Oncology 34 : 123-128 (1977)
5a-Reductase as a Target Enzyme for Anti-Prostatic Drugs in Organ Culture N. Kadohama, R. Y. Kirdani, G. P. Murphy, and A. A. Sandberg Roswell Park Memorial Institute, Buffalo, New York
Supported in part by a grant (CA-15436) from the National Cancer Institute
Key Words. Organ culture - Prostate 5a-reductase - Effects of drugs
Introduction
Since the 5a-reduction product of testosterone (T), dihydro testosterone (DHT), appears to be crucial for prostatic inte grity and function [1, 22, 24], a model system based on 5a-reductase enzyme activity (5a-RA) was developed in our laboratory for testing the action of chemotherapeutic agents potentially useful in prostatic cancer [8, 19, 21, 25]. This approach involved in vivo administration of the drugs to test animals and subsequent in vitro assay of the enzyme activity in prostatic homogenates. To complement and extend these studies, the present report deals with some very encouraging initial results obtained with a completely in vitro test system, i.e., short-term organ culture, whereby a more direct measure of the interaction of the drugs with isolated target tissue in chemically defined media is made possible and is applicable to the testing of drugs directly on human cancerous prostate, thus offering an approach which is not afforded by the few animals models available or other testing systems.
Materials and Methods
Adult Wistar rats (400-450 g) obtained from Charles River Laboratories, Wilmington, Mass, were used in some experi ments. Sexually immature rats of Fischer 344 strain were obtained from A.R.S./Sprague Dawley, Madison, Wis. and were used at the age of 27 days. In some experiments, orchi ectomy was performed via the scrotal route. Human tissues (BPH and carcinoma) were used as soon as possible after surgery. The methodology for organ culture was basically similar to that previously described by others [18, 20]. For the prepara tion of rat tissue, the ventral and dorsolateral lobes of the prostate were teased apart into 2 mm3 fragments and placed on siliconized lens paper. It was necessary to use fine scissors
to prepare explants from human prostate specimens. Culture medium (7.5 ml per dish) was Medium 199 and contained 35 pg/ml penicillin and 50 ug/ml streptomycin sulfate. No serum was present in the medium. Plastic disposable petri dishes, 60x15 mm, were products of Falcon Plastics, Inc. The explants were usually incubated for 24 hr. in hormone less medium and transferred to hormone-supplemented media for various lengths of time, (usually 2 days). The cultures were maintained at 37 ° C in an atmosphere of 95 % air and 5 % carbon dioxide. T (purchased from Schwartz/Mann) con centration was usually 0.5 pM, unless otherwise stated. Insulin (E. R. Squibb and Sons, New York, N.Y.) and cortisol (The Upjohn Co., Kalamazoo, Mich.) were used at a concentration of 0.08 units/ml and 0.5 uM, respectively. Media were changed every 2 days. Estradiol-17(3 (E 2 ) was purchased from Steraloids, Inc., Rawling, N.Y. and progesterone from Organon, West Orange, N.J. Estracyt was obtained from AB Leo, Helsingborg, Swe den, Compounds A and B from Dr. C. H. Robinson of the Johns Hopkins School of Medicine, and the Ea-flurodeoxyuridine complex from Dr. A. Bloch of our Institute. E2-4-14C (specific activity = 52 mCi/mMole) were purchased from the New England Nuclear Corp., as were T-4-14C specific activity = 57.5 mCi/mMole), and T-1,2,6,7-3H(N) (specific activity = 85 pCi/mMole). For 5a-RA measurement in organ culture, the dishes were pulsed for 2 h with medium containing 3H-T at a concentra tion of 1.2 nM. Explants from each set of dishes were com bined, thoroughly washed free of radioactive medium, weighed and processed for 5a-RA; the latter was determined according to the method of Shimazaki el al. [22] as modified by Kirdani et al. [8], The procedure was further modified by (a) scaling down the assay to handle 20-30 mg of tissue and (b) accounting for the formation of 5a-androstane-3a, 17(3diol metabolized from DHT. Since androstanediol was found to partially overlap with T in the initial paper chromatographic separation (in 34% benzene: 66% heptane/80% methanol in water) of the ethanol extract of the tissue homogenates, separation was achieved by acetylation of this mixture and re-chromatography in heptane/80 % methanol in water. Thus, after extraction of the steroids from the homogenized tissue, the 4 major metabolites ultimately separated and quantitated were: 1) unmetabolized T, 2) DHT, 3) 5a-androstane-3a,17(3-diol (A-diol) and 4) 4-androstene-3,17-dione (A-dione).
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Abstract. Short-term organ culture of rat and human prostatic tissues has been utilized as a means of testing drugs potentially useful in cancer of the prostate. Optimal conditions, particularly the concen tration of T, have been established for organ culture of such tissues and the effects of various drugs on 5a-reductase activity (5a-RA) have been utilized as a means of ascertaining antiprostatic actions of the various compounds and drugs tested. Thus, it was shown that estracyt, estradiol-17(5, progesterone, and novel steroids and steroidconjugates have definite effects on 5a-RA in this system.
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Figure I. Representative sections of explants of rat ventral prostate after organ culture. (A-B), from normal, adult rat, maintained for 6 days in control medium (A) or medium supplemented with 0.5 pM T (B); (C-D), from 7-day castrated animal, maintained for 6 days in control medium (C) or medium containing 0.5 iiM T (D); (E-F), from 27 day old rat, maintained in control medium for 3 days (E) or in control medium for 1 day, then in 0.5 uM T-supplemented medium for 2 days (F). Hematoxylin and eosin strain, x 500.
For systems containing T (unlabelled) in the growth medium, correction was made with respect to specific activity in calcu lating total 5a-reduction of T.
