinant ET-1 did not significantly affect the proliferation of SKNEP3 cells. Furthermore, this growth-suppressive effect of NEP was partially restored by the addition of phosphoramidon in SKNEP3 cells.

We next examined the effect of NEP overexpression on migratory and invasive potential in vitro. As shown in Fig. 5A, the number of cells that had invaded the Matrigel was significantly small for both SKNEP3 and SKNEP16 cells compared with parental SKOV3 or SKpcDNA cells (P < 0.05). This reduction was partially restored by the addition of 10 μmol/L of phosphoramidon in the upper chamber. Additionally, invasive potential was enhanced by recombinant ET-1 in SKpcDNA cells but not in SKNEP3 cells (Fig. 5A). A similar result was obtained in the migration assay (Fig. 5B). Gelatin zymography revealed that pro-MMP-2 activity was remarkably reduced in SKNEP3 cells, whereas SKpcDNA cells showed the same level of pro-MMP-2 activity as parental SKOV3 cells (Fig. 5C). Moreover, we measured the effect of exogenous ET-1 on the secretion of MMP-2 in SKpcDNA or SKNEP3 cells using the human MMP-2 ELISA kit. As shown in Fig. 5D, ET-1 enhanced the secretion in SKpcDNA cells but not in SKNEP3 cells.

We next examined soluble ET-1 levels in the culture medium to clarify whether the concentration of ET-1 was affected by overexpression of NEP. Concentrations of soluble ET-1 were significantly lower in SKNEP3 and SKNEP16 cells than in SKpcDNA cells (Fig. 6; P < 0.001). Additionally, the addition of 10 μmol/L of phosphoramidon partially restored the decrease in the concentration of ET-1 in the conditioned medium (P < 0.05). Furthermore, we examined ETAR or ECE-1 expression in mock and NEP-transfected cells by Western blotting (Fig. 7). In this analysis, levels of both the proteins were found to be slightly elevated in NEP-expressing cell lines by quantitating these bands (data not shown).

Paclitaxel Chemosensitivity Assay. A previous report showed that ET-1 protected ovarian carcinoma cells against paclitaxel-induced apoptosis (7). In the present study, we examined whether the ET-1-degrading enzyme NEP influenced the susceptibility of ovarian carcinoma cells to paclitaxel. Cell death with morphologic changes associated with apoptosis was more frequently observed in SKNEP3 cells than in SKpcDNA cells 12 hours after exposure to 100 ng/mL paclitaxel (Fig. 8A-D). Moreover, similar morphologic change was observed when the cells were cultured in serum-free conditions for more than 72 hours (Fig. 8E and F). Therefore, NEP-overexpressing cells were more sensitive to stress such as exposure to paclitaxel or serum starvation. We then treated these cells with 30 to 100 ng/mL of paclitaxel for 36 hours and measured the effect on cell viability (Fig. 9). In SKNEP3 cells, a markedly enhanced sensitivity was displayed at 10 to 100 ng/mL of paclitaxel compared with SKpcDNA cells (P < 0.001).

Effect of Neutral Endopeptidase 24.11 on Tumor Progression In vivo. We investigated whether NEP suppressed the formation of tumors in s.c. xenografts using nude mice. S.c. tumorigenesis was observed approximately 1 week after the inoculation of SKpcDNA or SKNEP3 cells into the mice. Figure 10A shows the general appearance of the mice 29 days after the inoculation with SKpcDNA (left) or SKNEP3 cells (right). Significantly, tumors were macroscopically larger following the inoculation of SKpcDNA cells than SKNEP3 cells (Fig. 10A). The comparison of the time course of tumor-growth curves by ANOVA was statistically significant (P < 0.001; Fig. 10B). Furthermore, immunohistochemical evaluation of the expression of ET-1, ETAR, MMP-2, and vascular endothelial growth factor (VEGF) done on tumor xenografts revealed a marked reduction in the percentage of ET-1-, MMP-2-, and VEGF-positive cells among SKNEP3 cells (Fig. 11). However, ETAR expression was not altered in tumor xenografts by the inoculation of SKNEP3 cells compared with that in control xenografts (mock injection; data not shown). These results are consistent with the finding made in vitro that NEP inhibits the growth and invasion of SKOV3 cells. We also investigated in vivo whether NEP suppressed the of peritoneal metastasis of ovarian carcinoma. SKpcDNA or SKNEP3 cells were injected i.p. to examine their metastatic potential. Mice were sacrificed 30 days after the injection and the i.p. dissemination was evaluated. The mice injected with SKpcDNA cells displayed a number of disseminated and enlarged tumor nodules throughout the peritoneal cavity (Fig. 12A and B). In contrast, the mice injected with SKNEP3 cells had a small number of tumors. As shown in Fig. 12C, the total weight of the disseminated tumors was smaller in SKNEP3-injected than in SKpcDNA-injected mice (P < 0.0001).

