Gastricsin and Cathepsin D in Normal and Hypertrophic Human Prostates

0022-5347 /82/1275-1027$02.00/0 Vol.127, May Printed in U.S.A.

THE JOURNAL OF UROLOGY

Copyright© 1982 by The Williams & Wilkins Co.

GASTRICSIN AND CATHEPSIN DIN NORMAL AND HYPERTROPHIC HUMAN PROSTATES PETER H. WARD,* LEDA CONTRERAS, MAFALDA MALDONADO, HERNAN BAEZA AND LUCIANO CHIANG From the Universidad de Concepcion, Facultad de Ciencias Biologicas y de Recursos Naturales, Departamento de Ciencias Fisiologicas, Concepcion, Chile

ABSTRACT

The relative contents of gastricsinogen, the inactive zymogen precursor of gastric gastricsin (EC 3.4.23.3), and cathepsin D (EC 3.4.23.5) in normal and benign hyperplasia of the prostate gland have been determined. Gastricsinogen levels are significantly lower (0.116 ± 0.02 U/gm. wet tissue) in the hyperplastic than in normal prostates (0.65 ± 0.06 U/gm.). Conversely, cathepsin D leves are higher in the diseased (0.705 ± 0.17 U/gm.) as opposed to normal prostatic tissue (0.39 ± 0.12 U/gm.). The average gastricsin-cathepsin D differences between the 2 tissues (0.26 ± 0.025 for normal prostates and -0.59 ± 0.057 SEM for hyperplastic tissue) are also significantly different (p < 0.001). It is suggested that the simple determination of these 2 acid proteinases in prostate homogenates could be used as alternative and complementary marker enzymes for the study of the physiopathologic status of the prostate gland. Lundquist and Seedorf1 reported the existence of a pepsinogen-like zymogen in human seminal fluid which was subsequently further purified and partially characterized by Ruenwongsa and Chulavatnatol.2 The presence of immunologically unrelated groups of pepsinogens in human gastric mucosa, 3- 6 which on acid activation gives rise to pepsins and gastricsins, 7•8 has been well established. Samloff and coworkers4 ' 9- 14 have classified these gastric mucosa! zymogens into 2 immunologically unrelated groups, namely, the pepsinogen I and the pepsinogen II group of zymogens. Each group can, in turn, be electrophoretically resolved into 5 distinct potentially active protein bands. 4 ' 13• 14 Group I pepsinogens give rise to pepsins and the group II proteins to gastricsins. 8 Both types of zymogens can be detected in blood serum, 12 but only group I pepsinogens can be found in human urine. 9 On the other hand, only group II zymogens are found in human seminal plasma. 2 ' 11 Recently, we reported that a gastricsinogen-like zymogen could be separated from cathepsin D in human prostates 15 and developed a method16 for assaying both enzymes in prostate homogenates using hemoglobin as substrate. The similarities between prostate gastricsinogen and the seminal zymogen suggests that the latter could be originated in this gland. In order to test this possibility and to evaluate the importance of the gastricsin-like enzyme as a possible new marker system for the study of the functional state of the prostate, which might complement the current use of prostatic acid phosphatase, 11-20 we measured and partially purified cathepsin D and gastricsin activities in homogenates from both normal and benign hypertrophic human prostates. MATERIALS AND METHODS

Normal human prostates (average weight: 13.6 gm.) were removed at autopsy from not more than 12-hour postmortem corpses and were kept frozen at -40C until used. In all cases the cause of death was sudden. Benign hypertrophic prostatic tissue was obtained from the Department of Urology of the Accepted for publication October 28, 1981. Supported by a grant from the Vicerrectoria de Investigaci6n of the University of Concepcion, Chile. * Requests for reprints: Universidad de Concepcion, Facultad de Ciencias Biologicas y de Recursos N aturales, Departamento de Ciencias Fisiologicas, Casilla 2407, Concepcion, Chile.