Results
5a-/?/4 in Organ Culture The response of prostatic cells to androgens in organ culture was verified by histological examination (figure I). In explants derived from either normal adult rats or those castrated for 7 days or young prepubertal rats, the presence of T in the medium at a concentration of 0.5 pM (144 ng/mg) resulted in the generation and preservation of prominent alveolar cells
surrounding the gland lumina. Explants from normal, intact rats and 7-day castrates responded to 0.08 units/ml insulin (not shown) almost as well as to T. Cells of the immature prostate, however, did not respond to insulin. The effect of varying the concentration of T in the medium on 5a-RA is shown in table 1. The concentration of T chosen for use in subsequent experiments was 0.5 pM, as it repre sented near optimal efficiency of conversion of T to DHT and, morphologically, yielded healthy luminar epithelium. In contrast, T at a concentration of 50 pM proved deleterious to cell structure. In figure 2 is shown the rate of DHT, A-diol and A-dione
Testosterone concen (ration
pMoles DHT/30 mg tissuc/2 h 1
0 5x10"14 Molar 5x 1 0-" 5 x 10"9 1x 10-8 5 x 10-" 1 x 10-7 5 x10-7 1 x lO-6 5x10-« lx lO -5 5 x l0 -5
0.09 0.08 0.11 0.44 0.78 4.04 10.08 40.00 85.83 304.10 433.30 666.60
'A fter 2 hr exposure to 1.2xlO_9M 3H-1,2,6,7-Testosterone and correction for specific activity.
Figure 2. Rate of metabolism of testosterone during varying periods of exposure to 3H-T. Explants were prepared from rat ventral pros tate. After 1 day in control medium, they were maintained in medium supplemented with 0.5 uM T for 2 days. On the 3rd day of organ culture, T-medium was replaced with new one also containing 3Hlabelled T for the duration of times indicated. Determination of formation of DHT (A), DHT plus A-diol (O) and A-dione (□) was performed as described in "Methods” . The values are averages of duplicate determinations.
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Table I. Effect of T-concentration in maintenance medium on 5 a reductase activity.
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Kadohama et al.: Organ Culture and 5a-RA
0
0.2pg/ml 0.5pg/ml
02pg/mt 0.5pg/ml
Figure 3. 5a-RA in normal and partially regressed rat ventral prostate in organ culture. Explants derived from normal and 4-day castrated rats were maintained in organ culture for 4 days in media supple mented with 0.5 aM T. 5a-RA determinations were performed on the 4th day as described in ’’Methods”. For comparison, in vivo 5a-RA values in normal and 4-day castrates were determined, using homogenates of freshly excised tissues. The values are averages of duplicate determinations.
Figure 5. Interaction of estradiol with rat prostate. Explants were prepared from ventral and dorsolateral lobes. After 1 day in control medium, they were exposed for 2 days to media containing l4Cestradiol-17(5. On the final day, 3H-testosterone was added to the dishes for the determination of 5a-RA. v, ventral; d, dorsolateral; T, testosterone; E 2, estradiol.
production by the rat ventral prostate in organ culture. This rate, especially that of DHT, is linear under the conditions and times employed in the experiments conducted. Explants derived from normal rats and four-day castrates were maintained in hormone-less and T-supplemented media, and 5a-RA measured (figure 3). The homogenate assays provided a measure of in vivo activity. Exposure of explants from four-day castrates to four days of T in culture resulted in the stimulation of enzyme activity beyond control (intact ani mals) value. In comparison to the homogenate assay, in organ culture a greater percentage of the product, DHT, was further metabolized to 5a-androstane-3a, 17(5-diol. On the other hand, the formation of 4-androstene-3,17-dione was less in organ culture than in the homogenate assay.
Effect of Drugs on 5a-RA In the initial studies, the effects of estrogen and progesterone on 5a-RA in the rat prostate were studied (figure 4). The results indicate that both E 2 and progesterone were stimu latory in the explants derived from the ventral lobe and strongly inhibitory in the dorsolateral tissue. In a follow-up experiment, the interaction of E 2 with rat prostate was further investigated (figure 5). The explants were exposed to 3H-E 2 for 2 days. At the concentrations used (0.2 pg/ml and 0.5 pg/ml), the estrogen had no effect on the ventral lobe 5a-RA. On the other hand, enzyme activity in the dorsolateral prostate was again markedly inhibited. The amount of uptake of 3H-E 2 roughly correlated with the degree of inhibition of 5«-RA.
Figure 4. Effect of estradiol-173 and progesterone on 5a-RA in rat prostate. Explants were prepared from ventral (v) and dorsolateral (d) lobes of rat prostates. After 1 day in control medium, they were ex posed for 6 days to steroid-containing media, with media change every 2 days. 5a-RA determinations were performed as described in ’’Methods” . T, testosterone; E 2, estradiol; P.progesterone.
Figure 6. Effect of estracyt on tes:osterone metabolism by rat prostate in organ culture. After 1 day in control medium, explants from ventral (v) and dorsolateral (d) lobes were exposed for 2 days to media containing 1 pM T plus 0.5 and 50 pg/ml estracyt. On the final day, the culture dishes were pulsed with media containing 1.2 nM 3H-T. Each bar represents mean value ± standard error for 3 deter minations.
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Figure 7. Effect of estracyt on 5a-RA in human prostatic cancer in organ culture. Results of 2 separate experiments are shown. Expiants were prepared from carcinomas of the prostate. After 1 day in control medium, the expiants were exposed for 4 days to T or T plus estracyt. 5a-RA measurements were done on the final day. Solid bar = for mation of DHT plus A-diol; Open bar = formation of androstenedione. The values are averages of duplicate determinations.
