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).
We thank Dr. Khan for the letter regarding our early evidence for a role of prostate-specific antigen (PSA) as an endogenous antiangiogenic protein (1). Dr. Khan questions the validity of our conclusions by mentioning that the concentrations of PSA used in our experiments are not physiologic and very uncommon in humans. We agree that one should exercise caution when extrapolating in vitro bioactivity to predict potential effects in an in vivo setting. Nonetheless, we believe that our data do demonstrate the potential physiologic relevance of PSA in the regulation of tumor growth.
It is critical to note that the concentrations of PSA in prostatic and breast exudates would be expected to be far higher than the diluted levels found in the general circulation and typically measured in patients. Indeed, levels of PSA leading to endothelial cell responses in our in vitro assays (8-120 µg/mL) approximate PSA levels found by Diamandis et al. in breast duct fluids (3 µg/mL), and are up to 200 times lower than PSA levels found in seminal fluid (2000 µg/mL) (2-4). Thus, elevated plasma concentrations of PSA represent only a minute amount of the total PSA that escapes from glands and is diluted into the bloodstream.
In addition, the concentrations of biologically active molecules in in vitro angiogenesis assays are typically an order of magnitude higher than effective in vivo concentrations, as we demonstrated for EndostatinTM protein (5). These differences in activity between in vitro and in vivo concentrations may be due to myriad factors, including cell line sensitivity (see Fig. 1, A and C, in our study) and variable responses to angiogenic stimulators (fibroblast growth factor-2 versus vascular endothelial growth factor; Fig. 2 in our study) (1). Our preliminary in vivo findings required the daily administration of 9 µM (about 300 µg) to demonstrate antitumor effects in the mouse B16BL6 metastatic melanoma model; blood levels of PSA administration were not measured in this study (1).
We recently obtained further data that reinforces our earlier observations. We expressed a recombinant human PSA in the yeast Pichia pastoris and compared its activity with that of PSA purified from seminal plasma in a modified Boyden chamber migration assay (6). This assay is more sensitive to the inhibitory effects of PSA than those assays used in our preliminary report and demonstrated that concentrations in the 100 nM (3 µg/mL) range for both forms of PSA resulted in 50% inhibition of endothelial cell migration.
As we suggested in our report (1), the local antiangiogenic effects of PSA may account for the slow progression of prostate cancer relative to other malignancies. Indeed, as estimated by Montie et al. (7), approximately 40% of men more than 60 years old showed evidence of subclinical prostate malignancies. Most important, we do not in any way recommend against the important use of PSA as a marker of prostate cancer or its progression, nor do we recommend that patients or clinicians modify present therapeutic strategies based on our preliminary findings. Instead, our findings indicate that PSA is an endogenous antiangiogenic molecule, it may have therapeutic relevance, and it is not prostate specific.
NOTE
We thank Dr. E. P. Diamandis for helpful discussions.
REFERENCES
1
Fortier AH, Nelson BJ, Grella DK, Holaday JW. Antiangiogenic
activity of prostate-specific antigen. J Natl Cancer Inst 1999;91:1635-40.
2 Rittenhouse HG, Finlay JA, Mikolajczyk SD, Partin AW. Human kallikrein 2 (hK2) and prostate-specific antigen (PSA): two closely related, but distinct, kallikreins in the prostate. Crit Revs Clin Lab Sci 1998;35:275-368.
3 Sauter ER, Daly M, Linahan K, Ehya H, Engstrom PF, Bonney G, et al. Prostate specific antigen levels in nipple aspirate fluid correlate with breast cancer risk. Cancer Epidemiol Biomarkers Prev 1996;5:967-70.[Abstract]
4 Diamandis EP, Yu H. Non-prostatic sources of prostate-specific antigen. In: Oesterling JE, editor. The urologic clinics of North America. Prostate-specific antigen: the best tumor marker. Vol. 24. Philadelphia (PA): Saunders; 1997. p. 275-82.
5 Sim BL, Fogler WE, Zhou XH, Liang H, Madsen JW, Luu K, et al. Zinc ligand-disrupted recombinant human Endostatin: potent inhibition of tumor growth, safety and pharmacokinetic profile. Angiogenesis 1999;3:41-5.
6 Yamaguchi N, Anand-Apte B, Lee M, Sasaki T, Fukai N, Shapiro R, et al. Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding. EMBO J 1999;16:4414-23.
7 Montie JE, Wood DP, Pontes E, Boyett JM, Levin HS. Adenocarcinoma of the prostate in cytoprostatectomy specimens removed for bladder cancer. Cancer 1989;63:381-5.[Medline]
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