Faculty of Medicine from patients undergoing corrective surgery of their prostates. Homogenization. Both tissues were separately homogenized at full speed for 2 minutes in a Waring Blender with 9 times their weight of ice cold 0.05-M Tris-HCl buffer, pH 7.2. The homogenates were centrifuged at 20,000 x g for 30 minutes and the clear supernatants were dialyzed against distilled water before assaying for proteolytic activity. Proteolytic activity. The proteolytic activity of prostate gastricsin and cathepsin D were determined by a modification of the procedure of Anson and Mirsky21 as previously described. 6 • The 2 activities can be independently determined when present in mixtures since gastricsin, but not cathepsin D, is active at pH 1.0 and both are active at pH 3.0. 16 The pH 1.0 and 3.0 incubation mixtures, containing 0.3 ml. of supernatant, were taken to 1.0 ml. by the addition of 0. 7 ml. of 0.2 M citric acidHCl pH 1.0 and 0.2 M citrate buffer, pH 3.0, respectively. The reactions were started by the addition of 1.0 ml. of a 2.5 per cent hemoglobin solution (Sigma Chemical Company, St. Louis, Missouri) adjusted to the same pH values with 0.1 M HCl. After a 90-minute incubation period at 37C the reactions were stopped by the addition of 3.0 ml. of 10 per cent trichloroacetic acid (TCA) and the absorbance at 280 nm. of the TCA filtrate was taken as a measure of proteolysis. Enzyme units are defined as the amount of gastricsin or cathepsin D capable of eliciting and absorbancy change of 1.0 in 10 minutes at 37C. Determination of protein concentration. Protein was determined according to the procedure of Lowry and associates, 22 with bovine serum albumin as standard. Ion exchange chromatography. Prostate homogenate supernatants, previously equilibrated by dialysis against 0.05-M TrisHCl buffer, pH 7.2, were absorbed onto a 40 X 3 cm. DEAESephadex column equilibrated with the same buffer. After the non-retained protein, containing the catheptic activity, had been washed out with the equilibrating buffer, the gastricsinlike activity was eluted by 0.75 M NaCl in the same buffer. Individual fractions were read at 280 nm. for protein and assayed at pH 1.0 and 3.0 for proteolytic activity as indicated above. Statistical analysis. Results are reported as the mean ± SEM. An initial F test was performed to determine the validity of using Student's t test to compare the results between normal and hypertrophic tissues. The F tests showed that the differences (G - C) and gastricsin contents (G) between the 2 tissues

1027

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WARD AND ASSOCIATES

had variances which were significantly different from each other. Consequently, in these 2 cases, the Wilcoxon rank sum test for 2 independent non-parametric samples was used. The cathepsin D contents of the 2 tissues showed an equality of variances and Student's t test was applied. The levels of significance were computed on the basis of a 2-tailed analysis of the results. RESULTS

Figure 1 shows the pH optimum profiles from both normal and hypertrophic human prostate homogenates. It can be seen clearly that the latter shows very little or no pH 1.0 activity and a pH optimum of 3.6 which resembles that of cathepsin D. 23 On the other hand, normal prostatic tissue homogenates are active at pH 1.0 and present a maximum at pH 3.0 due to the greater proportion of the gastricsin-like enzyme in normal

0.6

0.5

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tissue. Both gastric juice gastricsin8 and seminal fluid acid proteinase 15 exibit pH optima of 3.0. In tables 1 and 2, the pH 1.0 and 3.0 activities of normal and hypertrophic prostate homogenates are shown, as well as their gastricsin and cathepsin D contents. The average gastricsin content of normal tissue is 0.65 ± 0.06 U/gm. wet tissue and that of hyperplastic tissue is 0.116 ± 0.02 U/gm. A Wilcoxon rank sum test shows that the 2 are significantly different (p < 0.01). Cathepsin D levels in the 2 tissues were 0.39 ± 0.12 and 0.705 ± 0.17 U/gm., respectively. A Student's ttest shows that the 2 levels are highly different (p < 0.001). The average gastricsin-cathepsin D differences (G- C) between the 2 tissues are also significantly different (p < 0.01, Wilcoxon test). Figure 2 shows the chromatographic elution profiles obtained from a DEAE-Sephadex A50 column with homogenates from a randomly obtained normal and hypertrophic prostate. In both cases, catheptic activity is associated with the non-retained protein portion of the homogenates as indicated by the pH 3.0, but lack of pH 1.0 proteolytic activity. The gastricsin-like activity can be clearly observed in the normal prostate homogenate after elution from the column by 0.75 M NaCl, but not in the hypertrophic tissue where there is essentially no pH 1.0 activity, i.e., gastricsin-like activity. This latter tissue also shows very little pH 3.0 activity, which is a reflection of the sum of the activities of the 2 enzymes present in this tissue, but which in hypertrophic prostates is mainly composed of cathepsin D.