The effect of estracyt on rat ventral prostate 5a-RA was investigated (figure 6). There was 25 % inhibition of enzyme activity at both concentrations of the drug. Studies of the effect of estracyt on 5a-RA in cultured human prostatic tissue gave variable results. Figure 7 shows the results of 2 such experiments. In one case, 5a-reduction of T was inhibited by 50% with 10 pg/ml of estracyt. In another sample, no effect was observed. The 5a-RA assay in organ culture was further used to assess the properties of some novel steroidal compounds with cyto static properties. Figures 8 and 9 show the effect of 2 such steroids on prostatic tissue from 2 different specimens. In one experiment (figure 8), Compound A stimulated 50-reduc tion of T, whereas Compound B was slightly inhibitory. In
Figure 9. Effect of compounds A and B and E 2FUDR complex on testosterone metabolism by human BPH explants. Explants were prepared from specimens of benign prostatic hypertrophy. After 1 day in control medium, the explants were maintained for 2 days in the presence of T or T plus drugs. 5a-RA determinations were done on the final day. All systems contained 0.5 11M T. 1) no drugs; 2) 1 pm/ml compound A; 3) 0.1 pg/ml E 2-FUDR; 4) 10 gg/ml E 2FUDR. Hatched bar = formation of DHT + A-diol; open bar = formation of androstenedione. Each bar represents mean value ± standard error for 3 determinations.
another experiment using explants derived from a different human prostatic specimen (figure 9), both Compound A and Compound B were inhibitory of 5a-RA. On the other hand, both drugs stimulated (greater than 2-fold by Compound A and about 36% over control value by Compound B) the metabolism of T via the oxidative pathway, as reflected by increase in the formation of A4-androstenedione. Histologi cally, sections prepared from explants cultured in T or T plus Compound A (not shown) displayed hyperplasia of the epi thelial components, whereas degeneration of glandular acini characterized Compound B tested tissue. In the same experi ment (figure 9), 5a-reduction of T was also strongly inhibited by a third steroidal-cytostatic complex, composed of estradiol and fluorodeoxyuridine (at both concentrations of 0.1 pg/ml and 10 pg/ml). Again, the effect on oxidative metabolism was non-inhibitory at a high concentration of the drug (10 pg/ml) and slightly stimulatory at the lower concentra tion (0.1 tig/ml).
Figure 8. Effect of compounds A and B on 5a-RA in human prostate. Explants were prepared from specimens of benign prostatic hyper trophy. After 1 day in control medium, the explants were main tained for 2 days in media containing T or T plus compound A or B. 5a-RA determinations were done on the final day. Solid bars repre sent 5a-reduction products (DHT + A-diol); Open bars represent formation of androstenedione. The values are averages of duplicate determinations.
In view of the central role played by 5a-reduction of T in the regulation of prostatic growth and physiology [1, 22, 24], a model system for testing drugs against cancer of the prostate, based on 5a-RA, has been developed and previously reported [8, 19, 21, 25], This approach has been extended with the use of organ culture to an in vitro system, providing a more sensi tive and direct assay of the effects of agents on the target tissue, particularly human prostatic cancer. Short-term organ culture, as presently reported, provides a rapid and flexible tool for studying changes in biochemical parameters under various experimental conditions, while still maintaining nor mal morphological features of the intact tissue. It, thus, fulfills
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Discussion
Kadohama et al.: Organ Culture and 5a-RA
plication of organ culture becomes obvious, since the system exposes isolated target tissues directly to test drugs. Compared to rat prostate, human prostatic tissue is less amenable to organ culture. Nevertheless, short-term culture of human prostate has been successfully achieved [6, 13, 14, 16, 23], Specimens of benign hyperplasia and carcinoma are invariably lacking in cellular homogeneity, contributed by varying degrees of differentiation, atrophy, proliferative acti vity and relative content of glandular and stromal elements. Moreover, these tissues often show differences in responsive ness to hormones and drugs. As a result, the use of human prostate involves great risk in variability in results between specimens and even in replicate cultures of explants from the same specimen. Furthermore, since the level of 5a-RA may vary considerably from one cancer of the prostate to another [17], the results obtained with such tissues in organ culture may reflect this variability. However, there is little doubt that a significant proportion of cancers of the prostate does have 5a-RA and. hence, the system utilized in the present study should have relevance to the planning of drugs potentially useful in some cases of cancer of the prostate. Results obtained in the present studies illustrate the wide range of effects produced by steroidal agents on 5a-reduction of T in human prostate. Whereas estracyt consistently caused inhibition of 5a-reduction of T in rat dorsolateral prostate, its effect on 5a-RA in human prostatic tissue varied from specimen to specimen. Similar results were obtained with other steroid-cytostatic molecule complexes tested. These discrepencies in results between specimens may be accounted for by differences in advancement of the disease and degree of sensitivity to hormonal action, as often observed in varia tions in response to estrogen therapy. Whereas these results point to some limitations in the use of 5a-RA as the sole index of drug action, its measurement in conjunction with other biochemical and morphological parameters should aid in the development of a rational chemotherapeutic program for abnormal prostatic growth. With regard to the action of E 2 and E 2 -cytostatic molecule complexes, the observed effects on 5cx-RA in organ culture cannot be attributed solely to a decrease in circulating andro gens via an effect on the hypothalamic-hypophyseal system. Direct interaction with isolated prostatic tissue and inhibition of 5a-reduction of testosterone were demonstrated. The action of E 2 on rat prostate was highly tissue specific, in that 5a-RA was inhibited only in tissue from the dorsolateral lobe. Similarly, inhibition of 5a-reduction of T by estracyt was confined mainly to the dorsolateral prostate. E 2 and the various estrogen derivatives tested had a non-inhibitory or often stimulatory effect on the metabolism of T via the oxi dative pathway, in both rat and human prostatic tissues. The significance of these observations in terms of anti-prostatic effects requires further study.
