0013-7227/90/1272-3052$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 6 Printed in U.S.A.
Receptors for Luteinizing Hormone-Releasing Hormone (LHRH) in Dunning R3327 Prostate Cancers and Rat Anterior Pituitaries after Treatment with a Sustained Delivery System of LHRH Antagonist SB-75* G. SRKALOVIC, L. BOKSER, S. RADULOVIC, E. KORKUT, AND A. V. SCHALLY Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center, New Orleans, Louisiana 70146; and the Department of Experimental Medicine, Tulane University Medical School, New Orleans, Louisiana 70112
ABSTRACT. Membrane receptors for LHRH were evaluated in Dunning R3327 prostate cancers and rat anterior pituitaries. The receptors were characterized both in untreated animals and after the in vivo treatment with microcapsules of the agonist DTrp6-LHRH and a sustained delivery system releasing different doses (23.8, 47.6, 71.4 Mg/day) of LHRH antagonist [Ac-DNal(2)1-D-Phe(4Cl)2-D-Pal(3)3,D-Cit6, D-Ala10]-LHRH (SB-75). The therapy, which lasted 8 weeks, strongly inhibited tumor growth. A group of normal Sprague-Dawley male rats was also treated for 6 weeks with microcapsules of SB-75 releasing 25 tig/ day. In the Dunning tumors from the control group, ligand [125I, D-Trp6]-LHRH was bound to two classes of binding sites [dissociation constant, class a (Kda) = 1.01 ± 0.30 x 10~9 M; Kdb = 1.71 ± 0.41 x 10"6 M; maximal binding capacity of receptors, class a (Bmaxa) = 48.66 ± 22.13 fmol/mg of protein; Bmaxb = 92.10 ± 29.40 pmol/mg of protein] in both kinetic and equilibrium studies. Treatment with D-Trp6-LHRH produced down-regulation of membrane receptors for LHRH in Dunning tumors. Microcapsules of SB-75 resulted in dose-dependent up-regulation of binding sites for LHRH in Dunning tumors. Analysis of the binding data showed that interaction of labeled D-Trp6-
H
IGHLY active agonistic and antagonistic analogs of LHRH have been developed by the substitution of various amino-acid residues in the decapeptide sequence (1). A successful clinical use of agonistic analogs of LHRH for the treatment of androgen-dependent prostate cancers was documented in numerous clinical studies (2-8). Although a repeated, chronic administration of LHRH agonists is required to induce an inhibition of LH and FSH release and reduction in the levels of sex steroids, similar effects can be obtained with a single Received May 29, 1990. Address all correspondence to: Andrew V. Schally, Veterans Administration Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70146. * This work was supported by NIH Grant CA-40003, the Medical Research Service of the Veterans Administration, the G. Harold and Leila Y. Mathers Foundation, and the U.S. Cancer Research Council (to A.V.S.).
LHRH with binding sites in anterior pituitaries was consistent with the presence of a single class of noncooperative receptors (Kd = 43.75 x 10~9 M; BmBX = 5.25 pmol/mg membrane proteins). Prolonged treatment with microcapsules of D-Trp6-LHRH reduced both Bmax and Kd. Lower doses of SB-75 (23.8 and 47.6 /ig/day) produced up-regulation, whereas the highest dose (71.4 Mg/day) resulted in down-regulation of binding sites for LHRH in rat pituitaries. In normal Sprague-Dawley rats, treatment with microcapsules of SB-75 (25 /ig/day) for 6 weeks produced a slight increase in the number of available binding sites (Bmox = 2.35 ± 0.82 pmol/mg membrane protein) and a moderate decrease in affinity (Kd = 35.10 ± 15.19 X 10"9 M) of pituitary membrane receptors for LHRH. The findings provide additional support for the view that LHRH analogs exert direct effects on tumor cells. Our findings indicate that prolonged treatment with high doses of modern LHRH antagonists produces down-regulation of pituitary receptors. Our work in tumors also implies that some differences may exist between LHRH receptors, even in the same tissue, leading to the concept of subclassification of LHRH receptors. {Endocrinology 127: 3052-3060,1990)
administration of LHRH antagonists (9,10). The antagonistic analogs of LHRH cause an immediate inhibition of the pituitary-gonadal axis, and their use would avoid the initial stimulatory effects that occasionally results in flare-up phenomena (11, 12). Recent receptor-binding and in vitro studies revealed that LHRH analogs, in addition to their principal effects mediated through the suppression of the pituitary-gonadal axis, can also exert a direct effect on rat and human prostate cancers (13-15). Some controversies exist about changes in receptors for LHRH after prolonged exposure to LHRH antagonists. Several reports indicated that LHRH antagonists cause a prolonged reduction in available receptor sites (16-19). In contrast to these findings, Loumaye and Catt (20, 21) did not find any changes in LHRH binding capacity after prolonged exposure of pituitary cells to LHRH antagonist [D-Phe2, Pro3, D-
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RECEPTORS FOR LHRH
Phe6]-LHRH. In this study, a modern LHRH antagonist, SB-75, free of edematogenic effects (12), was administered in the form of microcapsules to normal rats and rats bearing the Dunning R-3327 prostate carcinoma. Binding characteristics of receptors for LHRH were defined in membrane preparations from Dunning prostate tumors and anterior pituitaries from normal rats and rats bearing Dunning tumors. This work was aimed at defining the pattern of changes in number and affinity of receptors for LHRH after prolonged in vivo treatment with potent LHRH antagonist SB-75. Materials and Methods Peptides and sustained delivery systems SB-75 ([Ac-D-Nal(2)1-D-Phe(4Cl)2-D-Pal(3)3,D-Cit6, D-Ala10] -LHRH) was synthesized by ASTA-Degussa (Frankfurt, West Germany). Microcapsules of D-Trp6-LHRH and SB-75 in poly (D,L-lactide-co-glycolide) were prepared by Dr. P. Orsolini at Cytotech (Martigny, Switzerland). Sustained release formulation of D-Trp6-LHRH (Debiopharm, Lausanne, Switzerland) in an aliquot of 36 mg maintained a continuous liberation of about 25 Mg/day of the analog for 30 days. D-Trp6-LHRH microcapsules were injected once a month. Microcapsules of SB-75 trifluoroacetate (ASTA-Degussa) Batch No. RCS-00035 were designed to liberate about 23.8, 47.6, and 71.4 jig/day from aliquots of 25, 50, and 75 mg, respectively. This batch of SB-75 microcapsules was injected every 3 weeks. Another batch of SB-75 microcapsules (No. RCS-0001) used to treat normal rats was designed to release 25 ^g/day for 21 days from an aliquot of 30 mg. Reagents for RIA of LH were provided by the National Hormone and Pituitary Program of NIDDK.