Peritoneal dissemination is the main metastatic process in cases of ovarian carcinoma besides direct extension of the carcinoma into adjacent tissues and lymphatic dissemination. In the initial stage of peritoneal dissemination, carcinoma cells detach from the primary lesions and are released in the ascites. Once the carcinoma cells attach to mesothelial cells, they invade local tissues with increased neovascularization. During each of these steps, bioactive peptides such as ET-1 and bombesin act as autocrine growth factors on specific receptors to stimulate the progression of ovarian carcinoma cells (4, 5, 8).

Cell-surface peptidases play a key role in controlling the growth, differentiation, and signal transduction of many cellular systems by modulating the activity of peptide factors and regulating their access to receptors (11). NEP was also reported to be involved in carcinoma cell differentiation and apoptosis by regulating the local concentration of possible bioactive peptides including ET-1 (13, 23). In ovarian carcinoma, our previous reports showed that NEP was specifically expressed in the stroma of borderline and malignant ovarian tumors but not in benign adenomas and that stromal NEP expression was down-regulated as the histologic grade advanced (20). We had previously thought that NEP was expressed only in stromal cells in ovarian carcinoma but found that it was also expressed in tumor cells. Chu and Arber (24) reported the expression of NEP in 505 nonhematopoietic neoplasms and found that in 3 of 27 (11%) ovarian carcinomas, NEP was expressed in tumor cells. Thus, NEP was expressed in both tumor cells and stromal cells in ovarian carcinoma although the staining intensity varied among the tissues. However, a functional analysis of NEP in ovarian carcinoma is yet to be conducted. Accordingly, it would be beneficial to establish a subline overexpressing NEP to clarify the role of NEP in ovarian carcinoma.

In this study, we showed that there was a significant decrease in cell proliferation and invasiveness in NEP-transfected ovarian carcinoma cells. In addition, recombinant ET-1 promoted the proliferation of SKOV3 cells that were completely inhibited by TAK-044, an ETAR antagonist. In contrast, no evident effects of recombinant ET-1 or TAK-004 on proliferation were found in SKNEP3 cells. Moreover, we showed that the concentration of ET-1 was markedly lower in the conditioned medium of NEP-overexpressing SKOV3 cells than in cells. In this respect, the addition of phosphoramidon also reversed the reduction, although only partially. On the other hand, it is possible that the overexpression of NEP affects ETAR or ECE-1 expression in SKOV3 cells. In the Western blot analysis, both proteins were slightly enhanced in the cell lines overexpressing NEP. Although details remain unclear, one of the molecular mechanisms may be a negative feedback system from ET-1 lacking due to NEP overexpression at the cell surface. These results suggest that NEP may directly influence local concentrations of ET-1 via its enzyme activity. Therefore, we hypothesize that NEP could degrade ET-1 enzymatically on the cell surface and negatively regulate the mitogenic effects of ET-1 on SKOV3 cells.

We also showed that the invasive potential of SKOV3 cells was markedly reduced by transfection of NEP with a decrease in MMP-2 activity and that the reduction was in part reversed by the addition of NEP inhibitors. Rosano et al. (8) that ET-1 induced activation of MMP-2, -9, -3, -7, and -13 through ETAR in ovarian carcinoma, leading to the degradation of all known extracellular matrix components and thus enabling tumor cells to break down the basement membrane. This is consistent with our current data and, in this regard, ovarian carcinoma cells presumably enhance their metastatic ability by secreting endogenous ET-1 as an autocrine factor. Thus, we assume that increased NEP activity might, at least in part, contribute to the suppressive effect on invasiveness and metastasis in ovarian carcinoma. However, other mechanisms independent of enzyme activity may result in the partial suppression of invasive potential by the NEP inhibitor. Sumitomo et al. (25) reported that NEP inhibited the phosphorylation of focal adhesion kinase and cell migration by blocking the association of focal adhesion kinase with phosphatidylinositol 3-kinase in prostate cancer cells independently of the enzyme activity of NEP.