References 1
Baulieu , E. E.; Lasnitzki, I., and Robel , P.: Metabolism of
2
testosterone and action of metabolites on prostate glands grown in organ culture. Nature 219: 1155-1156 (1968). Chung, L. W. K. and Coffey, D. S.: Biochemical characteri
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the need for a model in vitro system for testing drugs against cancer of the prostate. Present studies involved the use of prostatic tissues from both rat and human sources for investi gating the effects of various steroids on 5a-RA. One of the purposes of the present studies was to establish reproducible and standardization conditions for prostatic 5a-RA and morphology in organ culture, though it is realized that other factors in addition to T may be necessary to achieve in vivo levels of activity and morphology [2, 11]. However, sufficient variability in 5a-RA was observed with various other factors (e.g., insulin, cortisol) to lead us to believe that an assay in the presence of only T was preferable for the pur pose of our studies. Morphology and 5a-RA were used as indi cators of T activity in organ culture, even though these reflect primarily a permissive action of T in regulating prostatic growth [4, 12, 20], Thus, the general histological features of the tissue were adequately maintained for at least 4 days, with absence of significant regressive changes. The 5a-RA was preserved during this time and progressively diminished in parallel with tissue degeneration thereafter (8 to 14 days). One mode of the anti-androgenic action of estrogens may be at the level of T metabolism in target tissues. The adminis tration of E2 , however, caused little or no inhibition of 5a-reduction of T in dog [9] and rat ventral prostates [3, 26], but did stimulate the formation of products of the oxidative path way relative to 5«-reduced metabolites. On the other hand, 5a-RA was inhibited in the dorsolateral prostate of E 2 -treated rats [26]. E2 and various derivatives of E 2 and estriol have been reported to inhibit 5a-reduction of T when assayed in homogenates of rat prostate [10], but it was not specified which lobe of the prostate was used. In organ culture of dog and rat ventral prostates, the presence of 17 pM E2 produced inhibition of T metabolism [5], In the present studies, the addition of 1.8 pM E2 to the growth medium containing 0.5 pM T resulted in a differential effect on 5a-RA, viz. there was no effect in explants derived from the ventral prostate, whereas 5a-reduction by the dorsolateral tissue was greatly inhibited. The dorsolateral lobe of the rat prostate displays unique sensitivity to estrogenic action in other respects not seen in the ventral lobe. Thus, estrogen induced uptake of 65Zn by the rat dorsolateral prostate in a highly tissue-specific manner [15], Zinc is a potent inhibitor of 5a-RA, at least in nuclei of rat ventral prostate [7], Whether this relationship of estrogens, zinc and 5a-reduction is signi ficant to the mechanism of the anti-androgen action of estro gens remains to be determined. Synthetically modified steroids and steroid-cytostatic com plexes are being increasingly utilized in chemotherapy. With respect to cancer of the prostate, one such compound is estracyt. It has been shown that one of the properties in its anti-androgenic action is an influence on 5 a-R A [7, 8, 19]. In vivo administration of estracyt to dogs and rats resulted in diminished 5a-RA of their prostates [8], H isaeter [7] reported no effect by estracyt on the rat ventral prostate after in vitro incubation or short-term (half-hour) in vivo adminis tration of the drug. Marked inhibitory effect was observed only after long-term (3 days) administration of estracyt. These results point out the difficulty in assessing effects of drug action in vivo, due possibly to the metabolic transformation that a given agent undergoes in the body. The practical ap
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and G riffiths, K.: The effect of oestrogen on the prostatic metabolism of testosterone in tissue culture. Biochem. J. 122: 125-126(1971). H arbitz , T. B.: Organ culture of benign nodular hyperplasia of human prostate in chemically-defined medium. Scand. J. Urol Nephrol 7:6-13(1973). H isaeter , P. A.: The effect of ocstradiol-3N-bis-(2 chloroethyl)carbamate-17|3-phosphate (Estracyt) on the 5a-reductase in the rat ventral prostate. Acta Endocrinol. 80: 188-198 (1975). Kirdani, R. Y.; MOntzing, J.; Varkarakis, M. J.; Murphy, G. P., and Sandberg . A. A.: Studies on the antiprostatic action of estracyt, a nitrogen mustard of estradiol. Cancer Res. 34: 1031-1037(1974). Leav , I.; Morphin , R. F.; O fner, P.; Cavazos, J. F., and Leeds . E. B.: Estrogen and castration-induced effects on canine prostatic fine structure and Ci9-steroid metabolism. Endo crinol. 89:465^183(1971). Lee , D. K. H.; B ird , C. E., and Clark , A. F.: In vitro effects of estrogens on rat prostatic 5a-reduction of testosterone. Steroids 22:677-685 (1973). L esser, B. and B ruchovsky, N.: The effects of testosterone, 5a-dihydrotestosterone and adenosine 3’,5’-monophosphate on cell proliferation and differentiation in rat prostate. Biochim. biophys. Acta 308:426-437 (1973). Lostroh , A. J.: Effect of testosterone and insulin in vitro on maintenance and repair of the secretory epithelium of the mouse prostate. Endocrinol. 88:500-503 (1971). Mc Mahon , M. J. and T homas, G. H.: Morphological changes of benign prostatic hyperplasia in culture. Br. J. Cancer 27: 323-335(1973). Mc Ra e , C. U.; G hanadian, R.; Fotherby . K.. and C hisholm, G. D.: The effect of testosterone on the human prostate in organ culture. Br. J. Urol. 45 : 156-162 (1973). MOntzing, J.; Kirdani, R. Y.; M urphy , G. P., and Sandberg , A. A.: Hormonal control of zinc uptake and binding in the rat dorsolateral prostate (submitted for publication).