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dark schedule, were injected with another batch of SB-75 microcapsules every 3 weeks for 6 weeks. Four control animals were treated with the injection vehicle only. Three weeks after the second injection of microcapsules, rats from the treated and the untreated groups were killed and decapitated under light anesthesia (Metofane), and pituitary glands removed and processed. All animals were maintained in accordance with the NIH guide for the Care and Use of Laboratory Animals. Buffers The homogenization buffer for all membrane receptor assays consisted of 0.3 M sucrose, 25 mM Tris base, 0.25 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 10 mM monothioglycerol and Trasylol (aprotinin) at 10,000 kallikrein inactivator units/ liter (pH 7.5). For D-Trp6-LHRH-binding experiments, the assay buffer was 25 mM Tris base, 1 mM dithiothreitol, 1 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride, and 0.1% BSA (pH 7.5). The wash buffer was the same as the assay buffer, but did not contain BSA. The chemicals used for buffers were purchased from Sjgma Chemical Co. (St. Louis, MO). Preparation of membranes Samples were cleaned of fat and connective tissue, cut into small slices, and homogenized in 5 vol sucrose buffer using an Ultra-Turrax homogenizer (Tissumizer, Tekmar, Cincinnati, OH) at maximal speed 5 x 5-s strokes at 0 C. The homogenate was centrifuged at 500 X g for 10 min at 4 C. The supernatant containing the crude membrane fractions was then ultracentrifuged at 70,000 X. g for 45 min at 4 C (Beckman Preparative Ultracentrifuge, Beckman Instruments, Inc., Palo Alto, CA). The resulting pellet was resuspended in wash buffer and used for the receptor-binding studies. Protein concentration was determined by t|ie Bio-Rad protein assay kit according to Bradford (22).
Treatment of experimental animals Male rats of the Copenhagen X Fisher Fi strain, bearing the Dunning R-3327 prostate carcinoma, were provided by Dr. N. Altman (Papanicolau Cancer Research Institute, Miami, FL). These Fj hybrids were inoculated with tumors at 60 to 80 days of age. Nineteen weeks after implantation, tumors were well developed, and the animals were injected im with three graded doses of microcapsules of SB-75. The control group was treated with the injection vehicle (2% CM-cellulose and 1% Tween 20 in water). Two more injections of SB-75 microcapsules were given at 3-week intervals. D-Trp6-LHRH microcapsules were injected twice at 4-week intervals. The tumor volumes were measured weekly until the end of the eighth week from the beginning of the treatment. The percent change in tumor volume was calculated on the basis of individual response. At the end of the 8 weeks treatment, rats were decapitated under anesthesia (Metofane, Pittmann-Moore, Washington Crossing, NJ). Pituitary glands were removed and preserved for receptor studies. Tumors were carefully cleaned and weighed, and a sample was taken for histological studies and stored at —80 C for receptor studies. Four normal male Sprague-Dawley rats (Charles River), weighing 230 to 250 g and maintained on a 12-h light, 12-h
Radioiodination of peptides For the radioiodination of D-Trp6-LHRH and SB-75, a modification of the chloramine-T method was used. An aliquot of the peptide (5 ng in 5 n\ of 0.01 N acetic acid) was mixed with 40 ^1 0.5 M phosphate buffer and 1 mCi I125Na in a volume of 2 n\. Ten microliters of chloramine-T (1 mg/ml in 0.05 M phosphate buffer) were added. The procedure was performed at room temperature. The purification of the labeled peptide was performed using HPLC with automated gradient controller, a pair of M-6000-A pumps, a U6K injector (Waters, Milford, MA), and a C-18 column (W-Porex 5C18, 250 X 4.6 mm ID, Rancho Palos Verdes, CA). The specific activity was found to be 1131 Ci/mM for D-Trp6-LHRH and 1100 Ci/mM for SB-75 as calculated by the self-displacement method. Receptor binding of peptides The binding assay of D-Trp6-LHRH was conducted as described previously (15, 17). Binding reactions were performed in 12 X 75-mm polypropylene conical or round bottom culture tubes at 4 C using a competitive inhibition method. Displacement curves were obtained with 0.2 to 0.3 nmol iodinated [125I,
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RECEPTORS FOR LHRH