The current study showed that overexpression of NEP in SKOV3 cells enhanced susceptibility to paclitaxel, resulting in an increased occurrence of apoptotic morphologic change. Figure 9 shows that NEP transfectants (SKNEP3 and SKNEP16) displayed markedly increased sensitivity to paclitaxel compared with mock cells. Del Bufalo et al. (7) provided data that the addition of ET-1 markedly inhibited serum withdrawal and paclitaxel-induced apoptosis in a concentration-dependent manner via the phosphatidylinositol 3-kinase-Akt pathway, and that the blockade of ETAR by ETAR antagonists reversed the ET-1-induced protective effect in ovarian carcinoma cells. According to this, we can assume that the relative decrease in ET-1 due to degradation by NEP may lead to a down-regulation of Akt phosphorylation and, thus, apoptosis signaling may be activated through a Bcl-2-dependent pathway.

A recent report described the ETAR antagonist as a new target in the treatment of ovarian carcinoma treatment. Rosano et al. (26) showed that ABT-627, a new ETAR antagonist, had therapeutic effects in nude mice and synergistic effects in combination with paclitaxel. In this study, in vivo tumor formation was reduced when NEP was overexpressed with the down-regulation of both MMP-2 and VEGF expressions. Moreover, peritoneal metastatic potential was decreased by transfection of NEP. Owing to the majority of patients with ovarian carcinoma having advanced i.p. metastatic disease at diagnosis, the 5-year survival rates for disseminated cases remain poor. Our evidence suggests the potential of NEP for treating i.p. metastatic ovarian carcinoma although extensive work is required before this enzyme can become available for clinical use.

In summary, the current study indicates a possible functional role of NEP in ovarian carcinoma through its association with ET-1. However, NEP also degrades peptides and cytokines other than ET-1, which promotes tumor proliferation and invasion. Thus, the result of the current study may also be due to the decrease in other NEP substrates or another molecular mechanism independent of enzyme activity. It is possible that NEP expression in stromal fibroblasts acts similarly considering that a loss of stromal NEP was reported in poorly differentiated ovarian carcinoma tissues (20). The potential of NEP for treating ovarian carcinoma including gene therapy is promising although extensive study is required to elucidate the molecular mechanisms of its activity in tumor cell biology.