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human prostate in organ culture. Cancer Chemother. Rep., Pt. 1,59:67-71 (1975). P rout, G. R. jr. ; Kliman, B.; D aly , J. J.; Maclauchlin ,R . A., and G riffin, P. P.: In vitro uptake of 3H testosterone and its conversion to dihydrotestosterone by prostatic carcinoma and other tissues. J. Urol. 776:603-610(1976). Robel , P.; Lasnitzki, I., and Baulieu . E. E.: Hormone me tabolism and action: testosterone and metabolites in prostate organ culture. Biochimie 53. 81-96 ( 1971). Sandberg , A. A.; Kirdani, R. Y.; Yamanaka. H.; Varka rakis, M. J., and Murphy, G. P.: Potential test systems for drugs against prostatic cancer. Cancer Chemother. Rep., Pt. 1,
59: 175-184(1975). San iti . R. S. and Johansson, R.: Some biochemical effects of insulin and steroid hormones on the rat prostate in organ culture. Exp. Cell Res. 77: 111-120(1973). Saroff, J.; Yamanaka, H.; Kirdani, R. Y.; Sandberg , A. A., and Murphy , G. P.: in G oland Normal and Abnormal Growth of the Prostate, pp. 743-758 (Thomas, Springfield. III. 1975). Shimazaki, J.; Matsushita, I.; F uruya , N.; Yamanaka, H., and Shida , K.: Reduction of 5u-position of testosterone in the rat ventral prostate. Endocrinol. Japan. 76:453-458 (1969). Schrodt , G. R. and Foreman, C. D.: In Vitro maintenance of human hyperplastic prostate tissue. Invest. Urol. 6: 85-94 (1971). W ilson. J. D.: Recent studies on the mechanism of action of testosterone. N. Eng. J. Med. 287: 1284-1291 (1972). Yamanaka, H.; Kirdani, R. Y.; Saroff, J.; Murphy, G. P., and Sandberg , A. A.: Effects of testosterone and prolactin on on rat prostatic weight, 5a-reductase and arginase. Am. J. Physiol. 229: 1102-1109 (1975). Yamanaka, H.; Shimazaki, J.; Imai, K.; Sugiyama, Y., and Shida , K.: Effect of estrogen administration on activities of testosterone 5a-reductase, alkaline phosphatase and arginase in the rat ventral and dorsolateral prostates of rats. Endocrinol. Japan. 22:297-302 (1975).
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5a-Reductase as a Target Enzyme for Anti-Prostatic Drugs in Organ Culture N. Kadohama, R. Y. Kirdani, G. P. Murphy, and A. A. Sandberg Roswell Park Memorial Institute, Buffalo, New York
Supported in part by a grant (CA-15436) from the National Cancer Institute
Key Words. Organ culture - Prostate 5a-reductase - Effects of drugs
Introduction
Since the 5a-reduction product of testosterone (T), dihydro testosterone (DHT), appears to be crucial for prostatic inte grity and function [1, 22, 24], a model system based on 5a-reductase enzyme activity (5a-RA) was developed in our laboratory for testing the action of chemotherapeutic agents potentially useful in prostatic cancer [8, 19, 21, 25]. This approach involved in vivo administration of the drugs to test animals and subsequent in vitro assay of the enzyme activity in prostatic homogenates. To complement and extend these studies, the present report deals with some very encouraging initial results obtained with a completely in vitro test system, i.e., short-term organ culture, whereby a more direct measure of the interaction of the drugs with isolated target tissue in chemically defined media is made possible and is applicable to the testing of drugs directly on human cancerous prostate, thus offering an approach which is not afforded by the few animals models available or other testing systems.
Materials and Methods
Adult Wistar rats (400-450 g) obtained from Charles River Laboratories, Wilmington, Mass, were used in some experi ments. Sexually immature rats of Fischer 344 strain were obtained from A.R.S./Sprague Dawley, Madison, Wis. and were used at the age of 27 days. In some experiments, orchi ectomy was performed via the scrotal route. Human tissues (BPH and carcinoma) were used as soon as possible after surgery. The methodology for organ culture was basically similar to that previously described by others [18, 20]. For the prepara tion of rat tissue, the ventral and dorsolateral lobes of the prostate were teased apart into 2 mm3 fragments and placed on siliconized lens paper. It was necessary to use fine scissors
to prepare explants from human prostate specimens. Culture medium (7.5 ml per dish) was Medium 199 and contained 35 pg/ml penicillin and 50 ug/ml streptomycin sulfate. No serum was present in the medium. Plastic disposable petri dishes, 60x15 mm, were products of Falcon Plastics, Inc. The explants were usually incubated for 24 hr. in hormone less medium and transferred to hormone-supplemented media for various lengths of time, (usually 2 days). The cultures were maintained at 37 ° C in an atmosphere of 95 % air and 5 % carbon dioxide. T (purchased from Schwartz/Mann) con centration was usually 0.5 pM, unless otherwise stated. Insulin (E. R. Squibb and Sons, New York, N.Y.) and cortisol (The Upjohn Co., Kalamazoo, Mich.) were used at a concentration of 0.08 units/ml and 0.5 uM, respectively. Media were changed every 2 days. Estradiol-17(3 (E 2 ) was purchased from Steraloids, Inc., Rawling, N.Y. and progesterone from Organon, West Orange, N.J. Estracyt was obtained from AB Leo, Helsingborg, Swe den, Compounds A and B from Dr. C. H. Robinson of the Johns Hopkins School of Medicine, and the Ea-flurodeoxyuridine complex from Dr. A. Bloch of our Institute. E2-4-14C (specific activity = 52 mCi/mMole) were purchased from the New England Nuclear Corp., as were T-4-14C specific activity = 57.5 mCi/mMole), and T-1,2,6,7-3H(N) (specific activity = 85 pCi/mMole). For 5a-RA measurement in organ culture, the dishes were pulsed for 2 h with medium containing 3H-T at a concentra tion of 1.2 nM. Explants from each set of dishes were com bined, thoroughly washed free of radioactive medium, weighed and processed for 5a-RA; the latter was determined according to the method of Shimazaki el al. [22] as modified by Kirdani et al. [8], The procedure was further modified by (a) scaling down the assay to handle 20-30 mg of tissue and (b) accounting for the formation of 5a-androstane-3a, 17(3diol metabolized from DHT. Since androstanediol was found to partially overlap with T in the initial paper chromatographic separation (in 34% benzene: 66% heptane/80% methanol in water) of the ethanol extract of the tissue homogenates, separation was achieved by acetylation of this mixture and re-chromatography in heptane/80 % methanol in water. Thus, after extraction of the steroids from the homogenized tissue, the 4 major metabolites ultimately separated and quantitated were: 1) unmetabolized T, 2) DHT, 3) 5a-androstane-3a,17(3-diol (A-diol) and 4) 4-androstene-3,17-dione (A-dione).
