Affiliation of authors: EntreMed, Inc., Rockville, MD.
Correspondence to: John W. Holaday, Ph.D., EntreMed, Inc., 9640 Medical Center Dr., Rockville, MD 20850 (e-mail: johnh{at}entremed.com)
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ABSTRACT |
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INTRODUCTION |
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PSA is neither prostate specific nor made exclusively by prostate epithelium. PSA has been found in patients with breast, lung, and uterine cancers (7,8). Circulating serum concentrations of PSA have been documented in healthy women and in women with benign and malignant breast diseases (9-11). Furthermore, as with men, the PSA gene in female breast tissues is regulated by androgens and progestins (12). In one study of particular interest, patients with breast tumors with high levels of PSA had a better prognosis than those patients whose tumors had lower PSA levels (13).
In cancer, the growth of the tumor is dependent on the angiogenic growth of new blood vessels (14). Angiogenesis is a tightly regulated process, modulated by the dynamic interplay between angiogenic stimulators and inhibitors that control endothelial cell proliferation, migration, and invasion. This concept is reinforced by the earlier discovery of endogenous stimulators of angiogenesis, such as fibroblast growth factors (FGF) and vascular endothelial growth factors (VEGF), and, more recently, by the discovery of endogenous inhibitors of angiogenesis, including the Angiostatin® and EndostatinTM proteins (15-17). Preliminary results indicate that increased concentrations of the antiangiogenic EndostatinTM protein may occur in animals and patients with growing tumors, which may indicate that angiogenesis is taking place (18). These and other observations prompted our speculation that increasing PSA concentrations may not be a harbinger of bad news and prostate cancer progression but, rather, may indicate that the body is attempting to fight cancer by producing its own antiangiogenic proteins. If so, then PSA would be expected to demonstrate an inhibitory effect on the key elements of angiogenesis.
The process of angiogenesis is complex and involves a number of orchestrated steps that can be studied separately in vitro, such as FGF-2- and/or VEGF-stimulated endothelial cell proliferation and migration. For example, the Angiostatin® and EndostatinTM proteins inhibit these processes (15,19). We hypothesized that PSA may have antiangiogenic properties. To test this hypothesis, we systematically evaluated the effects of PSA on endothelial cell proliferation, migration, and invasion.
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MATERIALS AND METHODS |
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Single-donor human umbilical vein endothelial cells (HUVEC) were obtained frozen at passage 1 from Clonetics (San Diego, CA). The cells were maintained in endothelial cell growth medium (Clonetics) supplemented with bovine brain extract (Clonetics), cultured at 37 °C in 5% CO2 in moist air and used at passages 2-5 in all experiments. For proliferation assays, HUVEC were resuspended in endothelial cell basal medium-2 (EBM-2; Clonetics) supplemented with 2% heat-inactivated fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT) and 2 mM L-glutamine (BioWhittaker, Inc., Walkersville, MD) and cultured overnight. Cells were incubated with various concentrations, at least three concentrations (0.1-10 µM) in several repeat experiments, of purified human PSA (Vitro Diagnostics, Littleton, CO) or media alone for 30 minutes, and then stimulated with 10 ng/mL of FGF-2 R&D Systems, Inc., Minneapolis, MN) or media for an additional 48 hours. Cell proliferation was assessed with a colorimetric enzyme-linked immunosorbent assay (ELISA) kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) that measured the amount of bromodeoxyuridine (BrdU) incorporated during DNA synthesis. Results are expressed as the mean absorbance of triplicate cultures measured at 370 nm (reference wavelength, 492 nm).
Bovine adrenal capillary endothelial cells (BCEC) were obtained at passage 9 from J. Folkman (Children's Hospital, Harvard Medical School, Boston, MA). The cells were cultured and maintained as described previously (11). For evaluation of PSA inhibition of BCEC proliferation, cells were exposed to five different concentrations of purified PSA or media, in triplicate wells, for 30 minutes at 37 °C in 10% CO2 prior to stimulation with FGF-2. Cell proliferation was assessed by counting the number of cells per well with a Coulter Z1 particle counter (Coulter Corp., Hialeah, FL).