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0.2

0.1

1.0

2.0

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3.0

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FIG. 1. pH optimum profiles of normal (--------) and hypertrophic (e-----e) human prostate homogenates. TABLE

We have shown previously that normal prostate homogenates contain 2 acid proteinase activities which can be separated and purified by ion-exchange and affinity chromatography. 15 The 2 activities correspond to lysosomal cathepsin D and to a gastricsin-like zymogen (fig. 2, A). The latter is apparently secreted by the prostate gland since its presence can be detected in both human and dog prostatic juice (unpublished observations) and in seminal plasma. Figure 2, B clearly shows that in patients with benign prostatic hypertrophy, the gastricsin-like enzyme is greatly diminished as shown by the lack of the pH 1.0 activity profile. The pH optimum profile or' normal and hyperplastic prostate

1. Gastricsin and cathepsin D contents of normal human prostates Calculated*

Experimental A A2so

nm

Gastricsin

Cathepsin D

Prostate No.

Age (yrs.)

Protein (mg./ml.)

pH LO

pH 3.0

A A2Bo

U/g.

A A2so

U/g.

1 2 3 4 5 6 7

42 37 47

3.7 4.2 4.2 7.8 5.8 4.8 3.5

0.150 0.150 0.200 0.160 0.160 0.100 0.225

0.275 0.275 0.395 0.300 0.305 0.205 0.400

0.179 0.179 0.238 0.190 0.190 0.119 0.268

0.59 0.59 0.79 0.63 0.63 0.40 0.89

0.096 0.076 0.157 0.110 0.115 0.086 0.172

0.32 0.25 0.52 0.37 0.38 0.29 0.58

G-ct 0.27 0.34 0.27 0.26 0.25 0.11 0.31

* According to reference 16. t Symbols used: G: gastricsin., C: cathepsin D. TABLE 2.

Gastricsin and cathepsin D contents of human benign hypertrophic prostates Calculated*

Experimental Prostate No.

Age (yrs.)

Protein (mg./ml.)

1 2 3 4 5 6 7 8 9 10

73 72 53 54

2.4 2.8 3.8 2.3 3.5 2.2 7.2 3.5 5.5 2.9

64 69 66 62

M2so

nM

Gastricsin

Cathepsin D G-Ct

pH LO

pH3.0

AA2so

U/g.

M2so

U/g.

0.015 0.010 0.040 0.025 0.020 0.040 0.040 0.020 0.065 0.014

0.255 0.240 0.195 0.250 0.210 0.200 0.320 0.160 0.350 0.285

O.Dl8 0.012 0.048 0.030 0.024 0.048 0.048 0.024 0.077 0.017

0.06 0.04 0.16 0.10 0.08 0.16 0.16 0.08 0.26 0.06

0.237 0.228 0.147 0.220 0.186 0.152 0.272 0.136 0.273 0.268

0.78 0.76 0.49 0.73 0.62 0.51 0.91 0.45 0.91 0.89

• According to reference 16. t Symbols used: G: gastricsin., C: cathepsin D.

-0.72 -0.72 -0.33 -0.63 -0.54 -0.35 -0.75 -0.37 -0.65 -0.83

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ACID PROTEINASES IN HUMAN PROSTATES

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60

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after 7 days of castration and that the process can be reserved by the administration of testosterone, thus showing, that this enzyme is also androgen dependent and, since we have detected its presence in normal human prostatic juice and seminal plasma, we feel that the possibility of its use as an alternative marker enzyme for the study of the physiopathologic status of the prostate gland merits further study. Acid phosphatase levels do not diagnose prostatic hyperplasia since its levels in serum do not normally change in this disease. 28· 29 Our present study clearly shows that both gastricsin and cathepsin D contents are dramatically altered in the benign E hyperplastic prostate and the determination of these two proC ~ teinase activities in this disease might well serve as a differential diagnostic tool.

. C

B

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u

!

e

Q.

0.8

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06

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20

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100

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140

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FIG. 2. DEAE-Sephadex A50 chromatography of a normal (A) and a hypertrophic (B) prostate homogenate. Fraction size: 5 ml. protein (0---0)., pH LO activity (D---0)., pH 3.0 activity (x--x).