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Abstract. Short-term organ culture of rat and human prostatic tissues has been utilized as a means of testing drugs potentially useful in cancer of the prostate. Optimal conditions, particularly the concen tration of T, have been established for organ culture of such tissues and the effects of various drugs on 5a-reductase activity (5a-RA) have been utilized as a means of ascertaining antiprostatic actions of the various compounds and drugs tested. Thus, it was shown that estracyt, estradiol-17(5, progesterone, and novel steroids and steroidconjugates have definite effects on 5a-RA in this system.
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Figure I. Representative sections of explants of rat ventral prostate after organ culture. (A-B), from normal, adult rat, maintained for 6 days in control medium (A) or medium supplemented with 0.5 pM T (B); (C-D), from 7-day castrated animal, maintained for 6 days in control medium (C) or medium containing 0.5 iiM T (D); (E-F), from 27 day old rat, maintained in control medium for 3 days (E) or in control medium for 1 day, then in 0.5 uM T-supplemented medium for 2 days (F). Hematoxylin and eosin strain, x 500.
For systems containing T (unlabelled) in the growth medium, correction was made with respect to specific activity in calcu lating total 5a-reduction of T.
Results
5a-/?/4 in Organ Culture The response of prostatic cells to androgens in organ culture was verified by histological examination (figure I). In explants derived from either normal adult rats or those castrated for 7 days or young prepubertal rats, the presence of T in the medium at a concentration of 0.5 pM (144 ng/mg) resulted in the generation and preservation of prominent alveolar cells
surrounding the gland lumina. Explants from normal, intact rats and 7-day castrates responded to 0.08 units/ml insulin (not shown) almost as well as to T. Cells of the immature prostate, however, did not respond to insulin. The effect of varying the concentration of T in the medium on 5a-RA is shown in table 1. The concentration of T chosen for use in subsequent experiments was 0.5 pM, as it repre sented near optimal efficiency of conversion of T to DHT and, morphologically, yielded healthy luminar epithelium. In contrast, T at a concentration of 50 pM proved deleterious to cell structure. In figure 2 is shown the rate of DHT, A-diol and A-dione
Testosterone concen (ration
pMoles DHT/30 mg tissuc/2 h 1
0 5x10"14 Molar 5x 1 0-" 5 x 10"9 1x 10-8 5 x 10-" 1 x 10-7 5 x10-7 1 x lO-6 5x10-« lx lO -5 5 x l0 -5
0.09 0.08 0.11 0.44 0.78 4.04 10.08 40.00 85.83 304.10 433.30 666.60
'A fter 2 hr exposure to 1.2xlO_9M 3H-1,2,6,7-Testosterone and correction for specific activity.
Figure 2. Rate of metabolism of testosterone during varying periods of exposure to 3H-T. Explants were prepared from rat ventral pros tate. After 1 day in control medium, they were maintained in medium supplemented with 0.5 uM T for 2 days. On the 3rd day of organ culture, T-medium was replaced with new one also containing 3Hlabelled T for the duration of times indicated. Determination of formation of DHT (A), DHT plus A-diol (O) and A-dione (□) was performed as described in "Methods” . The values are averages of duplicate determinations.
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Table I. Effect of T-concentration in maintenance medium on 5 a reductase activity.
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0
0.2pg/ml 0.5pg/ml
02pg/mt 0.5pg/ml
Figure 3. 5a-RA in normal and partially regressed rat ventral prostate in organ culture. Explants derived from normal and 4-day castrated rats were maintained in organ culture for 4 days in media supple mented with 0.5 aM T. 5a-RA determinations were performed on the 4th day as described in ’’Methods”. For comparison, in vivo 5a-RA values in normal and 4-day castrates were determined, using homogenates of freshly excised tissues. The values are averages of duplicate determinations.
Figure 5. Interaction of estradiol with rat prostate. Explants were prepared from ventral and dorsolateral lobes. After 1 day in control medium, they were exposed for 2 days to media containing l4Cestradiol-17(5. On the final day, 3H-testosterone was added to the dishes for the determination of 5a-RA. v, ventral; d, dorsolateral; T, testosterone; E 2, estradiol.
production by the rat ventral prostate in organ culture. This rate, especially that of DHT, is linear under the conditions and times employed in the experiments conducted. Explants derived from normal rats and four-day castrates were maintained in hormone-less and T-supplemented media, and 5a-RA measured (figure 3). The homogenate assays provided a measure of in vivo activity. Exposure of explants from four-day castrates to four days of T in culture resulted in the stimulation of enzyme activity beyond control (intact ani mals) value. In comparison to the homogenate assay, in organ culture a greater percentage of the product, DHT, was further metabolized to 5a-androstane-3a, 17(5-diol. On the other hand, the formation of 4-androstene-3,17-dione was less in organ culture than in the homogenate assay.