Single-donor adult human microvascular dermal cells (HMVEC-d) were obtained frozen at passage 4 from Clonetics. The cells were maintained in microvascular endothelial cell growth medium-2 (EGM-2-MV; Clonetics). Cells were cultured as for HUVEC described above and were used at passages 5-8 in all experiments. For proliferation assays, HMVEC-d were resuspended in endothelial cell basal medium-2 (Clonetics) supplemented with 2% FBS and 2 mM L-glutamine. Cells were plated as described above for BCEC, and after 30 minutes' preincubation with at least five different concentrations of PSA in triplicate wells, cultured with FGF-2 (10 ng/mL) for an additional 48 hours. Cell proliferation was assessed as above by counting the number of cells per well.
B16BL6, a murine melanoma, obtained from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD) cell repository and human prostate cancer cell line, PC3, a gift from J. Folkman, were maintained in Dulbecco's modified Eagle medium (BioWhittaker, Inc.), supplemented with 5% FBS and 2 mM L-glutamine. The ability of PSA to inhibit proliferation of B16BL6 was assessed with a colorimetric ELISA kit (Boehringer Mannheim Biochemicals) for BrdU incorporation as described above for HUVEC, and inhibition of PC3 proliferation was assessed by cell counts from triplicate cultures; each experiment was performed with five doses of PSA.
Migration
To determine the ability of PSA to block HUVEC migration induced by recombinant FGF-2 or recombinant VEGF 165 (R&D Systems, Inc.), we performed a wound-migration assay as previously described (20). To date, the migration assay as described here has been established only with HUVEC; migration assays with bovine (BCEC) and human (HMVEC-d) endothelial cells are currently under development in our laboratory. In brief, HUVEC in endothelial cell growth medium were plated onto 1.5% gelatin-coated tissue culture dishes (Corning Costar, Inc., Cambridge, MA) and incubated for 72 hours at 37 °C in 5% CO2 in moist air. After incubation, confluent monolayers were wounded with a sterile, single-edged No. 9 razor blade (VWR Scientific, Media, PA), washed with phosphate-buffered saline (PBS; BioWhittaker, Inc.), and further incubated in endothelial cell basal medium supplemented with 1% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL fungizone. Wounded monolayers were exposed to 2 ng/mL FGF-2 or 10 ng/mL VEGF in the presence or absence of PSA or to media alone for 16-20 hours. The monolayers were fixed with absolute methanol and stained with hematoxylin solution, Gill No. 3 (Sigma Diagnostics, St. Louis, MO). Migration was quantified by counting the number of cells that migrated from the wound edge into the denuded area along a 1-cm distance in duplicate cultures. Dose-dependent inhibition of migration was assessed in several repeat experiments (greater than five experiments with FGF-2-stimulated cells and two separate experiments with VEGF-stimulated cells) using at least five different concentrations of PSA in duplicate cultures.
Biocoat 8-µm invasion chambers (Collaborative Biomedical Products, Bedford, MA) were precoated with Matrigel (Collaborative Biomedical Products). The lower chambers were filled with assay media containing 5 ng/mL FGF-2 or assay media alone, and the upper chamber had HUVEC pretreated for 30 minutes with PSA (5 µM) or media. Cells were incubated in the upper chambers for 24 hours at 37 °C in 5% CO2. After incubation, the noninvading cells were removed with a cotton swab, and cells on the lower surface of the membrane were stained with Diff-Quik (Dade Diagnostics, Aquado, Puerto Rico). The membrane was removed and mounted on a microscope slide, and the number of cells invaded was determined by counting the cells at x150 magnification in the central field of the membrane from triplicate cultures.