Grant support: Grants-in-Aid from the Ministry of Posts, Telecommunications of Japan for specific medical research (collaboration with Nagoya Teishin Hospital), and the fund of Showakai.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Kikkawa F, Matsuzawa K, Arii Y, et al. Randomized trial of cisplatin and carboplatin versus cisplatin, vinblastine and bleomycin in ovarian cancer.
Gynecol Obstet Invest
2000
;
50
:
269
–74.
2
Rubanyi GM, Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology.
Pharmacol Rev
1994
;
46
:
325
–415.
3
Spinella F, Rosano L, Di Castro V, Natali PG, Bagnato A. Endothelin-1 induces vascular endothelial growth factor by increasing hypoxia-inducible factor-1α in ovarian carcinoma cells.
J Biol Chem
2002
;
277
:
27850
–5.
4
Bagnato A, Tecce R, Di Castro V, Catt KJ. Activation of mitogenic signaling by endothelin 1 in ovarian carcinoma cells.
Cancer Res
1997
;
57
:
1306
–11.
5
Bagnato A, Tecce R, Moretti C, Di Castro V, Spergel D, Catt KJ. Autocrine actions of endothelin-1 as a growth factor in human ovarian carcinoma cells.
Clin Cancer Res
1995
;
1
:
1059
–66.
6
Bagnato A, Salani D, Di Castro V, et al. Expression of endothelin 1 and endothelin A receptor in ovarian carcinoma: evidence for an autocrine role in tumor growth.
Cancer Res
1999
;
59
:
720
–7.
7
Del Bufalo D, Di Castro V, Biroccio A, et al. Endothelin-1 protects ovarian carcinoma cells against paclitaxel-induced apoptosis: requirement for Akt activation.
Mol Pharmacol
2002
;
61
:
524
–32.
8
Rosano L, Varmi M, Salani D, et al. Endothelin-1 induces tumor proteinase activation and invasiveness of ovarian carcinoma cells.
Cancer Res
2001
;
61
:
8340
–6.
9
Salani D, Di Castro V, Nicotra MR, et al. Role of endothelin-1 in neovascularization of ovarian carcinoma.
Am J Pathol
2000
;
157
:
1537
–47.
10
Shipp MA, Vijayaraghavan J, Schmidt EV, et al. Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 (“enkephalinase”): direct evidence by cDNA transfection analysis.
Proc Natl Acad Sci U S A
1989
;
86
:
297
–301.
11
Shipp MA, Look AT.
Hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key! Blood
1993
;
82
:
1052
–70.
12
Ino K, Suzuki T, Uehara C, et al. The expression and localization of neutral endopeptidase 24.11/CD10 in human gestational trophoblastic diseases.
Lab Invest
2000
;
80
:
1729
–38.
13
Suzuki T, Kikkawa F, Ino K, Nagasaka T, Tamakoshi K, Mizutani S. Imbalance between neutral endopeptidase 24.11 and endothelin-1 expression in human endometrial carcinoma.
Oncology
2001
;
60
:
258
–67.
14
Turner AJ, Tanzawa K. Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX.
Faseb J
1997
;
11
:
355
–64.
15
Erdos EG, Skidgel RA. Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones.
Faseb J
1989
;
3
:
145
–51.
16
Burns DM, Walker B, Gray J, Nelson J. Breast cancer cell-associated endopeptidase EC 24.11 modulates proliferative response to bombesin.
Br J Cancer
1999
;
79
:
214
–20.
17
Cohen AJ, Bunn PA, Franklin W, et al. Neutral endopeptidase: variable expression in human lung, inactivation in lung cancer, and modulation of peptide-induced calcium flux.
Cancer Res
1996
;
56
:
831
–9.
18
Dai J, Shen R, Sumitomo M, et al. Tumor-suppressive effects of neutral endopeptidase in androgen-independent prostate cancer cells.
Clin Cancer Res
2001
;
7
:
1370
–7.
19
Shipp MA, Tarr GE, Chen CY, et al. CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.
Proc Natl Acad Sci U S A
1991
;
88
:
10662
–6.
20
Khin EE, Kikkawa F, Ino K, et al. Neutral endopeptidase/CD10 expression in the stroma of epithelial ovarian carcinoma.
Int J Gynecol Pathol
2003
;
22
:
175
–80.
21
Kajiyama H, Kikkawa F, Khin E, Shibata K, Ino K, Mizutani S. Dipeptidyl peptidase IV overexpression induces up-regulation of E-cadherin and tissue inhibitors of matrix metalloproteinases, resulting in decreased invasive potential in ovarian carcinoma cells.
Cancer Res
2003
;
63
:
2278
–83.
22
Kajiyama H, Kikkawa F, Suzuki T, Shibata K, Ino K, Mizutani S. Prolonged survival and decreased invasive activity attributable to dipeptidyl peptidase IV overexpression in ovarian carcinoma.
Cancer Res,
2002
;
62
:
2753
–7.
23
Sumitomo M, Shen R, Goldberg JS, Dai J, Navarro D, Nanus, DM. Neutral endopeptidase promotes phorbol ester-induced apoptosis in prostate cancer cells by inhibiting neuropeptide-induced protein kinase C δ degradation.
Cancer Res
2000
;
60
:
6590
–6.
24
Chu P, Arber DA. Paraffin-section detection of CD10 in 505 nonhematopoietic neoplasms. Frequent expression in renal cell carcinoma and endometrial stromal sarcoma.
Am J Clin Pathol
2000
;
113
:
374
–82.
25
Sumitomo M, Shen R, Walburg M, et al. Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling.
J Clin Invest
2000
;
106
:
1399
–407.
26
Rosano L, Spinella F, Salani D, et al. Therapeutic targeting of the endothelin A receptor in human ovarian carcinoma.
Cancer Res
2003
;
63
:
2447
–53.