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D-Trp6]-LHRH in the presence of increasing amounts of unlabeled D-Trp6-LHRH (600 pM-6 fM). The procedure was as follows: 50 fil unlabeled D-Trp6-LHRH in incubation buffer and 50 /xl labeled ligand were placed into the tube and vortexed, and 50 n\ of the suspension containing the membrane-receptor fraction were added. In each binding experiment, one set of tubes was used with 50 n\ wash buffer instead of membrane to determine the radioactivity bound to nonmembrane particles. Each assay point was performed in triplicate. The tubes were incubated for 60 min at 4 C. The reaction was terminated by rapid filtration through glass-fiber filters (Filtermats-Receptor Binding, Skatron, As., Lier, Norway), prewetted in assay buffer and presoaked for at least 3 h in 0.5% polyethylenimine solution to minimize filter adsorption. A semiautomatic cell-harvesting system (Skatron) with 10-s washing time was used. The radioactivity of filters was counted in an automatic 7-counter (Micromedic System, Inc., Huntsville, AL). LH serum levels were determined by RIA. The sensitivity of the standard curve was 13 pg, and the intraassay variance was 3.6%. Mathematical analysis of the binding data The counts of radioactivity obtained from filters or tubes containing membrane particles were corrected by deducting the background count from assays in which no membranes were added. The LIGAND-PC computerized curve-fitting program of Munson and Rodbard (23) was used to determine the types of receptor binding, dissociation constant (Kd), and the maximal binding capacity of receptors (Bmax). Analysis of kinetic studies was made using a collection of radioligand-binding analysis programs by G. A. McPherson (Elsevier-Biosoft, Cambridge, United Kingdom). Statistical evaluation of data was performed by Student's t test and Duncan's new multiple-range test, and P < 0.05 was accepted as statistically significant.
Results Effect on tumor growth Treatment with microcapsules of D-Trp6-LHRH and SB-75 significantly inhibited the growth of Dunning prostate tumors. D-Trp6-LHRH caused a significant reduction in tumor weight (1.27 ± 0.23 g) when compared with controls (6.07 ± 0.89 g) (P < 0.01). The highest dose of SB-75 exerted a maximal inhibitory activity on prostate cancer growth, equivalent or better than that induced by D-Trp-6-LHRH as based on measurement of tumor volume and percent change in tumor volume and tumor weight. The body weights were unchanged. Doses of 23.8 Mg/day and 47.6 /ug/day of SB-75 induced a partial and submaximal decrease in tumor weight and volume, respectively. Other oncological and endocrine details of this study and pathological results are fully described elsewhere (24, 25). Serum LH levels were significantly reduced in all treated groups compared with controls, which measured 0.7 ± 0.1 ng/ml. The greatest reduction was noticed in the groups treated with D-Trp6-LHRH (0.29 ± 0.01 ng/ml) (P < 0.01), whereas the levels in rats
Endo • 1990 Vol 127 • No 6
given a high dose of SB-75 were 0.365 ± 0.02 ng/ml (P LHRH receptors in Dunning R-3327 prostate cancers: DTrp6-LHRH and SB-75 binding kinetics As shown in Fig. 1A, binding of D-Trp6-LHRH in Dunning tumor membranes was time dependent. The biexponential model gave a significantly better fit than the monoexponential model (P < 0.01). Therefore, two different association rate constants were calculated to be Kia = 4 X 106 M"1 min"1 and K lb = 5.3 X 104 M"1 min"1. Dissociation of bound ligand was also dependent on time (Fig. 1A). The rate of decline of [125I-D-Trp6]-LHRH binding did not follow a simple order of kinetics. A least square fitting of the data gave a value of 0.0101 min"1 and 0.0801 min"1 for the dissociation rate constants K_la and K_lb, respectively. Again, a biexponential model was selected as the most appropriate. D-Trp6-LHRH was incompletely displaced from its binding sites in Dunning tumor membranes. The Kd values from kinetic behavior were calculated to be Kda = 2.5 x 10"9 M for high affinity binding sites and Kdb = 1.5 X 10~6 M for low affinity binding sites. Corresponding dissociation half-times were 69.3 min for high and 8.7 min for low affinity binding sites. Specific binding of [125I]-SB-75 to membranes of Dunning tumor was also time dependent (Fig. IB). Biexponential models gave a better fit in both association and dissociation experiments. Association rate constants (Kla and K lb ) were 2.3 X 107 M"1 min"1 for binding to high affinity and 1.9 X 105 M"1 min"1 for binding to low affinity binding sites. Dissociation rate constants were K_la = 0.0401 min"1 (t1/2a = 17.3 min) and K_lb = 0.0356 min"1 (t^b = 19.5 min), respectively. As shown in Fig. IB, SB-75 is almost completely displaced from its binding sites on Dunning tumors. Displacement analysis of f125!, D-Trp6]'-LHRH binding to Dunning tumors Displacement curve and Scatchard analysis of bindinginhibition data using control Dunning tumors, obtained with tracer D-Trp6-LHRH and increasing concentrations of the unlabeled peptide, also demonstrated the high affinity binding site as well as the putative low affinity site, with about Veooth of the Ka of the high affinity site (Fig. 2). The two-site model provided a better fit also in displacement experiments with antagonist SB-75 as labeled and unlabelled peptide (Kda = 1.4 x 10~9 M and Kdb = 0.14 X 10~6 M). In a separate experiment, antagonist SB-75 completely displaced labeled D-Trp6-LHRH from both binding sites on membranes of control Dunning tumors with Kda of 1.59 X 10"9 M and Kdb of 0.25 X 10~6 M.