homogenates (fig. 1) again show that there is very little pH 1.0 activity in the diseased organ and that the pH optimum of this tissue is displaced towards more alkaline values, reflecting the higher relative content of cathepsin D in this tissue. The secretory activity of the prostate and other male accessory organs of reproduction differ from that of other organs in that they are androgen dependent. 24. The importance of androgens in the development, growth and function of the prostate is now well recognized and it can be shown that after castration, the gland undergoes a process of involution which can be reversed by the administration of testosterone. 25 • 26 One of the stricking features of human prostatic secretion is its high content of citric acid and prostatic acid phosphatase. 24 The former decreases slightly in prostates affected by benign hypertrophy or adenocarcinoma, 27 whereas the latter seems to be unaffected. 28 ' 29 Human 17• 30 and rat ventral prostates25 ' 26 ' 31 contain a tartrate inhibitable lysosomal acid phosphatase as well as an immunologically distinct and non-inhibitable secretory acid phosphatase. The latter can be detected in both serum and seminal plasma and is androgen dependent as demonstrated by its dissapearence after castration and reappearence after androgen replacement therapy. 15' 26 Serum prostatic acid phosphatase levels have been used widely in the diagnosis of prostatic cancer and highly sensitive and specific radioimmunoassay systems have been developed for its quantitative assay. 20• 32• 33 Unfortunately, these methods, as well as the classical enzymatic procedures34• 35 do not diagnose all histologically verified prostatic cancers20 • 32• 33• 36• 37 and a fairly high incidence of false positive results are obtained in otherwise normal patients as well as in patients with prostates affected by benign hypertrophy in which acid phosphatase levels are not normally affected. 28 • 29 Furthermore, these methods are not specific for prostate carcinomas, since other tissues, especially leukocytes 17' 37 also contain this enzyme and high prostate specific acid phosphatase levels are detected in several types of leukemia. 17' 35' 36 Preliminary experiments in our laboratory38 show that the gastricsin-like levels in rat ventral prostates fall off dramatically

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30.

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Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 261, 1951. Barrett, A. J.: Proteinases in Mammalian Cells and Tissues. Amsterdam: North Holland Publishing Co., p. 209, 1977. Mann, T.: Secretory function of the prostate, seminal vesicles and other male accesory organs or reproduction. J. Reprod. Fertil., 37: 179, 1974. Tenniswood, M. D., Abrahams, P. P., Bird, C. E. and Clark, A. F.: Effects of castration and androgen replacement on acid phosphatase activity in the adult rat prostate gland. J. Endocrinol., 77: 301, 1978. Tenniswood, M. P., Abrahams, P. P., Bird, C. E. and Clark, A. F.: Effect of 5a-androstane-3/J, 17/J-diol and 5/J-dihydrotestosterone on acid phosphatase activity in the prostate gland of the castrated adult rat. J. Endocrinol., 79: 9, 1978. Marberger, H., Marberger, E., Mann, T. and Lutwar-Mann, C.: Citric acid in human prostatic secretion and metastasizing cancer of the prostate gland. Br. Med. J., 1: 835, 1962. Vihko, P., Kostama, A., Jiinne, 0., Sajanti, E. and Vihko, R.: Rapid radioimmunoassay for prostate-specific acid phosphatase in human serum. Clin. Cham., 26: 1544, 1980. Vihko, P., Lukkarinen, 0., Kontturi, M. and Vihko, R. The effect of manipulation of the prostate gland on serum prostate-specific acid phosphatase measureal by radioimmunoassay. Invest. Urol., 18:334, 1981. Vihko, P.: Human postatic acid phosphatases: purification of a minor enzyme and comparison of the enzymes. Invest. Urol., 16:

349, 1979. 31. Vanha-Perttula, T., Niemi, R. and Helminen, H.J.: Separate lysosomal and secretory acid phosphatases in the rat ventral prostate. Invest. Urol., 9: 345, 1972. 32. Cooper, J. F., Foti, A., Herschmann. and Finkle, W.: A solid phase radioimmunoassay for prostatic acid phosphatase. J. Urol., 119: 388, 1978. 33. Foti, A. G., Cooper, J. F., Herschman, H. and Malvaez, R. R.: Detection of prostatic cancer by solid-phase radioimmuno-assay of serum prostatic acid phosphatase. N. Engl. J. Med., 297: 1357, 1977. 34. Roy, A. V., Brower, M. E. and Hayden, J. E.: Sodium thymolphthalein monophosphate: a new acid phosphatase substrate with greater specificity for the prostate enzyme in serum. Clin. Chem., 17: 1093, 1971. 35. Li, C. Y., Chuda, R. A., Lam, W. K. W. and Yam, L. T.: Acid phosphatases in human plasma. J. Lab. Clin. Med., 82: 1285, 1979. 36. Chu, T. M., Wang, M. C., Kuciel, R., Valenzuela, L. and Murphy, G. P.: Enzyme markers in human prostatic carcinoma. Cancer Treat. Rep., 61: 193, 1977. 37. Li, C. Y., Yam, L. T. and Yam, L. T.: Studies of acid phosphatase isoenzymes in human leukocytes. J. Histochem. Cytochem., 18: 901, 1970. 38. Maldonado, M., Vivaldi, E., Ward, P.H. and Chiang, L.: Catepsina D y gastricsina en prostata de rata. Arch. Biol. Med. Exp; 13: 117, 1980.