Effect of Drugs on 5a-RA In the initial studies, the effects of estrogen and progesterone on 5a-RA in the rat prostate were studied (figure 4). The results indicate that both E 2 and progesterone were stimu latory in the explants derived from the ventral lobe and strongly inhibitory in the dorsolateral tissue. In a follow-up experiment, the interaction of E 2 with rat prostate was further investigated (figure 5). The explants were exposed to 3H-E 2 for 2 days. At the concentrations used (0.2 pg/ml and 0.5 pg/ml), the estrogen had no effect on the ventral lobe 5a-RA. On the other hand, enzyme activity in the dorsolateral prostate was again markedly inhibited. The amount of uptake of 3H-E 2 roughly correlated with the degree of inhibition of 5«-RA.
Figure 4. Effect of estradiol-173 and progesterone on 5a-RA in rat prostate. Explants were prepared from ventral (v) and dorsolateral (d) lobes of rat prostates. After 1 day in control medium, they were ex posed for 6 days to steroid-containing media, with media change every 2 days. 5a-RA determinations were performed as described in ’’Methods” . T, testosterone; E 2, estradiol; P.progesterone.
Figure 6. Effect of estracyt on tes:osterone metabolism by rat prostate in organ culture. After 1 day in control medium, explants from ventral (v) and dorsolateral (d) lobes were exposed for 2 days to media containing 1 pM T plus 0.5 and 50 pg/ml estracyt. On the final day, the culture dishes were pulsed with media containing 1.2 nM 3H-T. Each bar represents mean value ± standard error for 3 deter minations.
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E:
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Figure 7. Effect of estracyt on 5a-RA in human prostatic cancer in organ culture. Results of 2 separate experiments are shown. Expiants were prepared from carcinomas of the prostate. After 1 day in control medium, the expiants were exposed for 4 days to T or T plus estracyt. 5a-RA measurements were done on the final day. Solid bar = for mation of DHT plus A-diol; Open bar = formation of androstenedione. The values are averages of duplicate determinations.
The effect of estracyt on rat ventral prostate 5a-RA was investigated (figure 6). There was 25 % inhibition of enzyme activity at both concentrations of the drug. Studies of the effect of estracyt on 5a-RA in cultured human prostatic tissue gave variable results. Figure 7 shows the results of 2 such experiments. In one case, 5a-reduction of T was inhibited by 50% with 10 pg/ml of estracyt. In another sample, no effect was observed. The 5a-RA assay in organ culture was further used to assess the properties of some novel steroidal compounds with cyto static properties. Figures 8 and 9 show the effect of 2 such steroids on prostatic tissue from 2 different specimens. In one experiment (figure 8), Compound A stimulated 50-reduc tion of T, whereas Compound B was slightly inhibitory. In
Figure 9. Effect of compounds A and B and E 2FUDR complex on testosterone metabolism by human BPH explants. Explants were prepared from specimens of benign prostatic hypertrophy. After 1 day in control medium, the explants were maintained for 2 days in the presence of T or T plus drugs. 5a-RA determinations were done on the final day. All systems contained 0.5 11M T. 1) no drugs; 2) 1 pm/ml compound A; 3) 0.1 pg/ml E 2-FUDR; 4) 10 gg/ml E 2FUDR. Hatched bar = formation of DHT + A-diol; open bar = formation of androstenedione. Each bar represents mean value ± standard error for 3 determinations.
another experiment using explants derived from a different human prostatic specimen (figure 9), both Compound A and Compound B were inhibitory of 5a-RA. On the other hand, both drugs stimulated (greater than 2-fold by Compound A and about 36% over control value by Compound B) the metabolism of T via the oxidative pathway, as reflected by increase in the formation of A4-androstenedione. Histologi cally, sections prepared from explants cultured in T or T plus Compound A (not shown) displayed hyperplasia of the epi thelial components, whereas degeneration of glandular acini characterized Compound B tested tissue. In the same experi ment (figure 9), 5a-reduction of T was also strongly inhibited by a third steroidal-cytostatic complex, composed of estradiol and fluorodeoxyuridine (at both concentrations of 0.1 pg/ml and 10 pg/ml). Again, the effect on oxidative metabolism was non-inhibitory at a high concentration of the drug (10 pg/ml) and slightly stimulatory at the lower concentra tion (0.1 tig/ml).
Figure 8. Effect of compounds A and B on 5a-RA in human prostate. Explants were prepared from specimens of benign prostatic hyper trophy. After 1 day in control medium, the explants were main tained for 2 days in media containing T or T plus compound A or B. 5a-RA determinations were done on the final day. Solid bars repre sent 5a-reduction products (DHT + A-diol); Open bars represent formation of androstenedione. The values are averages of duplicate determinations.