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RECEPTORS FOR LHRH FIG. 1. Association (•—•) and dissociation (A—A) kinetics of [125I, D-Trp6] LHRH (A) and 125I-SB-75 (B) binding to membranes from rat Dunning R3327 prostate cancer. A, The membranes were incubated in 0.15 ml with 0.28 nmol [125I, D-Trp6]-LHRH for the time intervals indicated in the absence (total binding) and presence (nonspecific binding) of 6 Mmol unlabeled D-Trp6-LHRH. Dissociation was initiated by the addition of 12 MMol of unlabeled D-Trp6-LHRH after 60 min incubation at 4 C. B, The membranes were incubated in 0.15 ml with 0.25 nMol 125I-SB-75 at 4 C for the time intervals indicated in the absence (total binding) and presence (nonspecific binding) of 4 jtmol unlabeled SB-75. Dissociation was initiated by the addition of 8 jtmol unlabeled after 60 min incubation at 4 C. Specific binding represents the difference between total and nonspecific binding. All values given represent the extent of binding expressed as the percent of total radioactivity added to each tube. Each point is the average value ± SE of triplicate determinations.
cr
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3.0 2.5
JT-
2.0
1.5 1.0 0.5 0.0
60
120
180
240
TIME ( min ) 4.0
1.0
0.0' 60
120
160
240
TIME ( min )
2.10 T
receptors for LHRH on membranes of Dunning tumors. Administration of microcapsules of SB-75 (71.4 jug/day) highly significantly (P < 0.01) increased the binding capacity (130.40 ± 24.24 fmol/mg membrane protein) and the Kd (2.99 ± 0.32 X 10"9 M) for high affinity membrane binding sites (Table 1). Kd and Bmax of low affinity binding sites were slightly, but not significantly, increased after the highest dose of SB-75.
1.90--
1 . 7 0 ••
1.50 •
1.30 -11.0
LHRH receptors in pituitary membranes from rats bearing Dunning R-3327 prostate cancer
-9.0 -8.0 -7.0 -6.0 -5.0 [D-Trp6]LH-RH, LOG TOTAL (M) FIG. 2. A, Displacement of [125I, D-Trp6]LHRH by increasing amounts of unlabeled D-Trp6-LHRH using membranes of rat Dunning R3327 prostate cancer. The individual data points represent the average value ± SE of triplicate determinations. B, Scatchard plot analysis of data from displacement curves illustrated in A.
I
-10.0
The pooled data from six binding-inhibition experiments with D-Trp6-LHRH as labeled and unlabeled peptide using Dunning tumors from the control group gave an average Bmaxa of 48.76 ± 22.13 fmol/mg membrane proteins for high affinity, low capacity binding sites, and Bmaxb of 92.10 ± 29.40 pmol/mg membrane proteins for low affinity, high capacity binding sites (Table 1). Prolonged treatment with microcapsules of LHRH agonist D-Trp6-LHRH resulted in significant (P < 0.01) reduction of both Kd and Bmax of low affinity receptors for LHRH. The lower doses of SB-75 (23.8 and 47.6 /xg/day) did not significantly change binding characteristics of
Binding kinetics. Specific binding of [125I]-SB-75 to rat pituitary membrane particles was time dependent (Fig. 3). The dissociation followed the simple first order of kinetics. The dissociation rate constants (K-0 obtained by addition of 8 /uM unlabeled peptide after incubation of pituitary particles with 0.25 nM [125I]-SB-75 for 1 h at 4 C was 0.020422 min"1 (half-time, \>/i = 33.934 min). The value of K_i and the corresponding association rate constant (KJ value of 9.76 X 106 M"1 min"1 gave an estimate for the equilibrium Kd of 2.09 X 10"9 M. The monoexponential model gave significantly better (P < 0.05) fit than the biexponential model. SB-75 was completely displaceable from its binding sites on pituitary membranes in a time-dependent manner. The Ka of [125I-D-Trp6]-LHRH binding to pituitary membranes followed a complex kinetics order. The biexponential model gave better fit than the monoexponential model. Kds from kinetics behavior were calculated to
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Endo •1990 Vol 127•No 6
RECEPTORS FOR LHRH
TABLE 1. Characteristics of D-Trp6-LHRH binding in membranes of Dunning R3327 prostate cancer in rats after in vivo treatment with
microcapsules of LHRH analogs Treatment Control, untreated D-Trp6-LHRH (25 Mg/day) SB-75 (23.8 Mg/day) SB-75 (47.6 Mg/day) SB-75 (71.4 Mg/day)
Kda (X 10" 9 M) 1.01 0.40 2.59 1.64 2.99
± 0.30 ± 0.07 ± 1.33 ± 0.21 ± 0.32°
Bmaxa (fmol/mg protein) 48.66 16.25 70.34 55.50 130.42
6 ( X I L0~ M)
1.71 0.41 2.67 3.81 2.09
± 22.13 ± 4.83 ± 22.11 ± 10.53 ± 24.24*
Bma»b (pmol/mg protein)
±0.41 ± 0.16° ± 1.42 ± 1.10 ±0.88
92.10 ± 29.40 6.34 ± 2.40° 96.40 ± 33.20 153.10 ± 35.14 157.52 ± 39.13
Binding characteristics were obtained from 10-point displacement experiments in triplicate tubes. Significance was calculated with two-sided Student's t test. Values represent mean ± SE. °P
Vol. 127, No. 6 Printed in U.S.A.