Endothelial Tube Formation
For induction of endothelial tube formation, the following procedure was adapted from the protocol of Kubota et al. (21). In brief, Matrigel is aliquoted into a 96-well tissue culture plate and allowed to gel. PSA or 2-methoxyestradiol, as a positive control for inhibition (22), was added to Matrigel, followed by the addition of HUVEC. After 16 hours, endothelial cells were microscopically evaluated for tube formation.
In Vivo Metastatic Model
B16BL6 murine melanoma cells (5 x 104) were inoculated into C57BL/6J male mice (The Jackson Laboratory, Bar Harbor, ME) via the lateral tail vein on day 0. In two separate experiments, beginning on day 3, three groups of five mice each were treated subcutaneously for 11 consecutive days with either PBS (0.1 mL) or PSA (9 µM) or positive control EndostatinTM protein (15 µM). On day 14, mice were killed, lungs were removed and fixed in formalin (Sigma Chemical Co., St. Louis, MO), and melanoma lesions were counted with the aid of a dissecting microscope. Histologic examination of the fixed lung tissues confirmed the presence of tumor cells containing melanin. Animal care and use procedures were performed in accordance with standards described in the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Inhibition of PSA Enzymatic Activity
The ability of 1-antichymotrypsin (ACT) to inhibit the proteolytic activity of
PSA was measured by use of the synthetic substrate S-2586 (MeO-Suc-Arg-Pro-Tyr-pNA, where
MeO = methoxy, Suc = succinyl, and pNA = para-nitroaniline). The rate of
hydrolysis of S-2586 (1.3 mM) by 6 µg PSA (0.89 µM) with and
without pretreatment with an equimolar concentration of ACT (Sigma Chemical Co.) was
monitored at 405 nm in 50 mM Tris-HCl, 0.1 M NaCl (pH 7.8). Stable
complexes of PSA and ACT formed after a 4-hour incubation at 37 °C and were confirmed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results were plotted as an
increase in absorbance versus time in minutes. The ability of ACT to inhibit the antimigratory
activity of PSA was measured by preincubating PSA (5 µM) with an equimolar
concentration of ACT for 4 hours at 37 °C prior to addition to the HUVEC migration assay.
Statistical Analysis
Means were compared by use of the Student's t test (two-tailed). Differences were considered to be statistically significant at P<.05.
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RESULTS |
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For evaluation of the in vitro effects of PSA on endothelial cell migration in response
to FGF-2 or VEGF, confluent monolayers of HUVEC were scraped to remove a section of
monolayer and cultured for 24 hours with FGF-2 or VEGF in the presence or absence of purified
human PSA. The endothelial cell response was assessed by counting the number of cells that
migrated into the denuded area of the cell monolayer. The data in Fig. 2,
A and B, demonstrate the dose-response of HUVEC to the inhibitory effects of PSA on FGF-2-
and VEGF-stimulated migration, respectively, with an IC50 for PSA versus FGF-2 of
1.2 µM and for PSA versus VEGF of 4 µM. On a molar basis, the
inhibitory activity of purified PSA on both endothelial cell proliferation and migration was
approximately fivefold to 10-fold less potent than that of the antiangiogenic proteins
Angiostatin® protein and EndostatinTM protein (data not shown).
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We have been using the murine B16BL6 melanoma cell line in a model of experimental metastasis to assess the antitumor effects of the angiogenesis inhibitor EndostatinTM protein. In this model, mice were inoculated intravenously with B16BL6 cells on day 0. On day 3, the mice were treated subcutaneously once a day for 11 consecutive days with purified human PSA (9 µM). On day 14, the mice were killed, their lungs were removed, and the number of melanoma tumor nodules on the surface of the lungs were counted. The number of lung metastases in control mice treated with PBS was 115 ± 16 (mean ± standard deviation) and in PSA-treated mice 70 ± 8 (mean ± standard deviation), representing a 40% reduction in tumor number (P = .003). While the inhibition of B16BL6 development in the lung induced by PSA was not as great as that observed in EndostatinTM protein-treated mice (86% inhibition in the same experiment; mean number of lung metastasis was 16 ± 8; P = .0002), we also observed a fivefold to 10-fold difference in potency between the two proteins in our in vitro assays. Further studies in tumor models in which both vessel density and tumor growth can be assessed are needed to characterize the inhibition observed after PSA treatment.