In view of the central role played by 5a-reduction of T in the regulation of prostatic growth and physiology [1, 22, 24], a model system for testing drugs against cancer of the prostate, based on 5a-RA, has been developed and previously reported [8, 19, 21, 25], This approach has been extended with the use of organ culture to an in vitro system, providing a more sensi tive and direct assay of the effects of agents on the target tissue, particularly human prostatic cancer. Short-term organ culture, as presently reported, provides a rapid and flexible tool for studying changes in biochemical parameters under various experimental conditions, while still maintaining nor mal morphological features of the intact tissue. It, thus, fulfills
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Discussion
Kadohama et al.: Organ Culture and 5a-RA
plication of organ culture becomes obvious, since the system exposes isolated target tissues directly to test drugs. Compared to rat prostate, human prostatic tissue is less amenable to organ culture. Nevertheless, short-term culture of human prostate has been successfully achieved [6, 13, 14, 16, 23], Specimens of benign hyperplasia and carcinoma are invariably lacking in cellular homogeneity, contributed by varying degrees of differentiation, atrophy, proliferative acti vity and relative content of glandular and stromal elements. Moreover, these tissues often show differences in responsive ness to hormones and drugs. As a result, the use of human prostate involves great risk in variability in results between specimens and even in replicate cultures of explants from the same specimen. Furthermore, since the level of 5a-RA may vary considerably from one cancer of the prostate to another [17], the results obtained with such tissues in organ culture may reflect this variability. However, there is little doubt that a significant proportion of cancers of the prostate does have 5a-RA and. hence, the system utilized in the present study should have relevance to the planning of drugs potentially useful in some cases of cancer of the prostate. Results obtained in the present studies illustrate the wide range of effects produced by steroidal agents on 5a-reduction of T in human prostate. Whereas estracyt consistently caused inhibition of 5a-reduction of T in rat dorsolateral prostate, its effect on 5a-RA in human prostatic tissue varied from specimen to specimen. Similar results were obtained with other steroid-cytostatic molecule complexes tested. These discrepencies in results between specimens may be accounted for by differences in advancement of the disease and degree of sensitivity to hormonal action, as often observed in varia tions in response to estrogen therapy. Whereas these results point to some limitations in the use of 5a-RA as the sole index of drug action, its measurement in conjunction with other biochemical and morphological parameters should aid in the development of a rational chemotherapeutic program for abnormal prostatic growth. With regard to the action of E 2 and E 2 -cytostatic molecule complexes, the observed effects on 5cx-RA in organ culture cannot be attributed solely to a decrease in circulating andro gens via an effect on the hypothalamic-hypophyseal system. Direct interaction with isolated prostatic tissue and inhibition of 5a-reduction of testosterone were demonstrated. The action of E 2 on rat prostate was highly tissue specific, in that 5a-RA was inhibited only in tissue from the dorsolateral lobe. Similarly, inhibition of 5a-reduction of T by estracyt was confined mainly to the dorsolateral prostate. E 2 and the various estrogen derivatives tested had a non-inhibitory or often stimulatory effect on the metabolism of T via the oxi dative pathway, in both rat and human prostatic tissues. The significance of these observations in terms of anti-prostatic effects requires further study.
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Baulieu , E. E.; Lasnitzki, I., and Robel , P.: Metabolism of
2
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the need for a model in vitro system for testing drugs against cancer of the prostate. Present studies involved the use of prostatic tissues from both rat and human sources for investi gating the effects of various steroids on 5a-RA. One of the purposes of the present studies was to establish reproducible and standardization conditions for prostatic 5a-RA and morphology in organ culture, though it is realized that other factors in addition to T may be necessary to achieve in vivo levels of activity and morphology [2, 11]. However, sufficient variability in 5a-RA was observed with various other factors (e.g., insulin, cortisol) to lead us to believe that an assay in the presence of only T was preferable for the pur pose of our studies. Morphology and 5a-RA were used as indi cators of T activity in organ culture, even though these reflect primarily a permissive action of T in regulating prostatic growth [4, 12, 20], Thus, the general histological features of the tissue were adequately maintained for at least 4 days, with absence of significant regressive changes. The 5a-RA was preserved during this time and progressively diminished in parallel with tissue degeneration thereafter (8 to 14 days). One mode of the anti-androgenic action of estrogens may be at the level of T metabolism in target tissues. The adminis tration of E2 , however, caused little or no inhibition of 5a-reduction of T in dog [9] and rat ventral prostates [3, 26], but did stimulate the formation of products of the oxidative path way relative to 5«-reduced metabolites. On the other hand, 5a-RA was inhibited in the dorsolateral prostate of E 2 -treated rats [26]. E2 and various derivatives of E 2 and estriol have been reported to inhibit 5a-reduction of T when assayed in homogenates of rat prostate [10], but it was not specified which lobe of the prostate was used. In organ culture of dog and rat ventral prostates, the presence of 17 pM E2 produced inhibition of T metabolism [5], In the present studies, the addition of 1.8 pM E2 to the growth medium containing 0.5 pM T resulted in a differential effect on 5a-RA, viz. there was no effect in explants derived from the ventral prostate, whereas 5a-reduction by the dorsolateral tissue was greatly inhibited. The dorsolateral lobe of the rat prostate displays unique sensitivity to estrogenic action in other respects not seen in the ventral lobe. Thus, estrogen induced uptake of 65Zn by the rat dorsolateral prostate in a highly tissue-specific manner [15], Zinc is a potent inhibitor of 5a-RA, at least in nuclei of rat ventral prostate [7], Whether this relationship of estrogens, zinc and 5a-reduction is signi ficant to the mechanism of the anti-androgen action of estro gens remains to be determined. Synthetically modified steroids and steroid-cytostatic com plexes are being increasingly utilized in chemotherapy. With respect to cancer of the prostate, one such compound is estracyt. It has been shown that one of the properties in its anti-androgenic action is an influence on 5 a-R A [7, 8, 19]. In vivo administration of estracyt to dogs and rats resulted in diminished 5a-RA of their prostates [8], H isaeter [7] reported no effect by estracyt on the rat ventral prostate after in vitro incubation or short-term (half-hour) in vivo adminis tration of the drug. Marked inhibitory effect was observed only after long-term (3 days) administration of estracyt. These results point out the difficulty in assessing effects of drug action in vivo, due possibly to the metabolic transformation that a given agent undergoes in the body. The practical ap
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