Receptors for Luteinizing Hormone-Releasing Hormone (LHRH) in Dunning R3327 Prostate Cancers and Rat Anterior Pituitaries after Treatment with a Sustained Delivery System of LHRH Antagonist SB-75* G. SRKALOVIC, L. BOKSER, S. RADULOVIC, E. KORKUT, AND A. V. SCHALLY Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center, New Orleans, Louisiana 70146; and the Department of Experimental Medicine, Tulane University Medical School, New Orleans, Louisiana 70112
ABSTRACT. Membrane receptors for LHRH were evaluated in Dunning R3327 prostate cancers and rat anterior pituitaries. The receptors were characterized both in untreated animals and after the in vivo treatment with microcapsules of the agonist DTrp6-LHRH and a sustained delivery system releasing different doses (23.8, 47.6, 71.4 Mg/day) of LHRH antagonist [Ac-DNal(2)1-D-Phe(4Cl)2-D-Pal(3)3,D-Cit6, D-Ala10]-LHRH (SB-75). The therapy, which lasted 8 weeks, strongly inhibited tumor growth. A group of normal Sprague-Dawley male rats was also treated for 6 weeks with microcapsules of SB-75 releasing 25 tig/ day. In the Dunning tumors from the control group, ligand [125I, D-Trp6]-LHRH was bound to two classes of binding sites [dissociation constant, class a (Kda) = 1.01 ± 0.30 x 10~9 M; Kdb = 1.71 ± 0.41 x 10"6 M; maximal binding capacity of receptors, class a (Bmaxa) = 48.66 ± 22.13 fmol/mg of protein; Bmaxb = 92.10 ± 29.40 pmol/mg of protein] in both kinetic and equilibrium studies. Treatment with D-Trp6-LHRH produced down-regulation of membrane receptors for LHRH in Dunning tumors. Microcapsules of SB-75 resulted in dose-dependent up-regulation of binding sites for LHRH in Dunning tumors. Analysis of the binding data showed that interaction of labeled D-Trp6-
H
IGHLY active agonistic and antagonistic analogs of LHRH have been developed by the substitution of various amino-acid residues in the decapeptide sequence (1). A successful clinical use of agonistic analogs of LHRH for the treatment of androgen-dependent prostate cancers was documented in numerous clinical studies (2-8). Although a repeated, chronic administration of LHRH agonists is required to induce an inhibition of LH and FSH release and reduction in the levels of sex steroids, similar effects can be obtained with a single Received May 29, 1990. Address all correspondence to: Andrew V. Schally, Veterans Administration Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70146. * This work was supported by NIH Grant CA-40003, the Medical Research Service of the Veterans Administration, the G. Harold and Leila Y. Mathers Foundation, and the U.S. Cancer Research Council (to A.V.S.).
LHRH with binding sites in anterior pituitaries was consistent with the presence of a single class of noncooperative receptors (Kd = 43.75 x 10~9 M; BmBX = 5.25 pmol/mg membrane proteins). Prolonged treatment with microcapsules of D-Trp6-LHRH reduced both Bmax and Kd. Lower doses of SB-75 (23.8 and 47.6 /ig/day) produced up-regulation, whereas the highest dose (71.4 Mg/day) resulted in down-regulation of binding sites for LHRH in rat pituitaries. In normal Sprague-Dawley rats, treatment with microcapsules of SB-75 (25 /ig/day) for 6 weeks produced a slight increase in the number of available binding sites (Bmox = 2.35 ± 0.82 pmol/mg membrane protein) and a moderate decrease in affinity (Kd = 35.10 ± 15.19 X 10"9 M) of pituitary membrane receptors for LHRH. The findings provide additional support for the view that LHRH analogs exert direct effects on tumor cells. Our findings indicate that prolonged treatment with high doses of modern LHRH antagonists produces down-regulation of pituitary receptors. Our work in tumors also implies that some differences may exist between LHRH receptors, even in the same tissue, leading to the concept of subclassification of LHRH receptors. {Endocrinology 127: 3052-3060,1990)
administration of LHRH antagonists (9,10). The antagonistic analogs of LHRH cause an immediate inhibition of the pituitary-gonadal axis, and their use would avoid the initial stimulatory effects that occasionally results in flare-up phenomena (11, 12). Recent receptor-binding and in vitro studies revealed that LHRH analogs, in addition to their principal effects mediated through the suppression of the pituitary-gonadal axis, can also exert a direct effect on rat and human prostate cancers (13-15). Some controversies exist about changes in receptors for LHRH after prolonged exposure to LHRH antagonists. Several reports indicated that LHRH antagonists cause a prolonged reduction in available receptor sites (16-19). In contrast to these findings, Loumaye and Catt (20, 21) did not find any changes in LHRH binding capacity after prolonged exposure of pituitary cells to LHRH antagonist [D-Phe2, Pro3, D-
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RECEPTORS FOR LHRH
Phe6]-LHRH. In this study, a modern LHRH antagonist, SB-75, free of edematogenic effects (12), was administered in the form of microcapsules to normal rats and rats bearing the Dunning R-3327 prostate carcinoma. Binding characteristics of receptors for LHRH were defined in membrane preparations from Dunning prostate tumors and anterior pituitaries from normal rats and rats bearing Dunning tumors. This work was aimed at defining the pattern of changes in number and affinity of receptors for LHRH after prolonged in vivo treatment with potent LHRH antagonist SB-75. Materials and Methods Peptides and sustained delivery systems SB-75 ([Ac-D-Nal(2)1-D-Phe(4Cl)2-D-Pal(3)3,D-Cit6, D-Ala10] -LHRH) was synthesized by ASTA-Degussa (Frankfurt, West Germany). Microcapsules of D-Trp6-LHRH and SB-75 in poly (D,L-lactide-co-glycolide) were prepared by Dr. P. Orsolini at Cytotech (Martigny, Switzerland). Sustained release formulation of D-Trp6-LHRH (Debiopharm, Lausanne, Switzerland) in an aliquot of 36 mg maintained a continuous liberation of about 25 Mg/day of the analog for 30 days. D-Trp6-LHRH microcapsules were injected once a month. Microcapsules of SB-75 trifluoroacetate (ASTA-Degussa) Batch No. RCS-00035 were designed to liberate about 23.8, 47.6, and 71.4 jig/day from aliquots of 25, 50, and 75 mg, respectively. This batch of SB-75 microcapsules was injected every 3 weeks. Another batch of SB-75 microcapsules (No. RCS-0001) used to treat normal rats was designed to release 25 ^g/day for 21 days from an aliquot of 30 mg. Reagents for RIA of LH were provided by the National Hormone and Pituitary Program of NIDDK.