PSA has serine protease activity and, in serum, is predominantly bound to the protease
inhibitor ACT (23). We tested the ability of ACT to inhibit both serine
protease activity of our purified PSA (Fig. 3, A) as well as the
antimigratory effects of PSA on FGF-2-stimulated HUVEC (Fig. 3,
B).
By use of equimolar concentrations of ACT and PSA, preincubation of PSA with ACT
completely blocked serine protease activity (Fig. 3,
A) and significantly
blocked migration inhibition (Fig. 3,
B; P = .0038) when
compared with cells treated with PSA alone.
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DISCUSSION |
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As Folkman (24) noted, there are two prominent compartments in growing tumors: the cancer cells and the endothelial cells making up the blood vessels that provide the cancer cells with oxygen and nutrients. The balance between the positive and negative regulators produced by these two compartments determines the ultimate growth rate of the tumor. Many reports (25) have used morphimetric analysis to establish a predictive association between microvessel density (angiogenesis) and prostate cancer progression. Our results suggest that, in addition to its role as an indicator of prostate cancer, PSA may also inhibit the growth of blood vessels associated with cancer progression; however, when prostate cancer progression occurs in spite of elevations of PSA, the local angiogenic stimulators overcome the effects of PSA and dominate. Our observations are therefore consistent with other data indicating a "paradoxical" increase in PSA associated with disease cure in certain circumstances (26). Furthermore, in patients with prostate cancer, PSA values range between 4 and 15 000 ng/mL (equivalent to 0.1 nM-0.5 µM), with the highest concentrations in the range of our in vitro inhibitory results (0.3-5 µM). These data suggest that the production of PSA by prostate cancers is one reason for their characteristically slow growth and that PSA may help to maintain homeostasis in the face of progressing angiogenesis and cancer.
With the exception of data indicating a potential role as an IGFBP-3 protease (27), surprisingly little is known about the role of PSA in cancer. The antiangiogenic and antitumor effects of PSA described in this report may result from its actions as a serine protease, since ACT blocked both its enzymatic activity and its antiangiogenic activity in vitro. These data may point to a generalized action of serine proteases and suggest that other serine proteases and members of the kallikrein multigene family of enzymes should be evaluated for potential antiangiogenic actions.
Cohen et al. (27) have speculated that lowering PSA production or decreasing PSA enzymatic activity may inhibit the progression of prostate cancer. Likewise, a number of initiatives are under way to develop a vaccine against PSA (5,6). Our findings indicate that these strategies should be rethought in view of the evidence that PSA acts as an antiangiogenic agent that may play a physiologic role in slowing the progression of cancer.
Published reports indicating that PSA is not prostate specific and that higher PSA concentrations are associated with improved outcome in breast cancer are consistent with our findings. Furthermore, the antiangiogenic drugs thalidomide and TNP470 have recently been reported to produce statistically significant increases in PSA concentrations in vitro (28). We believe that such an effect may partially account for the antiangiogenic properties of these drugs in vivo. Taken together, our data may indicate that elevations of PSA in a variety of malignancies are part of a normal homeostatic process to fight cancer progression. Furthermore, the administration of PSA as a drug to augment endogenous concentrations could provide a rational therapeutic approach in the treatment of cancer.
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NOTES |
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We thank Stacy Plum and Hong Vu for technical support and the EntreMed staff for helpful discussions.
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Manuscript received August 10, 1999; revised August 25, 1999; accepted August 27, 1999.
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