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dark schedule, were injected with another batch of SB-75 microcapsules every 3 weeks for 6 weeks. Four control animals were treated with the injection vehicle only. Three weeks after the second injection of microcapsules, rats from the treated and the untreated groups were killed and decapitated under light anesthesia (Metofane), and pituitary glands removed and processed. All animals were maintained in accordance with the NIH guide for the Care and Use of Laboratory Animals. Buffers The homogenization buffer for all membrane receptor assays consisted of 0.3 M sucrose, 25 mM Tris base, 0.25 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 10 mM monothioglycerol and Trasylol (aprotinin) at 10,000 kallikrein inactivator units/ liter (pH 7.5). For D-Trp6-LHRH-binding experiments, the assay buffer was 25 mM Tris base, 1 mM dithiothreitol, 1 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride, and 0.1% BSA (pH 7.5). The wash buffer was the same as the assay buffer, but did not contain BSA. The chemicals used for buffers were purchased from Sjgma Chemical Co. (St. Louis, MO). Preparation of membranes Samples were cleaned of fat and connective tissue, cut into small slices, and homogenized in 5 vol sucrose buffer using an Ultra-Turrax homogenizer (Tissumizer, Tekmar, Cincinnati, OH) at maximal speed 5 x 5-s strokes at 0 C. The homogenate was centrifuged at 500 X g for 10 min at 4 C. The supernatant containing the crude membrane fractions was then ultracentrifuged at 70,000 X. g for 45 min at 4 C (Beckman Preparative Ultracentrifuge, Beckman Instruments, Inc., Palo Alto, CA). The resulting pellet was resuspended in wash buffer and used for the receptor-binding studies. Protein concentration was determined by t|ie Bio-Rad protein assay kit according to Bradford (22).
Treatment of experimental animals Male rats of the Copenhagen X Fisher Fi strain, bearing the Dunning R-3327 prostate carcinoma, were provided by Dr. N. Altman (Papanicolau Cancer Research Institute, Miami, FL). These Fj hybrids were inoculated with tumors at 60 to 80 days of age. Nineteen weeks after implantation, tumors were well developed, and the animals were injected im with three graded doses of microcapsules of SB-75. The control group was treated with the injection vehicle (2% CM-cellulose and 1% Tween 20 in water). Two more injections of SB-75 microcapsules were given at 3-week intervals. D-Trp6-LHRH microcapsules were injected twice at 4-week intervals. The tumor volumes were measured weekly until the end of the eighth week from the beginning of the treatment. The percent change in tumor volume was calculated on the basis of individual response. At the end of the 8 weeks treatment, rats were decapitated under anesthesia (Metofane, Pittmann-Moore, Washington Crossing, NJ). Pituitary glands were removed and preserved for receptor studies. Tumors were carefully cleaned and weighed, and a sample was taken for histological studies and stored at —80 C for receptor studies. Four normal male Sprague-Dawley rats (Charles River), weighing 230 to 250 g and maintained on a 12-h light, 12-h
Radioiodination of peptides For the radioiodination of D-Trp6-LHRH and SB-75, a modification of the chloramine-T method was used. An aliquot of the peptide (5 ng in 5 n\ of 0.01 N acetic acid) was mixed with 40 ^1 0.5 M phosphate buffer and 1 mCi I125Na in a volume of 2 n\. Ten microliters of chloramine-T (1 mg/ml in 0.05 M phosphate buffer) were added. The procedure was performed at room temperature. The purification of the labeled peptide was performed using HPLC with automated gradient controller, a pair of M-6000-A pumps, a U6K injector (Waters, Milford, MA), and a C-18 column (W-Porex 5C18, 250 X 4.6 mm ID, Rancho Palos Verdes, CA). The specific activity was found to be 1131 Ci/mM for D-Trp6-LHRH and 1100 Ci/mM for SB-75 as calculated by the self-displacement method. Receptor binding of peptides The binding assay of D-Trp6-LHRH was conducted as described previously (15, 17). Binding reactions were performed in 12 X 75-mm polypropylene conical or round bottom culture tubes at 4 C using a competitive inhibition method. Displacement curves were obtained with 0.2 to 0.3 nmol iodinated [125I,
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RECEPTORS FOR LHRH
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D-Trp6]-LHRH in the presence of increasing amounts of unlabeled D-Trp6-LHRH (600 pM-6 fM). The procedure was as follows: 50 fil unlabeled D-Trp6-LHRH in incubation buffer and 50 /xl labeled ligand were placed into the tube and vortexed, and 50 n\ of the suspension containing the membrane-receptor fraction were added. In each binding experiment, one set of tubes was used with 50 n\ wash buffer instead of membrane to determine the radioactivity bound to nonmembrane particles. Each assay point was performed in triplicate. The tubes were incubated for 60 min at 4 C. The reaction was terminated by rapid filtration through glass-fiber filters (Filtermats-Receptor Binding, Skatron, As., Lier, Norway), prewetted in assay buffer and presoaked for at least 3 h in 0.5% polyethylenimine solution to minimize filter adsorption. A semiautomatic cell-harvesting system (Skatron) with 10-s washing time was used. The radioactivity of filters was counted in an automatic 7-counter (Micromedic System, Inc., Huntsville, AL). LH serum levels were determined by RIA. The sensitivity of the standard curve was 13 pg, and the intraassay variance was 3.6%. Mathematical analysis of the binding data The counts of radioactivity obtained from filters or tubes containing membrane particles were corrected by deducting the background count from assays in which no membranes were added. The LIGAND-PC computerized curve-fitting program of Munson and Rodbard (23) was used to determine the types of receptor binding, dissociation constant (Kd), and the maximal binding capacity of receptors (Bmax). Analysis of kinetic studies was made using a collection of radioligand-binding analysis programs by G. A. McPherson (Elsevier-Biosoft, Cambridge, United Kingdom). Statistical evaluation of data was performed by Student's t test and Duncan's new multiple-range test, and P < 0.05 was accepted as statistically significant.
Results Effect on tumor growth Treatment with microcapsules of D-Trp6-LHRH and SB-75 significantly inhibited the growth of Dunning prostate tumors. D-Trp6-LHRH caused a significant reduction in tumor weight (1.27 ± 0.23 g) when compared with controls (6.07 ± 0.89 g) (P < 0.01). The highest dose of SB-75 exerted a maximal inhibitory activity on prostate cancer growth, equivalent or better than that induced by D-Trp-6-LHRH as based on measurement of tumor volume and percent change in tumor volume and tumor weight. The body weights were unchanged. Doses of 23.8 Mg/day and 47.6 /ug/day of SB-75 induced a partial and submaximal decrease in tumor weight and volume, respectively. Other oncological and endocrine details of this study and pathological results are fully described elsewhere (24, 25). Serum LH levels were significantly reduced in all treated groups compared with controls, which measured 0.7 ± 0.1 ng/ml. The greatest reduction was noticed in the groups treated with D-Trp6-LHRH (0.29 ± 0.01 ng/ml) (P < 0.01), whereas the levels in rats
Endo • 1990 Vol 127 • No 6
given a high dose of SB-75 were 0.365 ± 0.02 ng/ml (P LHRH receptors in Dunning R-3327 prostate cancers: DTrp6-LHRH and SB-75 binding kinetics As shown in Fig. 1A, binding of D-Trp6-LHRH in Dunning tumor membranes was time dependent. The biexponential model gave a significantly better fit than the monoexponential model (P < 0.01). Therefore, two different association rate constants were calculated to be Kia = 4 X 106 M"1 min"1 and K lb = 5.3 X 104 M"1 min"1. Dissociation of bound ligand was also dependent on time (Fig. 1A). The rate of decline of [125I-D-Trp6]-LHRH binding did not follow a simple order of kinetics. A least square fitting of the data gave a value of 0.0101 min"1 and 0.0801 min"1 for the dissociation rate constants K_la and K_lb, respectively. Again, a biexponential model was selected as the most appropriate. D-Trp6-LHRH was incompletely displaced from its binding sites in Dunning tumor membranes. The Kd values from kinetic behavior were calculated to be Kda = 2.5 x 10"9 M for high affinity binding sites and Kdb = 1.5 X 10~6 M for low affinity binding sites. Corresponding dissociation half-times were 69.3 min for high and 8.7 min for low affinity binding sites. Specific binding of [125I]-SB-75 to membranes of Dunning tumor was also time dependent (Fig. IB). Biexponential models gave a better fit in both association and dissociation experiments. Association rate constants (Kla and K lb ) were 2.3 X 107 M"1 min"1 for binding to high affinity and 1.9 X 105 M"1 min"1 for binding to low affinity binding sites. Dissociation rate constants were K_la = 0.0401 min"1 (t1/2a = 17.3 min) and K_lb = 0.0356 min"1 (t^b = 19.5 min), respectively. As shown in Fig. IB, SB-75 is almost completely displaced from its binding sites on Dunning tumors. Displacement analysis of f125!, D-Trp6]'-LHRH binding to Dunning tumors Displacement curve and Scatchard analysis of bindinginhibition data using control Dunning tumors, obtained with tracer D-Trp6-LHRH and increasing concentrations of the unlabeled peptide, also demonstrated the high affinity binding site as well as the putative low affinity site, with about Veooth of the Ka of the high affinity site (Fig. 2). The two-site model provided a better fit also in displacement experiments with antagonist SB-75 as labeled and unlabelled peptide (Kda = 1.4 x 10~9 M and Kdb = 0.14 X 10~6 M). In a separate experiment, antagonist SB-75 completely displaced labeled D-Trp6-LHRH from both binding sites on membranes of control Dunning tumors with Kda of 1.59 X 10"9 M and Kdb of 0.25 X 10~6 M.
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RECEPTORS FOR LHRH FIG. 1. Association (•—•) and dissociation (A—A) kinetics of [125I, D-Trp6] LHRH (A) and 125I-SB-75 (B) binding to membranes from rat Dunning R3327 prostate cancer. A, The membranes were incubated in 0.15 ml with 0.28 nmol [125I, D-Trp6]-LHRH for the time intervals indicated in the absence (total binding) and presence (nonspecific binding) of 6 Mmol unlabeled D-Trp6-LHRH. Dissociation was initiated by the addition of 12 MMol of unlabeled D-Trp6-LHRH after 60 min incubation at 4 C. B, The membranes were incubated in 0.15 ml with 0.25 nMol 125I-SB-75 at 4 C for the time intervals indicated in the absence (total binding) and presence (nonspecific binding) of 4 jtmol unlabeled SB-75. Dissociation was initiated by the addition of 8 jtmol unlabeled after 60 min incubation at 4 C. Specific binding represents the difference between total and nonspecific binding. All values given represent the extent of binding expressed as the percent of total radioactivity added to each tube. Each point is the average value ± SE of triplicate determinations.
cr
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3.0 2.5
JT-
2.0
1.5 1.0 0.5 0.0
60
120
180
240
TIME ( min ) 4.0
1.0
0.0' 60
120
160
240
TIME ( min )
2.10 T
receptors for LHRH on membranes of Dunning tumors. Administration of microcapsules of SB-75 (71.4 jug/day) highly significantly (P < 0.01) increased the binding capacity (130.40 ± 24.24 fmol/mg membrane protein) and the Kd (2.99 ± 0.32 X 10"9 M) for high affinity membrane binding sites (Table 1). Kd and Bmax of low affinity binding sites were slightly, but not significantly, increased after the highest dose of SB-75.
1.90--
1 . 7 0 ••
1.50 •
1.30 -11.0
LHRH receptors in pituitary membranes from rats bearing Dunning R-3327 prostate cancer
-9.0 -8.0 -7.0 -6.0 -5.0 [D-Trp6]LH-RH, LOG TOTAL (M) FIG. 2. A, Displacement of [125I, D-Trp6]LHRH by increasing amounts of unlabeled D-Trp6-LHRH using membranes of rat Dunning R3327 prostate cancer. The individual data points represent the average value ± SE of triplicate determinations. B, Scatchard plot analysis of data from displacement curves illustrated in A.
I
-10.0
The pooled data from six binding-inhibition experiments with D-Trp6-LHRH as labeled and unlabeled peptide using Dunning tumors from the control group gave an average Bmaxa of 48.76 ± 22.13 fmol/mg membrane proteins for high affinity, low capacity binding sites, and Bmaxb of 92.10 ± 29.40 pmol/mg membrane proteins for low affinity, high capacity binding sites (Table 1). Prolonged treatment with microcapsules of LHRH agonist D-Trp6-LHRH resulted in significant (P < 0.01) reduction of both Kd and Bmax of low affinity receptors for LHRH. The lower doses of SB-75 (23.8 and 47.6 /xg/day) did not significantly change binding characteristics of
Binding kinetics. Specific binding of [125I]-SB-75 to rat pituitary membrane particles was time dependent (Fig. 3). The dissociation followed the simple first order of kinetics. The dissociation rate constants (K-0 obtained by addition of 8 /uM unlabeled peptide after incubation of pituitary particles with 0.25 nM [125I]-SB-75 for 1 h at 4 C was 0.020422 min"1 (half-time, \>/i = 33.934 min). The value of K_i and the corresponding association rate constant (KJ value of 9.76 X 106 M"1 min"1 gave an estimate for the equilibrium Kd of 2.09 X 10"9 M. The monoexponential model gave significantly better (P < 0.05) fit than the biexponential model. SB-75 was completely displaceable from its binding sites on pituitary membranes in a time-dependent manner. The Ka of [125I-D-Trp6]-LHRH binding to pituitary membranes followed a complex kinetics order. The biexponential model gave better fit than the monoexponential model. Kds from kinetics behavior were calculated to
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Endo •1990 Vol 127•No 6
RECEPTORS FOR LHRH
TABLE 1. Characteristics of D-Trp6-LHRH binding in membranes of Dunning R3327 prostate cancer in rats after in vivo treatment with
microcapsules of LHRH analogs Treatment Control, untreated D-Trp6-LHRH (25 Mg/day) SB-75 (23.8 Mg/day) SB-75 (47.6 Mg/day) SB-75 (71.4 Mg/day)
Kda (X 10" 9 M) 1.01 0.40 2.59 1.64 2.99
± 0.30 ± 0.07 ± 1.33 ± 0.21 ± 0.32°
Bmaxa (fmol/mg protein) 48.66 16.25 70.34 55.50 130.42
6 ( X I L0~ M)
1.71 0.41 2.67 3.81 2.09
± 22.13 ± 4.83 ± 22.11 ± 10.53 ± 24.24*
Bma»b (pmol/mg protein)
±0.41 ± 0.16° ± 1.42 ± 1.10 ±0.88
92.10 ± 29.40 6.34 ± 2.40° 96.40 ± 33.20 153.10 ± 35.14 157.52 ± 39.13
Binding characteristics were obtained from 10-point displacement experiments in triplicate tubes. Significance was calculated with two-sided Student's t test. Values represent mean ± SE. °P
